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Fabrication of out-of-plane microneedles for drug delivery and biosensing Mansoor, Iman 2014

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FABRICATION	 ?OF	 ?OUT-??OF-??PLANE	 ?MICRONEEDLES	 ?FOR	 ?DRUG	 ?DELIVERY	 ?AND	 ?BIOSENSING	 ?	 ? by	 ?	 ?Iman	 ?Mansoor	 ?	 ?M.A.Sc.,	 ?The	 ?University	 ?of	 ?British	 ?Columbia,	 ?2009	 ?	 ?A	 ?THESIS	 ?SUBMITTED	 ?IN	 ?PARTIAL	 ?FULFILMENT	 ?OF	 ?THE	 ?REQUIREMENTS	 ?FOR	 ?THE	 ?DEGREE	 ?OF	 ?	 ?	 ?DOCTOR	 ?OF	 ?PHILOSOPHY	 ?in	 ?The	 ?Faculty	 ?of	 ?Graduate	 ?and	 ?Postdoctoral	 ?Studies	 ?(Electrical	 ?and	 ?Computer	 ?Engineering)	 ?	 ?THE	 ?UNIVERSITY	 ?OF	 ?BRITISH	 ?COLUMBIA	 ?(Vancouver)	 ?March	 ?2014	 ??	 ?Iman	 ?Mansoor,	 ?2014	 ?ii	 ?	 ?ABSTRACT	 ?Hollow	 ?microneedles	 ? can	 ? be	 ? used	 ? to	 ? painlessly	 ? inject	 ? drugs	 ? or	 ? extract	 ? dermal	 ? interstitial	 ?fluid	 ? for	 ?biosensing.	 ?However,	 ? their	 ? fabrication	 ?so	 ?far	 ?has	 ?been	 ?associated	 ?with	 ?costly	 ?and	 ?time-??consuming	 ? steps	 ? restricting	 ? their	 ? batch	 ? production	 ? as	 ? a	 ? viable	 ? option.	 ? This	 ? thesis	 ?presents	 ?novel	 ?methods	 ? for	 ? fabricating	 ? inexpensive	 ?hollow	 ?microneedles,	 ?and	 ? investigates	 ?new	 ? methods	 ? of	 ? characterizing	 ? the	 ? drug	 ? delivery	 ? and	 ? interstitial	 ? fluid	 ? sampling	 ? using	 ?microneedles.	 ?	 ?First,	 ? a	 ? method	 ? is	 ? presented	 ? for	 ? fabrication	 ? of	 ? hollow	 ? polymer	 ? microneedle	 ? arrays.	 ?Microneedles	 ? are	 ? formed	 ? during	 ? a	 ? solvent	 ? casting	 ? process,	 ?which	 ? leaves	 ? a	 ? polymer	 ? layer	 ?around	 ?pillars	 ? in	 ?a	 ?pre-??fabricated	 ?mold.	 ?Arrays	 ?of	 ?microneedles	 ?with	 ?lengths	 ?up	 ?to	 ?250	 ??m	 ?have	 ?been	 ? fabricated.	 ? The	 ? strength	 ?of	 ? the	 ?microneedles	 ?was	 ?evaluated	 ? to	 ?ensure	 ? reliable	 ?skin	 ? penetration	 ? and	 ? their	 ? suitability	 ? for	 ? drug	 ? delivery	 ? was	 ? demonstrated	 ? by	 ? injection	 ? of	 ?fluorescent	 ?beads	 ?into	 ?a	 ?skin	 ?sample.	 ?	 ?A	 ? second	 ? fabrication	 ? method	 ? is	 ? presented	 ? for	 ? making	 ? metallic	 ? microneedles	 ? with	 ? high	 ?aspect	 ?ratios.	 ?Solvent	 ?casting	 ?was	 ?used	 ?to	 ?coat	 ?a	 ?mold	 ?with	 ?a	 ?conductive	 ?polymer	 ?composite	 ?layer,	 ?which	 ?was	 ? then	 ? used	 ? as	 ? a	 ? seed	 ? layer	 ? in	 ? a	 ?metal	 ? electrodeposition	 ? process	 ? to	 ? form	 ?500	 ??m	 ? tall	 ? microneedles.	 ? Some	 ? fabrication	 ? process	 ? steps	 ? were	 ? characterized	 ? and	 ? the	 ?strength	 ? of	 ? the	 ?microneedles	 ? was	 ? evaluated.	 ? Their	 ? usefulness	 ? for	 ? drug	 ? delivery	 ? was	 ? also	 ?demonstrated	 ?by	 ?injection	 ?of	 ?fluorescent	 ?microspheres	 ?into	 ?animal	 ?skin.	 ?	 ?iii	 ?	 ?Designing	 ?effective	 ?microneedles	 ?requires	 ?understanding	 ?the	 ?drug	 ?diffusion	 ?process	 ?in	 ?skin.	 ?Here,	 ?a	 ?novel	 ?method	 ?is	 ?used	 ?to	 ?characterize	 ?diffusion	 ?of	 ?a	 ?chemotherapeutic	 ?drug	 ?injected	 ?with	 ?microneedles	 ? into	 ? skin.	 ?Using	 ? confocal	 ?microscopy,	 ? the	 ? concentration	 ?distribution	 ?of	 ?the	 ?drug	 ?was	 ?measured	 ?over	 ? time	 ?and	 ?then	 ?compared	 ?to	 ?an	 ?analytical	 ?diffusion	 ?model	 ? to	 ?obtain	 ?the	 ?drug?s	 ?diffusion	 ?coefficient.	 ?Using	 ?this	 ?method,	 ?different	 ?skin	 ?storage	 ?conditions	 ?were	 ?evaluated.	 ? It	 ?was	 ?concluded	 ? that	 ?using	 ?previously	 ? frozen	 ?skin	 ? should	 ?be	 ?avoided	 ? for	 ?transdermal	 ?drug	 ?delivery	 ?studies.	 ?	 ?Finally,	 ? using	 ? the	 ?proposed	 ?processes,	 ? hollow	 ?and	 ? solid	 ?microneedles	 ?were	 ? fabricated	 ? for	 ?sampling	 ? interstitial	 ? fluid	 ? for	 ? biosensing	 ? applications.	 ? Minimal	 ? removal	 ? of	 ? the	 ? interstitial	 ?fluid	 ?was	 ?achieved	 ?with	 ?a	 ?solid	 ?microneedle	 ?design	 ?as	 ?well	 ?as	 ?a	 ?hollow	 ?metallic	 ?microneedle	 ?array	 ? attached	 ? to	 ? a	 ? vacuum	 ?probe,	 ?while	 ? no	 ? trace	 ? of	 ? the	 ? fluid	 ?was	 ? observed	 ?when	 ? using	 ?hollow	 ?polymer	 ?microneedles.	 ?	 ?	 ? 	 ?iv	 ?	 ?PREFACE	 ?The	 ? research	 ? presented	 ? in	 ? this	 ? dissertation	 ? was	 ? carried	 ? out	 ? at	 ? the	 ? University	 ? of	 ? British	 ?Columbia	 ? (UBC),	 ? in	 ? the	 ? Stoeber	 ? laboratory	 ? at	 ? the	 ? department	 ? of	 ? Electrical	 ? and	 ?Computer	 ?Engineering,	 ?under	 ?supervision	 ?of	 ?Dr.	 ?Boris	 ?Stoeber.	 ?This	 ?research	 ?was	 ?performed	 ? in	 ?close	 ?collaboration	 ? with	 ? Dr.	 ? Urs	 ? O.	 ? H?feli	 ? from	 ? UBC?s	 ? Faculty	 ? of	 ? Pharmaceutical	 ? Sciences.	 ? A	 ?portion	 ?of	 ?the	 ?research	 ?was	 ?also	 ?conducted	 ?at	 ?the	 ?BC	 ?Child	 ?and	 ?Family	 ?Research	 ?Institute	 ?in	 ?collaboration	 ? with	 ? Dr.	 ? Jan	 ? Dutz	 ? and	 ? Dr.	 ? Jacqueline	 ? Lai	 ? from	 ? UBC?s	 ? Department	 ? of	 ?Dermatology	 ?&	 ?Skin	 ?Science.	 ?Chapter	 ?1	 ?of	 ?this	 ?thesis	 ?starts	 ?with	 ?a	 ?brief	 ?overview	 ?of	 ?the	 ?transdermal	 ?drug	 ?delivery	 ?and	 ?the	 ?anatomy	 ? of	 ? the	 ? mammalian	 ? skin.	 ? Next,	 ? after	 ? discussing	 ? the	 ? basic	 ? concepts	 ? of	 ?microelectromechanical	 ?system	 ?(MEMS)	 ?fabrication	 ?technologies,	 ?it	 ?is	 ?discussed	 ?how	 ?MEMS	 ?can	 ?be	 ?used	 ?to	 ?fabricate	 ?microneedle	 ?systems	 ?which	 ?can	 ?significantly	 ?enhance	 ?transdermal	 ?drug	 ? delivery	 ? by	 ? overcoming	 ? the	 ? barriers	 ? and	 ? limitations	 ? of	 ? traditional	 ? drug	 ? delivery	 ?systems.	 ? This	 ? discussion	 ? is	 ? then	 ? followed	 ? by	 ? a	 ? detailed	 ? literature	 ? review	 ? on	 ? the	 ? previous	 ?fabrication	 ? methods	 ? used	 ? to	 ? make	 ? microneedle	 ? devices	 ? with	 ? different	 ? shapes	 ? and	 ? from	 ?various	 ?structural	 ?materials.	 ?Chapter	 ?2	 ?is	 ?based	 ?on	 ?the	 ?work	 ?published	 ?as	 ?the	 ?following	 ?journal	 ?article:	 ?? I.	 ? Mansoor,	 ? U.	 ? O.	 ? H?feli,	 ? and	 ? B.	 ? Stoeber,	 ? ?Hollow	 ? Out-??of-??plane	 ? Polymer	 ?Microneedles	 ?Made	 ?by	 ? Solvent	 ? Casting	 ? for	 ? Transdermal	 ?Drug	 ?Delivery,?	 ? Journal	 ? of	 ?Microelectromechanical	 ?Systems,	 ?vol.	 ?21(1),	 ?pp.	 ?44	 ??	 ?52,	 ?2012.	 ?	 ?v	 ?	 ?This	 ?work	 ?was	 ?also	 ?presented	 ?in	 ?the	 ?following	 ?workshops	 ?and	 ?conferences:	 ?? I.	 ?Mansoor,	 ?U.O.	 ?H?feli,	 ?and	 ?B.	 ?Stoeber,	 ??Arrays	 ?of	 ?Solvent	 ?Cast	 ?Hollow	 ?Out-??of-??plane	 ?Polymer	 ? Microneedles	 ? for	 ? Drug	 ? Delivery?,	 ? Proceedings	 ? of	 ? 2011	 ? IEEE	 ? 24th	 ?International	 ?Conference	 ?on	 ?MEMS,	 ?pp.	 ?1027-??1030,	 ?2011.	 ?? I.	 ? Mansoor,	 ? U.	 ? O.	 ? H?feli,	 ? and	 ? B.	 ? Stoeber,	 ? ?Hollow	 ? Out-??of-??plane	 ? Polymer	 ?Microneedles	 ? for	 ? Drug	 ? Delivery,?	 ? ICICS	 ? Biomedical	 ? Engineering	 ? Workshop,	 ?Vancouver,	 ?Canada,	 ?2010.	 ?? J.	 ?Lai,	 ?I.	 ?Mansoor,	 ?B.	 ?Stoeber,	 ?T.	 ?Esposito,	 ?U.	 ?O.	 ?H?feli,	 ?and	 ?J.	 ?Dutz,	 ??The	 ?Use	 ?of	 ?Novel	 ?Delivery	 ?Methods	 ? for	 ? the	 ? Transcutaneous	 ? Delivery	 ? of	 ? CpG	 ? ODN	 ? Adjuvants	 ? to	 ? the	 ?Skin,?	 ?Immunological	 ?Mechanisms	 ?of	 ?Vaccination,	 ?Seattle,	 ?WA,	 ?USA,	 ?2010.	 ?	 ?These	 ? papers	 ? present	 ? novel	 ? fabrication	 ? methods	 ? for	 ? hollow	 ? out-??of-??plane	 ? polymer	 ?microneedle	 ?arrays	 ?used	 ?for	 ?painless	 ?administration	 ?of	 ?compounds	 ?into	 ?the	 ?skin.	 ?Being	 ?cost	 ?effective	 ? and	 ? applicable	 ? for	 ? batch	 ? processing,	 ? the	 ? presented	 ? process	 ? uses	 ? an	 ? inexpensive	 ?polymer	 ?solvent	 ?casting	 ?technique	 ?to	 ?form	 ?disposable	 ?microneedle	 ?arrays	 ?that	 ?can	 ?be	 ?used	 ?for	 ?a	 ?variety	 ?of	 ?transdermal	 ?drug	 ?delivery	 ?applications.	 ?The	 ?proposed	 ?procedure	 ?was	 ?used	 ?to	 ?make	 ?microneedles	 ?with	 ?heights	 ?of	 ?up	 ?to	 ?250	 ??m.	 ?The	 ?devices	 ?were	 ?characterized	 ?through	 ?drug	 ? delivery	 ? trials	 ? and	 ? their	 ? strength	 ? was	 ? evaluated	 ? to	 ? ensure	 ? reliable	 ? penetration	 ? into	 ?skin.	 ?Chapter	 ?3	 ?of	 ?this	 ?thesis	 ?is	 ?based	 ?on	 ?the	 ?work	 ?published	 ?as	 ?the	 ?following	 ?journal	 ?article:	 ?? I.	 ? Mansoor,	 ? Y.	 ? Liu,	 ? U.	 ? O.	 ? H?feli,	 ? and	 ? B.	 ? Stoeber,	 ? ?Arrays	 ? of	 ? Hollow	 ? Out-??of-??plane	 ?Microneedles	 ? Made	 ? by	 ? Metal	 ? Electrodeposition	 ? onto	 ? Solvent	 ? Cast	 ? Conductive	 ?vi	 ?	 ?Polymer	 ? Structures,?	 ? Journal	 ? of	 ? Micromechanics	 ? and	 ? Microengineering,	 ? vol.	 ? 23,	 ?085011,	 ?2013.	 ?This	 ?work	 ?was	 ?also	 ?presented	 ?in	 ?the	 ?following	 ?workshops	 ?and	 ?conferences:	 ?? I.	 ?Mansoor,	 ?J.	 ?Lai,	 ?D.	 ?Lambert,	 ?J.	 ?Dutz,	 ?U.	 ?O.	 ?H?feli,	 ?and	 ?B.	 ?Stoeber,	 ??Hollow	 ?Metallic	 ?Microneedles	 ? for	 ? Transdermal	 ? Delivery	 ? of	 ? Compounds,?	 ? in	 ? IEEE	 ? 35th	 ? International	 ?Conference	 ?on	 ?Engineering	 ?in	 ?Medicine	 ?and	 ?Biology	 ?Society,	 ?Osaka,	 ?Japan,	 ?2013.	 ?? I.	 ?Mansoor,	 ?Y.	 ?Liu,	 ?U.	 ?O.	 ?H?feli,	 ?and	 ?B.	 ?Stoeber,	 ??Fabrication	 ?of	 ?Hollow	 ?Microneedle	 ?Arrays	 ? Using	 ? Electrodeposition	 ? of	 ? Metal	 ? onto	 ? Solvent	 ? Cast	 ? Conductive	 ? Polymer	 ?Structures,?	 ? in	 ?17th	 ? International	 ?Conference	 ?on	 ?Solid-??State	 ?Sensors,	 ?Actuators	 ?and	 ?Microsystems,	 ?pp.	 ?373-??376,	 ?2013.	 ?? I.	 ? Mansoor,	 ? S.	 ? Ranamukhaarachchi,	 ? V.	 ? Schmitt,	 ? D.	 ? Lambert,	 ? U.	 ? O.	 ? H?feli,	 ? and	 ? B.	 ?Stoeber,	 ??Hollow	 ?Microneedles	 ?for	 ?Transdermal	 ?Drug	 ?Delivery	 ?and	 ?Biosensing,?	 ?ECE	 ?Open	 ?Research	 ?Day	 ?Workshop,	 ?Vancouver,	 ?Canada	 ?2013.	 ?In	 ? addition,	 ? a	 ? provisional	 ? patent	 ? application	 ? has	 ? been	 ? filed	 ? based	 ? on	 ? the	 ? work	 ? in	 ? this	 ?chapter:	 ?? I.	 ?Mansoor,	 ?B.	 ?Stoeber,	 ?and	 ?U.	 ?O.	 ?H?feli,	 ??Method	 ?for	 ?Forming	 ?Out-??of-??plane	 ?Metallic	 ?Microneedles	 ? and	 ? Devices	 ? Formed	 ? Thereby,?	 ? US	 ? patent	 ? pending	 ? (application	 ?#61834482),	 ?2013.	 ?In	 ? this	 ? chapter,	 ? a	 ? process	 ? is	 ? presented	 ? for	 ? fabricating	 ? inexpensive	 ? hollow	 ? out-??of-??plane	 ?microneedle	 ? materials	 ? from	 ? metals.	 ? The	 ? proposed	 ? process	 ? is	 ? a	 ? combination	 ? of	 ? solvent	 ?vii	 ?	 ?casting	 ?and	 ?metal	 ?electroplating.	 ?It	 ?allows	 ?fabricating	 ?microneedles	 ?with	 ?large	 ?aspect	 ?ratios.	 ?The	 ?microneedles	 ? can	 ?be	 ?much	 ? longer	 ? than	 ? the	 ?polymer	 ?needles	 ? in	 ? the	 ?previous	 ? chapter	 ?and	 ?their	 ? length	 ? is	 ?only	 ? limited	 ?by	 ? the	 ?height	 ?of	 ? the	 ?solvent	 ?casting	 ?mold	 ?structure.	 ?Some	 ?process	 ? parameters	 ?were	 ? characterized	 ? and	 ? the	 ? needles	 ? fabricated	 ?were	 ? tested	 ? for	 ? drug	 ?delivery	 ? and	 ? mechanical	 ? strength.	 ? My	 ? contributions	 ? in	 ? the	 ? corresponding	 ? journal	 ?publication	 ? as	 ? a	 ? first	 ? author	 ? are:	 ? designing	 ? the	 ? fabrication	 ? procedure,	 ? fabricating	 ?microneedles,	 ? characterizing	 ? the	 ? plasma	 ? etching	 ? process,	 ? carrying	 ? out	 ? some	 ? of	 ? the	 ?conductivity	 ?and	 ? resistivity	 ?measurements,	 ? carrying	 ?out	 ?mechanical	 ? tests,	 ?performing	 ? skin	 ?penetration	 ? and	 ? injection	 ? trials,	 ? performing	 ? a	 ? detailed	 ? literature	 ? review,	 ? and	 ? writing	 ? the	 ?manuscript.	 ?Chapter	 ?4	 ?of	 ?this	 ?thesis	 ?is	 ?based	 ?on	 ?a	 ?manuscript	 ?that	 ?is	 ?ready	 ?for	 ?submission	 ?to	 ?a	 ?journal:	 ?? I.	 ? Mansoor,	 ? J.	 ? Lai,	 ? S.	 ? Ranamukhaarachchi,	 ? V.	 ? Schmitt,	 ? D.	 ? Lambert,	 ? J.	 ? Dutz,	 ? U.	 ? O.	 ?H?feli,	 ? and	 ? B.	 ? Stoeber,	 ? ?Studying	 ? Diffusion	 ? of	 ? a	 ? Microneedle-??Injected	 ? Drug	 ? Inside	 ?Skin:	 ?Injection	 ?of	 ?Doxorubicin	 ?into	 ?Pig	 ?Skin.?	 ?This	 ? chapter	 ? focuses	 ? on	 ? the	 ? drug	 ? delivery	 ? application	 ? of	 ?microneedles.	 ?More	 ? specifically,	 ?metallic	 ?microneedles	 ?are	 ?used	 ?to	 ?inject	 ?a	 ?chemotherapeutic	 ?compound	 ?into	 ?the	 ?skin.	 ?Using	 ?confocal	 ?microscopy,	 ?the	 ?drug	 ?diffusion	 ?in	 ?skin	 ?is	 ?investigated	 ?and	 ?compared	 ?to	 ?an	 ?analytical	 ?diffusion	 ? model	 ? to	 ? measure	 ? the	 ? diffusion	 ? coefficient	 ? of	 ? the	 ? drug	 ? in	 ? the	 ? skin.	 ? My	 ?contributions	 ? in	 ? this	 ? journal	 ? publication	 ? as	 ? a	 ? first	 ? author	 ? would	 ? be:	 ? performing	 ? the	 ?calibration	 ? tests,	 ? preparing	 ? the	 ? injection	 ? setup,	 ? carrying	 ? out	 ? all	 ? of	 ? the	 ? injection	 ? tests,	 ?viii	 ?	 ?carrying	 ? out	 ? the	 ? imaging	 ? process,	 ? carrying	 ? out	 ? the	 ? diffusion	 ?measurements	 ? and	 ? analysis,	 ?performing	 ?a	 ?detailed	 ?literature	 ?review,	 ?and	 ?writing	 ?the	 ?manuscript.	 ?Chapter	 ? 5	 ? of	 ? this	 ? dissertation	 ? is	 ? based	 ? on	 ? the	 ? research	 ? performed	 ? towards	 ? using	 ? the	 ?microneedles	 ?for	 ?biosensing	 ?applications.	 ?More	 ?specifically,	 ?the	 ?needles	 ?fabricated	 ?through	 ?the	 ? processes	 ? similar	 ? to	 ? those	 ? presented	 ? in	 ? Chapters	 ? 2	 ? and	 ? 3	 ? were	 ? used	 ? to	 ? extract	 ?interstitial	 ?fluid	 ?from	 ?skin.	 ?A	 ?version	 ?of	 ?one	 ?of	 ?the	 ?techniques	 ?presented	 ?in	 ?this	 ?chapter	 ?was	 ?presented	 ?in	 ?the	 ?following	 ?conference:	 ?? I.	 ?Mansoor,	 ? U.	 ? O.	 ? H?feli,	 ? and	 ? B.	 ? Stoeber,	 ? ?Arrays	 ? of	 ? Solvent	 ? Cast	 ? Hollow	 ? Polymer	 ?Microneedles	 ?for	 ?Biosensing	 ?Applications,?	 ?Microneedles	 ?Conference,	 ?Cork,	 ?Ireland,	 ?2012.	 ?The	 ?last	 ?chapter	 ?gives	 ?a	 ?summary	 ?of	 ?the	 ?previous	 ?chapters	 ?as	 ?well	 ?as	 ?a	 ?discussion	 ?of	 ?possible	 ?future	 ?works	 ?than	 ?can	 ?be	 ?pursued	 ?to	 ?improve	 ?the	 ?presented	 ?microneedle	 ?technology.	 ?The	 ?scientific	 ?contributions	 ?of	 ?the	 ?work	 ?in	 ?this	 ?thesis	 ?can	 ?be	 ?summarized	 ?as	 ?following:	 ?I. Demonstrating	 ?a	 ?novel	 ?fabrication	 ?method,	 ?based	 ?on	 ?solvent	 ?casting	 ?technology,	 ?to	 ?make	 ? hollow	 ? polymer	 ? microneedle	 ? arrays	 ? for	 ? drug	 ? delivery	 ? and	 ? biosensing.	 ? The	 ?proposed	 ?process	 ? is	 ? among	 ? the	 ?most	 ? inexpensive	 ?proposed	 ? fabrication	 ? techniques	 ?for	 ? polymer	 ? microneedles	 ? and	 ? is	 ? suitable	 ? for	 ? commercial	 ? adoption	 ? of	 ? the	 ?microneedle	 ? technology.	 ? It	 ? allows	 ? fabricating	 ? microneedles	 ? with	 ? any	 ? polymer	 ?material	 ?that	 ?can	 ?be	 ?cast,	 ?and	 ?allows	 ?a	 ?wide	 ?range	 ?of	 ?dimensions.	 ?ix	 ?	 ?II. Demonstrating	 ?a	 ?novel	 ? fabrication	 ?process,	 ?based	 ?on	 ?metal	 ?electrodeposition	 ?onto	 ?cast	 ?conductive	 ?polymer	 ?film,	 ?to	 ?make	 ?hollow	 ?metallic	 ?microneedle	 ?arrays	 ?for	 ?drug	 ?delivery	 ? and	 ? biosensing.	 ? The	 ? proposed	 ? process	 ? is	 ? among	 ? the	 ? most	 ? inexpensive	 ?proposed	 ? fabrication	 ? techniques	 ? for	 ? metallic	 ? microneedles	 ? and	 ? is	 ? suitable	 ? for	 ?commercial	 ? adoption	 ? of	 ? the	 ? microneedle	 ? technology.	 ? It	 ? allows	 ? fabricating	 ?microneedles	 ? with	 ? any	 ? electrodepositable	 ? metal	 ? and	 ? allows	 ? a	 ? wide	 ? range	 ? of	 ?dimensions	 ?and	 ?spacing.	 ?III. Demonstrating	 ? a	 ? novel	 ? technique	 ? to	 ? characterize	 ? transdermal	 ? drug	 ? delivery	 ? with	 ?hollow	 ? microneedles.	 ? The	 ? proposed	 ? technique	 ? uses	 ? fluorescent	 ? imaging	 ? to	 ?investigate	 ?diffusion	 ?of	 ?a	 ?microneedle-??injected	 ?drug	 ?in	 ?skin.	 ?This	 ?process	 ?can	 ?be	 ?used	 ?to	 ? analyze	 ? the	 ? usefulness	 ? of	 ? microneedles	 ? for	 ? delivery	 ? of	 ? any	 ? stable	 ? fluorescent	 ?compound.	 ? This	 ? technique	 ? also	 ? allows	 ? measuring	 ? the	 ? diffusion	 ? coefficient	 ? of	 ? the	 ?compound	 ?in	 ?artificial	 ?and	 ?biological	 ?skin	 ?models.	 ?	 ?IV. Evaluating	 ? three	 ? potential	 ? interstitial	 ? fluid	 ? extraction	 ? techniques	 ? using	 ? solid	 ? and	 ?hollow	 ? microneedles,	 ? to	 ? demonstrate	 ? which	 ? technique	 ? is	 ? more	 ? promising	 ? for	 ?developing	 ?microneedle-??based	 ?biosensing	 ?systems.	 ?	 ?	 ?	 ?	 ? 	 ?x	 ?	 ?TABLE	 ?OF	 ?CONTENTS	 ?Abstract	 ?....................................................................................................................................	 ?ii	 ?Preface	 ?....................................................................................................................................	 ?iv	 ?Table	 ?of	 ?Contents	 ?.....................................................................................................................	 ?x	 ?List	 ?of	 ?Tables	 ?...........................................................................................................................	 ?xv	 ?List	 ?of	 ?Figures	 ?........................................................................................................................	 ?xvi	 ?List	 ?of	 ?Variables	 ?....................................................................................................................	 ?xxv	 ?Acknowledgements	 ?.............................................................................................................	 ?xxvi	 ?Dedication	 ?...........................................................................................................................	 ?xxvii	 ?Chapter	 ?1:	 ?Introduction	 ?............................................................................................................	 ?1	 ?1.1	 ? Transdermal	 ?drug	 ?delivery	 ?.......................................................................................................	 ?3	 ?1.1.1	 ? Structure	 ?of	 ?skin	 ?................................................................................................................	 ?3	 ?1.1.2	 ? Adhesive	 ?skin	 ?patches	 ?.......................................................................................................	 ?6	 ?1.2	 ? MEMS	 ?for	 ?biomedical	 ?applications,	 ?drug	 ?delivery,	 ?and	 ?biosensing	 ?........................................	 ?10	 ?1.3	 ? Microneedles	 ?as	 ?a	 ?minimally	 ?invasive	 ?interface	 ?with	 ?the	 ?body	 ?.............................................	 ?12	 ?1.3.1	 ? In-??plane	 ?solid	 ?and	 ?hollow	 ?microneedles	 ?.........................................................................	 ?14	 ?1.3.2	 ? Solid	 ?out-??of-??plane	 ?microneedles	 ?.....................................................................................	 ?16	 ?1.3.3	 ? Hollow	 ?out-??of-??plane	 ?microneedles	 ?.................................................................................	 ?17	 ?Chapter	 ?2:	 ?Fabrication	 ?of	 ?Polymer	 ?Microneedles	 ?Based	 ?on	 ?Solvent	 ?Casting	 ?.........................	 ?25	 ?2.1	 ? Fabrication	 ?of	 ?hollow	 ?out-??of-??plane	 ?polymer	 ?microneedles	 ?...................................................	 ?26	 ?2.1.1	 ? Microneedle	 ?material	 ?selection	 ?......................................................................................	 ?26	 ?2.1.2	 ? Fabrication	 ?process	 ?.........................................................................................................	 ?29	 ?xi	 ?	 ?2.1.3	 ? Contact	 ?angle	 ?measurement	 ?of	 ?NMP	 ?on	 ?a	 ?PDMS	 ?surface	 ?...............................................	 ?35	 ?2.2	 ? Experimental	 ?procedures	 ?for	 ?needle	 ?characterization	 ?...........................................................	 ?37	 ?2.2.1	 ? Microneedle	 ?robustness	 ?tests	 ?.........................................................................................	 ?37	 ?2.2.2	 ? Microneedle	 ?injection	 ?tests	 ?............................................................................................	 ?37	 ?2.3	 ? Results	 ?and	 ?Discussion	 ?...........................................................................................................	 ?39	 ?2.3.1	 ? Microneedle	 ?robustness	 ?.................................................................................................	 ?39	 ?2.3.2	 ? Results	 ?of	 ?injection	 ?tests	 ?.................................................................................................	 ?41	 ?2.4	 ? Conclusions	 ?............................................................................................................................	 ?43	 ?Chapter	 ?3:	 ?Fabrication	 ?of	 ?Metallic	 ?Microneedles	 ?using	 ?Electrodeposition	 ?of	 ?Metal	 ?onto	 ?Conductive	 ?Polymer	 ?Films	 ?......................................................................................................	 ?45	 ?3.1	 ? Fabrication	 ?of	 ?hollow	 ?metallic	 ?microneedles	 ?.........................................................................	 ?47	 ?3.1.1	 ? Fabrication	 ?of	 ?the	 ?mold	 ?...................................................................................................	 ?47	 ?3.1.2	 ? Deposition	 ?of	 ?the	 ?polymer-??based	 ?conductive	 ?seed	 ?layer	 ?...............................................	 ?49	 ?3.1.3	 ? Metal	 ?deposition	 ?.............................................................................................................	 ?51	 ?3.1.4	 ? Microneedle	 ?array	 ?lift-??off	 ?................................................................................................	 ?53	 ?3.2	 ? Experimental	 ?procedures	 ?.......................................................................................................	 ?54	 ?3.2.1	 ? Conductivity	 ?measurements	 ?of	 ?PMMA/CB	 ?composites	 ?..................................................	 ?54	 ?3.2.2	 ? Dry	 ?etching	 ?of	 ?the	 ?PMMA/CB	 ?layer	 ?.................................................................................	 ?55	 ?3.2.3	 ? Characterization	 ?of	 ?the	 ?nickel	 ?electroplating	 ?process	 ?.....................................................	 ?57	 ?3.2.4	 ? Mechanical	 ?compression	 ?tests	 ?on	 ?the	 ?fabricated	 ?microneedles	 ?.....................................	 ?57	 ?3.2.5	 ? Fluid	 ?delivery	 ?into	 ?sample	 ?skin	 ?using	 ?the	 ?fabricated	 ?microneedles	 ?................................	 ?58	 ?3.3	 ? Results	 ?and	 ?discussion	 ?............................................................................................................	 ?59	 ?3.3.1	 ? Conductivity	 ?measurements	 ?...........................................................................................	 ?59	 ?3.3.2	 ? Dry	 ?etching	 ?of	 ?the	 ?PMMA/CB	 ?layer	 ?.................................................................................	 ?60	 ?xii	 ?	 ?3.3.3	 ? Nickel	 ?electroplating	 ?process	 ?..........................................................................................	 ?62	 ?3.3.4	 ? Mechanical	 ?compression	 ?tests	 ?........................................................................................	 ?64	 ?3.3.5	 ? Transdermal	 ?fluid	 ?delivery	 ?into	 ?sample	 ?skin	 ?...................................................................	 ?65	 ?3.4	 ? Conclusions	 ?............................................................................................................................	 ?67	 ?Chapter	 ?4:	 ?Studying	 ?Diffusion	 ?of	 ?a	 ?Microneedle-??injected	 ?Drug	 ?inside	 ?Skin:	 ?Injection	 ?of	 ?Doxorubicin	 ?into	 ?Pig	 ?Skin	 ?........................................................................................................	 ?69	 ?4.1	 ? Experimental	 ?procedures	 ?.......................................................................................................	 ?71	 ?4.1.1	 ? Materials	 ?.........................................................................................................................	 ?71	 ?4.1.2	 ? Injection	 ?setup	 ?................................................................................................................	 ?74	 ?4.1.3	 ? Injection	 ?procedure	 ?.........................................................................................................	 ?77	 ?4.1.4	 ? Confocal	 ?imaging	 ?.............................................................................................................	 ?78	 ?4.2	 ? Data	 ?analysis	 ?..........................................................................................................................	 ?78	 ?4.2.1	 ? Diffusion	 ?model	 ?...............................................................................................................	 ?78	 ?4.2.2	 ? Data	 ?processing	 ?...............................................................................................................	 ?81	 ?4.3	 ? Results	 ?and	 ?discussions	 ?..........................................................................................................	 ?82	 ?4.3.1	 ? Confocal	 ?data	 ?..................................................................................................................	 ?82	 ?4.3.2	 ? Diffusion	 ?measurements	 ?.................................................................................................	 ?84	 ?4.4	 ? Conclusions	 ?............................................................................................................................	 ?91	 ?Chapter	 ?5:	 ?Extraction	 ?of	 ?Interstitial	 ?Fluid	 ?using	 ?Microneedles	 ?...............................................	 ?92	 ?5.1	 ? Experimental	 ?procedures	 ?.......................................................................................................	 ?94	 ?5.1.1	 ? ISF	 ?extraction	 ?using	 ?solid	 ?polymer	 ?microneedles	 ?............................................................	 ?94	 ?5.1.2	 ? ISF	 ?extraction	 ?using	 ?hollow	 ?polymer	 ?microneedles	 ?........................................................	 ?97	 ?5.1.3	 ? ISF	 ?extraction	 ?using	 ?hollow	 ?metallic	 ?microneedles	 ?.......................................................	 ?101	 ?5.2	 ? Results	 ?and	 ?discussion	 ?..........................................................................................................	 ?102	 ?xiii	 ?	 ?5.2.1	 ? Solid	 ?microneedles	 ?........................................................................................................	 ?102	 ?5.2.2	 ? Hollow	 ?polymer	 ?microneedles	 ?......................................................................................	 ?103	 ?5.2.3	 ? Hollow	 ?metallic	 ?microneedles	 ?.......................................................................................	 ?104	 ?5.3	 ? Conclusions	 ?..........................................................................................................................	 ?104	 ?Chapter	 ?6:	 ?Summary	 ?and	 ?Future	 ?Work	 ?................................................................................	 ?106	 ?6.1	 ? Summary	 ?..............................................................................................................................	 ?106	 ?6.2	 ? Future	 ?work	 ?..........................................................................................................................	 ?110	 ?6.2.1	 ? Microneedle	 ?process	 ?improvement	 ?..............................................................................	 ?110	 ?6.2.1.1	 ? Mold	 ?..........................................................................................................	 ?110	 ?6.2.1.2	 ? Structural	 ?polymers	 ?and	 ?polymer	 ?solutions	 ?..............................................	 ?111	 ?6.2.1.3	 ? The	 ?electroplating	 ?process	 ?........................................................................	 ?111	 ?6.2.2	 ? Investigating	 ?the	 ?optimum	 ?needle	 ?height,	 ?diameter,	 ?spacing,	 ?and	 ?array	 ?size	 ?..............	 ?112	 ?6.2.3	 ? Drug	 ?delivery	 ?using	 ?microneedles	 ?.................................................................................	 ?113	 ?6.2.4	 ? Biofluid	 ?sampling	 ?using	 ?microneedles	 ?...........................................................................	 ?113	 ?References	 ?............................................................................................................................	 ?114	 ?Appendices	 ?...........................................................................................................................	 ?135	 ?A.1	 ? Detailed	 ?photolithography	 ?parameters	 ?and	 ?mold	 ?fabrication	 ?....................................	 ?135	 ?A.2	 ? Photolithography	 ?masks	 ?used	 ?for	 ?mold	 ?fabrication	 ?....................................................	 ?139	 ?A.3	 ? SEM	 ?images	 ?of	 ?polymer	 ?microneedles	 ?........................................................................	 ?140	 ?A.4	 ? Conductivity	 ?measurements	 ?.......................................................................................	 ?142	 ?A.5	 ? Plasma	 ?etch	 ?rate	 ?measurements	 ?................................................................................	 ?143	 ?A.6	 ? Nickel	 ?electroplating	 ?tests	 ?..........................................................................................	 ?144	 ?xiv	 ?	 ?A.7	 ? Additional	 ?data	 ?for	 ?the	 ?mechanical	 ?tests	 ?....................................................................	 ?146	 ?A.8	 ? Doxorubicin	 ?intensity-??concentration	 ?calibration	 ?........................................................	 ?147	 ?A.9	 ? MATLAB	 ?code	 ?used	 ?to	 ?calculate	 ?intensity	 ?distribution	 ?in	 ?the	 ?confocal	 ?images	 ?..........	 ?152	 ?A.10	 ? Confocal	 ?data	 ?for	 ?all	 ?of	 ?the	 ?injection	 ?trials	 ?...............................................................	 ?153	 ?	 ?	 ? 	 ?xv	 ?	 ?LIST	 ?OF	 ?TABLES	 ?Table	 ?2.1:	 ?Mean	 ?failure	 ? loads	 ? for	 ?different	 ?clay	 ?content	 ? in	 ?polyimide,	 ?obtained	 ?from	 ?three	 ?tests	 ?per	 ?clay	 ?content.	 ?............................................................................................................	 ?29	 ?Table	 ?2.2:	 ?Mean	 ? injection	 ?depth	 ?and	 ?95%	 ?confidence	 ? interval	 ? for	 ? the	 ? fluorescent	 ? intensity	 ?distribution	 ?of	 ?injected	 ?beads	 ?as	 ?in	 ?Figure	 ?2.12.	 ?....................................................................	 ?42	 ?Table	 ?4.1:	 ?Doxorubicin	 ?diffusion	 ?coefficient	 ?in	 ?epidermal	 ?tissue	 ?with	 ?units	 ?of	 ?[cm2/s]	 ?.........	 ?87	 ?Table	 ?4.2:	 ?Comparison	 ?of	 ?doxorubicin	 ?diffusion	 ?coefficient	 ?in	 ?different	 ?media	 ?.....................	 ?88	 ?Table	 ?5.1:	 ?Summary	 ?of	 ?the	 ?ISF	 ?extraction	 ?tests	 ?using	 ?14	 ?solid	 ?microneedles	 ?with	 ?a	 ?cellulose	 ?functionalization;	 ? the	 ?net	 ? extracted	 ? volume	 ? is	 ? calculated	 ?by	 ? subtracting	 ?0.18	 ?mg	 ? (average	 ?mass	 ?transferred	 ?during	 ?the	 ?control	 ?tests)	 ?from	 ?the	 ?mass	 ?differences,	 ?and	 ?using	 ?density	 ?of	 ?water	 ?at	 ?25?C	 ?(0.997	 ?g/ml)	 ?instead	 ?of	 ?that	 ?of	 ?ISF	 ?.................................................................	 ?103	 ?Table	 ?A.1:	 ?Conductivity	 ?and	 ? resistivity	 ?data	 ? for	 ?different	 ?CB	 ?content	 ? (three	 ?samples	 ?each).	 ?..............................................................................................................................................	 ?142	 ?Table	 ?A.2:	 ?Etch	 ?rates	 ?for	 ?different	 ?CF4	 ?proportion	 ?in	 ?O2/CF4	 ?gas	 ?combinations.	 ?..................	 ?143	 ?Table	 ?A.3:	 ?Nickel	 ?thickness	 ?from	 ?electroplating	 ?for	 ?different	 ?supply	 ?currents.	 ?....................	 ?144	 ?Table	 ?A.4:	 ?Nickel	 ?thickness	 ?from	 ?electroplating	 ?for	 ?different	 ?process	 ?durations.	 ?................	 ?145	 ?	 ? 	 ?xvi	 ?	 ?LIST	 ?OF	 ?FIGURES	 ?Figure	 ?1.1:	 ?Structure	 ?of	 ?mammalian	 ?skin	 ?[2].	 ?...........................................................................	 ?3	 ?Figure	 ?1.2:	 ?Structure	 ?of	 ?epidermis	 ?[4].	 ?.....................................................................................	 ?5	 ?Figure	 ?1.3:	 ?Photolithography	 ?process	 ?....................................................................................	 ?12	 ?Figure	 ?1.4:	 ?Comparison	 ?of	 ?orientation	 ?of	 ?in-??plane	 ?and	 ?out-??of-??plane	 ?microneedles,	 ?illustrated	 ?on	 ?a	 ?wafer	 ?substrate	 ?in	 ?stereoscopic	 ?view.	 ?In-??plane	 ?needles	 ?are	 ?arranged	 ?along	 ?the	 ?plane	 ?of	 ?the	 ? substrate	 ?while	 ? out-??of-??plane	 ? needles	 ? are	 ? arranged	 ? perpendicular	 ? to	 ? the	 ? plane	 ? of	 ? the	 ?substrate.	 ?...............................................................................................................................	 ?13	 ?Figure	 ?1.5:	 ?Fabrication	 ?of	 ?sharp	 ?cone-??shaped	 ?structures	 ?using	 ?silicon	 ?etching.	 ?.....................	 ?17	 ?Figure	 ?1.6:	 ?Concept	 ?of	 ?drug	 ?delivery	 ?using	 ?hollow	 ?microneedles.	 ?.........................................	 ?18	 ?Figure	 ?1.7:	 ?a)	 ? silicon	 ?microneedle	 ? fabrication	 ?process,	 ?b)	 ?SEM	 ? image	 ?of	 ?microneedles	 ?with	 ?symmetrical	 ?tips,	 ?c)	 ?SEM	 ?image	 ?of	 ?microneedles	 ?with	 ?pointed	 ?tips	 ?[56].	 ?..............................	 ?19	 ?Figure	 ?2.1:	 ?Setup	 ?used	 ?for	 ?compression	 ?tests.	 ?.......................................................................	 ?28	 ?Figure	 ? 2.2:	 ? Typical	 ? compression	 ? test	 ? curve	 ? for	 ? a	 ? test	 ? structure	 ? with	 ? 2	 ? wt%	 ? clay	 ?reinforcement.	 ?.......................................................................................................................	 ?28	 ?Figure	 ? 2.3:	 ? Fabrication	 ? process	 ? using	 ? solvent	 ? casting	 ? for	 ? hollow	 ? out-??of-??plane	 ? polymer	 ?microneedles,	 ? a	 ? &	 ? b)	 ? fabrication	 ? of	 ? pillars	 ? from	 ? SU-??8,	 ? c)	 ? PDMS	 ? deposition,	 ? d)	 ? O2	 ? plasma	 ?xvii	 ?	 ?treatment	 ?of	 ?the	 ?mold,	 ?e)	 ?deposition	 ?of	 ?a	 ?clay/polyimide	 ?suspension	 ?in	 ?NMP,	 ?f)	 ?Evaporation	 ?of	 ? NMP,	 ? g)	 ? removing	 ? of	 ? the	 ? microneedle	 ? array	 ? from	 ? the	 ? mold,	 ? h)	 ? opening	 ? of	 ? the	 ?microneedle	 ?tips.	 ?....................................................................................................................	 ?32	 ?Figure	 ?2.4:	 ?SEM	 ?image	 ?of	 ?an	 ?array	 ?of	 ?pillars	 ?in	 ?a	 ?mold	 ?used	 ?for	 ?microneedle	 ?fabrication.	 ?....	 ?32	 ?Figure	 ?2.5:	 ?A	 ?complete	 ?mold	 ?used	 ?for	 ?microneedle	 ?fabrication.	 ?............................................	 ?33	 ?Figure	 ? 2.6:	 ? SEM	 ? image	 ? of	 ? an	 ? array	 ? of	 ? solid	 ?microneedles	 ? formed	 ? in	 ? a	 ?mold	 ? after	 ? solvent	 ?evaporation.	 ?...........................................................................................................................	 ?33	 ?Figure	 ? 2.7:	 ? SEM	 ? images	 ? of	 ? fabricated	 ?microneedles:	 ? a)	 ? Array	 ? of	 ? 250	 ??m	 ? long	 ? needles,	 ? b)	 ?microneedle	 ?channel	 ?openings,	 ?c)	 ?a	 ?needle	 ?tip	 ?opened	 ?by	 ?plasma	 ?etching,	 ?and	 ?d)	 ?a	 ?needle	 ?tip	 ?opened	 ?by	 ?sanding.	 ?For	 ?more	 ?SEM	 ?images	 ?see	 ?Appendix	 ?A.3.	 ?.........................................	 ?34	 ?Figure	 ?2.8:	 ?NMP	 ?contact	 ?angle	 ?on	 ?a	 ?PDMS	 ?surface;	 ?a)	 ?contact	 ?angle	 ?measured	 ?3	 ?days	 ?after	 ?the	 ? PDMS	 ? surface	 ? was	 ? coated	 ? with	 ? a	 ? polyimide	 ? layer,	 ? 13	 ? minutes	 ? after	 ? removing	 ? the	 ?polyimide	 ?layer;	 ?b)	 ?contact	 ?angle	 ?measurements	 ?for	 ?NMP	 ?on	 ?a	 ?PDMS	 ?at	 ?different	 ?times	 ?after	 ?removing	 ? a	 ? polyimide	 ? layer	 ? that	 ? was	 ? deposited	 ? between	 ? 6	 ? hours	 ? and	 ? 6	 ? days	 ? prior	 ? to	 ?removal.	 ?.................................................................................................................................	 ?36	 ?Figure	 ?2.9:	 ?a)	 ?A	 ?microneedle	 ?array	 ?attached	 ?to	 ?a	 ?syringe,	 ?b)	 ?rabbit	 ?ear	 ?skin	 ?after	 ?application	 ?of	 ?the	 ?microneedles.	 ?..............................................................................................................	 ?39	 ?Figure	 ?2.10:	 ?a)	 ?A	 ?needle	 ?displacement	 ?under	 ?axial	 ?loading,	 ?b)	 ?a	 ?failed	 ?needle	 ?after	 ?loading.40	 ?xviii	 ?	 ?Figure	 ?2.11:	 ?Confocal	 ?scan	 ?of	 ?skin	 ?after	 ?injection	 ?of	 ?fluorescent	 ?beads.	 ?The	 ?skin	 ?surface	 ?is	 ?at	 ?0	 ??m.	 ?The	 ?confocal	 ?slice	 ?thickness	 ?is	 ?13.4	 ??m.	 ?.......................................................................	 ?41	 ?Figure	 ?2.12:	 ?Histogram	 ?of	 ?the	 ?intensity	 ?distribution	 ?of	 ?the	 ?fluorescent	 ?beads	 ?obtained	 ?from	 ?the	 ?confocal	 ?scan	 ?in	 ?Figure	 ?2.11.	 ?............................................................................................	 ?42	 ?Figure	 ?3.1:	 ? 	 ?Fabrication	 ?process	 ? for	 ?making	 ?hollow	 ?metallic	 ?microneedles,	 ?a)	 ? fabrication	 ?of	 ?the	 ?mold	 ? containing	 ? cone	 ? shaped	 ? pillars	 ? using	 ? backside	 ? exposure	 ? of	 ? SU-??8	 ? photoresist,	 ? b)	 ?deposition	 ?of	 ?PMMA/CB	 ?+	 ?NMP	 ?solution,	 ?c)	 ?evaporation	 ?of	 ?NMP	 ?at	 ?80?C,	 ?d)	 ?O2-??CF4	 ?plasma	 ?etching	 ? of	 ? dried	 ? PMMA/CB,	 ? e)	 ? electrodeposition	 ? of	 ? the	 ? metal	 ? layer,	 ? f)	 ? removing	 ? the	 ?microneedle	 ?array	 ?by	 ?dissolving	 ?the	 ?PMMA/CB	 ?in	 ?NMP.	 ?.......................................................	 ?48	 ?Figure	 ? 3.2:	 ? 	 ? Image	 ? of	 ? a	 ? mold	 ? used	 ? for	 ? microneedle	 ? fabrication,	 ? a)	 ? before	 ? coating	 ? with	 ?PMMA/CB,	 ?b)	 ?coated	 ?with	 ?PMMA/CB.	 ?...................................................................................	 ?49	 ?Figure	 ?3.3:	 ?Schematic	 ?of	 ?the	 ?setup	 ?used	 ?for	 ?electroplating	 ?of	 ?nickel.	 ?The	 ?wire	 ?contact	 ?point	 ?to	 ?the	 ?PMMA/CB	 ?layer	 ?is	 ?kept	 ?out	 ?of	 ?solution	 ?to	 ?prevent	 ?uneven	 ?nickel	 ?deposition.	 ?...........	 ?52	 ?Figure	 ?3.4:	 ?Images	 ?of	 ?microneedles	 ?fabricated	 ?through	 ?the	 ?process	 ?shown	 ?in	 ?Figure	 ?3.1,	 ?a)	 ?a	 ?single	 ?500	 ??m	 ?tall	 ?microneedle	 ?with	 ?a	 ?tip	 ?lumen	 ?diameter	 ?of	 ?40	 ??m	 ?and	 ?tip	 ?wall	 ?thickness	 ?of	 ?15	 ??m,	 ?b)	 ?and	 ?c)	 ?arrays	 ?of	 ?microneedle	 ?500	 ??m	 ?tall	 ?with	 ?1	 ?mm	 ?spacing,	 ?d)	 ?the	 ?backside	 ?of	 ?a	 ?microneedle	 ?array	 ?showing	 ?the	 ?needle	 ?lumen	 ?openings.	 ?......................................................	 ?54	 ?Figure	 ?3.5:	 ?Schematic	 ?of	 ?the	 ?setup	 ?used	 ?for	 ?mechanical	 ?compression	 ?tests	 ?on	 ?microneedles.	 ?................................................................................................................................................	 ?58	 ?xix	 ?	 ?Figure	 ?3.6:	 ?	 ?The	 ?resistivity	 ?of	 ?the	 ?PMMA/CB	 ?polymer	 ?film	 ?as	 ?a	 ?function	 ?of	 ?CB	 ?content.	 ?Each	 ?data	 ?point	 ?represents	 ?the	 ?average	 ?value	 ?from	 ?three	 ?experiments	 ?and	 ?the	 ?error	 ?bars	 ?indicate	 ??	 ?standard	 ?deviations.	 ?............................................................................................................	 ?60	 ?Figure	 ?3.7:	 ?Etch	 ?rates	 ?for	 ?PMMA/CB	 ?composite	 ?(with	 ?30	 ?wt%	 ?CB)	 ?for	 ?different	 ?ratios	 ?of	 ?CF4	 ?to	 ? total	 ? gas	 ? flow	 ? rate.	 ? The	 ? total	 ? gas	 ? flow	 ? rate	 ?was	 ? kept	 ? constant	 ? at	 ? 100	 ?sccm.	 ? Each	 ? data	 ?point	 ? represents	 ? the	 ?average	 ?value	 ? from	 ? three	 ?experiments	 ?and	 ? the	 ?error	 ?bars	 ? indicate	 ??	 ?standard	 ?deviations.	 ?...............................................................................................................	 ?61	 ?Figure	 ?3.8:	 ?The	 ?thickness	 ?of	 ?electroplated	 ?nickel	 ?on	 ?a	 ?PMMA/CB	 ?layer	 ?over	 ?time,	 ?with	 ?2	 ?mA	 ?supply	 ?current.	 ?The	 ?slope	 ?of	 ?the	 ?linear	 ?trend	 ?line	 ?is	 ?0.49	 ??m/min.	 ?........................................	 ?62	 ?Figure	 ? 3.9:	 ? Thickness	 ? of	 ? electroplated	 ? nickel	 ? as	 ? a	 ? function	 ? of	 ? power	 ? source	 ? current,	 ? for	 ?process	 ?duration	 ?of	 ?90	 ?min.	 ?The	 ?slope	 ?of	 ?the	 ?linear	 ?trend	 ?line	 ?is	 ?21.9	 ??m/mA.	 ?....................	 ?63	 ?Figure	 ?3.10:	 ?A	 ?needle	 ?displacement	 ?under	 ?axial	 ?loading.	 ?.......................................................	 ?64	 ?Figure	 ?3.11:	 ?Histology	 ? image	 ?of	 ? a	 ?microneedle	 ? insertion	 ? site	 ?on	 ?pig	 ? skin	 ? showing	 ? the	 ? skin	 ?damage	 ?caused	 ?by	 ?a	 ?500	 ??m	 ?tall	 ?microneedle.	 ?......................................................................	 ?66	 ?Figure	 ? 3.12:	 ? Confocal	 ? scan	 ? (514	 ? ?m	 ? ?	 ? 514	 ? ?m)	 ? of	 ? injection	 ? site	 ? on	 ? pig	 ? skin	 ? showing	 ? the	 ?distribution	 ?of	 ? fluorescent	 ?beads	 ?under	 ? the	 ? skin	 ? surface.	 ?The	 ? test	 ?was	 ?carried	 ?out	 ?using	 ?a	 ?500	 ??m	 ?tall	 ?microneedle.	 ?.......................................................................................................	 ?66	 ?Figure	 ? 4.1:	 ? Doxorubicin	 ? fluorescence	 ? intensity	 ? for	 ? different	 ? concentrations;	 ? the	 ? inset	 ?corresponds	 ?to	 ?the	 ?linear	 ?concentration	 ?range	 ?in	 ?the	 ?plot.	 ?...................................................	 ?74	 ?xx	 ?	 ?Figure	 ?4.2:	 ?Microneedle	 ?syringe	 ?concept	 ?presented	 ?by	 ?Stoeber	 ?et	 ?al.	 ?[121].	 ?........................	 ?75	 ?Figure	 ?4.3:	 ?a)	 ?schematics	 ?and	 ?b)	 ?actual	 ?image	 ?of	 ?injection	 ?setup	 ?used	 ?for	 ?delivering	 ?drugs	 ?at	 ?defined	 ? flow	 ?rates.	 ?The	 ?syringe	 ? is	 ?placed	 ? in	 ?a	 ? commercial	 ? syringe	 ?pump	 ?were	 ? the	 ?plunger	 ?speed	 ?is	 ?controlled.	 ?................................................................................................................	 ?75	 ?Figure	 ?4.4:	 ?Concept	 ?of	 ?drug	 ?backflow	 ?along	 ?outside	 ?of	 ?the	 ?needles	 ?to	 ?the	 ?skin	 ?surface.	 ?......	 ?76	 ?Figure	 ?4.5:	 ?Schematic	 ?of	 ?setup	 ?used	 ?to	 ?stretch	 ?skin	 ?and	 ?keep	 ? it	 ?moist	 ?during	 ? injection	 ?and	 ?imaging	 ?processes.	 ?.................................................................................................................	 ?77	 ?Figure	 ? 4.6:	 ? Concept	 ? of	 ? diffusion	 ? of	 ? drugs,	 ? injected	 ? with	 ? microneedles,	 ? through	 ? the	 ?epidermis.	 ?...............................................................................................................................	 ?80	 ?Figure	 ?4.7:	 ?Calculating	 ?concentration	 ?distribution	 ? from	 ?confocal	 ? images,	 ?by	 ?measuring	 ? the	 ?average	 ? intensity	 ? within	 ? hypothetical	 ? thin	 ? rings	 ? (with	 ? thickness	 ? of	 ? 10	 ? pixels)	 ? at	 ? different	 ?distances	 ?originating	 ?from	 ?drug	 ?source.	 ?.................................................................................	 ?82	 ?Figure	 ? 4.8:	 ? Confocal	 ? images	 ? obtained	 ? for	 ? a	 ? fresh	 ? skin	 ? sample	 ? injected	 ? with	 ? 87	 ??M	 ?doxorubicin	 ?solution.	 ?The	 ? images	 ?are	 ?775	 ??m	 ??	 ?775	 ??m.	 ?The	 ?rows	 ?correspond	 ?to	 ?different	 ?scan	 ? depths	 ? while	 ? the	 ? columns	 ? correspond	 ? to	 ? the	 ? scans	 ? over	 ? time.	 ? The	 ? time	 ? difference	 ?between	 ?two	 ?columns	 ?is	 ?5	 ?minutes.	 ?......................................................................................	 ?83	 ?Figure	 ? 4.9:	 ? Cross-??section	 ? of	 ? the	 ? injection	 ? location,	 ? reconstructed	 ? from	 ? confocal	 ? image	 ?shown	 ?in	 ?Figure	 ?4.8,	 ?corresponding	 ?to	 ?t1.	 ?..............................................................................	 ?84	 ?xxi	 ?	 ?Figure	 ? 4.10:	 ? Intensity	 ? distribution	 ? for	 ? the	 ? confocal	 ? images	 ? corresponding	 ? to	 ? the	 ? depth	 ?142	 ??m	 ?in	 ?Figures	 ?4.8	 ?and	 ?4.9,	 ?and	 ?obtained	 ?through	 ?the	 ?method	 ?illustrated	 ?in	 ?Figure	 ?4.7.	 ?85	 ?Figure	 ?4.11:	 ?Doxorubicin	 ?concentration	 ?distribution	 ? in	 ?skin	 ?obtained	 ?from	 ?Figure	 ?4.10.	 ?The	 ?theoretical	 ?fitted	 ?curves	 ?(i.e.	 ?Gaussian	 ?diffusion,	 ?eq.	 ?(6))	 ?are	 ?included	 ?as	 ?red	 ?lines.	 ?.............	 ?86	 ?Figure	 ?4.12:	 ?Doxorubicin	 ?diffusion	 ?coefficient	 ?for	 ?different	 ?skin	 ?types	 ?(fresh,	 ?refrigerated	 ?for	 ?3	 ?days,	 ?frozen	 ?for	 ?12	 ?days).	 ?Two	 ?of	 ?the	 ?data	 ?points	 ?for	 ?fresh	 ?skin	 ?overlap.	 ?...........................	 ?87	 ?Figure	 ?4.13:	 ?Doxorubicin	 ?concentration	 ?distribution	 ?obtained	 ?from	 ?analytical	 ?model	 ?(shown	 ?as	 ?lines)	 ?and	 ?the	 ?experimental	 ?data	 ?for	 ?all	 ?the	 ?time	 ?steps	 ?(shown	 ?as	 ?scatter	 ?plots).	 ?.............	 ?90	 ?Figure	 ?5.1:	 ?Conceptual	 ?sketch	 ?of	 ?interstitial	 ?fluid	 ?(ISF)	 ?in	 ?body.	 ?.............................................	 ?92	 ?Figure	 ? 5.2:	 ? Fabrication	 ? process	 ? using	 ? solvent	 ? casting	 ? for	 ? solid	 ? out-??of-??plane	 ? polymer	 ?microneedles	 ?for	 ?ISF	 ?extraction;	 ?a	 ?&	 ?b)	 ?fabrication	 ?of	 ?pillars	 ?from	 ?SU-??8;	 ?c)	 ?clay/polyimide	 ?+	 ?NMP	 ? solution	 ? deposition;	 ? d)	 ? evaporation	 ? of	 ? NMP;	 ? e)	 ? solvent	 ? casting	 ? of	 ? hydroxyethyl	 ?cellulose	 ?layer;	 ?f)	 ?solvent	 ?casting	 ?of	 ?poly(ethylene-??co-??vinyl	 ?acetate)	 ?layer.	 ?...........................	 ?95	 ?Figure	 ?5.3:	 ?a)	 ?A	 ?base	 ?structure	 ?used	 ?for	 ?solvent	 ?casting	 ?of	 ?solid	 ?microneedles,	 ?consisting	 ?of	 ?an	 ? array	 ? of	 ? tall	 ? pillars	 ? made	 ? from	 ? SU-??8;	 ? the	 ? distance	 ? between	 ? the	 ? pillars	 ? in	 ? the	 ? array	 ? is	 ?500	 ??m,	 ? b)	 ? An	 ? array	 ? of	 ? 250	 ??m-??long	 ? microneedles	 ? made	 ? through	 ? the	 ? process	 ? shown	 ? in	 ?Figure	 ?5.2.	 ?...............................................................................................................................	 ?96	 ?xxii	 ?	 ?Figure	 ?5.4:	 ?Extraction	 ?of	 ?dermal	 ? ISF	 ?using	 ?the	 ?fabricated	 ?microneedles;	 ?a)	 ? insertion	 ?site	 ?on	 ?the	 ?inner	 ?rabbit	 ?ear	 ?skin;	 ?b)	 ?swelling	 ?of	 ?the	 ?hydroxyethyl	 ?cellulose	 ?layer	 ?upon	 ?application	 ?to	 ?the	 ?skin	 ?as	 ?a	 ?result	 ?of	 ?ISF	 ?absorption	 ?......................................................................................	 ?97	 ?Figure	 ? 5.5:	 ? Fabrication	 ? process	 ? for	 ? making	 ? hollow	 ? PVA	 ? microneedles;	 ? a)	 ? fabrication	 ? of	 ? a	 ?mold	 ?consisting	 ?of	 ?an	 ?array	 ?of	 ?SU-??8	 ?pillars	 ?coated	 ?with	 ?a	 ?PDMS	 ?layer,	 ?b)	 ?deposition	 ?of	 ?PVA	 ?+	 ?BA	 ? solution,	 ? c)	 ? solvent	 ? evaporation,	 ? d)	 ? casting	 ? of	 ? a	 ? PMMA	 ? layer,	 ? e)	 ? separation	 ? from	 ? the	 ?mold,	 ?	 ?f)	 ?opening	 ?the	 ?tips	 ?using	 ?O2/CF4	 ?plasma	 ?etching.	 ?........................................................	 ?98	 ?Figure	 ? 5.6:	 ? a)	 ? SEM	 ? image	 ? of	 ? a	 ? PVA	 ?microneedle	 ? array,	 ? b)	 ? optical	 ? microscope	 ? image	 ? of	 ? a	 ?single	 ?PVA	 ?microneedle.	 ?.........................................................................................................	 ?99	 ?Figure	 ? 5.7:	 ? Contact	 ? angle	 ? measurement	 ? of	 ? a	 ? 4	 ??L	 ? water	 ? droplet	 ? on	 ? a	 ? cross-??linked	 ? PVA	 ?surface	 ?shows	 ?that	 ?the	 ?surface	 ?is	 ?hydrophilic.	 ?.....................................................................	 ?100	 ?Figure	 ?5.8:	 ?Dyed	 ?water	 ?transfer	 ?through	 ?the	 ?microneedle	 ?lumens	 ?when	 ?the	 ?tips	 ?are	 ?exposed	 ?to	 ?the	 ?liquid,	 ?a)	 ?schematic	 ?of	 ?experiment,	 ?b)	 ?microneedle	 ?backside	 ?showing	 ?capillary	 ?driven	 ?flow	 ?reaching	 ?the	 ?channel	 ?openings.	 ?...................................................................................	 ?100	 ?Figure	 ?5.9:	 ?Setup	 ?used	 ?for	 ?ISF	 ?extraction	 ?with	 ?hollow	 ?polymer	 ?microneedles.	 ?....................	 ?101	 ?Figure	 ? 5.10:	 ? a)	 ? Schematic	 ? of	 ? custom	 ? vacuum	 ? device	 ? made	 ? for	 ? ISF	 ? extraction	 ? using	 ? the	 ?500	 ??m	 ?metallic	 ?microneedles,	 ?b)	 ?image	 ?of	 ?the	 ?actual	 ?vacuum	 ?device.	 ?..............................	 ?102	 ?Figure	 ?A.1:	 ?Setup	 ?used	 ?to	 ?perform	 ?backside	 ?photoresist	 ?exposure.	 ?....................................	 ?135	 ?Figure	 ?A.2:	 ?A	 ?microneedle	 ?mold	 ?array	 ?before	 ?plasma	 ?etching.	 ?............................................	 ?137	 ?xxiii	 ?	 ?Figure	 ?A.3:	 ?A	 ?microneedle	 ?mold	 ?array	 ?after	 ?plasma	 ?etching.	 ?...............................................	 ?137	 ?Figure	 ?A.4:	 ?A	 ?100	 ?mm	 ?mask	 ?used	 ?for	 ?making	 ?microneedle	 ?molds.	 ?......................................	 ?139	 ?Figure	 ?A.5:	 ?An	 ?array	 ?of	 ?polyimide/clay	 ?microneedles	 ?made	 ?through	 ?the	 ?process	 ?in	 ?Chapter	 ?2.	 ?..............................................................................................................................................	 ?140	 ?Figure	 ?A.6:	 ?An	 ?array	 ?of	 ?polyvinyl	 ?alcohol	 ?microneedles	 ?made	 ?through	 ?the	 ?process	 ?in	 ?Chapter	 ?2.	 ?...........................................................................................................................................	 ?140	 ?Figure	 ?A.7:	 ?An	 ?array	 ?of	 ?polyvinyl	 ?alcohol	 ?microneedles	 ?made	 ?through	 ?the	 ?process	 ?in	 ?Chapter	 ?2.	 ?...........................................................................................................................................	 ?141	 ?Figure	 ?A.8:	 ?Top	 ?view	 ?image	 ?of	 ?a	 ?hollow	 ?polymer	 ?microneedle	 ?showing	 ?the	 ?needle	 ?lumen.	 ?141	 ?Figure	 ? A.9:	 ? PDMS	 ? mold	 ? used	 ? to	 ? make	 ? cylindrical	 ? pieces	 ? for	 ? PMMA/CB	 ? conductivity	 ?measurements.	 ?....................................................................................................................	 ?142	 ?Figure	 ? A.10:	 ? Compression	 ? test	 ? plots	 ? for	 ? 500	 ??m	 ? tall	 ? microneedles	 ? (made	 ? through	 ? the	 ?process	 ? in	 ?Chapter	 ? 3)	 ? showing	 ? the	 ? compressive	 ? forces	 ? as	 ? a	 ? function	 ?of	 ? displacement;	 ? the	 ?first	 ?load	 ?peaks	 ?indicate	 ?tip	 ?failure.	 ?......................................................................................	 ?146	 ?Figure	 ?A.11:	 ?Schematic	 ?of	 ?the	 ?setup	 ?to	 ?measure	 ?doxorubicin	 ?intensity.	 ?Confocal	 ?setting	 ?was:	 ?objective	 ?10x,	 ?frame	 ?size:	 ?512x512	 ?pixels,	 ?1.55	 ?mm	 ?x	 ?1.55	 ?mm,	 ?speed:	 ?600	 ?Hz	 ?bidirectional,	 ?laser:	 ?488	 ?nm	 ?@	 ?10%	 ?intensity,	 ?detector:	 ?HyD1,	 ?step	 ?size:	 ?2.06	 ??m.	 ?..................................	 ?147	 ?xxiv	 ?	 ?Figure	 ?A.12:	 ?Examples	 ?of	 ? intensity	 ?measurements	 ?(in	 ?arbitrary	 ?units)	 ?from	 ?hemocytometer	 ?for	 ?different	 ?concentrations.	 ?................................................................................................	 ?148	 ?Figure	 ?A.13:	 ?Locations	 ?of	 ?scanning	 ?on	 ?an	 ?examples	 ?intensity	 ?plot.	 ?......................................	 ?149	 ?Figure	 ?A.14:	 ?Calibration	 ?curve	 ?for	 ?doxorubicin	 ?from	 ?three	 ?regions	 ?of	 ?hemocytometer.	 ?.....	 ?150	 ?Figure	 ?A.15:	 ?Initial	 ?concentration	 ?calculated	 ?from	 ?the	 ?average	 ?intensity	 ?at	 ?the	 ?bright	 ?center	 ?of	 ?the	 ?diffusion	 ?source.	 ?Measured	 ?average	 ?intensity	 ?was	 ?120.4	 ?for	 ?87	 ??M	 ?concentration.	 ?151	 ?Figure	 ?A.16:	 ?a)	 ?and	 ?b)	 ?confocal	 ?images	 ?for	 ?fresh	 ?skin	 ?samples	 ?injected	 ?with	 ?87	 ??M	 ?solution	 ?of	 ? doxorubicin	 ? using	 ? 500	 ??m	 ? tall	 ?microneedles,	 ? a)	 ? first	 ? image	 ? taken	 ? 9	 ?min	 ? and	 ? 30	 ?s	 ? after	 ?injection,	 ?time	 ?difference	 ?between	 ?images:	 ?5	 ?min.	 ?b)	 ?first	 ?image	 ?taken	 ?10	 ?min	 ?and	 ?45	 ?s	 ?after	 ?injection,	 ?time	 ?difference	 ?between	 ?images:	 ?5	 ?min.	 ?Images	 ?size:	 ?775	 ??m	 ??	 ?775	 ??m.	 ?...........	 ?153	 ?Figure	 ?A.17:	 ?a),	 ?b)	 ?and	 ?c)	 ?confocal	 ?images	 ?for	 ?refrigerated	 ?skin	 ?samples	 ?injected	 ?with	 ?87	 ??M	 ?solution	 ?of	 ?doxorubicin	 ?using	 ?500	 ??m	 ?tall	 ?microneedles,	 ?a)	 ?first	 ?image	 ?taken	 ?12	 ?min	 ?and	 ?20	 ?s	 ?after	 ?injection,	 ?time	 ?difference	 ?between	 ?images:	 ?5	 ?min.	 ?b)	 ?first	 ?image	 ?taken	 ?9	 ?min	 ?and	 ?40	 ?s	 ?after	 ? injection,	 ? time	 ? difference	 ? between	 ? images:	 ? 5	 ?min.	 ? c)	 ? first	 ? image	 ? taken	 ? 8	 ?min	 ? after	 ?injection,	 ?time	 ?difference	 ?between	 ?images:	 ?5	 ?min.	 ?Images	 ?size:	 ?775	 ??m	 ??	 ?775	 ??m.	 ?...........	 ?154	 ?Figure	 ? A.18:	 ? a),	 ? b)	 ? and	 ? c)	 ? confocal	 ? images	 ? for	 ? frozen	 ? skin	 ? samples	 ? injected	 ? with	 ? 87	 ??M	 ?solution	 ?of	 ?doxorubicin	 ?using	 ?500	 ??m	 ?tall	 ?microneedles,	 ?a)	 ?first	 ?image	 ?taken	 ?10	 ?min	 ?and	 ?29	 ?s	 ?after	 ?injection,	 ?time	 ?difference	 ?between	 ?images:	 ?5	 ?min.	 ?b)	 ?first	 ?image	 ?taken	 ?5	 ?min	 ?and	 ?47	 ?s	 ?after	 ? injection,	 ? time	 ?difference	 ?between	 ? images:	 ?5	 ?min.	 ? c)	 ? first	 ? image	 ? taken	 ?5	 ?min	 ?and	 ?4	 ?s	 ?after	 ?injection,	 ?time	 ?difference	 ?between	 ?images:	 ?5	 ?min.	 ?Images	 ?size:	 ?775	 ??m	 ??	 ?775	 ??m.	 ?..	 ?155	 ?xxv	 ?	 ?LIST	 ?OF	 ?VARIABLES	 ??	 ? Membrane	 ?thickness	 ??	 ? Resistivity	 ?? ? Laplace	 ?operator	 ?A	 ? Area	 ?of	 ?cast	 ?cylindrical	 ?structures	 ??	 ? Characteristic	 ?skin	 ?membrane	 ?constant	 ?C	 ? Concentration	 ?D0	 ?	 ? Diffusivity	 ?of	 ?a	 ?hypothetical	 ?drug	 ?molecule	 ?with	 ?zero	 ?volume	 ?D	 ? Diffusion	 ?coefficient	 ?of	 ?drug	 ?FN	 ? Microneedle	 ?failure	 ?load	 ?I	 ? Electrical	 ?current	 ?J	 ? Steady-??state	 ?flux	 ?of	 ?solute	 ?Km	 ? Solvent-??membrane	 ?distribution	 ?coefficient	 ?l	 ? Thickness	 ?of	 ?cast	 ?cylindrical	 ?structures	 ?N0	 ? Initial	 ?mass	 ?r	 ? Radial	 ?distance	 ?from	 ?diffusion	 ?source	 ?R	 ? Resistance	 ?t1	 ? Duration	 ? of	 ? time	 ? between	 ? the	 ? end	 ? of	 ? the	 ? injection	 ? process	 ?and	 ?taking	 ?the	 ?first	 ?confocal	 ?image	 ?t	 ? Time	 ?ti	 ? Duration	 ?of	 ?drug	 ?injection	 ?V	 ? Molecular	 ?volume	 ?of	 ?drug	 ?xxvi	 ?	 ?ACKNOWLEDGEMENTS	 ?First	 ? of	 ? all,	 ? I	 ? would	 ? like	 ? to	 ? thank	 ? my	 ? supervisor	 ? Dr.	 ? Boris	 ? Stoeber	 ? who	 ? gave	 ? me	 ? the	 ?opportunity	 ?to	 ?be	 ?part	 ?of	 ?his	 ?group	 ?and	 ?work	 ?on	 ?this	 ?exciting	 ?research.	 ?I?d	 ?like	 ?to	 ?thank	 ?him	 ?for	 ?his	 ?continuous	 ?support,	 ?patience,	 ?and	 ?encouragement,	 ?without	 ?which	 ?this	 ?dissertation	 ?would	 ?not	 ?have	 ?been	 ?possible.	 ?Also,	 ? I	 ?am	 ?very	 ?thankful	 ?to	 ?Dr.	 ?Urs	 ?H?feli	 ? for	 ?his	 ? invaluable	 ?guidance	 ?and	 ?support	 ? in	 ?the	 ?past	 ?several	 ?years.	 ? I	 ?am	 ?also	 ?thankful	 ?to	 ?Dr.	 ?Jan	 ?Dutz	 ?and	 ?Dr.	 ?Jacqueline	 ?Lai	 ?for	 ?their	 ?contributions	 ?and	 ?support.	 ?	 ?I	 ?would	 ?like	 ?to	 ?thank	 ?Dr.	 ?Mu	 ?Chiao,	 ?Dr.	 ?Hongshen	 ?Ma,	 ?and	 ?Dr.	 ?Karen	 ?Cheung	 ?for	 ?providing	 ?lab	 ?space	 ?and	 ?equipment.	 ?Also,	 ?I	 ?thank	 ?my	 ?defense	 ?committee	 ?members	 ?for	 ?their	 ?time	 ?and	 ?valuable	 ? comments.	 ? I?d	 ? like	 ? to	 ? thank	 ? Dr.	 ? H?feli?s	 ? students	 ? Veronika	 ? Schmitt	 ? and	 ? Dana	 ?Lambert	 ? for	 ? their	 ? useful	 ? contributions.	 ? I	 ? am	 ? grateful	 ? to	 ? my	 ? lab	 ? mates	 ? especially	 ? Sahan	 ?Ranamukhaarachchi,	 ? Ashkan	 ? Babaie,	 ? Vahid	 ? Bazargan,	 ? Benjamin	 ? Mustin,	 ? Ron	 ? Linklater,	 ?Nathan	 ? Wolfe,	 ? John	 ? Berring,	 ? Mazyar	 ? Jalayer,	 ? Kelly	 ? He,	 ? and	 ? my	 ? friends	 ? Ramin,	 ? Amir	 ? E.,	 ?Mahmoud,	 ?Omid,	 ?Babak,	 ?and	 ?Amir	 ?K.,	 ?for	 ?their	 ?support	 ?and	 ?encouragement.	 ?I	 ?am	 ?also	 ?very	 ?thankful	 ?to	 ?my	 ?brother	 ?Hadi	 ?for	 ?his	 ?valuable	 ?guidance	 ?in	 ?the	 ?past	 ?years.	 ?I	 ?am	 ?always	 ?grateful	 ?to	 ?my	 ?parents	 ?for	 ?their	 ?never-??ending	 ?sacrifices	 ?and	 ?unconditional	 ?love.	 ?I	 ?am	 ?grateful	 ?to	 ?my	 ?brother	 ?Mehdi	 ?and	 ?sister	 ?Soudeh	 ?for	 ?always	 ?supporting	 ?me.	 ?And	 ?finally,	 ?I	 ?am	 ?grateful	 ?to	 ?my	 ?beautiful	 ? and	 ? kind	 ?wife,	 ? Sarah,	 ? for	 ? her	 ? love	 ? and	 ? support	 ? and	 ? for	 ? always	 ? standing	 ? by	 ?me.	 ?	 ?Finally,	 ? I?d	 ? like	 ? to	 ? acknowledge	 ? financial	 ? support	 ? from	 ? Natural	 ? Sciences	 ? and	 ? Engineering	 ?Research	 ? Council	 ? of	 ? Canada	 ? (NSERC)	 ? and	 ? Canadian	 ? Institutes	 ? of	 ? Health	 ? Research	 ? (CIHR)	 ?through	 ?the	 ?Collaborative	 ?Health	 ?Research	 ?Projects	 ?Grant.	 ?xxvii	 ?	 ?DEDICATION	 ?	 ?	 ?	 ?	 ?To my parents & my wife 	 ?1	 ?	 ?CHAPTER	 ?1 	 ?	 ?	 ?INTRODUCTION	 ?	 ?	 ?Finding	 ? novel	 ? and	 ? effective	 ?methods	 ? of	 ? delivering	 ?medicinal	 ? compounds	 ? into	 ? the	 ? human	 ?body	 ? and	 ? monitoring	 ? and	 ? controlling	 ? these	 ? compounds	 ? has	 ? been	 ? an	 ? ongoing	 ? field	 ? of	 ?research	 ?since	 ?the	 ?emergence	 ?of	 ?the	 ?medicine.	 ?When	 ?it	 ?comes	 ?to	 ?delivering	 ?an	 ?agent,	 ?the	 ?most	 ?effective	 ?administration	 ?method	 ?depends	 ?on	 ?the	 ?type	 ?of	 ?the	 ?drug	 ?itself	 ?as	 ?well	 ?as	 ?the	 ?location	 ?and	 ?properties	 ?of	 ? the	 ?target	 ?medium.	 ? In	 ?another	 ?view,	 ? the	 ?pharmacokinetics	 ?and	 ?the	 ?pharmacodynamics	 ?of	 ?the	 ?drugs	 ?determine	 ?how	 ?they	 ?should	 ?be	 ?administered.	 ?Common	 ?methods	 ?of	 ?drug	 ?administration	 ?use	 ?parenteral,	 ?enteral,	 ?or	 ?topical	 ?routes	 ?or	 ?a	 ?combination.	 ?	 ?Parenteral	 ? administration	 ? is	 ? direct	 ? injection	 ?of	 ?drugs	 ? into	 ? specific	 ? tissues	 ?using	 ? traditional	 ?hypodermic	 ? needles	 ? attached	 ? to	 ? drug-??filled	 ? syringes.	 ? This	 ?method	 ? is	 ? usually	 ? invasive	 ? and	 ?painful	 ?as	 ?often	 ?no	 ?local	 ?or	 ?general	 ?anaesthesia	 ?is	 ?used.	 ?The	 ?main	 ?advantage	 ?of	 ?this	 ?method,	 ?however,	 ? is	 ? that	 ? it	 ?allows	 ?delivery	 ?of	 ? large	 ?and	 ?controlled	 ?quantities	 ?of	 ?drugs	 ? into	 ?specific	 ?target	 ? areas.	 ? Enteral	 ? administration	 ? is	 ? based	 ? on	 ? delivering	 ? drugs	 ? to	 ? the	 ? digestive	 ? system	 ?orally,	 ?rectally,	 ?or	 ?through	 ?feeding	 ?tubes.	 ?Oral	 ?and	 ?rectal	 ?administrations	 ?are	 ?often	 ?easy	 ?to	 ?2	 ?	 ?practice	 ?and	 ?can	 ?be	 ?carried	 ?out	 ?by	 ?the	 ?patients	 ?themselves.	 ?Most	 ?of	 ?the	 ?over	 ?the	 ?counter	 ?medication	 ?uses	 ? this	 ?method	 ?of	 ?administration.	 ? 	 ?The	 ?main	 ?disadvantage	 ?of	 ? this	 ?method	 ? is	 ?that	 ? there	 ? is	 ? less	 ? control	 ?over	 ?how	 ?efficiently	 ? the	 ?drug	 ? can	 ? target	 ?a	 ? specific	 ? region	 ?of	 ? the	 ?body.	 ?In	 ?addition,	 ?many	 ?drugs	 ?might	 ?cause	 ?damage	 ?to	 ?the	 ?digestive	 ?system	 ?before	 ?they	 ?are	 ?absorbed	 ?or	 ?disposed.	 ?Moreover,	 ?a	 ? lot	 ?of	 ?patients	 ?(especially	 ?small	 ?children)	 ?may	 ?find	 ?this	 ?method	 ? of	 ? administration	 ? uncomfortable.	 ? Topical	 ? administration	 ? routes	 ? are	 ? among	 ? the	 ?least	 ? invasive	 ?methods	 ? and	 ? therefore	 ?more	 ? comfortable	 ? for	 ? patients.	 ? Some	 ? examples	 ? of	 ?topical	 ? administration	 ?methods	 ? include:	 ? 	 ? transdermal,	 ? rectal,	 ? inhalational	 ? or	 ? through	 ? eye	 ?drops,	 ?eardrops,	 ?or	 ?mucus	 ?membranes.	 ?	 ?In	 ?general,	 ?a	 ?crucial	 ?aspect	 ?of	 ?drug	 ?delivery	 ?is	 ?determining	 ?the	 ?type	 ?of	 ?drug	 ?that	 ?the	 ?patients	 ?need	 ?in	 ?the	 ?first	 ?place,	 ?which	 ?is	 ?often	 ?done	 ?by	 ?physicians;	 ?next,	 ?is	 ?deciding	 ?on	 ?the	 ?amount	 ?to	 ?be	 ? administered.	 ? In	 ? some	 ? cases,	 ? deciding	 ? on	 ? the	 ? amount	 ? requires	 ? knowing	 ? how	 ?much	 ? a	 ?certain	 ?compound	 ?(ex.	 ? the	 ?same	 ?drug)	 ?already	 ?exists	 ? in	 ? the	 ?blood	 ?prior	 ? to	 ?administration.	 ?This	 ? is	 ? especially	 ? important	 ? for	 ? therapeutic	 ? drug	 ? monitoring	 ? (TDM)	 ? whose	 ? main	 ? focus	 ? is	 ?keeping	 ?drug	 ?concentrations	 ?within	 ?a	 ?therapeutic	 ?window	 ?in	 ?order	 ?to	 ?avoid	 ?under-??	 ?or	 ?over-??dosing.	 ?While	 ? there	 ? have	 ? been	 ? numerous	 ?methods	 ? of	 ? determining	 ? the	 ? concentrations	 ? of	 ?compounds	 ? in	 ? the	 ? body,	 ? the	 ? non-??invasive	 ? methods	 ? (such	 ? as	 ? optical	 ? methods)	 ? are	 ? often	 ?limited	 ? to	 ? a	 ? very	 ? small	 ? number	 ? of	 ? compounds.	 ? The	 ? most	 ? common	 ? method	 ? of	 ? analyzing	 ?compounds	 ? in	 ? blood	 ? relies	 ? on	 ? taking	 ? blood	 ? samples	 ? from	 ? patients	 ? using	 ? hypodermic	 ?needles,	 ?which	 ?can	 ?be	 ?painful	 ?and	 ?uncomfortable	 ? for	 ?many	 ?patients	 ?or	 ?even	 ?unethical	 ? for	 ?small	 ?children.	 ?3	 ?	 ?1.1 Transdermal	 ?drug	 ?delivery	 ?Among	 ? the	 ? topical	 ? drug	 ? administration	 ? methods,	 ? transdermal	 ? drug	 ? delivery	 ? is	 ? becoming	 ?increasingly	 ? popular	 ? because	 ? it	 ? is	 ? the	 ? easiest	 ? to	 ? practice	 ? and	 ? not	 ? associated	 ? with	 ? the	 ?potential	 ? risks	 ?and	 ?pain	 ?of	 ? traditional	 ?hypodermic	 ?needles	 ? [1].	 ? 	 ? This	 ?method	 ? relies	 ?on	 ? the	 ?transport	 ?of	 ?drugs	 ?across	 ?the	 ?skin	 ?into	 ?the	 ?blood	 ?stream.	 ?The	 ?structure	 ?and	 ?properties	 ?of	 ?the	 ?skin	 ?are	 ?important	 ?factors	 ?for	 ?this	 ?method	 ?of	 ?administration.	 ?	 ?	 ?1.1.1 Structure	 ?of	 ?skin	 ?Skin	 ?is	 ?a	 ?protective	 ?membrane	 ?that	 ?makes	 ?up	 ?the	 ?outer	 ?covering	 ?layer	 ?of	 ?animals?	 ?body	 ?and	 ?guards	 ? the	 ? underlying	 ? muscles,	 ? bones,	 ? and	 ? internal	 ? organs.	 ? The	 ? general	 ? structure	 ? of	 ?mammalian	 ?skin	 ?is	 ?shown	 ?in	 ?Figure	 ?1.1.	 ?	 ?Figure	 ?1.1:	 ?Structure	 ?of	 ?mammalian	 ?skin	 ?[2].	 ?4	 ?	 ?The	 ?skin	 ?mainly	 ?consists	 ?of	 ?two	 ?layers,	 ?the	 ?epidermis	 ?and	 ?the	 ?dermis,	 ?which	 ?are	 ?attached	 ?to	 ?the	 ?hypodermis	 ?that	 ?connects	 ?the	 ?skin	 ?to	 ?the	 ?underlying	 ?bone	 ?and	 ?muscle.	 ?The	 ?epidermis,	 ?(shown	 ?in	 ?more	 ?detail	 ?in	 ?Figure	 ?1.2)	 ?constitutes	 ?the	 ?superficial	 ?layer	 ?of	 ?the	 ?skin.	 ?It	 ?is	 ?mainly	 ?made	 ? of	 ? keratinocytes,	 ? protective	 ? cells	 ? that	 ? serve	 ? as	 ? building	 ? blocks	 ? of	 ? a	 ? barrier	 ? against	 ?physical	 ? environmental	 ? damage,	 ? pathogens,	 ? heat,	 ? radiation,	 ? and	 ? water	 ? loss.	 ? The	 ?keratinocytes	 ? in	 ? the	 ? epidermis	 ? are	 ? arranged	 ? in	 ? multiple	 ? layers,	 ? with	 ? the	 ? stratum	 ? basale	 ?being	 ? the	 ? innermost	 ?and	 ?the	 ?stratum	 ?corneum	 ?(SC)	 ?being	 ? the	 ?outermost	 ? layer.	 ?Through	 ?a	 ?process	 ?called	 ?cornification,	 ?the	 ?fresh	 ?live	 ?keratinocytes	 ?in	 ?the	 ?stratum	 ?basale	 ?gradually	 ?lose	 ?their	 ? internal	 ? nuclei	 ? and	 ? organelles,	 ? produce	 ? keratin	 ? (a	 ? structural	 ? fibrous	 ? protein),	 ? and	 ?become	 ?flat.	 ?Different	 ?stages	 ?of	 ?this	 ?process	 ?correspond	 ?to	 ?the	 ?different	 ?epidermal	 ? layers,	 ?with	 ? the	 ? SC	 ? being	 ? the	 ? fully	 ? keratinized,	 ? flat,	 ? and	 ? rough	 ? cells	 ? that	 ? are	 ? gradually	 ? pushed	 ?outwards.	 ?The	 ?average	 ?thickness	 ?of	 ?the	 ?human	 ?SC	 ?layer	 ?is	 ?14.8	 ??	 ?4.8	 ??m	 ?while	 ?the	 ?average	 ?thickness	 ?of	 ?the	 ?viable	 ?epidermis	 ?is	 ?68.9	 ??	 ?17.0	 ??m	 ?[3].	 ?The	 ?dermis	 ? is	 ? the	 ? layer	 ? underneath	 ? the	 ? epidermis,	 ? and	 ? is	 ?made	 ?of	 ? connective	 ? tissue	 ? that	 ?connects	 ?the	 ?epidermis	 ?to	 ?the	 ?hypodermis	 ?(or	 ?subcutaneous,	 ?fatty)	 ?tissue.	 ?The	 ?dermis	 ? is	 ?a	 ?flexible	 ?membrane	 ?that	 ?protects	 ?the	 ?body	 ?from	 ?stress	 ?and	 ?strain.	 ?The	 ?collagen	 ?and	 ?elastic	 ?fibers	 ?in	 ?the	 ?dermis	 ?provide	 ?tensile	 ?strength	 ?and	 ?give	 ?it	 ?elasticity.	 ?The	 ?dermis	 ?also	 ?contains	 ?blood	 ?capillaries,	 ?lymphatic	 ?vessels,	 ?sweat	 ?glands,	 ?hair	 ?follicle,	 ?and	 ?nerve	 ?fibers.	 ?The	 ?dermis	 ?layer	 ? is	 ? generally	 ? much	 ? thicker	 ? than	 ? the	 ? epidermis,	 ? and	 ? its	 ? thickness	 ? varies	 ? widely	 ?throughout	 ?the	 ?human	 ?body	 ?and	 ?can	 ?range	 ?anywhere	 ?from	 ?0.6	 ?mm	 ?up	 ?to	 ?3	 ?mm	 ?in	 ?adults.	 ?In	 ?addition	 ?to	 ?the	 ?structural	 ?membranes	 ?and	 ?fibers,	 ?both	 ?the	 ?viable	 ?epidermis	 ?and	 ?the	 ?dermal	 ?5	 ?	 ?layers	 ?contain	 ?interstitial	 ?fluid	 ?(ISF).	 ?The	 ?ISF	 ?is	 ?a	 ?water-??based	 ?medium	 ?that	 ?surrounds	 ?all	 ?the	 ?cells	 ?and	 ?is	 ?responsible	 ?for	 ?transferring	 ?nutrients	 ?and	 ?ions	 ?to	 ?and	 ?from	 ?the	 ?cells.	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ? 	 ?Figure	 ?1.2:	 ?Structure	 ?of	 ?epidermis	 ?[4].	 ?The	 ? elasticity	 ? and	 ? thickness	 ? of	 ? both	 ? the	 ? dermis	 ? and	 ? the	 ? epidermis	 ? are	 ? also	 ? a	 ? function	 ? of	 ?gender,	 ? age,	 ? and	 ? ethnicity	 ? [5-??7].	 ? The	 ? mechanical	 ? properties	 ? of	 ? the	 ? skin	 ? depend	 ? on	 ? the	 ?nature	 ?and	 ?organization	 ?of	 ?the	 ?dermal	 ?components	 ?such	 ?as	 ?the	 ?collagen	 ?and	 ?elastic	 ?fibers	 ?as	 ?well	 ?as	 ?the	 ?water,	 ?proteins,	 ?and	 ?the	 ?macromolecules	 ?embedded	 ?in	 ?the	 ?extracellular	 ?matrix	 ?[8].	 ? Skin	 ? exhibits	 ? viscoelastic	 ? behaviour	 ? when	 ? undergoing	 ? deformation.	 ? There	 ? have	 ? been	 ?many	 ?proposed	 ? techniques	 ? to	 ?measure	 ? the	 ?mechanical	 ? properties	 ?of	 ? skin.	 ?According	 ? to	 ? a	 ?recent	 ? study	 ? that	 ?used	 ? indentation	 ? test	 ?methods,	 ? the	 ?average	 ?value	 ?of	 ? the	 ? skin?s	 ? Young?s	 ?modulus	 ?is	 ?between	 ?4.5	 ?kPa	 ?to	 ?8	 ?kPa	 ?[9].	 ?	 ?Stratum	 ?corneum	 ?Stratum	 ?lucidum	 ?Stratum	 ?granulosum	 ?Stratum	 ?spinosum	 ?Stratum	 ?basale	 ?6	 ?	 ?1.1.2 Adhesive	 ?skin	 ?patches	 ?One	 ?method	 ? for	 ? transdermal	 ? drug	 ? delivery	 ? uses	 ? adhesive	 ? skin	 ? patches.	 ? In	 ? this	 ?method,	 ? a	 ?pharmaceutical	 ?compound	 ?is	 ?absorbed	 ?through	 ?the	 ?skin	 ?after	 ?attaching	 ?a	 ?patch	 ?(filled	 ?with	 ?the	 ? formulation)	 ? to	 ? the	 ? skin	 ? surface	 ? by	 ?means	 ? of	 ? an	 ? adhesive	 ? layer.	 ? It	 ? is	 ? estimated	 ? that	 ?currently	 ?more	 ?than	 ?one	 ?billion	 ?transdermal	 ?adhesive	 ?patches	 ?are	 ?being	 ?manufactured	 ?each	 ?year	 ? [10].	 ? Various	 ? types	 ? of	 ? pressure	 ? sensitive	 ? adhesive	 ? patches	 ? (PSA)	 ? made	 ? from	 ?polyisobutylene,	 ? acrylic,	 ? and	 ? silicone	 ? are	 ? commercially	 ? available	 ? for	 ? use	 ? on	 ? skin	 ? [11]	 ? for	 ?applications	 ? such	 ? as	 ? smoking	 ? cessation	 ? (nicotine	 ? delivery)	 ? [12],	 ? pain	 ? relief	 ? (morphine	 ? or	 ?fentanyl	 ?delivery)	 ? [13,	 ?14],	 ?and	 ?high	 ?blood	 ?pressure	 ?treatment	 ?(glyceryl	 ? trinitrate	 ?delivery)	 ?[15].	 ?	 ?Adhesive	 ?patches	 ?are	 ?associated	 ?with	 ?limitations	 ?that	 ?restrict	 ?their	 ?usage	 ?for	 ?drug	 ?delivery.	 ?Among	 ?the	 ?skin	 ?layers,	 ?the	 ?epidermis	 ?and	 ?more	 ?specifically	 ?the	 ?SC	 ?plays	 ?the	 ?most	 ?important	 ?role	 ?in	 ?governing	 ?the	 ?diffusion	 ?of	 ?compounds	 ?from	 ?these	 ?patches	 ?into	 ?the	 ?body,	 ?due	 ?to	 ?their	 ?lower	 ?permeability	 ?compared	 ?to	 ?the	 ?dermis	 ?[16-??18].	 ?	 ?The	 ?SC	 ?barrier	 ?contains	 ?mostly	 ?tightly	 ?arranged	 ? flat	 ? cells	 ? filled	 ?with	 ? keratin,	 ? and	 ? it	 ? is	 ? essentially	 ? lipophilic.	 ? It	 ? is	 ? impermeable	 ? to	 ?most	 ?hydrophilic	 ?compounds	 ?but	 ?lipophilic	 ?molecules	 ?are	 ?generally	 ?better	 ?accepted	 ?by	 ?the	 ?SC	 ?[19].	 ?Ideally,	 ?the	 ?drug	 ?molecules	 ?have	 ?to	 ?be	 ?small	 ?(less	 ?than	 ?500	 ?kDa)	 ?and	 ?have	 ?to	 ?exhibit	 ?both	 ?lipoidal	 ?and	 ?aqueous	 ?behaviour	 ?in	 ?order	 ?to	 ?be	 ?able	 ?to	 ?liberate	 ?from	 ?the	 ?formulation,	 ?go	 ?through	 ?the	 ?lipid	 ?cellular	 ?matrix	 ?of	 ?the	 ?SC,	 ?and	 ?then	 ?move	 ?to	 ?the	 ?more	 ?aqueous	 ?epidermis	 ?underneath	 ?[19].	 ?In	 ?other	 ?words,	 ?if	 ?the	 ?drug	 ?is	 ?too	 ?hydrophilic,	 ?it	 ?will	 ?not	 ?diffuse	 ?into	 ?the	 ?SC,	 ?and	 ?of	 ? it	 ? is	 ?too	 ? lipophilic,	 ? it	 ?will	 ?stay	 ? in	 ?the	 ?SC	 ?and	 ?not	 ?diffuse	 ?to	 ?the	 ?viable	 ?epidermis	 ?and	 ?7	 ?	 ?dermis.	 ?When	 ?it	 ?comes	 ?to	 ?mathematically	 ?modeling	 ?the	 ?diffusion,	 ?using	 ?the	 ?familiar	 ?Stokes-??Einstein	 ?equation	 ?is	 ?invalid	 ?since	 ?this	 ?model	 ?describes	 ?the	 ?movement	 ?of	 ?spherical	 ?particles	 ?in	 ? uniform	 ? fluid	 ?medium	 ?whereas	 ? for	 ? the	 ? skin,	 ? the	 ? biological	 ? cell	 ?membrane	 ? is	 ? a	 ? gel-??like	 ?viscous	 ? lipid	 ? matrix	 ? that	 ? does	 ? not	 ? move	 ? around	 ? the	 ? particles	 ? and	 ? contain	 ? a	 ? staggered	 ?arrangement	 ?of	 ?cells.	 ?A	 ?model	 ?for	 ?diffusion	 ?across	 ?skin	 ?membrane	 ?0 exp( )D D V?= ? ? ? 	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ? 	 ?	 ?	 ?	 ?	 ?	 ?(1)	 ?is	 ?presented	 ?by	 ?Potts	 ?et	 ?al.	 ?[17],	 ?where	 ??	 ?is	 ?the	 ?diffusivity	 ?of	 ?the	 ?drug	 ?within	 ?the	 ?membrane,	 ???	 ? is	 ? the	 ? membrane	 ? diffusivity	 ? of	 ? a	 ? molecule	 ? with	 ? zero	 ? volume,	 ? ?	 ? is	 ? a	 ? characteristic	 ?membrane	 ?constant,	 ? and	 ??	 ? is	 ? the	 ?molecular	 ? volume	 ?of	 ? the	 ?drug.	 ?The	 ?permeability	 ?of	 ? the	 ?skin	 ?membrane	 ?can	 ?then	 ?be	 ?described	 ?in	 ?the	 ?terms	 ?of	 ?the	 ?steady-??state	 ?flux	 ?of	 ?solute	 ?mK D CJ??= 	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?(2)	 ?obtained	 ? from	 ? Fick?s	 ? law	 ? [18],	 ? where	 ???	 ? is	 ? the	 ? drug	 ? concentration	 ? difference	 ? across	 ? the	 ?membrane,	 ? ?	 ? is	 ? the	 ? membrane	 ? thickness,	 ? and	 ???	 ? is	 ? the	 ? solvent-??membrane	 ? distribution	 ?coefficient	 ? obtained	 ? from	 ? the	 ? ratio	 ? of	 ? the	 ? drug	 ? concentration	 ? sorbed	 ? on	 ? the	 ? SC	 ? over	 ? the	 ?drug	 ? concentration	 ? in	 ? the	 ? patch.	 ? In	 ? general	 ? ?	 ? and	 ? ?	 ? are	 ? small	 ? for	 ? most	 ? therapeutic	 ?compounds	 ?for	 ?diffusion	 ?through	 ?the	 ?SC	 ?[16-??19],	 ?which	 ?limits	 ?the	 ?usefulness	 ?of	 ?skin	 ?patches.	 ?	 ?Another	 ? limitation	 ? is	 ? that	 ? to	 ? deliver	 ? compounds	 ? using	 ? patches	 ? at	 ? useful	 ? dosages,	 ? these	 ?devices	 ?often	 ?have	 ? to	 ? rest	 ?on	 ? the	 ?skin	 ? for	 ?prolonged	 ?durations,	 ? from	 ?a	 ? few	 ?hours	 ? to	 ?days	 ?and	 ?even	 ?weeks.	 ?	 ?Long	 ?exposure	 ?times	 ?can	 ?potentially	 ?induce	 ?skin	 ?sensitivity	 ?reactions	 ?[20].	 ?8	 ?	 ?One	 ?type	 ?of	 ?these	 ?skin	 ?reactions	 ?is	 ?the	 ?non-??immunological	 ?contact	 ?sensitivity	 ?such	 ?as	 ?irritant	 ?dermatitis	 ? [20].	 ? Long-??term	 ? applications	 ? of	 ? transdermal	 ? patches	 ? cause	 ? hydration	 ? of	 ? the	 ?epidermal	 ? layer,	 ? which	 ? leads	 ? to	 ? accumulation	 ? of	 ? sweat	 ? on	 ? the	 ? surface;	 ? such	 ? a	 ? moist	 ?environment	 ? promotes	 ? yeast	 ? and	 ? bacteria	 ? growth	 ? on	 ? the	 ? surface.	 ? Both	 ? the	 ? bacterial	 ?overgrowth	 ?and	 ?the	 ?sweat	 ?accumulation	 ?contribute	 ?to	 ?the	 ?irritation	 ?reactions.	 ?Another	 ?type	 ?of	 ? skin	 ? reaction	 ? is	 ? allergic	 ? contact	 ? sensitivity,	 ? which	 ? is	 ? caused	 ? by	 ? the	 ? response	 ? of	 ? the	 ?immune	 ? system	 ? to	 ? the	 ? transdermal	 ? patch	 ? components	 ? such	 ? as	 ? the	 ? adhesive,	 ? the	 ?membrane,	 ?the	 ?solvent,	 ?the	 ?enhancer,	 ?and	 ?the	 ?active	 ?drug	 ?[20].	 ?	 ?Different	 ? methods	 ? have	 ? been	 ? previously	 ? proposed	 ? for	 ? evaluating	 ? skin	 ? permeability	 ? for	 ?different	 ?drugs	 ?exposed	 ?to	 ?the	 ?skin	 ?surface.	 ?One	 ?method	 ?involves	 ?measuring	 ?the	 ?change	 ?in	 ?electric	 ?properties	 ?of	 ?skin	 ?due	 ?to	 ?compound	 ?absorption	 ?[21-??23].	 ?Kasting	 ?et	 ?al.	 ?[21]	 ?used	 ?this	 ?method	 ?to	 ?evaluate	 ?the	 ?change	 ? in	 ?electrical	 ?resistance	 ?of	 ?skin	 ?with	 ?respect	 ?to	 ?a	 ?change	 ? in	 ?sodium	 ? ion	 ?content.	 ?A	 ?similar	 ?method	 ?was	 ?used	 ?by	 ?Dick	 ?et	 ?al.	 ? [22]	 ?who	 ?measured	 ?pig	 ?ear	 ?skin	 ?conductivity	 ?to	 ?evaluate	 ?its	 ?usefulness	 ?as	 ?an	 ?in	 ?vitro	 ?model	 ?for	 ?human	 ?skin	 ?permeability	 ?studies.	 ?Karande	 ?et	 ?al.	 ?[23]	 ?also	 ?used	 ?skin	 ?electrical	 ? impedance	 ?measurements	 ?to	 ?evaluate	 ?the	 ?effect	 ?of	 ? chemical	 ? enhancers	 ?on	 ? skin	 ?permeability.	 ?A	 ?more	 ? common	 ?method	 ? involves	 ?using	 ?diffusion	 ?cell	 ? instruments	 ?where	 ?the	 ?skin	 ? is	 ?mounted	 ?between	 ?a	 ?donator	 ?bath	 ?and	 ?a	 ?receptor	 ?bath	 ?and	 ?the	 ?change	 ? in	 ?concentration	 ?of	 ? the	 ?baths	 ? is	 ?monitored	 ?over	 ? time.	 ?Bath	 ?concentrations	 ? are	 ? measured	 ? by	 ? different	 ? methods	 ? including	 ? fluorescence	 ? imaging,	 ?radioactivity	 ?measurements,	 ?or	 ?high-??performance	 ?liquid	 ?chromatography	 ?[24-??35].	 ?Using	 ?this	 ?method,	 ? Khalil	 ? et	 ? al.	 ? [31]	 ?measured	 ? glucose	 ? diffusivity	 ? in	 ? the	 ? viable	 ? epidermis	 ? of	 ? human	 ?skin.	 ? Williams	 ? et	 ? al.	 ? [32]	 ? used	 ? a	 ? diffusion	 ? cell	 ? to	 ? measure	 ? the	 ? diffusion	 ? coefficient	 ? of	 ?9	 ?	 ?fluorouracil	 ?(an	 ?anti-??cancer	 ?drug)	 ?in	 ?the	 ?human	 ?epidermis	 ?when	 ?administered	 ?with	 ?different	 ?penetration	 ?enhancers.	 ?Kreilgaard	 ?et	 ?al.	 ?[33]	 ?used	 ?a	 ?diffusion	 ?cell	 ?to	 ?evaluate	 ?the	 ?usefulness	 ?of	 ? microemulsions	 ? for	 ? delivery	 ? of	 ? a	 ? lipophilic	 ? compound	 ? (lidocaine)	 ? and	 ? a	 ? hydrophilic	 ?compound	 ? (prilocaine	 ? hydrochloride)	 ? across	 ? rat	 ? skin.	 ? Similarly,	 ? Hansen	 ? et	 ? al.	 ? [34]	 ? studied	 ?the	 ?permeability	 ?of	 ?different	 ?human	 ?skin	 ?layers	 ?to	 ?flufenamic	 ?acid	 ?and	 ?caffeine	 ?compounds.	 ?A	 ? diffusion	 ? cell	 ? was	 ? also	 ? used	 ? by	 ? Mitragotri	 ? [35]	 ? to	 ? evaluate	 ? the	 ? influence	 ? of	 ? various	 ?enhancers	 ? including	 ?ultrasound	 ?and	 ? chemicals	 ? on	 ? the	 ?delivery	 ?of	 ? the	 ? five	 ?drugs	 ? estradiol,	 ?naphthol,	 ?aldosterone,	 ?lidocaine,	 ?and	 ?testosterone	 ?across	 ?the	 ?human	 ?epidermis.	 ?Confocal	 ?microscopy	 ?is	 ?also	 ?a	 ?useful	 ?approach	 ?to	 ?image	 ?diffusion	 ?of	 ?fluorescent	 ?drugs	 ?into	 ?skin	 ?[36-??40].	 ?This	 ?method	 ?has	 ?been	 ?previously	 ?used	 ?by	 ?Sch?tzlein	 ?et	 ?al.	 ?[37]	 ?to	 ?characterize	 ?transfersome-??based	 ?delivery	 ?of	 ?fluorescently	 ?labeled	 ?compounds	 ?into	 ?mouse	 ?skin.	 ?Scarmato	 ?De	 ? Rosa	 ? et	 ? al.	 ? [38]	 ? used	 ? confocal	 ? microscopy	 ? to	 ? visualize	 ? the	 ? penetration	 ? of	 ? 5-??aminolevulinic	 ?acid	 ?(a	 ?compound	 ?used	 ?in	 ?photodynamic	 ?therapy	 ?(PDT)	 ?of	 ?skin	 ?cancers)	 ?into	 ?mouse	 ? skin.	 ? Verma	 ? et	 ? al.	 ? [39]	 ? used	 ? confocal	 ? microscopy	 ? to	 ? evaluate	 ? penetration	 ? of	 ?carboxyfluorescein	 ?into	 ?abdominal	 ?human	 ?skin	 ?incubated	 ?on	 ?a	 ?diffusion	 ?cell.	 ?A.	 ?P.	 ?Raphael	 ?et	 ?al.	 ?[40]	 ?used	 ?confocal	 ?microscopy	 ?to	 ?characterize	 ?diffusivity	 ?of	 ?macromolecules	 ?delivered	 ?using	 ?a	 ?microprojection	 ?array	 ?through	 ?epidermal	 ?and	 ?dermal	 ?skin	 ?layers.	 ?10	 ?	 ?1.2 MEMS	 ?for	 ?biomedical	 ?applications,	 ?drug	 ?delivery,	 ?and	 ?biosensing	 ?The	 ?field	 ?of	 ?microelectromechanical	 ?system	 ?(MEMS)	 ?research	 ?deals	 ?with	 ?the	 ?development	 ?of	 ?very	 ?small	 ?(often	 ?less	 ?than	 ?1	 ?mm)	 ?electromechanical	 ?structures	 ?[41-??43].	 ?This	 ?technology	 ?uses	 ? the	 ? conventional	 ? semiconductor	 ? processing	 ? tools	 ? used	 ? for	 ? electronic	 ? chip	 ?manufacturing	 ?to	 ?create	 ?systems	 ?that	 ?are	 ?used	 ?for	 ?a	 ?variety	 ?of	 ?applications	 ?such	 ?as	 ?power	 ?generation,	 ? sensing,	 ? and	 ? actuation.	 ? The	 ? small	 ? size	 ? of	 ? these	 ? devices	 ? provides	 ? many	 ?advantages	 ?over	 ? large-??scale	 ? systems	 ? in	 ? terms	 ?of	 ?power	 ?usage,	 ? sensitivity,	 ? cost,	 ? and	 ? space	 ?occupation	 ?[41-??43].	 ?Common	 ?materials	 ?used	 ? in	 ?MEMS	 ?technology	 ?are	 ?silicon,	 ?polymers,	 ?metals,	 ?and	 ?ceramics,	 ?and	 ?the	 ?basic	 ?techniques	 ?used	 ?in	 ?MEMS	 ?fabrication	 ?are	 ?material	 ?deposition,	 ?patterning,	 ?and	 ?etching.	 ?Material	 ? deposition	 ? leads	 ? to	 ? the	 ? formation	 ?of	 ? layers	 ?of	 ?materials	 ?with	 ? controlled	 ?thicknesses	 ?ranging	 ?from	 ?a	 ?few	 ?nanometers	 ?up	 ?to	 ?hundreds	 ?of	 ?micrometers	 ?on	 ?a	 ?substrate	 ?[41-??43].	 ?Physical	 ?vapor	 ?deposition	 ?(PVD)	 ?is	 ?a	 ?physical	 ?deposition	 ?technique	 ?that	 ?evaporates	 ?material	 ? and	 ? deposits	 ? the	 ? vapor	 ? onto	 ? a	 ? surface.	 ? Examples	 ? are	 ? deposition	 ? of	 ? metals	 ? by	 ?sputtering	 ?or	 ?evaporation.	 ?Another	 ?physical	 ?deposition	 ?method	 ?is	 ?solvent	 ?casting	 ?in	 ?which	 ?a	 ?polymer	 ?solution	 ?is	 ?applied	 ?to	 ?a	 ?surface	 ?and	 ?after	 ?evaporation	 ?of	 ?the	 ?solvent	 ?a	 ?polymer	 ?layer	 ?coats	 ?the	 ?surface.	 ?In	 ?chemical	 ?deposition	 ?methods	 ?(such	 ?as	 ?chemical	 ?vapor	 ?deposition,	 ?CVD)	 ?a	 ? stream	 ?of	 ? gas	 ?or	 ?plasma	 ? reacts	 ?on	 ?a	 ? substrate	 ? surface	 ? to	 ? grow	 ? the	 ?desired	 ?material,	 ? or	 ?alternatively	 ?the	 ?gas	 ?species	 ?first	 ?react	 ?and	 ?then	 ?deposit	 ?on	 ?the	 ?substrate.	 ?11	 ?	 ?The	 ?patterning	 ?process	 ? involves	 ? transferring	 ?a	 ?pattern	 ?onto	 ?a	 ?substrate	 ? [41-??43].	 ?The	 ?most	 ?common	 ? type	 ? is	 ? photolithography	 ? in	 ?which	 ? the	 ?pattern	 ? is	 ? transferred	 ? to	 ? a	 ?photosensitive	 ?material	 ?(Figure	 ?1.3).	 ?Through	 ?this	 ?process,	 ?an	 ?extruded	 ?form	 ?of	 ?the	 ?pattern	 ?is	 ?created	 ?with	 ?the	 ?photosensitive	 ?material	 ?(i.e.	 ?photoresist).	 ?The	 ?photoresist	 ?can	 ?be	 ?positive	 ?(light	 ?weakens	 ?the	 ? adhesion	 ? between	 ? polymer	 ? chains)	 ? or	 ? negative	 ? (light	 ? cross-??links	 ? the	 ? polymer	 ? chains).	 ?The	 ?photolithography	 ?process	 ?can	 ?be	 ?used	 ?for	 ?making	 ?etch	 ?masks	 ?(i.e.	 ?temporary	 ?layers)	 ?or	 ?permanent	 ? mold	 ? structures,	 ? and	 ? is	 ? a	 ? common	 ? fabrication	 ? step	 ? in	 ? almost	 ? all	 ? MEMS	 ?processes.	 ?	 ?The	 ?remaining	 ?MEMS	 ?fabrication	 ?step	 ?involves	 ?etching	 ?or	 ?removing	 ?material	 ?by	 ?dry	 ?etching	 ?or	 ?wet	 ?etching	 ?methods	 ?[41-??43].	 ?This	 ?is	 ?often	 ?performed	 ?to	 ?pattern	 ?functional	 ?layers	 ?or	 ?to	 ?remove	 ?a	 ?sacrificial	 ?material	 ?for	 ?facilitating	 ?the	 ?lift-??off	 ?of	 ?a	 ?structural	 ?layer.	 ?The	 ?wet	 ?etching	 ?process	 ?uses	 ? chemicals	 ? to	 ?dissolve	 ? the	 ?materials.	 ? Some	 ?examples	 ? are	 ? isotropic	 ? etching	 ?of	 ?silicon	 ? dioxide	 ? using	 ? hydrofluoric	 ? acid	 ? (HF)	 ? or	 ? anisotropic	 ? etching	 ? of	 ? pure	 ? silicon	 ? using	 ?potassium	 ? hydroxide	 ? (KOH).	 ? In	 ? the	 ? dry	 ? etching	 ? technique,	 ? chemically	 ? reactive	 ? gases	 ? are	 ?used	 ?to	 ?remove	 ?material	 ?from	 ?substrate	 ?surface.	 ?Reactive	 ?ion	 ?etching	 ?(RIE)	 ?of	 ?polymers	 ?is	 ?an	 ?example	 ? of	 ? dry	 ? etching	 ? process	 ? that	 ? uses	 ? high	 ? energy	 ? plasma.	 ? Deep	 ? reactive	 ? ion	 ? etching	 ?(DRIE)	 ? is	 ? a	 ? highly	 ? anisotropic	 ? etching	 ? process,	 ? based	 ? on	 ?multiple	 ? RIE	 ? steps,	 ? used	 ? to	 ? form	 ?deep	 ?channels	 ? in	 ? silicon.	 ?Xenon	 ?difluoride	 ? is	 ? an	 ?example	 ?of	 ? a	 ?highly	 ? reactive	 ?gas	 ?used	 ? for	 ?isotropic	 ?dry	 ?etching	 ?of	 ?silicon.	 ?	 ?	 ?12	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?Figure	 ?1.3:	 ?Photolithography	 ?process	 ?The	 ? area	 ? of	 ? MEMS	 ? with	 ? applications	 ? to	 ? the	 ? life	 ? sciences	 ? and	 ? biomedical	 ? engineering	 ? is	 ?referred	 ? to	 ? as	 ? BioMEMS	 ? [44].	 ? Cheaper	 ? and	 ? smaller	 ? devices	 ? can	 ? be	 ? fabricated	 ? using	 ? this	 ?technology	 ?for	 ?diagnosis	 ?and	 ?treatment	 ?of	 ?various	 ?conditions.	 ?Miniature	 ?medical	 ? implants,	 ?drug	 ?delivery	 ?systems,	 ?on-??chip	 ?assays,	 ?cell	 ? sorting	 ?devices,	 ?and	 ?DNA	 ?sequencers	 ?are	 ?some	 ?examples	 ?of	 ?BioMEMS	 ?devices.	 ?1.3 Microneedles	 ?as	 ?a	 ?minimally	 ?invasive	 ?interface	 ?with	 ?the	 ?body	 ?A	 ?transdermal	 ?approach	 ?to	 ?drug	 ?delivery	 ?using	 ?microneedles	 ?can	 ?potentially	 ?circumvent	 ?the	 ?limitations	 ?of	 ? adhesive	 ? skin	 ?patches.	 ?Microneedles	 ? are	 ? sharp	 ?mechanical	 ? structures	 ?made	 ?through	 ?MEMS	 ? technology.	 ? Microneedles	 ? have	 ? been	 ? used	 ? for	 ? transdermal	 ? drug	 ? delivery	 ?[45-??47]	 ? and	 ? extraction	 ? of	 ? biofluids	 ? for	 ? sensing	 ? applications	 ? [48],	 ? or	 ? as	 ? neural	 ? probes	 ? and	 ?Light	 ?source	 ?Lens	 ?Photomask	 ?with	 ?pattern	 ?Substrate	 ?Patterned	 ?photoresist	 ?13	 ?	 ?microelectrode	 ?arrays	 ?[49].	 ?They	 ?can	 ?have	 ?solid	 ?or	 ?hollow	 ?structures,	 ?and	 ?can	 ?be	 ?arranged	 ?in	 ?out-??of-??plane	 ?or	 ? in-??plane	 ?arrays	 ?(Figure	 ?1.4).	 ?Out-??of-??plane	 ?microneedles	 ?are	 ?often	 ?less	 ?than	 ?1	 ?mm	 ?tall	 ?while	 ?in-??plane	 ?devices	 ?are	 ?usually	 ?longer	 ?than	 ?1	 ?mm.	 ?	 ?	 ?	 ?Figure	 ?1.4:	 ?Comparison	 ?of	 ?orientation	 ?of	 ?in-??plane	 ?and	 ?out-??of-??plane	 ?microneedles,	 ?illustrated	 ?on	 ?a	 ?wafer	 ?substrate	 ?in	 ?stereoscopic	 ?view.	 ?In-??plane	 ?needles	 ?are	 ?arranged	 ?along	 ?the	 ?plane	 ?of	 ?the	 ?substrate	 ?while	 ?out-??of-??plane	 ?needles	 ?are	 ?arranged	 ?perpendicular	 ?to	 ?the	 ?plane	 ?of	 ?the	 ?substrate.	 ?For	 ?drug	 ?delivery	 ?applications,	 ?unlike	 ?traditional	 ?hypodermic	 ?needles,	 ?microneedles	 ?can	 ?be	 ?used	 ? in	 ? a	 ?way	 ? that	 ? does	 ? not	 ? require	 ? a	 ? high	 ? level	 ? of	 ? training,	 ? and	 ? they	 ? bear	 ? a	 ? low	 ? risk	 ? of	 ?device	 ? contamination	 ? through	 ? blood.	 ? Furthermore,	 ? microneedles	 ? can	 ? facilitate	 ? fluid	 ?injection	 ?and	 ?sampling	 ?painlessly	 ?or	 ?with	 ?minimal	 ?pain	 ?sensation	 ?(mostly	 ?from	 ?the	 ?applied	 ?pressure)	 ? if	 ? they	 ?do	 ?not	 ? penetrate	 ? deep	 ? enough	 ? to	 ? reach	 ? the	 ?nerve	 ? endings	 ? in	 ? the	 ? skin?s	 ?dermal	 ? layer.	 ?Previous	 ?studies	 ?have	 ?demonstrated	 ?painless	 ? injection	 ?of	 ?drugs	 ?into	 ?the	 ?skin	 ?using	 ? microneedles	 ? [50].	 ? Microneedles	 ? can	 ? potentially	 ? replace	 ? hypodermic	 ? needles	 ? for	 ?delivery	 ?of	 ?many	 ?compounds	 ?including	 ?vaccines	 ?and	 ?drugs	 ?that	 ?target	 ?the	 ?blood	 ?circulation	 ?system.	 ?Microneedles	 ?can	 ?also	 ?be	 ?used	 ?to	 ?substantially	 ?increase	 ?the	 ?rate	 ?of	 ?delivery	 ?of	 ?drugs	 ?currently	 ?administered	 ?via	 ?adhesive	 ?patches.	 ?In	 ?some	 ?case	 ?where	 ?the	 ?drug	 ?properties	 ?(such	 ?as	 ?molecular	 ?weight)	 ?make	 ?it	 ?difficult	 ?to	 ?administer	 ?via	 ?adhesive	 ?patches,	 ?microneedles	 ?can	 ?overcome	 ?the	 ?SC	 ?barrier	 ?and	 ?substantially	 ? improve	 ?the	 ?delivery	 ?rate	 ?of	 ? these	 ?compounds.	 ?In-??plane	 ?Out-??of-??plane	 ?In-??plane	 ?needles	 ?Out-??of-??plane	 ?needles	 ? Substrate	 ?14	 ?	 ?Microneedles	 ? are	 ? also	 ? useful	 ? for	 ? targeted	 ? drug	 ? delivery	 ? into	 ? the	 ? skin	 ? for	 ? treatment	 ? of	 ?various	 ?skin	 ?conditions	 ?such	 ?as	 ?skin	 ?cancers.	 ?	 ?Since	 ?the	 ?early	 ?1990s,	 ?different	 ?concepts	 ?for	 ?the	 ?fabrication	 ?of	 ?microneedles	 ?with	 ?a	 ?variety	 ?of	 ?dimensions	 ?and	 ?geometries	 ?have	 ?been	 ?developed.	 ?These	 ?devices	 ?have	 ?been	 ? fabricated	 ?out	 ? of	 ? single	 ? or	 ? polycrystal	 ? silicon	 ? [51-??65],	 ? glass	 ? or	 ? silica	 ? [66-??68],	 ? metals	 ? [69-??76],	 ? and	 ?polymers	 ?[77-??82].	 ?1.3.1 	 ?In-??plane	 ?solid	 ?and	 ?hollow	 ?microneedles	 ?In-??plane	 ?microneedles	 ?are	 ?planar	 ?structures	 ? formed	 ?along	 ? the	 ?surface	 ?of	 ?a	 ?base	 ?substrate	 ?and	 ? are	 ? developed	 ? for	 ? drug	 ? delivery,	 ? biosensing,	 ? and	 ? neural	 ? stimulation	 ? [51-??54].	 ? These	 ?devices	 ? are	 ? often	 ? longer	 ? than	 ? 1	 ?mm	 ? and	 ? some	 ? are	 ? designed	 ? for	 ? targeted	 ? delivery	 ? at	 ? the	 ?cellular	 ? level.	 ? They	 ? are	 ? usually	 ? integrated	 ? with	 ? additional	 ? microfluidic	 ? and	 ? electronic	 ?circuitry	 ?to	 ?make	 ?more	 ?complete	 ?lab-??on-??a-??chip	 ?systems.	 ?Chen	 ?et	 ?al.	 ?[51]	 ?has	 ?presented	 ?a	 ?4	 ?mm	 ?silicon	 ?in-??plane	 ?hollow	 ?probe	 ?for	 ?neural	 ?stimulation	 ?and	 ?targeted	 ?drug	 ?delivery.	 ?The	 ?fabrication	 ?process	 ?uses	 ?multiple	 ?boron	 ?doping	 ?and	 ?silicon	 ?dioxide	 ? patterning	 ? steps	 ? as	 ?well	 ? as	 ? anisotropic	 ? etching	 ? of	 ? silicon	 ? along	 ?wafer	 ? plane	 ? using	 ?ethylene-??diamine	 ? pyrocatechol	 ? (EDP),	 ? to	 ? create	 ? the	 ? hollow	 ? needle	 ? structure.	 ? A	 ? similar	 ?fabrication	 ? process	 ? is	 ? presented	 ? by	 ? Lin	 ? et	 ? al.	 ? [52]	 ? in	 ? which	 ? needles,	 ? made	 ? from	 ? silicon	 ?nitride,	 ? are	 ? formed	 ? through	 ? silicon	 ? dioxide	 ? patterning,	 ? boron	 ? doping,	 ? silicon	 ? nitride	 ?deposition,	 ?and	 ?EDP	 ?etching	 ?of	 ?silicon.	 ?	 ?15	 ?	 ?An	 ? alternative	 ? fabrication	 ? method	 ? for	 ? in-??plane	 ? microneedles	 ? is	 ? presented	 ? by	 ? Zahn	 ? et	 ? al.	 ?	 ?[33].	 ?This	 ?fabrication	 ?method	 ?is	 ?based	 ?on	 ?forming	 ?a	 ?polysilicon	 ?microneedle	 ?structures	 ?onto	 ?a	 ? silicon	 ?micromold,	 ?with	 ?silicon	 ?dioxide	 ?being	 ?used	 ?as	 ? sacrificial	 ? release	 ? layer.	 ?The	 ?silicon	 ?mold	 ? is	 ? a	 ? planar	 ? channel	 ? formed	 ? by	 ? aligning	 ? and	 ? attaching	 ? two	 ? patterned	 ? silicon	 ? wafers	 ?together.	 ? The	 ?main	 ?advantage	 ?of	 ? the	 ?process	 ? is	 ? that	 ? the	 ? silicon	 ?micromolds	 ?are	 ? reusable,	 ?but	 ?each	 ?fabrication	 ?run	 ?requires	 ?precise	 ?alignment	 ?of	 ?the	 ?wafers.	 ?Paik	 ?et	 ?al.	 ?[54]	 ?presented	 ?a	 ?similar	 ?fabrication	 ?technique	 ?to	 ?make	 ?polysilicon	 ?needles	 ?but	 ?without	 ?alignment,	 ?however,	 ?the	 ?process	 ? required	 ?multiple	 ?deep	 ? reactive	 ? ion	 ?etching	 ? steps	 ? (DRIE)	 ? to	 ? form	 ?deep	 ? silicon	 ?channels.	 ?Fabrication	 ?of	 ?polymer	 ?in-??plane	 ?microneedle	 ?has	 ?been	 ?demonstrated	 ?by	 ?Takeuchi	 ?et	 ?al.	 ?[77].	 ?A	 ? flexible	 ? single	 ? needle	 ? has	 ? been	 ?made	 ? by	 ? deposition	 ? of	 ? polyimide	 ? on	 ? an	 ? AZ	 ? photoresist	 ?sacrificial	 ? layer	 ? that	 ? corresponds	 ? to	 ? the	 ? inner	 ? channel.	 ? The	 ? proposed	 ? design	 ? reduces	 ? the	 ?fabrication	 ?costs	 ?compared	 ?to	 ?the	 ?previous	 ?designs,	 ?but	 ?the	 ?needle	 ?by	 ?itself	 ?was	 ?too	 ?flexible	 ?and	 ?weak	 ?and	 ?not	 ?capable	 ?of	 ?penetrating	 ?skin.	 ?To	 ?solve	 ?this,	 ?the	 ?authors	 ?proposed	 ?filling	 ?the	 ?needle	 ? channel	 ? with	 ? a	 ? temporary	 ? polyethylene	 ? glycol	 ? (PEG)	 ? layer	 ? to	 ? increase	 ? the	 ? needle	 ?rigidity	 ?for	 ?skin	 ?penetration.	 ?Finally,	 ? to	 ?make	 ?metallic	 ? in-??plane	 ? needles,	 ? Brazzle	 ? et	 ? al.	 ? [69]	 ? used	 ? electroplating	 ? to	 ? form	 ?needles	 ? on	 ? sacrificial	 ? photoresist	 ? layers.	 ? This	 ? process	 ? is	 ? among	 ? the	 ? cheapest	 ? fabrication	 ?methods	 ? for	 ? in-??plane	 ? needles,	 ? but	 ? still	 ? requires	 ? multiple	 ? masking	 ? and	 ? metal	 ? sputtering	 ?steps.	 ?16	 ?	 ?Although	 ?the	 ?usefulness	 ?of	 ?in-??plane	 ?needles	 ?have	 ?been	 ?demonstrated	 ?in	 ?these	 ?works,	 ?they	 ?have	 ?not	 ?been	 ?commercially	 ?adopted	 ?since	 ?the	 ?manufacturing	 ?processes	 ?are	 ?too	 ?expensive	 ?for	 ? single-??use	 ? systems.	 ? In	 ? addition,	 ? these	 ? processes	 ? are	 ? useful	 ? for	 ? making	 ? single	 ? needle	 ?devices	 ? or	 ? one-??dimensional	 ? arrays,	 ? which	 ? may	 ? not	 ? be	 ? sufficient	 ? for	 ? delivering	 ? larger	 ?quantities	 ?of	 ?drugs.	 ?Developing	 ?cheaper	 ?two-??dimensional	 ?arrays,	 ?containing	 ?larger	 ?numbers	 ?of	 ?out-??of-??plane	 ?microneedles,	 ?is	 ?thus	 ?crucial	 ?for	 ?overcoming	 ?these	 ?limitations.	 ?1.3.2 Solid	 ?out-??of-??plane	 ?microneedles	 ?Solid	 ?out-??of-??plane	 ?microneedles	 ?are	 ?designed	 ?to	 ?increase	 ?the	 ?skin	 ?permeability	 ?by	 ?piercing	 ?the	 ?SC	 ?and	 ?exposing	 ?the	 ?underlying	 ?skin	 ?layers	 ?to	 ?the	 ?drugs	 ?that	 ?are	 ?later	 ?applied	 ?to	 ?the	 ?skin	 ?surface,	 ? or	 ? alternatively	 ? to	 ? the	 ? drugs	 ? that	 ? already	 ? coat	 ? the	 ? surface	 ? of	 ? the	 ? needles	 ? or	 ? are	 ?embedded	 ? in	 ?a	 ?biodegradable	 ?polymer	 ? that	 ? is	 ? the	 ?structural	 ?material	 ?of	 ? the	 ?needles	 ? [10].	 ?Solid	 ?microneedles	 ?provide	 ?an	 ?improvement	 ?to	 ?the	 ?existing	 ?skin	 ?patches	 ?by	 ?overcoming	 ?the	 ?SC	 ?barrier.	 ?It	 ?is	 ?shown	 ?that	 ?solid	 ?microneedles	 ?can	 ?increase	 ?skin	 ?permeability	 ?by	 ?almost	 ?four	 ?orders	 ? of	 ? magnitude	 ? [20,	 ? 75].	 ? However,	 ? the	 ? disadvantage	 ? of	 ? these	 ? devices	 ? is	 ? that	 ? they	 ?provide	 ?a	 ?passive	 ?drug	 ?delivery	 ?method	 ?that	 ?is	 ?slow	 ?which	 ?may	 ?not	 ?be	 ?useful	 ?for	 ?delivery	 ?of	 ?many	 ?compounds.	 ?Griss	 ?et	 ?al.	 ?[83],	 ?Hashmi	 ?et	 ?al.	 ?[84],	 ?Shikida	 ?et	 ?al.	 ?[64],	 ?and	 ?Ji	 ?et	 ?al.	 ?[85]	 ?all	 ?present	 ?fabrication	 ?processes	 ? for	 ? making	 ? large	 ? arrays	 ? of	 ? solid	 ? out-??of-??plane	 ? microneedles	 ? from	 ? silicon.	 ? The	 ?needles	 ?are	 ?cone-??shaped	 ?and	 ?sharp,	 ?and	 ?formed	 ?through	 ?isotropic	 ?or	 ?anisotropic	 ?etching	 ?of	 ?silicon	 ?that	 ?created	 ?undercuts	 ?beneath	 ?silicon	 ?dioxide	 ?mask	 ?layers	 ?(Figure	 ?1.5).	 ?17	 ?	 ?	 ?	 ?	 ?Figure	 ?1.5:	 ?Fabrication	 ?of	 ?sharp	 ?cone-??shaped	 ?structures	 ?using	 ?silicon	 ?etching.	 ?Park	 ?et	 ?al.	 ?[79]	 ?present	 ?solid	 ?biodegradable	 ?microneedle	 ?arrays	 ?designed	 ?to	 ?dissolve	 ?inside	 ?the	 ?skin	 ?and	 ?release	 ?the	 ?drugs	 ?that	 ?are	 ?integrated	 ?within	 ?the	 ?structural	 ?polymer	 ?matrix.	 ?For	 ?the	 ? fabrication,	 ? a	 ? master	 ? mold	 ? is	 ? first	 ? made,	 ? often	 ? through	 ? photolithography,	 ? which	 ?contains	 ? an	 ? array	 ? of	 ? pillars.	 ? Next,	 ? the	 ? inverse	 ? of	 ? the	 ? master	 ? mold	 ? is	 ? made	 ? by	 ? a	 ? flexible	 ?molding	 ? material	 ? such	 ? as	 ? polydimethylsiloxane	 ? (PDMS).	 ? A	 ? solution	 ? of	 ? the	 ? biodegradable	 ?polymer	 ?(polyglycolic	 ?acid,	 ?PGA	 ?or	 ?polylactice-??co-??glycolic	 ?acid,	 ?PLGA)	 ?was	 ?then	 ?filled	 ?in	 ?this	 ?mold	 ? leading	 ? to	 ? the	 ? replicated	 ?biodegradable	 ?pillar	 ?arrays.	 ? Similar	 ? technique	 ?was	 ?used	 ?by	 ?Han	 ?et	 ?al.	 ?[80]	 ?but	 ?instead	 ?of	 ?casting	 ?the	 ?polymer	 ?solution,	 ?polycarbonate	 ?(PC)	 ?needles	 ?were	 ?made	 ?through	 ?hot	 ?embossing.	 ?Solid	 ?metallic	 ?microneedle	 ? arrays	 ? have	 ? also	 ? been	 ?made	 ? and	 ? already	 ? used	 ? for	 ? commercial	 ?applications	 ?[86].	 ?One	 ?fabrication	 ?method	 ?uses	 ?a	 ?laser	 ?to	 ?create	 ?needle-??shaped	 ?patterns	 ?in	 ?metallic	 ?sheets;	 ? the	 ?needles	 ?are	 ? then	 ?bent	 ?perpendicular	 ? to	 ? the	 ?needle	 ?plane	 ?to	 ? form	 ?the	 ?needle	 ?array.	 ?1.3.3 Hollow	 ?out-??of-??plane	 ?microneedles	 ?Hollow	 ?out-??of-??plane	 ?microneedles	 ?can	 ?pierce	 ?through	 ?the	 ?SC	 ?and	 ?provide	 ?a	 ?passage	 ?for	 ?the	 ?injection	 ?of	 ?lipophilic	 ?and	 ?hydrophilic	 ?compounds	 ?of	 ?small	 ?and	 ?large	 ?molecular	 ?weights	 ?into	 ?SiO2	 ?masks	 ?Silicon	 ?substrate	 ? Undercuts	 ?18	 ?	 ?the	 ?skin	 ?(Figure	 ?1.6).	 ? In	 ?contrast	 ?to	 ?adhesive	 ?patches,	 ?hollow	 ?microneedles	 ?are	 ?not	 ?limited	 ?by	 ?skin	 ?permeability,	 ?and	 ?unlike	 ?solid	 ?microneedles,	 ?they	 ?can	 ?be	 ?potentially	 ?used	 ?to	 ?deliver	 ?larger	 ?amounts	 ?of	 ?drugs	 ?into	 ?the	 ?skin.	 ?	 ?	 ?	 ?Figure	 ?1.6:	 ?Concept	 ?of	 ?drug	 ?delivery	 ?using	 ?hollow	 ?microneedles.	 ?Transdermal	 ? drug	 ? delivery	 ? through	 ? hollow	 ? out-??of-??plane	 ? microneedles	 ? has	 ? been	 ?demonstrated	 ? in	 ? clinical	 ? trials,	 ? where	 ? methyl	 ?nicotinate	 ? was	 ? injected	 ? using	 ? hollow	 ?microneedles	 ? [50].	 ? During	 ? this	 ? study,	 ? the	 ? volunteers	 ? confirmed	 ? that	 ? the	 ? method	 ? was	 ?painless	 ?and	 ?they	 ?only	 ?felt	 ?a	 ?slight	 ?pressure	 ?during	 ?the	 ?injection.	 ?In	 ?a	 ?different	 ?study,	 ?hollow	 ?metal	 ?microneedles	 ?were	 ?used	 ?to	 ?deliver	 ?insulin	 ?to	 ?diabetic	 ?hairless	 ?rats	 ?[73];	 ?a	 ?4-??hr	 ?delivery	 ?of	 ?insulin	 ?injection	 ?resulted	 ?in	 ?47%	 ?reduction	 ?in	 ?the	 ?blood	 ?glucose.	 ?Hollow	 ? silicon	 ?microneedle	 ? arrays	 ?were	 ? the	 ? first	 ? hollow	 ? designs	 ? that	 ?were	 ? demonstrated	 ?and	 ?tested	 ?in	 ?clinical	 ?applications.	 ?Stoeber	 ?et	 ?al.	 ?[55,	 ?56]	 ?present	 ?a	 ?process	 ?for	 ?making	 ?arrays	 ?of	 ?200-??250	 ??m	 ?tall	 ?microneedles	 ?with	 ?sharp	 ?tips	 ?and	 ?wide	 ?bases	 ?(Figure	 ?1.7).	 ?The	 ?fabrication	 ?Drug	 ?Injection	 ?Stratum	 ?corneum	 ?Epidermis	 ?Dermis	 ?Subcutaneous	 ?tissue	 ?Nerves	 ?Blood	 ?vessels	 ?Microneedle	 ?19	 ?	 ?process	 ?is	 ?based	 ?on	 ?first	 ?creating	 ?a	 ?deep	 ?channel	 ?(corresponding	 ?to	 ?the	 ?needle	 ?lumen)	 ?from	 ?one	 ?side	 ?of	 ?a	 ?silicon	 ?wafer	 ?using	 ?DRIE	 ?(Figure	 ?1.7a-??1?3)	 ?and	 ?then	 ?using	 ?isotropic	 ?etching	 ?of	 ?silicon	 ? on	 ? the	 ? other	 ? side	 ? of	 ? the	 ?wafer	 ? to	 ? create	 ? the	 ? cone-??shape	 ? needle	 ? structure	 ? (Figure	 ?1.7a-??4?7).	 ?The	 ?proposed	 ?process	 ?also	 ?allows	 ?producing	 ?sharper	 ?needles	 ?by	 ?dislocating	 ?the	 ?centerline	 ? of	 ? the	 ? patterned	 ? silicon	 ? dioxide	 ? layer	 ? in	 ? Figure	 ? 1.7a-??5	 ? with	 ? respect	 ? to	 ? the	 ?centerline	 ?of	 ?the	 ?channel.	 ?This	 ?results	 ?in	 ?one	 ?side	 ?of	 ?the	 ?needle	 ?to	 ?be	 ?etched	 ?more	 ?than	 ?the	 ?other	 ?side	 ?resulting	 ?in	 ?pointed	 ?tip	 ?shapes	 ?shown	 ?in	 ?Figure	 ?1.7c.	 ?	 ?	 ?Figure	 ?1.7:	 ?a)	 ?silicon	 ?microneedle	 ?fabrication	 ?process,	 ?b)	 ?SEM	 ?image	 ?of	 ?microneedles	 ?with	 ?symmetrical	 ?tips,	 ?c)	 ?SEM	 ?image	 ?of	 ?microneedles	 ?with	 ?pointed	 ?tips	 ?[56].	 ?Similar	 ?concepts	 ?were	 ?used	 ?by	 ?other	 ?groups	 ?such	 ?as	 ?Griss	 ?et	 ?al.	 ?[63],	 ?Gardeniers	 ?et	 ?al.	 ?[60],	 ?and	 ? Mukerjee	 ? et	 ? al.	 ? [48]	 ? for	 ? producing	 ? silicon	 ? microneedles.	 ? In	 ? these	 ? works,	 ? the	 ? deep	 ?needle	 ? lumens	 ? are	 ? created	 ? through	 ? masking	 ? steps	 ? and	 ? DRIE	 ? of	 ? silicon,	 ? and	 ? the	 ? needle	 ?shapes	 ? are	 ? formed	 ? through	 ? isotropic	 ? and	 ? anisotropic	 ? wet	 ? or	 ? dry	 ? etching	 ? steps.	 ? DRIE	 ? of	 ?a)	 ? b)	 ?c)	 ?20	 ?	 ?silicon	 ?has	 ?also	 ?been	 ?combined	 ?with	 ?multiple	 ?deposition	 ?and	 ?etching	 ?steps	 ?to	 ? form	 ?silicon	 ?dioxide	 ?microneedles	 ?[66].	 ?Hollow	 ?polymer	 ?microneedles	 ?are	 ?not	 ?generally	 ?as	 ?strong	 ?as	 ?silicon	 ?designs	 ?but	 ?they	 ?are	 ?less	 ?expensive	 ?and	 ?thus	 ?more	 ?suitable	 ?to	 ?be	 ?used	 ?as	 ?single-??use	 ?disposable	 ?devices.	 ?Moon	 ?et	 ?al.	 ?[78]	 ? have	 ? used	 ? two	 ? successive	 ? inclined	 ? photoresist	 ? exposure	 ? steps	 ? and	 ? a	 ? hot	 ? embossing	 ?step	 ? using	 ? a	 ? PDMS	 ? mold	 ? to	 ? make	 ? 750?1000	 ??m	 ? tall	 ? microneedles	 ? from	 ? poly(methyl	 ?methacrylate)	 ? (PMMA).	 ? This	 ? process	 ? is	 ? time-??intensive	 ? and	 ? requires	 ? sensitive	 ? alignment	 ?steps	 ?to	 ?make	 ?the	 ?needles.	 ?An	 ?even	 ?longer	 ?fabrication	 ?process	 ?is	 ?proposed	 ?by	 ?Huang	 ?et	 ?al.	 ?[81]	 ? that	 ? involved	 ? making	 ? a	 ? patterned	 ? PDMS	 ? base	 ? layer	 ? on	 ? another	 ? patterned	 ? glass	 ?assembly,	 ?and	 ?then	 ?forming	 ?needles	 ?on	 ?the	 ?PDMS	 ?layer	 ?from	 ?SU-??8	 ?(an	 ?epoxy	 ?type	 ?negative	 ?photoresist)	 ? through	 ?photolithography.	 ?The	 ?resulting	 ?needles	 ? (~200	 ??m	 ?tall)	 ?are	 ?not	 ?sharp	 ?and	 ?their	 ?mechanical	 ?strength	 ?for	 ?reliable	 ?skin	 ?penetration	 ?is	 ?not	 ?sufficiently	 ?demonstrated.	 ?To	 ?make	 ?hollow	 ?microneedles	 ?from	 ?metal,	 ?Kim	 ?et	 ?al.	 ?[70]	 ?first	 ?make	 ?a	 ?mold	 ?(containing	 ?an	 ?array	 ?of	 ?pillars)	 ?from	 ?SU-??8	 ?on	 ?a	 ?glass	 ?substrate	 ?and	 ?then	 ?after	 ?deposition	 ?of	 ?a	 ?metallic	 ?seed	 ?layer,	 ?a	 ?thick	 ?nickel	 ? layer	 ? is	 ?electroplated	 ?on	 ?the	 ?mold.	 ?The	 ?electroplating	 ?step	 ?also	 ?covers	 ?the	 ?pillar	 ?tips	 ?resulting	 ?in	 ?non-??hollow	 ?structures.	 ?To	 ?remove	 ?the	 ?nickel	 ?covering	 ?the	 ?tips,	 ?an	 ?additional	 ?mechanical	 ?polishing	 ?step	 ?is	 ?used	 ?with	 ?an	 ?extra	 ?layer	 ?of	 ?unexposed	 ?SU-??8	 ?used	 ?as	 ?planarizing	 ? layer	 ? (to	 ? assist	 ? with	 ? uniform	 ? polishing	 ? of	 ? the	 ? tips).	 ? After	 ? polishing,	 ? the	 ?planarizing	 ? SU-??8	 ? layer	 ? is	 ? removed	 ? in	 ? developer.	 ? The	 ? microneedle	 ? array	 ? (400	 ??m	 ? tall)	 ? is	 ?separated	 ? by	 ? first	 ? dry	 ? etching	 ? the	 ? SU-??8	 ?mold	 ? and	 ? then	 ?wet	 ? etching	 ? of	 ? the	 ? electroplating	 ?seed	 ?layer.	 ?The	 ?proposed	 ?process	 ?is	 ?time	 ?consuming	 ?and	 ?expensive	 ?since	 ?there	 ?are	 ?multiple	 ?21	 ?	 ?wet	 ?and	 ?dry	 ?etching	 ?steps	 ?and	 ?also	 ?the	 ?SU-??8	 ?molds	 ?are	 ?not	 ?reusable.	 ?In	 ?addition,	 ?the	 ?process	 ?yield	 ? is	 ? low	 ?since	 ? removing	 ?SU-??8	 ? in	 ?deep	 ?metallic	 ? channels	 ?using	 ?dry	 ?etching	 ? is	 ?not	 ?highly	 ?efficient,	 ?which	 ?results	 ?in	 ?a	 ?lot	 ?of	 ?the	 ?SU-??8	 ?pillars	 ?getting	 ?stuck	 ?in	 ?the	 ?nickel	 ?needles.	 ?A	 ? different	 ? process	 ? proposed	 ? by	 ? Davis	 ? et	 ? al.	 ? [73]	 ? creates	 ? metallic	 ? microneedles	 ? by	 ?electroplating	 ?nickel	 ?on	 ?cone	 ?shaped	 ?through-??holes	 ?(drilled	 ?by	 ?laser)	 ?in	 ?polymer	 ?substrates.	 ?The	 ?microneedle	 ?array	 ?is	 ?separated	 ?from	 ?the	 ?polymer	 ?substrate	 ?by	 ?dissolving	 ?the	 ?polymer	 ?in	 ?solvent.	 ?The	 ?resulting	 ?needles	 ?are	 ?500	 ??m	 ?tall.	 ?The	 ?main	 ?disadvantage	 ?of	 ?this	 ?process	 ?is	 ?that	 ?it	 ? requires	 ? drilling	 ? individual	 ? holes	 ? on	 ? single-??use	 ? polymer	 ? substrate,	 ? which	 ? is	 ? a	 ? time	 ?consuming	 ?process	 ?especially	 ?for	 ?large	 ?needle	 ?arrays.	 ?Fabrication	 ? of	 ? ultra-??high-??aspect-??ratio	 ? metallic	 ? microneedles	 ? (taller	 ? than	 ? 1	 ?mm)	 ? was	 ?presented	 ?by	 ?Li	 ?et	 ?al.	 ? [76]	 ?and	 ?Lee	 ?et	 ?al.	 ? [87].	 ?To	 ?create	 ?microneedle	 ?arrays,	 ? first	 ?drawing	 ?photolithography	 ?technique	 ?was	 ?used	 ?to	 ?make	 ?a	 ?base	 ?layer	 ?containing	 ?tall	 ?pillar	 ?arrays	 ?from	 ?SU-??8.	 ?Drawing	 ? lithography	 ? takes	 ?advantage	 ?of	 ? the	 ?higher	 ?viscosity	 ?of	 ? the	 ?unexposed	 ?SU-??8	 ?material	 ?to	 ?form	 ?elongated	 ?pillar	 ?structures	 ?between	 ?two	 ?parallel	 ?plates.	 ?After	 ?making	 ?the	 ?base	 ? layer,	 ? the	 ?pillars	 ?were	 ?used	 ?as	 ?mold	 ? for	 ?metal	 ?seed	 ? layer	 ?deposition	 ?and	 ?subsequent	 ?nickel	 ? electroplating.	 ? The	 ? hollow	 ? needle	 ? tips	 ? in	 ? [87]	 ? were	 ? achieved	 ? by	 ? deposition	 ? of	 ? a	 ?nonconductive	 ? material	 ? onto	 ? the	 ? pillar	 ? tips	 ? prior	 ? to	 ? nickel	 ? deposition,	 ? while	 ? in	 ? [76]	 ? it	 ? is	 ?achieved	 ?by	 ?cutting	 ?the	 ?individual	 ?needle	 ?tips	 ?with	 ?a	 ?laser.	 ?In	 ?both	 ?cases,	 ?the	 ?needles	 ?were	 ?removed	 ? from	 ? the	 ?mold	 ? by	 ? removing	 ? the	 ? base	 ? SU-??8	 ? layer.	 ? These	 ? processes	 ? are	 ? also	 ? long	 ?(due	 ?to	 ?processing	 ?individual	 ?tips	 ?with	 ?laser)	 ?and	 ?expensive	 ?(due	 ?to	 ?the	 ?single-??use	 ?of	 ?the	 ?SU-??8	 ?molds).	 ?	 ?22	 ?	 ?In	 ? addition	 ? to	 ? drug	 ? delivery	 ? applications,	 ? hollow	 ? microneedles	 ? have	 ? been	 ? shown	 ? to	 ? be	 ?promising	 ?devices	 ?for	 ?biosensing	 ?applications.	 ?For	 ?this	 ?purpose,	 ?the	 ?hollow	 ?needles	 ?are	 ?used	 ?to	 ? sample	 ? ISF	 ? or	 ? blood	 ? from	 ? the	 ? skin	 ? in	 ? order	 ? to	 ? analyze	 ? the	 ? sample	 ? for	 ? a	 ? particular	 ?compound.	 ? Biofluid	 ? extraction	 ? using	 ? hollow	 ? microneedles	 ? is	 ? either	 ? facilitated	 ? through	 ?capillary	 ? forces	 ? or	 ? by	 ? applying	 ? a	 ? vacuum	 ? through	 ? the	 ? needle	 ? lumen.	 ?Mukerjee	 ? et	 ? al.	 ? [48]	 ?used	 ? 200	 ??m	 ? tall	 ? silicon	 ? microneedles	 ? to	 ? collect	 ? ISF	 ? using	 ? capillary	 ? forces	 ? from	 ? human	 ?earlobe.	 ?The	 ?presence	 ?of	 ?glucose	 ?was	 ?demonstrated	 ?in	 ?the	 ?sampled	 ?fluid	 ?using	 ?commercial	 ?glucose	 ?strips.	 ?	 ?Extraction	 ?of	 ?blood	 ?requires	 ?longer	 ?needles	 ?in	 ?order	 ?to	 ?reach	 ?the	 ?blood	 ?vessels	 ?in	 ?the	 ?dermis	 ?or	 ?in	 ?the	 ?hypodermal	 ?layer.	 ?Due	 ?to	 ?penetration	 ?into	 ?deeper	 ?skin	 ?layers,	 ?this	 ?process	 ?is	 ?often	 ?associated	 ? with	 ? pain.	 ? The	 ? amount	 ? of	 ? pain	 ? partly	 ? depends	 ? on	 ? the	 ? number	 ? of	 ? nerves	 ?stimulated	 ?during	 ?the	 ?insertion	 ?and	 ?extraction	 ?process,	 ?which	 ?depends	 ?on	 ?the	 ?needle	 ?shaft?s	 ?outer	 ? diameter.	 ? Therefore,	 ? thin	 ? microneedles	 ? may	 ? still	 ? have	 ? an	 ? advantage	 ? over	 ? the	 ?traditional	 ?hypodermis	 ?needles	 ?in	 ?this	 ?application.	 ?Blood	 ?extraction	 ? is	 ? shown	 ?by	 ? Li	 ? et	 ? al.	 ? [76]	 ? in	 ?which	 ? the	 ? authors	 ?use	 ? a	 ? single	 ?1800	 ??m	 ?tall	 ?metallic	 ?microneedle	 ? to	 ? extract	 ? 20	 ??L	 ?of	 ? blood	 ? from	 ?a	 ?mouse	 ? tail.	 ? The	 ?extraction	 ? force	 ? is	 ?provided	 ?by	 ?a	 ?negative	 ?pressure	 ?of	 ?13.45	 ?kPa	 ?through	 ?the	 ?needle	 ?lumen.	 ?Another	 ?proposed	 ?approach	 ? for	 ?using	 ?microneedles	 ? to	 ?extract	 ?blood	 ?or	 ? ISF	 ? is	 ? to	 ?puncture	 ?holes	 ? on	 ? the	 ? surface	 ? of	 ? the	 ? skin	 ? using	 ? microneedles	 ? and	 ? then	 ? applying	 ? vacuum	 ? to	 ? the	 ?damaged	 ?surface	 ?after	 ?removal	 ?of	 ?the	 ?needles.	 ?Wang	 ?et	 ?al.	 ?[88]	 ?use	 ?a	 ?similar	 ?technique	 ?by	 ?creating	 ?holes	 ?in	 ?skin	 ?using	 ?700-??1500	 ??m	 ?long	 ?microneedles,	 ?and	 ?then	 ?extracting	 ?1-??10	 ??L	 ?of	 ?23	 ?	 ?ISF	 ?using	 ?a	 ?200-??500	 ?mm-??Hg	 ?vacuum	 ?source	 ?after	 ?2-??10	 ?min.	 ?This	 ?method	 ?does	 ?not	 ?directly	 ?use	 ? microneedles	 ? to	 ? sample	 ? the	 ? fluids	 ? and	 ? therefore	 ? it	 ? is	 ? not	 ? possible	 ? to	 ? integrate	 ? a	 ?detection	 ?system	 ?with	 ?the	 ?microneedles.	 ?To	 ? conclude	 ? this	 ? section,	 ? the	 ? hollow	 ? microneedle	 ? offer	 ? a	 ? better	 ? alternative	 ? to	 ? other	 ?microneedle	 ?designs	 ?for	 ?injection	 ?of	 ?drugs	 ?in	 ?terms	 ?of	 ?the	 ?amount	 ?of	 ?liquid	 ?delivered	 ?as	 ?well	 ?as	 ? the	 ? injection	 ? rate.	 ? They	 ? also	 ? can	 ? be	 ? used	 ? to	 ? sample	 ? biological	 ? fluids	 ? for	 ? sensing	 ?applications.	 ? However,	 ? until	 ? now,	 ? the	 ? commercial	 ? use	 ? of	 ? hollow	 ? microneedles	 ? for	 ?biomedical	 ? applications	 ? has	 ? been	 ? encumbered	 ? by	 ? the	 ? expensive	 ? fabrication	 ? techniques	 ?mentioned	 ? above,	 ? such	 ? as	 ? deep	 ? reactive	 ? ion	 ? etching	 ? of	 ? silicon	 ? needles	 ? [48,	 ? 56,	 ? 60,	 ? 63],	 ?sequential	 ? formation	 ?of	 ?disposable	 ?polymer	 ?molds	 ? for	 ?electroplating	 ? [70,	 ?73,	 ?76,	 ?87],	 ? and	 ?multiple	 ?UV	 ?exposure	 ?or	 ?mold	 ? transfer	 ? and	 ?assembly	 ? steps	 ? to	 ? form	 ?polymer	 ?needles	 ? [78-??82].	 ? An	 ? inexpensive	 ? alternative	 ? to	 ? the	 ? costly	 ? fabrication	 ? techniques	 ? proposed	 ? in	 ? the	 ?literature	 ?would	 ?facilitate	 ?faster	 ?adoption	 ?of	 ?these	 ?systems	 ?in	 ?the	 ?commercial	 ?market.	 ?In	 ?addition	 ?to	 ?the	 ?fabrication	 ?processes,	 ?previous	 ?literature	 ?on	 ?hollow	 ?microneedles	 ?lacks	 ?an	 ?in-??depth	 ?analysis	 ?of	 ?what	 ?happens	 ? to	 ? the	 ?drug	 ?once	 ? it	 ? is	 ? injected	 ? into	 ? the	 ? skin	 ? tissue	 ?and	 ?how	 ?long	 ?it	 ?takes	 ?the	 ?drug	 ?to	 ?reach	 ?target	 ?skin	 ?layers.	 ?This	 ?is	 ?mainly	 ?because	 ?there	 ?has	 ?not	 ?yet	 ?been	 ?a	 ?method	 ?proposed	 ?to	 ?quantify	 ?the	 ?drug	 ?diffusion	 ?process	 ?in	 ?skin	 ?when	 ?the	 ?drug	 ?is	 ?delivered	 ?using	 ?hollow	 ?microneedles.	 ?Only	 ? after	 ? utilizing	 ? such	 ? a	 ?method,	 ? effective	 ?hollow	 ?microneedle	 ?systems	 ?can	 ?be	 ?designed	 ?for	 ?clinical	 ?drug	 ?delivery	 ?applications,	 ?and	 ?the	 ?drugs	 ?that	 ?are	 ?suitable	 ?for	 ?transdermal	 ?injection	 ?with	 ?hollow	 ?microneedles	 ?can	 ?be	 ?chosen.	 ?24	 ?	 ?Furthermore,	 ?although	 ?the	 ?potential	 ?for	 ?painless	 ?biosensing	 ?has	 ?been	 ?revealed	 ?in	 ?a	 ?limited	 ?number	 ? of	 ? publications,	 ? there	 ? is	 ? still	 ? a	 ? need	 ? for	 ? further	 ? analysis	 ? of	 ? the	 ? potential	 ? ISF	 ?extraction	 ?methods	 ?and	 ?investigate	 ?which	 ?ones	 ?are	 ?the	 ?most	 ?promising.	 ?	 ?This	 ?thesis	 ?aims	 ?at	 ?addressing	 ?some	 ?of	 ?the	 ?gaps	 ?and	 ?limitations	 ?in	 ?the	 ?previous	 ?literature	 ?on	 ?hollow	 ? microneedles.	 ? More	 ? specifically,	 ? the	 ? objectives	 ? pursued	 ? in	 ? this	 ? work	 ? can	 ? be	 ?summarized	 ?as	 ?follows:	 ?I. Develop	 ?cost	 ?effective	 ?fabrication	 ?processes	 ?applicable	 ? for	 ? large	 ?scale	 ?manufacturing	 ?of	 ?robust	 ?microneedles.	 ?II. Use	 ?the	 ?microneedles	 ?for	 ?biomedical	 ?applications:	 ?a. Demonstrate	 ?transdermal	 ?drug	 ?delivery	 ?using	 ?the	 ?fabricated	 ?microneedles.	 ?b. Assess	 ?the	 ?possibility	 ?of	 ?using	 ?microneedles	 ?for	 ?ISF	 ?sampling	 ?and	 ?biosensing	 ?applications.	 ?III. Develop	 ?a	 ?method	 ?useful	 ?for	 ?studying	 ?drug	 ?transport	 ?in	 ?skin	 ?and	 ?comparing	 ?different	 ?skin	 ?models.	 ?	 ?	 ?	 ?	 ? 	 ?25	 ?	 ?CHAPTER	 ?2 	 ?	 ?	 ?FABRICATION	 ?OF	 ?POLYMER	 ?MICRONEEDLES	 ?BASED	 ?ON	 ?SOLVENT	 ?CASTING	 ?	 ?	 ?Here,	 ?we	 ?present	 ?a	 ?simple	 ?solvent	 ?casting	 ?method	 ?for	 ?the	 ?fabrication	 ?of	 ?hollow	 ?out-??of-??plane	 ?polymer	 ? microneedles.	 ? Solvent	 ? casting	 ? has	 ? been	 ? a	 ? common	 ? method	 ? for	 ? more	 ? than	 ? a	 ?century	 ?to	 ?form	 ?polymer	 ?films	 ?[89].	 ?In	 ?this	 ?method,	 ?a	 ?polymer	 ?solution	 ?is	 ?deposited	 ?onto	 ?a	 ?mold,	 ?and	 ?after	 ?evaporation	 ?of	 ?the	 ?solvent,	 ?a	 ?polymer	 ?layer	 ?remains	 ?on	 ?the	 ?mold	 ?which	 ?is	 ?then	 ?separated	 ?by	 ?physical	 ?or	 ?chemical	 ?means.	 ?In	 ?MEMS,	 ?solvent	 ?casting	 ?has	 ?been	 ?used	 ?to	 ?coat	 ? microchannels	 ? with	 ? polymers	 ? such	 ? as	 ? poly(vinyl	 ? alcohol),	 ? poly(ethylene	 ? oxide),	 ?polyacrylamide,	 ? and	 ? poly(N-??hydroxyethylacrylamide),	 ? mainly	 ? for	 ? modifying	 ? surface	 ?properties	 ?or	 ? for	 ?protein	 ?and	 ?DNA	 ? separation	 ? [90].	 ? Solvent	 ? casting	 ?has	 ?also	 ?been	 ?used	 ? to	 ?make	 ? polylactic	 ? acid	 ? microstructures	 ? for	 ? tissue	 ? engineering	 ? [91]	 ? as	 ? well	 ? as	 ? polystyrene	 ?microcantilever	 ?beams	 ?for	 ?sensing	 ?applications	 ?[92].	 ?Additionally,	 ?solvent	 ?casting	 ?has	 ?been	 ?used	 ?to	 ? fabricate	 ?microneedles	 ?with	 ?solid	 ?structures	 ? [93].	 ?We	 ?have	 ?previously	 ?shown	 ?that	 ?26	 ?	 ?the	 ?polymer	 ?profile	 ?formed	 ?in	 ?a	 ?mold	 ?during	 ?solvent	 ?casting	 ?can	 ?be	 ?adjusted	 ?by	 ?controlling	 ?the	 ?solvent	 ?casting	 ?process	 ?parameters	 ?such	 ?as	 ?temperature	 ?[94].	 ?	 ?Solvent	 ?casting	 ?is	 ?a	 ?fast	 ?and	 ?repeatable	 ?fabrication	 ?process	 ?for	 ?microneedles,	 ?and	 ?it	 ?requires	 ?only	 ?one	 ?step	 ?of	 ?photolithography,	 ?eliminating	 ?the	 ?need	 ?for	 ?mask	 ?alignment.	 ?In	 ?this	 ?chapter	 ?we	 ?present	 ?the	 ?selection	 ?of	 ?the	 ?clay	 ?concentration	 ?for	 ?maximum	 ?strength	 ?of	 ?the	 ?polymer-??clay	 ? composite	 ? that	 ? serves	 ? as	 ? the	 ? structural	 ? material	 ? for	 ? the	 ? needles,	 ? followed	 ? by	 ? the	 ?detailed	 ? fabrication	 ? process	 ? of	 ? the	 ?microneedles	 ?made	 ?using	 ? solvent	 ? casting.	 ? This	 ? is	 ? then	 ?followed	 ?by	 ?a	 ?study	 ?of	 ?the	 ?mechanical	 ?strength	 ?of	 ?the	 ?fabricated	 ?microneedles,	 ?and	 ?by	 ?the	 ?demonstration	 ?of	 ?fluid	 ?injection	 ?into	 ?rabbit	 ?ear	 ?skin.	 ?	 ?	 ?2.1 Fabrication	 ?of	 ?hollow	 ?out-??of-??plane	 ?polymer	 ?microneedles	 ?2.1.1 Microneedle	 ?material	 ?selection	 ?Polyimide	 ? was	 ? chosen	 ? as	 ? the	 ? structural	 ? material	 ? for	 ? the	 ? microneedles	 ? due	 ? to	 ? its	 ? high	 ?Young?s	 ?modulus	 ?(8.5	 ?GPa)	 ?relative	 ?to	 ?that	 ?of	 ?other	 ?polymer	 ?materials	 ?[95].	 ?The	 ?polyimide	 ?type	 ?PI-??2611	 ?(HD	 ?Microsystems,	 ?Parlin,	 ?NJ)	 ?used	 ?in	 ?this	 ?work	 ?was	 ?diluted	 ?with	 ?its	 ?solvent	 ?N-??methyl-??2-??pyrrolidone	 ?(NMP)	 ?for	 ?better	 ?deposition	 ?and	 ?handling,	 ?at	 ?a	 ?weight	 ?ratio	 ?of	 ?4:3	 ?(PI-??2611:NMP).	 ? In	 ? order	 ? to	 ? further	 ? increase	 ? the	 ? strength	 ? and	 ? rigidity	 ? of	 ? the	 ? polyimide,	 ?montmorillonite	 ?nanoclay	 ?powder	 ?(Nanocor,	 ?Hoffman	 ?Estates,	 ?IL)	 ?was	 ?added	 ?to	 ?the	 ?polymer	 ?as	 ? reinforcement.	 ? A	 ? series	 ? of	 ? tests	 ? was	 ? performed	 ? to	 ? determine	 ? the	 ? optimum	 ? clay	 ?percentage	 ? in	 ? the	 ?polyimide	 ? that	 ?would	 ? result	 ? in	 ? the	 ?highest	 ? strength	 ?under	 ? compressive	 ?loading.	 ?For	 ?preparing	 ?the	 ?composite	 ?mixtures,	 ?first,	 ?the	 ?nanoclay	 ?powder	 ?was	 ?mixed	 ?with	 ?27	 ?	 ?the	 ? polyimide	 ? solvent	 ?NMP	 ? for	 ? 5	 ?seconds	 ? using	 ? a	 ?Model	 ? 100	 ? Sonic	 ?Dismembrator	 ? (Fisher	 ?Scientific)	 ?at	 ?5	 ?W	 ?output	 ?power.	 ?Then,	 ?this	 ?suspension	 ?was	 ?added	 ?to	 ?the	 ?PI-??2611	 ?polyimide	 ?solution	 ? and	 ? mixed	 ? using	 ? a	 ? stir	 ? bar	 ? for	 ? several	 ? minutes.	 ? The	 ? mechanical	 ? tests	 ? were	 ?performed	 ? on	 ? cone	 ? shaped	 ? test	 ? structures	 ? made	 ? through	 ? solvent	 ? casting	 ? the	 ? polyimide-??nanoclay	 ? composite	 ? from	 ? a	 ? PDMS	 ? mold.	 ? For	 ? each	 ? investigated	 ? clay	 ? percentage	 ? in	 ? the	 ?composite,	 ? three	 ? test	 ? structures	 ?were	 ? created	 ?using	 ? the	 ? same	 ?mold.	 ? The	 ? structures	 ?were	 ?280	 ??m	 ?tall,	 ?and	 ?had	 ?tip	 ?and	 ?base	 ?diameters	 ?of	 ?65	 ??m	 ?and	 ?115	 ??m,	 ?respectively.	 ?In	 ?order	 ?to	 ?apply	 ? compressive	 ? loads,	 ? a	 ? Physica	 ?MCR	 ? rheometer	 ? (Anton	 ? Paar,	 ? Ashland,	 ? VA)	 ?was	 ? used,	 ?which	 ? recorded	 ? force	 ? vs.	 ? displacement	 ? for	 ? each	 ? compression	 ? test	 ? (Figure	 ? 2.1).	 ? The	 ?rheometer?s	 ?test	 ?geometry	 ?was	 ?set	 ?to	 ?move	 ?vertically	 ?downwards	 ?onto	 ?the	 ?test	 ?structures	 ?at	 ?a	 ?constant	 ?velocity	 ?of	 ?2	 ??m/s.	 ?The	 ?force	 ?was	 ?recorded	 ?at	 ?a	 ?resolution	 ?of	 ?1	 ?mN.	 ?	 ?An	 ?example	 ?of	 ?a	 ?force	 ?vs.	 ?displacement	 ?graph	 ?is	 ?shown	 ?in	 ?Figure	 ?2.2.	 ?In	 ?this	 ?plot,	 ?the	 ?sharp	 ?peak	 ? corresponds	 ? to	 ? the	 ? failure	 ? load	 ? that	 ? leads	 ? to	 ? bending	 ? of	 ? the	 ? structure	 ? under	 ?compressive	 ?stress.	 ?The	 ?test	 ?results	 ?showed	 ?that	 ?the	 ?drop	 ?in	 ?force	 ?after	 ?the	 ?peak	 ?is	 ?steeper	 ?for	 ?higher	 ?clay	 ?content,	 ? indicating	 ?a	 ?more	 ?brittle	 ?behaviour.	 ?The	 ?average	 ?of	 ? the	 ?maximum	 ?failure	 ? loads	 ? for	 ? the	 ?different	 ? clay	 ?content	 ?are	 ? shown	 ? in	 ?Table	 ?1,	 ?which	 ? indicates	 ? that	 ? the	 ?2	 ?wt%	 ?clay	 ? reinforced	 ?polyimide	 ?has	 ? the	 ?highest	 ? failure	 ? load	 ?and	 ?was	 ? therefore	 ?chosen	 ?as	 ?the	 ?composite	 ?for	 ?the	 ?fabrication	 ?of	 ?microneedles.	 ?28	 ?	 ?	 ?Figure	 ?2.1:	 ?Setup	 ?used	 ?for	 ?compression	 ?tests.	 ?	 ?Figure	 ?2.2:	 ?Typical	 ?compression	 ?test	 ?curve	 ?for	 ?a	 ?test	 ?structure	 ?with	 ?2	 ?wt%	 ?clay	 ?reinforcement.	 ?	 ?	 ?	 ?29	 ?	 ?Table	 ?2.1:	 ?Mean	 ?failure	 ?loads	 ?for	 ?different	 ?clay	 ?content	 ?in	 ?polyimide,	 ?obtained	 ?from	 ?three	 ?tests	 ?per	 ?clay	 ?content.	 ?Clay	 ?content	 ?[weight	 ?%]	 ?0	 ? 1	 ? 2	 ? 3	 ? 4	 ? 5	 ? 10	 ?Average	 ?failure	 ?load	 ?[N]	 ?0.144	 ? 0.109	 ? 0.169	 ? 0.130	 ? 0.127	 ? 0.107	 ? 0.153	 ?Standard	 ?deviation	 ?[N]	 ? 0.009	 ? 0.014	 ? 0.016	 ? 0.006	 ? 0.012	 ? 0.011	 ? 0.010	 ?	 ?2.1.2 Fabrication	 ?process	 ?SU-??8	 ?photoresist	 ? (Microchem,	 ?Newton,	 ?MA)	 ?was	 ?used	 ?for	 ?the	 ?mold.	 ?For	 ? fabrication	 ?of	 ?the	 ?mold,	 ?a	 ?450	 ??m	 ?layer	 ?of	 ?SU-??8	 ?2150	 ?was	 ?first	 ?spin-??coated	 ?onto	 ?a	 ?300	 ??m	 ?thick	 ?Pyrex?	 ?glass	 ?substrate,	 ?and	 ?then	 ?soft-??baked	 ?on	 ?a	 ?hotplate	 ?at	 ?65?C	 ?for	 ?10	 ?min	 ?and	 ?at	 ?95?C	 ?for	 ?2	 ?h.	 ?The	 ?SU-??8	 ?was	 ?then	 ?exposed	 ?to	 ?5300	 ?mJ	 ?cm-??2	 ?UV	 ?light	 ?(performed	 ?in	 ?several	 ?2	 ?minute	 ?intervals	 ?with	 ?30	 ?s	 ?cooling	 ?breaks	 ?in	 ?between)	 ?through	 ?a	 ?dark	 ?field	 ?mask	 ?that	 ?contained	 ?arrays	 ?of	 ?circular	 ?transparent	 ? regions	 ? with	 ? diameter	 ? of	 ? 40	 ??m.	 ? The	 ? exposure	 ? was	 ? performed	 ? through	 ? the	 ?Pyrex?	 ? substrate,	 ? as	 ? shown	 ? in	 ? Figure	 ? 2.3a,	 ? in	 ? order	 ? to	 ? take	 ? advantage	 ? of	 ? light	 ? diffraction	 ?caused	 ?by	 ? the	 ? gap	 ?between	 ? the	 ?mask	 ? and	 ? the	 ?photoresist	 ? [96].	 ? This	 ?method	 ?of	 ? exposure	 ?resulted	 ?in	 ?tapered	 ?pillar	 ?structures	 ?with	 ?the	 ?wider	 ?bases	 ?attached	 ?to	 ?the	 ?Pyrex?	 ?base	 ?plate.	 ?After	 ? exposure,	 ? the	 ? sample	 ? was	 ? baked	 ? on	 ? a	 ? hotplate	 ? at	 ? 65?C	 ? for	 ? 5	 ?min	 ? and	 ? at	 ? 95?C	 ? for	 ?25	 ?min.	 ?Next,	 ? the	 ? sample	 ?was	 ? immersed	 ? in	 ? SU-??8	 ?developer	 ? for	 ? approximately	 ?45	 ?min	 ?and	 ?then	 ?washed	 ?for	 ?5	 ?min	 ?with	 ?fresh	 ?developer	 ?and	 ?isopropanol.	 ? 	 ?The	 ?resulting	 ?structure	 ?was	 ?an	 ? array	 ? of	 ? slightly	 ? tapered	 ? cylindrical	 ? pillars	 ? (Figure	 ? 2.3b	 ? and	 ? Figure	 ? 2.4)	 ? with	 ? base	 ?30	 ?	 ?diameters	 ? of	 ? 60	 ??m,	 ? tip	 ? diameters	 ? of	 ? 40	 ??m,	 ? and	 ? a	 ? center-??to-??center	 ? spacing	 ? of	 ? 500	 ??m.	 ?These	 ? pillars	 ? constituted	 ? the	 ? mold	 ? and	 ? they	 ? would	 ? eventually	 ? form	 ? the	 ? lumens	 ? of	 ? the	 ?needles.	 ? The	 ? entire	 ? mold	 ? structure	 ? was	 ? then	 ? coated	 ? with	 ? a	 ? 4	 ??m	 ? layer	 ? of	 ? Parylene	 ? C	 ?(Specialty	 ?Coating	 ?Systems,	 ?Indianapolis,	 ?IN).	 ?This	 ?layer	 ?improved	 ?adhesion	 ?of	 ?the	 ?pillars	 ?to	 ?the	 ? Pyrex?	 ? substrate	 ? and	 ? provided	 ? a	 ? protective	 ? surface	 ? on	 ? the	 ? mold	 ? for	 ? consecutive	 ?fabrication	 ?steps.	 ?Consecutively,	 ?a	 ?thin	 ?layer	 ?of	 ?PDMS	 ?(Dow	 ?Corning,	 ?Midland,	 ?MI)	 ?was	 ?spin-??coated	 ?onto	 ?the	 ?mold	 ?structure	 ?at	 ?a	 ?base	 ?layer	 ?thickness	 ?of	 ?30	 ??m	 ?and	 ?cured	 ?at	 ?65?C	 ?for	 ?2	 ?h	 ?(Figure	 ?2.3c).	 ?This	 ? layer	 ? improved	 ?the	 ?strength	 ?and	 ?rigidity	 ?of	 ? the	 ?mold	 ?structure	 ?and	 ?also	 ?had	 ? a	 ? poor	 ? adhesion	 ? with	 ? the	 ? structural	 ? material	 ? of	 ? the	 ? microneedles,	 ? which	 ? therefore	 ?allowed	 ? easy	 ? removal	 ? of	 ? the	 ? microneedle	 ? array	 ? from	 ? the	 ? mold.	 ? The	 ? complete	 ? mold	 ?structure	 ?contained	 ?an	 ?array	 ?of	 ?14	 ?pillars	 ?and	 ?was	 ?surrounded	 ?by	 ?a	 ?square	 ?(8	 ?mm	 ??	 ?8	 ?mm)	 ?of	 ?vertical	 ?walls	 ?(Figure	 ?2.5).	 ?The	 ?mold	 ?structure	 ?at	 ?this	 ?stage	 ?could	 ?be	 ?used	 ?for	 ?consecutive	 ?solvent	 ? casting	 ? of	 ? microneedles.	 ? For	 ? more	 ? detail	 ? on	 ? mold	 ? fabrication	 ? and	 ? the	 ?photolithography	 ?process	 ?see	 ?Appendices	 ?A.1	 ?and	 ?A.2.	 ?For	 ? fabrication	 ? of	 ? the	 ? microneedle	 ? arrays,	 ? first,	 ? the	 ? mold	 ? was	 ? treated	 ? for	 ? 30	 ?s	 ? with	 ? O2	 ?plasma	 ? in	 ? a	 ? RIE/PECVD	 ? tool	 ? (Trion	 ? Technology,	 ? Clearwater,	 ? FL),	 ? with	 ? a	 ? power	 ? setting	 ? of	 ?30	 ?W,	 ?an	 ?oxygen	 ?flow	 ?rate	 ?of	 ?50	 ?sccm,	 ?and	 ?a	 ?chamber	 ?pressure	 ?of	 ?500	 ?mTorr.	 ?The	 ?plasma	 ?treatment	 ?temporarily	 ?improved	 ?the	 ?surface	 ?wetting	 ?of	 ?PDMS	 ?by	 ?the	 ?polymer	 ?solution	 ?used	 ?for	 ? solvent	 ? casting	 ? and	 ? therefore	 ? resulted	 ? in	 ? sharper	 ? and	 ? taller	 ? needles.	 ?Without	 ? plasma	 ?treatment,	 ?the	 ?polymer	 ?solution	 ?does	 ?not	 ?cover	 ?the	 ?pillars	 ?entirely	 ?and	 ?leads	 ?to	 ?shorter	 ?and	 ?blunter	 ? structures.	 ? Consecutively,	 ? 35	 ??L	 ? of	 ? the	 ? 2%	 ? clay	 ? polyimide/NMP	 ? mixture	 ? was	 ?deposited	 ?into	 ?the	 ?mold	 ?structure	 ?(Figure	 ?2.3e).	 ?During	 ?a	 ?soft	 ?baking	 ?process	 ?in	 ?an	 ?oven	 ?at	 ?31	 ?	 ?65?C	 ?for	 ?2	 ?h,	 ?NMP	 ?evaporated	 ?and	 ?the	 ?microneedles	 ?were	 ?formed	 ?around	 ?the	 ?pillars	 ?in	 ?the	 ?mold	 ?with	 ?wide	 ?bases	 ?and	 ?sharp	 ?tips	 ?(Figure	 ?2.3f,	 ?Figure	 ?2.6).	 ?The	 ?obtained	 ?structures	 ?would	 ?be	 ?suitable	 ?for	 ?use	 ?as	 ?solid	 ?out-??of-??plane	 ?microneedles,	 ? in	 ?which	 ?case	 ?the	 ?molds	 ?would	 ?not	 ?be	 ?reusable	 ?for	 ?consecutive	 ?fabrication.	 ?	 ?To	 ?achieve	 ?hollow	 ?structures,	 ?the	 ?array	 ?was	 ?separated	 ?from	 ?the	 ?mold	 ?by	 ?mechanical	 ?force	 ?(Figure	 ?2.3g).	 ?It	 ?was	 ?observed	 ?that	 ?the	 ?separation	 ?of	 ?the	 ?polyimide/clay	 ?layer	 ?became	 ?easier	 ?if	 ?sufficient	 ?time	 ?had	 ?passed	 ?after	 ?the	 ?soft	 ?baking	 ?process	 ?(more	 ?than	 ?48	 ?h),	 ?which	 ?may	 ?have	 ?been	 ? due	 ? to	 ? a	 ? slight	 ? swelling	 ? of	 ? the	 ? polymer	 ? film	 ? due	 ? to	 ? moisture	 ? uptake	 ? [97-??99]	 ? or	 ?deformation	 ?due	 ?to	 ?further	 ?solvent	 ?evaporation	 ?at	 ?room	 ?temperature.	 ?After	 ?separating	 ?the	 ?needle	 ?structure	 ?from	 ?the	 ?mold,	 ?a	 ?thin	 ?layer	 ?of	 ?the	 ?polyimide/clay	 ?composite	 ?resided	 ?on	 ?top	 ?of	 ? the	 ?needles	 ? from	 ? the	 ?polymer	 ? solvent	 ? casting	 ? step	 ?obstructing	 ? the	 ?needle	 ? lumen.	 ? This	 ?layer	 ?was	 ? removed	 ? (Figure	 ? 2.1h)	 ? by	 ? either	 ? an	 ?O2/CF4	 ? plasma	 ? etching	 ? step	 ? (200	 ?W	 ?power,	 ?700	 ?mTorr	 ? pressure,	 ? 80	 ?sccm	 ? O2,	 ? 20	 ?sccm	 ? CF4,	 ? and	 ? duration	 ? of	 ? 300-??500	 ? s	 ? at	 ? 25?C)	 ? or	 ? by	 ?polishing	 ?the	 ?tips	 ?using	 ?fine	 ?3	 ??m	 ?aluminum	 ?oxide	 ?polishing	 ?film	 ?(3M,	 ?St.	 ?Paul,	 ?MN).	 ?	 ?32	 ?	 ?	 ?Figure	 ?2.3:	 ?Fabrication	 ?process	 ?using	 ?solvent	 ?casting	 ?for	 ?hollow	 ?out-??of-??plane	 ?polymer	 ?microneedles,	 ?a	 ?&	 ?b)	 ?fabrication	 ?of	 ?pillars	 ?from	 ?SU-??8,	 ?c)	 ?PDMS	 ?deposition,	 ?d)	 ?O2	 ?plasma	 ?treatment	 ?of	 ?the	 ?mold,	 ?e)	 ?deposition	 ?of	 ?a	 ?clay/polyimide	 ?suspension	 ?in	 ?NMP,	 ?f)	 ?Evaporation	 ?of	 ?NMP,	 ?g)	 ?removing	 ?of	 ?the	 ?microneedle	 ?array	 ?from	 ?the	 ?mold,	 ?h)	 ?opening	 ?of	 ?the	 ?microneedle	 ?tips.	 ?	 ?Figure	 ?2.4:	 ?SEM	 ?image	 ?of	 ?an	 ?array	 ?of	 ?pillars	 ?in	 ?a	 ?mold	 ?used	 ?for	 ?microneedle	 ?fabrication.	 ?33	 ?	 ?	 ?Figure	 ?2.5:	 ?A	 ?complete	 ?mold	 ?used	 ?for	 ?microneedle	 ?fabrication.	 ?	 ?Figure	 ?2.6:	 ?SEM	 ?image	 ?of	 ?an	 ?array	 ?of	 ?solid	 ?microneedles	 ?formed	 ?in	 ?a	 ?mold	 ?after	 ?solvent	 ?evaporation.	 ?Figure	 ?2.7	 ?shows	 ?SEM	 ? images	 ?of	 ? the	 ?microneedles	 ? fabricated	 ? through	 ?this	 ?process.	 ?Figure	 ?2.7a	 ? shows	 ? an	 ? array	 ? of	 ? 250	 ??m	 ? long	 ? microneedles	 ? with	 ? tip	 ? diameters	 ? of	 ? 50.4	 ??	 ?4.2	 ? ?m	 ?(mean	 ??	 ?standard	 ?deviation)	 ?and	 ?the	 ?same	 ?pitch	 ?of	 ?500	 ??m	 ?as	 ?the	 ?pillars.	 ?The	 ?slight	 ?variation	 ?in	 ? the	 ? microneedle	 ? tip	 ? diameter	 ? is	 ? mainly	 ? due	 ? to	 ? the	 ? variation	 ? in	 ? thickness	 ? of	 ? the	 ? spin	 ?coated	 ? PDMS	 ? layer	 ? on	 ? the	 ? SU-??8	 ? pillars,	 ?which	 ?may	 ? lead	 ? to	 ? small	 ? differences	 ? in	 ? the	 ? force	 ?34	 ?	 ?required	 ?for	 ?the	 ?penetration	 ?of	 ?the	 ?microneedles	 ? into	 ?skin.	 ?The	 ?wider	 ?bases	 ?of	 ?the	 ?pillars,	 ?due	 ?to	 ?the	 ?backside	 ?exposure	 ?of	 ?the	 ?SU-??8,	 ?result	 ?in	 ?larger	 ?channel	 ?openings	 ?on	 ?the	 ?backside	 ?of	 ?the	 ?backing	 ?plate	 ?as	 ?shown	 ?in	 ?Figure	 ?2.7b.	 ?In	 ?Figure	 ?2.7c,	 ?a	 ?single	 ?microneedle	 ?is	 ?shown	 ?with	 ? its	 ?tip	 ?opened	 ?through	 ?plasma	 ?etching,	 ?while	 ?Figure	 ?2.7d	 ?shows	 ?a	 ?single	 ?microneedle	 ?with	 ?the	 ?tip	 ?opened	 ?by	 ?sanding.	 ?	 ?Figure	 ?2.7:	 ?SEM	 ?images	 ?of	 ?fabricated	 ?microneedles:	 ?a)	 ?Array	 ?of	 ?250	 ??m	 ?long	 ?needles,	 ?b)	 ?microneedle	 ?channel	 ?openings,	 ?c)	 ?a	 ?needle	 ?tip	 ?opened	 ?by	 ?plasma	 ?etching,	 ?and	 ?d)	 ?a	 ?needle	 ?tip	 ?opened	 ?by	 ?sanding.	 ?For	 ?more	 ?SEM	 ?images	 ?see	 ?Appendix	 ?A.3.	 ?The	 ? dimensions	 ? of	 ? the	 ?microneedles	 ? can	 ? be	 ? adjusted	 ? by	 ? changing	 ? the	 ? dimensions	 ? of	 ? the	 ?mold	 ?structure	 ?during	 ?the	 ?photolithography	 ?steps.	 ?For	 ?instance,	 ? longer	 ?pillars	 ?with	 ?smaller	 ?diameter	 ?result	 ?in	 ?longer	 ?needles	 ?with	 ?smaller	 ?channel	 ?diameters.	 ?35	 ?	 ?2.1.3 Contact	 ?angle	 ?measurement	 ?of	 ?NMP	 ?on	 ?a	 ?PDMS	 ?surface	 ?In	 ? order	 ? to	 ? characterize	 ? the	 ? polyimide-??PDMS	 ? interaction	 ? after	 ? plasma	 ? treatment	 ? of	 ? the	 ?mold	 ?and	 ?during	 ?solvent	 ?casting,	 ?a	 ?series	 ?of	 ?tests	 ?was	 ?performed	 ?to	 ?observe	 ?the	 ?change	 ?in	 ?contact	 ?angle	 ?of	 ?polyimide	 ?solvent	 ?on	 ?a	 ?PDMS	 ?surface	 ?over	 ?time	 ?after	 ?the	 ?PDMS	 ?is	 ?plasma	 ?treated	 ?and	 ?covered	 ?with	 ?a	 ?layer	 ?of	 ?polyimide,	 ?as	 ?during	 ?the	 ?casting	 ?step	 ?in	 ?the	 ?fabrication	 ?process	 ?as	 ?shown	 ?in	 ?Figure	 ?2.3e,	 ?f.	 ?For	 ?these	 ?tests,	 ?first	 ?a	 ?layer	 ?of	 ?PDMS	 ?was	 ?spin	 ?coated	 ?on	 ?a	 ?microscope	 ?slide	 ?and	 ?then	 ?baked	 ?to	 ?cure.	 ?The	 ?PDMS	 ?surface	 ?was	 ?then	 ?treated	 ?with	 ?oxygen	 ?plasma	 ?as	 ?explained	 ?above.	 ?A	 ? thin	 ? layer	 ?of	 ?polyimide	 ?was	 ? then	 ? spin	 ? coated	 ?on	 ? the	 ?PDMS	 ?surface	 ?and	 ?left	 ?in	 ?an	 ?oven	 ?to	 ?soft	 ?bake	 ?at	 ?65?C	 ?for	 ?2	 ?h.	 ?The	 ?polyimide	 ?layer	 ?was	 ?peeled	 ?off	 ?from	 ? different	 ? samples	 ? after	 ? different	 ? time	 ? intervals	 ? (6	 ?h,	 ? 8	 ?h,	 ? 1	 ?day,	 ? 2	 ?days,	 ? 3	 ?days,	 ? and	 ?6	 ?days)	 ?following	 ?solvent	 ?casting.	 ?The	 ?advancing	 ?contact	 ?angle	 ?was	 ?measured	 ? immediately	 ?after	 ? peeling	 ? off	 ? the	 ? polyimide	 ? layer	 ? from	 ? the	 ? PDMS	 ? surface	 ? (Figure	 ? 2.8a)	 ? using	 ? a	 ? Theta	 ?tensiometer	 ? (Attension	 ? /	 ? Biolin	 ? Scientific,	 ? Espoo,	 ? Finland).	 ? The	 ? measurements	 ? were	 ?performed	 ? on	 ? 4	 ??L	 ? NMP	 ? droplets	 ? and	 ?were	 ? repeated	 ? several	 ? times	 ? over	 ? the	 ? duration	 ? of	 ?30	 ?min,	 ?each	 ?time	 ?in	 ?a	 ?different	 ?location	 ?on	 ?the	 ?PDMS	 ?sample.	 ?Figure	 ?2.8b	 ?shows	 ? the	 ?measurement	 ? results.	 ? It	 ?was	 ?observed	 ? that	 ? regardless	 ?of	 ?when	 ?the	 ?polyimide	 ?layer	 ?was	 ?removed,	 ?the	 ?immediate	 ?contact	 ?angle	 ?of	 ?NMP	 ?on	 ?PDMS	 ?was	 ?constant	 ?and	 ?low	 ?(about	 ?10-??15?).	 ?The	 ?angle	 ?only	 ?increased	 ?when	 ?the	 ?surface	 ?was	 ?in	 ?contact	 ?with	 ?air,	 ?and	 ?this	 ?increase	 ?occurred	 ?at	 ?a	 ?similar	 ?rate	 ?regardless	 ?of	 ?how	 ?long	 ?after	 ?solvent	 ?casting	 ?the	 ?polyimide	 ?layer	 ?was	 ?removed.	 ?	 ?36	 ?	 ?a)	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ? 	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?.	 ?b) 	 ?Figure	 ?2.8:	 ?NMP	 ?contact	 ?angle	 ?on	 ?a	 ?PDMS	 ?surface;	 ?a)	 ?contact	 ?angle	 ?measured	 ?3	 ?days	 ?after	 ?the	 ?PDMS	 ?surface	 ?was	 ?coated	 ?with	 ?a	 ?polyimide	 ?layer,	 ?13	 ?minutes	 ?after	 ?removing	 ?the	 ?polyimide	 ?layer;	 ?b)	 ?contact	 ?angle	 ?measurements	 ?for	 ?NMP	 ?on	 ?a	 ?PDMS	 ?at	 ?different	 ?times	 ?after	 ?removing	 ?a	 ?polyimide	 ?layer	 ?that	 ?was	 ?deposited	 ?between	 ?6	 ?hours	 ?and	 ?6	 ?days	 ?prior	 ?to	 ?removal.	 ?The	 ?wetting	 ?behaviour	 ?of	 ?NMP	 ?on	 ?PDMS	 ?remains	 ?constant	 ?as	 ?long	 ?as	 ?the	 ?PDMS	 ?surface	 ?is	 ?in	 ?contact	 ?with	 ?the	 ?polyimide	 ?composite	 ?layer.	 ?The	 ?wetting	 ?only	 ?changes	 ?when	 ?the	 ?PDMS	 ?is	 ?in	 ?contact	 ?with	 ?air,	 ? regardless	 ?of	 ?when	 ? the	 ?polyimide	 ?composite	 ? is	 ? removed	 ? from	 ?the	 ?PDMS	 ?surface.	 ?This	 ?showed	 ?that	 ?the	 ?surface	 ?wetting	 ?behaviour	 ?of	 ?NMP	 ?on	 ?PDMS	 ?remains	 ?identical	 ?throughout	 ? the	 ? evaporation	 ? process,	 ? which	 ? would	 ? result	 ? in	 ? needles	 ? with	 ? the	 ? desired	 ?geometry.	 ?37	 ?	 ?2.2 Experimental	 ?procedures	 ?for	 ?needle	 ?characterization	 ?2.2.1 Microneedle	 ?robustness	 ?tests	 ?In	 ?order	 ?to	 ?test	 ?the	 ?robustness	 ?of	 ?the	 ?fabricate	 ?microneedles,	 ?a	 ?series	 ?of	 ?compression	 ?tests	 ?were	 ?performed	 ?on	 ?individual	 ?2	 ?wt%	 ?clay-??reinforced	 ?polyimide	 ?needles,	 ?with	 ?tips	 ?opened	 ?by	 ?plasma	 ?etching.	 ?The	 ? test	 ?procedures	 ?were	 ? identical	 ? to	 ? the	 ? robustness	 ? tests	 ?performed	 ?on	 ?test	 ?structures	 ?described	 ?in	 ?section	 ?2.1.1.	 ? Individual	 ?needles	 ?were	 ?made	 ?using	 ?the	 ?process	 ?shown	 ? in	 ?Figure	 ?2.3.	 ?A	 ? total	 ?of	 ? ten	 ?250	 ??m-??long	 ?needles	 ?where	 ? tested	 ?under	 ?compressive	 ?loading	 ?and	 ?the	 ?corresponding	 ?force	 ?vs.	 ?displacement	 ?plots	 ?were	 ?obtained	 ?for	 ?each	 ?test.	 ?2.2.2 Microneedle	 ?injection	 ?tests	 ?The	 ? capability	 ? of	 ? the	 ? fabricated	 ? devices	 ? for	 ? drug	 ? delivery	 ? and	 ? skin	 ? penetration	 ? was	 ?demonstrated	 ?through	 ? injection	 ?trials	 ?on	 ?rabbit	 ?ear	 ?skin.	 ?Rabbit	 ?ear	 ?skin	 ?was	 ?used	 ?for	 ?this	 ?study	 ? because	 ? it	 ? is	 ? a	 ? reasonable	 ? human	 ? skin	 ?model	 ? for	 ? in	 ? vitro	 ? transdermal	 ? permeation	 ?studies	 ?[100].	 ?Traditionally,	 ?pig	 ?skin	 ?has	 ?been	 ?used	 ?as	 ?a	 ?skin	 ?model	 ?for	 ?permeation	 ?studies	 ?due	 ?to	 ? its	 ?biochemical	 ?similarity	 ?with	 ?human	 ?skin	 ? [101,	 ?102].	 ?However,	 ?pig	 ?skin	 ?and	 ?other	 ?traditional	 ? skin	 ?models	 ? are	 ?associated	 ?with	 ?a	 ?high	 ?permeability	 ? compared	 ? to	 ?human	 ? skin,	 ?especially	 ?with	 ?respect	 ?to	 ?hydrophilic	 ?agents.	 ?In	 ?contrast,	 ?rabbit	 ?ear	 ?skin	 ?has	 ?a	 ?considerably	 ?lower	 ?permeability	 ?to	 ?hydrophilic	 ?compounds	 ?compared	 ?with	 ?traditional	 ?skin	 ?models,	 ?and	 ?is	 ?therefore	 ?considered	 ?to	 ?be	 ?a	 ?suitable	 ?human	 ?skin	 ?model	 ?for	 ?in	 ?vitro	 ?trials	 ?[100].	 ?	 ?38	 ?	 ?Arrays	 ?of	 ?250	 ??m-??long	 ?microneedles,	 ?with	 ?tips	 ?opened	 ?by	 ?plasma	 ?etching,	 ?were	 ?bonded	 ?to	 ?modified	 ?plastic	 ? female	 ? luer-??to-??barb	 ?fittings	 ? (McMaster-??Carr,	 ?Cleveland,	 ?OH),	 ?using	 ?Loctite	 ?Super	 ?Glue	 ? (Henkel,	 ?Avon,	 ?OH),	 ? and	 ? then	 ?attached	 ? to	 ? conventional	 ?1	 ?mL	 ? syringes	 ? (Becton	 ?Dickinson,	 ?Mississauga,	 ?ON)	 ? as	 ? shown	 ? in	 ? Figure	 ?2.9a.	 ?A	 ?0.025	 ?wt%	 ? suspension	 ?of	 ? 0.21	 ??m	 ?polystyrene	 ?fluorescent	 ?beads	 ?(Bangs	 ?Laboratories,	 ?Fishers,	 ?IN)	 ?in	 ?water	 ?was	 ?prepared.	 ?For	 ?each	 ? trial,	 ? the	 ? syringes	 ?were	 ? filled	 ?with	 ? the	 ? suspension	 ? containing	 ? fluorescent	 ?beads,	 ? and	 ?were	 ?then	 ?pressed	 ?against	 ?the	 ?inner	 ?rabbit	 ?ear	 ?skin	 ?for	 ?about	 ?10	 ?seconds	 ?while	 ?a	 ?moderate	 ?pressure	 ? was	 ? applied	 ? to	 ? the	 ? plunger.	 ? Figure	 ?2.9b	 ? shows	 ? the	 ? skin	 ? after	 ? application	 ? of	 ? the	 ?microneedles.	 ?A	 ?total	 ?of	 ?six	 ? injection	 ?trials	 ?were	 ?carried	 ?out.	 ?After	 ?each	 ?test,	 ? the	 ? injection	 ?site	 ? was	 ? imaged	 ? using	 ? a	 ? D-??Eclipse	 ? C1	 ? confocal	 ? microscope	 ? (Nikon).	 ? Using	 ? the	 ? confocal	 ?microscope,	 ? the	 ? skin	 ? was	 ? scanned	 ? down	 ? to	 ? a	 ? depth	 ? of	 ? 200	 ? ?m	 ? to	 ? investigate	 ? the	 ?penetration	 ?of	 ?the	 ?fluorescent	 ?beads	 ?into	 ?the	 ?skin.	 ?39	 ?	 ?	 ?Figure	 ?2.9:	 ?a)	 ?A	 ?microneedle	 ?array	 ?attached	 ?to	 ?a	 ?syringe,	 ?b)	 ?rabbit	 ?ear	 ?skin	 ?after	 ?application	 ?of	 ?the	 ?microneedles.	 ?2.3 Results	 ?and	 ?Discussion	 ?2.3.1 Microneedle	 ?robustness	 ?Figure	 ?2.10a	 ?shows	 ?an	 ?example	 ?of	 ?a	 ? force	 ?vs.	 ?displacement	 ?plot	 ?obtain	 ? for	 ?a	 ?compression	 ?test	 ? on	 ? a	 ? single	 ? needle.	 ? The	 ? plot	 ? shows	 ? a	 ? sudden	 ? increase	 ? in	 ? force	 ? upon	 ? contact	 ? of	 ? the	 ?40	 ?	 ?compression	 ? test	 ? tool	 ?with	 ? the	 ?microneedle	 ? tip	 ? followed	 ? by	 ? a	 ? sudden	 ? drop	 ? in	 ? force.	 ? The	 ?peak	 ?force	 ?corresponds	 ?to	 ?the	 ?failure	 ?load	 ?of	 ?the	 ?microneedle	 ?similar	 ?to	 ?the	 ?changing	 ?force	 ?seen	 ?for	 ?the	 ?solid	 ?structures	 ?in	 ?Figure	 ?2.1.	 ?A	 ?needle	 ?tip	 ?after	 ?failure	 ?is	 ?shown	 ?in	 ?Figure	 ?2.10b.	 ?In	 ?this	 ?Figure,	 ?the	 ?tip	 ?of	 ?the	 ?microneedle	 ?has	 ?collapsed	 ?and	 ?bent	 ?almost	 ?at	 ?the	 ?midpoint	 ?of	 ?the	 ? needle	 ? shaft,	 ?while	 ? the	 ? needle	 ? base	 ? is	 ? still	 ? rigid,	 ?which	 ? explains	 ? the	 ? increase	 ? in	 ? force	 ?again	 ? as	 ? the	 ? needle	 ? is	 ? compressed	 ? further.	 ? From	 ? these	 ? tests,	 ? it	 ? was	 ? observed	 ? that	 ? on	 ?average,	 ? the	 ?microneedles	 ?can	 ?sustain	 ?compressive	 ? loads	 ?of	 ?up	 ? to	 ?0.32	 ??	 ?0.06	 ?N	 ? (mean	 ??	 ?standard	 ?deviation).	 ?	 ?	 ?Figure	 ?2.10:	 ?a)	 ?A	 ?needle	 ?displacement	 ?under	 ?axial	 ?loading,	 ?b)	 ?a	 ?failed	 ?needle	 ?after	 ?loading.	 ?Davis	 ?et	 ?al.	 ?investigated	 ?the	 ?relationship	 ?between	 ?the	 ?cross	 ?sectional	 ?area	 ?of	 ?a	 ?microneedle	 ?tip	 ? (interfacial	 ? area)	 ? and	 ? the	 ? force	 ? required	 ? for	 ? penetration	 ? of	 ? the	 ?microneedle	 ? into	 ? skin	 ?[103].	 ?The	 ?experimental	 ?results	 ?presented	 ?by	 ?Davis	 ?et	 ?al.	 ?seem	 ?to	 ?suggest	 ?that	 ?the	 ?needle	 ?strength	 ? obtained	 ? experimentally	 ? for	 ? polymer/clay	 ? composite	 ? needles	 ? with	 ? a	 ? 50	 ??m	 ? tip	 ?diameter	 ?in	 ?this	 ?work,	 ?	 ? NF 	 ?=	 ?0.32	 ?N,	 ?is	 ?sufficient	 ?to	 ?penetrate	 ?human	 ?skin.	 ?	 ?41	 ?	 ?2.3.2 Results	 ?of	 ?injection	 ?tests	 ?Figure	 ?2.11	 ?shows	 ?the	 ?fluorescent	 ?beads	 ?at	 ?different	 ?depths	 ?under	 ?the	 ?skin	 ?surface	 ?after	 ?an	 ?injection	 ? test	 ? using	 ? microneedles.	 ? For	 ? each	 ? confocal	 ? scan,	 ? an	 ? intensity	 ? histogram	 ?distribution	 ?was	 ?obtained	 ?by	 ?measuring	 ?the	 ?total	 ?bead	 ?fluorescent	 ?intensity	 ?at	 ?each	 ?depth	 ?using	 ?MATLAB	 ? for	 ? image	 ? processing.	 ? Figure	 ? 2.12	 ? shows	 ? the	 ? intensity	 ? distribution	 ? for	 ? the	 ?confocal	 ?scan	 ?shown	 ?in	 ?Figure	 ?2.11.	 ?	 ?	 ?Figure	 ?2.11:	 ?Confocal	 ?scan	 ?of	 ?skin	 ?after	 ?injection	 ?of	 ?fluorescent	 ?beads.	 ?The	 ?skin	 ?surface	 ?is	 ?at	 ?0	 ??m.	 ?The	 ?confocal	 ?slice	 ?thickness	 ?is	 ?13.4	 ??m.	 ?42	 ?	 ?	 ?Figure	 ?2.12:	 ?Histogram	 ?of	 ?the	 ?intensity	 ?distribution	 ?of	 ?the	 ?fluorescent	 ?beads	 ?obtained	 ?from	 ?the	 ?confocal	 ?scan	 ?in	 ?Figure	 ?2.11.	 ?For	 ?each	 ? injection	 ?test,	 ?an	 ?average	 ? injection	 ?depth	 ?and	 ?an	 ? injection	 ?range	 ?were	 ?calculated	 ?from	 ? these	 ? intensity	 ?measurements	 ? corresponding	 ? to	 ? the	 ?mean	 ? and	 ? the	 ? 95%	 ? confidence	 ?interval,	 ?respectively.	 ?The	 ?results	 ?from	 ?six	 ?injection	 ?trials	 ?are	 ?shown	 ?in	 ?Table.	 ?2.	 ?The	 ?average	 ?delivery	 ? depth	 ? for	 ? all	 ? the	 ? trials	 ? was	 ? 104.8	 ? ?	 ? 15.6	 ??m	 ? with	 ? an	 ? average	 ? range	 ? of	 ? 119	 ? ?	 ?25.8	 ??m,	 ?indicating	 ?successful	 ?delivery	 ?into	 ?the	 ?epidermis,	 ?past	 ?the	 ?stratum	 ?corneum.	 ?Table	 ?2.2:	 ?Mean	 ?injection	 ?depth	 ?and	 ?95%	 ?confidence	 ?interval	 ?for	 ?the	 ?fluorescent	 ?intensity	 ?distribution	 ?of	 ?injected	 ?beads	 ?as	 ?in	 ?Figure	 ?2.12.	 ?Trial	 ? 1	 ? 2	 ? 3	 ? 4	 ? 5	 ? 6	 ? Average	 ?Mean	 ?depth	 ?[?m]	 ? 110.3	 ? 113.9	 ? 83.7	 ? 108.7	 ? 123.9	 ? 88.1	 ? 104.8	 ?Upper	 ?range	 ?[?m]	 ? 169.4	 ? 176.7	 ? 121.8	 ? 0.017	 ? 180.3	 ? 166.3	 ? 164.3	 ?Lower	 ?range	 ?[?m]	 ? 51.3	 ? 51.1	 ? 45.5	 ? 46.4	 ? 67.4	 ? 9.9	 ? 45.3	 ?	 ?43	 ?	 ?Since	 ?the	 ?microneedles	 ?used	 ?for	 ?these	 ?tests	 ?are	 ?250	 ??m	 ?long,	 ?the	 ?average	 ?delivery	 ?depth	 ?of	 ?104.8	 ??m	 ?indicates	 ?that	 ?the	 ?majority	 ?of	 ?the	 ?microneedle	 ?height	 ?could	 ?not	 ?penetrate	 ?due	 ?to	 ?the	 ?flexibility	 ?of	 ?the	 ?skin.	 ?This	 ?penetration	 ?behaviour	 ?might	 ?be	 ?different	 ?for	 ?human	 ?skin	 ?and	 ?additional	 ?experiments	 ?have	 ?to	 ?be	 ?done	 ?in	 ?vivo	 ?to	 ?observe	 ?the	 ?penetration	 ?depth	 ?in	 ?human	 ?skin.	 ? Additionally,	 ? in	 ? all	 ? of	 ? the	 ? confocal	 ? images,	 ? the	 ? distribution	 ? of	 ? the	 ? fluorescent	 ? beads	 ?was	 ? localized	 ?to	 ?distinct	 ?regions	 ?corresponding	 ?to	 ?each	 ? individual	 ?needle	 ? in	 ?the	 ?array.	 ?The	 ?distance	 ? between	 ? the	 ? needles	 ? therefore	 ? seems	 ? to	 ? be	 ? sufficient	 ? to	 ? have	 ? them	 ? act	 ?independently	 ? as	 ? individual	 ? needles,	 ? and	 ? injection	 ? of	 ? more	 ? material,	 ? with	 ? controlled	 ?volume,	 ?could	 ?thus	 ?be	 ?accomplished	 ?by	 ?proportionally	 ?adding	 ?more	 ?needles.	 ?	 ?2.4 Conclusions	 ?A	 ?new	 ?fabrication	 ?technique,	 ?based	 ?on	 ?solvent	 ?casting,	 ?has	 ?been	 ?presented	 ?for	 ?fabrication	 ?of	 ? hollow	 ? out-??of-??plane	 ? microneedles	 ? on	 ? a	 ? reusable	 ? mold.	 ? This	 ? fabrication	 ? procedure	 ? is	 ?inexpensive	 ? and	 ? applicable	 ? for	 ?mass	 ? production	 ? of	 ? polymer	 ?microneedles.	 ? The	 ? proposed	 ?process	 ? is	 ?flexible	 ? in	 ?terms	 ?of	 ?the	 ?possible	 ?dimensions	 ?of	 ?the	 ?microneedle	 ?array	 ?as	 ?well	 ?as	 ?the	 ? needle	 ? material.	 ? By	 ? adjusting	 ? the	 ? photolithography	 ? parameters	 ? and	 ? therefore	 ?controlling	 ?the	 ?pillar	 ?dimensions	 ?in	 ?the	 ?mold,	 ?microneedles	 ?with	 ?various	 ?heights	 ?and	 ?lumen	 ?diameters	 ? can	 ? be	 ? made.	 ? Also,	 ? other	 ? polymers	 ? can	 ? be	 ? used	 ? in	 ? this	 ? process	 ? for	 ? hollow	 ?microneedle	 ?fabrication	 ?by	 ?adjusting	 ?the	 ?process	 ?parameters.	 ?Clay-??reinforced	 ? polyimide	 ? was	 ? used	 ? in	 ? the	 ? proposed	 ? process	 ? to	 ? fabricate	 ? 250	 ??m	 ? long	 ?microneedles.	 ?A	 ?series	 ?of	 ?compression	 ?tests	 ?were	 ?carried	 ?out	 ?on	 ?solid	 ?structures	 ?to	 ?find	 ?the	 ?optimum	 ?clay	 ?percentage	 ?in	 ?the	 ?composite,	 ?leading	 ?to	 ?the	 ?maximum	 ?compressive	 ?strength.	 ?44	 ?	 ?The	 ?robustness	 ?of	 ?the	 ?fabricated	 ?microneedles	 ?has	 ?been	 ?measured,	 ?and	 ?the	 ?needles	 ?were	 ?found	 ?to	 ?be	 ?strong	 ?enough	 ?for	 ?penetration	 ?into	 ?human	 ?skin.	 ?The	 ?delivery	 ?of	 ?a	 ?suspension	 ?of	 ?polystyrene	 ?beads	 ?into	 ?the	 ?epidermis	 ?of	 ?rabbit	 ?skin	 ?was	 ?also	 ?successfully	 ?demonstrated.	 ?	 ?	 ?	 ? 	 ?45	 ?	 ?CHAPTER	 ?3 	 ?	 ?	 ?FABRICATION	 ?OF	 ?METALLIC	 ?MICRONEEDLES	 ?USING	 ?ELECTRODEPOSITION	 ?OF	 ?METAL	 ?ONTO	 ?CONDUCTIVE	 ?POLYMER	 ?FILMS	 ?	 ?	 ?	 ?The	 ? fabrication	 ? process	 ? demonstrated	 ? in	 ? the	 ? previous	 ? chapter	 ? allowed	 ? fabrication	 ? of	 ? the	 ?microneedles	 ?with	 ?a	 ?variety	 ?of	 ?polymeric	 ?materials	 ?with	 ?heights	 ?of	 ?up	 ?to	 ?250	 ??m.	 ?However,	 ?the	 ?proposed	 ?process	 ?did	 ?not	 ?allow	 ?fabrication	 ?of	 ?taller	 ?devices	 ?with	 ?sufficient	 ?strength	 ?for	 ?skin	 ? penetration.	 ? Taller	 ? microneedles	 ? (i.e.	 ? up	 ? to	 ? 1	 ? mm)	 ? may	 ? be	 ? ideal	 ? for	 ? some	 ? medical	 ?applications	 ?in	 ?which	 ?the	 ?target	 ?delivery	 ?site	 ?is	 ?far	 ?below	 ?the	 ?skin	 ?surface	 ?or	 ?the	 ?skin	 ?is	 ?too	 ?flexible	 ?or	 ?too	 ?thick.	 ?A	 ?good	 ?structural	 ?material	 ?for	 ?tall	 ?hollow	 ?microneedles	 ?is	 ?metal	 ?due	 ?to	 ?its	 ? high	 ? strength	 ? and	 ? low	 ? cost.	 ? However,	 ? previous	 ? fabrication	 ? processes	 ? for	 ? metallic	 ?microneedles	 ? often	 ? require	 ? sequential	 ? formation	 ? of	 ? disposable	 ? polymer	 ? molds	 ? for	 ?electroplating.	 ? In	 ?addition,	 ? in	 ?most	 ?cases,	 ?metallic	 ?microneedles	 ?are	 ?opened	 ?through	 ? laser	 ?46	 ?	 ?cutting	 ?of	 ? individual	 ?needle	 ? tips,	 ?which	 ? can	 ?be	 ? time	 ? costly	 ? for	 ?making	 ?arrays	 ? containing	 ?a	 ?large	 ?number	 ?of	 ?microneedles.	 ?In	 ?this	 ?chapter,	 ?we	 ?have	 ?used	 ?a	 ?solvent	 ?casting	 ?process	 ?to	 ?form	 ?a	 ?thin	 ?conductive	 ?polymeric	 ?layer	 ?on	 ?reusable	 ?molds	 ?that	 ?contain	 ?arrays	 ?of	 ?vertical	 ?pillar;	 ?the	 ?conductive	 ?polymer	 ?layer	 ?is	 ?then	 ?used	 ?as	 ?a	 ? seed	 ? layer	 ? in	 ?an	 ?electroplating	 ?process	 ? to	 ? fabricate	 ?metallic	 ?microneedles.	 ?Several	 ?polymers	 ?have	 ?been	 ?previously	 ?shown	 ?to	 ?be	 ?capable	 ?of	 ?conducting	 ?electricity	 ?[104,	 ?105].	 ?Although	 ?their	 ?conductivity	 ?is	 ?not	 ?as	 ?high	 ?as	 ?the	 ?conductivity	 ?of	 ?metals,	 ?they	 ?are	 ?used	 ?in	 ?many	 ? small-??scale	 ? industrial	 ? applications	 ? because	 ? they	 ? are	 ? easy	 ? to	 ? process.	 ? In	 ? addition,	 ?non-??conductive	 ?polymers	 ?can	 ?also	 ?be	 ?modified	 ?to	 ?conduct	 ?electricity	 ?by	 ?adding	 ?conducting	 ?particles	 ?to	 ?their	 ?matrix.	 ?Nickel-??coated	 ?particles,	 ?copper	 ?powder,	 ?carbon	 ?black,	 ?and	 ?graphite	 ?are	 ?some	 ?examples	 ?of	 ?fillers	 ?used	 ?to	 ?increase	 ?polymer	 ?conductivity	 ?[106?111].	 ?In	 ?order	 ?to	 ?further	 ?characterize	 ?the	 ?fabrication	 ?process,	 ?some	 ?experiments	 ?were	 ?carried	 ?out	 ?to	 ?investigate	 ?the	 ?conductivity	 ?of	 ?the	 ?polymer	 ?layer,	 ?its	 ?plasma	 ?etching	 ?rate,	 ?and	 ?the	 ?metal	 ?electroplating	 ? process.	 ? The	 ? fabricated	 ? metallic	 ? microneedles	 ? were	 ? also	 ? tested	 ? for	 ?robustness	 ?through	 ?a	 ?series	 ?of	 ?mechanical	 ?compression	 ?tests,	 ?and	 ?their	 ?failure	 ? loads	 ?were	 ?compared	 ?with	 ? forces	 ? required	 ? for	 ? skin	 ?penetration.	 ?The	 ?microneedles?	 ?adequacy	 ? for	 ? skin	 ?penetration	 ?and	 ?drug	 ?delivery	 ?was	 ?then	 ?proven	 ?by	 ?using	 ?them	 ?to	 ? inject	 ? fluorescent	 ?beads	 ?into	 ?animal	 ?skin.	 ?	 ?47	 ?	 ?3.1 Fabrication	 ?of	 ?hollow	 ?metallic	 ?microneedles	 ?3.1.1 Fabrication	 ?of	 ?the	 ?mold	 ?The	 ?fabrication	 ?process	 ?of	 ?microneedles	 ?is	 ?summarized	 ?in	 ?Figure	 ?3.1.	 ?It	 ?starts	 ?with	 ?making	 ?a	 ?mold	 ? through	 ? photolithography	 ? for	 ? which	 ? the	 ? epoxy-??type	 ? negative	 ? photoresist	 ? SU-??8	 ?(MicroChem,	 ? Newton,	 ? MA)	 ? was	 ? used.	 ? A	 ? 700	 ? ?m	 ? thick	 ? layer	 ? of	 ? SU-??8	 ? 2150	 ? was	 ? first	 ? spin	 ?coated	 ?onto	 ?a	 ?300	 ??m	 ?thick	 ?Pyrex?	 ?wafer.	 ?The	 ?wafer	 ?was	 ?then	 ?soft-??baked	 ?for	 ?10	 ?min	 ?at	 ?65?C	 ?and	 ?2.5	 ?h	 ?at	 ?95?C.	 ?The	 ?SU-??8	 ?was	 ?then	 ?exposed	 ?to	 ?9200	 ? ?? ?	 ?UV	 ?light	 ?(performed	 ?in	 ?multiple	 ?3	 ?min	 ?intervals	 ?with	 ?20	 ?s	 ?cooling	 ?breaks	 ?in	 ?between).	 ?The	 ?exposure	 ?was	 ?performed	 ?through	 ?a	 ? dark	 ? field	 ? mask	 ? containing	 ? arrays	 ? of	 ? transparent	 ? circles	 ? with	 ? 40	 ? ?m	 ? diameter.	 ? These	 ?circular	 ?regions	 ?lead	 ?to	 ?SU-??8	 ?pillars	 ?on	 ?the	 ?mold	 ?which	 ?are	 ?used	 ?as	 ?the	 ?basic	 ?structures	 ?for	 ?microneedle	 ?formation,	 ?and	 ?therefore,	 ?their	 ?spacing	 ?and	 ?size	 ?defines	 ?the	 ?final	 ?spacing	 ?and	 ?the	 ? approximate	 ? channel	 ? diameters	 ? of	 ? the	 ? microneedles	 ? in	 ? the	 ? array.	 ? In	 ? addition,	 ? the	 ?exposure	 ? was	 ? carried	 ? out	 ? from	 ? the	 ? back	 ? side	 ? of	 ? the	 ? Pyrex?	 ? substrate.	 ? This	 ? method	 ? of	 ?exposure	 ?takes	 ?advantage	 ?of	 ? the	 ?gap	 ?between	 ?the	 ?mask	 ?and	 ?the	 ?photoresist	 ?and	 ? leads	 ?to	 ?cone	 ? shaped	 ? pillars,	 ? which	 ? eventually	 ? translates	 ? into	 ? microneedles	 ? with	 ? wide	 ? channel	 ?openings	 ?and	 ?sharp	 ?tips	 ?[96].	 ?The	 ?sharpness	 ?of	 ?the	 ?pillars	 ?can	 ?also	 ?be	 ?further	 ?increased	 ?by	 ?performing	 ? additional	 ? dry	 ? etching	 ? steps	 ? on	 ? the	 ? pillars	 ? to	 ? isotropically	 ? reduce	 ? the	 ? pillars?	 ?width	 ? to	 ?achieve	 ?a	 ?desired	 ? sharpness.	 ?Characterization	 ?of	 ? such	 ?dry	 ?etching	 ? steps	 ?was	 ?not	 ?pursued	 ? for	 ? the	 ?purpose	 ?of	 ? this	 ?work.	 ?After	 ?exposure,	 ? the	 ?sample	 ?was	 ?baked	 ? for	 ?5	 ?min	 ?at	 ?65?C	 ?and	 ?35	 ?min	 ?at	 ?95?C.	 ?Finally,	 ?the	 ?sample	 ?was	 ?placed	 ?in	 ?a	 ?developer	 ?bath	 ?for	 ?50	 ?min	 ?to	 ?remove	 ?the	 ?unexposed	 ?photoresist.	 ?48	 ?	 ? Figure	 ?3.1:	 ?	 ?Fabrication	 ?process	 ?for	 ?making	 ?hollow	 ?metallic	 ?microneedles,	 ?a)	 ?fabrication	 ?of	 ?the	 ?mold	 ?containing	 ?cone	 ?shaped	 ?pillars	 ?using	 ?backside	 ?exposure	 ?of	 ?SU-??8	 ?photoresist,	 ?b)	 ?deposition	 ?of	 ?PMMA/CB	 ?+	 ?NMP	 ?solution,	 ?c)	 ?evaporation	 ?of	 ?NMP	 ?at	 ?80?C,	 ?d)	 ?O2-??CF4	 ?plasma	 ?etching	 ?of	 ?dried	 ?PMMA/CB,	 ?e)	 ?electrodeposition	 ?of	 ?the	 ?metal	 ?layer,	 ?f)	 ?removing	 ?the	 ?microneedle	 ?array	 ?by	 ?dissolving	 ?the	 ?PMMA/CB	 ?in	 ?NMP.	 ?A	 ?final	 ?hard-??baking	 ?step	 ?was	 ?carried	 ?out	 ? in	 ?an	 ?oven	 ?set	 ?to	 ?175?C	 ?for	 ?1	 ?h.	 ?For	 ?convenience,	 ?the	 ?Pyrex?	 ?wafer	 ?was	 ?cut	 ?into	 ?smaller	 ?1?1	 ?cm2	 ?pieces,	 ?with	 ?each	 ?piece	 ?containing	 ?an	 ?array	 ?of	 ? pillars	 ? corresponding	 ? to	 ? a	 ? single	 ? microneedle	 ? array.	 ? For	 ? more	 ? detail	 ? on	 ? the	 ?photolithography	 ?process	 ?and	 ?the	 ?mask	 ?design	 ?see	 ?Appendices	 ?A.1	 ?and	 ?A.2.	 ?Ni2+ Ni2+ e- 49	 ?	 ?In	 ?order	 ? to	 ? further	 ? increase	 ? the	 ? strength	 ?of	 ? the	 ?pillars	 ? and	 ? their	 ?bonding	 ?with	 ? the	 ?Pyrex?	 ?substrate	 ?an	 ?additional	 ?SU-??8	 ?layer	 ?was	 ?cast	 ?on	 ?the	 ?mold	 ?pieces.	 ?For	 ?this	 ?step,	 ?SU-??8	 ?3025	 ?was	 ?first	 ?diluted	 ?with	 ?cyclopentanone	 ?to	 ?make	 ?a	 ?6.7	 ?wt%	 ?solution,	 ?and	 ?then	 ?45	 ??L	 ?of	 ?the	 ?solution	 ?was	 ? cast	 ? on	 ? the	 ?mold	 ? at	 ? 95?C	 ? for	 ? 20	 ?min,	 ? leading	 ? to	 ? a	 ? 30	 ? ?m	 ? thick	 ? coating	 ? on	 ? the	 ? base	 ?substrate.	 ?This	 ? layer	 ?was	 ? then	 ?cured	 ?with	 ?900	 ? ?? ?	 ?of	 ?UV	 ? light	 ?and	 ? then	 ?baked	 ?at	 ?95?C	 ? for	 ?5	 ?min	 ?followed	 ?by	 ?190?C	 ?for	 ?1	 ?h.	 ?At	 ?this	 ?stage,	 ?the	 ?molds	 ?were	 ?complete	 ?and	 ?used	 ?multiple	 ?times	 ?for	 ?the	 ?fabrication	 ?of	 ?microneedles	 ?(Figures	 ?3.1a	 ?and	 ?3.2a).	 ? Figure	 ?3.2:	 ?	 ?Image	 ?of	 ?a	 ?mold	 ?used	 ?for	 ?microneedle	 ?fabrication,	 ?a)	 ?before	 ?coating	 ?with	 ?PMMA/CB,	 ?b)	 ?coated	 ?with	 ?PMMA/CB.	 ?3.1.2 Deposition	 ?of	 ?the	 ?polymer-??based	 ?conductive	 ?seed	 ?layer	 ?Poly(methyl	 ?methacrylate)	 ?(PMMA,	 ?Polysciences,	 ?Warrington,	 ?PA),	 ?with	 ?molecular	 ?weight	 ?of	 ?25	 ?kDa,	 ?seeded	 ?with	 ?carbon	 ?black	 ?(CB,	 ?VULCAN	 ?XC72R,	 ?Cabot,	 ?Boston,	 ?MA),	 ?with	 ?a	 ?primary	 ?particle	 ? size	 ? of	 ? about	 ? 150	 ?nm,	 ?was	 ?used	 ? as	 ? the	 ? conductive	 ?polymer	 ? composite.	 ?Although,	 ?other	 ?particles	 ?such	 ?as	 ?copper	 ?particles	 ?or	 ?graphite	 ?can	 ?be	 ?used	 ?as	 ?conducting	 ?fillers,	 ?CB	 ?was	 ?used	 ?is	 ?this	 ?work	 ?mainly	 ?due	 ?to	 ?availability	 ?of	 ?the	 ?material	 ?and	 ?its	 ?low	 ?cost.	 ?Using	 ?any	 ?other	 ?filler	 ? would	 ? suffice	 ? as	 ? long	 ? as	 ? the	 ? conductivity	 ? of	 ? the	 ? deposited	 ? film	 ? is	 ? high	 ? enough	 ? to	 ?a) b) 1 mm 1 mm 50	 ?	 ?facilitate	 ?the	 ?electrodeposition	 ?of	 ?the	 ?metal	 ?layer.	 ?However,	 ?particles	 ?of	 ?higher	 ?density	 ?than	 ?CB	 ? have	 ? been	 ? observed	 ? to	 ? settle	 ? quickly	 ? during	 ? the	 ? evaporation	 ? process	 ? leading	 ? to	 ? a	 ?depleted	 ?region	 ?near	 ?the	 ?top	 ?of	 ?the	 ?pillars.	 ?	 ?To	 ?prepare	 ?the	 ?PMMA/CB	 ?polymer	 ?solution	 ?/	 ?particle	 ?suspension,	 ?first	 ?0.3	 ?g	 ?of	 ?PMMA	 ?was	 ?dissolved	 ? in	 ?5	 ?g	 ?of	 ?N-??methyl-??2-??pyrrolidone	 ?(NMP).	 ?Next,	 ?0.135	 ?g	 ?of	 ?CB	 ?was	 ?mixed	 ?with	 ?the	 ?solution.	 ? Subsequently,	 ? 0.015	 ?g	 ? of	 ? sodium	 ?dodecyl	 ? sulphate	 ? (SDS,	 ? Sigma-??Aldrich,	 ?Oakville,	 ?ON)	 ?was	 ?added	 ?as	 ?surfactant.	 ?The	 ?solution	 ?was	 ?then	 ?placed	 ?in	 ?an	 ?ultrasonic	 ?bath	 ?for	 ?30	 ?min.	 ?The	 ?SDS	 ?surfactant	 ?prevents	 ?the	 ?formation	 ?of	 ?CB	 ?particle	 ?clusters	 ?and	 ?therefore	 ?leads	 ?to	 ?a	 ?uniform	 ?suspension	 ?of	 ?the	 ?particles	 ?in	 ?the	 ?solution,	 ?which	 ?consequently	 ?results	 ?in	 ?a	 ?uniform	 ?distribution	 ?of	 ?the	 ?particles	 ?within	 ?the	 ?polymer	 ?matrix	 ?once	 ?the	 ?material	 ?is	 ?cast	 ?on	 ?the	 ?mold;	 ?this	 ? is	 ? necessary	 ? for	 ? getting	 ? a	 ? uniform	 ? conductivity	 ? and	 ? therefore	 ? metal	 ? coating	 ? on	 ? its	 ?surface.	 ?The	 ?resulting	 ? fluid	 ?had	 ?a	 ?solid	 ?concentration	 ?of	 ?9	 ?wt%	 ?with	 ?the	 ?CB	 ?accounting	 ? for	 ?30%	 ?of	 ?the	 ?total	 ?solid	 ?content.	 ?After	 ?fabrication	 ?of	 ?the	 ?mold,	 ?20	 ??l	 ?of	 ?hexamethyldisilazane	 ?(HMDS,	 ?Sigma-??Aldrich,	 ?Oakville,	 ?ON)	 ?was	 ?applied	 ?to	 ?the	 ?mold	 ?at	 ?room	 ?temperature	 ?to	 ? improve	 ? its	 ?surface	 ?adhesion.	 ?Next,	 ?40	 ??l	 ?of	 ?the	 ?9	 ?wt%	 ?PMMA/CB	 ?mixture	 ?was	 ?deposited	 ?into	 ?the	 ?mold	 ?and	 ?then	 ?heated	 ?to	 ?80?C	 ?for	 ?3	 ?h	 ?to	 ?evaporate	 ?the	 ?NMP	 ?and	 ?fully	 ?dry	 ?the	 ?PMMA/CB	 ?composite	 ?(Figures	 ?3.1b,	 ?3.1c,	 ?and	 ?3.2b).	 ?The	 ?thickness	 ?of	 ?this	 ?layer	 ?was	 ?100	 ??m	 ?on	 ?the	 ?base	 ?plate	 ?which	 ?gradually	 ?decreased	 ?towards	 ?the	 ?tip	 ?of	 ?the	 ?pillars.	 ?The	 ?PMMA/CB	 ?composite	 ?layer	 ?was	 ?then	 ?used	 ?as	 ?a	 ?seed	 ?layer	 ?in	 ?a	 ?metal	 ?electroplating	 ?process.	 ?This	 ? layer	 ?also	 ?served	 ?a	 ?sacrificial	 ? layer,	 ?which	 ?was	 ? later	 ?removed	 ?to	 ?separate	 ?the	 ?metallic	 ?microneedle	 ?structures	 ?from	 ?the	 ?mold.	 ? It	 ?has	 ?previously	 ?51	 ?	 ?been	 ?shown	 ?that	 ? the	 ?thickness	 ?of	 ?a	 ?polymer	 ? layer	 ?on	 ?the	 ?pillars	 ? that	 ?was	 ?deposited	 ?using	 ?solvent	 ?casting	 ?can	 ?be	 ?adjusted	 ?by	 ?controlling	 ?the	 ?solvent	 ?casting	 ?process	 ?parameters	 ?such	 ?as	 ?the	 ?temperature	 ?and	 ?the	 ?dew	 ?point	 ?[94].	 ?The	 ?thickness	 ?of	 ?the	 ?PMMA/CB	 ?seed	 ?layer	 ?on	 ?the	 ? pillars	 ? can	 ? therefore	 ? be	 ? adjusted	 ? in	 ? order	 ? to	 ? tune	 ? the	 ?microneedle	 ? shapes.	 ? After	 ? the	 ?PMMA/CB	 ?casting	 ?step,	 ?a	 ?thin	 ?PMMA/CB	 ?layer	 ?covered	 ?the	 ?top	 ?surfaces	 ?of	 ?the	 ?pillars;	 ?this	 ?layer	 ? was	 ? removed	 ? using	 ? an	 ? O2/CF4	 ? plasma	 ? etching	 ? step	 ? (O2:	 ? 80	 ? sccm,	 ? CF4:	 ? 20	 ? sccm,	 ?pressure:	 ? 500	 ?mTorr,	 ? temperature:	 ? 25?C,	 ? power:	 ? 200	 ?W,	 ? and	 ? duration:	 ? 200	 ?s)	 ? in	 ? order	 ? to	 ?expose	 ?the	 ?SU-??8	 ?pillar	 ?tips	 ?(Figure	 ?3.1d).	 ?This	 ?etching	 ?step	 ?ensures	 ?that	 ?the	 ?non-??conducting	 ?needle	 ?tips	 ?would	 ?be	 ?open	 ?once	 ?the	 ?needles	 ?are	 ?formed	 ?through	 ?the	 ?electroplating	 ?process.	 ?3.1.3 	 ?Metal	 ?deposition	 ?The	 ? next	 ? fabrication	 ? step	 ? involved	 ? deposition	 ? of	 ? metallic	 ? layers	 ? that	 ? constitute	 ? the	 ?microneedle	 ?array	 ? structure.	 ?This	 ?process	 ?was	 ?done	 ? in	 ? two	 ?steps:	 ? first	 ?a	 ? thick	 ?nickel	 ? layer	 ?was	 ?electroplated	 ?onto	 ?the	 ?conductive	 ?polymer	 ?layer	 ?(Figure	 ?3.1e),	 ?which	 ?made	 ?up	 ?the	 ?main	 ?structural	 ?material;	 ?then,	 ?the	 ?nickel	 ?layer	 ?was	 ?coated	 ?with	 ?a	 ?thin	 ?layer	 ?of	 ?gold	 ?to	 ?cover	 ?the	 ?outer	 ? surface	 ? of	 ? the	 ? microneedles.	 ? Nickel	 ? is	 ? inexpensive	 ? and	 ? has	 ? a	 ? high	 ? compressive	 ?strength	 ? and	 ? Young?s	 ?modulus	 ? compared	 ? with	 ? polymers,	 ? silicon,	 ? and	 ?many	 ? other	 ?metals	 ?[112,	 ? 113].	 ? Electrodeposition	 ? of	 ? nickel	 ? has	 ? been	 ? used	 ? in	 ? numerous	 ? applications	 ? and	 ? is	 ? a	 ?well-??established	 ?process.	 ?Since	 ?nickel	 ?has	 ?been	 ? found	 ? to	 ?cause	 ?allergic	 ? reaction	 ?upon	 ?skin	 ?contact	 ?in	 ?some	 ?people	 ?[114],	 ?it	 ?is	 ?coated	 ?with	 ?a	 ?gold	 ?layer	 ?to	 ?improve	 ?its	 ?biocompatibility	 ?[115].	 ?52	 ?	 ?For	 ? nickel	 ? deposition,	 ? after	 ? the	 ?microneedle	 ?mold	 ? piece	 ?was	 ? coated	 ?with	 ? the	 ? conductive	 ?polymer,	 ?it	 ?was	 ?positioned	 ?parallel	 ?to	 ?a	 ?pure	 ?nickel	 ?anode	 ?at	 ?a	 ?distance	 ?of	 ?2.5	 ?cm	 ?inside	 ?an	 ?electroplating	 ?solution	 ?consisting	 ?of	 ?nickel	 ?chloride	 ?(25 ??? ),	 ?nickel	 ?sulfate	 ?(170	 ??? ),	 ?and	 ?boric	 ?acid	 ?(15	 ??? ).	 ?Figure	 ?3.3	 ?shows	 ?the	 ?schematic	 ?of	 ?the	 ?electroplating	 ?setup.	 ?Since	 ?the	 ?conductive	 ?layer	 ?had	 ?a	 ? lower	 ?conductivity	 ? than	 ? the	 ?contact	 ?wire	 ?connected	 ? to	 ? the	 ?power	 ? supply,	 ? the	 ?top	 ? portion	 ? of	 ? the	 ? coated	 ?mold	 ? piece	 ? was	 ? kept	 ? outside	 ? of	 ? the	 ? electroplating	 ? solution	 ? in	 ?order	 ?to	 ?prevent	 ?a	 ?point	 ?of	 ?high	 ?field	 ?strength	 ?resulting	 ?in	 ?an	 ?accumulation	 ?of	 ?nickel	 ?on	 ?the	 ?contact	 ? wire.	 ? The	 ? power	 ? source	 ? was	 ? set	 ? to	 ? provide	 ? a	 ? constant	 ? current	 ? of	 ? 2	 ?mA	 ? and	 ? the	 ?process	 ?was	 ? set	 ? to	 ? run	 ? for	 ? 150	 ?min,	 ?which	 ? resulted	 ? in	 ? a	 ? 70	 ??m	 ? thick	 ?backing	 ? layer.	 ?After	 ?deposition	 ? of	 ? nickel,	 ? a	 ? 20	 ?nm	 ? layer	 ? of	 ? gold	 ? was	 ? sputtered	 ? on	 ? the	 ? top	 ? surface	 ? of	 ? the	 ?microneedle	 ?array.	 ?	 ?Figure	 ?3.3:	 ?Schematic	 ?of	 ?the	 ?setup	 ?used	 ?for	 ?electroplating	 ?of	 ?nickel.	 ?The	 ?wire	 ?contact	 ?point	 ?to	 ?the	 ?PMMA/CB	 ?layer	 ?is	 ?kept	 ?out	 ?of	 ?solution	 ?to	 ?prevent	 ?uneven	 ?nickel	 ?deposition.	 ?53	 ?	 ?3.1.4 Microneedle	 ?array	 ?lift-??off	 ?Removing	 ?the	 ?microneedle	 ?array	 ?was	 ?finally	 ?facilitated	 ?by	 ?dissolving	 ?the	 ?PMMA/CB	 ?layer	 ?in	 ?NMP	 ?and	 ? in	 ?an	 ?ultra	 ?sonic	 ?bath	 ? for	 ?60	 ?min	 ? (Figure	 ?3.1f).	 ?Once	 ? the	 ?microneedle	 ?array	 ?was	 ?separated,	 ?the	 ?mold	 ?could	 ?be	 ?further	 ?cleaned	 ?with	 ?acetone	 ?or	 ?NMP	 ?to	 ?remove	 ?any	 ?leftover	 ?CB	 ?particles	 ?and	 ?used	 ?again	 ? for	 ? fabrication.	 ? In	 ?addition,	 ? the	 ?dissolved	 ?PMMA/CB	 ? layer	 ?can	 ?be	 ? potentially	 ? reused	 ? for	 ? fabrication	 ? after	 ? evaporating	 ? part	 ? of	 ? the	 ? NMP	 ? to	 ? reduce	 ? its	 ?concentration	 ? to	 ? an	 ? amount	 ? applicable	 ? for	 ? solvent	 ? casting.	 ? Figure	 ? 3.4	 ? shows	 ? images	 ? of	 ?microneedles	 ?fabricated	 ?using	 ?this	 ?process.	 ?	 ?54	 ?	 ?	 ?Figure	 ?3.4:	 ?Images	 ?of	 ?microneedles	 ?fabricated	 ?through	 ?the	 ?process	 ?shown	 ?in	 ?Figure	 ?3.1,	 ?a)	 ?a	 ?single	 ?500	 ??m	 ?tall	 ?microneedle	 ?with	 ?a	 ?tip	 ?lumen	 ?diameter	 ?of	 ?40	 ??m	 ?and	 ?tip	 ?wall	 ?thickness	 ?of	 ?15	 ??m,	 ?b)	 ?and	 ?c)	 ?arrays	 ?of	 ?microneedle	 ?500	 ??m	 ?tall	 ?with	 ?1	 ?mm	 ?spacing,	 ?d)	 ?the	 ?backside	 ?of	 ?a	 ?microneedle	 ?array	 ?showing	 ?the	 ?needle	 ?lumen	 ?openings.	 ?	 ?3.2 Experimental	 ?procedures	 ?3.2.1 Conductivity	 ?measurements	 ?of	 ?PMMA/CB	 ?composites	 ?A	 ? series	 ? of	 ? experiments	 ? were	 ? carried	 ? out	 ? to	 ? investigate	 ? the	 ? effect	 ? of	 ? CB	 ? content	 ? on	 ?conductivity	 ?of	 ?the	 ?PMMA/CB	 ?composite.	 ?For	 ?these	 ?experiments,	 ?PMMA/CB	 ?suspensions	 ?in	 ?55	 ?	 ?NMP	 ?were	 ?prepared	 ?with	 ?a	 ?CB	 ?concentration	 ?in	 ?the	 ?final	 ?solid	 ?varying	 ?between	 ?0	 ?to	 ?50	 ?wt%.	 ?All	 ? suspensions	 ?had	 ?a	 ? total	 ? solid	 ? concentration	 ? (CB,	 ? SDS,	 ? and	 ?PMMA)	 ?of	 ? 9	 ?wt%.	 ?After	 ? the	 ?solutions	 ? were	 ? prepared,	 ? they	 ? were	 ? deposited	 ? into	 ? cylindrical	 ? cavities	 ? with	 ? diameters	 ? of	 ?4	 ?mm	 ? to	 ? create	 ? disk-??shaped	 ? composite	 ? films	 ? for	 ? conductivity	 ? measurements.	 ? The	 ? film	 ?thicknesses	 ?were	 ?then	 ?obtained	 ?based	 ?on	 ?deposition	 ?volume	 ?and	 ?the	 ?solids	 ?volume	 ?fraction	 ?in	 ? the	 ? solution,	 ? and	 ? were	 ? also	 ? verified	 ? using	 ? a	 ? digital	 ? micrometer.	 ? A	 ? copper	 ? layer	 ? was	 ?evaporated	 ?on	 ?the	 ?top	 ?and	 ?bottom	 ?surfaces	 ?of	 ?the	 ?films.	 ?The	 ?electrical	 ?resistance	 ?values	 ?R	 ?of	 ? the	 ? films	 ?were	 ? then	 ?measured	 ? using	 ? a	 ? conventional	 ?multimeter,	 ? by	 ? placing	 ? the	 ?meter	 ?probes	 ?on	 ?the	 ?top	 ?and	 ?bottom	 ?copper	 ?surfaces.	 ?The	 ?resistivity	 ?values	 ?	 ?! = R Al 	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?(3)	 ?were	 ?calculated	 ? from	 ?the	 ? thickness	 ? l	 ?of	 ? the	 ?cast	 ? film,	 ? the	 ?measured	 ? resistance	 ?R,	 ? and	 ? the	 ?area	 ?A	 ?of	 ?the	 ?circular	 ?cross	 ?section	 ?of	 ?the	 ?cylinders.	 ?The	 ?conductivity	 ?values	 ?were	 ?obtained	 ?by	 ?inversing	 ?the	 ?resistivity	 ?values.	 ?See	 ?appendix	 ?A.4	 ?for	 ?more	 ?detail.	 ?3.2.2 Dry	 ?etching	 ?of	 ?the	 ?PMMA/CB	 ?layer	 ?The	 ?dry	 ?etching	 ?step	 ? in	 ? the	 ? fabrication	 ?process	 ? impacts	 ? the	 ?height	 ?of	 ? the	 ?microneedles	 ?as	 ?well	 ? as	 ? their	 ? tip	 ? diameter;	 ? it	 ? is	 ? therefore	 ? necessary	 ? to	 ? precisely	 ? control	 ? this	 ? process	 ? to	 ?ensure	 ?there	 ?will	 ?be	 ?enough	 ?polymer	 ?removed	 ?from	 ?the	 ?pillar	 ?tips	 ?so	 ?that	 ?the	 ?microneedle	 ?tips	 ?will	 ?be	 ?open,	 ?while	 ?maintaining	 ?the	 ?sharpness	 ?and	 ?aspect	 ?ratio	 ?of	 ?the	 ?microneedles.	 ?56	 ?	 ?Through	 ? a	 ? series	 ? of	 ? experiments,	 ? the	 ? plasma	 ? etch	 ? rate	 ? of	 ? the	 ? PMMA/CB	 ? composite	 ? films	 ?were	 ?measured	 ?for	 ?different	 ?gas	 ?flow	 ?settings.	 ?It	 ?had	 ?previously	 ?been	 ?shown	 ?that	 ?O2	 ?and	 ?CF4	 ?plasma	 ?etching	 ?is	 ?an	 ?effective	 ?dry	 ?etching	 ?technique	 ?for	 ?removal	 ?of	 ?many	 ?organic	 ?polymers	 ?including	 ?PMMA	 ?[116,	 ?117].	 ?Here,	 ?a	 ?Trion	 ?RIE/PECVD	 ?(Trion	 ?Technology,	 ?Clearwater,	 ?FL)	 ?was	 ?used	 ?to	 ?investigate	 ?the	 ?etch	 ?rate	 ?of	 ?the	 ?PMMA/CB	 ?composite	 ?in	 ?plasma.	 ?First,	 ?100	 ??m	 ?thick	 ?layers	 ?of	 ?PMMA/CB	 ?composite,	 ?with	 ?30	 ?wt%	 ?CB	 ?in	 ?solid,	 ?were	 ?cast	 ?on	 ?1?1	 ?cm2	 ?glass	 ?chips.	 ?The	 ? surfaces	 ? were	 ? then	 ? partially	 ? covered	 ? with	 ? an	 ? adhesive	 ? polyimide	 ? tape	 ? to	 ? partially	 ?protect	 ?them	 ?from	 ?the	 ?plasma.	 ?The	 ?glass	 ?chips	 ?were	 ?then	 ?placed	 ?in	 ?the	 ?plasma	 ?etcher	 ?and	 ?were	 ?etched	 ?with	 ?different	 ?combinations	 ?of	 ?O2	 ?and	 ?CF4	 ?volumetric	 ?flow	 ?rates.	 ?The	 ?total	 ?flow	 ?rate	 ?was	 ?adjusted	 ?to	 ?100	 ?sccm.	 ?The	 ?power	 ?was	 ?set	 ?to	 ?200	 ?W,	 ?and	 ?the	 ?chamber	 ?pressure	 ?and	 ?temperature	 ? to	 ?500	 ?mTorr	 ?and	 ?25?C,	 ? respectively.	 ?The	 ?etching	 ?process	 ?was	 ? set	 ? to	 ? run	 ? for	 ?300	 ?s	 ?for	 ?all	 ?the	 ?trials.	 ?For	 ?determining	 ?the	 ?etch	 ?rates,	 ?first,	 ?the	 ?amount	 ?of	 ?polymer	 ?that	 ?was	 ?removed	 ?by	 ?plasma	 ?was	 ?measured.	 ?For	 ? this	 ?purpose,	 ?once	 ?the	 ?process	 ?was	 ?complete,	 ? the	 ?polyimide	 ?tape	 ?was	 ?removed	 ? from	 ? the	 ? polymer	 ? surface,	 ? and	 ? then	 ? a	 ? 40	 ?nm	 ? layer	 ? of	 ? gold	 ? was	 ? uniformly	 ?evaporated	 ?onto	 ? the	 ?surface.	 ?A	 ?Wyko	 ?NT1100	 ?optical	 ?profilometer	 ? (Veeco,	 ?Plainview,	 ?NY)	 ?was	 ?then	 ?used	 ?to	 ?measure	 ?the	 ?height	 ?difference	 ?between	 ?the	 ?areas	 ?exposed	 ?to	 ?the	 ?plasma	 ?and	 ?the	 ?areas	 ?covered	 ?with	 ?the	 ?tape,	 ?which	 ?corresponded	 ?to	 ?the	 ?etch	 ?depth.	 ?The	 ?etch	 ?rate	 ?was	 ? then	 ? calculated	 ? by	 ? dividing	 ? this	 ? height	 ? difference	 ? by	 ? the	 ? duration	 ? of	 ? the	 ? etching	 ?process.	 ?See	 ?appendix	 ?A.5	 ?for	 ?more	 ?detail.	 ?57	 ?	 ?3.2.3 Characterization	 ?of	 ?the	 ?nickel	 ?electroplating	 ?process	 ?The	 ?electroplated	 ?nickel	 ? constitutes	 ? the	 ?main	 ?structural	 ?material	 ?of	 ? the	 ?microneedles	 ?and	 ?its	 ?deposition	 ?thickness	 ?controls	 ?the	 ?rigidity	 ?and	 ?wall	 ?thickness	 ?of	 ?the	 ?microneedles,	 ?as	 ?well	 ?as	 ?the	 ?back	 ?plate	 ?of	 ?the	 ?array.	 ?	 ?Through	 ?a	 ?series	 ?of	 ?tests,	 ?the	 ?effect	 ?of	 ?the	 ?process	 ?duration	 ?and	 ?the	 ?electric	 ?current	 ?density	 ?on	 ?the	 ?nickel	 ?thickness	 ?was	 ? investigated,	 ? in	 ?order	 ?to	 ?determine	 ?the	 ?deposition	 ?rate	 ?of	 ?the	 ?nickel	 ?with	 ?respect	 ?to	 ?these	 ?parameters.	 ?For	 ?this	 ?purpose,	 ?100	 ??m	 ?layers	 ?of	 ?PMMA/CB,	 ?with	 ?30%	 ?CB	 ?in	 ?solid,	 ?were	 ?cast	 ?on	 ?glass	 ?chips,	 ?which	 ?were	 ?then	 ?immersed	 ?in	 ?the	 ?electroplating	 ?solution	 ?and	 ?used	 ?as	 ?seed	 ?layers	 ?similar	 ?to	 ?the	 ?microneedle	 ?electroplating	 ?fabrication	 ?steps.	 ?Once	 ?the	 ?electroplating	 ?process	 ?was	 ?complete	 ?for	 ?each	 ?trial,	 ?an	 ?Alpha	 ?Step	 ?200	 ?profilometer	 ?(1%	 ? accuracy,	 ? KLA-??Tencor,	 ? Milpitas,	 ? CA)	 ? was	 ? used	 ? to	 ? measure	 ? the	 ? deposited	 ? metal	 ?thickness.	 ?See	 ?appendix	 ?A.6	 ?for	 ?more	 ?detail.	 ?3.2.4 Mechanical	 ?compression	 ?tests	 ?on	 ?the	 ?fabricated	 ?microneedles	 ?The	 ? strength	 ? of	 ? the	 ? microneedles	 ? was	 ? measured	 ? through	 ? a	 ? series	 ? of	 ? mechanical	 ?compression	 ? tests	 ? and	 ? compared	 ?with	 ? literature	 ? data.	 ? The	 ? schematic	 ? of	 ? the	 ? test	 ? setup	 ? is	 ?shown	 ?in	 ?Figure	 ?3.5.	 ?A	 ?Physica	 ?MCR	 ?301	 ?Rheometer	 ?(Anton	 ?Paar,	 ?Ashland,	 ?VA)	 ?was	 ?used	 ?to	 ?apply	 ?vertical	 ?compressive	 ?loads	 ?to	 ?500	 ??m	 ?tall	 ?microneedles.	 ?The	 ?rheometer	 ?tool	 ?was	 ?set	 ?to	 ?move	 ?at	 ?a	 ?constant	 ?velocity	 ?of	 ?5	 ? ? ? 	 ?throughout	 ?the	 ?tests,	 ?and	 ?force	 ?vs.	 ?displacement	 ?data	 ?was	 ? obtained	 ? for	 ? analysis	 ? of	 ? the	 ? failure	 ? loads.	 ? After	 ? each	 ? experiment,	 ? the	 ? needles	 ? were	 ?58	 ?	 ?visually	 ? observed	 ? to	 ? make	 ? sure	 ? they	 ? did	 ? not	 ? buckle.	 ? These	 ? tests	 ? were	 ? carried	 ? out	 ? on	 ? 5	 ?individual	 ?microneedle	 ?samples	 ?made	 ?through	 ?the	 ?process	 ?shown	 ?in	 ?Figure	 ?3.1.	 ?	 ?	 ?Figure	 ?3.5:	 ?Schematic	 ?of	 ?the	 ?setup	 ?used	 ?for	 ?mechanical	 ?compression	 ?tests	 ?on	 ?microneedles.	 ?	 ?3.2.5 Fluid	 ?delivery	 ?into	 ?sample	 ?skin	 ?using	 ?the	 ?fabricated	 ?microneedles	 ?The	 ?capability	 ?of	 ?the	 ?metallic	 ?microneedles	 ?for	 ?skin	 ?penetration	 ?was	 ?realised	 ?by	 ?applying	 ?a	 ?500	 ??m	 ? tall	 ?microneedle	 ? to	 ? pig	 ? skin	 ? and	 ? then	 ? taking	 ? histology	 ? sections	 ? to	 ? investigate	 ? the	 ?penetration	 ? depth	 ? and	 ? skin	 ? surface	 ? damage.	 ? The	 ? capability	 ? of	 ? the	 ? microneedles	 ? for	 ?transdermal	 ?drug	 ?delivery	 ?was	 ?assessed	 ?through	 ?an	 ?injection	 ?of	 ?fluorescent	 ?beads	 ? into	 ?pig	 ?skin.	 ?	 ?For	 ?this	 ?purpose,	 ?a	 ?500	 ??m	 ?tall	 ?microneedle	 ?was	 ?bonded	 ?to	 ?the	 ?tip	 ?of	 ?a	 ?conventional	 ?1	 ?ml	 ? syringe.	 ? After	 ? filling	 ? the	 ? syringe	 ?with	 ? a	 ? 0.01	 ?wt%	 ? suspension	 ? of	 ? 2.28	 ??m	 ? fluorescent	 ?beads	 ? in	 ? water,	 ? the	 ? microneedle	 ? was	 ? pressed	 ? against	 ? the	 ? skin,	 ? and	 ? then	 ? a	 ? force	 ? of	 ?approximately	 ?2	 ?N	 ?was	 ?applied	 ?to	 ?the	 ?syringe	 ?plunger	 ?for	 ?5	 ?min.	 ?The	 ?skin	 ?surface	 ?near	 ?the	 ?injection	 ? site	 ? was	 ? then	 ? washed	 ? with	 ? water	 ? and	 ? dried	 ? with	 ? a	 ? wipe.	 ? A	 ? Nikon	 ? Eclipse	 ? C1	 ?59	 ?	 ?confocal	 ?microscope	 ?(Melville,	 ?NY)	 ?was	 ?then	 ?used	 ?to	 ?scan	 ?the	 ?distribution	 ?of	 ?the	 ?fluorescent	 ?beads	 ? inside	 ? the	 ? skin.	 ?Pig	 ? skin	 ?was	 ?used	 ? for	 ? transdermal	 ?analysis	 ?due	 ? to	 ? its	 ? similarities	 ? to	 ?human	 ?skin	 ?in	 ?terms	 ?of	 ?skin	 ?layer	 ?thickness	 ?and	 ?elasticity,	 ?which	 ?makes	 ?it	 ?a	 ?suitable	 ?model	 ?for	 ?in	 ?vitro	 ?studies	 ?[118].	 ?3.3 Results	 ?and	 ?discussion	 ?3.3.1 Conductivity	 ?measurements	 ?The	 ?resistivity	 ?of	 ?the	 ?polymer	 ?is	 ?shown	 ?in	 ?Figure	 ?3.6	 ?as	 ?a	 ?function	 ?of	 ?CB	 ?content.	 ?This	 ?plot	 ?shows	 ?a	 ?significant	 ?decrease	 ?of	 ? the	 ? resistivity	 ?as	 ? the	 ?CB%	 ? increases	 ? from	 ?0	 ? to	 ?50%;	 ? this	 ? is	 ?consistent	 ?with	 ?previous	 ?works	 ?investigating	 ?resistivity	 ?for	 ?lower	 ?CB	 ?contents	 ?[106,	 ?107,	 ?and	 ?119].	 ?Although	 ?using	 ?the	 ?PMMA/CB	 ?composite	 ?with	 ?the	 ?highest	 ?conductivity	 ?could	 ?facilitate	 ?faster	 ?electroplating,	 ?using	 ?CB	 ?beyond	 ?30%	 ? leads	 ? to	 ?cracks	 ? in	 ? the	 ?polymer	 ? film	 ?during	 ? the	 ?casting	 ? step.	 ? As	 ? the	 ? higher	 ? CB	 ? content	 ? makes	 ? the	 ? polymer	 ? more	 ? brittle,	 ? the	 ? cast	 ? films	 ?become	 ?more	 ? sensitive	 ? to	 ? internal	 ? stresses	 ? that	 ? develop	 ? during	 ? the	 ? evaporation	 ? process.	 ?Using	 ?CB	 ?above	 ?30	 ?wt%	 ?creates	 ?cracks	 ?mostly	 ?around	 ?the	 ?pillar	 ?bases	 ?and	 ?around	 ?the	 ?edges	 ?of	 ?the	 ?mold	 ?in	 ?areas	 ?of	 ?high	 ?thickness	 ?variations.	 ?The	 ?30	 ?wt%	 ?CB	 ?concentration,	 ?therefore,	 ?was	 ? chosen	 ? in	 ? this	 ? process	 ? to	 ? prevent	 ? crack	 ? formation	 ?while	 ? providing	 ? a	 ? sufficiently	 ? high	 ?conductivity	 ?for	 ?electroplating.	 ?60	 ?	 ?0 10 20 30 40 501E-30.010.1110100100010000Resistivity [kOhm?cm]Carbon black concentration [wt%]  Figure	 ?3.6:	 ?	 ?The	 ?resistivity	 ?of	 ?the	 ?PMMA/CB	 ?polymer	 ?film	 ?as	 ?a	 ?function	 ?of	 ?CB	 ?content.	 ?Each	 ?data	 ?point	 ?represents	 ?the	 ?average	 ?value	 ?from	 ?three	 ?experiments	 ?and	 ?the	 ?error	 ?bars	 ?indicate	 ??	 ?standard	 ?deviations.	 ?	 ?3.3.2 Dry	 ?etching	 ?of	 ?the	 ?PMMA/CB	 ?layer	 ?The	 ?plasma	 ?etching	 ? rates	 ?of	 ? the	 ?PMMA/CB	 ?composite	 ? is	 ? shown	 ? in	 ?Figure	 ?3.7	 ? for	 ?different	 ?gas	 ? flow	 ? settings.	 ? It	 ? has	 ? been	 ? previously	 ? shown	 ? that	 ? the	 ? addition	 ? of	 ? fluorine-??containing	 ?gases	 ? can	 ? lead	 ? to	 ? an	 ? increase	 ? in	 ? the	 ? oxygen	 ? atom	 ? density	 ? in	 ? the	 ? plasma	 ? and	 ? therefore	 ?enhance	 ? the	 ?polymer	 ?etching	 ? rates	 ?up	 ? to	 ?a	 ?certain	 ? fluorine	 ?concentration	 ? [116].	 ? In	 ?Figure	 ?3.7,	 ? the	 ? etch	 ? rate	 ? peaks	 ? for	 ? CF4	 ? flow	 ? rate	 ? approaching	 ? 20%,	 ?which	 ? is	 ? consistent	 ?with	 ? the	 ?peak	 ?locations	 ?for	 ?some	 ?other	 ?polymers	 ?[116].	 ?	 ?The	 ?20%	 ?CF4	 ?flow	 ?rate	 ?has	 ?then	 ?been	 ?used	 ?for	 ?the	 ?microneedle	 ?fabrication.	 ?	 ?61	 ?	 ?0 20 40 60 80 1000.00.10.20.30.40.5Etch rate [?m/min]% CF4  Figure	 ?3.7:	 ?Etch	 ?rates	 ?for	 ?PMMA/CB	 ?composite	 ?(with	 ?30	 ?wt%	 ?CB)	 ?for	 ?different	 ?ratios	 ?of	 ?CF4	 ?to	 ?total	 ?gas	 ?flow	 ?rate.	 ?The	 ?total	 ?gas	 ?flow	 ?rate	 ?was	 ?kept	 ?constant	 ?at	 ?100	 ?sccm.	 ?Each	 ?data	 ?point	 ?represents	 ?the	 ?average	 ?value	 ?from	 ?three	 ?experiments	 ?and	 ?the	 ?error	 ?bars	 ?indicate	 ??	 ?standard	 ?deviations.	 ?The	 ?plasma	 ?etching	 ?step	 ?could	 ?potentially	 ?be	 ?eliminated	 ?from	 ?the	 ?fabrication	 ?process	 ?if	 ?the	 ?PMMA/CB	 ?solution	 ?concentration	 ?was	 ?kept	 ?below	 ?a	 ?certain	 ?value	 ?for	 ?specific	 ?pillar	 ?heights.	 ?It	 ?was	 ? seen	 ?during	 ? the	 ?experiments	 ? that	 ? for	 ? low	 ?solution	 ?concentrations	 ? (less	 ? than	 ?5	 ?wt%	 ?total	 ?solid	 ?concentration)	 ?the	 ?polymer	 ?would	 ?not	 ?cover	 ?the	 ?pillar	 ?tips	 ?and	 ?the	 ?solution	 ?does	 ?not	 ? wet	 ? the	 ? pillars	 ? entirely.	 ? This	 ? could	 ? be	 ? due	 ? to	 ? a	 ? moving	 ? contact	 ? line	 ? of	 ? the	 ? polymer	 ?solution	 ?during	 ?the	 ?evaporation	 ?process,	 ?which	 ?occurs	 ?for	 ?lower	 ?polymer	 ?concentrations	 ?or	 ?taller	 ?pillars.	 ?The	 ?resulting	 ?electroplated	 ?layer	 ?would	 ?be	 ?expected	 ?to	 ?be	 ?uniform	 ?on	 ?the	 ?base	 ?plate	 ?and	 ?metal	 ?would	 ?not	 ?cover	 ?the	 ?pillars	 ?tips.	 ?62	 ?	 ?3.3.3 Nickel	 ?electroplating	 ?process	 ?The	 ? thickness	 ?of	 ? the	 ?nickel	 ? layer	 ?over	 ? time	 ? is	 ? shown	 ? in	 ? Figure	 ?3.8,	 ? for	 ? a	 ? constant	 ? current	 ?source	 ?of	 ?2	 ?mA,	 ?and	 ?a	 ?cathode	 ?area	 ?of	 ?1?1	 ?cm2	 ?positioned	 ?2.5	 ?cm	 ?from	 ?the	 ?nickel	 ?anode.	 ?In	 ?Figure	 ? 3.9,	 ? the	 ? nickel	 ? thickness	 ? is	 ? shown	 ? for	 ? different	 ? current	 ? source	 ? settings	 ? and	 ? a	 ?deposition	 ? time	 ? of	 ? 90	 ?min	 ? with	 ? the	 ? same	 ? electrode	 ? size	 ? and	 ? orientation	 ? as	 ? the	 ? previous	 ?experiment.	 ?The	 ?change	 ? in	 ?nickel	 ? thickness	 ? is	 ? linear	 ? in	 ?both	 ?cases	 ?within	 ? the	 ? range	 ?of	 ? the	 ?tested	 ?parameters.	 ?The	 ?nickel	 ?deposition	 ?rate	 ?of	 ?0.49	 ? ???? 	 ?at	 ?2	 ?mA	 ?was	 ?found	 ?from	 ?the	 ?slope	 ?of	 ? the	 ? linear	 ? fit	 ? in	 ? Figure	 ? 3.8,	 ? and	 ? the	 ? thickness	 ? as	 ? a	 ? function	 ? of	 ? current	 ? of	 ? 21.9	 ? ??? 	 ? over	 ?90	 ?min	 ?was	 ?found	 ?from	 ?the	 ?linear	 ?fit	 ?in	 ?Figure	 ?3.9,	 ?yielding	 ?a	 ?current-??dependent	 ?deposition	 ?rate	 ?of	 ?0.24	 ? ???  ? ?? .	 ?0 20 40 60 80 100 120 140 160 180 200020406080100Thickness of nickel [?m]Time [min]  Figure	 ?3.8:	 ?The	 ?thickness	 ?of	 ?electroplated	 ?nickel	 ?on	 ?a	 ?PMMA/CB	 ?layer	 ?over	 ?time,	 ?with	 ?2	 ?mA	 ?supply	 ?current.	 ?The	 ?slope	 ?of	 ?the	 ?linear	 ?trend	 ?line	 ?is	 ?0.49	 ??m/min.	 ? 63	 ?	 ?0 1 2 3 4 5 6 7 8020406080100120140160Thickness of nickel [?m]Current [mA]  Figure	 ?3.9:	 ?Thickness	 ?of	 ?electroplated	 ?nickel	 ?as	 ?a	 ?function	 ?of	 ?power	 ?source	 ?current,	 ?for	 ?process	 ?duration	 ?of	 ?90	 ?min.	 ?The	 ?slope	 ?of	 ?the	 ?linear	 ?trend	 ?line	 ?is	 ?21.9	 ??m/mA.	 ?	 ?The	 ?recorded	 ?thickness	 ?values	 ?correspond	 ?to	 ?the	 ?thickness	 ?of	 ?the	 ?microneedle	 ?array	 ?backing	 ?plates	 ?and	 ?not	 ?the	 ?microneedle	 ?structures	 ?themselves;	 ?as	 ?seen	 ?in	 ?Figure	 ?3.4a,	 ?the	 ?thickness	 ?of	 ?the	 ?nickel	 ?at	 ?the	 ?microneedle	 ?tip	 ?is	 ?much	 ?less	 ?than	 ?that	 ?of	 ?the	 ?70	 ??m	 ?backing	 ?plate,	 ?which	 ?shows	 ? that	 ? the	 ?nickel	 ?coating	 ? is	 ?not	 ? isotropic.	 ?This	 ?could	 ?be	 ?due	 ? to	 ? the	 ?orientation	 ?of	 ? the	 ?mold	 ? pillars	 ? as	 ? well	 ? as	 ? the	 ? lower	 ? conductivity	 ? of	 ? the	 ? polymer	 ? at	 ? the	 ? pillar	 ? tips	 ? due	 ? to	 ?decreased	 ?thickness.	 ?Since	 ?the	 ?pillars?	 ?orientation	 ?in	 ?the	 ?electrolyte	 ?is	 ?perpendicular	 ?to	 ?the	 ?anode	 ? electrode	 ? plane,	 ? there	 ? could	 ? be	 ? less	 ? Ni2+	 ? accumulation	 ? on	 ? their	 ? tips	 ? due	 ? to	 ? the	 ?perpendicular	 ?movement	 ?of	 ?the	 ?charged	 ?particles	 ?to	 ?the	 ?microneedle	 ?plane.	 ?In	 ?addition,	 ?the	 ?lower	 ? conductivity	 ? at	 ? the	 ? pillar	 ? tips	 ? could	 ? lead	 ? to	 ? lower	 ? current	 ? density	 ? in	 ? those	 ? regions	 ?which	 ?results	 ?in	 ?a	 ?slower	 ?metal	 ?coating	 ?at	 ?the	 ?beginning	 ?of	 ?the	 ?electroplating	 ?process.	 ?	 ?64	 ?	 ?3.3.4 Mechanical	 ?compression	 ?tests	 ?Figure	 ?3.10	 ?shows	 ?an	 ?example	 ?of	 ?a	 ?force	 ?vs.	 ?displacement	 ?measurement	 ?used	 ?to	 ?determine	 ?the	 ?microneedle	 ?failure	 ?load.	 ?In	 ?this	 ?figure,	 ?the	 ?first	 ?peak	 ?in	 ?the	 ?force	 ?graph	 ?corresponds	 ?to	 ?the	 ? tip	 ? failure	 ? under	 ? compressive	 ? loading.	 ? From	 ? the	 ? five	 ? compression	 ? tests,	 ? the	 ? average	 ?failure	 ?load	 ?was	 ?4.2	 ??	 ?0.61	 ?N.	 ?A	 ?plot	 ?with	 ?all	 ?measurements	 ?is	 ?provided	 ?in	 ?Appendix	 ?A.7.	 ?In	 ?a	 ?previous	 ?study	 ?by	 ?Davis	 ?et	 ?al.	 ?[103],	 ?the	 ?authors	 ?have	 ?studied	 ?the	 ?fracture	 ?force	 ?of	 ?hollow	 ?metallic	 ?microneedles	 ?with	 ?respect	 ?to	 ?the	 ?needle	 ?tip	 ?radius,	 ?wall	 ?thickness,	 ?and	 ?wall	 ?angle.	 ?For	 ? this	 ? purpose,	 ? the	 ? authors	 ? have	 ? compared	 ? the	 ? experimentally	 ? obtained	 ? fracture	 ? force	 ?data	 ?with	 ?an	 ?analytical	 ?solution	 ?estimating	 ?the	 ?force,	 ?as	 ?well	 ?as	 ?a	 ?finite	 ?element	 ?model.	 ?	 ?0 50 100 150 200 250012345Force [N]Displacement [?m] 	 ?Figure	 ?3.10:	 ?A	 ?needle	 ?displacement	 ?under	 ?axial	 ?loading.	 ?The	 ?analytical	 ?model	 ?assumes	 ?thin-??shell	 ?needles,	 ?which	 ?requires	 ?the	 ?ratio	 ?of	 ?the	 ?tip	 ?radius	 ?to	 ? the	 ?wall	 ? thickness	 ? to	 ?be	 ? larger	 ? than	 ? ten.	 ? This	 ?model	 ? is	 ? therefore	 ?not	 ? applicable	 ? to	 ? the	 ?needles	 ?presented	 ?in	 ?this	 ?work,	 ?since	 ?this	 ?ratio	 ?is	 ?much	 ?smaller.	 ?	 ?Failure load 65	 ?	 ?The	 ? finite	 ? element	 ? analysis	 ?was	 ? also	 ? found	 ? to	 ? underestimate	 ? the	 ? fracture	 ? force	 ? due	 ? to	 ? a	 ?conservative	 ? failure	 ? criterion	 ? of	 ? the	 ? needle	 ? material.	 ? However,	 ? the	 ? experimental	 ? data	 ? in	 ?[103]	 ?for	 ?needles	 ?with	 ?a	 ?similar	 ?material,	 ?deposition	 ?method,	 ?and	 ?height	 ?as	 ? in	 ?the	 ?present	 ?work	 ?can	 ?be	 ?compared	 ?with	 ?the	 ?experimental	 ?results	 ?obtained	 ?here.	 ?It	 ?was	 ?found	 ?in	 ?[103]	 ?that	 ?the	 ?fracture	 ?force	 ?is	 ?insensitive	 ?to	 ?tip	 ?radius	 ?for	 ?radii	 ?ranging	 ?from	 ?40	 ??m	 ?to	 ?65	 ??m,	 ?the	 ?fracture	 ?force	 ?increases	 ?significantly	 ?with	 ?wall	 ?thickness	 ?from	 ?4	 ??m	 ?to	 ?15	 ??m	 ?and	 ?slightly	 ?with	 ?wall	 ?angle	 ?from	 ?60?	 ?to	 ?70?.	 ?Extrapolating	 ?the	 ?data	 ?in	 ?[103]	 ?following	 ?these	 ? trends	 ? would	 ? lead	 ? to	 ? a	 ? fracture	 ? force	 ? above	 ? 5	 ?N	 ? for	 ? the	 ? needle	 ? geometry	 ? in	 ? the	 ?present	 ?work	 ? (tip	 ? radius:	 ? 28	 ??m,	 ?wall	 ? thickness:	 ? 15	 ??m,	 ?wall	 ? angle:	 ? 80?),	 ?which	 ? is	 ? slightly	 ?higher	 ?than	 ?the	 ?measured	 ?4.2	 ??	 ?0.61	 ?N.	 ?This	 ?slight	 ?difference	 ?in	 ?strength	 ?can	 ?be	 ?a	 ?result	 ?of	 ?a	 ?different	 ?nickel	 ?nanocrystalline	 ?structure	 ?formed	 ?during	 ?the	 ?electrodeposition	 ?[113].	 ?	 ?In	 ? addition	 ? to	 ? the	 ? fracture	 ? force	 ? measurements	 ? in	 ? [103],	 ? the	 ? forces	 ? required	 ? for	 ? the	 ?penetration	 ? of	 ? microneedles	 ? into	 ? human	 ? skin	 ? have	 ? been	 ? investigated	 ? experimentally	 ? for	 ?microneedles	 ?with	 ?different	 ?tip	 ?diameters.	 ?According	 ?to	 ?this	 ?study,	 ?the	 ?forces	 ?required	 ?for	 ?tip	 ?diameters	 ?of	 ?less	 ?than	 ?50	 ??m	 ?is	 ?below	 ?1	 ?N;	 ?the	 ?failure	 ?load	 ?here	 ?is	 ?well	 ?above	 ?this	 ?force	 ?indicating	 ?that	 ?the	 ?needle	 ?will	 ?most	 ?likely	 ?not	 ?break	 ?upon	 ?insertion	 ?into	 ?skin.	 ?	 ?3.3.5 Transdermal	 ?fluid	 ?delivery	 ?into	 ?sample	 ?skin	 ?The	 ? histology	 ? image	 ? in	 ? Figure	 ? 3.11	 ? shows	 ? the	 ? skin	 ? surface	 ? damage	 ? caused	 ? by	 ? the	 ?microneedle	 ? penetration.	 ? The	 ? confocal	 ? scan	 ? of	 ? the	 ? injection	 ? site,	 ? shown	 ? in	 ? Figure	 ? 3.12,	 ?indicates	 ?delivery	 ?of	 ? the	 ? fluorescent	 ?beads	 ? to	 ? a	 ?depth	 ?of	 ? 250	 ??m	 ? into	 ? the	 ?pig	 ? skin,	 ?which	 ?66	 ?	 ?shows	 ? the	 ? usefulness	 ? the	 ? microneedles	 ? for	 ? transdermal	 ? delivery	 ? of	 ? drugs	 ? including	 ?suspensions.	 ?	 ?	 ?Figure	 ?3.11:	 ?Histology	 ?image	 ?of	 ?a	 ?microneedle	 ?insertion	 ?site	 ?on	 ?pig	 ?skin	 ?showing	 ?the	 ?skin	 ?damage	 ?caused	 ?by	 ?a	 ?500	 ??m	 ?tall	 ?microneedle.	 ?	 ?Figure	 ?3.12:	 ?Confocal	 ?scan	 ?(514	 ??m	 ??	 ?514	 ??m)	 ?of	 ?injection	 ?site	 ?on	 ?pig	 ?skin	 ?showing	 ?the	 ?distribution	 ?of	 ?fluorescent	 ?beads	 ?under	 ?the	 ?skin	 ?surface.	 ?The	 ?test	 ?was	 ?carried	 ?out	 ?using	 ?a	 ?500	 ??m	 ?tall	 ?microneedle.	 ?To	 ? verify	 ? that	 ? the	 ? skin	 ? is	 ? not	 ? permeable	 ? to	 ? the	 ? fluorescent	 ? beads	 ?without	 ? the	 ? aid	 ? of	 ? the	 ?microneedles,	 ?a	 ?series	 ?of	 ?control	 ? tests	 ?were	 ?performed	 ?by	 ?applying	 ? the	 ? fluorescent	 ?beads	 ?67	 ?	 ?solution	 ?to	 ?skin	 ?surface	 ?for	 ?10	 ?min	 ?and	 ?then	 ?scanning	 ?the	 ?skin	 ?after	 ?washing	 ?the	 ?surface.	 ?The	 ?control	 ? tests	 ? did	 ? not	 ? show	 ? any	 ? fluorescent	 ? microspheres	 ? penetration	 ? below	 ? the	 ? skin	 ?surface.	 ?3.4 Conclusions	 ?A	 ?new	 ?fabrication	 ?process	 ?was	 ?demonstrated	 ?for	 ?making	 ?metallic	 ?microneedles.	 ?The	 ?process	 ?uses	 ? a	 ? conductive	 ? polymer	 ? as	 ? the	 ? seed	 ? layer	 ? for	 ? the	 ? electrodeposition	 ? of	 ? metal.	 ? Open	 ?microneedle	 ?tips	 ?are	 ?achieved	 ?through	 ?a	 ?single	 ?plasma	 ?etching	 ?step	 ?prior	 ?to	 ?deposition	 ?of	 ?the	 ?metal	 ?layer.	 ?This	 ?process	 ?can	 ?potentially	 ?allow	 ?fabrication	 ?of	 ?microneedles	 ?with	 ?a	 ?wide	 ?range	 ? of	 ? dimensions	 ? and	 ? spacing.	 ? This	 ? process	 ? can	 ? also	 ? be	 ? used	 ? for	 ? batch	 ? fabrication	 ? of	 ?microneedles,	 ? since	 ? large	 ? arrays	 ? of	 ? hollow	 ? needles	 ? can	 ? be	 ? formed	 ? without	 ? the	 ? need	 ? of	 ?opening	 ?the	 ?individual	 ?needles?	 ?tips,	 ?a	 ?process	 ?which	 ?is	 ?often	 ?time	 ?and	 ?labour	 ?intensive	 ?and	 ?requires	 ?methods	 ?such	 ?as	 ? laser	 ?micromachining.	 ? In	 ?addition,	 ? the	 ?molds	 ?can	 ?be	 ? reused	 ? for	 ?multiple	 ?fabrication	 ?runs	 ?which	 ?would	 ?substantially	 ?reduce	 ?the	 ?materials	 ?and	 ?labor	 ?costs	 ?for	 ?mass	 ?production.	 ?The	 ?molds	 ?in	 ?this	 ?work,	 ?for	 ?instance,	 ?were	 ?used	 ?up	 ?to	 ?three	 ?times	 ?without	 ?seeing	 ?any	 ?degradation	 ?of	 ?the	 ?needle	 ?shape	 ?and	 ?structure.	 ?PMMA	 ?filled	 ?with	 ?CB	 ?has	 ?been	 ?used	 ?as	 ?the	 ?conductive	 ?polymer	 ?layer	 ?during	 ?the	 ?microneedle	 ?preparation.	 ? The	 ? conductivity	 ? of	 ? this	 ? polymer	 ? composite	 ? was	 ? characterized	 ? to	 ? find	 ? the	 ?optimum	 ?CB	 ?content	 ?which	 ?was	 ?30	 ?wt%.	 ?Also,	 ?from	 ?a	 ?series	 ?of	 ?O2-??CF4	 ?plasma	 ?tests,	 ?a	 ?20%	 ?CF4	 ? flow	 ? rate	 ? composition	 ?was	 ? found	 ? to	 ? result	 ? in	 ? the	 ? fastest	 ? etch	 ? rate	 ? for	 ? the	 ? PMMA/CB	 ?composite	 ?with	 ?30%	 ?CB.	 ?The	 ?thickness	 ?of	 ?electroplated	 ?nickel	 ?was	 ?found	 ?to	 ?be	 ? linear	 ?with	 ?68	 ?	 ?respect	 ?to	 ?both	 ?process	 ?time	 ?as	 ?well	 ?as	 ?supply	 ?current.	 ?The	 ?current-??dependant	 ?deposition	 ?rate	 ?was	 ?measured	 ?to	 ?be	 ?0.24 ? ???  ? ?? .	 ?Through	 ? mechanical	 ? compression	 ? tests,	 ? the	 ? average	 ? strength	 ? of	 ? the	 ? fabricated	 ?microneedles	 ?was	 ? found	 ?to	 ?be	 ?above	 ?4	 ?N,	 ? showing	 ? that	 ? these	 ?needles	 ?can	 ?pierce	 ? through	 ?human	 ?skin	 ?without	 ?breakage.	 ? In	 ?addition,	 ? the	 ?usefulness	 ?of	 ? the	 ?microneedle	 ?devices	 ?was	 ?demonstrated	 ?by	 ?successful	 ?delivery	 ?of	 ?2.28	 ??m	 ?fluorescent	 ?microspheres	 ?into	 ?pig	 ?skin	 ?to	 ?a	 ?depth	 ?of	 ?about	 ?250	 ??m,	 ?using	 ?500	 ??m	 ?long	 ?microneedles.	 ?	 ?	 ? 	 ?69	 ?	 ? CHAPTER	 ?4 	 ?	 ?	 ?STUDYING	 ?DIFFUSION	 ?OF	 ?A	 ?MICRONEEDLE-??INJECTED	 ?DRUG	 ?INSIDE	 ?SKIN:	 ?INJECTION	 ?OF	 ?DOXORUBICIN	 ?INTO	 ?PIG	 ?SKIN	 ?	 ?	 ?Drug	 ?transport	 ?across	 ?the	 ?epidermis	 ?is	 ?often	 ?the	 ?time	 ?limiting	 ?step	 ?for	 ?drug	 ?transport	 ?across	 ?the	 ? skin	 ? due	 ? to	 ? the	 ?dry	 ? and	 ?dense	 ? cellular	 ? structure	 ? of	 ? the	 ? epidermis,	 ?which	 ?makes	 ? drug	 ?diffusion	 ? difficult.	 ? The	 ? dermis,	 ? in	 ? contrast,	 ? is	 ? much	 ? less	 ? dense	 ? and	 ? has	 ? more	 ? ISF,	 ? which	 ?makes	 ?drug	 ?diffusion	 ?much	 ?easier	 ?compared	 ?to	 ?the	 ?epidermis.	 ?The	 ?dermis	 ?also	 ?contains	 ?the	 ?blood	 ?capillaries	 ?and	 ? is	 ? thus	 ?often	 ?considered	 ? the	 ? target	 ?medium	 ? in	 ? the	 ? transdermal	 ?drug	 ?delivery	 ? process.	 ? Previous	 ? methods	 ? investigated	 ? drug	 ? transport	 ? from	 ? the	 ? skin	 ? surface.	 ?Microneedles,	 ? however,	 ? release	 ? the	 ? drug	 ? below	 ? the	 ? SC	 ? layer,	 ? and	 ? thus	 ? require	 ? different	 ?methods	 ?of	 ?analysis	 ? to	 ? follow	 ?the	 ?drug	 ?movement.	 ?Most	 ?of	 ?the	 ?previous	 ?works	 ?on	 ?hollow	 ?microneedles	 ? focus	 ? on	 ? the	 ? fabrication	 ? technologies	 ? but	 ? not	 ? much	 ? has	 ? been	 ? invested	 ? on	 ?studying	 ?the	 ?drug	 ?delivery	 ?process.	 ?Although	 ?these	 ?devices	 ?overcome	 ?the	 ?SC	 ?barrier,	 ?drug	 ?70	 ?	 ?delivery	 ?using	 ?microneedles	 ?still	 ?relies	 ?on	 ?the	 ?diffusion	 ?of	 ?drugs	 ?through	 ?the	 ?epidermal	 ?and	 ?the	 ?dermal	 ?layers.	 ?	 ?Developing	 ?microneedle	 ?systems	 ?capable	 ?of	 ?replacing	 ?traditional	 ?injection	 ?methods	 ?require	 ?additional	 ? research	 ?on	 ? the	 ?drug	 ?absorption	 ?process	 ? in	 ? the	 ?skin	 ?and	 ?the	 ? factors	 ? that	 ?affect	 ?this	 ?process,	 ? such	 ?as	 ? the	 ? injection	 ? rate,	 ? the	 ?diffusion	 ?medium,	 ?and	 ? the	 ?drug	 ?chemistry.	 ? In	 ?the	 ? case	 ? of	 ? the	 ? diffusion	 ? medium	 ? (i.e.,	 ? the	 ? skin)	 ? many	 ? have	 ? proposed	 ? using	 ? the	 ? skin	 ? of	 ?animals	 ?as	 ?models	 ?for	 ?studying	 ?transdermal	 ?drug	 ?delivery.	 ? Ideally	 ?for	 ? in	 ?vitro	 ?experiments,	 ?freshly	 ?excised	 ?skin	 ?would	 ?be	 ? the	 ?most	 ? representative	 ?of	 ? in	 ?vivo	 ? skin.	 ?However,	 ?obtaining	 ?fresh	 ? skin	 ? may	 ? not	 ? be	 ? feasible	 ? or	 ? ethical	 ? when	 ? a	 ? study	 ? involves	 ? a	 ? large	 ? number	 ? of	 ?experiments.	 ?Researchers,	 ?therefore,	 ?often	 ?have	 ?to	 ?use	 ?skin	 ?specimens	 ?previously	 ?stored	 ?in	 ?refrigerators	 ? or	 ? freezers	 ? and	 ? it	 ? is	 ? not	 ? clear	 ? how	 ? the	 ? storage	 ? procedure	 ? will	 ? affect	 ? the	 ?absorption	 ?capability	 ?of	 ?the	 ?skin.	 ?Besides	 ?the	 ?diffusion	 ?medium,	 ?understanding	 ?the	 ?effects	 ?of	 ?the	 ?drug	 ?injection	 ?rate	 ?and	 ?its	 ?chemical	 ?and	 ?physical	 ?influences	 ?on	 ?the	 ?diffusion	 ?process	 ?is	 ?essential	 ?in	 ?order	 ?to	 ?maximize	 ?the	 ?process	 ?efficacy.	 ?Analysing	 ?all	 ?these	 ?factors,	 ?however,	 ?first	 ?requires	 ?developing	 ?a	 ?method	 ?that	 ?can	 ?facilitate	 ?studying	 ? injection	 ? with	 ? microneedles.	 ? So	 ? far,	 ? in	 ? the	 ? previous	 ? works	 ? on	 ? microneedles,	 ?researchers	 ?injected	 ?compounds	 ?into	 ?the	 ?skin	 ?with	 ?microneedles	 ?and	 ?then	 ?determined	 ?the	 ?compound	 ?concentrations	 ?in	 ?blood	 ?[10,	 ?50	 ?and	 ?120]	 ?or	 ?simply	 ?injected	 ?fluorescent	 ?dye	 ?(or	 ?particles)	 ? into	 ? an	 ? animal	 ? tissue	 ? and	 ? measured	 ? the	 ? penetration	 ? depth	 ? of	 ? the	 ? dye	 ? [121].	 ?However,	 ?it	 ?is	 ?instructive	 ?to	 ?investigate	 ?drug	 ?transport	 ?inside	 ?the	 ?skin	 ?right	 ?after	 ?injection	 ?to	 ?understand	 ?how	 ?fast	 ?the	 ?drug	 ?diffuses	 ?through	 ?the	 ?skin	 ?to	 ?reach	 ?blood	 ?vessels.	 ?	 ?71	 ?	 ?This	 ?chapter	 ?uses	 ?confocal	 ?microscopy	 ?to	 ?study	 ?drug	 ?delivery	 ?with	 ?hollow	 ?microneedles	 ?for	 ?the	 ? first	 ? time,	 ? by	 ? characterizing	 ? the	 ? diffusion	 ? of	 ? a	 ? fluorescent	 ? drug	 ? injected	 ? into	 ? pig	 ? skin	 ?using	 ? microneedles.	 ? Confocal	 ? microscopy	 ? is	 ? used	 ? to	 ? obtain	 ? the	 ? fluorescence	 ? intensity	 ?images	 ? at	 ? different	 ? skin	 ? depths	 ? after	 ? the	 ? drug	 ? is	 ? injected.	 ? The	 ? drug	 ? concentration	 ?distribution	 ? is	 ? calculated	 ? using	 ? an	 ? intensity-??concentration	 ? calibration.	 ? The	 ? change	 ? in	 ? the	 ?spatial	 ? concentration	 ? distribution	 ? is	 ?monitored	 ? over	 ? time	 ? and	 ? then	 ? used	 ? to	 ? calculate	 ? the	 ?diffusion	 ? coefficient	 ? of	 ? the	 ? drug	 ? in	 ? skin	 ? and	 ? to	 ? validate	 ? an	 ? analytical	 ? diffusion	 ?model.	 ? In	 ?addition,	 ? this	 ?method	 ?was	 ? used	 ? to	 ? compare	 ? drug	 ? diffusion	 ? in	 ? skin	 ? specimens	 ? from	 ? three	 ?different	 ? storage	 ? conditions	 ? (fresh	 ? skin,	 ? refrigerated	 ? skin,	 ? and	 ? frozen	 ? skin)	 ? in	 ? order	 ? to	 ?evaluate	 ?their	 ?usefulness	 ?for	 ?in	 ?vitro	 ?drug	 ?delivery	 ?studies.	 ?Many	 ?previous	 ?studies	 ?have	 ?used	 ?skin	 ?after	 ?any	 ?of	 ?these	 ?three	 ?storage	 ?methods;	 ?however,	 ?as	 ?it	 ?was	 ?observed	 ?through	 ?some	 ?experiments	 ? that	 ? they	 ? may	 ? not	 ? be	 ? equal	 ? in	 ? terms	 ? of	 ? skin	 ? permeation	 ? properties,	 ? a	 ?comparison	 ? study	 ? is	 ? pursued	 ? here	 ? to	 ? compare	 ? the	 ? refrigerated	 ? and	 ? frozen	 ? skin	 ? with	 ? the	 ?most	 ? physiologically	 ? correct	 ? case	 ? (i.e.	 ? fresh	 ? skin).	 ? This	 ? is	 ? achieved	 ? by	 ? comparing	 ? the	 ?diffusion	 ?coefficients	 ?obtained	 ?for	 ?refrigerated	 ?and	 ?frozen	 ?skin	 ?to	 ?that	 ?of	 ?fresh	 ?skin.	 ?	 ?4.1 Experimental	 ?procedures	 ?4.1.1 Materials	 ?For	 ?the	 ?injection	 ?tests,	 ?domestic	 ?pig	 ?skin	 ?was	 ?chosen,	 ?since	 ?it	 ? is	 ?a	 ?useful	 ?model	 ?for	 ?human	 ?skin	 ? in	 ? terms	 ? of	 ? the	 ? mechanical	 ? properties	 ? as	 ? well	 ? as	 ? cellular	 ? structure	 ? and	 ? drug	 ?permeability	 ?[118].	 ?The	 ?skin	 ?from	 ?the	 ?belly	 ?of	 ?one	 ?animal	 ?was	 ?freshly	 ?excised	 ?immediately	 ?72	 ?	 ?after	 ?animal	 ?sacrifice.	 ?Injections	 ?were	 ?carried	 ?out	 ?into	 ?skin	 ?specimens	 ?after	 ?storing	 ?them	 ?in	 ?three	 ? different	 ? ways;	 ? first	 ? as	 ? fresh	 ? skin	 ? (within	 ? eight	 ? hours	 ? after	 ? sacrifice),	 ? secondly	 ? as	 ?refrigerated	 ?skin	 ?(after	 ?three	 ?days	 ?of	 ?storage),	 ?and	 ?thirdly	 ?as	 ?frozen	 ?skin	 ?(after	 ?twelve	 ?days	 ?of	 ? storage).	 ? The	 ? skin	 ? pieces	 ? were	 ? placed	 ? in	 ? sealed	 ? vials	 ? before	 ? being	 ? placed	 ? in	 ? the	 ?refrigerator	 ?or	 ?the	 ?-??20	 ??C	 ?freezer	 ?in	 ?order	 ?to	 ?maintain	 ?their	 ?moisture.	 ?Before	 ?injection,	 ?the	 ?skin	 ?samples	 ?were	 ?removed	 ?from	 ?the	 ?refrigerator	 ?or	 ?the	 ?freezer,	 ?and	 ?they	 ?were	 ?kept	 ?sealed	 ?until	 ?they	 ?reached	 ?room	 ?temperature.	 ?Doxorubicin	 ?(or	 ?14-??hydroxydaunorubicin)	 ?was	 ?chosen	 ?as	 ?the	 ?drug	 ?for	 ?studying	 ?the	 ?diffusion	 ?process.	 ?Doxorubicin	 ?is	 ?a	 ?chemotherapeutic	 ?drug	 ?that	 ?works	 ?by	 ?intercalating	 ?DNA	 ?molecules	 ?(binding	 ?between	 ?DNA	 ?strands)	 ?to	 ?inhibit	 ?cellular	 ?replication	 ?[122-??124].	 ?It	 ?is	 ?a	 ?water-??soluble	 ?compound	 ?and	 ?has	 ?the	 ?chemical	 ?formula	 ?of	 ?C27H29NO11	 ?and	 ?a	 ?molar	 ?mass	 ?of	 ?579.98	 ?g/mol.	 ?Doxorubicin	 ? is	 ? commonly	 ? used	 ? to	 ? treat	 ? a	 ? large	 ? variety	 ? of	 ? cancer	 ? conditions	 ? including	 ?leukemias	 ?and	 ? lymphomas,	 ?as	 ?well	 ?as	 ?cancers	 ?of	 ?breast,	 ? lung,	 ?prostate,	 ?bladder,	 ?stomach,	 ?ovaries,	 ?thyroid,	 ?soft	 ?tissue	 ?sarcoma,	 ?and	 ?others	 ?[125,	 ?126].	 ?Doxorubicin	 ?is	 ?among	 ?the	 ?most	 ?cytotoxic	 ? compounds	 ? and	 ? is	 ? associated	 ? with	 ? many	 ? adverse	 ? side	 ? effects	 ? such	 ? as	 ? skin	 ?reactions,	 ? digestive	 ? system	 ? disorders,	 ? and	 ? even	 ? fatal	 ? heart	 ? damage	 ? [125,	 ? 126].	 ? Precise	 ?delivery	 ? of	 ? the	 ? drug	 ? is	 ? thus	 ? critical	 ? to	 ? avoid	 ? over-??	 ? or	 ? under-??	 ? dosing.	 ? In	 ? some	 ? cases,	 ? it	 ? is	 ?advantageous	 ? to	 ? deliver	 ? the	 ? drug	 ? via	 ? dermal	 ? routes	 ? especially	 ? in	 ? situations	 ? that	 ? involve	 ?targeting	 ? tissues	 ? close	 ? to	 ? the	 ? skin.	 ? In	 ? addition,	 ? reaching	 ? effective	 ? doses	 ? of	 ? the	 ? drug	 ?sometimes	 ? requires	 ?multiple	 ? hypodermic	 ? injections	 ? into	 ? the	 ? blood	 ? system	 ?within	 ? a	 ? short	 ?time	 ? period,	 ? which	 ? can	 ? be	 ? very	 ? uncomfortable	 ? for	 ? patients,	 ? especially	 ? small	 ? children.	 ?Microneedles	 ? can	 ? be	 ? useful	 ? in	 ? this	 ? case	 ? since	 ? they	 ? can	 ? provide	 ? a	 ? targeted	 ? delivery	 ?73	 ?	 ?mechanism	 ?to	 ?the	 ?skin	 ?while	 ?being	 ?painless.	 ?However,	 ?in	 ?order	 ?to	 ?determine	 ?the	 ?amount	 ?of	 ?doxorubicin	 ?that	 ?reaches	 ?the	 ?target	 ?area	 ?after	 ?injection	 ?with	 ?microneedles,	 ?it	 ?is	 ?important	 ?to	 ?measure	 ? the	 ?drug?s	 ?diffusion	 ? rate	 ?within	 ? the	 ? skin.	 ?Doxorubicin	 ? is	 ?a	 ? fluorescent	 ?compound	 ?with	 ?excitation	 ?and	 ?emission	 ?peaks	 ?of	 ?480	 ?nm	 ?and	 ?580	 ?nm,	 ?respectively	 ?[127].	 ?It	 ?is	 ?therefore	 ?possible	 ? to	 ? image	 ? the	 ? drug	 ? and	 ? investigate	 ? its	 ? diffusion	 ? within	 ? tissue	 ? using	 ? fluorescence	 ?microscopy.	 ?	 ?A	 ?series	 ?of	 ?experiments	 ?was	 ?performed	 ?to	 ?measure	 ?the	 ?fluorescence	 ?intensity	 ?of	 ?the	 ?drug	 ?at	 ?different	 ? concentrations	 ? and	 ? to	 ? construct	 ? a	 ? calibration	 ? curve.	 ? For	 ? this	 ? purpose,	 ? eight	 ?calibration	 ?solutions	 ?were	 ?prepared	 ?using	 ?distilled	 ?water	 ?and	 ?doxorubicin	 ?powder	 ?(Polymed	 ?Therapeutics,	 ?Houston,	 ?TX;	 ?assayed	 ?purity	 ?>98.0%;	 ?MW	 ?579.98	 ?g/mol).	 ?The	 ?solutions	 ?were	 ?deposited	 ?on	 ?a	 ?hemocytometer	 ?gridded	 ?region	 ?(to	 ?maintain	 ?a	 ?consistent	 ?volume)	 ?and	 ?then	 ?imaged	 ? using	 ? the	 ? confocal	 ? microscope	 ? to	 ? measure	 ? fluorescence	 ? intensity.	 ? The	 ?concentrations	 ?were	 ?0,	 ?25,	 ?50,	 ?150,	 ?300,	 ?900,	 ?1500,	 ?and	 ?3000	 ??M.	 ?To	 ?prepare	 ?the	 ?solutions,	 ?first,	 ?5.2	 ?mg	 ?of	 ?doxorubicin	 ?powder	 ?was	 ?weighed	 ?and	 ?dissolved	 ?in	 ?3.00	 ?mL	 ?of	 ?distilled	 ?water	 ?to	 ?make	 ?a	 ?3000	 ??M	 ?solution.	 ?The	 ?remaining	 ?solutions	 ?were	 ?then	 ?prepared	 ?via	 ?serial	 ?dilution	 ?with	 ?the	 ?appropriate	 ?volumes	 ?of	 ?distilled	 ?water.	 ?A	 ?vortex	 ?mixer	 ?was	 ?used	 ?after	 ?each	 ?dilution	 ?to	 ?ensure	 ?adequate	 ?mixing.	 ? After	 ?measuring	 ?the	 ?intensities,	 ?it	 ?was	 ?found	 ?that	 ?the	 ?drug?s	 ?fluorescence	 ?intensity	 ?changed	 ?linearly	 ? up	 ? to	 ? concentrations	 ? of	 ? 150	 ? ?M	 ? (Figure	 ? 4.1).	 ? Injected	 ? concentrations	 ? were	 ?therefore	 ? kept	 ? within	 ? this	 ? linear	 ? range	 ? to	 ? obtain	 ? quantitative	 ? distribution	 ? data	 ? from	 ? the	 ?intensity	 ?measurements.	 ?	 ?74	 ?	 ?0 500 1000 1500 2000 2500 3000020406080100120140160Intensity [a.u.]Concentration [?M] 	 ?Figure	 ?4.1:	 ?Doxorubicin	 ?fluorescence	 ?intensity	 ?for	 ?different	 ?concentrations;	 ?the	 ?inset	 ?corresponds	 ?to	 ?the	 ?linear	 ?concentration	 ?range	 ?in	 ?the	 ?plot.	 ?For	 ?further	 ?details	 ?on	 ?the	 ?calibration	 ?procedure,	 ?refer	 ?to	 ?Appendix	 ?A.8.	 ?For	 ?the	 ?experiments	 ?described	 ? here,	 ? a	 ? concentration	 ? of	 ? 87	 ??M	 ? (0.047	 ? kg/m3,	 ? prepared	 ? through	 ? a	 ? similar	 ?procedure	 ? as	 ? above)	 ? was	 ? chosen	 ? for	 ? the	 ? initial	 ? injected	 ? formulation	 ? to	 ? ensure	 ? that	 ? the	 ?drug?s	 ?concentration	 ?is	 ?well	 ?within	 ?the	 ?linear	 ?range.	 ?	 ?4.1.2 Injection	 ?setup	 ?Different	 ? systems	 ? have	 ? been	 ? used	 ? in	 ? the	 ? past	 ? to	 ? inject	 ? compounds	 ? with	 ? microneedles.	 ?Stoeber	 ? et	 ? al.	 ? [121]	 ? proposed	 ? a	 ? microneedle	 ? syringe	 ? concept	 ? in	 ? which	 ? the	 ? needles	 ? are	 ?attached	 ? to	 ?a	 ?drug	 ?container,	 ?and	 ? the	 ?drug	 ? is	 ?ejected	 ?by	 ?pushing	 ?on	 ?a	 ? flexible	 ?membrane	 ?(Figure	 ?4.2).	 ?This	 ?setup	 ?was	 ?used	 ?by	 ?H?feli	 ?et	 ?al.	 ? [120]	 ? to	 ? inject	 ?compounds	 ? into	 ? live	 ?mice	 ?with	 ?silicon	 ?microneedle	 ?arrays.	 ?R?	 ?=	 ?0.99382	 ?0	 ?20	 ?40	 ?60	 ?80	 ?0	 ? 50	 ? 100	 ? 150	 ?Intensity	 ?[a.	 ?u.]	 ?Concentra?n	 ?[ ?M	 ?]	 ?75	 ?	 ?	 ?Figure	 ?4.2:	 ?Microneedle	 ?syringe	 ?concept	 ?presented	 ?by	 ?Stoeber	 ?et	 ?al.	 ?[121].	 ?Another	 ? alternative	 ? method	 ? is	 ? to	 ? simply	 ? glue	 ? a	 ? microneedle	 ? array	 ? to	 ? the	 ? tip	 ? of	 ? a	 ?conventional	 ?syringe,	 ?and	 ?then	 ?injecting	 ?the	 ?drug	 ?by	 ?pushing	 ?the	 ?syringe	 ?plunger,	 ?similar	 ?to	 ?the	 ?injection	 ?tests	 ?described	 ?in	 ?Chapters	 ?2	 ?and	 ?3.	 ?The	 ?disadvantage	 ?of	 ?these	 ?methods	 ?is	 ?that	 ?there	 ? is	 ? less	 ? control	 ? over	 ? the	 ? rate	 ? of	 ? drug	 ? injection.	 ? For	 ? investigating	 ? drug	 ? delivery	 ? with	 ?microneedles	 ? it	 ? is	 ? better	 ? to	 ? use	 ? a	 ?more	 ? controlled	 ? injection	 ?mechanism	 ? in	 ? order	 ? to	 ? keep	 ?track	 ?of	 ?flow	 ?rates.	 ?For	 ?the	 ?injection	 ?tests	 ?here,	 ?the	 ?setup	 ?shown	 ?in	 ?Figure	 ?4.3	 ?was	 ?used	 ?to	 ?inject	 ?doxorubicin	 ?into	 ?pig	 ?skin.	 ?	 ? 	 ?Figure	 ?4.3:	 ?a)	 ?schematics	 ?and	 ?b)	 ?actual	 ?image	 ?of	 ?injection	 ?setup	 ?used	 ?for	 ?delivering	 ?drugs	 ?at	 ?defined	 ?flow	 ?rates.	 ?The	 ?syringe	 ?is	 ?placed	 ?in	 ?a	 ?commercial	 ?syringe	 ?pump	 ?were	 ?the	 ?plunger	 ?speed	 ?is	 ?controlled.	 ?Syringe	 ? Microneedle	 ?Steel	 ?needle	 ?luer	 ?end	 ?Pump	 ? Tube	 ?a)	 ?b)	 ?76	 ?	 ?Single	 ?500	 ??m	 ?long	 ?microneedles	 ?were	 ?bonded	 ?to	 ?the	 ?luer	 ?ends	 ?of	 ?23G	 ?steel	 ?needles,	 ?with	 ?the	 ? steel	 ? needle	 ? connected	 ? to	 ? a	 ? 1	 ?ml	 ? syringe	 ? via	 ? flexible	 ? capillary	 ? tubes.	 ? After	 ? filling	 ? the	 ?syringe	 ?with	 ?drug	 ?solution,	 ? it	 ?was	 ?placed	 ?on	 ?a	 ?syringe	 ?pump	 ?(PicoPlus,	 ?Harvard	 ?Apparatus,	 ?Holliston,	 ?MA,	 ?USA).	 ?	 ?Generally,	 ?when	 ?microneedles	 ?penetrate	 ?the	 ?skin	 ?and	 ?the	 ?injection	 ?process	 ?begins,	 ?the	 ?skin	 ?tends	 ?to	 ?resist	 ?fluid	 ?flow.	 ?As	 ?a	 ?result,	 ?if	 ?the	 ?injection	 ?rate	 ?is	 ?too	 ?high,	 ?the	 ?drug	 ?may	 ?leak	 ?along	 ?the	 ?side	 ?of	 ?the	 ?needle	 ?shafts	 ?to	 ?the	 ?skin	 ?surface	 ?(Figure	 ?4.4).	 ?	 ?	 ?Figure	 ?4.4:	 ?Concept	 ?of	 ?drug	 ?backflow	 ?along	 ?outside	 ?of	 ?the	 ?needles	 ?to	 ?the	 ?skin	 ?surface.	 ?To	 ? prevent	 ? the	 ? fluid	 ? flowing	 ? along	 ? the	 ? microneedle	 ? shafts	 ? to	 ? the	 ? surface,	 ? the	 ? injection	 ?pressure	 ? has	 ? to	 ? stay	 ? below	 ? a	 ? threshold	 ? that	 ? is	 ? controlled	 ? by	 ? skin	 ? elasticity	 ? around	 ? the	 ?microneedle	 ? tips.	 ? The	 ? injection	 ? rate	 ? of	 ? 200	 ? nL/min	 ? was	 ? chosen	 ? for	 ? our	 ? experiments	 ? to	 ?ensure	 ?there	 ?was	 ?no	 ?backflow.	 ?This	 ?rate	 ?was	 ?also	 ?found	 ?to	 ?be	 ?the	 ?effective	 ?rate	 ?for	 ?delivery	 ?of	 ?compounds	 ?into	 ?animal	 ?skin	 ?in	 ?vivo,	 ?using	 ?microneedles	 ?[120].	 ?A	 ?custom	 ?skin	 ?holder,	 ?made	 ?from	 ?the	 ?cap	 ?of	 ?a	 ?polypropylene	 ?microcentrifuge	 ?vial,	 ?keeps	 ?the	 ?skin	 ? in	 ? tension	 ? during	 ? the	 ? injection	 ? and	 ? the	 ? imaging	 ? process	 ? in	 ? order	 ? to	 ? maximize	 ?microneedle	 ?penetration	 ?depth	 ?into	 ?the	 ?skin	 ?(Figure	 ?4.5).	 ?	 ?	 ?Microneedle	 ?array	 ?Drug	 ?injection	 ?Skin	 ?77	 ?	 ?	 ?	 ?	 ?Figure	 ?4.5:	 ?Schematic	 ?of	 ?setup	 ?used	 ?to	 ?stretch	 ?skin	 ?and	 ?keep	 ?it	 ?moist	 ?during	 ?injection	 ?and	 ?imaging	 ?processes.	 ?After	 ?mounting	 ? the	 ?skin	 ?on	 ? the	 ?holder	 ?and	 ? injecting	 ? the	 ?drug,	 ? the	 ?holder	 ?was	 ? flipped	 ?and	 ?placed	 ?on	 ? the	 ? confocal	 ?microscope?s	 ? inverted	 ? stage.	 ?Water	 ?was	 ? added	 ? to	 ? the	 ?backside	 ? in	 ?order	 ?to	 ?keep	 ?the	 ?skin	 ?as	 ?moist	 ?as	 ?possible	 ?during	 ?imaging	 ?(Figure	 ?4.5).	 ?4.1.3 Injection	 ?procedure	 ?After	 ? preparing	 ? the	 ? solution	 ? and	 ? setting	 ? up	 ? the	 ? syringe	 ? pump	 ? and	 ? the	 ? skin,	 ? the	 ? injection	 ?process	 ? was	 ? carried	 ? out	 ? three	 ? times	 ? for	 ? each	 ? skin	 ? storage	 ? condition.	 ? For	 ? each	 ? trial,	 ? the	 ?microneedle	 ?was	 ?pressed	 ?against	 ? the	 ? skin	 ?with	 ?a	 ? force	 ?of	 ? approximately	 ?3	 ?N.	 ?The	 ? syringe	 ?pump	 ? was	 ? then	 ? set	 ? to	 ? run	 ? for	 ? 5	 ?min	 ? (~2	 ? min	 ? lag	 ? time	 ? before	 ? liquid	 ? came	 ? out	 ? of	 ? the	 ?microneedle,	 ?due	 ?to	 ?compliance	 ?of	 ? flexible	 ?tubing).	 ?The	 ?needles	 ?were	 ?then	 ?removed	 ?from	 ?the	 ? skin	 ? surface	 ? and	 ? the	 ? skin	 ? was	 ? transferred	 ? to	 ? a	 ? confocal	 ? microscope	 ? for	 ? imaging.	 ?Transferring	 ? the	 ? skin	 ? to	 ? the	 ? microscope,	 ? finding	 ? the	 ? injection	 ? spot,	 ? and	 ? adjusting	 ? the	 ?confocal	 ?range	 ?took	 ?between	 ?5	 ?-??	 ?12	 ?min.	 ?	 ?Water	 ?bath	 ?Skin	 ?support	 ?made	 ?from	 ?plastic	 ?vial	 ?caps	 ?Stretched	 ?skin	 ?Pin	 ? used	 ? to	 ? stretch	 ? and	 ?hold	 ?skin	 ?Through	 ?holes	 ?used	 ?to	 ?keep	 ?skin	 ?the	 ?skin	 ?moist	 ?78	 ?	 ?4.1.4 Confocal	 ?imaging	 ?The	 ?confocal	 ? images	 ?were	 ?obtained	 ?using	 ?a	 ?TCS	 ?SP5	 ?system	 ?(Leica	 ?Microsystems,	 ?Wetzlar,	 ?Germany).	 ?An	 ?argon	 ?laser	 ?source	 ?was	 ?used	 ?for	 ?excitation.	 ?The	 ?excitation	 ?wavelength	 ?was	 ?set	 ?at	 ?488	 ?nm	 ?at	 ?50%	 ?illumination	 ?intensity.	 ?A	 ?HyD1	 ?detector	 ?was	 ?used	 ?to	 ?collect	 ?fluorescence	 ?in	 ?the	 ?wavelength	 ?range	 ?of	 ?535-??625	 ?nm.	 ?The	 ?system?s	 ?smart	 ?gain	 ?was	 ?adjusted	 ?to	 ?280%	 ?to	 ?enhance	 ?the	 ?fluorescence	 ?brightness,	 ?and	 ?the	 ?pinhole	 ?size	 ?was	 ?set	 ?to	 ?60	 ??m.	 ?The	 ?confocal	 ?thickness	 ?was	 ?set	 ?at	 ?9.5	 ??m	 ?and	 ?the	 ?X-??Y	 ? image	 ?size	 ?was	 ?512?512	 ?pixels	 ? (corresponding	 ?to	 ?775	 ??m	 ??	 ?775	 ??m	 ?physical	 ?image	 ?size).	 ?4.2 Data	 ?analysis	 ?4.2.1 Diffusion	 ?model	 ?The	 ?steady-??state	 ?diffusion	 ?flux	 ? J = !D"C 	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?(4)	 ?can	 ?be	 ?described	 ?by	 ?Fick?s	 ?first	 ?law	 ?[128],	 ?where	 ? ? 	 ?is	 ?the	 ?concentration	 ?gradient	 ?in	 ?three-??dimensional	 ?space.	 ?The	 ?diffusion	 ?coefficient	 ??	 ?depends	 ?on	 ?the	 ?drug	 ?properties	 ?(i.e.,	 ?MW,	 ??)	 ?as	 ?well	 ? as	 ? the	 ?properties	 ?of	 ? the	 ? skin	 ? (i.e.,	 ? viscosity,	 ?porosity,	 ??).	 ? The	 ?Fick`s	 ? second	 ? law	 ?of	 ?diffusion	 ?	 ?2C D Ct?= ??	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?(5)	 ?79	 ?	 ?describes	 ?how	 ?the	 ?diffusion	 ?causes	 ?the	 ?concentration	 ?to	 ?change	 ?with	 ?time	 ?[128].	 ?Solving	 ?the	 ?differential	 ? equation	 ? (5)	 ? gives	 ? the	 ? spatial	 ? concentration	 ? distribution	 ? over	 ? time.	 ? The	 ? initial	 ?boundary	 ?condition	 ?describing	 ?the	 ?source	 ?can	 ?be	 ?continuous	 ?or	 ?limited,	 ?each	 ?resulting	 ?in	 ?a	 ?different	 ? solution.	 ? In	 ? addition,	 ? sometimes	 ? it	 ? is	 ? simpler	 ? to	 ? solve	 ? this	 ? equation	 ? in	 ? spherical	 ?coordinates	 ? rather	 ? than	 ? Cartesian.	 ? This	 ? is	 ? especially	 ? useful	 ? when	 ? the	 ? initial	 ? boundary	 ?condition	 ?is	 ?a	 ?point	 ?source	 ?or	 ?spherical	 ?source	 ?that	 ?spreads	 ?symmetrically	 ?in	 ?all	 ?directions.	 ?The	 ? solution	 ? to	 ? equations	 ? (4)	 ? and	 ? (5)	 ? in	 ? spherical	 ? coordinates	 ? for	 ? a	 ? limited	 ? point-??source	 ?deposited	 ?at	 ?time	 ?zero	 ?at	 ?the	 ?origin	 ?is	 ?given	 ?by	 ?the	 ?Gaussian	 ?distribution	 ?[129]	 ?	 ?	 ?2032( , ) exp( )4(4 )N rC r tDtDt?= ? ,	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?(6)	 ?where	 ???	 ?is	 ?the	 ?initial	 ?mass.	 ?Equation	 ?(6)	 ?assumes	 ?isotropic	 ?diffusion	 ?from	 ?the	 ?source	 ?with	 ?the	 ? same	 ? D	 ? in	 ? all	 ? directions.	 ? This	 ? equation	 ? describes	 ? how	 ? concentration	 ? is	 ? distributed	 ? in	 ?space,	 ? at	 ? different	 ? distances	 ? ?	 ? from	 ? the	 ? source.	 ? In	 ? addition,	 ? it	 ? describes	 ? how	 ? the	 ? spatial	 ?concentration	 ? distribution	 ? changes	 ? over	 ? time.	 ? The	 ? negative	 ? sign	 ? of	 ? the	 ? exponential	 ? term	 ?indicates	 ? how	 ? the	 ? concentration	 ? drops	 ? with	 ? distance.	 ? As	 ? the	 ? value	 ? of	 ? ? 	 ? increases	 ? over	 ?time,	 ? the	 ? term	 ? before	 ? the	 ? exponential	 ? decreases	 ? indicating	 ? a	 ? depleting	 ? source.	 ? For	 ? a	 ?continuous	 ? source,	 ? this	 ? term	 ? is	 ? a	 ? constant	 ???	 ?which	 ? does	 ? not	 ? change	 ? over	 ? time	 ? and	 ? the	 ?exponential	 ?function	 ?is	 ?replaced	 ?by	 ?the	 ?complementary	 ?error	 ?function.	 ?	 ?	 ?	 ?80	 ?	 ?The	 ?diffusion	 ?process	 ?for	 ?injection	 ?with	 ?microneedles	 ?can	 ?be	 ?described	 ?in	 ?two	 ?steps:	 ?1. A	 ?constant-??source	 ?diffusion	 ?step,	 ?while	 ?the	 ?microneedle	 ?is	 ?inserted	 ?into	 ?the	 ?skin	 ?and	 ?the	 ?drug	 ?is	 ?ejected.	 ?2. A	 ? limited-??source	 ?diffusion	 ?step,	 ?which	 ?starts	 ?upon	 ?removal	 ?of	 ? the	 ?needle	 ? from	 ?the	 ?skin.	 ?	 ?It	 ? is	 ?difficult	 ?to	 ?image	 ?the	 ?skin	 ?during	 ?the	 ?first	 ?step	 ?since	 ?the	 ?microneedle	 ?would	 ?block	 ?the	 ?optical	 ?path.	 ?The	 ?confocal	 ? imaging	 ? is,	 ? therefore,	 ?done	 ?only	 ?during	 ?the	 ?second	 ?step,	 ?where	 ?the	 ? diffusion	 ? process	 ? can	 ? be	 ? modeled	 ? as	 ? a	 ? spherical	 ? source,	 ? positioned	 ? below	 ? the	 ?microneedle	 ?insertion	 ?spot,	 ?spreading	 ?in	 ?all	 ?directions	 ?(Figure	 ?4.6).	 ?Equation	 ?(6)	 ?can	 ?then	 ?be	 ?used	 ?to	 ?describe	 ?the	 ?diffusion.	 ?Here	 ?the	 ?injection	 ?step	 ?is	 ?much	 ?shorter	 ?than	 ?the	 ?duration	 ?of	 ?the	 ?diffusion	 ?experiments.	 ?We	 ?therefore	 ?assume	 ?the	 ?injection	 ?step	 ?is	 ?associated	 ?with	 ?only	 ?a	 ?small	 ? amount	 ?of	 ?diffusion,	 ? so	 ? that	 ? the	 ?measured	 ?diffusion	 ?can	 ?be	 ?modeled	 ?with	 ?equation	 ?(6).	 ?	 ?	 ?	 ?Figure	 ?4.6:	 ?Concept	 ?of	 ?diffusion	 ?of	 ?drugs,	 ?injected	 ?with	 ?microneedles,	 ?through	 ?the	 ?epidermis.	 ?Equation	 ? (6)	 ? also	 ? assumes	 ? a	 ? constant	 ? D	 ? independent	 ? of	 ? direction	 ? of	 ? diffusion.	 ? In	 ? reality,	 ?since	 ?the	 ?density	 ?of	 ?skin	 ?layers	 ?changes	 ?with	 ?depth,	 ?the	 ?value	 ?of	 ?D	 ?also	 ?changes	 ?with	 ?depth.	 ?In	 ? addition,	 ? upon	 ? application	 ? of	 ? pressure	 ? from	 ? the	 ? injection	 ? process,	 ? the	 ? skin	 ? tissue	 ? gets	 ?Epidermis	 ?Microneedle	 ?insertion	 ?spot	 ?Dermis	 ?SC	 ?81	 ?	 ?squeezed	 ? which	 ?might	 ? result	 ? in	 ? a	 ? variation	 ? in	 ? D	 ? near	 ? the	 ? needle	 ? surface.	 ? However,	 ? D	 ? is	 ?expected	 ? to	 ? stay	 ? constant	 ? when	 ? spreading	 ? in	 ? planar	 ? direction	 ? at	 ? each	 ? depth.	 ? Confocal	 ?microscopy	 ? gives	 ?planar	 ? images;	 ? therefore,	 ? since	 ? the	 ? analysis	 ? is	 ? always	 ?done	 ? in	 ? the	 ? same	 ?depth,	 ?assumption	 ?of	 ?constant	 ?D	 ?is	 ?justified.	 ?4.2.2 Data	 ?processing	 ?In	 ?order	 ?to	 ?use	 ?equation	 ?(6)	 ?to	 ?describe	 ?the	 ?diffusion,	 ?the	 ?concentration	 ?distribution	 ?of	 ?the	 ?drug	 ?must	 ? be	 ? first	 ? obtained	 ? from	 ? the	 ? intensity	 ? data	 ? for	 ? the	 ? depth	 ? corresponding	 ? to	 ? the	 ?spherical	 ? source	 ? illustrated	 ? in	 ? Figure	 ? 4.6.	 ? For	 ? spherical	 ? diffusion,	 ? the	 ? concentration	 ? or	 ?intensity	 ? distribution	 ? should	 ? normally	 ? be	 ? the	 ? same	 ? in	 ? all	 ? directions	 ? originating	 ? from	 ? the	 ?source	 ? at	 ? each	 ? depth	 ? plane.	 ?However,	 ? in	 ? this	 ? case,	 ? the	 ? distribution	 ?may	 ? not	 ? be	 ? perfectly	 ?symmetrical	 ?due	 ?to	 ?the	 ?natural	 ?non-??uniformity	 ?of	 ?the	 ?skin`s	 ?cellular	 ?matrix	 ?which	 ?might	 ?lead	 ?to	 ?non-??uniform	 ?spreading	 ?of	 ? the	 ?drug.	 ?The	 ?data	 ? should	 ? thus	 ?be	 ?averaged.	 ?To	 ?do	 ? this,	 ? the	 ?total	 ?intensity	 ?is	 ?first	 ?obtained	 ?for	 ?narrow	 ?hypothetical	 ?rings	 ?spreading	 ?from	 ?the	 ?source,	 ?and	 ?then	 ? divided	 ? by	 ? the	 ? number	 ? of	 ? pixels	 ? in	 ? the	 ? rings	 ? to	 ? get	 ? the	 ? average	 ? intensity	 ? for	 ? each	 ?individual	 ? ring	 ? (Figure	 ?4.7).	 ? In	 ?order	 ? to	 ?measure	 ? the	 ? intensities	 ?with	 ? the	 ?process	 ?shown	 ? in	 ?Figure	 ?4.7,	 ?the	 ?MATLAB	 ?image	 ?processing	 ?toolbox	 ?was	 ?used.	 ?The	 ?code	 ?used	 ?to	 ?calculate	 ?the	 ?average	 ?values	 ?in	 ?rings	 ?spreading	 ?from	 ?the	 ?source	 ?is	 ?shown	 ?in	 ?Appendix	 ?A.9.	 ?	 ?	 ?	 ?82	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?Figure	 ?4.7:	 ?Calculating	 ?concentration	 ?distribution	 ?from	 ?confocal	 ?images,	 ?by	 ?measuring	 ?the	 ?average	 ?intensity	 ?within	 ?hypothetical	 ?thin	 ?rings	 ?(with	 ?thickness	 ?of	 ?10	 ?pixels)	 ?at	 ?different	 ?distances	 ?originating	 ?from	 ?drug	 ?source.	 ?4.3 Results	 ?and	 ?discussions	 ?4.3.1 Confocal	 ?data	 ?Figure	 ?4.8	 ?shows	 ?an	 ?example	 ?of	 ?a	 ?series	 ?of	 ?confocal	 ?images	 ?obtained	 ?for	 ?a	 ?fresh	 ?skin	 ?sample	 ?injected	 ?with	 ?the	 ?doxorubicin	 ?solution.	 ?In	 ?this	 ?image,	 ?the	 ?trace	 ?of	 ?the	 ?drug	 ?can	 ?be	 ?observed	 ?down	 ?to	 ?a	 ?depth	 ?of	 ?236	 ??m	 ?under	 ?the	 ?skin	 ?surface.	 ?The	 ?change	 ?in	 ? intensity	 ?of	 ?the	 ?drug	 ?at	 ?each	 ? depth	 ? indicates	 ? drug	 ? diffusion	 ? over	 ? time.	 ? The	 ? hole	 ? created	 ? by	 ? the	 ? microneedle	 ?insertion	 ?can	 ?be	 ?observed	 ? in	 ?the	 ? image	 ?series	 ?corresponding	 ?to	 ?the	 ?surface	 ?and	 ?depths	 ?of	 ?47	 ??m	 ?and	 ?95	 ??m.	 ?The	 ?high	 ?brightness	 ?of	 ?the	 ?skin	 ?surface	 ?is	 ?due	 ?to	 ?the	 ?exposure	 ?of	 ?the	 ?skin	 ?to	 ? some	 ? doxorubicin	 ? that	 ? was	 ? dried	 ? on	 ? the	 ? microneedle	 ? array`s	 ? outside	 ? surface	 ? before	 ?insertion.	 ?In	 ?addition,	 ?through	 ?a	 ?control	 ?test,	 ?it	 ?was	 ?verified	 ?that	 ?there	 ?is	 ?no	 ?drug	 ?diffusion	 ?through	 ?the	 ?SC	 ?when	 ?the	 ?drug	 ?was	 ?applied	 ?to	 ?the	 ?skin	 ?surface.	 ?	 ?	 ?	 ?R2	 ? R3	 ?R1	 ?R1	 ? R2	 ? R3	 ?Average	 ?intensity	 ?in	 ?ring	 ?	 ?	 ?Distance	 ?from	 ?source?s	 ?origin	 ?83	 ?	 ?	 ?	 ?Figure	 ?4.8:	 ?Confocal	 ?images	 ?obtained	 ?for	 ?a	 ?fresh	 ?skin	 ?sample	 ?injected	 ?with	 ?87	 ??M	 ?doxorubicin	 ?solution.	 ?The	 ?images	 ?are	 ?775	 ??m	 ??	 ?775	 ??m.	 ?The	 ?rows	 ?correspond	 ?to	 ?different	 ?scan	 ?depths	 ?while	 ?the	 ?columns	 ?correspond	 ?to	 ?the	 ?scans	 ?over	 ?time.	 ?The	 ?time	 ?difference	 ?between	 ?two	 ?columns	 ?is	 ?5	 ?minutes.	 ?The	 ? first	 ?column	 ?on	 ?the	 ? left,	 ?which	 ?correspond	 ?to	 ? the	 ?start	 ?of	 ? the	 ? imaging	 ?procedure	 ? (i.e.	 ?t1),	 ?was	 ?taken	 ?almost	 ?12	 ?min	 ?after	 ?the	 ?injection	 ?procedure	 ?stopped,	 ?which	 ?is	 ?the	 ?amount	 ?of	 ?time	 ? it	 ? took	 ? to	 ? take	 ? the	 ? sample	 ? to	 ? the	 ?microscope	 ? and	 ? prepare	 ? it	 ? for	 ? scanning.	 ? The	 ? row	 ?corresponding	 ?to	 ?the	 ?depth	 ?of	 ?142	 ??m	 ?is	 ?the	 ?depth	 ?corresponding	 ?to	 ?the	 ?spherical	 ?source	 ?of	 ?drug	 ?below	 ?the	 ?injection	 ?spot;	 ?this	 ?depth	 ?corresponds	 ?to	 ?the	 ?deeper	 ?epidermal	 ?layers	 ?or	 ?the	 ?47	 ??m	 ?95	 ??m	 ?Surface	 ?142	 ??m	 ?189	 ??m	 ?236	 ??m	 ?t1	 ? t1+5	 ?min	 ? t1+10	 ?min	 ? t1+15	 ?min	 ? t1+20	 ?min	 ? t1+25	 ?min	 ? t1+30	 ?min	 ?84	 ?	 ?upper	 ?dermis	 ?layers	 ?which	 ?is	 ?a	 ?suitable	 ?depth	 ?for	 ?releasing	 ?the	 ?drug	 ?since	 ?it	 ?is	 ?close	 ?to	 ?the	 ?target	 ?blood	 ?vessels.	 ?Figure	 ?4.9	 ?shows	 ?the	 ?cross-??section	 ?view	 ?that	 ?has	 ?been	 ?reconstructed	 ?from	 ? the	 ? confocal	 ? image	 ? in	 ? Figure	 ? 4.8,	 ? and	 ? the	 ? 142	 ??m	 ? depth	 ? images	 ? correspond	 ? to	 ? the	 ?yellow	 ?dashed	 ?line	 ?in	 ?the	 ?figure.	 ?	 ?Figure	 ?4.9:	 ?Cross-??section	 ?of	 ?the	 ?injection	 ?location,	 ?reconstructed	 ?from	 ?confocal	 ?image	 ?shown	 ?in	 ?Figure	 ?4.8,	 ?corresponding	 ?to	 ?t1.	 ?4.3.2 Diffusion	 ?measurements	 ?The	 ? resulting	 ? intensity	 ? distribution	 ? for	 ? all	 ? time	 ? steps,	 ? obtained	 ? through	 ? the	 ? procedure	 ?detailed	 ? in	 ? section	 ? 4.2.2,	 ? for	 ? the	 ? experiment	 ? corresponding	 ? to	 ? Figure	 ? 4.8	 ? is	 ? shown	 ? in	 ?Figure	 ?4.10.	 ? In	 ? this	 ?plot,	 ? the	 ?origin	 ?of	 ? the	 ?x-??axis	 ?corresponds	 ? to	 ? the	 ?origin	 ?of	 ? the	 ?spherical	 ?source.	 ?As	 ?expected	 ?from	 ?equation	 ?(6),	 ?for	 ?each	 ?time	 ?step,	 ?the	 ?intensity	 ?drops	 ?exponentially	 ?moving	 ? away	 ? from	 ? the	 ? source.	 ? As	 ? time	 ? passes,	 ? the	 ? intensity	 ? levels	 ? drop.	 ? The	 ? confocal	 ?images	 ? include	 ? a	 ? low	 ? level	 ? of	 ? noise	 ?with	 ? a	 ? standard	 ? deviation	 ? of	 ? the	 ?measured	 ? intensity	 ?around	 ? 5.8	 ? in	 ? regions	 ? of	 ? zero	 ? fluorescence. Figure	 ? 4.11	 ? shows	 ? the	 ? corresponding	 ?concentration	 ? distribution	 ? obtained	 ? from	 ? Figure	 ? 4.10.	 ? The	 ? concentration	 ? distributions	 ? for	 ?limited	 ? source	 ?diffusion	 ? in	 ?3D	 ?spherical	 ? coordinates	 ? look	 ?different	 ? from	 ?the	 ?more	 ? familiar	 ?concentration	 ? distribution	 ? for	 ? linear	 ? diffusion.	 ?In	 ? 3D	 ? diffusion,	 ? the	 ? curves	 ? don?t	 ? intersect	 ?while	 ?in	 ?the	 ?case	 ?of	 ?linear	 ?diffusion,	 ?the	 ?curves	 ?intersect. 100	 ??m	 ? Diffusion	 ?source	 ?142	 ??m	 ?depth	 ?Skin	 ?surface	 ?85	 ?	 ?The	 ?calibration	 ?curve	 ?in	 ?Figure	 ?4.1	 ?is	 ?found	 ?using	 ?a	 ?confocal	 ?setting	 ?different	 ?from	 ?what	 ?was	 ?used	 ?to	 ?detect	 ?the	 ?drug	 ?distribution	 ?in	 ?the	 ?skin	 ?in	 ?Figure	 ?4.8.	 ?The	 ?detected	 ?intensity	 ?level	 ?in	 ?the	 ?skin	 ?attenuates	 ?due	 ? to	 ? the	 ? light	 ?absorption	 ?and	 ?scattering	 ?effects,	 ?and	 ? is	 ? thus	 ?weaker	 ?than	 ? the	 ? intensity	 ? levels	 ? for	 ? the	 ? pure	 ? solution	 ? on	 ? the	 ? hemocytometer	 ? slide	 ? for	 ? the	 ? same	 ?confocal	 ?setting.	 ?The	 ?concentrations	 ?values	 ?in	 ?Figure	 ?4.11	 ?were	 ?estimated	 ?from	 ?the	 ?highest	 ?intensity	 ? region	 ? for	 ? the	 ? image	 ?with	 ? the	 ? smallest	 ? t1;	 ? the	 ? corresponding	 ? concentration	 ?was	 ?assumed	 ?to	 ?be	 ?equal	 ?to	 ?the	 ?initial	 ?injected	 ?concentration	 ?(see	 ?Appendix	 ?A.8	 ?for	 ?details).	 ?	 ?0 50 100 150 200051015202530354045	 ?t1	 ?t1 	 ?+ 	 ?5 	 ?m in	 ?t1 	 ?+ 	 ?10	 ?m in	 ?t1 	 ?+ 	 ?15	 ?m in	 ?t1 	 ?+ 	 ?20	 ?m in	 ?t1 	 ?+ 	 ?25	 ?m in	 ?t1 	 ?+ 	 ?30	 ?m inIntensity [a.u.]Distance [?m] 	 ?Figure	 ?4.10:	 ?Intensity	 ?distribution	 ?for	 ?the	 ?confocal	 ?images	 ?corresponding	 ?to	 ?the	 ?depth	 ?142	 ??m	 ?in	 ?Figures	 ?4.8	 ?and	 ?4.9,	 ?and	 ?obtained	 ?through	 ?the	 ?method	 ?illustrated	 ?in	 ?Figure	 ?4.7.	 ?Equation	 ?(6)	 ?can	 ?be	 ?used	 ?as	 ?the	 ?theoretical	 ?model	 ?to	 ?describe	 ?the	 ?change	 ?in	 ?concentrations	 ?in	 ? Figure	 ? 4.11.	 ? Using	 ? the	 ? MATLAB	 ? curve	 ? fitting	 ? toolbox,	 ? a	 ? Gaussian	 ? function	 ? describing	 ?diffusion	 ?is	 ?fitted	 ?to	 ?the	 ?data	 ?in	 ?Figure	 ?4.11.	 ?	 ?86	 ?	 ?The	 ? fit	 ?parameters	 ? then	 ?yield	 ? the	 ?diffusion	 ?coefficient	 ?and	 ? initial	 ?mass.	 ?The	 ?value	 ? for	 ?D	 ? in	 ?equation	 ?(6)	 ?can	 ?be	 ?obtained	 ?by	 ?comparing	 ?the	 ?exponential	 ?terms.	 ?Here,	 ?comparing	 ?the	 ?t1	 ?and	 ?t1	 ?+	 ?5	 ?min	 ?curves	 ?gives	 ?a	 ?value	 ?of	 ?5.86	 ??	 ?10-??9	 ?cm2/s	 ?for	 ?D	 ?while	 ?t1	 ?and	 ?t1	 ?+	 ?10	 ?min	 ?curves	 ?gives	 ? 8.23	 ? ?	 ? 10-??9	 ? cm2/s.	 ? However,	 ? for	 ? comparing	 ? the	 ? different	 ? skin	 ? types	 ? here,	 ? only	 ? the	 ?values	 ?of	 ?D	 ?for	 ?t1	 ?and	 ?t1	 ?+	 ?5	 ?min	 ?are	 ?used.	 ?This	 ?is	 ?because	 ?as	 ?time	 ?passes	 ?the	 ?skin	 ?properties	 ?such	 ? as	 ?moisture	 ? content	 ?might	 ? change	 ? due	 ? to	 ? the	 ? exposure	 ? to	 ? the	 ? heat	 ? of	 ? the	 ? confocal	 ?laser.	 ? Therefore	 ?using	 ? the	 ? initial	 ? time	 ? steps	 ? gives	 ? the	 ?most	 ? realistic	 ? representation	 ?of	 ? the	 ?diffusion	 ? value.	 ?Using	 ? this	 ? technique,	 ? the	 ? value	 ? for	 ?D	 ?was	 ? obtained	 ? for	 ? each	 ? trial	 ? and	 ? for	 ?each	 ?skin	 ?storage	 ?condition	 ?(see	 ?Appendix	 ?A.10	 ?for	 ?the	 ?confocal	 ?images	 ?for	 ?all	 ?trials).	 ?0.0 5.0x10-5 1.0x10-4 1.5x10-4 2.0x10-40.0000.0020.0040.0060.0080.0100.0120.0140.0160.018Concentration [kg/m3 ]Distance [m]t1	 ?t1	 ? t+ t5tmint1	 ?t+ t	 ?0tmint1	 ?t+ t	 ?5tmint1	 ?t+ t20tmint1	 ?t+ t25tmint1	 ?t+ t30tmin	 ?Figure	 ?4.11:	 ?Doxorubicin	 ?concentration	 ?distribution	 ?in	 ?skin	 ?obtained	 ?from	 ?Figure	 ?4.10.	 ?The	 ?theoretical	 ?fitted	 ?curves	 ?(i.e.	 ?Gaussian	 ?diffusion,	 ?eq.	 ?(6))	 ?are	 ?included	 ?as	 ?red	 ?lines.	 ?Table	 ?4.1	 ?summarizes	 ?the	 ?diffusion	 ?coefficients	 ?obtained	 ?for	 ?the	 ?different	 ?skin	 ?types.	 ?	 ?87	 ?	 ?Table	 ?4.1:	 ?Doxorubicin	 ?diffusion	 ?coefficient	 ?in	 ?epidermal	 ?tissue	 ?with	 ?units	 ?of	 ?[cm2/s]	 ?Skin	 ?type	 ? Trial	 ?1	 ?	 ? Trial	 ?2	 ?	 ? Trial	 ?3	 ?	 ? Average	 ? Standard	 ?Deviation	 ? Coefficient	 ?of	 ?Variation	 ?Fresh	 ? 5.82?10-??9	 ? 5.86?10-??9	 ? 2.16?10-??9	 ? 4.61?10-??9	 ? 2.12?10-??9	 ? 0.461	 ?Refrigerated	 ?(3	 ?Days)	 ? 6.02?10-??9	 ? 1.07?10-??8	 ? 2.25?10-??8	 ? 1.31?10-??8	 ? 8.47?10-??9	 ? 0.648	 ?Frozen	 ?(12	 ?Days)	 ? 1.44?10-??8	 ? 9.10?10-??8	 ? 2.09?10-??8	 ? 4.21?10-??8	 ? 4.24?10-??8	 ? 1.008	 ?The	 ?diffusion	 ?coefficient	 ?values	 ?in	 ?Table	 ?4.1	 ?are	 ?plotted	 ?in	 ?Figure	 ?4.12.	 ?	 ?	 ?Figure	 ?4.12:	 ?Doxorubicin	 ?diffusion	 ?coefficient	 ?for	 ?different	 ?skin	 ?types	 ?(fresh,	 ?refrigerated	 ?for	 ?3	 ?days,	 ?frozen	 ?for	 ?12	 ?days).	 ?Two	 ?of	 ?the	 ?data	 ?points	 ?for	 ?fresh	 ?skin	 ?overlap.	 ?Here,	 ? an	 ? increasing	 ? trend	 ? in	 ? D	 ? can	 ? be	 ? observed	 ? from	 ? the	 ? fresh	 ? skin	 ? to	 ? the	 ? frozen	 ? skin,	 ?indicating	 ? that	 ? the	 ? freezing	 ? and	 ? thawing	 ?process	 ? can	 ?have	 ?a	 ? significant	 ? impact	 ?on	 ?D.	 ? This	 ?observation	 ? is	 ? consistent	 ? with	 ? another	 ? study	 ? that	 ? showed	 ? how	 ? permeability	 ? of	 ? skin	 ? to	 ?sodium	 ? ion	 ?was	 ? slightly	 ? higher	 ? for	 ? previously	 ? frozen	 ? skin	 ? compared	 ? to	 ? fresh	 ? skin	 ? [130].	 ?A	 ?similar	 ?observation	 ?was	 ?reported	 ?by	 ?another	 ? investigator	 ?who	 ?compared	 ?the	 ?permeability	 ?88	 ?	 ?of	 ?chromone	 ?acid	 ?in	 ?fresh	 ?cadaver	 ?skin	 ?to	 ?that	 ?of	 ?frozen	 ?skin	 ?[131].	 ?A	 ?possible	 ?reason	 ?for	 ?the	 ?change	 ? in	 ?D	 ? could	 ? be	 ? a	 ? change	 ? in	 ? the	 ? skin?s	 ?water	 ? content	 ? as	 ? a	 ? result	 ? of	 ? freezing,	 ?which	 ?could	 ? influence	 ? drug	 ? diffusion	 ? through	 ? the	 ? cellular	 ? matrix.	 ? Another	 ? reason	 ? could	 ? be	 ? the	 ?formation	 ?of	 ?ice	 ?crystals	 ?during	 ?freezing	 ?in	 ?the	 ?cells,	 ?which	 ?damages	 ?the	 ?skin	 ?such	 ?that	 ?after	 ?thawing	 ?gaps	 ?and	 ?holes	 ? remain	 ?within	 ? the	 ? skin.	 ?To	 ?prevent	 ? that,	 ?a	 ?much	 ?more	 ?controlled	 ?freezing	 ?could	 ?be	 ?used.	 ?The	 ?difference	 ?between	 ?the	 ?refrigerated	 ?skin	 ?and	 ?the	 ?fresh	 ?skin	 ? is	 ?not	 ?large;	 ?however,	 ?the	 ?slight	 ?difference	 ?could	 ?be	 ?due	 ?to	 ?the	 ?skin?s	 ?partial	 ?water	 ?loss	 ?when	 ?stored	 ?in	 ?the	 ?refrigerator.	 ?The	 ?value	 ?for	 ?D	 ?for	 ?doxorubicin	 ?has	 ?been	 ?previously	 ?obtained	 ?in	 ?different	 ? media	 ? both	 ? analytically	 ? and	 ? experimentally.	 ? Table	 ? 4.2	 ? summarizes	 ? the	 ? values	 ?reported	 ?in	 ?the	 ?literature.	 ?	 ?Table	 ?4.2:	 ?Comparison	 ?of	 ?doxorubicin	 ?diffusion	 ?coefficient	 ?in	 ?different	 ?media	 ?Medium	 ? Diffusion	 ?Coefficient	 ?[cm2/s]	 ? Method	 ? Source	 ?PBS	 ? 2.96?10-??5	 ? Analytical	 ? [132]	 ?Mouse	 ?liver	 ?tumor	 ? 5.01?10-??7	 ? Experimental	 ? [133]	 ?Normal	 ?mouse	 ?liver	 ? 6.70?10-??7	 ? Experimental	 ? [134]	 ?macroscopic	 ?diffusion	 ?coefficient	 ?in	 ?interstitial	 ?network	 ? 1.20?10-??8	 ? Analytical	 ? [135]	 ?Pig	 ?belly	 ?skin	 ?(fresh)	 ? 4.61?10-??9	 ? Experimental	 ? this	 ?work	 ?macroscopic	 ?diffusion	 ?coefficient	 ?in	 ?cellular	 ?network	 ? 2.22?10-??9	 ? Analytical	 ? [135]	 ?MDA-??468	 ?cell	 ?nuclei	 ? 2.70?10-??10	 ? Experimental	 ? [135]	 ?89	 ?	 ?According	 ?to	 ?Table	 ?4.2,	 ?the	 ?value	 ?measured	 ?for	 ?doxorubicin	 ?diffusion	 ?in	 ?pig	 ?skin	 ?is	 ?between	 ?the	 ?macroscopic	 ?diffusion	 ?coefficients	 ?obtained	 ?for	 ?an	 ? interstitial	 ?network	 ?and	 ?the	 ?cellular	 ?network.	 ? As	 ? the	 ? skin?s	 ? epidermis	 ? is	 ? a	 ? relatively	 ? dry	 ? medium	 ? with	 ? a	 ? limited	 ? amount	 ? of	 ?interstitial	 ?fluid,	 ?it	 ?is	 ?expected	 ?that	 ?the	 ?measured	 ?value	 ?of	 ?D	 ?for	 ?skin	 ?would	 ?be	 ?closer	 ?to	 ?that	 ?of	 ?cellular	 ?network.	 ?The	 ?value	 ?of	 ?D	 ?for	 ?some	 ?other	 ?compounds	 ?in	 ?the	 ?skin	 ?has	 ?been	 ?obtained	 ?from	 ?diffusion	 ?cell	 ?experiments.	 ? One	 ? work	 ?measured	 ? 7.5?10-??8	 ?cm2/s	 ? for	 ? glucose	 ? diffusion	 ? in	 ? epidermis	 ? [31],	 ?which	 ?is	 ?larger	 ?than	 ?the	 ?value	 ?obtained	 ?here	 ?for	 ?doxorubicin.	 ?Although	 ?glucose	 ?has	 ?a	 ?smaller	 ?diffusion	 ? coefficient	 ? in	 ? PBS	 ? (6.8?10-??6	 ?cm2/s,	 ? obtained	 ? from	 ? the	 ? Einstein-??Stokes	 ? equation	 ?[110])	 ? its	 ?faster	 ?diffusion	 ?(i.e.	 ? larger	 ?D)	 ? in	 ?the	 ?epidermis	 ?can	 ?be	 ?associated	 ?with	 ? its	 ?smaller	 ?molecular	 ?weight	 ?giving	 ?the	 ?molecules	 ?more	 ?mobility	 ?within	 ?the	 ?cellular	 ?matrix.	 ?The	 ?value	 ?obtained	 ? analytically	 ? by	 ? the	 ? Einstein-??Stokes	 ? equation	 ? is	 ? not	 ? applicable	 ? here	 ? since	 ? it	 ? only	 ?considers	 ?the	 ?molecular	 ?size	 ?and	 ?describes	 ?diffusion	 ?in	 ?a	 ?purely	 ?liquid	 ?medium,	 ?while	 ?in	 ?this	 ?case	 ?the	 ?drug	 ?interacts	 ?with	 ?a	 ?solid	 ?cellular	 ?array.	 ?The	 ?diffusion	 ?coefficient	 ?in	 ?the	 ?SC	 ?has	 ?also	 ?been	 ? measured	 ? for	 ? fluorouracil,	 ? caffeine,	 ? and	 ? flufenamic	 ? acid	 ? to	 ? be	 ? 7.2?10-??12	 ?cm2/s,	 ?	 ?	 ?1.1?10-??11	 ?cm2/s,	 ?and	 ?	 ?2.2?10-??11	 ?cm2/s,	 ?respectively	 ?[32,	 ?34].	 ?All	 ?these	 ?compounds	 ?also	 ?have	 ?smaller	 ? molecular	 ? weights,	 ? but	 ? their	 ? smaller	 ? values	 ? of	 ? D	 ? can	 ? be	 ? explained	 ? by	 ? the	 ? lower	 ?permeability	 ?of	 ?the	 ?SC	 ?compared	 ?to	 ?the	 ?viable	 ?epidermis.	 ?By	 ?using	 ?the	 ?calculated	 ?diffusion	 ?coefficient	 ?D	 ?in	 ?equation	 ?(6),	 ?and	 ?using	 ?the	 ?curve	 ?fit	 ?terms	 ?for	 ?the	 ?concentration	 ?distribution	 ?curve	 ?in	 ?Figure	 ?4.11,	 ?the	 ?value	 ?for	 ?N0	 ?is	 ?calculated	 ?to	 ?be	 ?6.32	 ??	 ?10-??15	 ?kg.	 ?The	 ?analytical	 ?concentration	 ?distribution	 ?model	 ?from	 ?equation	 ?(6)	 ?can	 ?then	 ?90	 ?	 ?be	 ?plotted	 ?(Figure	 ?4.13).	 ?The	 ?drug	 ?distribution	 ?obtained	 ?experimentally	 ?(shown	 ?as	 ?scattered	 ?data	 ? in	 ?Figure	 ?4.13)	 ?has	 ?close	 ?resemblance	 ?to	 ?the	 ?analytical	 ?distribution,	 ?which	 ?shows	 ?the	 ?validity	 ?of	 ?model	 ?for	 ?prediction	 ?the	 ?drug	 ?concentration.	 ?	 ?0.0 5.0x10-5 1.0x10-4 1.5x10-4 2.0x10-40.0000.0020.0040.0060.0080.0100.0120.0140.0160.018Concentration [kg/m3 ]Distance [m]	 ?t1	 ?t1 	 ?+ 	 ?5min	 ?t1 	 ?+ 	 ?10min	 ?t1 	 ?+ 	 ?15min	 ?t1 	 ?+ 	 ?20min	 ?t1 	 ?+ 	 ?25min	 ?t1 	 ?+ 	 ?30min	 ?Figure	 ?4.13:	 ?Doxorubicin	 ?concentration	 ?distribution	 ?obtained	 ?from	 ?analytical	 ?model	 ?(shown	 ?as	 ?lines)	 ?and	 ?the	 ?experimental	 ?data	 ?for	 ?all	 ?the	 ?time	 ?steps	 ?(shown	 ?as	 ?scatter	 ?plots).	 ?A	 ?characteristic	 ?diffusion	 ? length	 ? (obtained	 ? from	 ? ???  ?	 ?where	 ???  ?~ ?3	 ?min	 ? is	 ? the	 ?duration	 ?of	 ?the	 ? injection	 ? process)	 ? for	 ? the	 ? first	 ? constant-??source	 ? diffusion	 ? step	 ? is	 ? also	 ? calculated	 ? to	 ? be	 ?9.11	 ??m.	 ?This	 ?length	 ?describes	 ?how	 ?much	 ?the	 ?drug	 ?progresses	 ?in	 ?the	 ?skin	 ?before	 ?the	 ?second	 ?limited-??source	 ?diffusion	 ?step	 ?starts.	 ?Given	 ?the	 ?much	 ?longer	 ?distances	 ?of	 ?drug	 ?progression	 ?in	 ?the	 ? second	 ? step	 ? (i.e.	 ?more	 ? than	 ?150	 ??m)	 ? the	 ?assumption	 ?of	 ? instantaneous	 ? source	 ? for	 ? the	 ?second	 ?diffusion	 ?step	 ?can	 ?be	 ?justified.	 ?The	 ?method	 ?presented	 ?here	 ?is	 ?used	 ?to	 ?study	 ?diffusion	 ?microscopically,	 ?while	 ?understanding	 ?the	 ?effect	 ? of	 ? drug	 ? chemical	 ? properties	 ?on	 ? the	 ?diffusion	 ?process	 ? requires	 ? investigating	 ? the	 ?91	 ?	 ?interaction	 ? of	 ? drug	 ? at	 ? the	 ?molecular	 ? level.	 ? The	 ? skin	 ? samples	 ? used	 ? here	 ?were	 ? excised	 ? and	 ?contained	 ?dead	 ?cells.	 ?Drug	 ?uptake	 ?might	 ?be	 ?different	 ?for	 ?live	 ?cells	 ?compared	 ?to	 ?dead	 ?cells,	 ?which	 ?would	 ? have	 ? an	 ? impact	 ? on	 ? the	 ? diffusion	 ? rate	 ? of	 ? the	 ? drug.	 ? A	 ? useful	 ? study	 ?would	 ? be	 ?investigating	 ?the	 ?drug	 ?diffusion	 ?behaviour	 ?in	 ?presence	 ?of	 ?live	 ?epidermal	 ?and	 ?dermal	 ?cells	 ?at	 ?the	 ?microscopic	 ?level,	 ?which	 ?should	 ?be	 ?considered	 ?for	 ?future	 ?studies.	 ?4.4 Conclusions	 ?This	 ?chapter	 ?presents	 ?an	 ?optical	 ?method	 ?to	 ?investigate	 ?the	 ?diffusion	 ?of	 ?a	 ?drug	 ?injected	 ?with	 ?microneedles	 ? inside	 ? the	 ? skin.	 ? Single	 ? 500	 ??m	 ? tall	 ? microneedles	 ? were	 ? used	 ? to	 ? inject	 ?doxorubicin	 ? into	 ? pig	 ? skin.	 ? The	 ? drug	 ? intensity	 ? distribution	 ? was	 ? then	 ? measured	 ? using	 ? a	 ?confocal	 ? microscope.	 ? By	 ? obtaining	 ? the	 ? concentration	 ? distribution,	 ? using	 ? intensity-??concentration	 ?calibration	 ?curves,	 ?and	 ?then	 ?fitting	 ?the	 ?analytical	 ?diffusion	 ?model	 ?to	 ?the	 ?data,	 ?the	 ?diffusion	 ?coefficients	 ?were	 ?obtained.	 ?A	 ?theoretical	 ?model	 ?was	 ?then	 ?compared	 ?with	 ?the	 ?experimental	 ?data,	 ? indicating	 ?that	 ?the	 ?model	 ?is	 ?valid	 ?for	 ?predicting	 ?the	 ?drug	 ?concentration	 ?distribution	 ?in	 ?skin.	 ?Using	 ?this	 ?technique,	 ?the	 ?doxorubicin	 ?diffusion	 ?coefficient	 ?was	 ?obtained	 ?for	 ?three	 ?skin	 ?conditions:	 ?fresh	 ?skin,	 ?refrigerated	 ?skin	 ?(for	 ?three	 ?days),	 ?and	 ?frozen	 ?skin	 ?(for	 ?twelve	 ?days).	 ?Overall,	 ?there	 ?was	 ?no	 ?significant	 ?difference	 ?between	 ?the	 ?values	 ?for	 ?fresh	 ?skin	 ?versus	 ?the	 ?refrigerated	 ?skin,	 ?while	 ?the	 ?diffusion	 ?coefficient	 ?for	 ?frozen	 ?skin	 ?was	 ?considerably	 ?larger,	 ? suggesting	 ? that	 ? the	 ?use	 ?of	 ? frozen	 ? skin	 ? should	 ?be	 ?avoided	 ?when	 ?performing	 ? similar	 ?diffusion	 ?studies.	 ?	 ?92	 ?	 ?CHAPTER	 ?5 	 ?	 ?	 ?EXTRACTION	 ?OF	 ?INTERSTITIAL	 ?FLUID	 ?USING	 ?MICRONEEDLES	 ?	 ?	 ?Microneedles	 ? can	 ? be	 ? used	 ? for	 ? painless	 ? or	 ? less	 ? painful	 ? (compared	 ? to	 ? hypodermic	 ? needle)	 ?sampling	 ? of	 ? biological	 ? liquids	 ? from	 ? the	 ? body	 ? for	 ? therapeutic	 ? measurements.	 ? One	 ? target	 ?liquid	 ? is	 ? ISF,	 ? which	 ? is	 ? present	 ? all	 ? over	 ? the	 ? body	 ? and	 ? is	 ? a	 ? water-??based	 ? medium.	 ? Its	 ?composition	 ?is	 ?similar	 ?to	 ?blood	 ?plasma	 ?(the	 ?liquid	 ?component	 ?of	 ?blood	 ?that	 ?holds	 ?the	 ?blood	 ?cells	 ?in	 ?suspension),	 ?and	 ?only	 ?lacks	 ?the	 ?large	 ?proteins	 ?that	 ?cannot	 ?pass	 ?through	 ?the	 ?capillary	 ?membrane.	 ?	 ?	 ?	 ?	 ?	 ?Figure	 ?5.1:	 ?Conceptual	 ?sketch	 ?of	 ?interstitial	 ?fluid	 ?(ISF)	 ?in	 ?body.	 ?Cells	 ?Interstitial	 ?fluid	 ?Hydrostatic	 ?pressure	 ?Osmotic	 ?pressure	 ?Blood	 ?flow	 ?Capillary	 ?93	 ?	 ?ISF	 ? is	 ? responsible	 ? for	 ? transferring	 ?nutrients	 ?and	 ? ions	 ? from	 ?the	 ?blood	 ?vessels	 ? to	 ?tissue	 ?cells	 ?and	 ?vice	 ?versa	 ?(Figure	 ?5.1),	 ?and	 ?its	 ?composition	 ?is	 ?regulated	 ?by	 ?diffusion	 ?of	 ?these	 ?nutrients	 ?through	 ?the	 ?semipermeable	 ?vessel	 ?wall.	 ?Due	 ?to	 ?its	 ?similar	 ?composition	 ?compared	 ?to	 ?blood	 ?plasma	 ?and	 ?the	 ?constant	 ?exchange	 ?of	 ?compounds	 ?between	 ?the	 ?ISF	 ?and	 ?the	 ?blood,	 ?ISF	 ?can	 ?be	 ?used	 ? to	 ? indirectly	 ?measure	 ? the	 ? concentration	 ? of	 ?many	 ? blood	 ? components.	 ? Previous	 ?work	 ?has	 ?demonstrated	 ? the	 ? ISF/blood	 ? correlation	 ? for	 ? various	 ? compounds	 ? [136].	 ? In	 ?many	 ? cases,	 ?the	 ?change	 ?in	 ?concentration	 ?in	 ?ISF	 ?lags	 ?that	 ?of	 ?blood	 ?by	 ?several	 ?minutes	 ?and	 ?should	 ?be	 ?taken	 ?into	 ?account	 ?when	 ?estimating	 ?the	 ?blood	 ?concentrations	 ?[136].	 ?	 ?Hollow	 ?microneedles	 ?can	 ?be	 ?used	 ?to	 ?sample	 ?ISF	 ?or	 ?blood	 ?from	 ?the	 ?skin	 ?using	 ?capillary	 ?forces	 ?or	 ?by	 ?applying	 ?a	 ?negative	 ?pressure	 ? through	 ?the	 ?needle	 ? lumens.	 ?Apart	 ? from	 ?the	 ?SC,	 ?all	 ? the	 ?skin	 ?layers	 ?contain	 ?live	 ?cells	 ?that	 ?depend	 ?on	 ?nutrients;	 ?therefore,	 ?the	 ?presence	 ?of	 ?ISF	 ?in	 ?skin	 ?is	 ?crucial	 ?to	 ?maintain	 ?the	 ?cells?	 ?life	 ?cycle.	 ?The	 ?epidermis	 ?is	 ?however,	 ?a	 ?relatively	 ?dry	 ?medium	 ?compared	 ?to	 ?other	 ?body	 ?tissues	 ?with	 ?the	 ?ISF	 ?making	 ?up	 ?about	 ?25%	 ?of	 ?its	 ?composition	 ?[137].	 ?In	 ?contrast,	 ?ISF	 ?makes	 ?up	 ?a	 ?larger	 ?portion	 ?of	 ?the	 ?dermal	 ?tissue,	 ?40%	 ?[137],	 ?which	 ?is	 ?mainly	 ?due	 ? to	 ? the	 ?presence	 ?of	 ?blood	 ?capillaries	 ? in	 ? this	 ? layer.	 ?Extraction	 ?of	 ? ISF	 ? from	 ?the	 ?dermis	 ? is	 ?therefore	 ?easier	 ?than	 ?from	 ?the	 ?epidermis,	 ?but	 ?the	 ?presence	 ?of	 ?nerves	 ?may	 ?cause	 ?a	 ?sensation	 ?of	 ?pain	 ?upon	 ?needle	 ?penetration.	 ?ISF	 ?extraction	 ?from	 ?the	 ?skin	 ?using	 ?microneedles	 ?was	 ?first	 ?demonstrated	 ?by	 ?Mukerjee	 ?et	 ?al.	 ?[48],	 ?where	 ?200	 ??m	 ?tall	 ?silicon	 ?microneedles	 ?were	 ?used	 ?to	 ?collect	 ?the	 ?liquid	 ?using	 ?capillary	 ?forces	 ?from	 ?human	 ?earlobe.	 ?To	 ?prove	 ?successful	 ?extraction	 ?of	 ?ISF,	 ?the	 ?authors	 ?used	 ?commercial	 ?glucose	 ?strips	 ?and	 ?demonstrated	 ?a	 ?color	 ?change	 ?in	 ?the	 ?presence	 ?of	 ?the	 ?collected	 ?sample.	 ?	 ?94	 ?	 ?This	 ? chapter	 ? presents	 ? some	 ? ISF	 ? extraction	 ? procedures	 ? carried	 ? out	 ? with	 ? the	 ? needles	 ?produced	 ? in	 ? the	 ? previous	 ? chapters	 ? as	 ? well	 ? as	 ? a	 ? new	 ? solid	 ? microneedle	 ? design.	 ? Hollow	 ?polymer	 ?and	 ?metallic	 ?microneedles	 ?were	 ?used	 ?on	 ?animal	 ?skin	 ?to	 ?evaluate	 ?their	 ?usefulness	 ?for	 ? ISF	 ? extraction.	 ? In	 ? addition,	 ? the	 ? solvent	 ? casting	 ? process	 ? in	 ? Chapter	 ? 2	 ? was	 ?modified	 ? to	 ?make	 ? solid	 ? microneedles	 ? with	 ? a	 ? water	 ? absorbent	 ? layer	 ? embedded	 ? in	 ? its	 ? structure,	 ? and	 ?needles	 ?were	 ?then	 ?applied	 ?to	 ?animal	 ?skin	 ?for	 ?ISF	 ?sampling.	 ?5.1 Experimental	 ?procedures	 ?5.1.1 ISF	 ?extraction	 ?using	 ?solid	 ?polymer	 ?microneedles	 ?A	 ?new	 ?method	 ? is	 ?presented	 ? for	 ? the	 ?extraction	 ?of	 ?dermal	 ? ISF	 ? that	 ?uses	 ? solid	 ?microneedles	 ?with	 ? an	 ? absorbent	 ? polymer	 ? layer	 ? embedded	 ? in	 ? its	 ? structure.	 ? Through	 ? a	 ? solvent	 ? casting	 ?process	 ? solid	 ? microneedles	 ? were	 ? formed,	 ? composed	 ? of	 ? mainly	 ? three	 ? layers	 ? and	 ? a	 ? base	 ?support	 ?structure	 ?to	 ?facilitate	 ? insertion	 ?of	 ?the	 ?needles	 ? into	 ?the	 ?skin.	 ?The	 ?solvent	 ?casting	 ? is	 ?performed	 ?on	 ?a	 ?support	 ?structure	 ?made	 ?from	 ?SU-??8	 ?photoresist	 ?that	 ?consists	 ?of	 ?an	 ?array	 ?of	 ?cone	 ?shaped	 ?pillars	 ?(Figure	 ?5.2a,	 ?b,	 ?and	 ?Figure	 ?5.3a).	 ?95	 ?	 ?	 ?Figure	 ?5.2:	 ?Fabrication	 ?process	 ?using	 ?solvent	 ?casting	 ?for	 ?solid	 ?out-??of-??plane	 ?polymer	 ?microneedles	 ?for	 ?ISF	 ?extraction;	 ?a	 ?&	 ?b)	 ?fabrication	 ?of	 ?pillars	 ?from	 ?SU-??8;	 ?c)	 ?clay/polyimide	 ?+	 ?NMP	 ?solution	 ?deposition;	 ?d)	 ?evaporation	 ?of	 ?NMP;	 ?e)	 ?solvent	 ?casting	 ?of	 ?hydroxyethyl	 ?cellulose	 ?layer;	 ?f)	 ?solvent	 ?casting	 ?of	 ?poly(ethylene-??co-??vinyl	 ?acetate)	 ?layer.	 ?	 ?The	 ? cone	 ? shaped	 ? pillars	 ? are	 ? created	 ? by	 ? backside	 ? exposure	 ? of	 ? the	 ? SU-??8	 ? layer	 ? through	 ? a	 ?Pyrex?	 ?base	 ?plate,	 ?similar	 ?to	 ?the	 ?mold	 ?fabrication	 ?steps	 ?in	 ?Chapters	 ?2	 ?and	 ?3.	 ?Then	 ?a	 ?polymer	 ?composite	 ?layer	 ?is	 ?deposited,	 ?made	 ?of	 ?2%	 ?clay-??reinforced	 ?polyimide	 ?(Figures	 ?5.2c	 ?and	 ?5.2d)	 ?with	 ?high	 ? rigidity	 ? and	 ?mechanical	 ? strength.	 ?Next,	 ? a	 ?water	 ? soluble/absorbent	 ?hydroxyethyl	 ?cellulose	 ? (HEC)	 ? layer	 ? is	 ? cast	 ? on	 ? top	 ? of	 ? the	 ? polyimide	 ? layer	 ? (Figure	 ? 5.2e).	 ? Finally,	 ? a	 ? thin	 ?poly(ethylene-??co-??vinyl	 ?acetate)	 ?(PEVA)	 ?layer	 ?is	 ?cast	 ?on	 ?top	 ?of	 ?everything	 ?(Figure	 ?5.2f).	 ?Since	 ?PEVA	 ?is	 ?hydrophobic,	 ?when	 ?cast	 ?on	 ?top	 ?of	 ?the	 ?hydrophilic	 ?HEC	 ?layer,	 ?it	 ?does	 ?not	 ?cover	 ?the	 ?needles	 ?during	 ?the	 ?drying	 ?process	 ?and	 ?only	 ?covers	 ?the	 ?backing	 ?plate.	 ?This	 ?layer	 ?improves	 ?the	 ?96	 ?	 ?biocompatibility	 ?of	 ?the	 ?microneedle	 ?array	 ?[138]	 ?and	 ?also	 ?provides	 ?a	 ?protective	 ?layer	 ?for	 ?the	 ?HEC	 ?layer	 ?from	 ?the	 ?dead	 ?cells,	 ?fat,	 ?and	 ?hair	 ?on	 ?the	 ?skin	 ?surface;	 ?and	 ?it	 ?can	 ?be	 ?later	 ?removed	 ?either	 ? by	 ? its	 ? solvent	 ? or	 ? by	 ? peeling	 ? it	 ? off	 ? from	 ? the	 ? HEC	 ? layer.	 ? Figure	 ? 5.3b	 ? shows	 ? the	 ? final	 ?microneedle	 ?array	 ?made	 ?through	 ?this	 ?process.	 ?	 ?Once	 ?the	 ?microneedle	 ?array	 ?is	 ?inserted	 ?into	 ?the	 ?skin,	 ?the	 ?dermal	 ?ISF	 ?is	 ?absorbed	 ?by	 ?the	 ?HEC	 ?layer,	 ?which	 ?is	 ?highly	 ?water	 ?absorbent,	 ?causing	 ?it	 ?to	 ?swell	 ?and	 ?expand.	 ?The	 ?agent	 ?of	 ?interest,	 ?which	 ?includes	 ?biomarkers	 ?and	 ?drugs,	 ?can	 ?then	 ?be	 ?analyzed	 ?either	 ?directly	 ?in	 ?the	 ?HEC	 ?layer,	 ?or	 ?after	 ?dissolving	 ?the	 ?HEC	 ?in	 ?water.	 ?	 ?Figure	 ?5.3:	 ?a)	 ?A	 ?base	 ?structure	 ?used	 ?for	 ?solvent	 ?casting	 ?of	 ?solid	 ?microneedles,	 ?consisting	 ?of	 ?an	 ?array	 ?of	 ?tall	 ?pillars	 ?made	 ?from	 ?SU-??8;	 ?the	 ?distance	 ?between	 ?the	 ?pillars	 ?in	 ?the	 ?array	 ?is	 ?500	 ??m,	 ?b)	 ?An	 ?array	 ?of	 ?250	 ??m-??long	 ?microneedles	 ?made	 ?through	 ?the	 ?process	 ?shown	 ?in	 ?Figure	 ?5.2.	 ?	 ?Mechanical	 ?tests,	 ?similar	 ?to	 ?the	 ?tests	 ?demonstrated	 ?in	 ?Chapters	 ?2	 ?and	 ?3,	 ?were	 ?performed	 ?on	 ?the	 ?microneedles	 ?to	 ?evaluate	 ?their	 ?strength.	 ?The	 ?average	 ?failure	 ?load	 ?of	 ?single	 ?needles	 ?from	 ?three	 ?compressions	 ?tests	 ?was	 ?measured	 ?to	 ?be	 ?0.28	 ?N,	 ?which	 ?should	 ?be	 ?sufficient	 ?to	 ?pierce	 ?human	 ?skin	 ?given	 ?the	 ?needle	 ?dimensions	 ?[103].	 ? In	 ?addition	 ?to	 ?strength	 ?tests,	 ?extraction	 ?of	 ?dermal	 ? ISF	 ? was	 ? demonstrated	 ? using	 ? the	 ? fabricated	 ? microneedles.	 ? For	 ? this	 ? purpose,	 ? the	 ?a)	 ? b)	 ?97	 ?	 ?microneedle	 ?arrays	 ?were	 ?applied	 ?to	 ?the	 ?inner	 ?rabbit	 ?ear	 ?skin	 ?and	 ?they	 ?were	 ?held	 ?in	 ?place	 ?for	 ?5	 ?min.	 ?Figure	 ?5.4	 ?shows	 ?a	 ?microneedle	 ?insertion	 ?site	 ?for	 ?an	 ?extraction	 ?experiment,	 ?as	 ?well	 ?as	 ?a	 ? swollen	 ? 14-??microneedle	 ? array	 ? after	 ? absorption	 ? of	 ? the	 ? dermal	 ? ISF.	 ? Three	 ? ISF	 ? extraction	 ?experiments	 ? were	 ? carried	 ? out	 ? on	 ? the	 ? rabbit	 ? ear	 ? skin;	 ? and	 ? the	 ? microneedle	 ? arrays	 ? were	 ?weighed	 ?before	 ?and	 ?after	 ?application	 ?to	 ?the	 ?skin	 ?(with	 ?measurement	 ?resolution	 ?of	 ?0.01	 ?mg)	 ?to	 ? measure	 ? the	 ? amount	 ? of	 ? the	 ? extracted	 ? fluid.	 ? Control	 ? tests	 ? were	 ? also	 ? performed	 ? with	 ?polymer	 ?surfaces	 ?having	 ?no	 ?needles,	 ?to	 ?estimate	 ?the	 ?amount	 ?of	 ?fat,	 ?dead	 ?cells,	 ?and	 ?hair	 ?that	 ?accumulates	 ?on	 ?the	 ?surface.	 ?	 ?	 ?Figure	 ?5.4:	 ?Extraction	 ?of	 ?dermal	 ?ISF	 ?using	 ?the	 ?fabricated	 ?microneedles;	 ?a)	 ?insertion	 ?site	 ?on	 ?the	 ?inner	 ?rabbit	 ?ear	 ?skin;	 ?b)	 ?swelling	 ?of	 ?the	 ?hydroxyethyl	 ?cellulose	 ?layer	 ?upon	 ?application	 ?to	 ?the	 ?skin	 ?as	 ?a	 ?result	 ?of	 ?ISF	 ?absorption	 ?5.1.2 ISF	 ?extraction	 ?using	 ?hollow	 ?polymer	 ?microneedles	 ?Removing	 ? ISF	 ? using	 ? capillary	 ? forces	 ? requires	 ? a	 ? hydrophilic	 ? surface	 ? in	 ? the	 ? microneedle	 ?lumens.	 ? In	 ? this	 ? work,	 ? we	 ? have	 ?modified	 ? the	 ? casting	 ? process	 ? to	 ? make	 ?microneedles	 ? with	 ?hydrophilic	 ? channels	 ? to	 ? extract	 ? ISF	 ? from	 ? the	 ? skin	 ? through	 ? capillary	 ? action.	 ? Hydrophilic	 ?98	 ?	 ?polymers	 ? are	 ? often	 ? water-??soluble	 ? or	 ? swell	 ? in	 ? the	 ? presence	 ? of	 ? water	 ? (or	 ? ISF),	 ? which	 ? is	 ? a	 ?problem	 ? since	 ? microneedles	 ? can	 ? soften	 ? during	 ? the	 ? extraction	 ? procedure,	 ? thus	 ? lose	 ? their	 ?mechanical	 ?rigidity.	 ?To	 ?solve	 ?this,	 ?they	 ?can	 ?be	 ?further	 ?cross-??linked	 ?by	 ?chemical	 ?treatment	 ?to	 ?improve	 ?their	 ?mechanical	 ?strength	 ?and	 ?substantially	 ?reduces	 ?swelling	 ?caused	 ?by	 ?the	 ?ISF.	 ?	 ?Figure	 ?5.5:	 ?Fabrication	 ?process	 ?for	 ?making	 ?hollow	 ?PVA	 ?microneedles;	 ?a)	 ?fabrication	 ?of	 ?a	 ?mold	 ?consisting	 ?of	 ?an	 ?array	 ?of	 ?SU-??8	 ?pillars	 ?coated	 ?with	 ?a	 ?PDMS	 ?layer,	 ?b)	 ?deposition	 ?of	 ?PVA	 ?+	 ?BA	 ?solution,	 ?c)	 ?solvent	 ?evaporation,	 ?d)	 ?casting	 ?of	 ?a	 ?PMMA	 ?layer,	 ?e)	 ?separation	 ?from	 ?the	 ?mold,	 ?	 ?f)	 ?opening	 ?the	 ?tips	 ?using	 ?O2/CF4	 ?plasma	 ?etching.	 ?Here,	 ? the	 ?microneedles	 ? are	 ? fabricated	 ? from	 ? cross-??linked	 ? polyvinyl	 ? alcohol	 ? (PVA)	 ? (average	 ?MW:	 ? 124,000	 ? to	 ? 186,000,	 ? 99%	 ? hydrolyzed)	 ?with	 ? boric	 ? acid	 ? (BA)	 ? used	 ? as	 ? the	 ? cross-??linking	 ?agent	 ?(Figure	 ?5.5).	 ?A	 ?thin	 ?poly(methyl	 ?methacrylate)	 ?(PMMA)	 ?layer	 ?is	 ?cast	 ?on	 ?top	 ?of	 ?the	 ?PVA	 ?in	 ?order	 ?to	 ?protect	 ?the	 ?PVA	 ?during	 ?the	 ?plasma	 ?etching	 ?step	 ?that	 ?opens	 ?the	 ?tips.	 ?Figure	 ?5.6	 ?shows	 ?the	 ?PVA	 ?microneedles	 ?made	 ?using	 ?solvent	 ?casting.	 ?with a PDMS layer 99	 ?	 ?	 ?Figure	 ?5.6:	 ?a)	 ?SEM	 ?image	 ?of	 ?a	 ?PVA	 ?microneedle	 ?array,	 ?b)	 ?optical	 ?microscope	 ?image	 ?of	 ?a	 ?single	 ?PVA	 ?microneedle.	 ?	 ?A	 ? series	 ? of	 ? mechanical	 ? tests	 ? were	 ? performed	 ? to	 ? evaluate	 ? the	 ? strength	 ? of	 ? the	 ? fabricated	 ?microneedles	 ? under	 ? compressive	 ? loading.	 ? It	 ? was	 ? found	 ? that	 ? the	 ? needles	 ? can	 ? sustain	 ?compressive	 ?loads	 ?of	 ?up	 ?to	 ?0.21	 ??	 ?0.04	 ?N	 ?which	 ?is	 ?sufficient	 ?to	 ?pierce	 ?human	 ?skin	 ?given	 ?the	 ?needle	 ?dimensions	 ?[103].	 ?A	 ?series	 ?of	 ?contact	 ?angle	 ?measurements	 ?were	 ?also	 ?carried	 ?out	 ?to	 ?evaluate	 ? the	 ?surface	 ?hydrophilicity	 ?of	 ? the	 ?cross-??linked	 ?PVA.	 ?For	 ? this	 ?purpose,	 ? the	 ?material	 ?was	 ? spin	 ? coated	 ? on	 ? a	 ? microscope	 ? slide.	 ? After	 ? baking	 ? the	 ? polymer,	 ? the	 ? contact	 ? angle	 ?measurements	 ?were	 ?carried	 ?out	 ?(Figure	 ?5.7).	 ?From	 ?five	 ?measurements,	 ?the	 ?average	 ?contact	 ?angle	 ? was	 ? observed	 ? to	 ? be	 ? 48.6?	 ? showing	 ? sufficient	 ? hydrophilicity.	 ? This	 ? is	 ? confirmed	 ? in	 ?Figure	 ?5.8	 ?where	 ?an	 ?array	 ?of	 ?microneedles	 ? is	 ?exposed	 ? to	 ?a	 ?dyed	 ?water	 ? solution	 ?at	 ? its	 ? tips	 ?and	 ?capillary	 ?action	 ?has	 ?transported	 ?the	 ?liquid	 ?from	 ?the	 ?microneedle	 ?tips	 ?along	 ?their	 ?lumens	 ?to	 ?the	 ?backside	 ?of	 ?the	 ?chip.	 ?200	 ??m	 ?100	 ?	 ?	 ?Figure	 ?5.7:	 ?Contact	 ?angle	 ?measurement	 ?of	 ?a	 ?4	 ??L	 ?water	 ?droplet	 ?on	 ?a	 ?cross-??linked	 ?PVA	 ?surface	 ?shows	 ?that	 ?the	 ?surface	 ?is	 ?hydrophilic.	 ?	 ?Figure	 ?5.8:	 ?Dyed	 ?water	 ?transfer	 ?through	 ?the	 ?microneedle	 ?lumens	 ?when	 ?the	 ?tips	 ?are	 ?exposed	 ?to	 ?the	 ?liquid,	 ?a)	 ?schematic	 ?of	 ?experiment,	 ?b)	 ?microneedle	 ?backside	 ?showing	 ?capillary	 ?driven	 ?flow	 ?reaching	 ?the	 ?channel	 ?openings.	 ?	 ?Finally,	 ? extraction	 ?of	 ? ISF	 ? from	 ? skin	 ?using	 ? the	 ?microneedles	 ?was	 ? attempted	 ?by	 ? applying	 ? an	 ?array	 ?of	 ?14	 ?microneedles	 ?attached	 ?to	 ?a	 ?glucose	 ?test	 ?strip	 ?(BETACHEK	 ?VISUAL	 ?from	 ?National	 ?Diagnostic	 ? Products	 ? Pty	 ? Ltd,	 ? Sydney,	 ? Australia)	 ? against	 ? rabbit	 ? ear	 ? skin	 ? for	 ? 30	 ?min	 ? (Figure	 ?5.9).	 ?The	 ?strip	 ?was	 ?tested	 ?several	 ?times	 ?to	 ?ensure	 ?it	 ?only	 ?changes	 ?color	 ?in	 ?response	 ?to	 ?blood	 ?101	 ?	 ?or	 ? interstitial	 ? fluid	 ?and	 ?not	 ?sweat	 ?or	 ?other	 ? fluids.	 ?To	 ?evaluate	 ?sampling,	 ? the	 ?glucose	 ?strips	 ?were	 ?observed	 ?under	 ?a	 ?microscope	 ?for	 ?signs	 ?of	 ?color	 ?change.	 ?	 ?Figure	 ?5.9:	 ?Setup	 ?used	 ?for	 ?ISF	 ?extraction	 ?with	 ?hollow	 ?polymer	 ?microneedles.	 ?5.1.3 ISF	 ?extraction	 ?using	 ?hollow	 ?metallic	 ?microneedles	 ?The	 ?500	 ??m	 ?tall	 ?microneedles	 ?fabricated	 ?in	 ?Chapter	 ?3	 ?were	 ?used	 ?for	 ?extracting	 ?ISF	 ?from	 ?pig	 ?skin	 ?using	 ?vacuum.	 ?For	 ?this	 ?purpose,	 ?a	 ?vacuum-??operating	 ?extraction	 ?device	 ?was	 ?made	 ?which	 ?contained	 ?a	 ?hollow	 ?solid	 ?tube	 ?attached	 ?to	 ?a	 ?microneedle	 ?array	 ?on	 ?one	 ?end	 ?and	 ?to	 ?a	 ?vacuum	 ?source	 ? in	 ? the	 ? other	 ? end	 ? (Figure	 ? 5.10).	 ? The	 ? microneedle	 ? array	 ? contained	 ? six	 ? 500	 ??m	 ? tall	 ?needles.	 ?The	 ?microneedle	 ?array	 ?needle	 ?opening	 ?side	 ?rested	 ?on	 ?a	 ?small	 ?cone-??shaped	 ?cavity	 ?that	 ?was	 ?connected	 ?to	 ?a	 ?capillary	 ?tube	 ?on	 ?the	 ?narrow	 ?end.	 ?	 ?The	 ?capillary	 ?tube	 ?was	 ?directed	 ?at	 ?a	 ?commercial	 ?glucose	 ?strip	 ?(BETACHEK	 ?VISUAL	 ?from	 ?National	 ?Diagnostic	 ?Products	 ?Pty	 ?Ltd,	 ?Sydney,	 ? Australia).	 ? The	 ? vacuum	 ? was	 ? set	 ? to	 ? 10	 ?psi	 ? and	 ? was	 ? chosen	 ? based	 ? on	 ? a	 ? previous	 ?literature	 ?that	 ?used	 ?vacuum	 ?to	 ?sample	 ?ISF	 ?from	 ?the	 ?skin	 ?surface	 ?[88].	 ?The	 ?device	 ?was	 ?then	 ?pressed	 ?against	 ?pig	 ?skin	 ?for	 ?10	 ?min.	 ?After	 ?this	 ?period,	 ?the	 ?glucose	 ?strip	 ?was	 ?removed	 ?from	 ?the	 ?tube	 ?and	 ?observed	 ?under	 ?microscope.	 ?To	 ?ensure	 ?no	 ?blood	 ?was	 ?extracted	 ?instead	 ?of	 ?ISF,	 ?the	 ?capillary	 ?tube	 ?was	 ?carefully	 ?observed	 ?under	 ?a	 ?microscope	 ?to	 ?ensure	 ?there	 ?was	 ?no	 ?sign	 ?of	 ?blood.	 ?This	 ?process	 ?was	 ?repeated	 ?five	 ?times.	 ?Glucose	 ?strip	 ?Rabbit	 ?ear	 ?102	 ?	 ?	 ?	 ?Figure	 ?5.10:	 ?a)	 ?Schematic	 ?of	 ?custom	 ?vacuum	 ?device	 ?made	 ?for	 ?ISF	 ?extraction	 ?using	 ?the	 ?500	 ??m	 ?metallic	 ?microneedles,	 ?b)	 ?image	 ?of	 ?the	 ?actual	 ?vacuum	 ?device.	 ?5.2 Results	 ?and	 ?discussion	 ?5.2.1 Solid	 ?microneedles	 ?The	 ?results	 ?of	 ?the	 ?three	 ?experiments	 ?(Table	 ?5.1)	 ?show	 ?an	 ?average	 ?extracted	 ? ISF	 ?volume	 ?of	 ?0.77	 ??L.	 ?Extraction	 ?of	 ?larger	 ?amounts	 ?of	 ?ISF	 ?can	 ?be	 ?achieved	 ?by	 ?proportionally	 ?increasing	 ?the	 ?number	 ? of	 ? microneedles	 ? in	 ? the	 ? array,	 ? which	 ? can	 ? be	 ? realized	 ? by	 ? simply	 ? increasing	 ? the	 ?number	 ?of	 ?pillars	 ?in	 ?the	 ?base	 ?structure.	 ?Although	 ?this	 ?technique	 ?may	 ?be	 ?useful	 ?for	 ?removing	 ?the	 ? liquid	 ? from	 ?the	 ?skin,	 ?analyzing	 ? the	 ? ISF	 ?absorbed	 ? in	 ? the	 ?cellulose	 ? layer	 ? is	 ?more	 ?difficult	 ?Image of vacuum device made for ISF 	 ?	 ?Microneedles array Tube Pressure valves To vacuum pump Glucose strip in vial a) b) 103	 ?	 ?compared	 ?to	 ?analyzing	 ?the	 ?pure	 ?ISF.	 ?One	 ?approach	 ?can	 ?be	 ?dissolving	 ?the	 ?layer	 ?in	 ?a	 ?solvent	 ?and	 ? then	 ? investigating	 ? the	 ? concentrations.	 ? Another	 ? approach	 ? can	 ? be	 ? developing	 ? systems	 ?that	 ?directly	 ?measure	 ?the	 ?compound	 ?in	 ?the	 ?cellulose	 ?layer.	 ?Table	 ?5.1:	 ?Summary	 ?of	 ?the	 ?ISF	 ?extraction	 ?tests	 ?using	 ?14	 ?solid	 ?microneedles	 ?with	 ?a	 ?cellulose	 ?functionalization;	 ?the	 ?net	 ?extracted	 ?volume	 ?is	 ?calculated	 ?by	 ?subtracting	 ?0.18	 ?mg	 ?(average	 ?mass	 ?transferred	 ?during	 ?the	 ?control	 ?tests)	 ?from	 ?the	 ?mass	 ?differences,	 ?and	 ?using	 ?density	 ?of	 ?water	 ?at	 ?25?C	 ?(0.997	 ?g/ml)	 ?instead	 ?of	 ?that	 ?of	 ?ISF	 ?Experiment	 ?No.	 ?	 ? Mass	 ?before	 ?[mg]	 ?	 ?Mass	 ?after	 ?[mg]	 ?	 ?Mass	 ?difference	 ?[mg]	 ?	 ?Net	 ?mass	 ?difference	 ?[mg]	 ? Net	 ?extracted	 ?volume	 ?[?L]	 ?1	 ? 167.70	 ? 168.56	 ? 0.86	 ? 0.68	 ? 0.68	 ?2	 ? 116.01	 ? 117.07	 ? 1.06	 ? 0.88	 ? 0.88	 ?3	 ? 130.73	 ? 131.67	 ? 0.94	 ? 0.76	 ? 0.76	 ?Control	 ?test	 ?1	 ? 112.87	 ? 113.06	 ? 0.19	 ? 	 ? -??	 ?Control	 ?test	 ?2	 ? 114.23	 ? 114.40	 ? 0.17	 ? 	 ? -??	 ?	 ?5.2.2 Hollow	 ?polymer	 ?microneedles	 ?No	 ? signs	 ? of	 ? color	 ? change	 ? were	 ? observed	 ? after	 ? inspecting	 ? the	 ? glucose	 ? strips.	 ? Successful	 ?extraction,	 ?therefore,	 ?was	 ?not	 ?demonstrated	 ?with	 ?these	 ?needles.	 ?Possible	 ?reasons	 ?could	 ?be	 ?the	 ?uptake	 ?of	 ?the	 ?liquid	 ?by	 ?the	 ?polymer	 ?upon	 ?application	 ?or	 ?simply	 ?the	 ?duration	 ?required	 ?for	 ?sampling	 ?the	 ?liquid	 ?is	 ?much	 ?longer	 ?than	 ?what	 ?was	 ?tried.	 ?A	 ?possible	 ?approach	 ?to	 ?solve	 ?this	 ?is	 ?to	 ? replace	 ? the	 ?material	 ?with	 ?a	 ?polymer	 ? that	 ?has	 ?no	 ? liquid	 ?uptake	 ?upon	 ?application	 ? to	 ? the	 ?skin.	 ?	 ?104	 ?	 ?5.2.3 Hollow	 ?metallic	 ?microneedles	 ?After	 ? observing	 ? the	 ? glucose	 ? strips	 ? under	 ? a	 ?microscope,	 ? two	 ? trials	 ? showed	 ? traces	 ? of	 ? color	 ?change	 ? due	 ? to	 ? presence	 ? of	 ? ISF	 ? on	 ? the	 ? glucose	 ? strip.	 ? However,	 ? the	 ? area	 ? on	 ? the	 ? strip	 ? that	 ?changed	 ? color	 ?was	 ? small	 ? and	 ? not	 ? enough	 ? for	 ? estimating	 ? the	 ? exact	 ? concentrations.	 ? These	 ?experiments	 ? demonstrate	 ? the	 ? potential	 ? and	 ? the	 ? usefulness	 ? of	 ? the	 ? vacuum	 ? and	 ? metallic	 ?microneedles	 ? for	 ? ISF	 ? sampling.	 ? However,	 ? only	 ? very	 ? small	 ? amounts	 ? of	 ? ISF	 ? were	 ? extracted	 ?which	 ?may	 ?not	 ?be	 ?sufficient	 ?for	 ?many	 ?applications.	 ?To	 ?improve	 ?this,	 ?it	 ?is	 ?possible	 ?to	 ?increase	 ?the	 ? number	 ? of	 ? needles	 ? in	 ? the	 ? array	 ? to	 ? target	 ? a	 ? larger	 ? skin	 ? area.	 ? Other	 ? possibilities	 ? are	 ?increasing	 ?the	 ?duration	 ?of	 ?application	 ?as	 ?well	 ?as	 ?the	 ?vacuum	 ?pressure	 ?in	 ?the	 ?tube.	 ?5.3 Conclusions	 ?In	 ?this	 ?chapter,	 ?ISF	 ?sampling	 ?was	 ?attempted	 ?with	 ?three	 ?microneedle	 ?designs:	 ?solid	 ?polymer	 ?array,	 ? hollow	 ? polymer	 ? array,	 ? and	 ? hollow	 ? metallic	 ? array.	 ? Solid	 ? microneedles	 ? showed	 ?successful	 ? removal	 ?of	 ? the	 ? ISF	 ?with	 ?an	 ?average	 ?volume	 ?of	 ?55	 ?nL	 ?per	 ?needle.	 ?However,	 ? it	 ? is	 ?difficult	 ? to	 ? measure	 ? the	 ? compounds	 ? in	 ? the	 ? ISF	 ? removed	 ? with	 ? this	 ? technique	 ? since	 ? it	 ? is	 ?embedded	 ? in	 ? the	 ? cellulose	 ? layer.	 ? Hollow	 ? polymer	 ? microneedles	 ? did	 ? not	 ? show	 ? any	 ? liquid	 ?removal	 ?while	 ?the	 ?hollow	 ?metallic	 ?microneedles	 ?showed	 ?minimal	 ?removal	 ?of	 ?the	 ?ISF	 ?using	 ?vacuum.	 ?	 ?Overall	 ? the	 ? proposed	 ? methods	 ? suggest	 ? the	 ? potential	 ? of	 ? using	 ? microneedles	 ? for	 ? this	 ?application;	 ?however,	 ?additional	 ?investigation	 ?should	 ?be	 ?carried	 ?out	 ?to	 ?study	 ?the	 ?limitation	 ?105	 ?	 ?of	 ?this	 ?technique	 ?before	 ?implementing	 ?experimental	 ?procedures.	 ?Also,	 ?other	 ?methods	 ?that	 ?enhance	 ?ISF	 ?sampling	 ?should	 ?be	 ?investigated	 ?and	 ?combined	 ?with	 ?the	 ?proposed	 ?techniques.	 ? 	 ?106	 ?	 ?	 ?CHAPTER	 ?6 	 ?	 ?	 ?SUMMARY	 ?AND	 ?FUTURE	 ?WORK	 ?	 ?	 ?6.1 Summary	 ?This	 ?dissertation	 ?presents	 ?research	 ?performed	 ?towards	 ?developing	 ?manufacturing	 ?processes	 ?for	 ?hollow	 ?microneedle	 ?devices	 ?used	 ?for	 ?drug	 ?delivery	 ?and	 ?biosensing	 ?applications.	 ?A	 ?major	 ?focus	 ?of	 ?the	 ?research	 ?objectives	 ?was	 ?developing	 ?cost	 ?effective	 ?processes	 ?that	 ?are	 ?applicable	 ?for	 ? batch	 ? manufacturing,	 ? thus	 ? providing	 ? the	 ? possibility	 ? of	 ? widespread	 ? adoption	 ? of	 ?microneedles	 ?and	 ?replacing	 ?traditional	 ?painful	 ?hypodermic	 ?needles.	 ? In	 ?addition,	 ?this	 ?thesis	 ?presents	 ? a	 ? novel	 ? method	 ? of	 ? studying	 ? drug	 ? delivery	 ? using	 ? microneedles,	 ? which	 ? can	 ? help	 ?future	 ?researchers	 ?to	 ?evaluate	 ?the	 ?usefulness	 ?of	 ?microneedle	 ?for	 ?administration	 ?of	 ?various	 ?clinical	 ?compounds.	 ?Chapter	 ? 1	 ? of	 ? this	 ? thesis,	 ? first,	 ? provides	 ? an	 ? overview	 ? of	 ? the	 ? skin	 ? anatomy	 ? as	 ? well	 ? as	 ? the	 ?transdermal	 ?drug	 ?delivery.	 ? It	 ? is	 ?shown	 ?how	 ?adhesive	 ?skin	 ?patches	 ? facilitate	 ?drug	 ?transport	 ?107	 ?	 ?across	 ?skin	 ?and	 ?what	 ?are	 ?the	 ? important	 ?parameters	 ?and	 ?formulas	 ?controlling	 ?this	 ?process.	 ?Next,	 ? this	 ? chapter	 ? discusses	 ? the	 ? emerging	 ?microneedle	 ? technology	 ? and	 ? how	 ? they	 ? can	 ? be	 ?more	 ?effective	 ?over	 ?the	 ?traditional	 ?adhesive	 ?patches	 ?for	 ?transdermal	 ?drug	 ?delivery.	 ?It	 ?is	 ?also	 ?shown	 ?how	 ?they	 ?can	 ?be	 ?useful	 ?over	 ?traditional	 ?needles	 ?due	 ?to	 ?the	 ?minimal	 ?pain	 ?associated	 ?with	 ?them.	 ?	 ?In	 ?Chapter	 ?2,	 ?we	 ?present	 ?a	 ?process	 ?based	 ?on	 ?solvent	 ?casting	 ?process	 ?to	 ?make	 ?hollow	 ?out-??of-??plane	 ? polymer	 ? microneedles.	 ? Through	 ? photolithography	 ? of	 ? SU-??8	 ? photoresist,	 ? molds	 ? are	 ?created	 ?containing	 ?arrays	 ?of	 ?cone-??shaped	 ?pillars	 ?450	 ??m	 ?tall.	 ?A	 ?thin	 ? layer	 ?of	 ?PDMS	 ?is	 ?then	 ?deposited	 ? and	 ? cured	 ? followed	 ?by	 ? a	 ? plasma	 ? treatment	 ? step.	 ?Next,	 ? a	 ? solution	 ? of	 ? polyimide	 ?and	 ? nanoclay	 ? suspension	 ? is	 ? cast	 ? on	 ? the	 ?mold.	 ? The	 ? optimum	 ? clay	 ? percentage	 ? of	 ? 2	 ?wt%	 ? in	 ?solid	 ?was	 ?found	 ?from	 ?a	 ?series	 ?of	 ?compression	 ?tests	 ?on	 ?pillar	 ?(made	 ?of	 ?polyimide	 ?with	 ?varying	 ?clay	 ? content)	 ? of	 ? similar	 ? dimension	 ? scale	 ? as	 ? microneedles.	 ? The	 ? polyimide/nanoclay	 ? layer	 ?constitutes	 ?the	 ?microneedle	 ?structure	 ?and	 ?is	 ?separated	 ?from	 ?the	 ?mold	 ?by	 ?mechanical	 ?force.	 ?Through	 ?an	 ?additional	 ?plasma	 ?etching	 ?step,	 ?their	 ?tips	 ?are	 ?opened.	 ?An	 ?alternative	 ?technique	 ?is	 ?demonstrated	 ?for	 ?opening	 ?the	 ?tips	 ?using	 ?mechanical	 ?polishing	 ?of	 ?the	 ?tips	 ?with	 ?fine	 ?lapping	 ?paper.	 ? The	 ? microneedles	 ? prepared	 ? through	 ? this	 ? process	 ? are	 ? 250	 ??m	 ? tall.	 ? The	 ? needle	 ?strength	 ?of	 ?0.32	 ?N,	 ?found	 ?from	 ?compression	 ?tests,	 ?is	 ?shown	 ?to	 ?be	 ?sufficient	 ?for	 ?skin	 ?insertion	 ?without	 ? failure.	 ? To	 ? test	 ? the	 ? needles	 ? for	 ? drug	 ? delivery,	 ? fluorescent	 ? beads	 ? are	 ? injected	 ? six	 ?times	 ? into	 ? rabbit	 ? ear	 ? skin	 ? and	 ? their	 ? distribution	 ? is	 ? observed	 ? in	 ? deep	 ? skin	 ? tissue	 ? using	 ?confocal	 ? microscopy.	 ? The	 ? average	 ? injection	 ? depth	 ? of	 ? 104.8	 ??m	 ? is	 ? found	 ? from	 ? the	 ? mean	 ?value	 ?of	 ? the	 ? intensity	 ? distribution.	 ? It	 ? is	 ? shown	 ? that	 ? the	 ?microneedles	 ? clearly	 ? facilitate	 ? the	 ?delivery	 ? of	 ? the	 ? compounds	 ? past	 ? the	 ? stratum	 ? corneum.	 ? The	 ? process	 ? in	 ? Chapter	 ? 2	 ? allows	 ?108	 ?	 ?making	 ?needles	 ?up	 ?to	 ?a	 ?height	 ?of	 ?250	 ??m	 ?which	 ?may	 ?not	 ?be	 ?sufficient	 ?for	 ?some	 ?applications;	 ?the	 ?natural	 ?result	 ?of	 ?the	 ?polymer	 ?casting	 ?process	 ?is	 ?a	 ?curved-??shaped	 ?profile	 ?that	 ?gets	 ?thinner	 ?closer	 ?to	 ?the	 ?pillar	 ?tips.	 ?For	 ?taller	 ?than	 ?250	 ??m	 ?structures,	 ?this	 ?thickness	 ?is	 ?so	 ?small	 ?that	 ?the	 ?microneedles	 ? would	 ? not	 ? be	 ?mechanically	 ? rigid	 ? and	 ? thus	 ? their	 ? tips	 ? would	 ? bend	 ? or	 ? buckle	 ?upon	 ?attempting	 ?skin	 ?insertion.	 ?Chapter	 ?3	 ?of	 ? this	 ? thesis	 ?presents	 ?a	 ? fabrication	 ?procedure	 ? for	 ?making	 ?metallic	 ?microneedle	 ?with	 ?taller	 ?structures	 ?compared	 ?to	 ?the	 ?polymer	 ?ones	 ?in	 ?Chapter	 ?2.	 ?The	 ?solvent	 ?casting	 ?step	 ?is	 ?used	 ?to	 ?create	 ?an	 ?electrically	 ?conductive	 ?seed	 ?layer	 ?for	 ?subsequent	 ?metal	 ?deposition,	 ?to	 ?create	 ? metallic	 ? microneedles	 ? strong	 ? enough	 ? for	 ? skin	 ? penetration	 ? even	 ? at	 ? very	 ? tall	 ?dimensions.	 ?The	 ? fabrication	 ?process	 ?uses	 ?a	 ?similar	 ?array	 ?of	 ?molds	 ?presented	 ? in	 ?Chapter	 ?2,	 ?but	 ?with	 ?an	 ?additional	 ?structural	 ?layer	 ?added	 ?to	 ?the	 ?molds	 ?to	 ?improve	 ?their	 ?strength.	 ?PMMA	 ?filled	 ? with	 ? carbon	 ? black	 ? is	 ? used	 ? as	 ? the	 ? cast	 ? seed	 ? layer.	 ? The	 ? optimum	 ? carbon	 ? black	 ?percentage	 ?of	 ?30	 ?wt%	 ?in	 ?solid	 ?is	 ?found	 ?from	 ?a	 ?series	 ?of	 ?conductivity	 ?measurements.	 ?Through	 ?a	 ?plasma	 ?etching	 ?process,	 ?the	 ?conductive	 ?layer	 ?covering	 ?the	 ?pillar	 ?tips	 ?are	 ?physically	 ?etched	 ?exposing	 ?the	 ?pillar	 ?tips	 ?which	 ?later	 ?resulted	 ?in	 ?hollow	 ?needles.	 ?Next,	 ?nickel	 ?is	 ?electroplated	 ?on	 ?the	 ?conductive	 ?layer.	 ?Through	 ?some	 ?tests,	 ?the	 ?electroplating	 ?process	 ?is	 ?characterized	 ?for	 ?optimum	 ?deposition	 ?rate.	 ?After	 ?the	 ?electroplating,	 ?the	 ?microneedles	 ?are	 ?lifted	 ?off	 ?from	 ?the	 ?molds	 ?by	 ?chemically	 ?etching	 ? the	 ?conductive	 ? layer.	 ?Using	 ? this	 ?process,	 ?500	 ??m	 ?tall	 ?needles	 ?were	 ?made.	 ? The	 ?mechanical	 ? strength	 ?of	 ? the	 ?needles	 ?was	 ?evaluated	 ? to	 ?be	 ?4.2	 ?N	 ?and	 ?drug	 ?delivery	 ?was	 ?demonstrated	 ?by	 ?injection	 ?of	 ?fluorescent	 ?beads	 ?into	 ?pig	 ?skin.	 ?109	 ?	 ?Chapter	 ?4	 ?of	 ? this	 ? thesis	 ? presents	 ? a	 ?novel	 ?method	 ?used	 ? to	 ? characterize	 ?drug	 ?delivery	 ?with	 ?hollow	 ?microneedles.	 ?	 ?For	 ?this	 ?purpose,	 ?a	 ?fluorescent	 ?drug	 ?(doxorubicin)	 ?is	 ?first	 ?injected	 ?into	 ?pig	 ? skin	 ? using	 ? metallic	 ? microneedles,	 ? and	 ? then	 ? using	 ? confocal	 ? microscopy	 ? the	 ? intensity	 ?distribution	 ?of	 ?the	 ?drug	 ?is	 ?measured	 ?over	 ?time	 ?in	 ?the	 ?skin.	 ?The	 ?concentration	 ?distribution	 ?of	 ?the	 ?drug	 ?was	 ?then	 ?calculated	 ?from	 ?intensity	 ?data,	 ?and	 ?then	 ?compared	 ?to	 ?an	 ?analytical	 ?model	 ?based	 ?on	 ?Fick?s	 ? laws	 ?of	 ?diffusion.	 ?Through	 ?this	 ?comparison,	 ?the	 ?diffusion	 ?coefficient	 ?of	 ?the	 ?drug	 ? in	 ? the	 ? skin?s	 ? epidermal	 ? layer	 ? is	 ? obtained	 ? (4.61?10-??9	 ? cm2/s	 ? for	 ? fresh	 ? skin)	 ? and	 ? then	 ?compared	 ? with	 ? the	 ? values	 ? reported	 ? in	 ? the	 ? literature	 ? for	 ? other	 ? tissues.	 ? The	 ? diffusion	 ?coefficient	 ? of	 ? doxorubicin	 ? is	 ? also	 ? compared	 ? in	 ? skin	 ? specimens	 ? treated	 ?with	 ? three	 ? storage	 ?conditions	 ? (fresh,	 ? refrigerated,	 ? and	 ? frozen).	 ? It	 ? is	 ? found	 ? that	 ? freezing	 ? the	 ? skin	 ? may	 ?considerably	 ? alter	 ? the	 ? rate	 ? of	 ? drug	 ? diffusion	 ?within	 ? the	 ? skin,	 ? and	 ? thus	 ? should	 ? be	 ? avoided	 ?when	 ?performing	 ?similar	 ?studies.	 ?In	 ? the	 ? last	 ?chapter,	 ?we	 ?evaluate	 ?some	 ?potential	 ?ways	 ?of	 ? removing	 ? ISF	 ? from	 ?the	 ?skin	 ?using	 ?microneedles,	 ? through	 ? experimental	 ? trials.	 ? First,	 ? the	 ? solvent	 ? casting	 ? process	 ? used	 ? in	 ?Chapters	 ?2	 ?and	 ?3	 ?was	 ?modified	 ?to	 ?make	 ?solid	 ?microneedles	 ?from	 ?a	 ?highly	 ?water	 ?absorbent	 ?polymer	 ?material	 ? (HEC).	 ? Extraction	 ? of	 ? ISF	 ? was	 ? then	 ? demonstrated	 ? by	 ? applying	 ? the	 ? water	 ?absorbing	 ? microneedles	 ? to	 ? skin	 ? samples	 ? in	 ? vitro.	 ? Using	 ? arrays	 ? of	 ? microneedles	 ? (with	 ? 14	 ?needles),	 ?an	 ?average	 ?volume	 ?of	 ?0.77	 ??L	 ? is	 ?removed	 ?from	 ?skin.	 ?The	 ?solvent	 ?casting	 ?process	 ?was	 ?also	 ?used	 ?to	 ?make	 ?hollow	 ?microneedle	 ?made	 ?of	 ?a	 ?hydrophilic	 ?polymer	 ?(PVA)	 ?that	 ?can	 ?withstand	 ?swelling	 ?upon	 ?exposure	 ?to	 ?water	 ?or	 ?ISF.	 ?The	 ?purpose	 ?of	 ?developing	 ?these	 ?systems	 ?was	 ?using	 ? capillary	 ? forces	 ? to	 ? attract	 ? ISF	 ? from	 ? skin.	 ?However,	 ? successful	 ? sampling	 ?was	 ?not	 ?demonstrated	 ? through	 ? several	 ? trials.	 ? And	 ? finally,	 ? the	 ? metallic	 ? microneedles	 ? (array	 ? of	 ? 6	 ?110	 ?	 ?needles)	 ?made	 ?in	 ?Chapter	 ?3	 ?are	 ?used	 ?to	 ?remove	 ?small	 ?amount	 ?of	 ?ISF	 ?from	 ?skin	 ?by	 ?applying	 ?vacuum	 ? pressure	 ? using	 ? a	 ? custom	 ? made	 ? vacuum	 ? probe.	 ? Traces	 ? of	 ? ISF	 ? are	 ? observed	 ? on	 ?glucose	 ? strips	 ? positioned	 ? in	 ? the	 ? probe.	 ?However,	 ? the	 ? probe	 ?was	 ? not	 ? able	 ? to	 ? attract	 ? large	 ?enough	 ?amounts	 ?required	 ?for	 ?glucose	 ?concentration	 ?measurements.	 ?6.2 Future	 ?work	 ?This	 ? section	 ? discusses	 ? some	 ? future	 ? work	 ? that	 ? can	 ? be	 ? pursued	 ? to	 ? help	 ? improve	 ? the	 ?microneedle	 ? fabrication	 ? repeatability	 ? and	 ? yield,	 ? as	 ?well	 ? as	 ? its	 ? efficiency	 ? for	 ? drug	 ? delivery	 ?sampling	 ?of	 ?biological	 ?liquids.	 ?	 ?	 ?6.2.1 Microneedle	 ?process	 ?improvement	 ?6.2.1.1 Mold	 ?The	 ?mold	 ? is	 ? the	 ? backbone	 ? of	 ? the	 ?microneedle	 ? formation	 ? process	 ? and	 ? the	 ? shape	 ? of	 ?mold	 ?pillars	 ? affects	 ? the	 ? final	 ? shape	 ? and	 ? sharpness	 ?of	 ? the	 ?microneedles.	 ?At	 ? this	 ? stage,	 ? the	 ?mold	 ?structure	 ? is	 ? the	 ? most	 ? important	 ? factor	 ? that	 ? influences	 ? the	 ? process	 ? repeatability.	 ? 	 ? The	 ?inconsistency	 ? in	 ?the	 ?photolithography	 ?process	 ? is	 ?reflected	 ? in	 ?the	 ?variation	 ?observed	 ? in	 ?the	 ?height	 ? and	 ?width	 ? of	 ? the	 ? pillars	 ? even	 ? on	 ? the	 ? same	 ?wafer.	 ? This	 ? is	 ?mainly	 ? due	 ? to	 ? the	 ? poor	 ?performance	 ? of	 ? the	 ? photolithography	 ? equipment	 ? used	 ? in	 ? the	 ? cleanroom	 ? facility	 ? (i.e.	 ? the	 ?spinner,	 ?hotplates,	 ?and	 ?mask	 ?aligner).	 ?Therefore,	 ?for	 ?better	 ?molds,	 ?better	 ?photolithography	 ?equipment	 ?must	 ?be	 ?used.	 ?Alternatively,	 ? the	 ?mold	 ? can	 ?be	 ?made	 ? from	 ?other	 ?materials	 ? and	 ?through	 ?other	 ?fabrication	 ?techniques,	 ?such	 ?as	 ?the	 ?ones	 ?used	 ?for	 ?making	 ?solid	 ?microneedle	 ?systems.	 ?111	 ?	 ?In	 ?addition,	 ?as	 ?the	 ?mold	 ?pillars	 ?are	 ?subject	 ?to	 ?mechanical	 ?stress	 ?upon	 ?needle	 ?lift-??off,	 ?using	 ?a	 ?stronger	 ?mold	 ?alternative	 ?is	 ?recommended.	 ?Right	 ?now,	 ?the	 ?molds	 ?may	 ?be	 ?usable	 ?up	 ?to	 ?four	 ?or	 ?five	 ?runs,	 ?but	 ? increasing	 ?the	 ?usability	 ?can	 ?substantially	 ?reduce	 ?the	 ?costs	 ? in	 ?an	 ? industrial	 ?setting.	 ?In	 ?addition,	 ?having	 ?less	 ?number	 ?of	 ?pillars	 ?on	 ?the	 ?mold	 ?would	 ?increase	 ?the	 ?chances	 ?of	 ?more	 ?successful	 ?microneedle	 ?lift-??off	 ?leaving	 ?intact	 ?mold	 ?for	 ?subsequent	 ?fabrication.	 ?6.2.1.2 Structural	 ?polymers	 ?and	 ?polymer	 ?solutions	 ?Although	 ? the	 ? current	 ? material	 ? used	 ? for	 ? polymer	 ? microneedle	 ? fabrication	 ? is	 ? among	 ? the	 ?strongest	 ?polymers,	 ?investigating	 ?other	 ?options	 ?for	 ?the	 ?structural	 ?material	 ?would	 ?be	 ?useful	 ?especially	 ? for	 ? developing	 ? polymer	 ? microneedle	 ? taller	 ? than	 ? 250	 ??m.	 ? Possible	 ? options	 ? are	 ?using	 ? carbon	 ? nanotube-??based	 ? composites.	 ? In	 ? addition,	 ? for	 ? the	 ? conductive	 ? polymer	 ? in	 ?Chapter	 ?3,	 ?using	 ?other	 ?solvents	 ?(such	 ?as	 ?PM	 ?Acetate)	 ? instead	 ?of	 ?NMP	 ?eliminates	 ?the	 ?need	 ?for	 ?surfactant	 ?and	 ?also	 ?results	 ?in	 ?a	 ?more	 ?uniformly	 ?coated	 ?layer	 ?on	 ?the	 ?pillars.	 ?6.2.1.3 The	 ?electroplating	 ?process	 ?Nickel,	 ?which	 ?is	 ?the	 ?current	 ?metal	 ?used	 ?for	 ?microneedle	 ?fabrication,	 ?is	 ?not	 ?biocompatible	 ?as	 ?discussed	 ? in	 ? Chapter	 ? 3,	 ? and	 ? the	 ? needles	 ? are	 ? coated	 ? with	 ? an	 ? extra	 ? layer	 ? of	 ? gold	 ? for	 ?biocompatibility.	 ?While	 ?most	 ?electrodepositable	 ?pure	 ?metals	 ? that	 ?are	 ? inexpensive	 ?are	 ?not	 ?biocompatible,	 ? it	 ? is	 ?possible	 ? to	 ? form	 ?biocompatible	 ?alloys	 ? through	 ? the	 ?electroplating.	 ?This	 ?would	 ? eliminate	 ? a	 ? final	 ? gold	 ? coating	 ? step	 ? and	 ? reduce	 ? the	 ? fabrication	 ? duration.	 ? Some	 ?possible	 ?options	 ?for	 ?the	 ?metals	 ?are	 ?nickel-??chromium	 ?or	 ?cobalt-??chromium	 ?alloys.	 ?In	 ?addition	 ?to	 ?the	 ?material,	 ?it	 ?is	 ?recommended	 ?to	 ?use	 ?an	 ?electroplating	 ?station	 ?designed	 ?for	 ?MEMS	 ?application,	 ?for	 ?better	 ?control	 ?of	 ?the	 ?distances,	 ?electrode	 ?sizes,	 ?supply	 ?currents,	 ?and	 ?112	 ?	 ?electrolyte	 ? solution	 ? (i.e.	 ? its	 ? pH	 ? and	 ? purity).	 ? 	 ? In	 ? addition,	 ? it	 ? was	 ? observed	 ? that	 ? adding	 ? a	 ?brightener	 ? component	 ? to	 ? the	 ? nickel	 ? bath	 ? would	 ? not	 ? facilitate	 ? coating	 ? of	 ? the	 ? thinner	 ?conductive	 ?polymer	 ?parts,	 ?which	 ?should	 ?be	 ?taken	 ?into	 ?account.	 ?6.2.2 Investigating	 ?the	 ?optimum	 ?needle	 ?height,	 ?diameter,	 ?spacing,	 ?and	 ?array	 ?size	 ?Until	 ? now,	 ? the	 ? hollow	 ? microneedle	 ? technology	 ? has	 ? mostly	 ? focused	 ? on	 ? the	 ? fabrication	 ?techniques.	 ?Little	 ?has	 ?been	 ?done	 ?to	 ?study	 ?the	 ?optimum	 ?height,	 ?diameter	 ?(outer	 ?and	 ?inner),	 ?and	 ?spacing	 ?of	 ? the	 ?microneedle	 ?arrays	 ? that	 ?could	 ? facilitate	 ?effective	 ?drug	 ?delivery	 ?or	 ? fluid	 ?extraction.	 ?This	 ?also	 ?requires	 ?studying	 ?the	 ?skin	 ?mechanics	 ? in	 ?detail.	 ?Using	 ?taller	 ?needles	 ?to	 ?reach	 ? the	 ? dermis	 ? might	 ? be	 ? beneficial	 ? for	 ? both	 ? injection	 ? and	 ? extraction	 ? due	 ? to	 ? the	 ? low	 ?density	 ?of	 ?the	 ?dermis	 ?and	 ?its	 ?large	 ?amount	 ?of	 ?ISF,	 ?but	 ?at	 ?the	 ?same	 ?time	 ?it	 ?may	 ?cause	 ?pain.	 ?Using	 ?needles	 ?with	 ? larger	 ?diameters	 ?may	 ?be	 ?useful	 ? for	 ? injecting/extracting	 ?at	 ? faster	 ? rates	 ?but	 ? it	 ? may	 ? cause	 ? more	 ? nerve	 ? damage	 ? and	 ? be	 ? painful.	 ? In	 ? addition,	 ? using	 ? more	 ? densely	 ?arranged	 ?needles	 ?might	 ?be	 ?beneficial	 ?in	 ?terms	 ?of	 ?space	 ?occupation	 ?but	 ?at	 ?the	 ?same	 ?time	 ?it	 ?could	 ? lead	 ? to	 ? a	 ? bed-??of-??nails	 ? effect	 ? causing	 ?no	 ? skin	 ?penetration	 ?by	 ? the	 ?needles.	 ?One	 ?main	 ?advantage	 ? of	 ? the	 ? proposed	 ? processes	 ? here	 ? is	 ? that	 ? they	 ? are	 ? flexible	 ? and	 ? allow	 ? making	 ?microneedles	 ?with	 ?a	 ?wide	 ?range	 ?of	 ?dimensions,	 ?which	 ?would	 ?be	 ?useful	 ?when	 ?investigating	 ?the	 ?optimum	 ?dimensions.	 ?	 ?	 ?113	 ?	 ?6.2.3 Drug	 ?delivery	 ?using	 ?microneedles	 ?As	 ?discussed	 ?in	 ?Chapter	 ?4,	 ?the	 ?microneedle	 ?technology	 ?requires	 ?extensive	 ?research	 ?into	 ?the	 ?drugs	 ?and	 ?pharmaceutical	 ?agents	 ?to	 ?be	 ?delivered.	 ?As	 ?this	 ?technology	 ?relies	 ?on	 ?drug	 ?diffusion	 ?through	 ? the	 ? skin,	 ? understanding	 ? the	 ? chemical	 ? structure	 ? of	 ? drugs	 ? and	 ? their	 ? physical	 ?properties	 ?is	 ?important	 ?to	 ?design	 ?useful	 ?systems.	 ?In	 ?addition,	 ?additional	 ?research	 ?has	 ?to	 ?be	 ?carried	 ?out	 ?to	 ?find	 ?optimum	 ?injection	 ?pressures,	 ?rates,	 ?and	 ?volumes	 ?for	 ?specific	 ?skin	 ?layers.	 ?	 ?6.2.4 Biofluid	 ?sampling	 ?using	 ?microneedles	 ?The	 ? studies	 ? of	 ? fluid	 ? sampling	 ? with	 ? microneedles	 ? are	 ? at	 ? a	 ? preliminary	 ? stage.	 ? To	 ? develop	 ?consistent	 ?systems,	 ?more	 ?research	 ?has	 ?to	 ?be	 ?done	 ?to	 ?find	 ?the	 ?optimum	 ?target	 ?layer	 ?in	 ?the	 ?skin	 ?as	 ?well	 ?as	 ?the	 ?right	 ?needle	 ?material	 ?and	 ?dimensions.	 ?In	 ?addition,	 ?further	 ?research	 ?has	 ?to	 ?be	 ? carried	 ? out	 ? to	 ? investigate	 ? possible	 ? ways	 ? of	 ? treating	 ? skin	 ? to	 ? facilitate	 ? faster	 ? and	 ?more	 ?reliable	 ?fluid	 ?sampling.	 ?In	 ?addition,	 ?since	 ?we	 ?are	 ?dealing	 ?with	 ?very	 ?small	 ?amounts	 ?of	 ?liquid,	 ?very	 ?sensitive	 ?assays	 ?have	 ?to	 ?be	 ?developed	 ?in	 ?order	 ?to	 ?extract	 ?useful	 ?information	 ?from	 ?the	 ?sampled	 ?liquids.	 ? 	 ?114	 ?	 ?REFERENCES	 ?[1]	 ? M.	 ?Sch?fer-??Korting,	 ??Drug	 ?Delivery,?	 ?Handbook	 ?of	 ?Experimental	 ?Pharmacology,	 ?vol.	 ?197,	 ?Springer-??Verlag,	 ?Berlin,	 ?Heidelberg,	 ?2010.	 ?[2]	 ? National	 ? Cancer	 ? Institute.	 ? (1989).	 ? Skin	 ? [Online].	 ? Available	 ? FTP:	 ?http://visualsonline.cancer.gov/details.cfm?imageid=2496	 ?	 ?[3]	 ? J.	 ?Sandby-??M?ller	 ?,	 ?T.	 ?Poulsen	 ?and	 ?H.	 ?C.	 ?Wulf,	 ??Epidermal	 ?thickness	 ?at	 ?different	 ?body	 ?sites:	 ? relationship	 ? to	 ? age,	 ? gender,	 ? pigmentation,	 ? blood	 ? content,	 ? skin	 ? type	 ? and	 ?smoking	 ?habits,?	 ?Acta	 ?Dermato-??Venereologica.	 ?vol.	 ?83,	 ?pp.	 ?410?413,	 ?2003.	 ?[4]	 ? National	 ?Cancer	 ?Institute.	 ?(2001).	 ?Skin:	 ?Epidermis	 ?and	 ?Dermis	 ?[Online].	 ?Available	 ?FTP:	 ?http://visualsonline.cancer.gov/details.cfm?imageid=1785	 ?[5]	 ? A.	 ? B.	 ? Cua,	 ? K.	 ? P.	 ? Wilhelm	 ? and	 ? H.	 ? I.	 ? Maibach,	 ? ?Elastic	 ? properties	 ? of	 ? human	 ? skin:	 ?relation	 ? to	 ? age,	 ? sex,	 ? and	 ?anatomical	 ? region,?	 ?Archives	 ?of	 ?Dermatological	 ?Research,	 ?vol.	 ?282,	 ?pp.	 ?283?288,	 ?1990.	 ?[6]	 ? A.	 ? B.	 ? Cua,	 ? K.	 ? P.	 ? Wilhelm	 ? and	 ? H.	 ? I.	 ? Maibach,	 ? ?Skin	 ? surface	 ? lipid	 ? and	 ? skin	 ? friction:	 ?relation	 ?to	 ?age,	 ?sex	 ?and	 ?anatomical	 ?region,?	 ?Skin	 ?Pharmacology	 ?&	 ?Physiology,	 ?vol.	 ?8,	 ?pp.	 ?246?251,	 ?1995.	 ?[7]	 ? A.	 ? B.	 ? Cua,	 ? K.	 ? P.	 ?Wilhelm	 ? and	 ? H.	 ? I.	 ?Maibach,	 ? ?Frictional	 ? properties	 ? of	 ? human	 ? skin:	 ?relation	 ? to	 ? age,	 ? sex	 ? and	 ? anatomical	 ? region,	 ? stratum	 ? corneum	 ? hydration	 ? and	 ?transepidermal	 ?water	 ? loss,?	 ?British	 ? Journal	 ?of	 ?Dermatology,	 ? vol.	 ? 123,	 ?pp.	 ?473?479,	 ?115	 ?	 ?1990.	 ?[8]	 ? P.	 ?G.	 ?Agache,	 ?C.	 ?Monneur,	 ?J.	 ?L.	 ?Leveque	 ?and	 ?J.	 ?De	 ?Rigal,	 ??Mechanical	 ?properties	 ?and	 ?young?s	 ?modulus	 ?of	 ?human	 ?skin	 ? in	 ?vivo,?	 ?Archives	 ?of	 ?Dermatological	 ?Research,	 ?vol.	 ?269,	 ?pp.	 ?221-??232,	 ?1980.	 ?[9]	 ? C.	 ? Pailler-??Mattei,	 ? S.	 ? Bec	 ? and	 ? H.	 ? Zahouani,	 ? ?In	 ? vivo	 ? measurements	 ? of	 ? the	 ? elastic	 ?mechanical	 ?properties	 ?of	 ?human	 ?skin	 ?by	 ? indentation	 ?tests,?	 ?Medical	 ?Engineering	 ?&	 ?Physics,	 ?vol.	 ?30(5),	 ?pp.	 ?599-??606,	 ?2008.	 ?[10]	 ? M.	 ?R.	 ?Prausnitz	 ?and	 ?R.	 ?Langer,	 ??Transdermal	 ?drug	 ?delivery,?	 ?Nature	 ?Biotechnology,	 ?vol.	 ?26,	 ?pp.	 ?1261-??1268,	 ?2008.	 ?[11]	 ? S.	 ? Venkatraman	 ? and	 ? R.	 ? Gale,	 ? ?Skin	 ? adhesives	 ? and	 ? skin	 ? adhesion:	 ? 1.	 ? Transdermal	 ?drug	 ?delivery	 ?systems,?	 ?Biomaterials,	 ?vol.	 ?19,	 ?pp.	 ?1119-??1136,	 ?1998.	 ?[12]	 ? S.	 ? K.	 ? Govil	 ? and	 ? P.	 ? Kohiman,	 ? ?Transdermal	 ? Delivery	 ? of	 ? Nictoine,?	 ? US	 ? Patent:	 ?4908213,	 ?1990.	 ?[13]	 ? K.	 ? J.	 ? Miller,	 ? S.	 ? K.	 ? Govil,	 ? and	 ? K.	 ? S.	 ? Bhatia,	 ? ?Fentanyl	 ? Suspension-??based	 ? Silicone	 ?Adhesive	 ? Formulations	 ? and	 ? Devices	 ? for	 ? Transdermal	 ? Delivery	 ? of	 ? Fentanyl,?	 ? US	 ?Patent:	 ?7556823,	 ?2009.	 ?[14]	 ? Takeshi,	 ? T.	 ? Tetsuro,	 ? and	 ? H.	 ? Naruhito,	 ? ?Transdermal	 ? Patch	 ? for	 ? External	 ? Use	 ?Compromising	 ?Fentanyl,?	 ?International	 ?Patent:	 ?WO/2004/035054,	 ?2004.	 ?[15]	 ? S.	 ?K.	 ?Govil,	 ?E.	 ?M.	 ?Rudnic,	 ?and	 ?D.	 ?G.	 ?Sterner,	 ??Transdermal	 ?nitroglycerin	 ?patch	 ?with	 ?116	 ?	 ?penetration	 ?enhancers,?	 ?US	 ?Patent:	 ?5262165,	 ?1993.	 ?	 ?	 ?[16]	 ? R.	 ? J.	 ? Scheuplein,	 ? ?Permeability	 ? of	 ? the	 ? skin,?	 ? Comprehensive	 ? Physiology,	 ? Wiley,	 ?2011,	 ?pp.	 ?299-??322.	 ?[17]	 ? R.	 ?O.	 ?Potts	 ?and	 ?R.	 ?H.	 ?Guy,	 ??Predicting	 ?skin	 ?permeability,?	 ?Pharmaceutical	 ?Research,	 ?vol.	 ?9(5),	 ?pp.	 ?663-??669,	 ?1992.	 ?[18]	 ? R.	 ?J.	 ?Scheuplein	 ?and	 ? I.	 ?H.	 ?Blank,	 ??Permeability	 ?of	 ?the	 ?skin,?	 ?Physiological	 ?Reviews,	 ?vol.	 ?51(4),	 ?pp.	 ?702-??747,	 ?1971.	 ?[19]	 ? A.	 ?Naik,	 ?Y.	 ?N.	 ?Kalia	 ?and	 ?R.	 ?H.	 ?Guy,	 ??Transdermal	 ?drug	 ?delivery:	 ?overcoming	 ?the	 ?skin	 ?barrier?s	 ?function,?	 ?Pharmaceutical	 ?Science	 ?&	 ?Technology	 ?Today,	 ?vol.	 ?3,	 ?pp.	 ?318-??326,	 ?2000.	 ?[20]	 ? M.	 ?Murphy	 ? and	 ? A.	 ? J.	 ? Carmichael,	 ? ?Transdermal	 ? Drug	 ? Delivery	 ? Systems	 ? and	 ? Skin	 ?Sensitivity	 ? Reactions:	 ? Incidence	 ? and	 ? Management,?	 ? American	 ? Journal	 ? of	 ? Clinical	 ?Dermatology,	 ?vol.	 ?1,	 ?pp.	 ?361-??368,	 ?2000.	 ?[21]	 ? G.	 ?B.	 ?Kasting	 ?and	 ?L.	 ?A.	 ?Bowman,	 ??DC	 ?electrical	 ?properties	 ?of	 ?frozen,	 ?excised	 ?human	 ?skin,?	 ?Pharmaceutical	 ?Research,	 ?vol.	 ?7(2),	 ?pp	 ?134-??143,	 ?1990.	 ?[22]	 ? I.	 ? P.	 ? Dick	 ? and	 ? R.	 ? C.	 ? Scott,	 ? ?Pig	 ? ear	 ? skin	 ? as	 ? an	 ? in-??vitro	 ? model	 ? for	 ? human	 ? skin	 ?permeability,?	 ? Journal	 ? of	 ? Pharmacy	 ? and	 ? Pharmacology,	 ? vol.	 ? 44(8),	 ? pp.	 ? 640?645,	 ?1992.	 ?[23]	 ? P.	 ? Karande,	 ? A.	 ? Jain,	 ? and	 ? S.	 ? Mitragotri,	 ? ?Relationships	 ? between	 ? skin's	 ? electrical	 ?117	 ?	 ?impedance	 ? and	 ? permeability	 ? in	 ? the	 ? presence	 ? of	 ? chemical	 ? enhancers,?	 ? Journal	 ? of	 ?Controlled	 ?Release,	 ?vol.	 ?110(2),	 ?pp.	 ?307?313,	 ?2006.	 ?[24]	 ? M.	 ? R.	 ? Prausnitz,	 ? V.	 ? G.	 ? Bose,	 ? R.	 ? Langer,	 ? and	 ? J.	 ? C.	 ? Weaver,	 ? ?Electroporation	 ? of	 ?mammalian	 ?skin:	 ?A	 ?mechanism	 ?to	 ?enhance	 ?transdermal	 ?drug	 ?delivery,?	 ?Proceedings	 ?of	 ?the	 ?National	 ?Academy	 ?of	 ?Sciences,	 ?vol.	 ?90,	 ?pp.	 ?10504-??10508,	 ?1993.	 ?[25]	 ? F.	 ? P.	 ? Schmook,	 ? J.	 ? G.	 ?Meingassner,	 ? and	 ? A.	 ? Billich,	 ? ?Comparison	 ? of	 ? human	 ? skin	 ? or	 ?epidermis	 ?models	 ?with	 ?human	 ?and	 ?animal	 ?skin	 ?in	 ?in-??vitro	 ?percutaneous	 ?absorption,?	 ?International	 ?Journal	 ?of	 ?Pharmaceutics,	 ?vol.	 ?215(1?2),	 ?pp.	 ?51?56,	 ?2001.	 ?[26]	 ? M.	 ? Kirjavainen,	 ? A.	 ? Urtti,	 ? R.	 ? Valjakka-??Koskela,	 ? J.	 ? Kiesvaara,	 ? and	 ? J.	 ? M?nkk?nen,	 ??Liposome?skin	 ? interactions	 ? and	 ? their	 ? effects	 ? on	 ? the	 ? skin	 ? permeation	 ? of	 ? drugs,?	 ?European	 ?Journal	 ?of	 ?Pharmaceutical	 ?Sciences,	 ?vol.	 ?7(4),	 ?pp.	 ?279?286,	 ?1999.	 ?[27]	 ? E.	 ? R.	 ? Cooper,	 ? ?Increased	 ? skin	 ? permeability	 ? for	 ? lipophilic	 ? molecules,?	 ? Journal	 ? of	 ?Pharmaceutical	 ?Sciences,	 ?vol.	 ?73(8),	 ?pp.	 ?1153-??1156,	 ?1984.	 ?[28]	 ? H.	 ?Herai,	 ?T.	 ?Gratieri,	 ?J.	 ?A.	 ?Thomazine,	 ?M.	 ?V.	 ?Bentley,	 ?and	 ?R.	 ?F.	 ?Lopez,	 ??Doxorubicin	 ?skin	 ? penetration	 ? from	 ? monoolein-??containing	 ? propylene	 ? glycol	 ? formulations,?	 ?International	 ?Journal	 ?of	 ?Pharmaceutics,	 ?vol.	 ?329(1?2),	 ?pp.	 ?88?93,	 ?2007.	 ?[29]	 ? P.	 ?Odraska,	 ? E.	 ?Mazurova,	 ? L.	 ? Dolezalova,	 ? and	 ? L.	 ? Blaha,	 ? ?In	 ? vitro	 ? evaluation	 ? of	 ? the	 ?permeation	 ? of	 ? cytotoxic	 ? drugs	 ? through	 ? reconstructed	 ? human	 ? epidermis	 ? and	 ? oral	 ?epithelium,?	 ?Klin	 ?Onkol,	 ?vol.	 ?24(3):	 ?pp.	 ?195?202,	 ?2011.	 ?118	 ?	 ?[30]	 ? S.	 ?F.	 ?Taveira,	 ?A.	 ?Nomizo,	 ?and	 ?R.	 ?F.	 ?Lopez,	 ??Effect	 ?of	 ?the	 ?iontophoresis	 ?of	 ?a	 ?chitosan	 ?gel	 ?on	 ?doxorubicin	 ?skin	 ?penetration	 ?and	 ?cytotoxicity,?	 ?Journal	 ?of	 ?Controlled	 ?Release,	 ?vol.	 ?134(1),	 ?pp.	 ?35?40,	 ?2009.	 ?	 ?[31]	 ? E.	 ?Khalil,	 ?K.	 ?Kretsos,	 ?and	 ?G.	 ?B.	 ?Kasting,	 ??Glucose	 ?partition	 ?coefficient	 ?and	 ?diffusivity	 ?in	 ?the	 ?lower	 ?skin	 ?layers,?	 ?Pharmaceutical	 ?Research,	 ?vol.	 ?23(6),	 ?pp.	 ?1227-??1234,	 ?2006.	 ?[32]	 ? A.	 ?C.	 ?Williams	 ?and	 ?B.	 ?W.	 ?Barry,	 ??Terpenes	 ?and	 ?the	 ?lipid-??protein-??partitioning	 ?theory	 ?of	 ? skin	 ? penetration	 ? enhancement,?	 ? Pharmaceutical	 ? Research,	 ? vol.	 ? 8(1),	 ? pp.	 ? 17-??24,	 ?1991.	 ?[33]	 ? M.	 ?Kreilgaarda,	 ?E.	 ?J.	 ?Pedersenb,	 ?and	 ?J.	 ?W.	 ?Jaroszewski,	 ??NMR	 ?characterisation	 ?and	 ?transdermal	 ? drug	 ? delivery	 ? potential	 ? of	 ? microemulsion	 ? systems,?	 ? Journal	 ? of	 ?Controlled	 ?Release,	 ?vol.	 ?69(3),	 ?pp.	 ?421?433,	 ?2000.	 ?	 ?[34]	 ? S.	 ?Hansen,	 ?A.	 ?Henning,	 ?A.	 ?Naegel,	 ?M.	 ?Heisig,	 ?G.	 ?Wittum,	 ?D.	 ?Neumann,	 ?K.	 ?Kostka,	 ?J.	 ?Zbytovska,	 ?C.	 ?Lehr,	 ?and	 ?U.	 ?F.	 ?Schaefer,	 ??In-??silico	 ?model	 ?of	 ?skin	 ?penetration	 ?based	 ?on	 ?experimentally	 ?determined	 ?input	 ?parameters.	 ?Part	 ?I:	 ?Experimental	 ?determination	 ?of	 ?partition	 ? and	 ? diffusion	 ? coefficients,?	 ? European	 ? Journal	 ? of	 ? Pharmaceutics	 ? and	 ?Biopharmaceutics,	 ?vol.	 ?68(2),	 ?pp.	 ?352?367,	 ?2008.	 ?[35]	 ? S.	 ? Mitragotri,	 ? ?Effect	 ? of	 ? therapeutic	 ? ultrasound	 ? on	 ? partition	 ? and	 ? diffusion	 ?coefficients	 ? in	 ?human	 ? stratum	 ?corneum,?	 ? Journal	 ? of	 ? Controlled	 ?Release,	 ? vol.	 ? 71(1),	 ?pp.	 ?23?29,	 ?2001.	 ?	 ?[36]	 ? R.	 ?Alvarez-??Rom?n,	 ?A.	 ?Naik,	 ?Y.	 ?N.	 ?Kalia,	 ?H.	 ?Fessi,	 ?and	 ?R.	 ?H.	 ?Guy,	 ??Visualization	 ?of	 ?skin	 ?119	 ?	 ?penetration	 ? using	 ? confocal	 ? laser	 ? scanning	 ? microscopy,?	 ? European	 ? Journal	 ? of	 ?Pharmaceutics	 ?and	 ?Biopharmaceutics,	 ?vol.	 ?58,	 ?pp.	 ?301?316,	 ?2004.	 ?[37]	 ? A.	 ?Sch?tzlein	 ?and	 ?G.	 ?Cevc,	 ??Non-??uniform	 ?cellular	 ?packing	 ?of	 ?the	 ?stratum	 ?corneum	 ?and	 ? permeability	 ? barrier	 ? function	 ? of	 ? intact	 ? skin:	 ? a	 ? high-??resolution	 ? confocal	 ? laser	 ?scanning	 ? microscopy	 ? study	 ? using	 ? highly	 ? deformable	 ? vesicles	 ? (Transfersomes),?	 ?British	 ?Journal	 ?of	 ?Dermatology,	 ?vol.	 ?138,	 ?pp.	 ?583?592,	 ?1998.	 ?[38]	 ? F.	 ?Scarmato	 ?De	 ?Rosa,	 ?J.	 ?M.	 ?Marchetti,	 ?J.	 ?A.o	 ?Thomazini,	 ?A.	 ?C.	 ?Tedesco,	 ?M.	 ?V.	 ?Lopes,	 ?and	 ? B.	 ? Bentley,	 ? ?A	 ? vehicle	 ? for	 ? photodynamic	 ? therapy	 ? of	 ? skin	 ? cancer:	 ? influence	 ? of	 ?dimethylsulphoxide	 ?on	 ?5-??aminolevulinic	 ? acid	 ? in	 ? vitro	 ? cutaneous	 ?permeation	 ?and	 ? in	 ?vivo	 ?protoporphyrin	 ? IX	 ? accumulation	 ?determined	 ?by	 ? confocal	 ?microscopy,?	 ? Journal	 ?of	 ?Controlled	 ?Release,	 ?vol.	 ?65,	 ?pp.	 ?359?366,	 ?2000.	 ?[39]	 ? D.	 ? D.	 ? Verma,	 ? S.	 ? Verma,	 ? G.	 ? Blume,	 ? and	 ? A.	 ? Fahr,	 ? ?Liposomes	 ? increase	 ? skin	 ?penetration	 ? of	 ? entrapped	 ? and	 ? non-??entrapped	 ? hydrophilic	 ? substances	 ? into	 ? human	 ?skin:	 ? a	 ? skin	 ? penetration	 ? and	 ? confocal	 ? laser	 ? scanning	 ?microscopy	 ? study,?	 ? European	 ?Journal	 ?of	 ?Pharmaceutics	 ?and	 ?Biopharmaceutics,	 ?vol.	 ?55,	 ?pp.	 ?271?277,	 ?2003.	 ?[40]	 ? A.	 ? P.	 ? Raphaela,	 ? S.	 ? C.	 ? Meligaa,	 ? X.	 ? Chena,	 ? G.	 ? J.	 ? P.	 ? Fernandoa,	 ? C.	 ? Flaima,	 ? M.	 ? A.	 ? F.	 ?Kendalla,	 ??Depth-??resolved	 ?characterization	 ?of	 ?diffusion	 ?properties	 ?within	 ?and	 ?across	 ?minimally-??perturbed	 ?skin	 ?layers,?	 ?Journal	 ?of	 ?Controlled	 ?Release,	 ?vol.	 ?166(2),	 ?pp.	 ?87?94,	 ?2013.	 ?[41]	 ? N.	 ?Maluf,	 ?An	 ?Introduction	 ?to	 ?Microelectromechanical	 ?Systems	 ?Engineering,	 ?2nd	 ?Ed.,	 ?120	 ?	 ?Artech	 ?House,	 ?2000.	 ?[42]	 ? S.	 ?D.	 ?Senturia,	 ?Microsystem	 ?Design,	 ?Springer,	 ?2005.	 ?[43]	 ? M.	 ?J.	 ?Madou,	 ?Fundamentals	 ?of	 ?Microfabrication	 ?and	 ?Nanotechnology,	 ?3rd	 ?Ed.,	 ?CRC	 ?Press,	 ?2011.	 ?[44]	 ? A.	 ?Folch,	 ?Introduction	 ?to	 ?BioMEMS,	 ?CRC	 ?Press,	 ?2012.	 ?[45]	 ? M.	 ? Bernadete	 ? Riemma	 ? Pierre	 ? and	 ? F.	 ? Cristina	 ? Rossetti,	 ? ?Microneedle-??Based	 ? Drug	 ?Delivery	 ?Systems	 ?for	 ?Transdermal	 ?Route,?	 ?Current	 ?Drug	 ?Targets,	 ?vol.	 ?15(3),	 ?pp.	 ?281-??291,	 ?2014.	 ?[46]	 ? 	 ?J.	 ?J.	 ?Norman,	 ?S.	 ?O.	 ?Choi,	 ?N.	 ?T.	 ?Tong,	 ?A.	 ?R.	 ?Aiyar,	 ?S.	 ?R.	 ?Patel,	 ?M.	 ?R.	 ?Prausnitz,	 ?and	 ?M.	 ?G.	 ? Allen,	 ? ?Hollow	 ? microneedles	 ? for	 ? intradermal	 ? injection	 ? fabricated	 ? by	 ? sacrificial	 ?micromolding	 ?and	 ?selective	 ?electrodeposition,?	 ?Biomedical	 ?Microdevices,	 ? vol	 ?15(2),	 ?pp.	 ?203-??210,	 ?2013.	 ?	 ?[47]	 ? K.	 ?van	 ?der	 ?Maaden,	 ?S.	 ?J.	 ?Trietsch,	 ?H.	 ?Kraan,	 ?E.	 ?M.	 ?Varypataki,	 ?S.	 ?Romeijn,	 ?R.	 ?Zwier,	 ?H.	 ?J.	 ?van	 ?der	 ?Linden,	 ?G.	 ?Kersten,	 ?T.	 ?Hankemeier,	 ?W.	 ?Jiskoot,	 ?and	 ?J.	 ?Bouwstra,	 ??Novel	 ?Hollow	 ? Microneedle	 ? Technology	 ? for	 ? Depth-??Controlled	 ? Microinjection-??Mediated	 ?Dermal	 ?Vaccination:	 ?A	 ?Study	 ?with	 ?Polio	 ?Vaccine	 ? in	 ?Rats,?	 ?Pharmaceutical	 ?Research,	 ?10.1007/s11095-??013-??1288-??9,	 ?2014.	 ?[48]	 ? E.V.	 ? Mukerjee,	 ? S.D.	 ? Collins,	 ? R.R.	 ? Isseroff	 ? and	 ? R.L.	 ? Smith,	 ? ?Microneedle	 ? array	 ? for	 ?transdermal	 ?biological	 ?fluid	 ?extraction	 ?and	 ?in	 ?situ	 ?analysis,?	 ?Sensors	 ?and	 ?Actuators	 ?A:	 ?121	 ?	 ?Physical,	 ?vol.	 ?114,	 ?pp.	 ?267-??275,	 ?2004.	 ?[49]	 ? M.	 ? HajjHassan,	 ? V.	 ? Chodavarapu	 ? and	 ? S.	 ? Musallam,	 ? ?NeuroMEMS:	 ? neural	 ? probe	 ?microtechnologies,?	 ?Sensors,	 ?vol.	 ?8(10),	 ?pp.	 ?6704-??6726,	 ?2008.	 ?[50]	 ? R.	 ?K.	 ?Sivamani,	 ?B.	 ?Stoeber,	 ?G.	 ?C.	 ?Wu,	 ?H.	 ?Zhai,	 ?D.	 ?Liepmann	 ?and	 ?H.	 ?Maibach,	 ??Clinical	 ?Microneedle	 ? Injection	 ? of	 ? Methyl	 ? Nicotinate:	 ? Stratum	 ? Corneum	 ? Penetration?,	 ? Skin	 ?Research	 ?and	 ?Technology,	 ?vol.	 ?11,	 ?pp.	 ?152-??156,	 ?2005.	 ?[51]	 ? J.	 ?Chen,	 ?K.	 ?D.	 ?Wise,	 ?J.	 ?F.	 ?Hetke	 ?and	 ?S.	 ?C.	 ?Bledsoe,	 ?Jr.,	 ?"A	 ?multichannel	 ?neural	 ?probe	 ?for	 ?selective	 ?chemical	 ?delivery	 ?at	 ?the	 ?cellular	 ?level,"	 ?IEEE	 ?Transactions	 ?on	 ?Biomedical	 ?Engineering,	 ?vol.	 ?44,	 ?pp.	 ?760-??769,	 ?1997.	 ?[52]	 ? L.	 ? Lin	 ? and	 ? A.	 ? P.	 ? Pisano,	 ? ?Silicon	 ? processed	 ? microneedles,?	 ? Journal	 ? of	 ?Microelectromechanical	 ?Systems,	 ?vol.	 ?8(1),	 ?pp.	 ?78-??84,	 ?1999.	 ?[53]	 ? J.	 ?D.	 ?Zahn,	 ?N.	 ?H.	 ?Talbot,	 ?D.	 ?Liepmann,	 ?and	 ?A.	 ?P.	 ?Pisano,	 ??Microfabricated	 ?Polysilicon	 ?Microneedles	 ?for	 ?Minimally	 ?Invasive	 ?Biomedical	 ?Devices,?	 ?Biomedical	 ?Microdevices,	 ?vol	 ?2,	 ?pp.	 ?295-??303,	 ?2000.	 ?[54]	 ? S.	 ?J.	 ?Paik,	 ?S.	 ?Byun,	 ?J.	 ?M.	 ?Lim,	 ?Y.	 ?Park,	 ?A.	 ?Lee,	 ?S.	 ?Chung,	 ?J.	 ?Chang,	 ?K.	 ?Chun,	 ?and	 ?D.	 ?Cho,	 ??In-??plane	 ? single-??crystal-??silicon	 ? microneedles	 ? for	 ? minimally	 ? invasive	 ? microfluid	 ?systems,?	 ?Sensors	 ?and	 ?Actuators	 ?A:	 ?Physical,	 ?vol.	 ?114,	 ?pp.	 ?276-??284,	 ?2004.	 ?[55]	 ? B.	 ?Stoeber	 ?and	 ?D.	 ?Liepmann,	 ??Fluid	 ?injection	 ?through	 ?out-??of-??plane	 ?microneedles,?	 ?1st	 ?Annual	 ?International,	 ?Conference	 ?On	 ?Microtechnologies	 ?in	 ?Medicine	 ?and	 ?Biology,	 ?122	 ?	 ?pp.	 ?224-??228,	 ?2000.	 ?[56]	 ? B.	 ?Stoeber	 ?and	 ?D.	 ?Liepmann,	 ??Arrays	 ?of	 ?hollow	 ?out-??of-??plane	 ?microneedles	 ?for	 ?drug	 ?delivery,?	 ?Journal	 ?of	 ?Microelectromechanical	 ?Systems,	 ?vol.	 ?14,	 ?pp.	 ?472-??479,	 ?2005.	 ?[57]	 ? S.	 ? Henry,	 ? D.	 ? V.	 ? Mcallister,	 ? M.	 ? G.	 ? Allen,	 ? and	 ? M.	 ? R.	 ? Prausnitz,	 ? ?Microfabricated	 ?microneedles:	 ? A	 ? novel	 ? approach	 ? to	 ? transdermal	 ? drug	 ? delivery,?	 ? Journal	 ? of	 ?Pharmaceutical	 ?Sciences,	 ?vol.	 ?87,	 ?pp.	 ?922-??925,	 ?1998.	 ?[58]	 ? W.	 ?Martanto,	 ?S.	 ?Davis,	 ?N.	 ?Holiday,	 ?J.	 ?Wang,	 ?H.	 ?Gill,	 ?and	 ?M.	 ?Prausnitz,	 ??Transdermal	 ?delivery	 ?of	 ?insulin	 ?using	 ?microneedles	 ?in	 ?vivo,?	 ?Pharmaceutical	 ?Research,	 ?vol.	 ?21,	 ?pp.	 ?947-??952,	 ?2004.	 ?[59]	 ? J.A.	 ?Mikszta,	 ?J.B.	 ?Alarcon,	 ?J.M.	 ?Brittingham,	 ?D.E.	 ?Sutter,	 ?R.J.	 ?Pettis,	 ?and	 ?N.G.	 ?Harvey,	 ??Improved	 ? genetic	 ? immunization	 ? via	 ? micromechanical	 ? disruption	 ? of	 ? skinbarrier	 ?function	 ? and	 ? targeted	 ? epidermal	 ? delivery,?	 ?Nature	 ?Medicine,	 ? vol.	 ? 8,	 ? pp.	 ? 415-??	 ? 419,	 ?2002.	 ?[60]	 ? H.	 ?J.	 ?G.	 ?E.	 ?Gardeniers,	 ?R.	 ?Luttge,	 ?E.	 ?J.	 ?W.	 ?Berenschot,	 ?M.	 ?J.	 ?de	 ?Boer,	 ?S.	 ?Y.	 ?Yeshurun,	 ?M.	 ? Hefetz,	 ? R.van?t	 ? Oever,	 ? and	 ? A.	 ? van	 ? den	 ? Berg,	 ? ?Silicon	 ? micromachined	 ? hollow	 ?microneedles	 ? for	 ? transdermal	 ? liquid	 ? transport,?	 ? Journal	 ? of	 ?Microelectromechanical	 ?Systems,	 ?vol.	 ?12,	 ?pp.	 ?855-??862,	 ?2003.	 ?[61]	 ? J.	 ?Brazzle,	 ?D.	 ?Bartholomeusz,	 ?R.	 ?Davies,	 ?and	 ?J.	 ?Andrade,	 ??Active	 ?microneedles	 ?with	 ?integrated	 ?functionality,?	 ?Proceeding	 ?of	 ?Solid	 ?State	 ?Sensors	 ?and	 ?Actuators	 ?Workshop,	 ?Hilton	 ?Head,	 ?pp.	 ?199-??202,	 ?2000.	 ?123	 ?	 ?[62]	 ? J.	 ?Ji,	 ?F.	 ?E.H.	 ?Tay,	 ?and	 ?J.	 ?Miao,	 ??Microfabricated	 ?Hollow	 ?Microneedle	 ?Array	 ?Using	 ?ICP	 ?Etcher,?	 ?Journal	 ?of	 ?Physics:	 ?Conference	 ?Series,	 ?vol.	 ?34,	 ?pp.	 ?1132-??1136,	 ?2006.	 ?[63]	 ? P.	 ?Griss	 ?and	 ?G.	 ?Stemme,	 ??Side-??opened	 ?out-??of-??plane	 ?microneedles	 ? for	 ?microfluidic	 ?transdermal	 ?liquid	 ?transfer,?	 ?Journal	 ?of	 ?Microelectromechanical	 ?Systems,	 ?vol.	 ?12,	 ?pp.	 ?296-??301,	 ?2003.	 ?[64]	 ? M.	 ? Shikida,	 ? M.	 ? Ando,	 ? Y.	 ? Ishihara,	 ? T.	 ? Ando,	 ? K.	 ? Sato,	 ? and	 ? K.	 ? Asaumi,	 ??Nonphotolithographic	 ? pattern	 ? transfer	 ? for	 ? fabricating	 ? pen-??shaped	 ? microneedle	 ?structures,?	 ? Journal	 ? of	 ? Micromechanics	 ? and	 ? Microengineering,	 ? vol.	 ? 14,	 ? pp.	 ? 1462-??1467,	 ?2004.	 ?[65]	 ? P.	 ?Jur???ek,	 ?H.	 ?Zou,	 ?S.	 ?Zhang,	 ?C.	 ?Liu,	 ??Design	 ?and	 ?fabrication	 ?of	 ?hollow	 ?out-??of-??plane	 ?silicon	 ?microneedles,?	 ?Micro	 ?&	 ?Nano	 ?Letters,	 ?vol.	 ?8(2),	 ?pp.	 ?78-??81,	 ?2013.	 ?[66]	 ? K.	 ?Takei,	 ?T.	 ?Kawashima,	 ?T.	 ?Kawano,	 ?H.	 ?Kaneko,	 ?K.	 ?Sawada	 ?and,	 ?M	 ?Ishida,	 ??Out-??of-??plane	 ?microtube	 ?arrays	 ?for	 ?drug	 ?delivery	 ??	 ?liquid	 ?flow	 ?properties	 ?and	 ?an	 ?application	 ?to	 ?the	 ?nerve	 ?block	 ?test,?	 ?Biomedical	 ?Microdevices,	 ?vol.	 ?11,	 ?pp.	 ?539?545,	 ?2009.	 ?[67]	 ? K.	 ?van	 ?der	 ?Maaden,	 ?S.	 ?J.	 ?Trietsch,	 ?H.	 ?Kraan,	 ?E.	 ?M.	 ?Varypataki,	 ?S.	 ?Romeijn,	 ?R.	 ?Zwier,	 ?H.	 ?J.	 ?van	 ?der	 ?Linden,	 ?G.	 ?Kersten,	 ?T.	 ?Hankemeier,	 ?W.	 ?Jiskoot,	 ?and	 ?J.	 ?Bouwstra,	 ??Novel	 ?Hollow	 ? Microneedle	 ? Technology	 ? for	 ? Depth-??Controlled	 ? Microinjection-??Mediated	 ?Dermal	 ?Vaccination:	 ?A	 ?Study	 ?with	 ?Polio	 ?Vaccine	 ? in	 ?Rats,?	 ?Pharmaceutical	 ?Research,	 ?DOI:	 ?10.1007/s11095-??013-??1288-??9,	 ?pp.	 ?1-??9,	 ?2014.	 ?[68]	 ? K.	 ? T.	 ? Brown	 ? and	 ? D.	 ? G.	 ? Flaming,	 ? Advanced	 ? Micropipette	 ? Techniques	 ? for	 ? Cell	 ?124	 ?	 ?Physiology,	 ?Wiley,	 ?New	 ?York,	 ?1986.	 ?[69]	 ? J.	 ? D.	 ? Brazzle,	 ? I.	 ? Papautsky,	 ? and	 ? A.	 ? B.	 ? Frazier,	 ? ?Hollow	 ? Metallic	 ? Micromachined	 ?Needle	 ?Arrays,?	 ?Biomedical	 ?Microdevices,	 ?vol.	 ?2,	 ?pp.	 ?197-??205,	 ?2000.	 ?[70]	 ? K.	 ?Kim,	 ?D.	 ?S.	 ?Park,	 ?H.	 ?M.	 ?Lu,W.	 ?Che,	 ?K.	 ?Kim,	 ?J.	 ?Lee,	 ?and	 ?C.	 ?H.	 ?Ahn,	 ??A	 ?tapered	 ?hollow	 ?metallic	 ? microneedle	 ? array	 ? using	 ? backside	 ? exposure	 ? of	 ? SU-??8,?	 ? Journal	 ? of	 ?Micromechanics	 ?and	 ?Microengineering,	 ?vol.	 ?14,	 ?pp.	 ?597-??603,	 ?2004.	 ?	 ?[71]	 ? J.A.	 ?Matriano,	 ?M.	 ?Cormier,	 ?J.	 ?Johnson,	 ?W.A.	 ?Young,	 ?M.	 ?Buttery,	 ?K.	 ?Nyam,	 ?and	 ?P.E.	 ?Daddona,	 ? ?Macroflux	 ?microprojection	 ? array	 ? patch	 ? technology:	 ? a	 ? new	 ?and	 ? efficient	 ?approach	 ? for	 ? intracutaneous	 ? immunization,?	 ?Pharmaceutical	 ?Research,	 ? vol.	 ? 19,	 ?pp.	 ?63-??70,	 ?2002.	 ?[72]	 ? S.	 ?Chandrasekaran	 ?and	 ?A.	 ?B.	 ?Frazier,	 ? ?Characterization	 ?of	 ?Surface	 ?Micromachined	 ?Metallic	 ?Microneedles,?	 ?Journal	 ?of	 ?Microelectromechanical	 ?Systems,	 ?vol.	 ?12,	 ?pp.	 ?289-??295,	 ?2003.	 ?[73]	 ? S.	 ?P.	 ?Davis,	 ?W.	 ?Martanto,	 ?M.	 ?Allen	 ?and	 ?M.	 ?Prausnitz,	 ?"Hollow	 ?metal	 ?microneedles	 ?for	 ? insulin	 ? delivery	 ? to	 ? diabetic	 ? rats,"	 ? IEEE	 ? Transactions	 ? on	 ? Biomedical	 ? Engineering,	 ?vol.	 ?52,	 ?pp.	 ?909-??915,	 ?2005.	 ?[74]	 ? K.	 ?Kobayashi	 ?and	 ?H.	 ?Sizuki,	 ??A	 ?sampling	 ?mechanism	 ?employing	 ?the	 ?phase	 ?transition	 ?of	 ?a	 ?gel	 ?and	 ?its	 ?application	 ?to	 ?a	 ?micro	 ?analysis	 ?system	 ?imitating	 ?a	 ?mosquito,?	 ?Sensors	 ?and	 ?Actuators	 ?B:	 ?Chemical,	 ?vol.	 ?80,	 ?pp.	 ?1-??8,	 ?2001.	 ?125	 ?	 ?[75]	 ? D.	 ?V.	 ?McAllister,	 ?P.	 ?M.Wang,	 ?S.	 ?P.	 ?Davis,	 ?J.H.	 ?Park,	 ?P.	 ?J.	 ?Canatella,	 ?M.	 ?G.	 ?Allen,	 ?and	 ?M.	 ? R.	 ? Prausnitz,	 ? ?Microfabricated	 ? needles	 ? for	 ? transdermal	 ? delivery	 ? of	 ?macromolecules	 ? and	 ? nanoparticles:	 ? fabrication	 ? methods	 ? and	 ? transport	 ? studies,?	 ?Proceedings	 ?of	 ?National	 ?Academy	 ?of	 ? Sciences	 ?of	 ? the	 ?United	 ?States	 ?of	 ?America,	 ? vol.	 ?100,	 ?pp.	 ?13755-??13760,	 ?2003.	 ?[76]	 ? C.	 ? G.	 ? Li,	 ? C.	 ? Y.	 ? Lee,	 ? K.	 ? Lee	 ? and	 ? H.	 ? Jung,	 ? ?An	 ? optimized	 ? hollow	 ? microneedle	 ? for	 ?minimally	 ?invasive	 ?blood	 ?extraction,?	 ?Biomedical	 ?Microdevices,	 ?vol.	 ?15(1),	 ?pp.	 ?17-??25,	 ?2013.	 ?[77]	 ? S.	 ? Takeuchi,	 ? D.	 ? Ziegler,	 ? Y.	 ? Yoshida,	 ? K.	 ?Mabuchi,	 ? and	 ? T.	 ? Suzuki,	 ? "Parylene	 ? flexible	 ?neural	 ?probes	 ?integrated	 ?with	 ?microfluidic	 ?channels,"	 ?Lab	 ?on	 ?a	 ?Chip,	 ?vol.	 ?5,	 ?pp.	 ?519-??523,	 ?2005.	 ?[78]	 ? S.	 ?J.	 ?Moon,	 ?S.	 ?S.	 ?Lee,	 ?H.	 ?S.	 ?Lee,	 ?and	 ?T.	 ?H.	 ?Kwon,	 ??Fabrication	 ?of	 ?microneedle	 ?array	 ?using	 ?LIGA	 ?and	 ?hot	 ?embossing	 ?process,?	 ?Microsystem	 ?Technologies,	 ?vol.	 ?11,	 ?pp.	 ?311-??318,	 ?2005.	 ?[79]	 ? J.	 ? Park,	 ? M.	 ? G.	 ? Allen,	 ? M.	 ? R.	 ? Prausnitz,	 ? ?Biodegradable	 ? polymer	 ? microneedles:	 ?Fabrication,	 ? mechanics	 ? and	 ? transdermal	 ? drug	 ? delivery,?	 ? Journal	 ? of	 ? Controlled	 ?Release,	 ?vol.	 ?104,	 ?pp.	 ?51-??66,	 ?2005.	 ?[80]	 ? M.	 ?Han,	 ?D.	 ?Hyun,	 ?H.	 ?Park,	 ?S.	 ?S	 ?Lee,	 ?C.	 ?Kim,	 ?and	 ?C.	 ?Kim,	 ??A	 ?novel	 ?fabrication	 ?process	 ?for	 ? out-??of-??plane	 ? microneedle	 ? sheets	 ? of	 ? biocompatible	 ? polymer,?	 ? Journal	 ? of	 ?Micromechanics	 ?and	 ?Microengineering,	 ?vol.	 ?17,	 ?pp.	 ?1184-??1191,	 ?2007.	 ?126	 ?	 ?[81]	 ? H.	 ?Huang	 ?and	 ?C.	 ?Fu,	 ??Different	 ?fabrication	 ?methods	 ?of	 ?out-??of-??plane	 ?polymer	 ?hollow	 ?needle	 ? arrays	 ? and	 ? their	 ? variations,?	 ? Journal	 ? of	 ? Micromechanics	 ? and	 ?Microengineering,	 ?vol.	 ?17,	 ?pp.	 ?393-??402,	 ?2007.	 ?[82]	 ? S.	 ?Kuo	 ?and	 ?Y.	 ?Chou,	 ??A	 ?Novel	 ?Polymer	 ?Microneedle	 ?Arrays	 ?and	 ?PDMS	 ?Micromolding	 ?Technique,?	 ?Tamkang	 ?Journal	 ?of	 ?Science	 ?and	 ?Engineering,	 ?vol.	 ?7,	 ?pp.	 ?95-??98,	 ?2004.	 ?[83]	 ? P.	 ? Griss,	 ? P.	 ? Enoksson,	 ? H.	 ? K.	 ? Tolvanen-??Laakso,	 ? P.	 ? Meril?inen,	 ? S.	 ? Ollmar	 ? and	 ? G.	 ?Stemme,	 ? ?Micromachined	 ? electrodes	 ? for	 ? biopotential	 ? measurements,?	 ? Journal	 ? of	 ?Microelectromechanical	 ?Systems,	 ?vol.	 ?10(1),	 ?pp.	 ?10-??16,	 ?2001.	 ?[84]	 ? S.	 ?Hashmi,	 ? P.	 ? Ling,	 ?G.	 ?Hashmi,	 ?M.	 ? L.	 ? Reed,	 ?R.	 ?Gaugler	 ? and	 ?W.	 ? Trimmer,	 ? ?Genetic	 ?transformation	 ?of	 ?nematodes	 ?using	 ?arrays	 ?of	 ?micromechanical	 ?piercing	 ?structures,?	 ?BioTechniques,	 ?vol.	 ?19(5),	 ?pp.766?70,	 ?1995.	 ?[85]	 ? J.	 ?Ji,	 ?F	 ?E	 ?H	 ?Tay,	 ?J.	 ?Miao	 ?and	 ?C.	 ? Iliescu,	 ??Microfabricated	 ?microneedles	 ?with	 ?porous	 ?tip	 ?for	 ?drug	 ?delivery,?	 ?Journal	 ?of	 ?Micromechanics	 ?and	 ?Microengineering,	 ?vol.	 ?16,	 ?pp.	 ?958?964,	 ?2006.	 ?[86]	 ? J.	 ?A.	 ?Matriano,	 ?M.	 ?Cormier,	 ?J.	 ?Johnson,	 ?W.	 ?A.	 ?Young,	 ?M.	 ?Buttery,	 ?K.	 ?Nyam,	 ?and	 ?P.	 ?E.	 ?Daddona,	 ??Macroflux?	 ?Microprojection	 ?Array	 ?Patch	 ?Technology:	 ?A	 ?New	 ?and	 ?Efficient	 ?Approach	 ? for	 ? Intracutaneous	 ? Immunization,?	 ?Pharmaceutical	 ? Research,	 ? vol	 ? 19,	 ? pp.	 ?63-??70,	 ?2002	 ?[87]	 ? K.	 ? Lee,	 ?H.	 ?C.	 ? Lee,	 ?D.	 ?S.	 ? Lee	 ?and	 ?H.	 ? Jung,	 ? ?Drawing	 ? lithography:	 ? three-??dimensional	 ?fabrication	 ? of	 ? an	 ? ultrahigh-??aspect-??ratio	 ? microneedle,?	 ? Advanced	 ? Materials,	 ? vol.	 ?127	 ?	 ?22(4),	 ?pp.	 ?483-??486,	 ?2010.	 ?[88]	 ? P.	 ?M.	 ?Wang,	 ?M.	 ? Cornwell	 ? and	 ?M.	 ? R.	 ? Prausnitz,	 ? ?Minimally	 ? invasive	 ? extraction	 ? of	 ?dermal	 ? interstitial	 ? fluid	 ? for	 ? glucose	 ? monitoring	 ? using	 ? microneedles,?	 ? Diabetes	 ?Technology	 ?&	 ?Therapeutics,	 ?vol.	 ?7(1),	 ?pp	 ?131-??141,	 ?2005.	 ?[89]	 ? U.	 ?Siemann,	 ??Solvent	 ?Cast	 ?Technology?A	 ?Versatile	 ?Tool	 ?for	 ?Thin	 ?Film	 ?Production,?	 ?Progress	 ?in	 ?Colloid	 ?and	 ?Polymer	 ?Science,	 ?vol.	 ?130,	 ?pp	 ?1-??14,	 ?2005.	 ?[90]	 ? E.	 ?A.	 ?S.	 ?Doherty,	 ?R.	 ?J.	 ?Meagher,	 ?M.	 ?N.	 ?Albarghouthi	 ?and	 ?A.	 ?E.	 ?Barron,	 ??Microchannel	 ?Wall	 ? Coatings	 ? for	 ? Protein	 ? Separations	 ? by	 ? Capillary	 ? and	 ? Chip	 ? Electrophoresis?,	 ?Electrophoresis,	 ?vol.	 ?24,	 ?pp.	 ?34-??54,	 ?2003.	 ?	 ?[91]	 ? G.	 ? J.	 ? Wang,	 ? K.	 ? H.	 ? Ho	 ? and	 ? C.	 ? C.	 ? Hsueh,	 ? ?Biodegradable	 ? Polylactic	 ? Acid	 ?Microstructures	 ?for	 ?Scaffold	 ?Applications?,	 ?Microsystem	 ?Technologies,	 ?vol.	 ?14(7),	 ?pp.	 ?989-??993,	 ?2008.	 ?	 ?[92]	 ? A.	 ?W.	 ?McFarland,	 ?M.	 ? A.	 ? Poggi,	 ?L.	 ? A.	 ? Bottomley	 ? and	 ?J.	 ? S.	 ? Colton,	 ? ?Production	 ? and	 ?Characterization	 ?of	 ?Polymer	 ?Microcantilevers?,	 ?Review	 ?of	 ?Scientific	 ?Instruments,	 ?vol.	 ?75(8),	 ?pp.	 ?2756-??2758,	 ?2004.	 ?[93]	 ? Jeong	 ?W.	 ?Lee,	 ?Jung-??Hwan	 ?Park	 ?and	 ?Mark	 ?R.	 ?Prausnitz,	 ??Dissolving	 ?microneedles	 ?for	 ?transdermal	 ?drug	 ?delivery?,	 ?Biomaterials,	 ?vol.	 ?29(13),	 ?pp.	 ?2113-??2124,	 ?2008.	 ?	 ?[94]	 ? I.	 ?Mansoor	 ?and	 ?B.	 ?Stoeber,	 ??PIV	 ?Measurements	 ?of	 ?Flow	 ?in	 ?Drying	 ?Polymer	 ?Solutions	 ?during	 ?Solvent	 ?Casting?,	 ?Experiments	 ?in	 ?Fluids,	 ?vol.	 ?50(5),	 ?pp.	 ?1409-??1420,	 ?2010.	 ?128	 ?	 ?[95]	 ? HD	 ? Microsystems.	 ? (2009).	 ? ?PI-??2600	 ? Series	 ? ?	 ? Low	 ? Stress	 ? Applications?,	 ? Product	 ?Bulletin.	 ? [Online].	 ? Available:	 ?http://hdmicrosystems.com/HDMicroSystems/en_US/pdf/PI-??2600_ProcessGuide.pdf.	 ?	 ?	 ?[96]	 ? M.	 ? C.	 ? Peterman,	 ? P.	 ? Hulie,	 ? D.	 ? M.	 ? Bloom	 ? and	 ? H.	 ? A.	 ? Fishman,	 ? ?Building	 ? Thick	 ?Photoresist	 ? Structures	 ? From	 ? the	 ? Bottom	 ? Up?,	 ? Journal	 ? of	 ? Micromechanics	 ? and	 ?Microengineering,	 ?vol.	 ?13(3),	 ?pp.	 ?380-??382,	 ?2003.	 ?[97]	 ? C.	 ?Jang,	 ?S.	 ?Yoon	 ?and	 ?B.	 ?Han,	 ??Measurement	 ?of	 ?the	 ?Hygroscopic	 ?Swelling	 ?Coefficient	 ?of	 ? Thin	 ? Film	 ? Polymers	 ? Used	 ? in	 ? Semiconductor	 ? Packaging?,	 ? IEEE	 ? Transactions	 ? on	 ?Components	 ?and	 ?Packaging	 ?Technologies,	 ?vol.	 ?33,	 ?pp.	 ?340-??346,	 ?2010.	 ?[98]	 ? S.	 ? C.	 ? Noe,	 ? J.	 ? Y.	 ? Pan,	 ? and	 ? S.	 ? D.	 ? Senturia,	 ? ?Optical	 ? waveguiding	 ? as	 ? a	 ? method	 ? for	 ?characterizing	 ? the	 ? effect	 ? of	 ? extended	 ? cure	 ? and	 ? moisture	 ? on	 ? polyimide	 ? films?,	 ?Polymer	 ?Engineering	 ?&	 ?Science,	 ?vol.	 ?32,	 ?pp.	 ?1015-??1020,	 ?1992.	 ?[99]	 ? H.	 ? L.	 ? Tyan,	 ? C.	 ? Y.	 ?Wu,	 ? and	 ? K.	 ? H.	 ?Wei,	 ? ?Effect	 ? of	 ?montmorillonite	 ? on	 ? thermal	 ? and	 ?moisture	 ? absorption	 ? properties	 ? of	 ? polyimide	 ? of	 ? different	 ? chemical	 ? structures?,	 ?Journal	 ?of	 ?Applied	 ?Polymer	 ?Science,	 ?vol.	 ?81,	 ?pp.	 ?1742-??1747,	 ?2001.	 ?[100]	 ? S.	 ?Nicoli,	 ?C.	 ?Padula,	 ?V.	 ?Aversa,	 ?B.	 ?Vietti,	 ?P.W.	 ?Wertz,	 ?A.	 ?Millet,	 ?F.	 ?Falson,	 ?and	 ?R.	 ?P.	 ?Feynman,	 ? ?Characterization	 ? of	 ? Rabbit	 ? Ear	 ? Skin	 ? as	 ? a	 ? Skin	 ? Model	 ? for	 ? in	 ? vitro	 ?Transdermal	 ? Permeation	 ? Experiments:	 ? Histology,	 ? Lipid	 ? Composition	 ? and	 ?Permeability?,	 ?Skin	 ?Pharmacology	 ?and	 ?Physiology,	 ?vol.	 ?21,	 ?pp.	 ?218-??226,	 ?2008.	 ?129	 ?	 ?[101]	 ? P.	 ? Dick	 ? and	 ? R.	 ? C.	 ? Scott	 ? RC,	 ? ?Pig	 ? Ear	 ? Skin	 ? as	 ? an	 ? In-??vitro	 ?Model	 ? for	 ? Human	 ? Skin	 ?Permeability?,	 ?Journal	 ?of	 ?Pharmacy	 ?and	 ?Pharmacology,	 ?vol.	 ?44,	 ?pp.	 ?640-??645,	 ?1992.	 ?[102]	 ? N.	 ?Sekkat,	 ?Y.	 ?N.	 ?Kalia,	 ?R.	 ?H.	 ?Guy,	 ??Biophysical	 ?Study	 ?of	 ?Porcine	 ?Ear	 ?Skin	 ?In	 ?Vitro	 ?and	 ?Its	 ?Comparison	 ?to	 ?Human	 ?Skin	 ?In	 ?Vivo?,	 ?Journal	 ?of	 ?Pharmaceutical	 ?Sciences,	 ?vol.	 ?91,	 ?pp.2376-??2381,	 ?2002.	 ?[103]	 ? S.	 ?P.	 ?Davis,	 ?B.	 ? J.	 ? Landis,	 ? Z.	 ?H.	 ?Adams,	 ?M.	 ?G.	 ?Allen,	 ?M.	 ?R.	 ?Prausnitz,	 ? ?Insertion	 ?of	 ?Microneedles	 ?Into	 ?Skin:	 ?Measurement	 ?and	 ?Prediction	 ?of	 ?Insertion	 ?Force	 ?and	 ?Needle	 ?Fracture	 ?Force?,	 ?Journal	 ?of	 ?Biomechanics,	 ?vol.	 ?37(8),	 ?pp.	 ?1155-??1163,	 ?2004.	 ?[104]	 ? D.	 ? Kumar	 ? R.	 ? C.	 ? and	 ? Sharma,	 ? ?Advances	 ? in	 ? conductive	 ? polymers,?	 ? European	 ?Polymer	 ?Journal,	 ?vol.	 ?34,	 ?	 ?pp.	 ?1053?1060,	 ?1998.	 ?[105]	 ? M.	 ? Gerard	 ? M,	 ?A.	 ? Chaubey	 ? and	 ? B.	 ? D.	 ? Malhotra,	 ? ?Application	 ? of	 ? conductive	 ?polymers	 ?to	 ?biosensors,?	 ?Biosensors	 ?and	 ?Bioelectronics,	 ?vol.	 ?17,	 ?pp.	 ?345?359,	 ?2002.	 ?[106]	 ? R.	 ? Ou,	 ?S.	 ? Gupta,	 ?C.	 ? A.	 ? Parker	 ? and	 ?R.	 ? A.	 ? Gerhardt,	 ? ?Fabrication	 ? and	 ? electrical	 ?conductivity	 ? of	 ? poly(methyl	 ? methacrylate)	 ? (pmma)/carbon	 ? black	 ? (cb)	 ? composites:?	 ?comparison	 ? between	 ? an	 ? ordered	 ? carbon	 ? black	 ? nanowire-??like	 ? segregated	 ? structure	 ?and	 ? a	 ? randomly	 ? dispersed	 ? carbon	 ? black	 ? nanostructure,?	 ? Journal	 ? of	 ? Physical	 ?Chemistry	 ?B,	 ?vol.	 ?110(45),	 ?pp.	 ?22365?22373,	 ?2006.	 ?[107]	 ? M.	 ?Sumita,	 ?K.	 ?Sakata,	 ?S.	 ?Asai,	 ?K.	 ?Miyasaka	 ?and	 ?H.	 ?Nakagawa,	 ??Dispersion	 ?of	 ?fillers	 ?and	 ? electrical	 ? conductivity	 ? of	 ? polymer	 ? blends	 ? filled	 ? with	 ? carbon	 ? black,?	 ? Polymer	 ?Bulletin,	 ?vol.	 ?25,	 ?pp.	 ?265?271,	 ?1991.	 ?130	 ?	 ?[108]	 ? W.	 ? Zheng	 ? and	 ? S.	 ? C.	 ? Wong,	 ? ?Electrical	 ? conductivity	 ? and	 ? dielectric	 ? properties	 ? of	 ?PMMA/expanded	 ? graphite	 ? composites,?	 ? Composites	 ? Science	 ? and	 ? Technology,	 ? vol.	 ?63,	 ?pp.	 ?225?235,	 ?2003.	 ?[109]	 ? G.	 ? H.	 ? Chen,	 ?D.	 ? J.	 ? Wu,	 ?W.	 ? G.	 ? Weng	 ? and	 ?W.	 ? L.	 ? Yan,	 ? ?Preparation	 ? of	 ?polymer/graphite	 ? conducting	 ? nanocomposite	 ? by	 ? intercalation	 ? polymerization,?	 ?Journal	 ?of	 ?Applied	 ?Polymer	 ?Science,	 ?	 ?vol.	 ?82	 ?pp.	 ?2506?2513,	 ?2001.	 ?[110]	 ? G.	 ?Jiang,	 ?M.	 ?Gilbert,	 ?D.	 ?J.	 ?Hitt,	 ?G.	 ?D.	 ?Wilcox	 ?and	 ?K.	 ?Balasubramanian,	 ??Preparation	 ?of	 ?nickel	 ?coated	 ?mica	 ?as	 ?a	 ?conductive	 ?filler,?	 ?Composites	 ?Part	 ?A:	 ?Applied	 ?Science	 ?and	 ?Manufacturing,	 ?vol.	 ?33,	 ?pp.	 ?745?751,	 ?2002.	 ?[111]	 ? Y.	 ? P.	 ? Mamunya,	 ? V.	 ? V.	 ? Davydenko,	 ? P.	 ? Pissis	 ? and	 ? E.	 ? V.	 ? Lebedev,	 ? ?Electrical	 ? and	 ?thermal	 ? conductivity	 ? of	 ? polymers	 ? filled	 ? with	 ? metal	 ? powders,?	 ? European	 ? Polymer	 ?Journal,	 ?vol.	 ?38,	 ?pp.	 ?1887?1897,	 ?2002.	 ?[112]	 ? W.	 ?N.	 ?Sharpe	 ?Jr,	 ?D.	 ?A.	 ?LaVan	 ?and	 ?R.	 ?L.	 ?Edwards,	 ??Mechanical	 ?properties	 ?of	 ?LIGA-??deposited	 ?nickel	 ? for	 ?MEMS	 ? transducers,?	 ?Proc.	 ? of	 ? International	 ? Solid	 ? State	 ? Sensors	 ?and	 ?Actuators	 ?Conference	 ?(Transducers	 ?`97),	 ?Chicago,	 ?IL,	 ?USA,	 ?June	 ?16?19	 ?1997,	 ?pp.	 ?604?610,	 ?1997.	 ?[113]	 ? F.	 ?Ebrahimi,	 ?G.	 ?R.	 ?Bourne,	 ?M.	 ?S.	 ?Kelly	 ?and	 ?T.	 ?E.	 ?Matthews	 ??Mechanical	 ?properties	 ?of	 ?nanocrystalline	 ?nickel	 ?produced	 ?by	 ?electrodeposition,?	 ?Nanostructured	 ?Materials,	 ?vol.	 ?11,	 ?343?350,	 ?1999.	 ?[114]	 ? L.	 ?Peltonen,	 ??Nickel	 ?sensitivity	 ?in	 ?general	 ?population,?	 ?Contact	 ?Dermatitis,	 ?vol.	 ?5,	 ?131	 ?	 ?pp.	 ?27?32,	 ?1979.	 ?[115]	 ? G.	 ?Voskerician,	 ?M.	 ? S.	 ? Shive,	 ? R.	 ? S.	 ? Shawgo,	 ?H.	 ? von	 ?Recum,	 ? J.	 ?M.	 ? Anderson,	 ?M.	 ? J.	 ?Cima	 ? and	 ? R.	 ? Langer,	 ? ?Biocompatibility	 ? and	 ? biofouling	 ? of	 ? MEMS	 ? drug	 ? delivery	 ?devices,?	 ?Biomaterials,	 ?vol.	 ?24,	 ?pp.	 ?1959?1967,	 ?2003.	 ?[116]	 ? F.	 ? D.	 ? Egitto,	 ? ?Plasma	 ? etching	 ? and	 ? modification	 ? of	 ? organic	 ? polymers,?	 ? Pure	 ? and	 ?Applied	 ?Chemistry,	 ?vol.	 ?62,	 ?pp.	 ?1699?1708,	 ?1990.	 ?[117]	 ? K.	 ?Harada,	 ??Plasma	 ?etching	 ?durability	 ?of	 ?poly	 ? (methyl	 ?methacrylate),?	 ? Journal	 ?of	 ?Applied	 ?Polymer	 ?Science,	 ?vol.	 ?26,	 ?pp.	 ?1961?1973,	 ?1981.	 ?[118]	 ? A.	 ?M.	 ?Barbero	 ?and	 ?H.	 ?F.	 ?Frasch,	 ??Pig	 ?and	 ?guinea	 ?pig	 ?skin	 ?as	 ?surrogates	 ?for	 ?human	 ?in	 ?vitro	 ?penetration	 ?studies:	 ?a	 ?quantitative	 ?review,?	 ?Toxicology	 ?in	 ?Vitro,	 ?vol.	 ?23,	 ?pp.	 ?1?13,	 ?2009.	 ?[119]	 ? J.	 ? G.	 ? Mallette,	 ? A.	 ? M?rquez,	 ?O.	 ? Manero	 ? and	 ? R.	 ? Castro-??Rodr?guez,	 ? ?Carbon	 ? black	 ?filled	 ?PET/PMMA	 ?blends:	 ?electrical	 ?and	 ?morphological	 ?studies,?	 ?Polymer	 ?Engineering	 ?and	 ?Science,	 ?vol.	 ?40,	 ?pp.	 ?2272?2278,	 ?2000.	 ?[120]	 ? U.	 ?O.	 ?H?feli,	 ? A.	 ?Mokhtari,	 ? D.	 ? Liepmann	 ? and	 ?B.	 ? Stoeber,	 ? ?In	 ? vivo	 ?evaluation	 ? of	 ? a	 ?microneedle-??based	 ? miniature	 ? syringe	 ? for	 ? intradermal	 ? drug	 ? delivery,?	 ? Biomedical	 ?Microdevices,	 ?vol.	 ?11(5),	 ?pp.	 ?943-??950,	 ?2009.	 ?[121]	 ? B.	 ?Stoeber	 ?and	 ?D.	 ?Liepmann,	 ??Design,	 ?fabrication,	 ?and	 ?testing	 ?of	 ?a	 ?MEMS	 ?syringe.?	 ?Proceedings	 ? of	 ? Solid-??State	 ? Sensor,	 ? Actuator	 ? and	 ? Microsystems	 ? Workshop,	 ? Hilton	 ?132	 ?	 ?Head,	 ?SC,	 ?2002.	 ?	 ?[122]	 ? V.	 ? G.	 ? S.	 ? Box,	 ? ?The	 ? intercalation	 ? of	 ? DNA	 ? double	 ? helices	 ? with	 ? doxorubicin	 ? and	 ?nagalomycin,?	 ? Journal	 ? of	 ?Molecular	 ? Graphics	 ? and	 ?Modeling,	 ? vol.	 ? 26(1),	 ? pp.	 ? 14-??19,	 ?2007.	 ?[123]	 ? N.	 ?T.	 ?Chen,	 ?C.	 ?Y.	 ?Wu,	 ?C.	 ?Y.	 ?Chung,	 ?Y.	 ?Hwu,	 ?S.	 ?H.	 ?Cheng,	 ?C.	 ?Y.	 ?Mou,	 ?and	 ?L.	 ?W.	 ?Lo,	 ??Probing	 ?the	 ?dynamics	 ?of	 ?doxorubicin-??DNA	 ?intercalation	 ?during	 ?the	 ?initial	 ?activation	 ?of	 ? apoptosis	 ? by	 ? fluorescence	 ? lifetime	 ? imaging	 ?microscopy	 ? (FLIM),?	 ?PLOS	 ?ONE,	 ? vol.	 ?7(9),	 ?pp.	 ?1-??8,	 ?2012.	 ?[124]	 ? D.	 ?Hynek,	 ? L.	 ? Krejcova,	 ?O.	 ? Zitka,	 ?V.	 ?Adam,	 ? L.	 ? Trnkova,	 ? J.	 ? Sochor,	 ?M.	 ? Stiborova,	 ? T.	 ?Eckschlager,	 ? J.	 ? Hubalek,	 ? and	 ? R.	 ? Kizek,	 ? ?Electrochemical	 ? study	 ? of	 ? doxorubicin	 ?interaction	 ? with	 ? different	 ? sequences	 ? of	 ? single	 ? stranded	 ? oligonucleotides,	 ? part	 ? I,?	 ?International	 ?Journal	 ?of	 ?Electrochemical	 ?Science,	 ?vol.	 ?7,	 ?pp.	 ?13	 ??	 ?33,	 ?2012.	 ?[125]	 ? NCBI	 ? Pubchem,	 ? Doxorubicin	 ? ?	 ? Compound	 ? Summary	 ? [online],	 ? Available	 ? FTP:	 ?http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=31703	 ?[126]	 ? Cancer	 ? Care	 ? Ontario,	 ? Doxorubicin	 ? [online],	 ? Available	 ? FTP:	 ?https://www.cancercare.on.ca/search/default.aspx?q=doxorubicin&sortby=Relevance&type=-??1,1377-??78|0,6-??76,6-??40484&pg=1	 ?[127]	 ? D.	 ?Missirlis,	 ?R.	 ?Kawamura,	 ?N.	 ?Tirelli,	 ?and	 ?J.	 ?A.	 ?Hubbell,	 ??Doxorubicin	 ?encapsulation	 ?and	 ? diffusional	 ? release	 ? from	 ? stable,	 ? polymeric,	 ? hydrogel	 ? nanoparticles,?	 ? European	 ?Journal	 ?of	 ?Pharmaceutical	 ?Sciences,	 ?vol.	 ?29(2),	 ?pp.	 ?120-??129,	 ?2006.	 ?133	 ?	 ?[128]	 ? G.Bormann,	 ? F.	 ? Brosens,	 ? and	 ? E.	 ? De	 ? Schutter,	 ? ?Modeling	 ? molecular	 ? diffusion,?	 ?Computational	 ? methods	 ? in	 ? molecular	 ? and	 ? cellular	 ? biology:	 ? from	 ? genotype	 ? to	 ?phenotype,	 ?MIT	 ?Press,	 ?Boston,	 ?2001,	 ?pp.	 ?189-??224.	 ?[129]	 ? Oregon	 ?Medical	 ? Laser	 ?Center,	 ?Time-??resolved	 ?Diffusion	 ?Theory	 ? [online],	 ?Available	 ?FTP:	 ?http://omlc.ogi.edu/education/ece532/class5/trdt.html	 ?[130]	 ? G.	 ?B.	 ?Kasting	 ?and	 ?L.	 ?A.	 ?Bowman,	 ??Electrical	 ?analysis	 ?of	 ?fresh,	 ?excised	 ?human	 ?skin:	 ?a	 ?comparison	 ?with	 ? frozen	 ?skin,?	 ?Pharmaceutical	 ?Research,	 ? vol.	 ?7(11),	 ?pp.	 ?1141-??1146,	 ?1990.	 ?[131]	 ? J.	 ?Swarbrick,	 ?G.	 ?Lee,	 ?and	 ?J.	 ?Brom,	 ??Drug	 ?permeation	 ?through	 ?human	 ?skin:	 ?I.	 ?Effect	 ?of	 ? storage	 ?conditions	 ?of	 ? skin,?	 ? Journal	 ?of	 ? Investigative	 ?Dermatology,	 ? vol.	 ?78(1),	 ?pp.	 ?63-??66,	 ?1982.	 ?[132]	 ? W.	 ? Kaowumpai,	 ? D.	 ? Koolpiruck,	 ? and	 ? K.	 ? Viravaidya,	 ? ?Development	 ? of	 ? a	 ? 3D	 ?mathematical	 ?model	 ?for	 ?a	 ?doxorubicin	 ?controlled	 ?release	 ?system	 ?using	 ?pluronic	 ?gel	 ?for	 ? breast	 ? cancer	 ? treatment,?	 ? World	 ? Academy	 ? of	 ? Science,	 ? Engineering	 ? and	 ?Technology,	 ?vol.32,	 ?pp.287-??292,	 ?2007.	 ?[133]	 ? B.	 ? D.	 ? Weinberg,	 ? R.	 ? B.	 ? Patel,	 ? A.	 ? A.	 ? Exner,	 ? G.	 ? M.	 ? Saidel,	 ? and	 ? J.	 ? Gao,	 ? ?Modeling	 ?doxorubicin	 ?transport	 ?to	 ?improve	 ?intratumoral	 ?drug	 ?delivery	 ?to	 ?RF	 ?ablated	 ?tumors,?	 ?Journal	 ?of	 ?Controlled	 ?Release,	 ?vol.	 ?124,	 ?pp.	 ?11-??19,	 ?2007.	 ?[134]	 ? F.	 ?Qiana	 ?,	 ?N.	 ?Stoweb,	 ?E.	 ?H.	 ?Liua,	 ?G.	 ?M.	 ?Saidela,	 ?and	 ?J.	 ?Gaoa,	 ??Quantification	 ?of	 ?in	 ?vivo	 ?doxorubicin	 ?transport	 ?from	 ?PLGA	 ?millirods	 ?in	 ?thermoablated	 ?rat	 ?livers,?	 ?Journal	 ?134	 ?	 ?of	 ?Controlled	 ?Release,	 ?vol.	 ?91,	 ?pp.	 ?157?166,	 ?2003.	 ?[135]	 ? Jan	 ?Lankelma,	 ?Rafael	 ?Fern?ndez	 ?Luque,	 ?Henk	 ?Dekker,	 ?Wim	 ?Schinkel,	 ?and	 ?Herbert	 ?M.	 ? Pinedo,	 ? ?A	 ? Mathematical	 ? Model	 ? of	 ? Drug	 ? Transport	 ? in	 ? Human	 ? Breast	 ? Cancer?	 ?Microvascular	 ?Research,	 ?vol.	 ?59,	 ?pp.	 ?149?161,	 ?2000.	 ?[136]	 ? G.	 ?D.	 ?Chisholm,	 ?P.	 ?M.	 ?Waterworth,	 ?J.	 ?S.	 ?Calnan,	 ?and	 ?L.	 ?P.	 ?Garrod,	 ??Concentration	 ?of	 ? Antibacterial	 ? Agents	 ? in	 ? Interstitial	 ? Tissue	 ? Fluid,?	 ? British	 ? Medical	 ? Journal,	 ? vol.	 ?1(5853),	 ?pp.	 ?569-??573,	 ?1973.	 ?[137]	 ? W.	 ?Groenendaal,	 ?G.	 ?von	 ?Basum,	 ?K.	 ?A.	 ?Schmidt,	 ?P.	 ?A.	 ?J.	 ?Hilbers,	 ?and	 ?N.	 ?A.	 ?W.	 ?van	 ?Riel,	 ??Quantifying	 ?the	 ?composition	 ?of	 ?human	 ?skin	 ?for	 ?glucose	 ?sensor	 ?development,?	 ?J.	 ?	 ?Diabetes	 ?Science	 ?and	 ?Technology,	 ?vol.	 ?4(5),	 ?pp.	 ?1032-??1040,	 ?2010.	 ?[138]	 ? S.	 ? Defrere,	 ? M.	 ? Mestagdt,	 ? R.	 ? Riva,	 ? F.	 ? Krier,	 ? A.	 ? Van	 ? Langendonckt,	 ? P.	 ? Drion,	 ? C.	 ?Jerome,	 ? B.	 ? Evrard,	 ? J.	 ? P.	 ? Dehoux,	 ? J.	 ? M.	 ? Foidart,	 ? and	 ? J.	 ? Donnez,	 ? ?In	 ? vivo	 ?biocompatibility	 ? of	 ? three	 ? potential	 ? intraperitoneal	 ? implants,?	 ? Macromolecular	 ?Bioscience,	 ?vol.	 ?11(10),	 ?pp.	 ?1336-??1345,	 ?2011.	 ?	 ?	 ? 	 ?135	 ?	 ?APPENDICES	 ?	 ?A.1 Detailed	 ?photolithography	 ?parameters	 ?and	 ?mold	 ?fabrication	 ?Fabrication	 ?of	 ?the	 ?SU-??8	 ?molds	 ?for	 ?polymer	 ?microneedles	 ?in	 ?Chapter	 ?2:	 ?Equipment	 ?used	 ?(located	 ?in	 ?UBC	 ?Nanofabrication	 ?Facility):	 ?Spinner:	 ?LAURELL	 ?WS-??400-??6NPP-??LITE	 ?Mask	 ?aligner:	 ?CANON	 ?DOUBLE	 ?SIDE	 ?100MM	 ?ALIGNER	 ?Dicing	 ?saw:	 ?MA1006	 ?Disco	 ?Saw	 ?Plasma	 ?etching:	 ?Trion	 ?RIE/PECVD	 ?	 ?Parameters	 ?	 ? Substrate:	 ?	 ?300	 ??m	 ?thick	 ?Pyrex?	 ?SU-??8	 ?type:	 ?2150	 ?Spinner:	 ?500	 ?RPM	 ?for	 ?10	 ?s	 ?(110	 ?RPM/s),	 ?1100	 ?RPM	 ?for	 ?35	 ?s	 ?(330	 ?RPM/s),	 ?0	 ?RPM	 ?for	 ?10	 ?s	 ?(110	 ?RPM/s)	 ?Soft	 ?bake:	 ?65?C	 ?for	 ?10	 ?min,	 ?95?C	 ?for	 ?2	 ?h	 ?Rest:	 ?5	 ?min	 ?Exposure:	 ? 5300	 ?mJ	 ?cm-??2	 ? UV	 ? light;	 ? backside	 ? exposure	 ? as	 ? described	 ? in	 ? Figure	 ? A.1.	 ?(Performed	 ?in	 ?multiple	 ?3	 ?min	 ?intervals	 ?with	 ?20	 ?s	 ?cooling	 ?breaks	 ?in	 ?between)	 ?	 ? 	 ?	 ?	 ?	 ?	 ?	 ?	 ?Figure	 ?A.1:	 ?Setup	 ?used	 ?to	 ?perform	 ?backside	 ?photoresist	 ?exposure.	 ?Mask	 ?UV	 ?transparent	 ?glass	 ?to	 ?put	 ?weight	 ?on	 ?mask	 ?Pyrex?	 ?wafer	 ?SU-??8	 ?resist	 ?Non-??reflective	 ?surface	 ?(Ex.	 ?silicon	 ?wafer	 ?backside)	 ?UV	 ?Light	 ?136	 ?	 ?Post	 ?exposure	 ?bake:	 ?65?C	 ?for	 ?5	 ?min,	 ?95?C	 ?for	 ?25	 ?min	 ?Rest:	 ?5	 ?min	 ?Developer:	 ?~50	 ?min	 ?Rinse:	 ?Fresh	 ?Developer	 ?+	 ?IPA	 ?Hard	 ?bake	 ?in	 ?oven:	 ?ramp	 ?up	 ?temperature	 ?to	 ?175?C,	 ?maintain	 ?for	 ?30	 ?min,	 ?ramp	 ?down	 ?to	 ?RT	 ?Resulting	 ?thickness:	 ?450	 ??m	 ??	 ?10	 ??m	 ?Wafer	 ?dicing	 ?The	 ?wafer	 ?was	 ?first	 ?glued	 ?to	 ?a	 ?silicon	 ?wafer	 ?(as	 ?base	 ?support)	 ?using	 ?AZ	 ?photoresist.	 ?It	 ?was	 ?then	 ?cut	 ?along	 ?the	 ?+	 ?patterns	 ?on	 ?the	 ?wafer	 ?to	 ?give	 ?square	 ?mold	 ?chips.	 ?The	 ?mold	 ?chips	 ? were	 ? then	 ? separated	 ? from	 ? the	 ? silicon	 ? wafer	 ? using	 ? acetone	 ? to	 ? remove	 ? AZ	 ?photoresist.	 ?Pillar	 ?diameter	 ?reduction	 ?To	 ?achieve	 ?better	 ?aspect	 ?ratio	 ?for	 ?the	 ?pillars	 ?which	 ?leads	 ?to	 ?sharper	 ?microneedles,	 ?a	 ?plasma	 ?etching	 ?tool	 ?can	 ?be	 ?used	 ?after	 ?or	 ?before	 ?cutting	 ?the	 ?wafer	 ?into	 ?square	 ?pieces	 ?to	 ? etch	 ? the	 ? pillar	 ? structures.	 ? The	 ? etching	 ? process	 ? is	 ? not	 ? fully	 ? anisotropic	 ? or	 ? fully	 ?isotropic.	 ? But	 ? it	 ? leads	 ? to	 ? the	 ? decrease	 ? in	 ? the	 ? height	 ? of	 ? the	 ? pillars	 ? as	 ?well	 ? but	 ? not	 ?substantial	 ?with	 ?respect	 ?to	 ?the	 ?reduction	 ?in	 ?diameter.	 ?One	 ?should	 ?take	 ?into	 ?account	 ?that	 ? having	 ? thinner	 ? pillar	 ? structure	 ? also	 ? means	 ? less	 ? mechanical	 ? strength	 ? for	 ? the	 ?pillars,	 ? which	 ? would	 ? increase	 ? the	 ? chance	 ? of	 ? pillars	 ? getting	 ? damaged	 ? during	 ?microneedle	 ?lift-??off.	 ?	 ?Images	 ? below	 ? (Figure	 ? A.2	 ? and	 ? A.3)	 ? compare	 ? arrays	 ? of	 ? pillars	 ? before	 ? and	 ? after	 ? the	 ?plasma	 ?etching	 ?process:	 ?	 ? 	 ?	 ?137	 ?	 ?	 ?Figure	 ?A.2:	 ?A	 ?microneedle	 ?mold	 ?array	 ?before	 ?plasma	 ?etching.	 ?	 ?Figure	 ?A.3:	 ?A	 ?microneedle	 ?mold	 ?array	 ?after	 ?plasma	 ?etching.	 ?For	 ?the	 ?needles	 ?produced	 ? in	 ?chapter	 ?2	 ?and	 ?3,	 ? the	 ?following	 ?parameters	 ?were	 ?used	 ?with	 ?the	 ?Trion	 ?RIE/PECVD	 ?to	 ?achieve	 ?pillars	 ?with	 ?35	 ??m	 ?tip	 ?diameter	 ?(Figure	 ?A.3):	 ?O2:	 ?90	 ?sccm;	 ?CF4:	 ?10	 ?sccm;	 ?temperature:	 ?25?C;	 ?power:	 ?200W;	 ?pressure:	 ?500	 ?mTorr;	 ?duration	 ?=	 ?1200	 ?s	 ?Fabrication	 ?of	 ?the	 ?SU-??8	 ?molds	 ?for	 ?metallic	 ?microneedles	 ?in	 ?Chapter	 ?3:	 ?Similar	 ? process	 ? as	 ? the	 ? molds	 ? described	 ? for	 ? polymer	 ? microneedles	 ? but	 ? with	 ? the	 ?following	 ?parameters:	 ?Substrate:	 ?	 ?300	 ??m	 ?thick	 ?Pyrex?	 ?SU-??8	 ?type:	 ?2150	 ?138	 ?	 ?	 ? Spinner:	 ?800	 ?RPM	 ?for	 ?45	 ?s	 ?(110	 ?RPM/s),	 ?0	 ?RPM	 ?for	 ?10	 ?s	 ?(110	 ?RPM/s)	 ?Rest:	 ?3	 ?min	 ?Soft	 ?bake:	 ?65?C	 ?for	 ?10	 ?min,	 ?95?C	 ?for	 ?2.5	 ?h	 ?Rest:	 ?5	 ?min	 ?Exposure:	 ? 9200	 ?mJ	 ?cm-??2	 ? UV	 ? light;	 ? backside	 ? exposure	 ? as	 ? described	 ? in	 ? Figure	 ? A.1.	 ?(Performed	 ?in	 ?multiple	 ?3	 ?min	 ?intervals	 ?with	 ?20	 ?s	 ?cooling	 ?breaks	 ?in	 ?between)	 ?Post	 ?exposure	 ?bake:	 ?65?C	 ?for	 ?5	 ?min,	 ?95?C	 ?for	 ?30	 ?min	 ?Rest:	 ?5	 ?min	 ?Developer:	 ?~50	 ?min	 ?Rinse:	 ?Fresh	 ?Developer	 ?+	 ?IPA	 ?Hard	 ?bake	 ?in	 ?oven:	 ?ramp	 ?up	 ?temperature	 ?to	 ?175?C,	 ?maintain	 ?for	 ?30	 ?min,	 ?ramp	 ?down	 ?to	 ?RT	 ?Resulting	 ?thickness:	 ?680	 ??m	 ??	 ?30	 ??m	 ?	 ?	 ? 	 ?139	 ?	 ?A.2 Photolithography	 ?masks	 ?used	 ?for	 ?mold	 ?fabrication	 ?For	 ? mold	 ? fabrication	 ? 100	 ?mm	 ? dark	 ? field	 ? masks	 ? were	 ? used	 ? (Figure	 ? A.4).	 ? The	 ? masks	 ? were	 ?divided	 ? into	 ? square	 ? sections	 ? by	 ? ?+?	 ?marker,	 ? each	 ? square	 ? corresponding	 ? to	 ? a	 ? single	 ?mold	 ?chip.	 ? Each	 ? square	 ? contains	 ? an	 ? array	 ? of	 ? 40	 ??m	 ? circular	 ? regions.	 ?Masked	 ?were	 ? designed	 ? in	 ?Clewin	 ? 4	 ? software.	 ?Once	 ? the	 ?mold	 ?wafer	 ?was	 ?made,	 ? it	 ?wass	 ? cut	 ? into	 ? the	 ? square	 ? sections	 ?along	 ?the	 ?+	 ?marks,	 ?using	 ?a	 ?dicing	 ?saw.	 ?	 ?Figure	 ?A.4:	 ?A	 ?100	 ?mm	 ?mask	 ?used	 ?for	 ?making	 ?microneedle	 ?molds.	 ?	 ? 	 ?140	 ?	 ?A.3 SEM	 ?images	 ?of	 ?polymer	 ?microneedles	 ?This	 ? section	 ? includes	 ? some	 ? additional	 ? SEM	 ? images	 ? of	 ? molds	 ? and	 ? polymer	 ? microneedles	 ?made	 ?through	 ?the	 ?process	 ?in	 ?Chapter	 ?2.	 ?	 ?Figure	 ?A.5:	 ?An	 ?array	 ?of	 ?polyimide/clay	 ?microneedles	 ?made	 ?through	 ?the	 ?process	 ?in	 ?Chapter	 ?2.	 ?	 ?Figure	 ?A.6:	 ?An	 ?array	 ?of	 ?polyvinyl	 ?alcohol	 ?microneedles	 ?made	 ?through	 ?the	 ?process	 ?in	 ?Chapter	 ?2.	 ?141	 ?	 ?	 ?Figure	 ?A.7:	 ?An	 ?array	 ?of	 ?polyvinyl	 ?alcohol	 ?microneedles	 ?made	 ?through	 ?the	 ?process	 ?in	 ?Chapter	 ?2.	 ?	 ?Figure	 ?A.8:	 ?Top	 ?view	 ?image	 ?of	 ?a	 ?hollow	 ?polymer	 ?microneedle	 ?showing	 ?the	 ?needle	 ?lumen.	 ?	 ?	 ? 	 ?142	 ?	 ?A.4 Conductivity	 ?measurements	 ?The	 ?effect	 ?of	 ?CB	 ?content	 ?on	 ?conductivity	 ?of	 ?the	 ?PMMA/CB	 ?composite	 ?was	 ?investigated.	 ?For	 ?these	 ?experiments,	 ?PMMA/CB	 ?suspensions	 ?in	 ?NMP	 ?were	 ?prepared	 ?with	 ?a	 ?CB	 ?concentration	 ?in	 ?the	 ?final	 ?solid	 ?varying	 ?between	 ?0	 ?to	 ?50	 ?wt%.	 ?After	 ?the	 ?solutions	 ?were	 ?prepared,	 ?they	 ?were	 ?cast	 ?into	 ?cylindrical	 ?cavities	 ?with	 ?diameters	 ?of	 ?4	 ?mm	 ?to	 ?create	 ?disk-??shaped	 ?composite	 ?films	 ?for	 ?conductivity	 ?measurements	 ?(as	 ?shown	 ?in	 ?Figure	 ?A.9).	 ?	 ?Figure	 ?A.9:	 ?PDMS	 ?mold	 ?used	 ?to	 ?make	 ?cylindrical	 ?pieces	 ?for	 ?PMMA/CB	 ?conductivity	 ?measurements.	 ?Using	 ? an	 ? e-??beam	 ? evaporator	 ? a	 ? thin	 ? copper	 ? layer	 ? was	 ? deposited	 ? on	 ? the	 ? top	 ? and	 ? bottom	 ?surfaces.	 ?The	 ?resistance	 ?of	 ?the	 ?pieces	 ?was	 ?then	 ?measured	 ?using	 ?a	 ?multimeter	 ?and	 ?then	 ?used	 ?to	 ?calculate	 ?conductivity	 ?and	 ?resistivity	 ?values	 ?(Table	 ?A.1).	 ?Table	 ?A.1:	 ?Conductivity	 ?and	 ?resistivity	 ?data	 ?for	 ?different	 ?CB	 ?content	 ?(three	 ?samples	 ?each).	 ?Carbon	 ?black	 ?content	 ? 0%	 ? 5%	 ? 10%	 ? 15%	 ? 20%	 ? 30%	 ? 40%	 ? 50%	 ?Resistivity	 ?[ohm	 ?x	 ?cm]	 ? ?	 ? 1.55E+06	 ? 2.81E+03	 ? 1.63E+03	 ? 9.36E+01	 ? 3.54E+01	 ? 1.53E+01	 ? 1.45E+00	 ?Conductivity	 ?[S/cm]	 ? 0	 ? 6.50E-??07	 ? 3.99E-??04	 ? 8.44E-??04	 ? 1.10E-??02	 ? 3.02E-??02	 ? 8.61E-??02	 ? 7.07E-??01	 ?Cylindrical	 ?cavity	 ?created	 ?with	 ?PDMS	 ?Cast	 ?piece	 ?used	 ?to	 ?measure	 ?conductivity	 ?1	 ?cm	 ?143	 ?	 ?A.5 Plasma	 ?etch	 ?rate	 ?measurements	 ?The	 ?etch	 ?rate	 ?for	 ?PMMA/CB	 ?composite,	 ?with	 ?30	 ?wt%	 ?CB	 ?in	 ?solid,	 ?was	 ?measured	 ?for	 ?different	 ?gas	 ? flow	 ? settings	 ? (Table	 ? A.2).	 ? The	 ? O2	 ? and	 ? CF4	 ? flow	 ? rates	 ? were	 ? varied	 ? while	 ? the	 ? other	 ?parameters	 ?were	 ?kept	 ?constant:	 ?	 ?Equipment:	 ?TRION	 ?RIE/PECVD	 ?at	 ?UBC	 ?Nanofabrication	 ?Facility	 ?Power:	 ?200	 ?W	 ?Pressure:	 ?500	 ?mTorr	 ?Temperature:	 ?20?C	 ?	 ?Table	 ?A.2:	 ?Etch	 ?rates	 ?for	 ?different	 ?CF4	 ?proportion	 ?in	 ?O2/CF4	 ?gas	 ?combinations.	 ?CF4	 ?content	 ?%	 ? etch	 ?rate	 ?[?m/min]	 ? Standard	 ?deviation	 ?[?m/min]	 ?0	 ? 0.290	 ? 0.069	 ?10	 ? 0.415	 ? 0.032	 ?20	 ? 0.423	 ? 0.036	 ?30	 ? 0.337	 ? 0.051	 ?40	 ? 0.102	 ? 0.023	 ?60	 ? 0.032	 ? 0.026	 ?80	 ? 0.010	 ? 0.001	 ?100	 ? 0.008	 ? 0.005	 ?	 ?	 ?144	 ?	 ?A.6 Nickel	 ?electroplating	 ?tests	 ?The	 ?change	 ? in	 ? thickness	 ?of	 ?nickel	 ?was	 ?measured	 ? for	 ?different	 ? source	 ?currents	 ? (Table	 ?A.3).	 ?The	 ?setting	 ?used	 ?for	 ?the	 ?plating	 ?process	 ?was	 ?as	 ?follows:	 ?Time:	 ?90	 ?min	 ?Area:	 ?1	 ?cm	 ??	 ?1	 ?cm	 ?Electrode-??electrode	 ?distance:	 ?2.5	 ?cm	 ?Solution:	 ?nickel	 ?chloride	 ?(25 ??? ),	 ?nickel	 ?sulfate	 ?(170	 ??? ),	 ?and	 ?boric	 ?acid	 ?(15	 ??? )	 ?	 ?Table	 ?A.3:	 ?Nickel	 ?thickness	 ?from	 ?electroplating	 ?for	 ?different	 ?supply	 ?currents.	 ?Electric	 ?Current	 ?[mA]	 ? Nickel	 ?thickness	 ?[?m]	 ?0	 ? 0	 ?1	 ? 22.94	 ?2	 ? 46.88	 ?3	 ? 70.8	 ?4	 ? 92.35	 ?5	 ? 108.1	 ?6	 ? 131.5	 ?7	 ? 156.1	 ?The	 ? thickness	 ? of	 ? nickel	 ? was	 ? measured	 ? for	 ? different	 ? process	 ? durations	 ? (Table	 ? A.4).	 ? The	 ?setting	 ?used	 ?for	 ?the	 ?plating	 ?process	 ?was	 ?as	 ?follows:	 ?Current:	 ?2	 ?mA	 ?Area:	 ?1	 ?cm	 ??	 ?1	 ?cm	 ?Electrode-??	 ?electrode	 ?distance:	 ?2.5	 ?cm	 ?145	 ?	 ?Solution:	 ?nickel	 ?chloride	 ?(25 ??? ),	 ?nickel	 ?sulfate	 ?(170	 ??? ),	 ?and	 ?boric	 ?acid	 ?(15	 ??? )	 ?	 ?Table	 ?A.4:	 ?Nickel	 ?thickness	 ?from	 ?electroplating	 ?for	 ?different	 ?process	 ?durations.	 ?Process	 ?duration	 ?[min]	 ? Nickel	 ?thickness	 ?[?m]	 ?0	 ? 0	 ?15	 ? 6.19	 ?30	 ? 11.45	 ?60	 ? 31.04	 ?90	 ? 46.88	 ?120	 ? 58.69	 ?150	 ? 72.18	 ?180	 ? 85.84	 ?	 ?	 ? 	 ?146	 ?	 ?A.7 Additional	 ?data	 ?for	 ?the	 ?mechanical	 ?tests	 ?Figure	 ? A.10	 ? shows	 ? all	 ? the	 ? curves	 ? obtained	 ? for	 ? the	 ? compression	 ? tests	 ? performed	 ? on	 ? the	 ?500	 ??m	 ?metallic	 ?needles	 ?presented	 ?in	 ?Chapter	 ?3.	 ?0 50 100 150 200 250 3000123456Force [N]Displacement [?m]	 ?N eedle 	 ?1	 ?Needle 	 ?2	 ?Needle 	 ?3	 ?Needle 	 ?4	 ?Needle 	 ?5	 ?Figure	 ?A.10:	 ?Compression	 ?test	 ?plots	 ?for	 ?500	 ??m	 ?tall	 ?microneedles	 ?(made	 ?through	 ?the	 ?process	 ?in	 ?Chapter	 ?3)	 ?showing	 ?the	 ?compressive	 ?forces	 ?as	 ?a	 ?function	 ?of	 ?displacement;	 ?the	 ?first	 ?load	 ?peaks	 ?indicate	 ?tip	 ?failure.	 ?	 ?	 ?	 ? 	 ?147	 ?	 ?A.8 Doxorubicin	 ?intensity-??concentration	 ?calibration	 ?Finding	 ?the	 ?linear	 ?region	 ?in	 ?the	 ?intensity-??concentration	 ?plots	 ?Calibration	 ? experiments	 ? performed	 ? to	 ? measure	 ? the	 ? intensity	 ? of	 ? various	 ? doxorubicin	 ?concentrations	 ?on	 ?a	 ?hemocytometer	 ?slide.	 ?For	 ?each	 ?test,	 ?a	 ?droplet	 ?is	 ?placed	 ?on	 ?the	 ?gridded	 ?region	 ?and	 ?then	 ?covered	 ?with	 ?a	 ?coverslip.	 ?The	 ?Hemocytometer	 ?is	 ?then	 ?flipped	 ?and	 ?placed	 ?on	 ?the	 ?confocal	 ?microscope	 ?stage.	 ?Figure	 ?A.11	 ?below	 ?shows	 ?the	 ?setup	 ?and	 ?the	 ?slide	 ?orientation	 ?with	 ?respect	 ?to	 ?the	 ?objective.	 ?	 ?Figure	 ?A.11:	 ?Schematic	 ?of	 ?the	 ?setup	 ?to	 ?measure	 ?doxorubicin	 ?intensity.	 ?Confocal	 ?setting	 ?was:	 ?objective	 ?10x,	 ?frame	 ?size:	 ?512x512	 ?pixels,	 ?1.55	 ?mm	 ?x	 ?1.55	 ?mm,	 ?speed:	 ?600	 ?Hz	 ?bidirectional,	 ?laser:	 ?488	 ?nm	 ?@	 ?10%	 ?intensity,	 ?detector:	 ?HyD1,	 ?step	 ?size:	 ?2.06	 ??m.	 ?For	 ? each	 ? concentration,	 ? up	 ? to	 ? five	 ? measurements	 ? are	 ? carried	 ? out,	 ? and	 ? for	 ? each	 ?measurement,	 ? two	 ? scans	 ? are	 ? obtained	 ? and	 ? averaged.	 ? The	 ? Hemocytometer	 ? is	 ? washed	 ?carefully	 ? with	 ? soap	 ? and	 ? DI	 ? water	 ? between	 ? each	 ? test.	 ? Figure	 ? A.12	 ? shows	 ? examples	 ? of	 ?intensity	 ?plots	 ?obtained	 ?for	 ?some	 ?of	 ?the	 ?concentrations.	 ?	 ?148	 ?	 ?	 ?	 ?	 ?Figure	 ?A.12:	 ?Examples	 ?of	 ?intensity	 ?measurements	 ?(in	 ?arbitrary	 ?units)	 ?from	 ?hemocytometer	 ?for	 ?different	 ?concentrations.	 ?0	 ?50	 ?100	 ?150	 ?200	 ?0	 ? 50	 ? 100	 ? 150	 ? 200	 ? 250	 ?Mean	 ?Intensity	 ?Ver?al	 ?Distance	 ?[?m] 	 ?0	 ??M	 ?0	 ?50	 ?100	 ?150	 ?200	 ?0	 ? 50	 ? 100	 ? 150	 ? 200	 ? 250	 ?Mean	 ?Intensity	 ?Ver?al	 ?Distance	 ?[?m]	 ?25	 ??M	 ?0	 ?50	 ?100	 ?150	 ?200	 ?0	 ? 50	 ? 100	 ? 150	 ? 200	 ? 250	 ?Mean	 ?Intensity	 ?Ver?al	 ?Distance	 ?[?m] 	 ?50	 ??M	 ?0	 ?50	 ?100	 ?150	 ?200	 ?0	 ? 100	 ? 200	 ? 300	 ?Mean	 ?Intensity	 ?Ver?al	 ?Distance	 ?[?m] 	 ?150	 ??M	 ?0	 ?50	 ?100	 ?150	 ?200	 ?0	 ? 100	 ? 200	 ? 300	 ?Mean	 ?Intensity	 ?Ver?al	 ?Distance	 ?[?m] 	 ?300	 ??M	 ?0	 ?50	 ?100	 ?150	 ?200	 ?0	 ? 100	 ? 200	 ? 300	 ?Mean	 ?Intensity	 ?Ver?al	 ?Distance	 ?[?m] 	 ?900	 ??M	 ?0	 ?50	 ?100	 ?150	 ?200	 ?0	 ? 50	 ? 100	 ? 150	 ? 200	 ? 250	 ?Mean	 ?Intensity	 ?Ver?al	 ?Distance	 ?[?m] 	 ?1500	 ??M	 ?0	 ?50	 ?100	 ?150	 ?200	 ?0	 ? 100	 ? 200	 ? 300	 ?Mean	 ?Intensity	 ?Ver?al	 ?Distance	 ?[?m] 	 ?3000	 ??M	 ?149	 ?	 ?Moving	 ? from	 ? left	 ? to	 ? right	 ?on	 ? the	 ? x-??axis	 ? correspond	 ? to	 ?moving	 ? from	 ? top	 ? (grids)	 ? to	 ?bottom	 ?(away	 ?from	 ?the	 ?hemocytometer)	 ?as	 ?described	 ?in	 ?Figure	 ?A.13.	 ?	 ?	 ?Figure	 ?A.13:	 ?Locations	 ?of	 ?scanning	 ?on	 ?an	 ?examples	 ?intensity	 ?plot.	 ?	 ?Results:	 ?Based	 ? on	 ? the	 ? curves	 ? in	 ? Figure	 ?A.12,	 ? three	 ? calibration	 ? curves	 ?were	 ? plotted:	 ? one	 ? based	 ? on	 ?intensity	 ?readings	 ?from	 ?the	 ?grid	 ?region,	 ?one	 ?from	 ?near	 ?the	 ?coverslip,	 ?and	 ?one	 ?in	 ?the	 ?middle	 ?of	 ?the	 ?two	 ?(Figure	 ?A.14).	 ?The	 ?linear	 ?regions	 ?(0-??150	 ??M)	 ?are	 ?the	 ?same	 ?for	 ?all	 ?the	 ?curves	 ?and	 ?beyond	 ?the	 ?linear	 ?region,	 ?the	 ?curves	 ?diverge.	 ?0	 ?50	 ?100	 ?150	 ?200	 ?0	 ? 100	 ? 200	 ? 300	 ?Mean	 ?Intensity	 ?Ver?al	 ?Distance	 ?[?m] 	 ?1500	 ??M	 ?Grids	 ?on	 ?Hemocytometer	 ? Near	 ?coverslip	 ?150	 ?	 ?	 ?Figure	 ?A.14:	 ?Calibration	 ?curve	 ?for	 ?doxorubicin	 ?from	 ?three	 ?regions	 ?of	 ?hemocytometer.	 ?	 ?Finding	 ?the	 ?intensity	 ?corresponding	 ?to	 ?the	 ?initial	 ?concentration	 ?for	 ?the	 ?tests	 ?in	 ?Chapter	 ?4	 ?The	 ?calibration	 ?curve	 ? in	 ?Figure	 ?A.14	 ? is	 ? found	 ?using	 ?a	 ? confocal	 ? setting	 ?different	 ? from	 ?what	 ?was	 ? used	 ? to	 ? detect	 ? the	 ? drug	 ? distribution	 ? in	 ? skin.	 ? The	 ? detected	 ? intensity	 ? level	 ? in	 ? skin	 ? is	 ?influenced	 ?by	 ?the	 ?light	 ?absorption	 ?and	 ?scattering	 ?effects	 ?in	 ?skin,	 ?and	 ?is	 ?thus	 ?weaker	 ?than	 ?the	 ?intensity	 ?levels	 ?in	 ?hemocytometer	 ?slide	 ?for	 ?the	 ?same	 ?setting.	 ?Fluorescence	 ?detection	 ?in	 ?skin,	 ?thus,	 ? requires	 ? a	 ? higher	 ? laser	 ? power	 ? setting	 ? for	 ? excitation.	 ? The	 ? calibration	 ? curve	 ? here	 ? is,	 ?therefore,	 ? only	 ? useful	 ? for	 ? finding	 ? the	 ? linear	 ? range	 ? of	 ? intensity	 ? change	 ? with	 ? respect	 ? to	 ?concentration	 ?but	 ?not	 ?useful	 ?for	 ?calculating	 ?the	 ?concentration	 ?values	 ?in	 ?confocal	 ?images.	 ?	 ?To	 ? estimate	 ? the	 ? concentration	 ? values	 ? in	 ? confocal	 ? images	 ? taken	 ? from	 ? skin,	 ? the	 ? t1	 ? image	 ?taken	 ?for	 ?the	 ?experiment	 ?with	 ?the	 ?smallest	 ?t1	 ?(5	 ?min)	 ?was	 ?used	 ?to	 ?estimate	 ?the	 ?intensity	 ?of	 ?0	 ?50	 ?100	 ?150	 ?200	 ?250	 ?0	 ? 500	 ? 1000	 ? 1500	 ? 2000	 ? 2500	 ? 3000	 ? 3500	 ?Intensity	 ?[a.u.]	 ?Doxorubicin	 ?Concentra?on	 ?[?M]	 ?Hemocytometer	 ?Surface	 ?Close	 ?to	 ?Coverslide	 ?Middle	 ?Region	 ?151	 ?	 ?the	 ? injected	 ? drug	 ? concentration	 ? at	 ? the	 ? start	 ? of	 ? the	 ? diffusion	 ? process	 ? (Figure	 ? A.15).	 ? The	 ?concentration	 ?of	 ?the	 ?very	 ?bright	 ?center	 ?of	 ?the	 ?diffusion	 ?source	 ?was	 ?assumed	 ?to	 ?correspond	 ?to	 ?roughly	 ?the	 ?initial	 ?concentration	 ?injected	 ?into	 ?the	 ?skin	 ?(i.e.	 ?87	 ??M).	 ?	 ?Figure	 ?A.15:	 ?Initial	 ?concentration	 ?calculated	 ?from	 ?the	 ?average	 ?intensity	 ?at	 ?the	 ?bright	 ?center	 ?of	 ?the	 ?diffusion	 ?source.	 ?Measured	 ?average	 ?intensity	 ?was	 ?120.4	 ?for	 ?87	 ??M	 ?concentration.	 ?	 ?	 ?	 ?	 ? 	 ?Bright	 ?region	 ?in	 ?the	 ?center	 ?corresponding	 ?to	 ?the	 ?initial	 ?concentration	 ?152	 ?	 ?A.9 MATLAB	 ? code	 ? used	 ? to	 ? calculate	 ? intensity	 ? distribution	 ? in	 ? the	 ?confocal	 ?images	 ?aveintensity(1,numImages) = 0; %aveintensity = resulting average intensity distribution of rings  %numImages = number of images for timesteps  i = 1; p = 2; while i<6      I = imread(strcat('T= ',int2str(i),'.tif')); %Igray = image in grayscale Igray = rgb2gray(I); %Image size SZ = size(Igray); %circular boundary under investigation: boundary=min(min(c),min(SZ)-max(c)); %c = matrix coordinates for the center of source (found independantly)   ix=SZ(1);iy=SZ(2);   %center coordinates of the mask from c (being the center of interest) cx=c(1,1);cy=c(1,2); %ringthickness: rt = 10;   %circular mask generating function %http://www.mathworks.com/matlabcentral/newsreader/view_thread/146031     numcircles = int64(floor((boundary/rt)));   %previous masked images; previousmasked = Igray.*0;   %ring average intensity ringav = 0;   k=1; for n=1:numcircles          [x,y]=meshgrid(-(cx-1):(ix-cx),-(cy-1):(iy-cy));     c_mask=((x.^2+y.^2)<=(n*rt)^2);     maskedring = im2uint8(c_mask)/255 - previousmasked;     maskedimage = Igray.*maskedring;     ringav = sum(sum(maskedimage))/nnz(maskedring);     previousmasked = im2uint8(c_mask)/255;     aveintensity(k,p) = ringav;     aveintensity(n,1) = n;     k = k+1;   end   i = i + 1; p = p + 1; end 153	 ?	 ?A.10 Confocal	 ?data	 ?for	 ?all	 ?of	 ?the	 ?injection	 ?trials	 ?Figures	 ?below	 ?show	 ? the	 ? confocal	 ? images	 ? corresponding	 ? to	 ? the	 ?depth	 ?where	 ? the	 ? spherical	 ?source	 ?below	 ?the	 ? injection	 ?spot	 ?was	 ? located.	 ?The	 ?average	 ? injection	 ?depth	 ? for	 ?all	 ? the	 ?trials	 ?was	 ?121.9	 ??	 ?22.5	 ??m.	 ?	 ?	 ?Figure	 ?A.16:	 ?a)	 ?and	 ?b)	 ?confocal	 ?images	 ?for	 ?fresh	 ?skin	 ?samples	 ?injected	 ?with	 ?87	 ??M	 ?solution	 ?of	 ?doxorubicin	 ?using	 ?500	 ??m	 ?tall	 ?microneedles,	 ?time	 ?difference	 ?between	 ?images:	 ?5	 ?min;	 ?a)	 ?first	 ?image	 ?taken	 ?9	 ?min	 ?and	 ?30	 ?s	 ?after	 ?injection;	 ?b)	 ?first	 ?image	 ?taken	 ?10	 ?min	 ?and	 ?45	 ?s	 ?after	 ?injection.	 ?Size	 ?of	 ?images:	 ?775	 ??m	 ??	 ?775	 ??m.	 ?	 ?	 ?	 ?	 ?	 ?a)	 ?b)	 ?154	 ?	 ?	 ?	 ?	 ?	 ?Figure	 ?A.17:	 ?a),	 ?b)	 ?and	 ?c)	 ?confocal	 ?images	 ?for	 ?refrigerated	 ?skin	 ?samples	 ?injected	 ?with	 ?87	 ??M	 ?solution	 ?of	 ?doxorubicin	 ?using	 ?500	 ??m	 ?tall	 ?microneedles,	 ?time	 ?difference	 ?between	 ?images:	 ?5	 ?min;	 ?a)	 ?first	 ?image	 ?taken	 ?12	 ?min	 ?and	 ?20	 ?s	 ?after	 ?injection;	 ?b)	 ?first	 ?image	 ?taken	 ?9	 ?min	 ?and	 ?40	 ?s	 ?after	 ?injection;	 ?c)	 ?first	 ?image	 ?taken	 ?8	 ?min	 ?after	 ?injection.	 ?Size	 ?of	 ?images:	 ?775	 ??m	 ??	 ?775	 ??m.	 ?	 ?	 ?	 ?	 ?a)	 ?b)	 ?c)	 ?155	 ?	 ?	 ?	 ?	 ?Figure	 ?A.18:	 ?a),	 ?b)	 ?and	 ?c)	 ?confocal	 ?images	 ?for	 ?frozen	 ?skin	 ?samples	 ?injected	 ?with	 ?87	 ??M	 ?solution	 ?of	 ?doxorubicin	 ?using	 ?500	 ??m	 ?tall	 ?microneedles,	 ?time	 ?difference	 ?between	 ?images:	 ?5	 ?min;	 ?a)	 ?first	 ?image	 ?taken	 ?10	 ?min	 ?and	 ?29	 ?s	 ?after	 ?injection;	 ?b)	 ?first	 ?image	 ?taken	 ?5	 ?min	 ?and	 ?47	 ?s	 ?after	 ?injection;	 ?c)	 ?first	 ?image	 ?taken	 ?5	 ?min	 ?and	 ?4	 ?s	 ?after	 ?injection;	 ?Size	 ?of	 ?images:	 ?775	 ??m	 ??	 ?775	 ??m.	 ?	 ?	 ?	 ?	 ?a)	 ?b)	 ?c)	 ?

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