@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Medicine, Faculty of"@en, "Biochemistry and Molecular Biology, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Harrigan, Paul Richard"@en ; dcterms:issued "2010-07-14T19:46:36Z"@en, "1987"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description "Plant seeds, yeasts, bacterial spores, rotifers, and other organisms are capable of suspending their metabolism and entering a state of latency when dehydrated. These organisms may maintain this state for extremely long periods of time, yet upon rehydration resume normal metabolism, without evidence of severe membrane disruption. With many of these organisms, the ability to survive dehydration has been correlated to the production of large amounts of carbohydrates, including glycerol, glycogen and the disaccharide trehalose. Trehalose has been shown to protect isolated sarcoplasmic reticulum microsomes and phospholipid vesicles from dehydration damage, implying that the site of protective action of trehalose and other carbohydrates is the lipid portion of membranes. In this thesis, the effects of carbohydrate composition, vesicle size, and lipid composition on the protection of liposomes from dehydration was investigated, as was the structure of the solid lipid-trehalose complex. Electron microscopy of dried liposomes indicated that vesicles protected with trehalose remain essentially intact even when dry, while vesicles not protected by sugar are severely disrupted by drying . ³¹-P and ¹³-C NMR results suggested that the lipid of protected vesicles is in a similar phase as that of unprotected vesicles, and that this state is similar to powdered anhydrous phospholipid. Using carboxyfluorescein as a probe, it was demonstrated that trehalose, other sugars can prevent vesicle disruption upon dehydration. Different lipid compositions of the liposomes showed nearly identical behavior, with the exception of vesicles composed of dipalmitoylphosphatidylcholine and egg phosphatidylcholine, which showed greater and lower stability to dehydration respectively. Light scattering experiments indicated that a wide variety of carbohydrate and lipid vesicle combinations can withstand dehydration and maintain their original size when protected by sugars. The implications of these results in the development of liposomes as pharmaceuticals are discussed, and a hypothesis is advanced regarding the role of carbohydrates in the preservation of dry lipid membranes."@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/26416?expand=metadata"@en ; skos:note "FACTORS INFLUENCING THE' STABILITY OF DEHYDRATED LIPOSOMAL SYSTEMS by PAUL RICHARD HARRIGAN B.Sc. The U n i v e r s i t y of B r i t i s h Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST 1987 (cT) PAUL RICHARD HARRIGAN 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date foXP&gfc V* , ABSTRACT Plant seeds, yeasts, bacterial spores, rotifers, and other organisms are capable of suspending their metabolism and entering a state of latency when dehydrated. These organisms may maintain this state for extremely long periods of time, yet upon rehydration resume normal metabolism, without evidence of severe membrane disruption. With many of these organisms, the ability to survive dehydration has been correlated to the production of large amounts of carbohydrates, including glycerol, glycogen and the disaccharide trehalose. Trehalose has been shown to protect isolated sarcoplasmic reticulum microsomes and phospholipid vesicles from dehydration damage , implying that the site of protective action of trehalose and other carbohydrates is the lipid portion of membranes . In this thesis, the effects of carbohydrate composition, vesicle size, and lipid composition on the protection of liposomes from dehydration was investigated, as was the structure of the solid lipid-trehalose complex. Electron microscopy of dried liposomes indicated that vesicles protected with trehalose remain essentially intact even when dry ,while vesicles not 31 13 protected by sugar are severely disrupted by drying . -P and -C NMR results suggested that the lipid of protected vesicles is in a similar phase as that of unprotected vesicles, and that this state is similar to powdered anhydrous phospholipid. Using carboxyfluorescein as a probe, it was demonstrated that trehalose, other sugars can prevent vesicle disruption upon dehydration. Different lipid compositions of the liposomes showed - i i -nearly identical behavior, with the exception of vesicles composed of dipalmitoylphosphatidylcholine and egg phosphatidylcholine, which showed greater and lower stability to dehydration respectively. Light scattering experiments indicated that a wide variety of carbohydrate and lipid vesicle combinations can withstand dehydration and maintain their original size when protected by sugars. The implications of these results in the development of liposomes as pharmaceuticals are discussed, and a hypothesis is advanced regarding the role of carbohydrates in the preservation of dry lipid membranes. -iii-TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v L I S T OF TABLES v i i L I S T OF FIGURES v i i i ABBREVIATIONS X ACKNOWLEDGEMENTS x i 1. INTRODUCTION 1.1 H i s t o r i c a l I n t r o d u c t i o n 1 1.2 H y p o t h e s e s o f C a r b o h y d r a t e P r o t e c t i o n o f Membranes... 4 1. 3 L i p o s o m e s a s M o d e l Membranes 5 31 13 1.4 U s e o f -P a n d -C NMR i n Membrane B i o c h e m i s t r y . . . . 6 1.5 G e l t o L i q u i d - C r y s t a l l i n e P h a s e T r a n s i t i o n 7 1.6 Membrane F l u i d i t y 9 1.7 O u t l i n e o f t h i s T h e s i s 10 - i v -2. MATERIALS AND METHODS 2.1 M a t e r i a l s 11 2.2 P u r i f i c a t i o n o f C a r b o x y f l u o r e s c e i n 11 2.3 V e s i c l e P r e p a r a t i o n and Work-up 12 2.4 Dehydration of Liposomes 12 2.5 F l u o r i m e t r y 13 2.6 Membrane P o t e n t i a l Determination 13 2.7 Glucose Release 14 2.8 Phosphorous Assay 14 31 13 2.9 -P and -C NMR P r e p a r a t i o n and Set-up 15 2.10 V e s i c l e S i z e Determination 16 2.11 S o l i d s t a t e F r e e z e - f r a c t u r e E l e c t r o n Microscopy 16 2.12 Determination of Doxorubicin Leakage 16 2.13 D e f i n i t i o n o f Dryness 17 2.14 S t r u c t u r e of Key Chemicals Used 17 3. RESULTS 3.1 S t r u c t u r e of the Dry T r e h a l o s e - V e s i c l e Complex 19 31 3.1.1 -P NMR Study of D r i e d V e s i c l e s 19 13 3.1.2 -C NMR Study of D r i e d V e s i c l e s 21 3.1.3 E l e c t r o n Microscopy of Dry V e s i c l e s 24 3.2 E f f e c t o f V a r i o u s Parameters on the Dehydration 26 -v-3.2.1 V a l i d i t y o f CF as a Marker of Contents 26 3.2.2 Dehydration of Liposomes w i t h and without T r e h a l o s e 32 3.2.3 P r o t e c t i v e E f f e c t o f Other Carbohydrates 35 3.2.4 Dehydration of liposomes of V a r y i n g F a t t y A c i d Composition 37 3.2.5 I n f l u e n c e of C h o l e s t e r o l on Dehydration 39 3.2.6 Dehydration of Liposomes of V a r y i n g P h o s p h o l i p i d Headgroups 39 3.2.7 E f f e c t o f V e s i c l e S i z e on Dehydration 41 3.3 A p p l i c a t i o n t o Liposomal Drug Therapy - Do x o r u b i c i n 43 3.3.1 Uptake of Doxorubicin - Response t o pH Gr a d i e n t 44 3.3.2 P r e p a r a t i o n of Dehydrated Liposomal D o x o r u b i c i n 46 4. CONCLUSIONS 4.1 An Hypothesis Regarding the P r e s e r v a t i o n of Dehydrated Membranes by Carbohydrates 48 4.2 General C o n c l u s i o n s 50 5. BIBLIOGRAPHY 51 - v i -LIST OF TABLES I. S i z e of Rehydrated V e s i c l e s 33 I I . P r o t e c t i v e e f f e c t of d i f f e r e n t carbohydrates on d e h y d r a t i o n of p h o s p h o l i p i d v e s i c l e s 36 I I I . Dehydration of liposomes of v a r y i n g f a t t y a c i d composition i n the presence of t r e h a l o s e 36 IV. I n f l u e n c e of c h o l e s t e r o l i n c o r p o r a t i o n on d e h y d r a t i o n 3 8 - v i i -LIST OF FIGURES 1 S t r u c t u r e of T r e h a l o s e and C a r b o x y f l u o r e s c e i n 18 31 2 S o l i d s t a t e -P NMR of egg PC v e s i c l e s i n the presence and absence of t r e h a l o s e 20 13 3 S o l i d s t a t e -C NMR of egg p h o s p h a t i d y l c h o l i n e v e s i c l e s i n the presence and absence of t r e h a l o s e 2 2 4 F r e e z e - f r a c t u r e e l e c t r o n microscopy of dehydrated v e s i c l e s i n the presence and absence of t r e h a l o s e 25 5 I n f l u e n c e of c a r b o x y f l u o r e s c e i n on g l u c o s e r e l e a s e from liposomes 27 6 Determination of membrane p o t e n t i a l due t o carboxyf l u o r e s c e i n 29 7 E f f e c t of ionophores on c a r b o x y f l u o r e s c e i n r e l e a s e from liposomes 30 - v i i i -8 Retention of Trapped Contents i n Rehydrated Vesicles 34 9 E f f e c t of d i f f e r e n t phospholipid headgroups on the dehydration of liposomes 4 0 10 Dehydration of liposomes of various si z e i n the presence of trehalose 42 11 Uptake of doxorubicin i n response to a proton gradient 45 12 Retention of doxorubicin upon dehydration i n the presence of varying amounts of glucose 47 - i x -ABBREVIATIONS USED C Carbon CF 5 ( 6 ) - c a r b o x y f l u o r e s c e i n CCCP Carbonyl cyanide m-chlorophenylhydrazone DOPC 1 , 2 , - d i o l e o y l - s n - g l y c e r o - 3 - p h o s p h o r y l c h o l i n e DPPC 1 , 2 , - d i p a l m i t o y l - s n - g l y c e r o - 3 - p h o s p h o r y l c h o l i n e E ( e 1000 250 mM trehalose on both sides of 107 + 19 105 + 30 vesicle membrane 250 mM trehalose added to outside of vesicles 104 + 36 112 + 56 only 250 mM trehalose 108 + 21 > 1000 trapped only inside vesicles 1 2 - Standard deviations from NICOMP Gaussian Analysis - Vesicles too large to be sized by NICOMP Sizer ( >1000 nm) -34-Figure 8. Retention of entrapped contents i n rehydrated v e s i c l e s . Retention of CF by large u n i l a m e l l a r v e s i c l e s dehydrated and rehydrated as i n S e c t i o n 2.4. V e s i c l e s were dehydrated l n the absence of t r e h a l o s e ( a ) ; i n the presence of 250 mM t r e h a l o s e on both s i d e s of. the membrane ( b ) ; with 250 t r e h a l o s e only on the outside of the v e s i c l e s ( c ) ; or with 250 mM t r e h a l o s e only on the i n s i d e o.f the v e s i c l e s ( d ) . -35-3.2.3 PROTECTIVE EFFECT OF OTHER CARBOHYDRATES The ability to protect phospholipid vesicles is not unique to trehalose, though it appears to be one of the few sugars that can protect at physiological concentrations (Crowe et al., 1984) . While physiological concentrations of trehalose in anhydrobiotic organisms are very high (up to 20% by weight of the organism) (Clegg, 1965), trehalose also appears to be more effective than other sugars at low concentrations. Therefore it was of interest to examine the effect of different sugars at the same concentration, to determine whether there is any difference in the degree of protection afforded by a given carbohydrate. From Table II , there is little difference in the level of retention of CF among the sugars tested, and all samples retained their original size distribution. Thus, a range of carbohydrates share the remarkable stabilizing effects of trehalose. Trehalose was used to protect the liposomes in most of the rest of this thesis since its interactions with dry membranes have been the most thoroughly investigated. The range of carbohydrates effective in protecting these vesicles suggests that the prevention of vesicle disruption upon dehydration may be a general property of sugars, and that any proposed mechanism for this protection should take account of this relative non-specificity. As well, it indicates that alternative sugars can replace the expensive, and non-injectable, sugar trehalose as an agent to preserve liposomes from damage from dehydration. This could be of importance if liposomally encapsulated drugs become a common dosage form. (See Section 3.3). -36-TABLE II PROTECTIVE EFFECT OF DIFFERENT SUGARS ON DEHYDRATION OF LIPOSOMES SUGAR RETENTION OF CF UPON REHYDRATION (%) Trehalose 73 Glucose 69 Lactose 71 Galactose 70 TABLE II: 40 /zmol egg PC vesicles (100 nm mean diameter) was dehydrated in the presence of 250 mM of the sugar listed, and the retention of carboxyfluorescein upon rehydration was monitored as in Methods. Results shown are the average of two experiments. TABLE III DEHYDRATION OF LIPOSOMES OF VARYING FATTY ACID COMPOSITION IN THE PRESENCE OF TREHALOSE FATTY ACID RETENTION OF CF UPON REHYDRATION (%) DPPC 88 + 2 POPC 69 + 6 EPC 73 + 4 DOPC 72 + 2 TABLE III: Large unilamellar vesicles (mean diameter 100 nm) were prepared as in Methods using 40 umol of the lipid shown, then dehydrated in the presence of 250 mM trehalose. Results and standard deviations are the average of four experiments. -37-3.2.4 DEHYDRATION OF LIPOSOMES OF VARYING FATTY ACID COMPOSITION The influence of the phospholipid acyl chain composition on the ability of trehalose to protect against dehydration is examined in Table III. These samples, and all others in this thesis unless otherwise stated, maintained their original size distribution after dehydration in the presence of trehalose, but showed dramatic increases in size and loss of trapped CF when dehydrated without trehalose. As the degree of unsaturation of the acyl chains increases (ie. DPPC