Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Synthesis of a model lipid and observation of behaviour of model liposomes by electrophoresis Song, Xu Chun 1994

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1994-0300.pdf [ 1.41MB ]
Metadata
JSON: 831-1.0059587.json
JSON-LD: 831-1.0059587-ld.json
RDF/XML (Pretty): 831-1.0059587-rdf.xml
RDF/JSON: 831-1.0059587-rdf.json
Turtle: 831-1.0059587-turtle.txt
N-Triples: 831-1.0059587-rdf-ntriples.txt
Original Record: 831-1.0059587-source.json
Full Text
831-1.0059587-fulltext.txt
Citation
831-1.0059587.ris

Full Text

SYNTHESIS OF A MODEL LIPID AND OBSERVATION OF BEHAVIOUR OFMODEL LIPOSOMES BY ELECTROPHORES ISByXU CHUN SONGB.Sc.., University of Science and Technology of ChinaHefei, Anhui, P.R.China, 1986A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF CHEMISTRYWe accept this thesis as conformingApril 1994© Xu Chun Song, 1994THE UNIVERSITY OF BRITISH COLUMBIAIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)__________________________Department of CftkLSTP1The University of British ColumbiaVancouver, CanadaDate Apr. 4jDE-6 (2188)ABSTRACTA model lipid consisting of a cholesterol base, tetraethoxy— spacerand glucuronic acid head group was synthesized. First, the head group wasprepared by acetylation and esterification of glucuronolactone to producemethyl (1, 2,3,4-tetra-O— acetyl——D—glucopyran)uronate (j.) which was thenbrominated to produce methyl (2,3,4—tri-O—acetyl—--D—glucopyranosylbromide)uronate (Z). The combination of the cholesterol part and tetra—ethoxy chain was made by reacting cholesteryl—p—toluenesulfonate andtetraethylene glycol, to produce 3—O—(i1-hydroxy—3, 6, 9—trioxaundecyl)cholest—5-ene (tetra-EC) (3). The above steps were carried out with methodsreported previously. The coupling of the head group and tetra-EC employeda different method, which had been used by others in the coupling reactionof the same head group and cholesterol, by using silver oxide as thecatalyst instead of silver carbonate. Methyl t3—O--(3,6,9—trioxaundecyl)cholest—5—en—3-yl—2,3, 4—tri—O—acetyl——D—glucopyranosid3uronate () wasproduced from the coupling reaction with a yield estimated to be — 50’!.,higher than that of the reaction with silver carbonate as the catalyst.The final step was to remove the methyl group and the acetyl protectinggroups on the head group by using excess MaCH in a specific solventsystem and acidifying with HC1, to obtain crude 3—O—(3,6,9—trioxundecyl)cholest—5—en—3—yl——D—glucopyranosiduronic acid (). The crude acidproduct was primarily purified by adjusting the pH of the suspension of theacid in warn ethanol and water and the salt form was obtained. The saltproduct, (i), was precipitated pure from chloroform solution by addition ofmixed ethyl acetate arid hexane.iiThe product (tetra—ECG), which has a negative charge on the headgroup, was used with other lipids to prepare liposomes. The liposomes,which vary in poly(ethylene glycol) (PEG) chain density and charge locationwere made for the purpose of mimicking the glycocalyx region in actualbiomembranes. Particle electrophoresis was used to measure the mobilitiesof the liposomes in the solutions with variation of pH (1.8—9.9), ionicstrength (O.OO1M—O.1H) and PEG chain density (O-60’A by molar ratio). Theclassical theory for particle electrophoresis was applied to calculate themobility and the apparent charge density of the liposoznes. The pKa oftetra—ECG was determined with a plot of the mobility of the tetra—ECG-containing liposome against pH on the surface of the particle. A numericalmodel, which has been developed as a computational program, was used tointerpret the results of electrophoresis as a function of ionic strength,in terms of several parameters which describe the surface properties of theliposomes.iiiTABLE OF CONTENTSPageABSTRACT jjTABLE OF CONTENTS ivLIST OF FIGURES viLIST OF TABLES viiSYMBOLS AND ABBREVIATIONS viiiACKNOWLEDGEMENTS ixChapter 1 Synthesis of Lipids 11.0 Objectives 11.1 Introduction 11.1.1 Lipids in Biomembranes 21.1.2 Chemical Structure of Lipids in Biomembranes 21.1.3 The Model Lipid 41.2 Methods 51.2.1 Synthesis of Tetraethoxycholesterol(tetra—EC) 71.2.2 Synthesis of Protected and Brominated Glucuronate 91.2.3 Coupling Reaction 101.2.4 Deprotection 131.3 Experimental 17Preparation of Methyl (1,2,3, 4—tetra—O—acetyl——D—glucopyran)uronate 18Preparation of Methyl (2,3,4-tri—O—acetyl——D—glucopyranosyl bromide)uronate 18Preparation of 3—O—(11—hydroxy—3, 6, 9—trioxaundecyl)cholest—5—ene 19Preparation of Methyl [3—O—(3,6,9—trioxaundecyl)cholest—5—en—3—yl—2, 3, 4-tri—O-acetyl—-D—glucopyranosid]uronate .. 20Preparation of (3—O—(3,6,9—trioxaundecyl)cholest—5—en—3—-yl--D-glucopyranosid]uronic acidand Sodium [3—O-(3,6,9-trioxaundecyl)cholest—5-en-3-yl--D-glucopyranosid]uronate 21iv1.4 Discussion 24Chapter 2 Electrophoresis of Liposomes 262.1 Introduction 262.2 Theories of Particle Electrophoresis 282.2.1 The Theory for Charged Particle with A Smooth Surface 282.2.2 pH at Surface of the Liposomes 322.2.3 The Theory for NHairy Model Liposomes 332.3 Methods 352.3.1 Preparation of Liposomes 352.3.2 Electrophoresis Equipment 372.4 Experimental 392.4.1 Liposome Preparation 392.4.2 Electrophoresis 412.4.2.1 pH Dependence Studies 412.4.2.2 Ionic Strength Dependence Studies 412.4.2.3 Different Compositions of Liposomes 432.5 Result and Discussion 432.5.1 pH Dependence 452.5.2 Composition Dependence 472.5.3 Ionic Strength Dependence 492.6 Conclusions 52REFERENCES 53LIST OF APPENDICES 56VLIST OF FIGURESPageFigure 1.1 The Modified Fluid Mosaic Model of Biomembrane 3Figure 1.2 The Model Lipid 4Figure 1.3 Procedure of Lipid Synthesis 6Figure 1.4 Two Anomers of Protected Sugar 9Figure 1.5 Koenig—Knorr Coupling Reaction 11Figure 1.6 Scheme of Schneider’s Coupling Reaction iiFigure 1.7 Procedure of Deprotection of tetra—ECPG 14Figure 1.8 TLC Result for Side Product in Deprotection 16Figure 2.1 Structure of Liposome 27Figure 2.2 Concepts of Electric Double Layer andElectrokinetic Potential (C) 30Figure 2.3 The Liposome Model of Glycocalyx 34Figure 2.4 Preparation of Liposomes 36Figure 2.5 Equipment for Particle Electrophoresis 38Figure 2.6 Deducted Mobility of Liposome EL* 2vs pH at Surface of Liposome 48Figure 2.7 Mobility of Liposomes EL* 7—1, 7—2, 7—3,and EL* 8-1, 8-2, 8-3vs PEG Chain Concentration 48Figure 2.8 Mobility of Liposomes EL* 3 — 6vs Ionic Strength 50viLIST OF TABLESPageTable 1.1 Brief Review of Syntheses of PEG—CholesterolDerivatives 8Table 1.2 Yields of Products 25Table 2.1 Composition of Liposomes for Electrophoresis .... 40Table 2.2 Mobility and Apparent Surface Charge Density ofEL* 1 and EL* 2 with Variation of pH 44Table 2.3 Mobility and Apparent Surface Charge Density ofEL* 7 — EL* 8 with Variation of Composition 44Table 2.4 Mobility and Apparent Surface Charge Density ofEL* 3 — EL* 6 with Variation of Ionic Strength .. 45Table 2.5 Deducted Mobility, Net Surface Charge Density andpH at Particle Surface of EL* 2with Variation of pH 46Table 2.6 Comparison of Mobility Model Parametersfor Four Liposome Preparation 51viiABBREVIATIONSCI chemical ionizationChol. cholesteryl or cholesterold doublet (In N)ffi results)DCI desorption chemical ionizationDPPG dipalmitoyl phosphatidyl glycerolegg PC egg phosphatidyicholineEl electron ionizationHPLC high pressure liquid chromatographyLSIMS liquid secondary ion mass spectrometrym multiple (In NMR results)NNR nuclear magnetics resonancePEG poly(ethylene glycol)s singlet (In NMR results)tetra—EC tetraethoxycholesteroltetra—ECG sodium [3—O—(3,69—trioxauridecyl)cholest—5-en-3-yl---D-glucopyranosid)uronatetetra—ECPG Methyl [3—O—(3,6,9—trioxaundecyl)cholest—5—en-3—yl—2, 3, 4—tri—O—acetyl—-D—glucopyranosidluronateTLC thin layer chromatographytr triplet (in NNR results)tri—EC triethoxycholesterolqt quartet (in N)4R results)viiiAcKNOWLEDGE24ENTSI would like to express my special thaiilcs to my supervisor, DonBrooks, for his patience, assistance and encouragement throughout this work.I am especially grateful to Johan Janzen, who has helped me so muchduring almost all y project. Thanks to everyone in this group fortheir valuable suggestions and assistance.ixChapter 11.0 ObjectivesThe objectives of this project are:1) to modify the synthesis of a model lipid which has a cholesterylbase, poly(ethylene glycol) (PEG) chain and glucuronic acid head group;2) to prepare liposomes, which have different (negative) chargelocation and different densities of PEG chains, as models of the glycocalyxregion in actual biomembranes;3) to use particle electrophoresis to measure the mobilities of thecharged liposomes and apply both the classical theory and a numerical modelof liposomes to interpret the results.1.1 IntroductionBiological membranes are directly involved in many biologicalprocesses of living organisms. They are composed primarily of lipids andproteins, with some associated oligosaccharides and small amounts of mono—and divalent ions binding to ionic groups of lipids and proteins, and water.For more than 50 years, biomembranes have been extensively studied in orderto understand the relationship between their structure and the function(1, 2). In 1925 Gorter and Grendell (3) first described the structure of abiomembrane as a bilayer lipid matrix. Since then, various models havebeen proposed to describe membrane organization, resulting in our presentunderstanding of membranes described by the Singer and Nicholson fluidmosaic model (4), which has been proved to be useful for presentation of1the gross organization and structure of lipids and proteins inbiomembranes. Figure 1.1 is a diagram which is based on the model given bySinger et al. and modified to include a description of carbohydrates onthe surface of the membranes (2).1.1.1 Lipids in BiomembranesBiological membranes contain an astonishing variety of lipids, both inamount and in kind. Phospholipids are usually found in biological systems,as well as sphingolipids, glycolipids and steroids. Not only do lipidsfunction as a matrix for association of membrane proteins and provide apermeability barrier between the exterior and interior of a cell, they alsoparticipate in a variety of specialized biological processes (5).1.1.2 Chemical Structure of Lipids in BiomembranesGenerally the structure of lipids in biomembranes consists of twoparts: a polar or hydrophilic region, and a nonpolar or hydrophobic region.The chemical nature of these two sections can vary substantially. In waterthe polar regions tend to orient toward the aqueous phase while thenonpolar regions are withdrawn from water. For phospholipids bearing twoalkyl chains the lowest free energy is achieved through formation of a twodimensional bilayer which is the basis of biological membranes.In some cases, models of lipids may be described to have a thirdpart, the spacer region, between the terminal of the hydrophilic head groupand the hydrophobic tail in a bilayer or micelle (6). In recent work on thesynthesis of artificial lipids, poly(ethylene glycol) (PEG) has been appliedas a spacer (7).2Figure 1.1 A modified fluid mosaic model of biomembrane(2)It offers a three—dimensional and cross—sectional description ofprotein, lipid and carbohydrate in the model of a biomembrane. Lipidand protein form a structural bilayer matrix and carbohydrate moietiesextend from the surface of membranes into the aqueous solution.3Another characteristics of most lipids is that they have charged headgroups, such as phosphate, sulfate, and carboxylate (anionic), as well asammonium groups (cationic) (8). These provide biomembranes which containsuch lipids with net surface charges.1.1.3 The Model LipidOur synthesis of lipids is aimed at making liposomes to mimicbiomembrane structures to allow studies of the properties of a modelbiomembrane surface. The detailed purpose is described in Chapter 2.The model lipid consists of three sections:Figure 1.2 The Model LipidR = H (3—O—(3, 6, 9—trioxaundecyl)cholest—5-en—3—yl—-D-glucopyranosid]uronic acidR = Na sodium [3—O—(3,6,9—trioxaundecyl)cholest—5—en--3—yl--D—glucopyranosid)uronate4The cholesteryl group is placed as a hydrophobic anchor in this lipid,because it is present in a wide variety of biological membranes andparticipates actively in many biological processes (9).The hydrophilic section is glucuronic acid, which provides thesaccharide character. It has been widely observed that carbohydrates onthe surfaces of biomembranes are common groups in living organisms. Ingeneral they can act as recognition reactants, structural materials andenergy stores and are involved in a multitude of interactions with otherorganisms and biological reagents (10, 11). They are also considered tostabilize the membranes against disruption in some biological systems (12).The spacer section of the model lipid is PEG, because it is soluble inwater and most organic solvents and apparently has a high compatibilitywith biological systems (13). A compound with four ethylene glycol unitswas chosen. This particular lipid had been synthesized by Paula J. Sather,a previous student in our laboratory, in 1990 (7). However, she obtained alow yield and was unable to isolate enough product to be used in modelmembrane studies.1.2 MethodsThe synthesis of the model lipid employed modifications of theprocedure described in P.J.Sather’s thesis (7). It consisted of threemajor steps:50UHO\ o OH HOrjY/—OHtretraathylena qlycolH OHqlucuroo1acton.A0Ac‘oAc I cholesteryl-p-toluerie sulfonateHmethyl t.tra-O-&cety].— -Dqlueopyranuronata (1)1Me000ACH +‘OAc IBrCompound ta> Compound (1)MeOOCOAo HCompound CL)0—1-----Compound () & (>Figure 1.3 Procedure of Lipid Synthesis6The first step is connection of tetra—ethylene glycol to thecholesteryl group by displacing the tosylate from cholesteryl—p—toluenesulfonate. The second step is coupling between compound () (tetra—EC)and the protected and brominated glucuronic acid - compound (a), and thefinal step is removal of the protecting acetyl groups from the glucuronicmoiety.1.2.1 Synthesis of Tetraethoxycholesterol (tetra—EC)The starting materials, tetraethylene glycol and cholesteryl—p—toluenesulfonate, are commercially available. Cholesteryl—p—toluene sulfonate hasvery high reactivity with the compounds which contain hydroxyl groups,especially with water. For this reason, the reaction between tetraethyleneglycol and cholesteryl—p—toluene sulfonate is carried out under anhydrousconditions and in the absence of oxygen. This method was reported byBrockerhoff and Ramsammy (14). Patel et al. (15) used it to synthesizetriethoxycholesterol (tri—EC) and the yield was >90X. Sather (7) appliedthe same procedures to make a series of oligo—ethoxy—cholesterols andreported the yields given in Table 1.1.From Sather’s work, a key to a high coupling yield was to useanhydrous 2,4-dioxane (which had been dried with sodium metal and thendistilled prior to the reaction) as the solvent.Liquid chromatography of mixed organic solvent systems on silica gelcolumns is the usual way of purification of these compounds. The yield ofthis procedure was 58.9’/.7Table 1.1 Brief Review of Recent Syntheses of PEG—CholesterolDerivativesHf[OEthoxy Reaction Molar Ratio ReactionREF4CE Unit Pathway of Reagents: Time Yield(n) cTS*/pEG (hr) (•/.)(14) 3 a -- -— ——(15) 3 b 1/25.3 2 92(16) 1 b 1 / 25—30 2—4 813 b 1/25-30 2—4 92(20) 3 b -— 2—3 ——(13) 3 b 1 / 25.4 24 —1004 b 1/4.5 24 56.56 b 1/5 60 26(18) 3 b —- —— ——a reported by Fong et al in Lipids, Vol.12, iQ 857—62(1977)b excess PEG and cholesteryl—p—toluenesulfonate were stirredin ref lux of dioxane(dried by ref lux with Na) for severalhours under N2 [Patel et al, 1984, Ref. (15)]* Cl’S: cholesteryl—p—toluene sulfonate81.2.2 Synthesis of Protected and Brominated Glucuronate (2)Two steps are taken to obtain the compound (2) (See Figure 1.3). Theywere reported initially by Bollenback et al. (17) in 1955 and utilizedwidely since then as the traditional method to make this compound (7).The first step includes esterification of glucuronolactone (to producemethyl glucuronate) arid acetylation of the four hydroxyl groups with aceticanhydride. The solvents are anhydrous methanol and pyridine, the latteracting as a catalyst of acetylation. In this part, the crude product isobtained by crystallization at — 4C from the solution of the reactionmixture and the pure crystals are obtained by recrystallization in absoluteethanol. Sometimes the color of the reaction mixture is so dark that thesolution of crude product needs to be decolorized by carbon (ref lux underreduced pressure) to remove the colored impurities before recrystallization.Compound (1) has two anomers: and (See Figure 1.4). The producthas been reported to be the anomer based on the NNR evidence (7). In ourwork the yield was 33.5%.typeFigure 1.4 Two Anomers of Compound (1)type9The second step of the synthesis of compound (Z) is bromination at theanomeric carbon of compound(). Hydrobromic acid in glacial acetic acid(3O% by weight) reacts with the acetyl group, eliminating one molecule ofAcOH and forming the C-Br bond. The crude product is crystallized withabsolute ethanol.The product was anomer of compound () (because of the anomericeffect of the suger ring) and the yield was 857. in Bollenback et al.’s workand 737. in our synthesis. The NMR spectrum of the product in Figure 1.5indicates, from the coupling constant between the protons connecteddirectly to the sugar ring, that the bromine is axial in product (18) —that is the anomer of compound (a). (See Appendix II)The procedure of bromination had also been used by Sather (7) withvery satisfactory results. An important point is that the brominatedcompound (2) should be used in the next coupling reaction as soon aspossible because the Br is sensitive to both oxygen and water, especiallyin light and heat, which might produce the impurities that could complicatesubsequent reactions.1.2.3 Coupling ReactionTypically, catalysts are used in the coupling reaction between thebrominated sugar and tetra—EC to activate the bromine first; then the sugaris attacked by the hydroxyl group on tetra—EC to form the ether linkage.Koenig Knorr’s method was the first one dealing with this type of coupling,with silver carbonate as the catalyst (See Figure 1.5). Again, Satherfollowed this method in her work, but the yields reported were very low,around 30% or less, after purification by column chromatography (7).10MeOOCAcOO\HBrCompound ()Figure 1.6 Scheme of Schneider et al.’s Coupling Reaction (20)+Compound (2)Ag1CO3BenzeneAcO3Compound (i.)Figure 1.5Mo00Koenig-Knorr Coupling Reaction+Compound (2.) CholesterolAgOAcOAcO.11This method was considered and tried in our project. The coupledproduct did form, but the yields varied from a few percent to less than 30°A,similar to Sather’s results. Even though silver carbonate was freshly madeand dried in the dark (which did affect its catalytic efficiency), therewas no significant improvement in the yield.A mixed catalytic system of mercury bromide and mercury oxide, basedon Goodrich et al.’s work (19), was tried in our system, but the results werealso not satisfactory.Another approach was an older method reported in 1969 by Schneider andBhacca (20), which had been used in Goodrich et al.’s previous work (21) (SeeFigure 1.6). In this method, silver oxide (freshly made) had been usedinstead of silver carbonate. The reaction was between exactly the samesugar used in our system, and simple cholesterol. The yield reported bySchneider et al. was very high (and so was the yield of their deprotectionreactions).This method was applied in our system and brought the yield of couplingreaction of the sugar and tetra-EC up to 50%. (The yield was estimatedfrom 1H—NMR spectra of several samples of impure products, because of thedifficulty of purification).The crude product was a syrup which was easily dissolved in an organicsolvent. Silica gel provided an appropriate stationary phase to separatethe compounds in liquid chromatography. (Aluminum oxide column was triedand proved inadequate) The problem in the purification is that bothtetra-EC and the product (3), tetra—ECPG, have very strong affinity forsilica gel. Even though they can be separated very well on TLC silicaplates in small scale, with the mixed solvent system of ethyl acetate,chloroform and hexane, the separation on a column was quite differentbecause the product, tetra—ECPG, always came off contaminated with a small12amount of tetra—EC (as detected by TLC). Controlling the amount and mannerof loading material did not improve the separation. The pure product hadto be collected from only a few of fractions in each run of chromatography,since most of the fractions remained as mixtures of the product andtetra—EC (by TLC results). The practical reaction yield, >50%, wasestimated roughly from the sum of the pure product and the product whichremained in the mixture with tetra—EC. For determination of the molarratio of tetra—ECPG and tetra—EC in the mixtures, the method is describedin Appendix II. TLC analysis of the mixtures indicated that most of thecomponents were those two compounds.The yield from the purification was so low that several chromatographyruns had to be made, starting from a few grams of the crude product, toprovide sufficient material for the next step.1.2.4 DeprotectionIn organic syntheses, it is an old problem to make specificfimctionalized compounds by retaining some of the functional groups whilechanging others in reactions. Many reagents have been applied inprotection, deprotection or catalysis for both of these two reactionsunder different circumstances.In our project, deacetylation and deesterification were required.The latter is easy to be approached by hydrolysis (13, 19, 20). However,the deacetylation step took much more effort. Although there have beenmany methods of deacetylation used, with sodium methoxide in methanol asthe most commonly used reagent system (22), most of them were applied onlyfor small molecule systems.13compound (4)(i) addition of NaOH—arthydrous MeOH, methylene chloride(5 hrs)(ii) addition of THF, aqueous NaOH, MeOH and water(1.5 hrs)(iii)pH -* 3, concentration of the mixture to 10—30 ml to givethe precipitate of the acid productcompound ()(crude)(i) suspension in warm ethanol—water, pH - 7(ii) addition of ethanol to precipitate the salt productcompound () compound ()(crude) recrystallization from (pure)chloroform solutionethyl acetate/hexaneFigure 1.7 A diagrammatic description of the procedure of thedeprotection of the compound(4)(tetra—ECPG)A procedure, which had been reported by Schneider et al. (20) and usedin Sather’s deacetylation step (7), was first applied in deacetylation ofcompound (4). It is a complex system of NaOH in anhydrous methanol (sodiummethoxide), methylene chloride, THF and then aqueous NaOH solution. Theprocedure is described in Figure 1.7. Following adjustment of the pH to 3,the crude acid product is separated from the mixture by centrifugation.The primary purification is transformation of the crude acid () to thesalt () by adjusting pH to 7 in aqueous ethanol. This step removes theimpurities which are not soluble in water and ethanol.14The procedures, including the quantities of reagents given, were exactlyfollowed, but the reactions did not go to completion (See the TLC results inFigure 1.8). A series of other compounds appeared, making purificationvery difficult and greatly lowering the deprotection yield. Longerreaction times produced no improvement in the results. It seems that someuncompletely deprotected compounds form producing poor yields. Thesecompounds were identified because the amounts were very small.After several failures, the sodium hydroxide concentration in thefirst part of the reaction was increased. The yield of crude material wasimproved. After the primary purification, further purification againbecame a significant problem because of the polyhydroxylated nature of thecompounds () and (i). (See Section 1.3 TLC results).TLC showed that the deprotected product interacted strongly withsilica gel for both acidic and salt forms. In the usual (not highly polar)solvent systems, the product (acid or salt) almost always remained at theorigin of the plate. The product dot moved from the origin when a polarsolvent, e.g., methanol, was added, but the solubilities of the productswere limited in methanol. Hence, chromatography on a silica gel column wasnot considered for the purification.Instead, recrystallization of the salt product, compound (i), fromchloroform (which is the best solvent for the products) was attempted, withsolvents which were expected to lower the solubility of the product andcrystallize them from solution. A precipitate, which was the pure product,resulted upon addition of a mixed solvent of ethyl acetate and hexane. TLCgave an indication of its purity. The integration information from protonNMR and LSIMS results in different matrices were also useful.15Column 1 2 3 4 5 6 7 8 9 109....L.i. Apr.F6A1Figure 1.8 TLC Results for the Compounds in the Deprotection Step(solvent system: ethyl acetate/chioroform/hexane 3:3:1)Columns 1 -‘ 7 are the results of TLC monitoring of the first 5—hourreaction (See Figure 1.7), with sampling times indicated below. Column 8is the result for the sample taken from the reaction system afteraddition of aqueous NaOH solution. Column 9 is for the mixture extractedfrom the finished reaction solution with chloroform, in which there are aseries of compounds. Column 10 is the reference of the compound beforedeprotect ion.16The yield from this procedure was 32.6%, but not all of the product wasrecovered. A portion of the product remained in solution with theimpurities.The practical yield of the reaction was not estimated, therefore,because it was difficult to determine the type and concentration of theimpurities. The NMR analysis of pure deprotected product (6) is inAppendix II. The product (i), called tetra—ECG, was used to prepareliposome in the next part of this project.1.3 ExperimentalGeneral DetailsProton NNR spectra were recorded on Bruker 141-1—400 MHz and BrukerAC—200E (200 MHz) spectrometers in CDC].3 or CD3O as the solvent. Massspectra were recorded on a Kratos MS—40 (El), a Delsi I4ermag Rb—b C MassSpectrometer (CI & DCI) with ammonia reagent grade gas and a Kratos ConceptII HQ Mass Spectrometer(LSIMS) with matrices of glycerol and thioglycerol.Solvents and reagents were used as purchased with the exception ofthose listed below:Chloroform (solvent for making tetra—EC), benzene, pyridine,dichioromethane and tetrahydrofuran (THF) were distilled prior to use.Dioxane was distilled from a ref lux with sodium for 1 hour prior to use.Methanol was distilled from a ref lux with magnesium and iodine for 0.5hours, stored over 4A molecular sieves, and distilled again from calciumhydride or sodium hydride, prior to use. Cholesteryl—p—toluene sulfonate,tetra—ethylene glycol, and glucuronolactone were dried under vacuum. Ag2017was made from hot NaOH and AgNO3 solution in the dark and dried prior touse. Ag CO was made from AgNO and Na CO solutions in the dark.23 3 23SYNTHESESPreparation of Methyl (1,2,3, 4-tetra-O-acetyl-j3-D-glucopyran)uronate (1)This reaction was based on the procedure described by Bollenback etal. (17), and also by Sather (7). The reagents and amounts were:Glucuronolactone 10 g, 56.78 mmolSodium hydroxide 0.08 g(in MeOH)Anhydrous methanol 75 mlAcetic anhydride 60 mlPyridine 25 ml in 5 ml acetic anhydrideThe product was decolorized with carbon if necessary and recrystallized fromabsolute ethanol. The yield was 7.15 g, 19.00 inmol.NMR (400 MHz) CDC1 2.03(2s,9H), 2.11(s,3H), 3.73(s,3H), 4.16(d,1H),5.12(tr,1H), 5.26(m,2H), 5.76(d,1H)Mass Spectrum DCI: m/e 394(M+NH), m/e 317(M—59), m/e 257(317—59—H)m/e 228(257—O—CH), m/e 114(317—3x59-2CH) or (257—0-CH)44Preparation of Methyl (2,3,4-tri-O-acetyl-a-D-glucopyranosyl bromide)uronate (2)This reaction procedure was also from Bollenback et al. (17). Thereagents and amounts were:Compound (1) 1.8114 g, 4.813 inmolHydrobromic acid (30% in acetic acid) 7.2 ml(in 3.6ml)Chloroform 30 mlSodium bicarbonate (saturated) 30 ml18After reaction and removal of acetic acid, the crude product dissolved inchloroform was washed with NaHIDO saturated solution and then water to3remove the residual acid. The product was recrystallized from 100%ethanol. The yield was 1.389 g, 3.497 mmol.NNR (400 MHz) CDC13 2.04(2s,6H), 2.09(s,3H), 3.75(s,3H), 4.57 (d,1H),4.84(qt1H), 5.22(tr1H), 5.59(tr,1H), 6.62(d,1H)Preparation of 3-O-(11-hydroxy--3, 6, 9-trioxaundecyl)cholest-5-ene (3)Tetraethylene glycol (11.0162 g, 56.7 mmol), cholesteryl—p--toluenesulfonate (0.9928 g, 1.836 mniol) and dioxane (20 ml) were added to a 50 mlround bottom flask. A condenser was added and the mixture was stirred atref lux under a nitrogen atmosphere for 6 hours (after 3 hours the reactionwas finished according to the results of TLC). The dioxane was removed byrotary evaporation and the residue was dissolved in 30 ml water andextracted with diethyl ether (5 x 80 ml) in a 250 ml separatory furmel. Theorganic extracts were combined, washed with 10°!. NaC03solution (1 x 40 ml),and then water (5 x 60 ml). The organic phase was dried over anhydroussodium sulfate, filtered and the filtrate was evaporated under reducedpressure. The residue was loaded onto a silica gel column (20 g), packedwith 1:1 (volume ratio) ethyl acetate/chloroform. The column was elutedwith the same solvent system and the product was collected in 1 mlfractions (each fraction was analyzed by mc). The fractions containingproduct were combined and evaporated under reduced pressure to give0.609 g, 1.082 mmol of product.19TLC R= 0.24 , silica 60, ethyl acetate/chloroform 1:1(by volume ratio)NMR CDC1 (400 MHz) 0.64(s,3H), 0.84(qt,6H), 0.88(d,3H), 0.97(s,3H),0.98”2.40 (m, -29H), 3.16(m,1H), 3.60-’3.75(m, “-16H), 5.32(d,1H)Mass Spectrum El nile 560(M—2)4, nile 368(M—194), mle 353(368_CH),nile 255(368ll3side chain), mle 247(368—CH13), nile 195(M—368+H)DCI (NH3) nile 581 & 580(M+NH3), mle 563(M)’,nile 369(M-193), nile 353(369-CH—1), nile 195(M-368+H)’Preparation of Methyl [3-O-(3, 6, 9-trioxaundecyl)cholest-5-en-313--yl--2, 3,4-tri-O-acetyl-13-D-glucopyranosid]uronate ()Compound (2) (0.4642 g, 0.8247 mmol), () (1.2415 g, 3.126 mmol),freshly made silver oxide (0.3919 g, 1.691 mmol) and benzene (10 ml), werestirred together at room temperature in a 25 ml round bottom flask in thedark. After 24 hours, adding a few grams of Celite, the mixture wasfiltered and the filtrate evaporated under reduced pressure to removesolvent. The residue was separated on a silica gel column (100 g) packedwith ethyl acetate/chloroformlhexane 3:1:2 (by volume ratio). The columnwas eluted with the same solvent system, and 1 ml fractions were collectedand monitored by TLC. The crude product, which by TLC contained mostlyunreacted (3) and the product (4), was 0.6523 g. From 1H NMR, the molarratio of (3) and (4) was calculated to be 1.15—1.4. The practical yield ofthe product (4) was 0.397-0.433 mmol, 0.349-0.381 g.The crude product was purified with about 10 runs of the samechromatography as above, and the pure product was obtained.20111 NMR CDC13 (200 MHz) 0.65(s, 3H), 0.83(d, 3H), 0.85(d, 3H), 0.89(d,311), 0.98(s, 3H), 1.0 2.4( 29H), 1.98,2.00 & 2.02(3s. 9H), 3.62( 16H),3.74(s, 3H), 4.03(d, 1H), 4.65(d, 1H), 4.99(tr, 1H), 5.22(2tr, 2H),5.32(d, 1H)TLC (pure product) Rf = 0.45, silica 60,ethyl acetatelchloroformlhexane 3:1:2 (by volume ratio)Mass Spectrum (pure product) DCI (Nil3)nile 929(M+2NH3+NH4)”, nile 913(M+NH+NH4), nile 897(M+NH4)”,nile 877(M—1)’, nile 837(M—CHCO+1)”, nile 528(M+NH—368), nile396(528—3C2H40)’, nile 368(Chol.—16)”, nile 352(396—C40)”,mle 336(352—0)’,nile 334(352-NH)’, nile 317(334—0—1)”, nile 308(352—CHCO—1)”, nile292(308—0)’, nile 276(292—20)”, nile 257(276—NH4—1)”, nile 234(276—CHCO+1)”,nile 218(234-0)”, nile 216(234-NH4)”Preparations of [3-0-(3,6, 9-trioxaundecyl)cholest-5-en-313-yl-13-D-glucopyranosid]uronic acid (5)and Sodium [3-O-(3, 6, 9-trioxaundecyl)cholest-5-en-3(3-yl-3-D-glucopyranosid Juronate (0(4) (0.460 g, 0.524 mmol) was dissolved in anhydrous ether (6 ml) anddichloromethane (6 ml). 0.5 N sodium hydroxide in anhydrous methanol (0.48ml, 0.24mmol) was added and the reaction was carried out at roomtemperature, monitored by TLC. After 4 hours, it was found by TLC withsolvent system of ethyl acetatelchlorofornilhexane 3:1:2 that a series ofcompounds formed . 0.7m1 sodium hydroxide—methanol (0.66 N, 0.462mmol) wasadded. After 1 hour further, TLC showed only one product. THF(40 ml) wasadded with initiation of stirring, followed by addition of aqueous 1 Msodium hydroxide (12 ml, l2mmol) in one portion and methanol (35 ml).21Finally water (75 ml) was added slowly. Stirring was continued for another1.5 hours at room temperature. The pH of the solution was adjusted to 3with the addition of 10°!. HC1. The solution was concentrated withdifficulty by rotary evaporation under reduced pressure to about 10—30 ml.The precipitate was centrifuged and the supernatant was concentrated againto provide more material. The precipitate, the crude acid (5), wassuspended in warm aqueous ethanol and sufficient aqueous sodium hydroxidewas added to adjust the pH to around 7 at which point all the acid haddissolved. Ethanol was added and the salt precipitate formed and separatedwith centrifuging. The yield of crude salt () was 295.9 mg. The crudeproduct () was dissolved in chloroform and then precipitated by ethylacetate and hexane (3:1, volume ratio). The precipitate was proven by TLC,proton NMR and LSIMS to be pure product (h), 130 ing, 0.171 mmol.TLC R1[Salt, (6)] = 0 for:ethyl acetate/chloroform/hexane 3:1:2, chloroform/ethyl acetate 3:1,ethyl acetate/chloroform/benzene 10: 10:3, chloroform,ethyl acetate/CHC1/ethanol 2:2:1,R[Salt, (6)] = 0.56 for methanol, 0.55 for ethanol,0.17—0.6 for chloroform/methanol 7:1,0.57—0.86 for chloroform/methanol 1:1,0.39—0.48 for ethyl acetate/methanol 6:1;Rf[Acid. (i)] = 0 for chloroform/methanol 7:1,0.07 for ethyl acetate/methanol 6:11H NMR COC1 0.65—Z.40(cholesteryl section, —46H by integration),3.0—4.8(broad, —24H), 5.34(d, 1H)LSIMS (low resolution) matrix: thioglycerol22nile 761(M+1)4’, ni/e 693(761— COONa), ni/e 585(M—l76sugar part+2C),ni/e 475(chol.+CH CII OCH CH O+2H), ni/e 443(chol.i-CH CII OCH )‘, ni/e22 22 22 2413(chol.+CHCH)’, ni/e 369(chol.—16)4LSIMS (high resolution) matrix: thioglycerolMass range: 761—761 No. peaks: 1 Base mt.: 168501Mass: 761.48241 Carbon 41 Hydrogen 70 Oxygen 11 Sodium 1(The theoretical formula for the product isC41H6O1Na)231.4 DiscussionIn this part of the work, compounds (4), () and () were produced.Table 1.2 is the list of all the products synthesized. However, there aresome problems which remained unsolved. The first problem was purificationof these types of lipids. Problems occurred in almost all the procedures,from synthesis of tetra—EC through production of the deprotected lipid.Since the purifications of some of the products are on a hundred—milligram scale, HPLC seems appropriate but optimizing the solvent systemswill be very important for improvement of the purification.Better methods of purification on a milligram scale need to bedeveloped for the deprotected product, tetra—ECG, and an effort should bemade to increase the yield of the reaction. Although it has been improvedin this work, several methods suggested themselves, based on more recentpublications:(i) OH—resin as a reagent for deacetylation (22).In this reference, IRA—400(OH) resin was applied in sugar and nucleosidesystems to remove acetyl groups with yields of 70°!. up to 91%.(ii) Enzymes (23).It was reported that seven enzymes could catalyze the partialdeacetylation of sucrose derivatives in phosphate buffer or phosphatebuffer—organic co—solvent with variable yields.Basically, these two methods were again effective for small moleculesand the conditions of the reactions were selected only for the particularcircumstances in their syntheses. However, these could be good startingpoints to improve the deprotection of tetra—ECPG.24Table 1.2 Yield of Products(See Figure 1.3, the procedure of the synthesis)Product Catalyst/Solvent Purification Yield(%)(1) pyridine/(AcO)20 crystallization 33.5(from reactionsolution)() ——/acetic acid recrystallization 72.7(from 100% EtOH)(3) dioxane, N2 chromatography 58.9tetra—EC (ethyl acetate/chloroform 1:1,silica gel)(4) Ag20/benzene chromatography 48.1_52.5*tetra—ECPG (ethyl acetate/chloroform/hexane 3: 1:2, silica gel)(6) ——/methanol(etc.) precipitation 32.6**tetra—ECG (from chloroform solutionby ethyl acetate/hexane)* the yield was estimated from 1H NMR results** the yield was for the pure product obtained in the experiment25Chapter 22.1 IntroductionLiposomes are artificial lipid vesicles which are composed of oneor more bilayers and have an aqueous interior or interlameller phase (SeeFigure 2.1). They can be classified into small unilamellar vesicles(SUV), large unilamellar vesicles (LUV) and multilamellar vesicles (MV) bytheir size and the number of concentric bilayers present (24).Liposomes are structurally similar to biomembranes and have beenutilized as models for studying many aspects of biological membraneproperties, e.g., membrane lipid chemistry, lipid—protein interactions,transport phenomena and ligand binding to membranes. They also have beendeveloped as an approach to controlled drug—delivery systems (25).The liposomes in this work were made from the model lipid synthesizedin Chapter 1, tetra-ECG, which has a cholesteryl base, a PEG spacer and anacidic monosaccharide head group. While cholesterol does not form bilayersspontaneously, addition of PEG chains allows these molecules to formbilayers and liposomes (15). The liposomes not only have a bilayer matrixof cholesterol derivatives, but also have carbohydrates as well as PEGchains linked on the surface. They therefore can be employed as a model tomimic the behavior of actual membranes using pure molecules with welldefined properties. The liposomes are then examined by a technique whichis thought to be sensitive to the properties of the head group region,particle electrophoresis. The result is interpreted in terms of a theoryfor the electrophoretic mobility which contains parameters incorporatingthe chain concentration, thickness and charge location within the surfaceregion (26).26. .• . •• .•I •VVATER • • • • . •• I• • •I• I0. •I•I•I•III•.•• . •I••f(_o’••-.- • :_•_•____________• I___I.I.• • •.cz.•a • : • •ê • • • • •.•I•I•I I.I•1• •II ••I •••I •I•II if• -‘• Liposome00• ••IIpFigure 2.1 Liposome structure (24)27Using the method of particle electrophoresis, the electrophoreticmobility of charged particles in an electric field can be measured. Themobility, which is the particle velocity per unit electric field strength,is related to the physico—chemical properties of the particle surface bythe theory described below. Since biological surfaces are normallynegatively charged, particle electrophoresis has been used in research onmany biological organisms and mammalian cells, especially red blood cells(27). There has been a lot of work which focused on the relationshipbetween the surface properties and behavior of single red cells, usingparticle electrophoresis. Usually the mobilities are interpreted usingtheories which apply to smooth charged surfaces. Cell membranes, however,contain many glycolipids and glycoproteins whose head groups extend intosolution some distance and carry charged residues which are distributedthroughout the depth of the head group region, or glycocalyx. Hence, it isnot appropriate to interpret the electrophoretic mobilities of cells withtheories which assume smooth charged surfaces.In this project, particle electrophoresis was applied to liposomesof known compositions with head groups whose properties modeled those foundon biological cells. A theory expressly designed for surfaces bearing suchstructures was used to interpret the results.2.2 Theories of Particle Electrophoresis2.2.1 The Theory for Charged Particles with A Smooth SurfaceThe classical theory for dealing with the behaviour of smooth chargedparticles, assumed to be locally flat, in electrophoresis is as follows:28An electrical double layer forms near the surface of charged particlesbecause the counterions in solution are attracted to the surface. (SeeFigure 2.2) The electrostatic potential at the surface of chargedparticles, (o), is related to the surface charge density, a’, by (29):= (I) _1{c’12(5)2}[11which reduces to (27):ifZF4’o)<< 1 [21ice RTwhere a’ surface charge density(esu/cm2)ic Debye-Hückel parameter:r 2 ,1/2‘8irNave Ii -iK= [ j (cm ),1O3ckTis called the electric double layer thickness(cm)e dielectric constant of water (78.5)k Boltzmann’s constant (1.3805 x 1O’6erg/e), 23Nay Avogadro s number (6.025 x 10 )T temperature (°K) (298°K in our experiments)e electron charge(4.8 x 10esu)I ionic strength I = 4- CZ (M)ionsC1 molar concentration of i—th ionic speciesZ1 valence of i—th ionic speciesF Faraday constant F = Nay eThe electrostatic potential decays with distance from the surface ofZFo .particles; ifRT1, (o) is given by (27):-KxW(x) = ‘I’(O) e [3]29abI ISOLUTION-l--’V-——-’4±)— --T”H’— ‘I Ia b distance_______ _______from solidFigure 2.2 Concepts of Electric Double Layerand Electrokinetic Potentail()The electric potential, ‘, decays with the distance.a—-the Stern layer of “fixed” charges; b-—shearing boundaryof the solution when solid moves; 1/K——electric double layer;C——electrokinetic potential or zeta potntial (28)II1/K30The motion of the charged particle is determined by the directelectrical force, the fluid drag (from the viscosity of the solution) andthe electroosmotic retardation (due to motion of the electric double layerin the electric field in the direction opposite to velocity of theparticles). The famous Helmholtz—Smoluchowski formula describes thisbehaviour, for the case in which the radius of curvature of the particlesurface is large compared with 1/ic, the electric double layer thickness:[4]E 4inwhere U mobility of particlesdielectric constantr medium viscosity (0.009 poise)E electric field? (zeta potential) is the electrostatic potential atshear plane(or the nonslip surface)The electric field, E, can be calculated from either the voltage orcurrent. Although the total voltage in the experiments was controlled to beconstant (40V) , because of differences in cross—sectional area, it isbest to calculate E from the conductivity of the solution in which theliposomes are suspended.From [4], the electric field was calculated from the measured current:E—_iR — 1[5]1 1 — aAcwhere V , i and R are voltage, current and resistance respectively. 1 isthe electric length related to the distance between two electrodes (27).a is the cross—sectional area of the electrophoresis chamber in the viewingregion (the radius of the viewing section of the chamber is 1.362 mm, givenby the manufactor) and A is the equivalent conductivity of the solution(30). c is the concentration of the electrolyte solution.31Generally, for smooth charged particles, an assumption is made thatzeta potential is equal to the potential at particle surface, P(O). From[2] and [3], an expression for the surface charge density is obtained (forlow surface potentials):[6]2.2.2 pH at Surface of the LiposomesIn aqueous systems, pH is one of the most important parameters thataffect the electrostatic behaviour of the liposomes because H or OH ionsparticipate in the processes of ion binding to surface molecules on theliposomes.According to the model for smooth charged particles, the concentrationof any ion at the surface of liposome, Cs, is different with that of theion in bulk solution, Cb. The relationship between these two concentrationsis (27):ZF’I’ (0)Cs=Cbexp{—RT ]Considering the H distribution, applying [7] and solving forpH = -log H gives:Fo) epHs = pHb+ RT= pHb+ 2.3O3kT[8]where pHb is the value of pH in the bulk solution (measured).2.2.3 The Theory for “Hairy” Model LiposomesThe above relations have been widely used to describe theelectrophoresis of biological cells, even though the surfaces of cells areactually more complicated than that of a smooth particle (31). The regionof the glycocalyx in the real cells, as mentioned in Section 2.1, containsthe polyelectrolyte or polymer chains which are penetrated by ions or small32electrolytes, such as carbohydrates. It causes a distribution of fixedcharges throughout the glycocalyx (rather than a uniform chargedistribution over the smooth surface particles) and hydrodynamic resistanceproduced by polymer segments.There has been some work published which focuses on the description ofthe behaviour of real cells in electrophoresis (32). Levine et al. (31)offered a mathematical treatment of the cell surface, which considered thecharge distribution (within the surface layer) and the hydrodynamic flow.This treatment was improved by Sharp et al. (32) and a model to representthe behaviour of liposomes bearing charged glycolipids in electrophoresis wasdeveloped by McDaniel et al (26).The approach used to interpret the electrophoretic mobilities ofliposomes containing tetra—EC or tetra—ECG is an adaptation of thatdescribed by Sharp and Brooks (32). Briefly, for particles large enoughthat their radius is large compared to the double layer thickness, theelectrophoretic mobility is calculated from the electroosmotic velocityresulting from motion of the electrical double layer adjacent to thecharged particle surface in the electric field. The resulting fluidvelocity a long way from the particle surface is set equal and opposite tothe particle mobility (31). For smooth particles the velocity distributionnear the surface is determined only by the ion concentration profile in thedouble layer, increasing in magnitude with surface charge and with decreasingionic strength. When the surface carries polymer chains which extend awayfrom the surface into solution, however, they exert an additional drag whichreduces the electroosmotic velocity and hence the mobility. At lower ionicstrengths where the double layer is expanded, if the double layer, and hencethe region in which electroosmosis takes place, extends beyond the surfacepolymer layer the effective drag associated with the chains will be reduced33C.)ITj0p.0 I I-.. 0 z U)CDw0 0 rt CD Ii I....0 (I)r 0 ‘rJ G)*0I-..‘IC)(DO0Ii,wp,•p.(DI-CDop0ij(DOo iift‘.3‘:s‘-3CDmIt-0‘t-Ii‘-I)Ph1,.Jp’CD::iLiip(APIC).Q—00tl C)and the mobility will tend to behave more like that of a smooth particle.Hence, the ionic strength dependence is a function of the depth of thesurface layer, which allows its estimation.If the fixed surface charge is located within the region occupied bythe chains the effect on the velocity profile is to reduce the overall dragrelative to the case in which the charge is distributed over the smoothsurface to which the polymer chains are anchored. This increases themobility.In the calculations utilized here, the numerical integration programdescribed by Sharp and Brooks (32) was used. The hydrodynamic drag exertedby the polymer chains is considered to be equal to the Stokes drag exerted bysegments of hydrodynamic radius a present at a uniform density throughout athickness (3. The larger the segment concentration, a or (3, the greater thedrag and the lower the predicted mobility. The parameters used in thisprogram are: i) thickness of the glycocalyx, (3; ii) polymer chain density(number per unit area); iii) the polymer segment radius, a (A); iv) thefixed charge density, o (esu/cm2); v) the location of the fixed charge;vi) the location of the shear plane.2.3 Methods2.3.1 Preparation of LiposomesLiposome behaviour and stability depend on particle size, number ofbilayers, chemical composition and the composition of the aqueous phase inwhich liposomes are formed. It is essential to control all these factorsif one is to understand the relationship between each of these propertiesand liposome function (33).There have been many techniques for preparation of liposomes. Theprocedure (33) can be divided into three stages (See Figure 2.4):35Preparation ofAqueous PhaseHydration of SecondaryLipids: Processing:Formation of Formation ofLiposomes Specified(primary) LiposomesPreparation ofMixture of Lipids:Removal of SolventsFigure 2.4 Procedure of Liposome Preparationfirst, preparation of the aqueous phase and lipid mixture; second, lipidhydration and third, optional steps to make specific sorts of liposomes.In preparation of the aqueous phase, several factors must be considered:osniolarity, ionic strength, pH, choice of materials and their concentrationfor both interior and exterior phases and contaminants.For making a particular molecular mixture of lipids, each of them isfirst dissolved in a single or mixed solvent. Generally, all the lipidsshould be soluble at the desired concentrations in the chosen solvent system.A dry lipid mixture is made by removing the solvent uniformly since theform of dry lipids can seriously affect the hydration and formation ofliposomes. Usually, a continuous film on the container wall is consideredoptimal.The usual hydration method is to disperse the dried lipid mixture intothe aqueous phase by shaking. Following this step, particular techniques36are applied for making specific liposomes, for instance of a particularsize (34). In this work, however, only shaken, multilayered liposomes wereused.2.3.2 Electrophoresis EquipmentThere have been many kinds of equipment used in particleelectrophoresis research for various purposes. The equipment in our projectwas described by Seaman et al. (27) in their work on electrophoresis ofred cells (See Figure 2.5a & 2.5b).In Figure 2.5a, a constant temperature is maintained in a water bathcontaining a stirring device and a thermostated heater(e, thermostat). Thecalibrated vertical traverse, b, and the dial test indicator, k, providethe readings of the vertical and horizontal position of the chamber. Theocular with a fitted graticule, h, serves for the measurement of thedistance that a particle moves in a measured interval.The most important part in the electrophoresis apparatus is thechamber, which is mounted horizontally (between a and g); its verticaland horizontal positions are adjusted until the axis of the microscope islocated at right angles and passes through the center line of the chamber.There are two kinds of chambers, cylindrical and rectangular, whichcan provide accurate and reliable results. In our experiment, a cylindricalchamber similar to that described in Figure 2.5b was used, except that thestopcocks were replaced by plugs in ground glass joints.The chamber can be divided functionally into two parts: the endscontaining two compartments for the electrodes (in KC1 solution) and thecentral observing section, separated by two pieces of sintered glass discs.37Figure 2.5a Cylindrical microelectrophoresis apparatus:a, Tube holder; b calibrated vertical traverse; c, crossbar;d, locking screw; e, thermostat; f, microscope tube; g,objective;h, ocular with fitted graticule/reticule; i, light source;j, microscope fine adjustment; k, dial test indicator. (27)Figure 2.Sb All glass small volume cylindrical electrophoresischamber incorporating a Ag/AgC1/KC1 electrode system withfused—in sintered glass discs. (27)11 IGloss lmpregnoledRT.F.E. (Fluon)Stopcock Key38Current was supplied to the electrodes by a power supply operated inconstant voltage mode; the voltage and current were read from two digitalmultimeters.Ag/AgC1 (/KC1) electrodes have been used previously and proven to be themost satisfactory system. They were treated as described in Section 2.4.The refurbishing of Ag/AgC1 electrode is performed with nitric acid andammonia respectively (to recover Ag), followed by replating in a KC1solution (the detailed procedures are described in Section 2.4.3.1.).2.4 ExperimentalGeneral DetailsCentrifugation was carried out with a Micro Centaur Centrifuge (JohnsScientific Inc.). Solution pH was measured with an Acumet pH Meter 915(Fisher Scientific) to ±0.02 units. Current was supplied with a HewlettPackard 6212A Power Supply; the voltage and current were read from twoHewlett Packard 3438A Digital Multimeters. The water used in preparationof aqueous solutions was from a Millipore Milli—Q Plus ultra—pure watersystem.All organic solvents and reagents were used as purchased. Tetra—ECand tetra-ECG were synthesized in Chapter 1 (See Section 1.4). ParticularpH solutions were made by adjusting pH prior to usage with HC1, NaOH orNaHCO3 solutions of the same ionic strength as the solution being adjusted.2.4.1 Liposome PreparationThe same procedure for preparing liposomes was used for all lipidcompositions(See Table 2.1)39Table 2.1 Composition of Liposomes in Particle ElectrophoresisComposition of LiposomesLiposome (molar ratio)Numberegg PC DPPG Cholesterol tetra—EC tetra—ECGEL*1 60 —- —— 40 ——EL*2 60 —- —— 30 10EL*3 50 10 40 —— ——EL*4 50 10 10 30 ——EL*5 60 —- 10 20 10EL*6 55 5 10 25 5EL* 7-1 30 10 —— 60 ——EL* 7-2 30 10 30 30 ——EL* 7-3 30 10 50 10 —-EL* 8-1 40 —— —— 50 10EL* 8-2 40 —— 30 20 10EL* 8-3 40 —- 50 —— 10Sodium chloride 0.6867 g(11.75 mmol) and sodium azide (NaN3) 0.049 g(0.75 mmol) were dissolved in a 250 ml volumetric flask as the aqueousphase for liposomes. The lipids in desired concentration were dissolved inchloroform (DPPG was warmed up to around 43°C), and transferred into a 250ml round bottom flask (which was cleaned with chromic acid, rinsedthoroughly in water and dried prior to usage). The volume of solution wasadjusted to 5 — 6 ml with chloroform. The mixture then was dried slowlywith rotary evaporation under reduced pressure at room temperature.Addition of about 5 ml NaC1-NaN3 solution was followed by incubation of themixture in a 42°C water bath. The suspension was centrifuged for 10 to 30minutes (13000 IPM; radius 65 mm) and the supernatant was removed. Theliposomes were washed twice in 5 ml of the medium in which they were to beexamined by electrophoresis. The procedure was to pipette the supernatant40carefully, resuspending the liposomes with addition of the solution of thedesired pH and ionic strength, followed by centrifugation. The sameresuspension and centrifugation were repeated to ensure the completereplacement by medium of the desired pH and ionic strength.2.4.2 ElectrophoresisGeneral Preparation(See Figure 2.5 for details)All solutions were de—gassed before they were used. The cylindricalchamber was cleaned with chromic acid (Cr03/112S04) and thoroughly rinsedbefore use. Ag/AgC1 electrodes were washed with nitric acid and ammoniasequentially, and replated in KC1 solutions at a current density of0.43 mA/cm2.The water bath tank was filled and temperature was set up to becontrolled at 25.00+0.02°C. The optical microscope was focused at thestationary level, equal to 0.293 x (radius ,i.362 mm), from the innerwall (35). At this location the fluid velocity caused by electroosmosisalong the walls of the chamber is zero.The electrode cells at both ends of the chamber were filled with KC1solution and stoppered by electrodes. (The center cell was filled with KC1solution as well when not in use.) The voltage was adjusted and controlledat nominally 40 V.2.4.2.1 pH Dependence StudiesThe experimental salt solution (NaCl 50 mM) with adjusted pH, whichwas also used to wash the liposomes (EL* 1 & EL# 2), was degassed andwarmed in the water bath. The center chamber was filled, after being rinsedseveral times, with the warmed solution (See Section 2.3.2 & Figure 2.3b).One of the outlets of the center chamber was stoppered. The liposome41sample was taken in suspension in the same solution in a 1.0 ml syringeand approximately 0.1 ml injected into the viewing region (“Optical Flat”)by a tubing coimected to the tip of the syringe. The sample was stirred todilute it and provide a roughly uniform distribution near the viewingregion of the chamber. The other outlet was stoppered to keep the systemstable during the measurement. The electric field was switched on and themotion of particles in the field was observed through the microscope at amagnification of x 320. The time taken by a particular particle to transita fixed distance on the eyepiece graticule was recorded. The direction ofthe current was reversed, producing migration of the same particle in theopposite direction; the mobility of the particle was calculated from theaverage of the velocities which were first calculated from the pair ofreadings.Usually, ten particles were observed for each pH solution and thevalues of voltage and current were read before and after each measurement,as well as the pH values of the NaCl solution. In the pH dependenceexperiments, the range from 1.8-9.9 was examined.When an experiment was finished, the sample could be recovered with thesyringe and the center chamber was washed with the next salt solution threetimes. For these experiments, the concentration of KC1 solution in theelectrode cells was kept constant (0.050 M), equal to that of NaCl invarious pH solutions.2.4.2.2 Ionic Strength Dependence StudiesThe procedure for the ionic strength experiments was almost the sameas the previous one, except that the concentration of KC1 solution in theelectrode cells was changed to be the same as that of the NaCl solution.42The ionic strength of the salt solution varied from 0.001 M to 0.100 M,with a constant pH — 7. The liposome samples were EL* 3 — EL* 6.2.4.2.3 Different Compositions of LiposoesThe procedure was as described above. The salt solution was 0.050 MNaCl with pH 7. The liposomes were EL* 7—1, EL* 7—2, EL* 7-3, EL* 8—1,EL* 8-2, and EL* 8—3. From EL* 7—1 to 7-3, the molar ratio of PEG chainsdecreased, while the charged lipid, DPPG, was held constant at 10%. FromEL# 8-1 to EL* 8—3, the molar ratio of PEG chain concentration variedwhile the charged lipid, tetra-ECG, was held constant at 10%.2.5 Result and DiscussionThe mobility and apparent charge density of liposomes for all theexperiments were calculated from the velocity data in Appendix III, withthe classical theory of electrophoresis of smooth particles (See Section 2.2),giving Table 2.2, Table 2.3 and Table 2.4. The constants are given inSection 2.2.1. In calculations of the apparent charge densities, theequivalent conductivity A is given below for NaC1 solution at 298°K (30):concentration (M) 0.15 0.1 0.05 0.02 0.005 0.001A (cm21equiv1) 103.89 106.74 111.06 115.76 120.64 123.7443Table 2.2 Mobility and Charge Density with Variation of pHat Constant Ionic Strength I = 0.050 Mfor Liposomes EL* 1 & ELI 2C’—” and 11+11 are the signs of charges)Electrophoretic Mobility Apparent Charge DensitypH (jim-cm/sec-volt: ±S.D.) (esu/cm2: ±S.D.)xO.001(±)Liposome *1 Liposome *2 Liposome *1 Liposome *29.88±0. 14 —0.77±0.10 —2.23±0.09 —2.64±0.34 -7.67±0.329.43±0.09 -1.53±0.19 —3.36±0.19 —5.27±0.66 —11.56±0.657. 97±0. 17 —0.48±0.11 —2.40±0.24 —1.66±0.36 -8.24±0.817. 30±0. 11 0.34±0.12 —2.34±0.26 1. 18±0.39 -8.05±0.907.03±0.05 0.07±0.07 —1.92±0.15 0.23±0.26 -6.60±0.516.87±0.06 0.16±0.05 —2.08±0.19 0.55±0.19 —7.15±0.675.99±0.02 0.24±0. 15 —2.03±0.26 0.83±0.50 —6.99±0.894.95±0.02 0.24±0.07 —1.48±0.24 0.82±0.23 —5.07±0.824.91±0.02 0.23±0.03 —1.47±0.21 0.80±0.11 -5.07±0.733.98±0.02 0.35±0.06 —0.64±0.17 1.20±0.20 —2.18±0.602.97±0.02 0.60±0.07 0.17±0.09 2.07±0.24 0.58±0.291.90±0.02 0.80±0.05 0.74±0.06 2.75±0.16 2.55±0.211.89±0.03 0.88±0.04 0.82±0.09 3.01±0.15 2.83±0.32Table 2.3 Mobility and Apparent Charge Density withVariation of Compositions at Constant pH — 7and Constant Ionic Strength 0.05 M* A and B are two measurements for one compositionComposition (molar ratio):Liposome egg—PC/cholesterol/tetra—EC Electrophoretic Mobility/DPPG/tetra-ECG (pm-cm/sec-volt: ±S.D.)EL*7-1 30/ 0/60/10/ 0 —3.47±0.45ELI 7_2A* 30 / 30 / 30 / 10 / 0 —3.46 ± 0.39ELI 7_2B* 30 / 30 / 30 / 10 / 0 —2.93 ± 0.83EL*7—3 30/50/10/10/0 —3.13±0.31EL*8—1 40/ 0/50/ 0/10 —1.53±0.16EL* 8—2 40 / 30 / 20 / 0 / 10 —1.85 ± 0.02EL*8—3 40/50/ 0/ 0/10 —2.29±0.1144Table 2.4 Mobility and Apparent Charge Densitywith Variation of Ionic Strengthat Constant pH around 7 for Liposomes *3,4,5 & 6* repeated measurementElectrophoretic Mobility Apparent Charge DensityIonic (pm—cm/sec-volt: ±S.D.) (esu/cm’2: S.D. )xO.001Strength(M) Liposome *3 Liposome *4 Liposome *3 Liposome *40.001 -4.39±0.40 —3.78±0.40 -1.23±0.11 —1.06±0.110.005 -2.85±0.32 —2.14±0.31 -1.79±0.20 —1.34±0.190.005* —3.58±0.33 —3.14±0.38 -2.24±0.21 -1.97±0.240.020 —3.13±0.26 —1.78±0.19 -3.92±0.33 -2.23±0.230.050 —2.71±0.20 —1.20±0.19 -5.37±0.41 -2.39±0.380.100 -1.55±0.27 —0.91±0.14 -4.35±0.77 -2.56±0.39Electrophoretic Mobility Apparent Charge DensityIonic (pm—cm/sec—volt: ±S.D.) (esu/cm2: S.D. )xO.001Strength(M) Liposome *5 Liposome *6 Liposome 415 Liposome *60.001 —3.79±0. 16 —4. 11±0. 18 —1.06±0.04 -1.15±0.050.005 —3.50±0. 13 —3.54±0.35 —2.20±0.08 —2.22±0.220.005* —3.27±0. 18 —3.67±0.25 —2.05±0.11 —2.30±0. 160.020 —3.27±0.16 —2.71±0.14 —4.10±0.20 —3.40±0.170. 050 —2.65±0.19 -2.11±0.15 —5.25±0.38 —4.18±0.310.100 —1.90±0.18 -1.60±0.45 —5.33±0.52 —4.49±1.262.5.1 pH DependenceSince the liposome *2 (EL* 2) has the composition of tetra—ECG insteadof tetra—EC in the liposome *1 (EL* 1), the mobility should differ due tothe presence of PEG chains and the location of the charges, even though thenet charge density is expected to be the same.From Table 2.2, a phenomenon was observed that EL*1 with a neutralsurface had low mobilities in all the pH range, because the surface ofliposomes can adsorb ions which are concentrated in solution by van derWaals interactions. The net charge density was calculated with deductionof the results of EL* 1 from those of EL* 2.(See Table 2.5)45Table 2.5 Calculation of Net Electrophoretic Mobility, Net ChargeDensity of Liposome *2 and pH at Surface of Liposomeswith Variation of pH at Constant I = 0.050 Mfor Charged Liposome EL* 2 (o’=o’—c)pH Electrophoretic Mobility Net Surface pH(measured) U’ = U2 - Ui Charge Density at surface(aim—cm/sec—volt: ±S.D.) (esu/cm’2)x 0.001 of liposome9.88±0. 14 —1.46 ± 0.14 —5.03 ± 0.47 9.56±0. 149.43±0.09 -1.83 ± 0.27 —6.28 ± 0.93 9.03±0.107.97±0.17 -1.92 ± 0.26 —6.59 ± 0.89 7.55±0.187.30±0. 11 -2.69 ± 0.29 —9.23 ± 0.98 6.71±0. 137.03±0.05 -1.99 ± 0.17 —6.83 ± 0.57 6.59±0.066.87±0.06 -2.24 ± 0.20 —7.70 ± 0.70 6.38±0.075.99±0.02 -2.27 ± 0.30 —7.82 ± 1.02 5.49±0.074.95±0.02 -1.72 ± 0.25 —5.90 ± 0.85 4.57±0.064.91±0.02 -1.71 ± 0.21 —5.87 ± 0.73 4.54±0.053.98±0.02 -0.99 ± 0.18 —3.38 ± 0.63 3.76±0.042.97±0.02 —0.44 ± 0.11 —1.50 ± 0.38 2.87±0.031.90±0.02 —0.06 ± 0.08 —0.29 ± 0.27 1.89±0.031.89±0.03 —0.05 ± 0.10 —0.18 ± 0.35 1.88±0.04Theoretically, the surface charge density of liposome *2 is constantand can be calculated from its lipid composition. The surface chargedensity is expressed as the net charge on the surface divided by thesurface area. The composition of EL* 2 is known as 60Y. egg PC, 30°?.tetra-EC and 10°!. tetra—ECG. The surface area occupied per molecule ofegg PC was estimated by De Young et al. (36) as 50 A2(in presence of 40%cholesterol). With the assumption that the surface area taken per lipidfor tetra—EC or tetra—ECG is the same as that of cholesterol, which wasreported to be 37 A2 (36), the surface charge density was calculated to be4 21.07 x 10 esu/cm . It is obvious from Table 2.2 that the results46calculated from electrophoresis were lower than this estimate.The pH at the particle surface was calculated with the classical theoryand the mobilities of the liposome particles were plotted as a function ofsurface pH (see Table 2.5 & Figure 2.6).The pKa of the charged liposome, which is also the pKa of the chargedlipid (tetra-ECG), calculated from Figure 2.6, was equal to 3.9.2.5.2 Composition DependenceTwo groups of liposomes, with variation of PEG chain density at thesurface of the particles, were observed. The plot of the mobility vsthe molar percentage of PEG chains in each liposome is given in Figure 2.7.In the liposomes *7—1, 7-2 and 7-3, the negative charge was contributedfrom lOX (molar) DPPG and located on the surface of the bilayer. Themobilities can be considered unchanged within the experimental error whenthe PEG chain density was varied.In the results of the liposomes *8—1, 8—2 and 8—3, the mobilityslightly decreased while the PEG chain concentration increased. (The charge,contributed from tetra—ECG in these liposomes was located at the outerplane of the glycocalyx.) This is the usual case when the polymer chaindensity increases. The resistance of the motion of liposomes increases,causing mobility to decrease; while the charge density is kept constant.For the liposomes *7—1, 7-2 and 7—3 (with constant lOX DPPG), however,the mobility was almost unchanged while the PEG chain density increased.471•---------U) ‘ I03—4 4 I I 4 1 4 4 j 4 I I I 4 I 4 4 I I 41 2 3 4 5 6 7 8 9 10 11pH(surface)Figure 2.6 Net Liposome Mobility vs pH at Surface of Liposomesfor EL*2 at constant ionic strength 0.050 H0EL#7-1,7-2 & 7-3-0.5x EIA8-1,8-2 & 8-3-1o 48_i-I.eE-28-2E .8-3a.-2.5zo 7—2B-34.572A cf7-4 4 4 4 I0 10 20 30 40 50 70PEG Chain Concentration (molar%)Figure 2.7 Electrophoretic Mobility vs PEG Chain Concentrationfor Different Compositions at ionic strength 0.050 H and pH ‘- 7482.5.3 Ionic Strength DependenceThe mobilities of the liposomes EL* 3, EL* 4, EL* 5 & EL* 6 areplotted as a function of ionic strength in Figure 2.8.EL*3 and ELM have the same charge locations (DPPG, inner plane) andcharge density, but different PEG chain concentrations. The EL*3 particle,which has lower PEG chain density, moves more quickly in the electric fieldthan EL*4 at all ionic strengths.EL*4, EL*5 and EL*6 have the same PEG chain density and chargedensity, but different charge locations. The mobilities of EL*6 arebetween those of EL*4 and ELlIS at high ionic strength, as expected sincethe charges in EL*4, ELliS and EL*6 are distributed at the inner plane, outerplane and both positions, respectively. At low ionic strength, themobilities are almost the same within experimental error.The numerical model was used to fit the data. The results are listedin Table 2.6 and the figures with a more detailed description of thefitting procedure in Appendix IV.49L#34 AL#4[2.4+-4-o I I I0.00 0.02 0.04 0.06 0.08 0.10 0.12Ionic Strength (M)Figure 2.8a Electrophoretic Mobility vs Ionic Strengthfor EL*3 and *4 at constant pH — 70•A-0.5 L#4+•I0.00 0.02 0.04 0.06 0.08 0.12Ionic Strength (M)Figure 2.8b Electrophoretic Mobility vs Ionic Strengthfor EL*4, *5 and *6 at constant pH — 750Table 2.6 Comparison of Mobility Model Parametersfor Four Liposome Preparation (EL*3,4,5 & 6)Ionic Charge Molecular ExtensionPreparation Strength Density Density2 2(mM) (esu/cm ) (molec./cm ) (A)EL*3:CHEMI CALDPPG 10% 10700 2.2E+13 8egg PC 50% 1.1E+14 8MOBILITY >10 8500 1.3E+14 8<10 8500 1.3E+14 >8EL*4:CHEMI CALDPPG 10% 10700 2.2E+-13 8egg PC 50°!. 1.1E+14 8tetra-EC 30% 6.6E+13 15MOBILITY 1—150 9500 1.3E+14 86.6E+13 15—73EL*5:CHEMICALegg PC 60% 1.3E+14 8tetra-EC 20% 4.4E+13 15tetra—ECG 10% 10700 2.2E+13 21MOBILITY >10 5750 2.2E+13 214.4E+13 15<10 <5000 2.2E+13 214.4E+13 15EL*6:CHEMICALDPPG 5°!. 5350 1.1E+13 8egg PC 55°!. 1.2E+14 8tetra—EC 25% 5.5E+13 15tetra-ECG 5% 5350 1.1E+13 21NOBILITY >10 4250 6.2E+13 152875 2.2E+14 21512.6 ConclusionTetraethoxycholesterol and tetra-EC terminated with glucuronic acid,tetra—ECG, were successfully synthesized and purified in sufficient yieldto allow their investigation in model membranes. A series of liposomescomposed of egg PC, DPPG, cholesterol, tetra—EC and tetra-ECG were made andobserved with particle electrophoresis. The pKa of tetra—ECG was estimatedto be 3.9 from the pH dependence of tetra—ECG—containing liposomes, takinginto account the difference in pH between the surface region and the bulkphase when the surface is negatively charged.The electrophoretic mobilities of the liposomes were measured as afunction of ionic strength of the suspending medium. The classical theoryfor smooth particles was found not to describe the data, particularly whenPEG chains were anchored in the surface. The model of Sharp and Brooks wasfound to be more successful in describing the general effects of tetra—EC andtetra-ECG, allowing the experimental data to be fit with physicallyreasonable parameter values for chain extension and charge density at ionicstrengths above 10 mM. The data taken at low ionic strengths did not fiteither theory in the presence or absence of surface polymer chains,however, suggesting that the surface charge density was not constant underthese conditions, possibly due to the adsorption phenomena.52REFERENCES1. Jam, M.K., Nonrandom Lateral Organization in Bilayers andBiomembranes, in Membrane Fluidity in the Biolov Vol.1, ed. Aloia,R.C., Academic Press, New York, 19832. Cullis, P.R., Hope, M.J., Physical Properties and Functional Rolesof Lipids in Membranes, in Biochemistry of Lipids and Membranes , ed.Vance, D.E., Vance, J.E., 19923. Gorter, E., Grendel, F., Journal of Experimental Medicine, 41, 439-£143pp, 19254. Singer, S.J., Nicolson, G.L., Science, 175, 72O—73Opp, 19725. Brockerhoff, H., Molecular Designs of Membrane Lipids, in BioorganicChemistry, lpp, 19776. Ponpipom, M.M., Shen, T.Y., Baldeschwieler, J.D., Wu, P.-S.,Liposome Technology, Vol. III, 95—ll5pp, 197. Sather, P.J., “Synthesis of Cholesterol Based Model Glycolipids”,Master Thesis, The University of British Columbia, Canada, 19908. (a)Kunitake, T., Okahata, Y., Bulletin of Chemical Society of Japan,Vol.51(6), 18T1—1879pp, 1978(b)Farhood, H., Bottega, R., Epand, R. M., Huang, L., Biochimica etBiophysica Acta, 1111, 239—246pp, 19929. Demel, R.A., De Kruyff, B., Biochimica et Biophysica Acta, 457, 109-132pp, 197610. Johnston, D. S., Coppard, E., Parera, G. V., Chapman, D., Biochemistry,23, 69l2-69l9pp, 198411. Crowe, J.H., Crowe, L.M., Carpenter, J.F., Rudolph, A.S., Wistrom,C.A., Spargo, B.J., Anchordoguy, T.J., Biochimica et Biophysica Acta,974(2), 367—384pp, 198812. Chandrasekhar, I., Gaber, B.P., Journal of Biomolecular Structure& Dynamics, Vol.5, ll63—ll7lpp, 198813. Harris, J.M., Introduction to Biotechnical and BiomedicalApplications of Poly(ethylene glycol), in Poly(Ethylene Glycol)Chemistry: Biotechnical and Biomedical Applications, ed. Harris, J.M.,Pleum, New York, 199214. Brockerhoff, H., Ramsammy, L.S., Biochimica et Biophysica Acta, 691,22’T—232pp, 198215. Patel, K.R., Li, M.P., Schuh, J.R., Baldeschwieler, J.D., Biochimicaet Biophysica Acta, 797, 2O-26pp, 19845316. Patel, K.R., Li, M.P., Schuh, J.R., Baldeschwieler, J.D., Biochimicaet Biophysica Acta, 814, 256-264pp, 198517. Bollenback, G.N., Long, J.W., Benjamin, D.G., Lindquist, J.A.,Journal of the American Chemical Society, 77, 33lOpp, 195518. Paudler, W.W., Nuclear Magnetic Resonance, Chapter 3, Allyn andBacon, Boston, 197119. Goodrich, R.P., Crowe, J.H., Crowe, L.M., Baldeschwieler, J.D.,Biochemistry, 30, 5313-S3l8pp, 199120. Schneider, J. J., Bhacca, N. S., Journal of Organic Chemistry, 34(6),199O—1993pp, 196921. Goodrich, R.P., Handel, T.M., Baldeschwieler, J.D., Biochimica etBiophysica Acta, 938, l4Z3—lS4pp, 198822. Pathak, V.P., Synthetic Communications, 23(1), 83-SSpp, 199323. (a)Wu, S.-H., Ong, G.-T., Hsiao, K.-F., Wang, K.-T., Journal of theChinese Chemical Society, 39, 6’TS—682pp, 1992(b)Chang, K. -Y., Wu, S. -H., Wang, K. -T., Carbohydrate Research, 222,l2lpp, 199124. Bangham, A.D., Liposomes: An Historical Perspective, in Liposomes,ed. Ostro, M.J., 1—26pp, Marcel Deckker Inc., New York, 198325. Juliano, R.L., Interaction of Proteins and Drugs with Liposomes, inLiposomes, ed. Ostro, N.J., Marcel Dekker Inc., New York, 198326. McDaniel, R.V., Sharp, K., Brooks, D.E., McLaughlin, A.C., Winiski,A.P., Cafiso, D., MaLaughlin, S., Biophysical J., 49, 74l-752pp, 198627. Seaman, G.V., Electrokinetics Behavior of Red Cells, in The RedBlood Cell, Vol.2, ed. Surgenor, D.M., Academic Press, New York, 197528. Popiel, W.J., Introduction to Colloid Science, Chapter 9, lS2pp,Exposition Press, New York, 197829. Davies, J.T., Rideal, E.K., Interfacial Phenomena, Chapter 2, 75pp,Academic Press, London, 196330. Robinson, R.A., Stokes, R.H., Electrolyte Solutions, 4l—45pp &46S—466pp, Butterworth, London, 195931. Levine, S., Levine, M., Sharp, K.A., Brooks, D.E., BiophysicalJournal, 42, l27-l35pp, 198332. Sharp, K. A., Brooks, D. E., Biophysical Journal, 4i S63-566pp, 198533. Woodle, M.C., Papahadjopoulos, D., Liposome Preparation and SizeCharacterization, in Methods in Enzvmology, Vol.171, ed. Colowick, S.P.,Kaplan, N.0., 19895434. Jousma, H., Taisma, H., Spies, F., Joosten, J.G.H., Junginger, H.E.,Crommelin, D. J. A., International Journal of Pharmaceutics, 35,263—274pp, 198735. From personal communication with Robert J. Knox, Western BiomedicalResearch Institute, P.O.Box 22510, Eugene, Oregon 97402-041936. De Young, L.R., Dill, K.A., Biochemistry, , 5281—5289pp, 198855LIST OF APPENDICESPageAppendix I Intel III: View of the Model Lipid AlAppendix II (a) Proton NMR analysis of Brominated Sugar A2(b) Estimation of the Coupling Reaction Yieldof Tetra-ECPG with Proton NNR A3(c) Proton NMR Result of Tetra—ECG A4Appendix III Velocity Data Table of Electrophoresis ASAppendix IV Data Fit to Equations for Mobilities ofHairy Particles A856Appendix I Molecular Model of Tetra-ECG(Builded and studied with INSIGHT II Modeling System)AlAppendix II (a) 1H NMR Analysis of Brominated and Protected Sugar()Part of the whole spectrum of proton NMR 400 MHz is shown with thechemical shift varying from 4.3 to 7.3 ppm. The single peak around 7.2is from the solvent deuterium chloroform. The signals at 4.57, 4.84,5.22, 5.59 and 6.62 ppm refer tollS, H2, H4, 113 and Hi respectively.The coupling constant of Hi and 112, .312, is measured and calculated to be4 Hz, smaller than that of the axial—axial coupling, 9—14 Hz (17); while.323, .334 and J45 are the same 11Hz.114 CH0 OC1640 4.6i . I I I I I I I I I I I I I I ‘7.0 6.5 6.0 5.5 5.0 45A2Appendix II (b) ‘H NNR Analysis of the Compounds in Coupling ReactionThe estimation of the molar ratio of tetra—EC and tetra—ECPG isbased on the assumption that the mixture only composes of these twocompounds. At the left below a table shows whether or not and whichproton(s) of tetra—EC or tetra—ECPG contribute to the NNR integral ofa particular signal.Assume the molar ratio of tetra—ECand tetra-ECPG is Xi : X2. Assign therelations as:44x1 + 53X2 = ITGL 1xi =ITGL216i + 2OX2 = ITGL 3Xi 5X2=ITGL4The ratio x’ : X2 can be given by anytwo of these equations.Shift(ppm)tetra—EC tetra-ECPG0.4-2.6 44Hc 44Hc+9Ha3,2 iHo3.44.5 l6Hp l6Hp+3Ha+lHs4.5—5.6 lHcd 4Hs+lHcda acety]. group c cholesterolcd double bond in cholesterols methyl group on sugar parta hydroxyl groupp PEG chain s sugar ringTjTi2’oi’oPPMI-JwI-.8.0 7.0 6.0 0.0A3A4Appendix II (c) 200 MHz I1 NMRChemicalShift (ppm)Integral/Ratio tetra-ECG0.4—2.6 412.7/45.9 44Hc3.0—4.8 217.4/24.2 l6Hp+3Ho+5Hs5.4 9.00/1 lHcdc cholesterol • sugar ringcd double bond in cholesterolo hydroxy group p PEG chainAnalysis of Deprotected Compound ()trJj-<CD‘IiIz14• I I I I I10.0 9.0K11 /•‘IL A6.0 5.uPPM0.06.0 0 1.0Appendix III Particle Velosity Data TablesVelosity of liposome particle is: v = dlt =____where d is the distance the particle moves in particular time; D Is thelength per division, 0.024mm; n is the number of division.The average velosity is obtained from the average of the inversionsof all the time readings from motion of different particles, multipliedby d.Liposoine 1 1 (EL* 1) (constant Ionic strength at 0.05014)pH Charge Average Velosity Current Voltage(±) Sign (cmlsec:±S.D.)(+1—) x 10000 (mA) CV)9.88 ± 0.14 — —2.63 ± 0.34 1.11 40.019.43 ± 0.09 — —5.26 ± 0.66 1.11 39.917.97±0.17 — —1.67±0.37 1.12 40.007.30 ± 0.11 + 1.12 ± 0.47 1.10 39.917.03 ± 0.05 + 0.23 ± 0.26 1.12 40.016.87 ± 0.06 + 0.55 ± 0.19 1.11 40.025.99 ± 0.02 + 0.80 ± 0.52 1.11 40.014.95 ± 0.02 + 0.82 ± 0.23 1.11 40.014.91 ± 0.02 + 0.81 ± 0.11 1.12 40.023.98 ± 0.02 + 1.21 ± 0.20 1.12 40.012.97 ± 0.02 + 2.19 ± 0.25 1.18 39.981.90 ± 0.02 + 4.63 ± 0.28 1.87 39.911.89 ± 0.03 + 5.12 ± 0.25 1.89 40.00Liposome * 2 (EL* 2) (constant ionic strength at 0.05CM)pH Charge Average Velosity Current Voltage(±) Sign (cm/sec:±S.D.)(+1—) x 10000 (mA) CV)9.88 ± 0.14 — —7.66 ± 0.32 1.11 40.009.43 ± 0.09 — —11.53 ± 0.65 1.11 39.917.97 ± 0.17 — —8.23 ± 0.81 1.11 40.007.30 ± 0.11 — —7.94 ± 0.88 1.10 39.957.03 ± 0.05 — —6.58 ± 0.51 1.12 40.026.87 ± 0.06 — —7.14 ± 0.67 1.11 40.025.99 ± 0.02 — —6.98 ± 0.87 1.11 40.024.95 ± 0.02 — —5.06 0.82 1.11 40.024.91 ± 0.02 — —5.08 ± 0.73 1.12 40.013.98 ± 0.02 — —2.20 ± 0.63 1.12 40.012.97 ± 0.02 + 0.61 ± 0.31 1.18 39.991.90 ± 0.02 + 4.31 ± 0.36 1.88 39.911.89 ± 0.03 + 4.80 ± 0.53 1.89 40.00ASAppendix III Particle Velosity Data TablesLiposome *3 (EL* 3) (constant pH)Ionic pH Average Velosity Current VoltageStrength (cm/sec: S.D.)(M) (±0.02) x 10000 (mA) (my)0.005 6.94 -15.00 ± 1.37 0.127 39.820.020 6.91 —11.89 ± 0.99 0.461 39.890.050 6.88 - 9.27 ± 0.70 1.11 39.900.100 6.95 - 5.36 ± 0.94 2.10 39.94Liposome *4 (EL* 4) (constant pH)Ionic pH Average Velosity Current VoltageStrength (cm/sec: S.D.)CM) (±0.02) x 10000 (mA) (mV)0.005 6.94 -13.16 ± 1.60 0.127 39.840.020 6.91 - 6.77 ± 0.70 0.462 39.910.050 6.88 - 4.12 ± 0.65 1.11 39.920.100 6.95 - 3.81 ± 0.49 2.11 39.95Liposome *5 (EL* 5) (constant pH)Ionic pH Average Velosity Current VoltageStrength (cm/sec: S.D.)(H) (±0.02) x 10000 (mA) (mV)0.005 6.97 -15.03 ± 0.56 0.130 39.990.020 6.91 -12.51 ± 0.60 0.464 39.990.050 6.88 — 9.08 ± 0.65 1.11 39.930.100 6.95 — 6.57 ± 0.63 2.10 39.93Liposome *6 (EL* 6) (constant pH)Ionic pH Average Velosity Current VoltageStrength (cm/sec: S.D.)CM) (±0.02) x 10000 (mA) (mV)0.005 6.94 -15.40 ± 1.06 0.127 39.840.020 6.91 —10.34 ± 0.52 0.463 39.970.050 6.88 — 7.24 ± 0.53 1.11 39.940.100 6.95 — 5.59 ± 1.56 2.12 39.99A6Appendix III Particle Velosity Data TablesVelosity of Electrophoresis forLiposome Composition Dependence(constant pH arid ionic strength)Liposome with Average Velosity Current Voltagedifferent (cm/sec: S.D.)composition x 10000 (mA) (mV)EL* 7—1 —11.79 ± 1.54 1.10 40.00EL* 7_2A* —11.76 ± 1.31 1.10 39.91ET_ 7—2B — 9.97 ± 2.82 1.10 39.92EL* 7—3 —10.65 ± 2.19 1.10 39.89EL* 8—1 — 5.19 ± 0.54 1.10 39.93EL* 8-2 — 6.31 ± 0.69 1.10 39.93EL 8-3 - 7.79 ± 0.36 1.10 39.92PH 6.96 ± 0.02, Ionic Strength 0.050 HA & B are two measurements for composition EL* 7—2A7Appendix IV Data Fit to Equations for )4obilities of Hairy ParticlesContributed by X. Song, J. Janzen and D. E. BrooksModel ParametersThe mobility model assumes a region, the glycocalyx, exterior to thebilayer which contains surface associated molecules and suspending medium.Some of these associated molecules bear charged groups. The mobility iscalculated as a function of the ionic strength of the suspending medium.The composition data for the liposomes and structural data for themolecular species were used to calculate parameter values which wereexpected to fit the mobility data. The parameter values were then varied toimprove the fit. Assessment of the degree of fit was qualitative.The mole percent data for the liposome components were first convertedinto molecular surface densities, i.e., molecules/cm2 This calculationused the reported areas per molecule for egg PC (50 A2) and cholesterol(37 A2) in 40 mole h cholesterol bilayers (36). Surface charge densitieswere calculated from molecular densities assuming one ionized group percarboxylic acid and phosphodiester residue. Ten mole percent of a specieswith a single charge per molecule corresponds to 2.2 x 1013 molecules/cm2and 10700 esujcm2.In the mobility calculation the electrostatic charge densitycontributes a positive term to the magnitude of the particle mobility whilethe interaction of anchored molecular chains with the suspending medium isresistive. In the current program, electrostatic charge may be specifiedindependently at the inner and outer interfaces of the glycocalyx and as aA8diffuse charge within the glycocalyx. In the latter case the volumedensity of the charge is calculated from the surface charge density andeither a gaussian distribution function or a uniform distribution function.Diffuse charge distributions were not used in these calculations.Stokes resistance is characterized by a radius which is estimated herefrom structural data. In reality this parameter is a hydrodynamiccharacteristic. This parameter is referred to here as the segment radiusand was set equal to one—half the unit cell length of the head group chainof tetraethoxycholesterol (tetra—EC), 1.85 A.The resistive term varies with distance from the bilayer due to thevelocity gradient across the glycocalyx and changes in the volume density ofresistive segments. In the particle frame of reference the hydrodynamicshear plane determines the zero velocity location. In the calculations thiswas set at the bilayer to glycocalyx interface (0 A). The volume density ofsegments is calculated from the surface density of segments and the lengthover which these are distributed. Two resistive elements, independentexcept for the common segment radius parameter, may be used. One (polymerI), associated with the glycocalyx, assumes a uniform distribution over theglycocalyx limits. The other (polymer II) assumes a uniform distributionover lower and upper limits freely set between 0 X and 120 V. of theglycocalyx’s depth. The latter was originally included to model adsorbedpolymer but is used here to include the effect of different dimensions fortetra-EC and the glucuronic acid derivative of tetra—EC (tetra-ECG) whenthey are present in the same liposome preparation.The surface density of segments is calculated from the surfaceA9density of molecular species and the number of segments per molecule. Thelatter was calculated from molecular lengths and the assumed segmentradius.Fitting ResultsThe mobilities of three liposome preparations EL*3, EL*4 and EL*5 werefitted as functions of ionic strength. A fourth, EL*6, was intermediate incomposition and behaviour to EL*4 and EL*5.The EL*3 preparation was 50 7. egg PC (PC), 107. DPPG (PG) and 40 ‘4cholesterol. The model for the surface region has the charge at thebilayer—glycocalyx interface, i.e., inner charge at 0 A. The combinedsurface density was 1.32 x 1014 molecules/cm2 Phospholipid and cholesterolhead group resistance was not considered initially but its effect wassubsequently examined.First, the charge density was varied while the head group resistancewas held at zero (Fig. A4-1). Under this condition mobilities above 10 mMNaC1 were best fit by a surface charge of 4500 esu/cm2. This is much lowerthan the value calculated from the chemical composition of the lipidmixture from which the liposomes were formed, 10,700 esu/cm2. Below 10 mMNaC1 successively lower charge densities were required.The head group resistance was then included (Figure A4—2) by allowingthe PC and PG molecules to extend from the bilayer 8 A, as estimated fromthe molecular structure. Two cases were considered. First, the number ofsegments per unit volume was varied in proportion to the depth. Thus theAlOsegment volume density was held constant, implying that changing the lengthof the molecule changes the molecular mass. This does not coincide withexperimental reality, however. Secondly, the segment volume density washeld constant at the value used for the BA depth. In this case the segmentvolume density varies inversely with the extension, but the molecular massis constant. This models a molecule which could collapse towards thesurface, for instance. Comparison of the curves for the two case showedalmost no difference over the length variation so in subsequent fittingonly the constant molecular mass case was calculated.The mobility was significantly reduced at all ionic strength by addingthe head group resistance. The charge density had to be increased to8,500 esulcm2 to match the high ionic strength data, a value near to thatcalculated from the assumed chemical composition.The EL*4 preparation was 50 % PC, 10’!. PG. 10°!. cholesterol and 30%tetra-EC. The model for the surface region has the charge at 0 A. The PCand PG parameters are as in preparation EL*3. The PEG chain on tetra-EC wasestimated from molecular models to extend about 15 A and the surfacedensity was 6.6 x 10 molecules/cm2.The data was fit by assuming = 15 A and varying the charge density(Figure A4—3). A value of 9,000 esu/cin2, very close to that used to fit thebare PC/PG surface, fit the mobility value at 0.10 M. With the chargedensity constant had to be varied to fit the points at the lower ionicstrengths. The thicknesses had to be continuously increased to fit thedata as lower ionic strength data was considered, the range being 15 to73 A.AllA set of calculations was also carried out with both an 8 A layer forthe PC/PG head groups and a greater extension for the tetra—EC chains asdescribed above. It was found that the resistance increase was small.A similar mobility fitting to that achieved above required the chargedensity to be increased to 9,500 esulcm2. However, the same range ofthicknesses fit the data at lower ionic strengths when the charge densitywas held at 9,500 esuJcm2.The EL*5 preparation was 60 X PC, 10% cholesterol, 20% tetra—EC and10% tetra-ECG. The model for the surface region has the charge at theoutside of the glycocalyx adjacent to the free medium. The PC surfacedensity is 1.32 x 1014 molecules/cm2. The tetra—EC surface density was4.4 x 1013 molecules/cm and its extension taken to be 15 A. The tetra—ECGhead group was estimated to extend about 21 A and the surface density was2.2 x 1013 molecules/cm2. The tetra—ECG surface charge density was varied(Figure A4—4) and found to fit the mobility in 0.10 M NaC1 at 5,750 esulcm2located at the extreme end of the molecule. The next two ionic strengthpoints were likewise fit with this parameter set, but the two lowest valuesfell well above the line.It is evident from the curves for other values that the reduction inmobility with increase in molecular extension is much less in this instancethan that obtained when the surface is modeled with all the charge at thebase of the chains. The mobility is less sensitive to the chain extensionwhen the charge is moved away from the the solid surface, presumablybecause the chains near the fixed charge plane offer less drag than thesolid surface, at which the relative velocity must be zero.A12The EL*6 preparation contained 25% tetra—EC and 5% tetra—ECG with 5% PG,55% PC and 10% cholesterol. Hence, the overall chain density was as abovebut the charge was distributed half on the ends of the tetra-ECG chains andhalf at their base. In this case (Figure A4—5) an acceptable fit to thethree highest ionic strength points was obtained if the charge valuesdetermined from best fits for EL*4 and ELN5 were each halved and assignedto the inner and outer charge layers in EL*6 and the polymer extensionstaken as 15 A and 21 A for the tetra—EC and tetra—ECG chains respectively,demonstrating the consistency of the modeling at moderate ionic strengths.DiscussionIt is clear from the above results that the model of Sharp and Brookscan explain the general features of the effect on the electrophoreticmobility of adding neutral polymer chains to charged liposomes providingthe data is not taken at ionic strengths below 10 mM. The values for thecharge density agree reasonably well with those expected from the lipidcomposition provided the charge is located at the plane of the solidsurface. In order to fit data obtained when the charge was located at theend of the chains on tetra—ECG the value was somewhat lower, however.The inability of the theory to fit the data in any system at lowionic strength, in the presence or absence of surface—associated polymerchains, suggests that the charge density in fact varied as a function ofionic strength. This could reflect the binding of cations or impurities tothe phosphate and acidic sugar moieties, a possibility that was notexamined quantitatively. The fact that B had to be increased as the ionicA13strength was decreased when the charge was located at the base of the PEGchains could be interpreted to mean the chain extension increased in lowerionic strength media. This seems an unlikely explanation, however, as thechain is electrically neutral and and its configuration would not beexpected to be very sensitive to ion concentration in the range examinedhere. It seems more likely the effect is associated with the relativelylow values of mobility observed in low ionic strengths, the reason forwhich is not known.A14C)a)0?0E9ED>-Q0Figure A4-1 LIPOSOME MOBILITYin pH 6.9 NaCIA150.0100Ionic Strength (I)(3ci)0Eci)E>4-,00Figure A4-2 LIPOSOME MOBILITYin pH 69 NaCIA16-1EL3glycocalyx thickness (A) 20.7polymer II between (A) 0.0, 8.0polymer II #segments/cm2 2.9E+14inner charge (esu/cm2): variable++Ionic Strength (I)A17C-)G)C?0292D0Figure A4-3 LIPOSOME MOBILITYin pH 6.9 NaCIEL4-1glycocalyx thickness (A) 15.0polymer I #segments/cm2 2.7E+14inner charge (esu/cm2): variable8000 900010700++-4.0. 0.0010 0.0100Ionic Strength (I)0. 1000A18()a)4-,0E(I)2D>010.0001I I liii0.0010I I I III1.0000Figure A4-4 LIPOSOME MOBILITYin pH 69 NaCI0--2--3--4-EL5glycocalyx thickness (A) 20.7polymer I #segments/cm2 1.2E+14polymer II between (A) 0.0, 15.0polymer II #segments/cm2 1.8E+14outer charge (esu/cm2): variable+- 50005500600065007000/ 10700Ionic Strength (I)I I I I III0.1000A19Figure A4-5 LIPOSOME MOBILITYin pH 69 NaCI0-EL6glycocalyx thickness (A) 20.7polymer I #segments/cm2 0.6E+14- 1- polymer II between (A) 0.0, 15.0polymer II #segments/cm2 2.2E+14inner,outer charge (esu/cm2): 4250, 2875 /0E92:3%-.-.-3->-Q+—— I 111111 I I 111111 I I 1111110.0001 0.0010 0.0100 0.1000 1.0000Ionic Strength (I)

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0059587/manifest

Comment

Related Items