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Influence of calcium on the transfection properties of lipid-based gene delivery systems Lam, Angela Man Iu 2000

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INFLUENCE OF CALCIUM ON THE TRANSFECTION PROPERTIES OF LIPID-B A S E D G E N E DELIVERY S Y S T E M S by Angela Man lu Lam B . S c , M o l e c u l a r a n d C e l l B io logy , Univers i ty of Ca l i fo rn ia , B e r k e l e y , U S A , 1994 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S D E P A R T M E N T O F B I O C H E M I S T R Y A N D M O L E C U L A R B I O L O G Y W e a c c e p U h i s t h e s i s a s c o n f o r m i n g j i r e ^ s t a n d ^ d " to the rec f T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A D e c e m b e r , 2 0 0 0 © A n g e l a M a n lu L a m , 2 0 0 0 I n p r e s e n t i n g th i s thes is i n par t i a l f u l f i l m e n t o f the r e q u i r e m e n t s fo r a n a d v a n c e d degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree that the L i b r a r y s h a l l m a k e it f ree ly a v a i l a b l e for re ference a n d s tudy . I further agree that p e r m i s s i o n for ex tens ive c o p y i n g o f this thes is fo r s c h o l a r l y p u r p o s e s m a y be g ranted b y the h e a d o f m y d e p a r t m e n t o r b y h i s o r h e r representat ives . It is u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f this thesis fo r f i n a n c i a l g a i n sha l l n o t b e a l l o w e d w i t h o u t m y w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f B i o c h e m i s t r y a n d M o l e c u l a r B i o l o g y T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r , C a n a d a D a t e D e c e m b e r 19, 2 0 0 0 ABSTRACT This thesis is focused on examining the influence of calcium on the transfection potencies and the intracellular processing of two lipid-based gene delivery systems: plasmid DNA-cationic lipid complexes, and stabilized plasmid-lipid particles (SPLP). Results in Chapter 2 demonstrate that calcium can increase the in vitro transfection potency of plasmid DNA-cationic liposome complexes up to 20-fold. The effect is Ca2+-specific: other cations such as Mg 2 + and Na + did not give rise to enhanced transfection and the presence of EGTA inhibited the stimulatory effect. It was shown that C a 2 + increased cellular uptake of the DNA-lipid complexes, suggesting that increased transfection potency arose from increased intracellular delivery of both cationic lipid and plasmid DNA in the presence of Ca 2 + . In particular, the levels of intact intracellular plasmid DNA were significantly enhanced when C a 2 + was present. The generality of the C a 2 + effect for enhancing complex-mediated transfection is demonstrated for a number of different cell lines and different cationic lipid formulations. The influence of C a 2 + on SPLP, a system in which plasmid DNA is encapsulated within unilamellar lipid vesicles, is investigated in Chapter 3. It was shown that the transfection potency of SPLP in baby hamster kidney cells could be enhanced several hundred-fold by the presence of Ca 2 + . Interestingly, Ca 2 + -enhanced transfection did not result from enhanced uptake of SPLP into the baby hamster kidney cells. Evidence from fluorescent microscopy studies employing rhodamine-labeled SPLP, Southern blots analysis of delivered DNA, and 3 1 P NMR i i studies of appropriate model membrane systems suggested that C a enhances the transfection potency of the encapsulated system by assist ing in the destabil ization of the endosomal membrane. This work was extended to .encapsulated sys tems that contained higher amounts of the cationic lipid and that contained a cationic pegylated lipid on its surface. Increases in potency of 3 to 5 orders of magnitude at the optimal C a 2 + concentrations were observed, indicating that C a 2 + a n d cationic lipid act synergistically to increase the transfection potency of l ip id-based gene transfer systems. In summary, results of this work show that C a 2 + acts as an efficient cofactor for enhancing the transfection properties of l ipid-based gene delivery systems. i i i TABLE OF CONTENTS A B S T R A C T ii T A B L E O F C O N T E N T S iv L I S T O F T A B L E S vii L I S T O F F I G U R E S viii A B B R E V I A T I O N S x A C K N O W L E D G M E N T S xiv D E D I C A T I O N xv CHAPTER 1: INTRODUCTION 1 1.1 Pro jec t overv iew: G e n e t h e r a p y a n d current D N A de l ivery m e t h o d s 1 1.1.1 V i r a l - b a s e d v e c t o r s 1 1.1.2 N o n v i r a l - b a s e d v e c t o r s 2 1.1.2.1 G e n e de l ivery by p h y s i c a l or c h e m i c a l m e t h o d s 3 1.1.2.2 G e n e de l ivery e m p l o y i n g l i p o s o m e s 3 1.2 L i p i d s 7 1.2.1 C h e m i s t r y a n d p h y s i c s of l ipids 7 1.2.1.1 P h o s p h o l i p i d s 9 1.2.1.2 S p h i n g o l i p i d s 9 1.2.1.3 C h o l e s t e r o l 11 1.2.1.4 C a t i o n i c l ipids '..11 1.2.1.5 P o l y e t h y l e n e glycol ) ( P E G ) l ipids 12 1.2.2 St ructura l b e h a v i o r of l ipids 14 1.2.2.1 G e l a n d liquid crysta l l ine p h a s e t ransi t ion 14 1.2.2.2 Lip id p o l y m o r p h i s m 15 1.2.2.3 M e m b r a n e f u s i o n 17 1.3 L i p o s o m e s 2 2 1.3.1 C l a s s i f i c a t i o n of l i p o s o m e s 2 2 1.3.1.1 Mul t i lamel lar v e s i c l e s ( M L V s ) '...22 1.3.1.2 L a r g e un i lamel la r v e s i c l e s ( L U V s ) 24 1.3.1.3 S m a l l un i lamel la r v e s i c l e s ( S U V s ) 2 4 1.3.2 P r e p a r a t i o n of L U V s 2 4 1.3.2.1 E x t r u s i o n t e c h n i q u e s 2 5 i v 1.3.2.2 D e t e r g e n t d ia lys is t e c h n i q u e s 2 5 1.4 L i p i d - m e d i a t e d D N A t ransfec t ion 2 6 1.4.1 D N A - c a t i o n i c lipid c o m p l e x e s 2 6 1.4.2 S t a b i l i z e d P l a s m i d - L i p i d Par t i c les 2 9 1.4.3 C o f a c t o r s e m p l o y e d in l i p i d - b a s e d m e d i a t e d t rans fec t ion 31 1.5 C a l c i u m ( C a 2 + ) 3 3 1.5.1 B i o l o g i c a l act ivi t ies of C a 2 + 3 3 1.5.2 Inf luence of C a 2 + o n lipid p o l y m o r p h i s m 34 1.5.3 R o l e s of C a 2 + in t ransfect ion 36 1.6 T h e s i s ob jec t i ves 38 C H A P T E R 2: CALCIUM ENHANCES THE TRANSFECTION P O T E N C Y OF PLASMID DNA-CATIONIC LIPOSOME C O M P L E X E S 39 2.1 Introduction 39 2.2 Mate r ia ls a n d m e t h o d s 4 2 2.2.1 P l a s m i d s 4 2 2 .2 .2 C e l l l ines 4 2 2 .2 .3 L i p i d s a n d C h e m i c a l s 4 2 2 .2 .4 P r e p a r a t i o n of L U V s 4 3 2 .2 .5 In vitro t ransfect ion in the p r e s e n c e of C a 2 + 4 3 2 .2 .6 De te rmina t ion of t r a n s g e n e e x p r e s s i o n 4 4 2 .2 .7 Intracellular u p t a k e s t u d i e s e m p l o y i n g 1 4 C - r a d i o l a b e l e d l ipids 4 5 2 .2 .8 Dot blot a n d S o u t h e r n blot a n a l y s e s 4 6 2 .3 R e s u l t s 4 7 2.3.1 T h e t ransfect ion p o t e n c y of p l a s m i d D N A - l i p i d c o m p l e x e s is i n c r e a s e d in the p r e s e n c e of C a 2 + 4 7 2 .3 .2 T h e propor t ion of ce l ls t rans fec ted is i n c r e a s e d in the p r e s e n c e of C a 2 + 4 9 2 .3 .3 C a t i o n - d e p e n d e n t t ransfect ion e n h a n c e m e n t is C a 2 + s p e c i f i c 51 2 .3 .4 C a 2 + i n c r e a s e s t h e rate of t rans fec t ion by c o m p l e x e s 51 2 .3 .5 I n c r e a s e d intracel lular de l ivery of lipid is o b s e r v e d in the p r e s e n c e of C a 2 + 53 2 .3 .6 I n c r e a s e d intracel lular de l ivery of intact p l a s m i d D N A is o b s e r v e d in the p r e s e n c e of C a 2 + . . . 56 2 .3 .7 T h e level of t ransfect ion e n h a n c e m e n t is d e p e n d e n t o n the t ime of C a 2 + addi t ion 5 8 2 .3 .8 C a 2 + - e n h a n c e d t ransfect ion for c o m p l e x e s is o b s e r v e d in a var ie ty of cel l l ines 6 0 2 .3 .9 C a e n h a n c e s the t ransfect ion p o t e n c y of c o m p l e x e s c o n t a i n i n g a var iety of ca t ion ic l ipids 6 2 2 .4 D i s c u s s i o n 6 5 C H A P T E R 3: CALCIUM DRAMATICALLY E N H A N C E S T H E TRANSFECTION P O T E N C Y OF STABILIZED PLASMID-LIPID PARTICLES 6 9 3.1 Introduction 6 9 3.2 M a t e r i a l s a n d m e t h o d s 71 3.2.1 Mater ia ls 71 3.2.2 P r e p a r a t i o n of S P L P 72 3 .2 .3 T r a n s f e c t i o n in the p r e s e n c e of C a 2 + 72 3 .2 .4 D e t e r m i n a t i o n of S P L P up take into ce l ls 7 3 3 .2 .5 F l u o r e s c e n c e m i c r o s c o p y s t u d i e s 74 3.2 .6 Intracellular p r o c e s s i n g of p l a s m i d D N A 74 3 .2 .7 3 1 P N M R s p e c t r o s c o p y 7 5 3.2.8 E n t r a p m e n t of C a 2 + ins ide S P L P 76 3 .2 .9 Insertion of C P L 7 7 3.3 R e s u l t s 7 9 3.3.1 T h e t ransfect ion p o t e n c i e s o f S P L P are dramat ica l ly e n h a n c e d in the p r e s e n c e of C a 2 + 7 9 3.3.2 S P L P a r e s tab le in the p r e s e n c e of C a 2 + 81 3 .3 .3 C a 2 + d o e s not in f luence the ce l lu lar up take of S P L P 81 3 .3 .4 F l u o r e s c e n c e s t u d i e s indicate e n h a n c e d e n d o s o m a l des tab i l i za t ion fo l lowing uptake of L U V in the p r e s e n c e of C a 2 + 8 3 3 .3 .5 Intracellular p r o c e s s i n g of de l i ve red p l a s m i d D N A 85 3 .3 .6 C a 2 + d e s t a b i l i z e s b i layer lipid s t ructures in a m a n n e r c o n s i s t e n t with a n ability to d e s t a b i l i z e e n d o s o m a l m e m b r a n e s 87 3 .3 .7 E x t e r n a l C a 2 + is requ i red to e n h a n c e S P L P t ransfec t ion p o t e n c y 91 3.3.8 E f fec t of C a 2 + o n S P L P s y s t e m s conta in ing ca t ion ic P E G l ipids a n d h igher leve ls of D O D A C 91 3.3 D i s c u s s i o n 95 C H A P T E R 4: SUMMARY AND FUTURE DIRECTIONS 100 4.1 S u m m a r y 100 4 .2 F u t u r e d i rec t ions 104 R E F E R E N C E S 106 LIST O F T A B L E S T a b l e 1 P o l y m o r p h i c p h a s e p r e f e r e n c e s of c o m m o n l ipids in b io log ica l m e m b r a n e 18 T a b l e 2 T y p e s a n d a c t i o n s of c o f a c t o r s c o m m o n l y e m p l o y e d in l i p i d - b a s e d m e d i a t e d t ransfect ion 32 T a b l e 3 E f fec t of C a 2 + in v a r i o u s m i x e d lipid s y s t e m s 36 vii LIST OF FIGURES F i g u r e 1.1 S c h e m a t i c illustration of the cel lu lar barr iers e n c o u n t e r e d dur ing l i p o s o m e - m e d i a t e d t ransfect ion in vitro 6 F i g u r e 1.2 L i p i d s a s c o m p o n e n t s of m e m b r a n e b i layer 8 F i g u r e 1.3 S t r u c t u r e s of natural ly o c c u r r i n g l ipids u s e d in this work 10 F i g u r e 1.4 S t r u c t u r e s of synthet ic ca t ion ic l ipids D O T M A a n d D O D A C 13 F i g u r e 1.5 P o l y m o r p h i c p h a s e b e h a v i o r of l ipids 16 F i g u r e 1.6 P r o p o s e d m o d e l s of m e m b r a n e f u s i o n a n d in termedia te s t ruc tures . . 20 F i g u r e 1.7 C l a s s i f i c a t i o n of l i p o s o m e s 2 3 F i g u r e 1.8 S c h e m a t i c representa t ion of p l a s m i d D N A - c a t i o n i c l i p o s o m e c o m p l e x e s 2 7 F i g u r e 1.9 S c h e m a t i c s t ructure of s tab i l i zed p lasmid - l ip id par t ic les 3 0 F i g u r e 2.1 E f fec t of i n c r e a s i n g C a 2 + c o n c e n t r a t i o n s o n the t ransfec t ion act ivi t ies of p l a s m i d D N A - c a t i o n i c l i p o s o m e c o m p l e x e s 4 8 F i g u r e 2 .2 F l u o r e s c e n c e m i c r o s c o p y of B H K ce l ls t r a n s f e c t e d with p C M V G F P u s i n g c o m p l e x e s in the p r e s e n c e of C a 2 + 5 0 F i g u r e 2.3 Spec i f ic i ty of C a 2 + for e n h a n c i n g t ransfect ion 52 F i g u r e 2 .4 E f fec t of incuba t ion t ime o n t rans fec t ion e m p l o y i n g contro l D N A - l i p i d a n d C a 2 + - e n h a n c e d D N A - l i p i d c o m p l e x e s 54 F i g u r e 2 .5 K inet ic a n a l y s i s of the intracel lular de l ivery of l ipids in the p r e s e n c e of C a 2 + 5 5 F i g u r e 2 .6 A n a l y s i s of p l a s m i d D N A u p t a k e u s i n g dot blot a n d intracel lular integrity u s i n g S o u t h e r n blot 5 7 v i i i F i g u r e 2.7 E f fec t of the o r d e r of C a 2 + addi t ion o n t ransfec t ion p o t e n c y 5 9 F i g u r e 2.8 E f fec t of C a 2 + o n t ransfect ion for different cel l l ines 61 F i g u r e 2 .9 E f fec t of C a 2 + o n t rans fec t ion p roper t i es o f di f ferent l i p o s o m a l fo rmu la t ions 6 3 F i g u r e 3.1 C a 2 + e n h a n c e s the t ransfect ion p o t e n c y of S P L P in a s p e c i f i c m a n n e r . . 8 0 F i g u r e 3.2 Stabi l i ty of S P L P in the p r e s e n c e of C a 2 + 8 2 F i g u r e 3.3 In f luence of C a 2 + o n the ce l lu lar up take of S P L P 84 F i g u r e 3.4 Inf luence of C a 2 + o n cel l m o r p h o l o g y fo l lowing u p t a k e of L U V labe l led with R h - D O P E a s d e t e c t e d u s i n g f l u o r e s c e n c e m i c r o s c o p y 86 F i g u r e 3 .5 In f luence of C a 2 + o n t h e integrity o f S P L P p l a s m i d fo l lowing u p t a k e of S P L P into B H K ce l ls 88 F i g u r e 3.6 3 1 P N M R s p e c t r a of v a r i o u s m o d e l m e m b r a n e s y s t e m s in the p r e s e n c e of C a 2 + 9 0 F i g u r e 3.7 E x t e r n a l C a 2 + w a s r e s p o n s i b l e for st imulat ing t rans fec t ion 9 2 F i g u r e 3.8 E f fec t of C a 2 + o n S P L P s y s t e m s conta in ing ca t ion ic P E G l ipids or h i g h e r a m o u n t s of D O D A C 94 ix ABBREVIATIONS P-gal (3 -galactosidase B H K b a b y h a m s t e r k i d n e y B C A b i c i n c h o n i n i c a c i d C h o i c h o l e s t e r o l C L cardio l ip in c m c critical mice l lar c o n c e n t r a t i o n C M V c y t o m e g a l o v i r u s C P L ca t ion ic p e g y l a t e d lipid C P R G c h l o r o p h e n o l red g a l a c t o p y r a n o s i d e D C - C H O L 3 - B - [A / - (A / ' ,A / -d imethy laminoethy l )carbamoyl ] -cho lestero l D D A B d i m e t h y l d i o c t a d e c y l a m m o n i u m b r o m i d e D E A E - S e p h a r o s e d i e t h y l a m i n o e t h y l - S e p h a r o s e D M E M D u l b e c c o mod i f i ed E a g l e m e d i u m D M T A P 1 , 2 - d i m y r i s t o y l o x y - 3 - t r i m e t h y l a m m i n o p r o p a n e D N A d e o x y r i b o n u c l e i c a c i d D O D A B A / ,A / -d io leoy l -A / , /V -d imethy lammonium b r o m i d e D O D A C A/ ,A / -d io ley l -A / ,A / -d imethy lammonium c h l o r i d e D O D M A - A N A/- [2,3-(dioleyloxy)propyl] -A/ ,A/-dimethyl-A/-c y a n o m e t h y l a m m o n i u m ch lor ide D O P C 1 , 2 - d i o l e o y l - 3 - p h o s p h a t i d y l c h o l i n e D O P E 1 , 2 - d i o l e o y l - 3 - p h o s p h a t i d y l e t h a n o l a m i n e D O P S 1 , 2 - d i o l e o y l - 3 - p h o s p h a t i d y l s e r i n e X D O T A P 1 ,2 -d io leoy l -3 - (oxy t r ime thy lammino )propane D O T M A A/ - [2 ,3- (d io leyloxy)propyl ] -A/ ,A/ ,A/ - t r imethylammonium c h l o r i d e D S C differential s c a n n i n g ca lor imetry D S D A C A/ ,A / -d is teary l -A / ,A / -d imethy lammonium c h l o r i d e E D T A e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d E G T A e t h y l e n e g lyco l -b is (p -aminoethy le ther ) -A / ,A / ,A / ' ,A / - te t raacet ic a c i d E M e lec t ron m i c r o s c o p y E S R e lec t ron s p i n r e s o n a n c e F B S fetal b o v i n e s e r u m F I D f ree induct ion d e c a y G F P g r e e n f l u o r e s c e n c e protein H B S H E P E S buf fered s a l i n e H E P E S A / - (2 -hydroxye thy l )p iperaz ine -A / ' - 2 -e thanesu l fon ic a c i d H i , h e x a g o n a l H M G high mobil i ty g r o u p i.v. i n t r a v e n o u s IMI inverted mice l lar in te rmedia tes L B P A l y s o b i s p h o s p h a t i d i c a c i d L u c luc i fe rase L U V s large uni lamel lar v e s i c l e s y s t e m M L V s mult i lamel lar v e s i c l e s N P - 4 0 N o n i d e t P 4 0 O G P n - o c t y l - p - D - g l u c o p y r a n o s i d e P A p h o s p h a t i d i c a c i d P B S p h o s p h a t e buf fered s a l i n e P C p h o s p h a t i d y l c h o l i n e P E p h o s p h a t i d y l e t h a n o l a m i n e P E G p o l y e t h y l e n e g l y c o l P E G - C e r C 2 o 1-0-(2 ' - (co-methoxypolyethyleneglycol( 2 ooo))succinoyl) -2-A/-a r a c h i d o y l s p h i n g o s i n e P e n - S t r e p penc i l l in -s t rep tomyc in P G p h o s p h a t i d y l g l y c e r o l PI phosphat idy l inos i to l 3 1 P - N M R p h o s p h o r u s - 3 1 n u c l e a r m a g n e t i c r e s o n a n c e P S p h o s p h a t i d y l s e r i n e Q E L S q u a s i - e l a s t i c light scat ter ing R d r h o d a m i n e R E S re t icu loendothe l ia l s y s t e m R h - P E A/ - ( l issamine r h o d a m i n e B s u l f o n y l ) - 1 , 2 - d i o l e o y l - s n -p h o s p h a t i d y l e t h a n o l a m i n e S D S s o d i u m d o d e c y l sul fate S K - O V 3 h u m a n o v a r y a d e n o c a r c i n o m a S P L P s tab i l i zed p lasmid- l ip id part ic les S S C s o d i u m ch lor ide , s o d i u m citrate S U V s s m a l l un i lamel la r v e s i c l e s TH t ransit ion t e m p e r a t u r e x i i T M C t r a n s - m o n o l a y e r c o n t a c t T R C - T E M t i m e - r e s o l v e d c r y o - t r a n s m i s s i o n e lec t ron m i c r o s c o p y T r i s t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e Tr i ton X - 1 0 0 t - o c t y l p h e n o x y p o l y e t h o x y e t h a n o l x i i i A C K N O W L E D G M E N T S First and foremost, I would like to thank my supervisor, Dr. Pieter Cull is , for allowing me to work in his laboratory. Dr. Cull is has inspired me to work hard, to think creatively, and to keep trying new experiments. I w i l l always remember those fun-filled parties and ski-trips. I have learned so much from you and it has really been a great pleasure to work with you. I would also like to extend my appreciation to my committee members: Dr. Roger Brownsey and Dr. Michel Roberge, who have provided me with helpful advice and guidance throughout the progress of my study at U B C . I would like to thank Dr. David Fenske, not just for being the first person to come across my graduate school application, but especially for his friendship. I've enjoyed the openness during all of our spiritual and philosophical discussions. A big thank you to the past and present members of the Cull is lab: especially K i m for his excellent memories and wisdom; Lome for always exercising with me and allowing me to play jokes on; Ismail, I have always admired your outstanding knowledge and interests in science; Lenore, for always being so pleasant to talk to; Ammen, for sharing wedding tips; John, for being "fresh and green"; Tabitha, for delivering my thesis; Norbert, for being "tall and handsome"; and Peter for helpful research advice; Al ison, Atsu, Austin, Benny, Conrad, Dora, Elizabeth, Ken , Myrna, Pat, and Tao: I had fun working with all o f you as well . Thank you for the friendships which all of you had shown me during my time in the Cul l i s ' lab. O f course, Science Council of B . C . for providing part of the funding in support of my research. I am also grateful to my parents for their decision to immigrate into Canada and to settle in the beautiful city of Vancouver, as well as for their never-ending support; my husband, Wilson M o k , for always believing in me and for making my life so much more amusing; and my family and friends, for their love and understanding. Finally, let us give thanks to our God, because every discovery is itself a witness to the amazing miracle of life which He has created out of His love for us. While scientists are discoverers, God is the ultimate creator. Thank you especially for having listened to my prayers and for always being there throughout my studies in science. x i v To my parents: Jimmy C . K . Lam & Rosa L M . Kuok And my siblings: Charles, Salina, and Stella XV C H A P T E R 1 INTRODUCTION 1.1 Project overview: Gene therapy and current DNA delivery methods M o r e t h a n 4 5 0 0 h u m a n g e n e t i c d i s e a s e s result f rom a d e f e c t in a s i n g l e g e n e ( M c k u s i c k , 1992) . Wi th the g rowing k n o w l e d g e o n the g e n e t i c b a s i s of d i s e a s e s a n d the a d v a n c e in e n g i n e e r i n g g e n e t ransfer v e c t o r s , g e n e t h e r a p y is e m e r g i n g a s a m e t h o d o f t rea tment for both g e n e t i c a n d a c q u i r e d d i s e a s e s . T h e pr inc ip le i n v o l v e s the cor rec t ion of g e n e t i c d i s o r d e r s by a d d i n g , de le t ing , or r e p l a c i n g g e n e s . A ma jor l imitation, h o w e v e r , l ies in the inability to de l iver the g e n e of interest in a s a f e a n d ef fect ive m a n n e r . T h e current g e n e t ransfer m e t h o d s c o n s i s t of both v i r a l - b a s e d a n d n o n v i r a l - b a s e d v e c t o r s . T h i s s e c t i o n will p r e s e n t a n o v e r v i e w of t h e s e g e n e de l ive ry s y s t e m s a n d d i s c u s s their a s s o c i a t e d p r o b l e m s . 1.1.1 Viral-based vectors Vi ra l v e c t o r s a r e rep l ica t ion-defect ive v i r u s e s that conta in t h e r a p e u t i c g e n e s within the viral g e n o m e . C o m m o n viral v e c t o r s inc lude re t rov i ruses , a d e n o v i r u s , a d e n o - a s s o c i a t e d v i rus , a n d h e r p e s s i m p l e x v i rus . V i ra l v e c t o r s a r e h ighly eff icient, a s they p o s s e s s the m a c h i n e r y n e c e s s a r y for g e n e de l ivery . R e t r o v i r u s e s utilize viral e n z y m e s to c o p y their g e n o m e s into D N A a n d integrate into the host c h r o m o s o m e ( V a r m u s , 1988) . L o n g - t e r m g e n e e x p r e s s i o n e m p l o y i n g retroviral v e c t o r s c a n b e a c h i e v e d but their infect ion is l imited to d iv id ing ce l ls ( A n d e r s o n , 1992) . H o w e v e r , the ability to integrate into the host g e n o m e c o n t a i n s a risk of l inser t ional m u t a g e n e s i s . A l t h o u g h the a d e n o v i r a l g e n o m e is not in tegrated into the hos t D N A , a d e n o v i r u s h a s the a d v a n t a g e that it c a n infect both d iv id ing a n d n o n d i v i d i n g ce l l s ( G r u n h a u s a n d Horwitz , 1992) . A d e n o - a s s o c i a t e d v i rus c a n a l s o infect both d iv id ing a n d non-d iv id ing ce l l s , but its activity requ i res the p r e s e n c e of a h e l p e r v i rus s u c h a s a d e n o v i r u s or h e r p e s v i rus ( M u z y c z k a , 1992) . H e r p e s s i m p l e x v i rus is a d o u b l e - s t r a n d e d D N A v i rus with a g e n o m e m e a s u r i n g m o r e t h a n 150 kb ( R o i z m a n a n d S e a r s , 1990) . A l t h o u g h h e r p e s s i m p l e x v i rus c a n infect a var ie ty of d iv id ing a n d non-d iv id ing ce l ls , it a l s o c a u s e s s o m e cytotoxici ty ( G l o r i o s o et a l . , 1994) . D e s p i t e b e i n g efficient g e n e t ransfer m e d i a t o r s , a n u m b e r of d i s a d v a n t a g e s a r e a s s o c i a t e d with viral v e c t o r s . A high viral titer c a n b e difficult to p r o d u c e for m a n y of the viral v e c t o r s . F u r t h e r m o r e , viral v e c t o r s h a v e a limitation in g e n e p a c k a g i n g : a d e n o - a s s o c i a t e d viral v e c t o r s c a n on ly carry 5 kb , retroviral v e c t o r c a n car ry up to 7 kb , a n d a d e n o v i r a l v e c t o r s c a n p a c k a g e a r o u n d 10 to 12 kb. M o s t important ly , this s y s t e m h a s the c o m p l i c a t i o n of b e c o m i n g i m m u n o g e n i c , c y t o p a t h i c , or r e c o m b i n o g e n i c . S u c h d r a w b a c k s h a v e p r o m p t e d the d e v e l o p m e n t of nonvi ra l g e n e car ry ing s y s t e m s . 1.1.2 Nonviral-based vectors N o n v i r a l v e c t o r s h a v e a d v a n t a g e s o v e r the viral v e c t o r s a s they h a v e low a c u t e toxicity a n d a r e n o n i m m u n o g e n i c . T h e y c a n a l s o b e p r o d u c e d o n a la rge s c a l e a n d d o not h a v e a g e n e p a c k a g i n g limitation. D r a w b a c k s with the nonv i ra l v e c t o r s i n c l u d e their lower t ransfect ion e f f ic iency t h a n viral v e c t o r s a n d their 2 t rans ient g e n e e x p r e s s i o n , a s they d o not conta in the m a c h i n e r y c a p a b l e to m e d i a t e g e n e de l ive ry or g e n e integrat ion. 1.1.2.1 Gene delivery by physical or chemical methods P l a s m i d D N A conta in ing therapeut ic g e n e s c a n b e in t roduced into ce l l s by e i ther p h y s i c a l ( s u c h a s microin ject ion a n d e lect roporat ion) or c h e m i c a l ( s u c h a s D E A E - d e x t r a n , c a l c i u m p h o s p h a t e ( C a P 0 4 ) , a n d ca t ion ic l i p o s o m e s ) t e c h n i q u e s . T h e p h y s i c a l m e t h o d s de l iver D N A into ce l l s th rough d isrupt ing the p l a s m a m e m b r a n e . A l t h o u g h t h e s e m e t h o d s c a n b e efficient in s o m e cel l l ines , they of ten p r o d u c e i n c o n s i s t e n t t ransfect ion results a n d a r e toxic to ce l ls . In add i t ion , the p h y s i c a l m e t h o d s requi re that the target t i s s u e s b e e x p o s e d in o r d e r to a c h i e v e D N A t ransfer . C h e m i c a l m e t h o d s u s i n g ei ther D E A E - d e x t r a n (Vaher i a n d P a g a n o , 1965) or C a P 0 4 ( G r a h a m a n d V a n d e r E b , 1973) h a v e b e e n p o p u l a r m e t h o d s for t ransfec t ing ce l ls in vitro. P l a s m i d D N A c o m p l e x e s with D E A E - d e x t r a n or c o p r e c i p i t a t e s with C a P 0 4 a r e a b s o r b e d onto the cel l m e m b r a n e s a n d s u b s e q u e n t l y in terna l i zed into the ce l l s . A l t h o u g h c o m m o n l y e m p l o y e d , g e n e de l ive ry u s i n g t h e s e m e t h o d s suf fers f r o m low a n d incons is ten t t ransfect ion e f f i c ienc ies . 1.1.2.2 Gene delivery employing liposomes T h e d e v e l o p m e n t of ca t ion ic l ipids a s a g e n t s for t ransfec t ion ( F e i g n e r et a l . 1987) h a s p r o v e n to b e the m o s t ef fect ive non-v i ra l g e n e t ransfer m e t h o d in vitro. T h e first g e n e r a t i o n of l i p i d - b a s e d g e n e carr ier s y s t e m c o n t a i n s two w o r k i n g c o m p o n e n t s : a posi t ive ly c h a r g e d ca t ion ic lipid a n d a h e l p e r lipid w h i c h is 3 " f u s o g e n i c " . A d v a n t a g e s a s s o c i a t e d with this l i p o s o m a l g e n e t ransfer s y s t e m are : 1) p l a s m i d D N A readi ly c o m p l e x e s with the ca t ion ic l i p o s o m e s , 2) the posi t ive ly c h a r g e d ca t ion ic l i p o s o m e - D N A c o m p l e x a l lows for interact ion with the nega t i ve ly c h a r g e d cel l p l a s m a m e m b r a n e , 3) the f u s o g e n i c c o m p o n e n t of ca t ion ic l i p o s o m e s c o u l d p r o m o t e destab i l i za t ion of the e n d o s o m e m e m b r a n e , 4 ) there is e s s e n t i a l l y n o limitation o n the s i z e of a g e n e that c a n b e p a c k a g e d , a n d 5) potent ia l target ing to s p e c i f i c t i s s u e s a n d cel l t y p e s . T h i s s y s t e m is a l s o b e i n g invest iga ted a s a v e c t o r for in v ivo g e n e t h e r a p y for t rea tments f o c u s e d o n m e l a n o m a ( N a b e l et a l . 1993) a n d c y s t i c f ib ros is ( F a s b e n d e r et a l . 1995 , Y o s h i m u r a et a l . 1992) . S y s t e m i c a l l y a d m i n i s t e r e d c o m p l e x e s of ca t ion ic l i p o s o m e s a n d p l a s m i d D N A h a v e b e e n s h o w n to t ransfec t ce l l s in v ivo ( H o n g et a l . , 1997; Li et a l . , 1997; L iu et a l . , 1995 ; L iu et a l . , 1997 ; S o n g et a l . , 1997; T e m p l e t o n et a l . , 1997; Th ie r ry et a l . , 1995; Z h u et a l . , 1993) . U n l i k e v i r a l - b a s e d s y s t e m s , l i p i d - b a s e d s y s t e m s d o not h a v e the c o m p l i c a t i o n s of mutagen ic i ty a n d immuni ty . H o w e v e r , their t rans fec t ion p o t e n c i e s a r e relat ively lower. A s u c c e s s f u l g e n e t ransfer requ i res that the integrity of a t r a n s f e c t e d g e n e of interest is re ta ined until it r e a c h e s the n u c l e u s , i.e., the D N A m u s t t r a v e r s e the cel l m e m b r a n e , the c y t o p l a s m a n d the n u c l e a r m e m b r a n e without c o n s i d e r a b l e d e g r a d a t i o n o c c u r r i n g . O n e major limitation in the l i p i d - b a s e d s y s t e m is the lack of a c o m p o n e n t that a s s i s t s in med ia t ing the r e l e a s e a n d d i rect ing the g e n e t i c mater ia ls into the nuc le i . F i g u r e 1.1 d e p i c t s the major ce l lu lar bar r ie rs that h i n d e r the p l a s m i d D N A f rom b e i n g t r a n s c r i b e d dur ing l i p o s o m e - m e d i a t e d g e n e de l ivery . 4 T h e p r e s e n t s t u d y d e s c r i b e s the d i s c o v e r y that the ca t ion c a l c i u m ( C a ) is a potent c o f a c t o r in e n h a n c i n g the t ransfect ion e f f i c ienc ies of l i p i d - b a s e d t rans fec t ion s y s t e m s . T w o l i p o s o m a l s y s t e m s w e r e e x a m i n e d : the c o m m o n p l a s m i d D N A -ca t ion ic l i p o s o m e c o m p l e x e s a n d the recent ly d e v e l o p e d s tab i l i zed p lasmid - l ip id par t ic les ( S P L P ) . B o t h t ransfect ion activit ies a s wel l a s intracel lular p r o c e s s i n g of l i p o s o m e s a n d p l a s m i d D N A w e r e d e t e r m i n e d . F u r t h e r m o r e , w e h a v e a l s o a t t e m p t e d to e x a m i n e the m e c h a n i s m by w h i c h C a 2 + faci l i tated the e n h a n c e d t rans fec t ion . 5 P l a s m i d D N A -lipid c o m p l e x e s 1. C e l l u l a r up take f Cytosol \ F i g u r e 1.1. S c h e m a t i c il lustration of the ce l lu lar barr iers e n c o u n t e r e d dur ing l i p o s o m e - m e d i a t e d t ransfect ion in vitro. 6 1.2 Lipids L i p i d s a r e d e f i n e d a s the wa te r - inso lub le m o l e c u l e s in ce l ls that a r e s o l u b l e in o r g a n i c s o l v e n t s . L i p i d s in the fo rm of fatty a c i d s a r e s t o r e d a s a n e n e r g y r e s e r v e . M o r e important ly , l ipids p rov ide the b a s i c b i layer s t ructure of the ce l l p l a s m a m e m b r a n e , w h i c h s e r v e s a s a relatively i m p e r m e a b l e barr ier to the p a s s a g e of m o s t w a t e r - s o l u b l e m o l e c u l e s . It is the a m p h i p a t h i c nature of lipid m o l e c u l e s that permi ts t h e m to f o r m b i layers s p o n t a n e o u s l y in a q u e o u s so lu t ion , with their h y d r o p h o b i c tails b u r i e d in the interior a n d their hydrophi l ic h e a d s e x p o s e d to w a t e r (F ig . 1 .2A) . B e s i d e s the p l a s m a m e m b r a n e , o r g a n e l l e s s u c h a s the m i t o c h o n d r i o n , n u c l e u s , e n d o p l a s m i c re t icu lum a n d golg i a p p a r a t u s a l s o e m p l o y l ipids to f o r m a m e m b r a n e barr ier b e t w e e n the c y t o s o l a n d their internal content . L ipid c o m p o s i t i o n c a n v a r y d r a m a t i c a l l y b e t w e e n the different s i d e s of the s a m e m e m b r a n e , a s wel l a s a m o n g different c e l l s or o r g a n e l l e m e m b r a n e s . F inal ly , lipid b i l ayers s e r v e a s m a t r i c e s with w h i c h m e m b r a n e prote ins a s s o c i a t e . T h e fluid m o s a i c m o d e l ( S i n g e r a n d N i c o l s o n , 1972) d e p i c t s the a s y m m e t r y of the lipid bi layer , w h i c h p r o v i d e s the f luid-l ike f r a m e w o r k for the a t t a c h m e n t of v a r i o u s m e m b r a n e prote ins (F ig . 1.2B) 1.2.1 Chemistry and physics of lipids Fat ty a c i d s a n d t r iacy lg lycero ls a r e the f o r m s of l ipids u s e d for e n e r g y s t o r a g e a n d e n e r g y m e t a b o l i s m . T h e three major t y p e s of l ipids in cel l m e m b r a n e s a r e p h o s p h o l i p i d s , c h o l e s t e r o l , a n d g lyco l ip ids . T h e fo l lowing s e c t i o n d e s c r i b e s the l ipids re levant to the invest igat ion of this t h e s i s . 7 Hydrophil ic head ft A q u e o u s buffer ™ \ / v / \ / v / \ / \ / \ / V / Hydrophobic tail Lipid molecule Lipid bilayer B Figure 1.2. Lipids as components of membrane bilayer. (A) Amphipathic nature of lipids in bilayer configuration. (B) Fluid mosa ic model depicting a membrane bilayer containing proteins, lipids, and glycosylated proteins (#). 8 1.2.1.1 Phospholipids T h e ma jor lipid const i tuent of e u k a r y o t i c cel l m e m b r a n e s is p h o s p h o l i p i d s , w h i c h c o n t a i n a g l y c e r o l - b a s e d b a c k b o n e a n d p h o s p h a t e - c o n t a i n i n g h e a d g r o u p s . P h o s p h o l i p i d s a r e a m p i p h i l e s with both hydrophi l ic a n d h y d r o p h o b i c m o i e t i e s , w h i c h p r o m o t e t h e m to a s s e m b l e into b i layer s t ructures . T h e major p h o s p h o l i p i d s f o u n d in b io log ica l m e m b r a n e s inc lude: p h o s p h a t i d y l c h o l i n e ( P C ) , p h o s p h a t i d y l e t h a n o l a m i n e ( P E ) , p h o s p h a t i d y l s e r i n e ( P S ) , p h o s p h a t i d i c a c i d ( P A ) , p h o s p h a t i d y l g l y c e r o l ( P G ) , p h o s p h a t i d y l i n o s i t o l (PI), a n d cardio l ip in ( C L ) . T h e s t ructures of the p h o s p h o l i p i d s u s e d in this work a r e s h o w n in F i g u r e 1.3A. T h e s t ruc tures of both the c h a i n a n d h e a d g r o u p c o m p o n e n t s a m o n g l ipids contr ibute to the different func t ions . T h e a c y l c h a i n s of p h o s p h o l i p i d s v a r y in their c h a i n length a n d d e g r e e of unsatura t ion . Increas ing unsatura t ion in the h y d r o c a r b o n reg ion t e n d to main ta in the b i layer in a fluid state , w h e r e a s m o r e s a t u r a t e d m e m b r a n e s a r e l e s s f lexible s i n c e they tend to b e m o r e tightly p a c k e d . P h o s p h o l i p i d s a l s o differ in their h e a d g r o u p s in that P S , P G , PI a n d P A a r e a n i o n i c whi le P C a n d P E a r e zwitterionic. T h e h e a d g r o u p s i z e s a l s o affect the d e g r e e of p a c k i n g in the m e m b r a n e : s m a l l e r s i z e h e a d g r o u p s a l low for t ighter p a c k i n g t h a n la rger o n e s . H e a d g r o u p s t ructures c a n a l s o h a v e a n effect o n the part icular lipid p h a s e o r g a n i z a t i o n that a n individual lipid a d o p t s u p o n hydra t ion , w h i c h will b e a d d r e s s e d in the fo l lowing lipid p o l y m o r p h i s m s e c t i o n . 1.2.1.2 Sphingolipids S p h i n g o s i n e - b a s e d l ipids a re a n o t h e r ma jor s p e c i e s in the e u k a r y o t i c 9 A. Phospholipids 0 1 A-X R 2 0 KJ Headgroups (X) P h o s p h a t i d y l c h o l i n e ( P C ) : ^ N ( C H 3 ) 3 -P h o s p h a t i d y l e t h a n o l a m i n e ( P E ) : 0 ^ ^ N H 3 + 0 P h o s p h a t i d y l s e r i n e ( P S ) : 0 ^ N H , Acyl chains (Rl, R2): O l e o y l (O): H C 8 H 1 7 H O C v H ^ i 7' '14 v B. Sphingolipid C e r a m i d e : R = C O R " , R' = H (R" = h y d r o c a r b o n ) C. Cholesterol F i g u r e 1 . 3 . S t r u c t u r e s of natural ly o c c u r r i n g l ipids u s e d in this work . 10 M e m b r a n e s . S p h i n g o l i p i d s a r e d e r i v e d f r o m the l o n g - c h a i n a m i n o a l c o h o l s p h i n g o s i n e . T h e h y d r o p h o b i c port ion, c a l l e d c e r a m i d e , c o n t a i n s a mixture of fatty a c i d s that a r e a m i d e - l i n k e d to the a m i n o g r o u p of s p h i n g o s i n e (F ig . 1 .3B) . T h e c e r a m i d e mo ie ty c a n b e e m b e d d e d a l o n g with the h y d r o p h o b i c c h a i n s of p h o s p h o l i p i d s a n d o ther m e m b r a n e l ipids. 1.2.1.3 Cholesterol E u k a r y o t i c p l a s m a m e m b r a n e s conta in large a m o u n t s of c h o l e s t e r o l (F ig . 1 .3C) , up to o n e m o l e c u l e for e v e r y p h o s p h o l i p i d m o l e c u l e . C h o l e s t e r o l m o l e c u l e s or ient t h e m s e l v e s in the b i layer with their hydroxy l g r o u p s c l o s e to the p o l a r h e a d g r o u p s of the p h o s p h o l i p i d m o l e c u l e s . A s a result , the fluidity of the m e m b r a n e is partly a f fec ted by the p r e s e n c e of c h o l e s t e r o l . T h o s e r e g i o n s of the h y d r o c a r b o n c h a i n s that a r e c l o s e s t to the po lar h e a d g r o u p s a r e i m m o b i l i z e d , whi le the rest of the c h a i n s r e m a i n f lexible. In addi t ion to affect ing fluidity, c h o l e s t e r o l d e c r e a s e s the permeab i l i ty of lipid b i layers to s m a l l w a t e r - s o l u b l e m o l e c u l e s . It is a l s o s u g g e s t e d that c h o l e s t e r o l e n h a n c e s both the flexibility a n d the m e c h a n i c a l stabil ity of the bi layer . 1.2.1.4 Cationic lipids T h e on ly b io logica l ly exist ing ca t ion ic l ipids a r e s p h i n g o s i n e a n d s t e a r y l a m i n e . T h e first syn the t ic ca t ion ic l ipids w e r e A / , A / - d i d o d e c y l - A / , A / - d i m e t h y l a m m o n i u m b r o m i d e (Kun i take a n d O k h a t a , 1977) a n d 1 ,2 -d imyr is toy loxy -3 -t r i m e t h y l a m m i n o p r o p a n e ( D M T A P ) (Eibl a n d W o o l e y , 1979) . It w a s not until the n s y n t h e s i s of the ca t ion ic lipid d i o l e y l o x y p r o p y l - t r i m e t h y l a m m o n i u m ch lo r ide , D O T M A , by F e i g n e r a n d c o w o r k e r s that the app l ica t ion of ca t ion ic l i p o s o m e s jn t rans fec t ion w a s d e m o n s t r a t e d ( F e i g n e r et a l . , 1987) . D O T M A c o n t a i n s a p e r m a n e n t pos i t ive c h a r g e in the q u a t e r n a r y a m i n e h e a d g r o u p (F ig . 1.4), w h i c h a l lows for e lec t ros ta t ic interact ion with n u c l e i c a c i d s s u c h a s p l a s m i d D N A a n d o l i g o n u c l e o t i d e s , a s wel l a s interact ion with cel l p l a s m a m e m b r a n e . T o g e t h e r with a h e l p e r l ipid, d i o l e o y l p h o s p h a t i d y l e t h a n o l a m i n e ( D O P E ) , F e i g n e r ' s g r o u p repor ted the t rans fec t ion activity of the ca t ion ic lipid c o n s i s t s of the p l a s m i d - D N A l i p o s o m e c o m p l e x e s . S i n c e t h e n , a var ie ty of ca t ion ic l ipids h a v e b e e n p r e p a r e d inc lud ing the o n e u s e d in this s t u d y ( d i o l e y l d i m e t h y l a m m o n i u m ch lor ide or D O D A C ) (F ig . 1.4). T h e s t ruc tu res of t h e s e ca t ion ic l ipids c a n b e var ied with the a m o u n t of pos i t ive c h a r g e s , t y p e s of q u a t e r n a r y a m i n e s , p ro tonatab le h e a d g r o u p s , e s t e r l i n k a g e s b e t w e e n the a l c o h o l of g l y c e r o l a n d the c a r b o x y l of the fatty a c i d , or a n e s t e r l inkage b e t w e e n the tails a n d the g l y c e r o l ( F e i g n e r et a l . , 1994; G a o a n d H u a n g , 1991; L iu e t a l . , 1997) . 1.2.1.5 Polyethylene glycol) (PEG) lipids P E G is a f lexible hydrophi l ic p o l y m e r with repea t ing units of e t h y l e n e g l y c o l (-[ 0 - C H 2 - C H 2 ] n - ) c o u p l e d to the h e a d g r o u p of a c o m m o n p h o s p h o l i p i d . P E G lipids a r e d e s i g n e d to a l low the l i p o s o m a l s y s t e m to a v o i d the re t icu loendothe l ia l s y s t e m ( R E S ) , resul t ing in i n c r e a s e d circulat ion l i fet imes ( L a s i c et a l , 1991 ; W o o d l e a n d L a s i c , 1992) . P E G lipids c r e a t e a highly s o l v a t e d p o l y m e r layer at the m e m b r a n e s u r f a c e , w h i c h s e r v e s a s ster ic barr ier that p r e v e n t s the b ind ing of s e r u m pro te ins on to v e s i c l e s . S u c h ster ic h i n d r a n c e a l s o r e d u c e s the b o d y ' s m o l e c u l a r 12 A/-[2,3-(dioleyloxy)propyl]-A/,A/,A/-trimethylammonium chloride (DOTMA) W,N-dioleyl-A/,A/-dimethylammonium chloride (DODAC) F i g u r e 1.4. S t r u c t u r e s of the synthe t ic ca t ion ic l ipids D O T M A a n d D O D A C . 13 recogn i t ion p r o c e s s e s f rom label ing the l i p o s o m e a s fore ign for s u b s e q u e n t u p t a k e a n d r e m o v a l by R E S ce l ls (Al len, 1994) . In addi t ion , it h a s b e e n f o u n d that P E G c a n ster ical ly inhibit m e m b r a n e f u s i o n b e t w e e n m e m b r a n e v e s i c l e s ( H o l l a n d et a l . , 1996) . T h e p r e s e n c e of P E G c a n a l s o stabi l ize n o n - b i l a y e r fo rming l ipids, s u c h a s P E , in v a r i o u s b i layer lipid mix tures ( H o l l a n d et a l . , 1996b) . 1.2.2 Structural behavior of lipids L i p i d s a d o p t part icular o r g a n i z a t i o n s or p h a s e s u p o n hydra t ion . S u c h structura l b e h a v i o r of l ipids c a n b e in f luenced by both lipid t y p e s a n d the s u r r o u n d i n g e n v i r o n m e n t . D e p e n d i n g o n the t e m p e r a t u r e , p h o s p h o l i p i d s in the b i layer c a n b e in two dif ferent s ta tes , the ge l or liquid crysta l l ine state . In add i t ion , indiv idual l ipids or lipid mix tures c a n a d o p t different lipid p h a s e s , c o m m o n l y k n o w n a s lipid p o l y m o r p h i s m . B o t h ge l to liquid p h a s e transit ion a n d lipid p o l y m o r p h i s m a r e d i s c u s s e d in the fo l lowing s e c t i o n s . F u r t h e r m o r e , the re la t ionship b e t w e e n lipid p o l y m o r p h i s m a n d m e m b r a n e f u s i o n is a l s o p r e s e n t e d . 1.2.2.1 Gel and liquid crystalline phase transition W h e n a synthe t ic lipid b i layer m a d e f rom a s i n g l e t y p e of p h o s p h o l i p i d c h a n g e s f r o m a l iquid s ta te to a rigid crysta l l ine (or gel ) s tate , th is c h a n g e of s ta te is c a l l e d a p h a s e transi t ion. M o s t lipid s p e c i e s in isolat ion c a n u n d e r g o a t ransi t ion f r o m ge l to the fluid state at a charac ter is t ic mel t ing point. S u c h p h a s e t rans i t ions c a n b e m o n i t o r e d by a var iety of t e c h n i q u e s : inc luding n u c l e a r m a g n e t i c r e s o n a n c e ( N M R ) , e l e c t r o n s p i n r e s o n a n c e ( E S R ) , differential s c a n n i n g ca lor imet ry ( D S C ) , a n d 14 fluorescence. At physiological temperatures most, and usually all, membrane lipids are fluid (Cullis and Hope, 1985). 1.2.2.2 Lipid polymorphism The ability of lipids to adopt different structures on hydration is commonly referred to as lipid polymorphism (Gruner et al., 1985). Extrinsic factors such as divalent cations, ionic strength, pH, and membrane protein are known to have an influence on lipid structural preferences (Cullis and de Kruijff, 1979; Killian and de Kruijff, 1986; Tilcock et al., 1988). Lipid polymorphism can be studied using X-ray diffraction, 3 1 P - and 2H-NMR, and freeze-fracture techniques (Cullis et al., 1985). The three general features include bilayer, hexagonal (Hu), and micelles (Fig. 1.5). The characteristics of bilayers include a broad, asymmetric 3 1 P NMR spectrum with a low-field shoulder and high-field peak, and a flat, featureless fracture plane in freeze fracture studies. Hu phase system exhibits 3 1 P NMR spectra with reversed asymmetry which are narrower by a factor of two. Freeze fracture techniques also give rise to a regular corrugated pattern as the fracture plane cleaves between the hexagonally packed cylinders. In general, unsaturated lipids with small headgroups ("cone" shape) such as dioleoyl-phosphatidylethanolamine (DOPE) adopt hexagonal structures in isolation, while lipids with symmetrical headgroup and chain ("cylindrical" shape) such as PC adopts the bilayer phase (Cullis et al., 1986; Rilfors et al., 1984). In biological membranes, a significant number of lipid species prefer or could adopt the HM arrangement at physiological condition or in the presence of 15 Phase 3 1P NMR line shape Molecular shape Mice l la r Isotropic Inverted C o n e B i l ayer i i 4 0 p p m Bilayer Cy l indr ica l H e x a g o n a l (H M ) Hexagonal C o n e g u r e 1.5 P o l y m o r p h i c p h a s e b e h a v i o r of l ipids. 16 extr ins ic fac tors s u c h a s low p H a n d the p r e s e n c e of C a (T i l cock , 1986) ( T a b l e 1). It is s u g g e s t e d that t h e s e l ipids p lay a role in e v e n t s s u c h a s m e m b r a n e f u s i o n , e x o c y t o s i s a n d e n d o c y t o s i s . 1.2.2.3 Membrane fusion M e m b r a n e f u s i o n is a n e v e n t that is vital to the funct ion ing of m a n y ce l l s , o c c u r r i n g in p r o c e s s e s s u c h a s intracel lular m e m b r a n e traff icking, e x o c y t o s i s a n d e n d o c y t o s i s , cel l d iv is ion , viral infect ion, a n d intracel lular m e m b r a n e t ranspor t (Wi lschut a n d H o e k s t r a , 1991) . M e m b r a n e prote ins c o u l d facil i tate f u s i o n by i n d u c i n g loca l d e h y d r a t i o n ( Z i m m e r b e r g et a l . , 1991) a n d per turbat ions in the lipid b i layer t h r o u g h their insert ion into m e m b r a n e s ( C h e r n o m o r d i k et a l . , 1997 ; K a n a s e k i et a l . , 1997; M o n c k a n d F e r n a n d e z , 1994) . P r o t e i n s invo lved in m e d i a t i n g intracel lular m e m b r a n e f u s i o n inc lude the A T P a s e A / - e t h y l m a l e i m i d e - s e n s i t i v e fac tor ( N S F ) , s o l u b l e N S F a t t a c h m e n t prote ins ( S N A P s ) , a n d v e s i c l e a n d target m e m b r a n e - a s s o c i a t e d S N A P r e c e p t o r s ( d e s i g n a t e d v e s i c u l a r (v)- a n d target (t)-S N A R E s , respect ive ly ) ( C h a p m a n , 1995; H a y a n d S c h e l l e r , 1997; R o t h m a n , 1994) . S N A P s b ind direct ly to the m e m b r a n e r e c e p t o r s a n d m e d i a t e the b ind ing of N S F . T h e A T P a s e N S F ac t iva tes the S N A R E c o m p l e x e s , w h i c h br ing m e m b r a n e s into c l o s e proximity. T h i s p r o c e s s e x c l u d e s water f r o m the inter face b e t w e e n the m e m b r a n e s a n d p r o m o t e s s p o n t a n e o u s f u s i o n b e t w e e n the d e h y d r a t e d b i l ayers ( M a y e r , 1999) . It is h y p o t h e s i z e d that f u s i o n prote ins ac t to facil i tate the m e r g e r of the p h o s p h o l i p i d s of living m e m b r a n e s by the s a m e p a t h w a y f o u n d in p h o s p h o l i p i d 17 Table 1. Polymorphic phase preferences of common lipids in biological membrane (Tilcock, 1986). (L = lamellar; H M = hexagonal ; M = micellar). Types of lipids Phase preference at physiological conditions Other conditions Phospatidylchol ine L Sphingomyel in L Phosphat idylethanolamine H i , Phosphat idylser ine L Hu: divalent cations, pH<3.5 Phosphatidylglycerol L Hu: at high salt and temp. Phosphatidyl inositol L Cardiol ipin L Hu: divalent cations, pH<3, high salts Phosphat id ic acid L Hu: divalent cations, pH<3.5, high salts Cholestero l Induces Hu in mixed lipid systems 18 bi layer f u s i o n . T h e fo l lowing s e c t i o n d e s c r i b e s the m e c h a n i s m s i n v o l v e d in f u s i o n p r o p o s e d f r o m s tudy ing m o d e l l i p o s o m a l s y s t e m s . A l t h o u g h it is difficult to s h o w f u s i o n p r o c e s s e s in v ivo , s t u d i e s o n m o d e l m e m b r a n e s y s t e m s h a v e p r o v i d e d c i rcumstant ia l e v i d e n c e in s u p p o r t of the h y p o t h e s i s that f u s i o n is d e p e n d e n t o n the p o l y m o r p h i c capab i l i t i es of l ip ids. It is s u g g e s t e d that in te rmed ia tes in b i l aye r -nonb i l aye r p h a s e t ransi t ions a l s o p r o v i d e in te rmedia te s t ruc tures for m e m b r a n e f u s i o n ( S i e g e l et a l . , 1989) . T h r e e m o d e l s h a v e b e e n p r o p o s e d that a d d r e s s the m e m b r a n e f u s i o n m e c h a n i s m (F ig . 1.6). T h e first m o d e l d e s c r i b e s the m e c h a n i s m invo lved in m e m b r a n e f u s i o n v i a inver ted mice l la r in te rmed ia tes (IMI), w h i c h a r e f o r m e d b e t w e e n the two c l o s e l y a p p o s e d b i l ayers in the f u s i o n p r o c e s s . E v i d e n c e i n c l u d e s the p r e s e n c e of a n isot rop ic 3 1 P N M R s i g n a l toge ther with s t ructures referred to a s "lipidic par t ic les" in the f rac tured s u r f a c e s of f u s e d v e s i c l e s in f r e e z e f racture e l e c t r o n m i c r o s c o p y ( E M ) (Cul l is a n d d e Kruijff, 1979; Cu l l i s et a l . , 1986; Verk le i j , 1984) . T h e s e c o n d m o d e l d e p i c t s a "stalk" in termedia te ( C h e r n o m o r d i k et a l . , 1985; M a r k i n et a l . , 1984) that is f o r m e d by c o a l e s c e n c e of the outer m o n o l a y e r s of the a p p o s i n g m e m b r a n e s . T h e i n t e r m e d i a t e s a r e thought to e x p a n d radial ly to fo rm a t r a n s - m o n o m e r c o n t a c t ( T M C ) . T h e T M C c o n t i n u e to radial ly e x p a n d to a c h i e v e a s i n g l e b i layer d i a p h r a g m b e t w e e n the two a p p o s e d v e s i c l e s , t e r m e d a "hemi fus ion intermediate" . T h e resul t ing t e n s i o n w o u l d l ead to rupture of the h e m i f u s i o n in te rmedia te a n d m e m b r a n e f u s i o n ( C h e r n o m o r d i k a n d Z i m m e r b e r g , 1995) . T h e last m o d e l , k n o w n a s the "modi f ied stalk m o d e l " , a l s o i n v o l v e s format ion of T M C ( S i e g e l , 1993) . T h e m o d e l p r o p o s e s that ins tead of e x p a n d i n g radial ly, m e m b r a n e f u s i o n c o u l d b e 19 Apposed bilayer Interlamellar micelle Stalk-pore model Modified stalk model intermediate F i g u r e 1.6. P r o p o s e d m o d e l s of m e m b r a n e f u s i o n a n d in termedia te s t ruc tures . 20 directly mediated by local diaphragm rupture in the TMC. Evidence for this comes mainly from different intermediates observed in TRC-TEM at various temperature below and above the transition temperature (TH) (Siegel, 1993; Siegel et al., 1994; Siegel and Epand, 1997). 21 1.3 Liposomes D u r i n g the late 1 9 6 0 s , it w a s f o u n d that iso la ted p h o s p h o l i p i d s of living ce l l s s p o n t a n e o u s l y r e f o r m e d into v e s i c l e s w h i c h later c a m e to b e k n o w n a s l i p o s o m e s ( B a n g h a m et a l . , 1965) . L i p o s o m e s h a v e b e e n e m p l o y e d a s m o d e l m e m b r a n e s y s t e m in s t u d i e s of l ipids b e h a v i o r , m e m b r a n e permeabi l i ty , a s wel l a s m e m b r a n e protein f u n c t i o n s a n d l ipid-protein interact ions. T h e c a p a c i t y of l i p o s o m e s to e n t r a p m o l e c u l e s a l s o r e n d e r t h e m usefu l a s d rug de l ivery s y s t e m s for t h e r a p e u t i c p u r p o s e s ( G r e g o r i a d i s , 1993) . 1.3.1 Classification of liposomes In g e n e r a l , l i p o s o m e s c a n b e c l a s s i f i e d into th ree c a t e g o r i e s : mult i lamel lar , l a rge uni lamel lar , a n d s m a l l un i lamel lar v e s i c l e s (F ig . 1.7). 1.3.1.1 Multilamellar vesicles (MLVs) H y d r a t i o n of a lipid film s p o n t a n e o u s l y g i v e s r ise to M L V s , w h i c h a r e h e t e r o g e n e o u s in s i z e with d i a m e t e r s rang ing f rom 0.5 to 10 u.m. M L V s a r e c o m p o s e d of a s e r i e s of c o n c e n t r i c b i layers s e p a r a t e d by nar row a q u e o u s s p a c e s ( B a n g h a m et a l . , 1965) . A s a result , M L V s h a v e v e r y low t rapp ing v o l u m e s (0.5 uJ/u.mol) a n d a r e not usefu l for d r u g l o a d i n g . T r a n s b i l a y e r d istr ibut ions of s o l u t e s c a n b e equ i l ib ra ted by a f r e e z e - t h a w p r o c e s s ( M a y e r et a l . , 1985) . T h e regu la r a r r a y s of b i l ayers in M L V s , o n the other h a n d , m a d e t h e m idea l for p h y s i c a l a n d m o t i o n a l a n a l y s i s o n b i layer o rgan iza t ion of indiv idual l ipids within a m e m b r a n e s t ructure e m p l o y i n g N M R (Cul l is et a l . , 1985) . 22 Multilamellar vesicles (MLVs) Figure 1.7. Classification of liposomes. Schematic representation and freeze fracture electron micrographs of (A) MLVs, (B) LUVs, and (C) SUVs are shown. The bar in the EM picture represents 200 nm. 23 1.3.1.2 Large unilamellar vesicles (LUVs) L U V s a r e un i lamel la r s y s t e m with d i a m e t e r s rang ing f rom 5 0 to 2 0 0 n m . L U V s c a n b e p r e p a r e d f rom M L V s th rough the u s e of d e t e r g e n t s or o r g a n i c s o l v e n t s by a n u m b e r of m e t h o d s w h i c h inc lude r e v e r s e p h a s e e v a p o r a t i o n ( S z o k a a n d P a p a h a d j o p o u l o s , 1978) , de te rgent d ia lys is ( M i m m s et a l . , 1981) , a n d e x t r u s i o n ( M a y e r et a l . , 1986; S z o k a a n d P a p a h a d j o p o u l o s , 1980) . A s L U V s h a v e a s i n g l e b i layer a n d a la rge t r a p p e d v o l u m e (1-2 uJ/u.mole for 100 n m s i z e l i p o s o m e s ) , they a r e m o s t c o m m o n l y e m p l o y e d a s m o d e l m e m b r a n e s y s t e m s a n d a s d r u g car r ie rs . 1.3.1.3 Small unilamellar vesicles (SUVs) S U V s c a n b e p r e p a r e d f rom M L V s by sub jec t ing the M L V s to u l t rason ic irradiat ion ( H u a n g , 1969) or by p a s s a g e th rough a F r e n c h p r e s s ( B a r e n h o l z t et a l . , 1979) . T h e d i a m e t e r s of S U V s a r e typical ly a r o u n d 2 5 to 4 0 n m . T h e i r s m a l l s i z e limits their u s e in m o d e l m e m b r a n e s t u d i e s . In addi t ion , S U V s ' t r a p p e d v o l u m e (0.2-0 .5 uJ/u.mol) is of ten too s m a l l to a l low for s t u d i e s of permeabi l i ty or ion d ist r ibut ions b e t w e e n the internal a n d external a q u e o u s c o m p a r t m e n t s . T h e s m a l l c u r v a t u r e of S U V s a l s o g i v e s r ise to a h igh d e g r e e of instability ( P a r e n t e a n d L e n t z , 1984; S c h u h e t a l . , 1982) . 1.3.2 Preparation of LUVs A n u m b e r of m e t h o d s exist for p repara t ion of L U V s , w h i c h invo lve so lub i l i z ing the l ipids in a n o r g a n i c s o l v e n t a n d s u b s e q u e n t l y hydrat ing t h e m in a q u e o u s buffer. 24 1.3.2.1 Extrusion techniques E x t r u s i o n is the m o s t c o n v e n i e n t m e t h o d for p r o d u c i n g L U V s ( M a y e r et a l . , 1986) . M L V s a r e r e p e a t e d l y e x t r u d e d u n d e r m o d e r a t e p r e s s u r e s ( less t h a n 5 0 0 psi) t h r o u g h p o l y c a r b o n a t e filters of d e f i n e d p o r e s i z e . L U V s in the r a n g e of 5 0 to 2 0 0 n m c a n b e g e n e r a t e d , d e p e n d i n g o n the p o r e s i z e of the filter e m p l o y e d . 1.3.2.2 Detergent dialysis techniques D e t e r g e n t s m a y a l s o b e u s e d to form L U V s ( A l m o g et a l . , 1986; K i w a d a et a l . , 1985 ; L a s c h et a l . , 1983; S z o k a a n d P a p a h a d j o p o u l o s , 1980) . D e t e r g e n t s ex is t a s m o n o m e r s d i s p e r s e d in solut ion at low de te rgen t c o n c e n t r a t i o n s . W h e n the c o n c e n t r a t i o n i n c r e a s e s to a s p e c i f i c level , referred to a s the crit ical m ice l l a r c o n c e n t r a t i o n (cmc) , "mice l les" or c lus te rs of de te rgen t m o l e c u l e s a r e f o r m e d . D e t e r g e n t s p r e s e n t in o n e mice l le c a n constan t ly e x c h a n g e with o ther m o n o m e r s p r e s e n t in the bulk solut ion or in o ther m i c e l l e s ( L i n d m a n a n d W e n n e r s t r o m , 1980) . D u r i n g fo rmat ion of L U V s , the lipid is first d i s s o l v e d in d e t e r g e n t m i c e l l e s a n d the d e t e r g e n t is t h e n r e m o v e d by d ia lys is . D e t e r g e n t s m o s t su i tab le for d ia lys is a r e t h o s e with h igh c m c v a l u e s (>1 m M ) a n d s m a l l mice l le s i z e ( D e n c h e r a n d H e y n , 1978; Fur th , 1980) . 25 1.4 Lipid-mediated DNA transfection Lipid-mediated gene transfer is gaining in popularity in both in vitro and in vivo cell transfection studies (Feigner et al . , 1994; Feigner et al . , 1987; Liu et al . , 1995; Liu et al . , 1997; Templeton et al . , 1997; V a n der W o u d e et al . , 1995). This thesis employs two l ipid-based gene delivery systems. One system involves complexing plasmid D N A to the external bilayer of the l iposomes, commonly referred to as plasmid DNA- l ip id complexes (Feigner et al . , 1987). In the other system, the stabi l ized plasmid-l ipid particles ( S P L P ) , D N A is entrapped inside the l iposomal bilayer (Wheeler et al . , 1999). The following section descr ibes the characterist ics of the two systems, the mechan isms involved in the transfection process, as well as cofactors that have been used in conjunction with the l iposomes for achieving higher transfection efficiencies. 1.4.1 DNA-cationic lipid complexes A s mentioned in the introductory section, the l iposomal mixtures in the complex system usually contain a cationic lipid and a helper lipid. D N A assoc ia tes with the cationic l iposomes through electrostatic interactions, and the resulting plasmid DNA- l ip id complexes are heterogeneous in structures and s izes . Severa l structural models have been proposed: including the "bead on string" complexes (Feigner and Ringold, 1989), the "lamellar" complexes (Gustafsson et a l . , 1995), and the "cylindrical" complexes (Sternberg et al . , 1994) (Fig. 1.8). The "bead on string" model w a s first observed using metal shadowing E M (Gershon et al . , 1993), which depicted several positively charged l iposomes attaching to the D N A as a string. 26 A Bead on string complexes t ionic l i posomes B Lamellar complexes mmmmammmm D N A Cat ion ic lipid b i layers iiSiiliiilyiiiy i i i i i i y i C Cylindrical complexes F i g u r e 1.8. S c h e m a t i c representa t ion of p l a s m i d D N A - c a t i o n i c l i p o s o m e c o m p l e x e s . 27 E v i d e n c e of the "lamellar" c o m p l e x c a m e f rom c r y o - T E M ( t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y ) ( G u s t a f s s o n et a l . , 1995) , X - r a y diffraction a n d opt ica l m i c r o s c o p y ( R a d l e r et a l . , 1997; S p e c t o r a n d S c h n u r , 1997) . In this m o d e l , D N A is c o n d e n s e d by the ca t ion ic l i p o s o m e s a n d is t r a p p e d b e t w e e n f u s e d lipid b i l ayers a r r a n g e d in a typ ica l M L V pattern. S t e r n b e r g ' s g r o u p p r o p o s e d the "cyl indr ical" m o d e l , in w h i c h a s t rand of D N A w a s c o a t e d by a m o n o l a y e r of ca t ion ic l ipids a n d / o r c o - l i p i d s a s e v i d e n c e d u s i n g f r e e z e f racture E M (S te rnberg et a l . , 1994) . S i n c e the structural f e a t u r e s of p l a s m i d D N A - l i p i d c o m p l e x e s a r e c o m p l i c a t e d , there is current ly n o c o n s e n s u s a s to w h i c h structure is ult imately r e s p o n s i b l e for med ia t ing t rans fec t ion . S t u d i e s h a v e b e e n d o n e to invest igate the m e c h a n i s m by w h i c h p l a s m i d D N A -ca t ion ic l i p o s o m e c o m p l e x e s t ransfect ce l l s . E M p ic tures revea l that the t rans fec t ion c o m p l e x e s a r e in terna l ized into ce l ls ma in ly th rough e n d o c y t o s i s a n d that f u s i o n t a k e s p l a c e ins ide the e n d o s o m e s ( F a r h o o d et a l . , 1995; F r i e n d et a l . , 1996; H u i et a l . , 1996; S t e g m a n n a n d L e g e n d r e , 1997; W r o b e l a n d C o l l i n s , 1995; Z a b n e r et a l . , 1995) . T h e m e c h a n i s m invo lved in the c o m p l e x e s c a p i n g f r o m e n d o s o m e s a n d p l a s m i d D N A r e l e a s i n g f rom l i p o s o m e s is still not c lear . It h a s b e e n s u g g e s t e d that D N A c o u l d b e r e l e a s e d th rough e n d o s o m a l destab i l i za t ion m e d i a t e d by e i ther the f u s o g e n i c lipid D O P E ( F a r h o o d et a l . , 1995; Hu i et a l . , 1996; L i tz inger a n d H u a n g , 1992) or v ia a n i o n / c a t i o n lipid e x c h a n g e b e t w e e n the ca t ion ic l ipids in the l i p o s o m e s a n d a n i o n i c l ipids in the e n d o s o m a l m e m b r a n e s ( B h a t t a c h a r y a a n d M a n d a l , 1998; E l O u a h a b i et a l . , 1997; X u a n d S z o k a , 1996) . A n u m b e r of p h y s i c a l charac te r is t i cs limit the app l ica t ion of p l a s m i d D N A - l i p i d c o m p l e x e s for in v ivo g e n e t ransfer . C o m p l e x e s a r e c l e a r e d rapidly by the R E S in 28 v i v o ( H u a n g a n d L i , 1997) a n d the t rans fec ted g e n e is ma in ly d e l i v e r e d to "first p a s s " o r g a n s s u c h a s lung , liver, a n d s p l e e n ( H u a n g a n d Li , 1997) . A s D N A is on ly partially p r o t e c t e d , it is s u s c e p t i b l e to D N a s e a n d s e r u m d e g r a d a t i o n (Li et a l . , 1999) . In add i t ion , c o m p l e x e s a r e toxic to ce l ls both in vitro ( F e i g n e r et a l . , 1994) a n d in v ivo (Li a n d H u a n g , 1997) . A s a result, m u c h effort h a s b e e n d e v o t e d to the d e v e l o p m e n t of s m a l l l i p i d - b a s e d D N A formula t ions for s y s t e m i c in v ivo a p p l i c a t i o n s . 1.4.2 Stabilized Plasmid-Lipid Particles (SPLP) S i n c e D N A is a large m o l e c u l e , s tab le a n d eff icient e n t r a p m e n t of D N A ins ide l i p o s o m e s h a v e b e e n f o u n d to b e quite a c h a l l e n g i n g p r o c e s s ( B a r u et a l . , 1995 ; F r a l e y e t a l . , 1979; F r a l e y e t a l . , 1980; L u r q u i n , 1979; N a k a n i s h i e t a l . , 1985 ; N i c o l a u a n d R o t t e m , 1982) . R e c e n t l y , a n o v e l s y s t e m conta in ing D N A within the lipid b i layer k n o w n a s S P L P h a v e b e e n d e v e l o p e d a n d c h a r a c t e r i z e d ( W h e e l e r et a l . , 1999) . P r e p a r e d by a de te rgen t d ia lys is t e c h n i q u e , S P L P c o n t a i n h igh leve ls of the f u s o g e n i c lipid D O P E (>80%) a n d low leve ls of D O D A C (<10%), a n d a r e s tab i l i zed by the p r e s e n c e of a P E G c o a t i n g . T h i s s y s t e m h a s high D N A e n t r a p m e n t e f f i c iency (>70%), is h o m o g e n e o u s a n d s m a l l in s i z e (~70 nm) , a n d offers full protect ion for the p l a s m i d D N A a g a i n s t n u c l e a s e d e g r a d a t i o n ( W h e e l e r et a l . , 1999) . A l t h o u g h the p h y s i c a l charac te r is t i cs a p p e a r ideal for in v ivo a p p l i c a t i o n , the t rans fec t ion p o t e n c y of S P L P is qui te l imited ( W h e e l e r et a l . , 1999) . F u r t h e r s t u d i e s h a v e f o u n d that S P L P a re def ic ient in two m a i n a r e a s : the P E G coa t ing a n d low level of ca t ion ic lipid limits the level of a s s o c i a t i o n with ce l ls a n d u p t a k e into ce l l s , a n d 29 Figure 1.9. Schemat ic structure of stabil ized plasmid-l ipid particles. 30 it m a y inhibit eff icient r e l e a s e f rom the e n d o s o m e s a n d s u b s e q u e n t de l i ve ry of the D N A into the nuc le i ( M o k et a l . , 1999) . 1.4.3 Cofactors employed in lipid-based mediated transfection Efforts to i m p r o v e the t ransfect ion proper t ies of l i p i d - b a s e d g e n e t ransfer s y s t e m s h a v e led to the incorporat ion of c o f a c t o r s , w h i c h c o u l d b e g e n e r a l l y c l a s s i f i e d into the fol lowing c a t e g o r i e s ( T a b l e 2): 1) l y s o s o m o t r o p i c a g e n t s that interfere with the e n d o s o m a l - l y s o s o m a l pa thway , 2) p o l y c a t i o n s that a s s i s t in c o n d e n s i n g a n d protect ing the p l a s m i d D N A f rom n u c l e a s e d i g e s t i o n , a n d 3) D N A -b i n d i n g pro te ins that a s s i s t in d i rect ing D N A n u c l e a r import . A g e n t s s u c h a s c h l o r o q u i n e a n d a m m o n i u m ch lor ide interfere with the maturat ion of the e n d o s o m e s to l y s o s o m e s by prevent ing the acidi f icat ion p r o c e s s ins ide the e n d o s o m e s ( H a e n s l e r a n d S z o k a , 1993) . H o w e v e r , it h a s b e e n repor ted that t h e s e l y s o s o m o t r o p i c a g e n t s c o u l d b e incons is ten t in their e n h a n c i n g effect, a s t h e y h a v e b e e n s h o w n to actua l ly inhibit t ransfect ion activity with cer ta in t y p e s of ca t ion ic l i p o s o m e s ( F a r h o o d et a l . , 1995) . P o l y l y s i n e s a n d p r o t a m i n e s a r e a m o n g the D N A -c o n d e n s i n g a g e n t s e m p l o y e d a s c o f a c t o r s with l i p o s o m e s . A 3- to 9- fold i n c r e a s e d in t rans fec t ion h a s b e e n repor ted ( G a o a n d H u a n g , 1996; Li a n d H u a n g , 1997) . P o l y c a t i o n s a r e c y t o p l a s m i c prote ins a n d t e n d to r e m a i n in the c y t o s o l (Whi te et a l . , 1989) . A s a result, the c o m p l e x n e e d s to d i s s o c i a t e b e f o r e t ranscr ip t ion of the p l a s m i d D N A c a n take p l a c e . H i s t o n e s s e r v e a s both a D N A - c o n d e n s i n g a g e n t a n d n u c l e a r d i rect ing e l e m e n t ; a ~5- fo ld i n c r e a s e in t ransfect ion h a s b e e n o b s e r v e d by inc lud ing the h is tone H1 in the t ransfect ion c o m p l e x ( B o u l i k a s a n d Mar t in , 1997) . 31 T a b l e 2. T y p e s a n d a c t i o n s of c o m m o n c o f a c t o r s e m p l o y e d in l i p i d - b a s e d m e d i a t e d t rans fec t ion . Cofactors Action of cofactors Transfection enhancement C h l o r o q u i n e A m m o n i u m c h l o r i d e P r e v e n t entry into l y s o s o m e s V a r y with different l i p o s o m a l fo rmu la t ions ( F a r h o o d et a l . , 1995) P o l y l y s i n e P r o t a m i n e s C o n d e n s e D N A ~ 3 to 9-fold ( G a o a n d H u a n g , 1996) H i s t o n e s N u c l e a r loca l iza t ion p e p t i d e s C o n d e n s e D N A , facil itate n u c l e a r en t ry? ~ 5-fold ( B o u l i k a s a n d Mar t in , 1997) 32 1.5 Calcium (Ca 2 +) E u k a r y o t i c ce l ls mainta in v e r y low c o n c e n t r a t i o n s of f ree C a 2 + in their c y t o s o l (~10" 7 M) , whi le the ext race l lu lar C a 2 + c o n c e n t r a t i o n s is v e r y m u c h h i g h e r (~10" 3 M) . S u c h C a 2 + g r a d i e n t s a r e m a i n t a i n e d by C a 2 + - b i n d i n g prote ins within the c y t o s o l , a s wel l a s C a 2 + p u m p s in the p l a s m a m e m b r a n e , m i t o c h o n d r i o n , a n d intracel lular C a 2 + -s e q u e s t e r i n g c o m p a r t m e n t s . A s m a l l influx of C a 2 + i n c r e a s e s s igni f icant ly the c o n c e n t r a t i o n of f ree C a 2 + in the c y t o s o l . It is wel l k n o w n that the influx of C a 2 + d o w n its s t e e p c o n c e n t r a t i o n grad ient in r e s p o n s e to ext race l lu lar s i g n a l s rapidly a c t i v a t e s C a 2 + - s e n s i t i v e r e s p o n s e m e c h a n i s m s in the ce l l s . 1.5.1 Biological activities of C a 2 + C a 2 + a c t s a s a s t rong intracel lular m e s s e n g e r in a w i d e var iety of ce l lu lar r e s p o n s e s . T h e r e a r e two major C a 2 + s igna l ing p a t h w a y s : o n e u s e d ma in ly by electr ical ly ac t ive ce l l s a n d the other u s e d by a l m o s t all e u k a r y o t i c ce l ls ( B a k e r a n d Knight , 1984; B e r r i d g e , 1993; Fewtre l l , 1993; T s i e n a n d T s i e n , 1990) . D u r i n g m e m b r a n e d e p o l a r i z a t i o n in e lec t ro -exc i tab le ce l l s s u c h a s n e u r o n s , a n i n c r e a s e in c y t o s o l i c C a 2 + t r iggers r e l e a s e of the neurot ransmi t ter a c e t y l c h o l i n e . In e u c a r y o t i c ce l l s , the b ind ing of extracel lu lar s igna l ing m o l e c u l e s to c e l l - s u r f a c e r e c e p t o r s a r e c o u p l e d to the r e l e a s e of C a 2 + s e q u e s t e r e d in intracel lular c o m p a r t m e n t s , w h i c h in turn act ivate s u b s e q u e n t s igna l t r ansduc t ion p a t h w a y s . S p e c i f i c C a 2 + - b i n d i n g pro te ins in the ce l ls s u c h a s c a l m o d u l i n rely o n the p r e s e n c e of C a 2 + to f o r m a func t iona l c o m p l e x , w h i c h b i n d s to other target prote ins a n d tr iggers their ce l lu lar func t ion . O t h e r activit ies s u c h a s cel l prol i ferat ion a n d g e n e e x p r e s s i o n a r e a l s o 33 r e g u l a t e d by C a (Ber r idge , 1993; H a r d i n g h a m et a l . , 1997) . In add i t ion , at tent ion h a s b e e n d e v o t e d to e x a m i n i n g the ro les of C a 2 + in regulat ing c y t o p l a s m i c - n u c l e a r t ranspor t a n d its role in g e n e e x p r e s s i o n ( B a d i n g et a l . , 1997 ; H a r d i n g h a m et a l . , 1998; P e t e r s e n et a l . , 1998; S a n t e l l a a n d C a r a f o l i , 1997) . O f par t icular interest is the role of C a 2 + in m e m b r a n e f u s i o n p r o c e s s e s . It h a s b e e n f o u n d that the i n c r e a s e in C a 2 + c o n c e n t r a t i o n c a n b e loca l i zed to the site of e x o c y t o s i s ( E r x l e b e n et a l . , 1997; L l i n a s et a l . , 1992; T s e et a l . , 1997) . Intracellular m e m b r a n e traff icking s u c h a s E R to G o l g i t ranspor t ( B a k e r et a l . , 1990; B e c k e r s a n d B a l c h , 1989) , e n d o s o m e f u s i o n ( C o l o m b o et a l . , 1997) , a n d v a c u o l e f u s i o n (Pe te rs a n d M a y e r , 1998) a l s o requi re the p r e s e n c e of C a 2 + . F inal ly , s e v e r a l C a 2 + b ind ing prote ins , s u c h a s s y n a p t o t a g m i n a n d s y n c o l l i n , h a v e b e e n s h o w n to interact with S N A R E s in a C a 2 + d e p e n d e n t m a n n e r dur ing intracel lular m e m b r a n e f u s i o n ( E d w a r d s o n et a l . , 1997; S u d h o f a n d R i z o , 1996) . It h a s b e e n s u g g e s t e d that C a 2 + f u n c t i o n s a s a m e d i a t o r of protein- l ip id in teract ions at m e m b r a n e f u s i o n in ter faces (Monta l , 1999) . 1.5.2 Influence of C a 2 + on lipid polymorphism T h e m o s t w ide ly s t u d i e d m o d e l s y s t e m for m e m b r a n e f u s i o n is the C a 2 + / P S s y s t e m ( P a p a h a d j o p o u l o s et a l . , 1977) . C a 2 + h a s the c a p a c i t y to i n d u c e the fo rmat ion of a rigid d e h y d r a t e d c o m p l e x with P S , c a u s i n g the v e s i c l e s to a g g r e g a t e , to r e l e a s e their internal a q u e o u s c o n t e n t s , a n d to fo rm large s t ruc tures c a l l e d " c o c h l e a t e " c y l i n d e r s . It h a s a l s o b e e n p r o p o s e d that C a 2 + a c t s e i ther by neutra l iz ing the c h a r g e repu ls ion b e t w e e n negat ive ly c h a r g e d l i p o s o m e s , t h e r e b y p r o m o t i n g d e h y d r a t i o n a n d b i layer c o n t a c t th rough the format ion of interbi layer C a 2 + - P S 34 c o m p l e x e s (Pom's et a l . , 1979) , or by i n d u c i n g format ion of n o n - b i l a y e r f u s i o n i n t e r m e d i a t e s (T i lcock et a l . , 1984) . O n the o ther h a n d , s t u d i e s of lipid p o l y m o r p h i s m in m i x e d lipid s y s t e m s , w h i c h h a v e p roper t i es that m o r e c l o s e l y r e s e m b l e t h o s e of b io log ica l m e m b r a n e s , c o u l d offer g r e a t e r insight to m e m b r a n e fus ion p r o c e s s e s . P h o s p h o l i p i d s that a d o p t the b i layer p h a s e in isolat ion c a n stabi l ize h e x a g o n a l - p r e f e r r i n g l ipids into a n overa l l b i layer o r g a n i z a t i o n in m i x e d lipid s y s t e m s . It h a s b e e n f o u n d that addi t ion of C a 2 + to m i x e d lipid s y s t e m s c o u l d tr igger Hu p h a s e fo rmat ion , by i n d u c i n g e i ther p h a s e s e p a r a t i o n or p h a s e transit ion (T i lcock et a l . , 1984; T i l c o c k a n d Cu l l i s , 1981) . T h e ability of C a 2 + to s e q u e s t e r a c i d i c lipid c o m p o n e n t s into s e p a r a t e b i layer d o m a i n s is re fer red to a s p h a s e s e p a r a t i o n , whi le p h a s e transit ion i n v o l v e s C a 2 + i n d u c i n g the fo rmat ion f r o m bi layer to Hu. In s y s t e m s conta in ing neutral l ipids s u c h a s P E a n d P C a n d a c i d i c l ipids s u c h a s P S a n d P A , it h a s b e e n d e m o n s t r a t e d that C a 2 + c o u l d s e g r e g a t e the a c i d i c c o m p o n e n t into b i layer d o m a i n s that usua l ly exhibi t ge l -s ta te c h a r a c t e r i s t i c s (T i lcock et a l . , 1988) . In P E / P S s y s t e m s , C a 2 + c o u l d s e g r e g a t e the P S c o m p o n e n t into the " c o c h l e a t e " m o r p h o l o g y , l eav ing P E to a d o p t the Hu o r g a n i z a t i o n it f a v o r s in isolat ion (Bal ly et a l . , 1983) . In u n s a t u r a t e d m o d e l s y s t e m s c o n t a i n i n g lipid s p e c i e s s u c h a s D O P E a n d D O P G , C a 2 + is u n a b l e to i n d u c e p h a s e s e p a r a t i o n but ins tead t r iggers a b i layer to h e x a g o n a l H M p h a s e transi t ion in both l ipids (T i l cock et a l . , 1988) . In u n s a t u r a t e d lipid s y s t e m s conta in ing c h o l e s t e r o l , C a 2 + a l s o p r o m o t e s Hu format ion by induc ing lipid p h a s e c h a n g e s in all part ic ipat ing l ipids (Cul l is a n d d e Kruijff, 1978; T i l c o c k et a l . , 1982; T i l c o c k et a l . , 1984) . T a b l e 3 s u m m a r i e s the effect of C a 2 + in m i x e d lipid s y s t e m s in w h i c h a lamel la r to Hu p h a s e 35 T a b l e 3. E f fec t of C a o n m i x e d lipid s y s t e m s . ( P E = p h o s p h a t i d y l e t h a n o l a m i n e , P S = p h o s p h a t i d y l s e r i n e , P G = p h o s p h a t i d y l g l y c e r o l , PI = p h o s p h a t i d y l i n o s i t o l , C L = card io l ip in , D O P E = d i o l e o y l p h o s p h a t i d y l e t h a n o l a m i n , D O P S = d i o l e o y l p h o s p h a t i d y l s e r i n e , C h o i = cho les tero l ) . Types of mixed lipid system C a 2 + / P S (mol ratio) at bilayer -> H„ P S / P E (30:70, mo l /mo l ) 1 P G / P E (30:70, mol /mol ) 1 P I / P E (15:85, mol /mol ) 1 C L / P E (30:70, mol /mol ) 0.5 D O P E / D O P S / C h o l (1:1:1, mo l /mo l /mo l ) 0.5 3 6 t ransi t ion is i n d u c e d . F inal ly , the ability of C a to p r o m o t e b i layer to H M p h a s e t ransi t ion in l ipids s y s t e m s conta in ing P E G lipids h a s a l s o b e e n inves t iga ted (Ho l l and et a l . , 1996) . In mixtures of n o n - b i l a y e r fo rming l ipids s u c h a s P S / P E l i p o s o m e s , the p r e s e n c e of P E G c o u l d actua l ly p reven t C a 2 + - i n d u c e d f u s i o n (Ho l l and et a l . , 1996) . H o w e v e r , the C a 2 + - i n d u c e d f u s o g e n i c activity is res to red u p o n addi t ion of a c c e p t o r l i p o s o m e s ( P O P C ) to w h i c h the P E G - l i p i d c a n e x c h a n g e . S i n c e it h a s b e e n p r o p o s e d that the in te rmed ia tes b e t w e e n b i layer a n d Hu p lay a role in m e m b r a n e f u s i o n , a n d that C a 2 + is often requi red for f u s i o n e v e n t s to p r o c e e d in v ivo , s t u d y i n g the effect of C a 2 + o n lipid p o l y m o r p h i s m is of s igni f icant v a l u e in u n d e r s t a n d i n g m e m b r a n e f u s i o n . 1.5.3 Roles of C a 2 + in transfection C a 2 + h a s b e e n u s e d together with p h o s p h a t e in the C a P 0 4 - m e d i a t e d t rans fec t ion ( G r a h a m a n d V a n d e r E b , 1973) . D e s p i t e its popular i ty , relat ively little w o r k h a s b e e n d o n e to invest igate the m e c h a n i s m by w h i c h C a P 0 4 m e d i a t e s t rans fec t ion . It is b e l i e v e d that p l a s m i d D N A c o p r e c i p i t a t e s with C a P 0 4 a n d the c o m p l e x is in terna l ized into the ce l ls v i a the e n d o c y t i c p a t h w a y (Loyter et a l . , 1982) . H o w the D N A is r e l e a s e d f rom the C a P 0 4 c o m p l e x a n d h o w it e n t e r s the nuc le i for t ranscr ip t ion is not c lear . It is on ly recent ly that C a 2 + h a s b e e n identif ied to b e a n eff icient c o f a c t o r of p o l y c a t i o n - m e d i a t e d g e n e t ransfer ( H a b e r l a n d et a l . , 1999 ; Z a i t s e v et a l . , 1997) . T h e s e p o l y c a t i o n s , w h i c h inc lude h is tone H 1 , h igh mobil i ty g r o u p ( H M G ) D N A - b i n d i n g prote ins H M G 1 a n d H M G 1 7 , a r e n u c l e a r p ro te ins that c o u l d b ind a n d c o n d e n s e D N A a s wel l a s a s s i s t in facilitating n u c l e a r import . 3 7 1.6 Thesis Objectives T h e s t u d i e s of this t h e s i s f o c u s o n e x a m i n i n g the effect of C a 2 + o n l i p i d - b a s e d t rans fec t ion proper t ies . C h a p t e r 2 d e s c r i b e s the in f luence of C a 2 + o n p l a s m i d D N A -ca t ion ic l i p o s o m e c o m p l e x e s . S t u d i e s w e r e car r ied out to con f i rm the s p e c i f i c activity of C a 2 + a n d the effect it h a s o n the c o m p l e x - m e d i a t e d t rans fec t ion . C h a p t e r 3 d i s c u s s e s the in f luence of C a 2 + o n the e n c a p s u l a t e d or S P L P s y s t e m , in w h i c h a d r a m a t i c t rans fec t ion e n h a n c e m e n t w a s o b s e r v e d . E x p e r i m e n t s w e r e p e r f o r m e d to e l u c i d a t e the m e c h a n i s m by w h i c h C a 2 + faci l i tated the S P L P t rans fec t ion p o t e n c y . Ul t imately , the g o a l is to d e v e l o p l i p i d - b a s e d g e n e carr ier s y s t e m s with e n h a n c e d t rans fec t ion p o t e n c y . 38 C H A P T E R 2 CALCIUM E N H A N C E S THE TRANSFECTION POTENCY OF PLASMID DNA-CATIONIC LIPOSOME C O M P L E X E S 2.1 INTRODUCTION P l a s m i d D N A - c a t i o n i c l i p o s o m e "complex" s y s t e m s f o r m e d by i n c u b a t i o n of p l a s m i d with posi t ive ly c h a r g e d l i p o s o m e s h a v e b e e n u s e d w ide ly a s t rans fec t ion a g e n t s in vitro a n d h a v e p r o m i s i n g potent ial for in v ivo a p p l i c a t i o n s (Alton & G e d d e s , 1995 ; L iu et a l . , 1995; N a b e l et a l . , 1993) . S t u d i e s o n the m e c h a n i s m of ca t ion ic l i p o s o m e - m e d i a t e d g e n e t ransfer indicate that c o m p l e x e s en te r c e l l s ma in ly by e n d o c y t o s i s (F r iend et a l . , 1996; L e g e n d r e & S z o k a , J r . , 1992; W r o b e l & C o l l i n s , 1995 ; Z a b n e r et a l . , 1995; Z h o u & H u a n g , 1994) a n d that the p l a s m i d e s c a p e s into the c y t o p l a s m a s a result of e n d o s o m a l destab i l i za t ion faci l i tated by a f u s o g e n i c "helper" lipid s u c h a s d i o l e o y l p h o s p h a t i d y l e t h a n o l a m i n e ( D O P E ) ( F a r h o o d et a l . , 1995 ; L i t z inger & H u a n g , 1992). It h a s b e e n s u g g e s t e d that a n i o n i c l ipids in the e n d o s o m a l m e m b r a n e s a l s o p lay a role in facil itating p l a s m i d D N A r e l e a s e by interact ing with ca t ion ic lipid in the c o m p l e x e s (Watt iaux et a l . , 1997; X u & S z o k a , J r . , 1996) . T h e e f f ic iency of cel l t ransfect ion by p l a s m i d D N A is great ly e n h a n c e d by the p r e s e n c e of the ca t ion ic l i p o s o m e s , h o w e v e r t ransfect ion by c o m p l e x e s r e m a i n s a relat ively inefficient p r o c e s s . Par t icu lar o b s t a c l e s inc lude e s c a p e f r o m the e n d o s o m e fol lowing e n d o c y t o s i s , intracel lular n u c l e a s e d e g r a d a t i o n of p l a s m i d ( L e c h a r d e u r et a l . , 1999) , a s wel l a s entry of the p l a s m i d D N A into the cel l n u c l e u s ( Z a b n e r e t a l . , 1995) . 39 Efforts to i m p r o v e l i p o s o m a l g e n e t ransfer s y s t e m s h a v e f o c u s e d pr imari ly o n d e s i g n i n g n o v e l ca t ion ic l ipids a n d d e v e l o p i n g l i p o s o m e fo rmula t ions c o n t a i n i n g different "helper" l ipids ( F e i g n e r et a l . , 1994; G a o & H u a n g , 1991; L e g e n d r e & S z o k a , J r . , 1992; P a u k k u et a l . , 1997; T e m p l e t o n et a l . , 1997) . O t h e r a p p r o a c h e s i n c l u d e the incorpora t ion of f u s o g e n i c p e p t i d e s , D N A - c o n d e n s i n g a g e n t s or target ing l i g a n d s s u c h a s n u c l e a r local izat ion s i g n a l s to i m p r o v e D N A t ransfer ( D z a u et a l . , 1996; G a o & H u a n g , 1996; H a g s t r o m et a l . , 1996; Ibanez et a l . , 1996; Li & H u a n g , 1997 ; M i z u g u c h i et a l . , 1996) . Re la t ive ly little work h a s f o c u s e d o n the ef fect of C a 2 + o n the t rans fec t ion p o t e n c y of p l a s m i d D N A - c a t i o n i c l i p o s o m e c o m p l e x e s . Trad i t iona l ly , C a 2 + h a s b e e n u s e d to e n h a n c e in vitro t rans fec t ion by the C a 2 + -p h o s p h a t e precipi tat ion m e t h o d ( G r a h a m & V a n d e r E b , 1973) . T h e u p t a k e of D N A by cu l tured ce l l s is b e l i e v e d to b e faci l i tated by the format ion of a C a 2 + p h o s p h a t e -D N A co -p rec ip i t a te , w h i c h en te rs the cel l by e n d o c y t o s i s a n d s u b s e q u e n t l y is t rans fe r red into the n u c l e u s after e s c a p e f rom the e n d o s o m a l c o m p a r t m e n t (Loyter et a l . , 1982; O r r a n t i a & C h a n g , 1990) . O t h e r s t u d i e s h a v e invest iga ted the role of C a 2 + in i n c r e a s i n g the rate of e n d o c y t o s i s a n d m a c r o - m o l e c u l a r n u c l e a r u p t a k e ( E l i a s s o n et a l . , 1996; E p s t e i n et a l . , 1992; P e r e z - T e r z i c et a l . , 1996) . R e c e n t l y , a n o n - l i p o s o m a l g e n e t ransfer s y s t e m e m p l o y i n g D N A - b i n d i n g e l e m e n t s h a s s h o w n e n h a n c e d t rans fec t ion e f f icacy in the p r e s e n c e of C a 2 + (Bot tger et a l . , 1998; H a b e r l a n d et a l . , 1999; Z a i t s e v et a l . , 1997) . It w a s s u g g e s t e d that C a 2 + c o u l d i n c r e a s e the rate of e n d o c y t o s i s (Za i tsev et a l . , 1997) or facil itate e n d o s o m a l r e l e a s e ( H a b e r l a n d e t a l . , 1999) . In this work w e h a v e s tud ied the effect of C a 2 + o n ca t ion ic l i p o s o m e - m e d i a t e d 40 cel l t rans fec t ion u s i n g a b o v i n e h a m s t e r k idney ( B H K ) cel l l ine. E x p e r i m e n t s w e r e c o n d u c t e d to d e m o n s t r a t e the speci f ic i ty of C a 2 + - e n h a n c e d t rans fec t ion a n d to e x a m i n e the m e c h a n i s m w h e r e b y C a 2 + i m p r o v e s t rans fec t ion . T h e resul ts ind icate that the p r e s e n c e of C a 2 + dur ing c o m p l e x - m e d i a t e d t ransfec t ion faci l i tates i m p r o v e d intracel lular de l ivery of ca t ion ic lipid a n d p l a s m i d D N A , l ead ing to e n h a n c e m e n t s in t r a n s g e n e e x p r e s s i o n of 3 to 20 - fo ld in a n u m b e r of different cel l l ines . 41 2.2 MATERIALS AND METHODS 2.2.1 Plasmids T h e pCMV (3 p l a s m i d , w h i c h e n c o d e s the l a c Z g e n e for the p - g a l a c t o s i d a s e ((3-gal) prote in a n d the p C M V G F P p l a s m i d , w h i c h e x p r e s s e s the g r e e n f l u o r e s c e n c e prote in ( G F P ) , w e r e both dr iven by a c y t o m e g a l o v i r u s ( C M V ) p r o m o t e r to a s s e s s e x p r e s s i o n . A l l p l a s m i d s w e r e o b t a i n e d f rom J . T h o m p s o n at Inex P h a r m a c e u t i c a l s C o r p o r a t i o n ( B u r n a b y , B C ) . 2.2.2 Cell lines 2 9 3 ( t rans formed pr imary e m b r y o n a l h u m a n k idney) , B H K ( B o v i n e h a m s t e r k idney) , C V - 1 (Afr ican G r e e n M o n k e y k idney) , H S 5 7 8 T ( h u m a n b r e a s t duc ta l c a r c i n o m a ) , a n d S K - O V 3 ( h u m a n o v a r y a d e n o c a r c i n o m a ) cel l l ines w e r e o b t a i n e d f r o m A m e r i c a n T i s s u e C u l t u r e C o l l e c t i o n ( A T C C C C L - 1 0 , R o c k v i l l e , M D ) . W i th the e x c e p t i o n of H S 5 7 8 T ce l ls w h i c h requi red a n addi t ional s u p p l e m e n t of b o v i n e insul in (10pg /ml ) , all cel l l ines w e r e cul tured in D u l b e c c o mod i f i ed E a g l e m e d i u m ( D M E M ) s u p p l e m e n t e d with 1 0 % fetal b o v i n e s e r u m ( F B S ) , 100 U /ml of penici l l in a n d 1 0 0 p g / m l of s t r e p t o m y c i n ( P e n - S t r e p ) . A l l cel l l ines w e r e m a i n t a i n e d a s a m o n o l a y e r at 3 7 ° C in a humid i f ied a t m o s p h e r e conta in ing 5 . 0 % C O 2 . 2.2.3 Lipids and Chemicals /V , /V -d io ley l - /V , /V -d imethy lammonium ch lo r ide ( D O D A C ) , 1 4 C - l a b e l l e d D O D A C , a n d N, A / -d is teary l -A / ,A / -d imethy lammonium ch lor ide ( D S D A C ) w e r e o b t a i n e d f r o m 42 Dr. S . A n s e l l at Inex P h a r m a c e u t i c a l s C o r p o r a t i o n . A/ - [2 ,3- (d io leyloxy)propyl ] -A / ,A / ,A / - t r imethy lammonium ch lo r ide ( D O T M A ) w a s p u r c h a s e d f r o m G i b c o B R L (Bur l ington, O N ) . 1 , 2 - d i o l e o y l - 3 - p h o s p h a t i d y l c h o l i n e ( D O P C ) a n d 1 ,2 -d io leoy l -3 -p h o s p h a t i d y l e t h a n o l a m i n e ( D O P E ) w e r e o b t a i n e d f rom Nor thern l ipids ( V a n c o u v e r , B C ) . D i m e t h y l - d i o c t a d e c y l a m m o n i u m B r o m i d e ( D D A B ) w a s p u r c h a s e d f r o m A v a n t i P o l a r L i p i d s (A labas te r , A L ) . C a l c i u m ch lor ide ( C a C I 2 ) , m a g n e s i u m c h l o r i d e ( M g C I 2 ) , a n d s o d i u m c h l o r i d e ( N a C l ) w e r e o b t a i n e d f rom F i s h e r Sc ient i f ic (Fa i r l awn , N J ) . E t h y l e n e g l y c o l - b i s ( p - a m i n o e t h y l ether) -A/ ,A/ ,A/ ' ,A/ - tetraacet ic a c i d ( E G T A ) w a s p u r c h a s e d f r o m S i g m a (St. L o u i s , M O ) . 2.2.4 Preparation of LUVs Al l l a rge un i lamel la r v e s i c l e s ( L U V s ) conta in ing D O D A C / D O P E w e r e p r e p a r e d in a 1:1 m o l a r ratio. Briefly, mix tures of l ipids d i s p e n s e d in c h l o r o f o r m w e r e dr ied u n d e r a s t r e a m of n i t rogen g a s a n d the res idua l s o l v e n t w a s r e m o v e d u n d e r h igh v a c u u m for 2 h. T h e result ing lipid f i lms w e r e h y d r a t e d with d H 2 0 a n d L U V s w e r e o b t a i n e d by f r e e z e - t h a w i n g a n d ext ruding ( M a y e r et a l . , 1986) . T h e s i z e s of L U V s w e r e c h e c k e d with a N i c o m p M o d e l 2 7 0 s u b m i c r o n part ic le s i z e r u s i n g q u a s i e l a s t i c light scat ter ing t e c h n i q u e s . P h o s p h o r u s a s s a y s w e r e u s e d to quant i fy lipid c o n c e n t r a t i o n s ( F i s k e & S u b b a r o w , 1925) . 2.2.5 In vitro transfection in the presence of C a 2 + Pr ior to t ransfec t ion with D N A - l i p i d c o m p l e x e s , ce l ls w e r e p la ted at a d e n s i t y of 2 x 1 0 4 ce l l s p e r wel l of a 96-wel l plate overn ight . 1 M C a C I 2 s t o c k so lu t ion w a s 43 p r e p a r e d in d H 2 0 a n d ster i l ized by filtering. L i p o s o m e s w e r e first a d d e d to p l a s m i d D N A a n d m i x e d briefly by pipett ing up a n d d o w n s e v e r a l t i m e s , after w h i c h the mix tures w e r e i m m e d i a t e l y a d d e d to t u b e s con ta in ing a p p r o p r i a t e C a C I 2 c o n c e n t r a t i o n s a n d i n c u b a t e d at r o o m t e m p e r a t u r e for 30 min b e f o r e diluting to the f inal v o l u m e with D M E M cul ture m e d i a conta in ing 1 0 % F B S , w h i c h w e r e t h e n u s e d to t ransfec t ce l l s . C a C I 2 c o n c e n t r a t i o n s a d d e d to the t ransfec t ion c o m p l e x e s w e r e c a l c u l a t e d with r e s p e c t to the final v o l u m e of the t ransfec t ion m e d i u m a p p l i e d to c e l l s . C e l l s w e r e i n c u b a t e d with the t ransfect ion c o m p l e x e s for 4 h u n l e s s o t h e r w i s e n o t e d , after w h i c h cul ture m e d i a w a s r e p l a c e d a n d ce l ls w e r e further i n c u b a t e d at 3 7 ° C , 5 % C 0 2 overn ight until a s s a y i n g for g e n e e x p r e s s i o n . 2.2.6 Determination of transgene expression p-gal e x p r e s s i o n w a s a s s a y e d by us ing the s u b s t r a t e C h l o r o p h e n o l red g a l a c t o p y r a n o s i d e ( C P R G ) ( B o e h r i n g e r M a n n h e i m , G e r m a n y ) at 1 m g / m l to i n d u c e c o l o r d e v e l o p m e n t , w h i c h w a s m e a s u r e d at 5 4 0 n m with a m i c r o p l a t e E L - 3 0 9 a u t o r e a d e r ( B i o - T e k Instruments) a s d e s c r i b e d p r e v i o u s l y ( M o k & C u l l i s , 1997) . A b s o r b a n c e r e a d i n g s w e r e ca l ibra ted a c c o r d i n g to a p-gal s t a n d a r d ( S i g m a ) . T o t a l ce l lu lar protein w a s d e t e r m i n e d by us ing the M i c r o B C A Pro te in A s s a y R e a g e n t Kit ( P i e r c e , Illinois), w h i c h w a s s u b s e q u e n t l y u s e d to n o r m a l i z e the p-gal act iv i t ies of e a c h t rans fec t ion . F o r the f l u o r e s c e n c e a n a l y s i s of G F P e x p r e s s i o n , ce l l s w e r e p la ted at 5 x 1 0 5 ce l ls p e r wel l of a 100 x 2 0 m m t i s s u e cul ture d i s h the night prior to t rans fec t ion . C e l l s e x p r e s s i n g G F P w e r e directly v i s u a l i z e d a n d p h o t o g r a p h e d at the ind ica ted t ime point (24, 4 8 , a n d 72 h) u s i n g a Z e i s s (Axiovert S 1 0 0 ) f l u o r e s c e n c e 44 m i c r o s c o p e with a n X F 1 0 0 filter set ( E x = 4 7 5 n m , E m = 5 3 5 nm) f r o m O m e g a O p t i c a l ( V e r m o n t , U S A ) . 2.2.7 Intracellular uptake studies employing 1 4 C-radiolabeled lipids B H K ce l ls w e r e p la ted at 7.5 x 1 0 5 ce l l s p e r 2 5 c m 2 cu l tured f l a s k s the d a y b e f o r e t rans fec t ion . T r a c e a m o u n t s of 1 4 C - r a d i o l a b e l e d D O D A C w e r e i n c o r p o r a t e d into D O D A C / D O P E l i p o s o m e s . F o r e a c h t ransfec t ion , 2 jag p C M V p w a s c o m p l e x e d to the l i p o s o m e s at a 1.5 c h a r g e ratio (mole of posi t ive c h a r g e s p e r m o l e of n e g a t i v e c h a r g e s ) in the p r e s e n c e or a b s e n c e of 5 m M C a 2 + . T r a n s f e c t i o n m e d i a w a s r e m o v e d f r o m the ce l ls at 1, 2, o r 4 h p o s t t ransfec t ion . F o r the 2 4 h t ime point, t rans fec t ion mix tures w e r e a l l o w e d to i n c u b a t e o n ce l l s for 4 h, after w h i c h c o m p l e t e m e d i a w a s r e p l a c e d for the r e m a i n i n g t ime. C e l l s w e r e d e t a c h e d by t ryps in iza t ion a n d w a s h e d in iso ton ic buffer (250 m M s u c r o s e , 3 m M M g C I 2 , 50 m M H E P E S , p H 7.2). O n e quar te r of the ce l l s w a s a n a l y z e d a s the w h o l e ce l l f ract ion , wh i le the r e m a i n i n g c e l l s w e r e s u b j e c t e d to n u c l e a r f ract ionat ion a c c o r d i n g to a p r e v i o u s l y d e s c r i b e d pro toco l with modi f ica t ions ( O l n e s & Kur l , 1994) . Brief ly, ce l l s w e r e t reated with h y p o t o n i c buffer (10 m M H E P E S p H 8, 10 m M KCI , 3 m M M g C I 2 , 0.1 m M E D T A , 0.1 m M E G T A ) o n ice for 15 min . C e l l s w e r e then l y s e d by the addi t ion of 0 . 6 % N o n i d e t P 4 0 ( N P - 4 0 ) ( B D H L a b o r a t o r y S u p p l i e s , E n g l a n d ) a n d m i x e d by gent le i n v e r s i o n s . N u c l e i w e r e pel le ted by centr i fuging at 5 0 0 x g for 2 m i n , a n d pel le ts w e r e w a s h e d u s i n g nuc le i r e s u s p e n s i o n buffer (20 m M H E P E S p H 8, 2 5 % g l y c e r o l , 1.5 m M K C I , 0.2 m M E D T A ) . B o t h cel l a n d nucle i pe l le ts w e r e l y s e d by the 45 addi t ion of 2 5 0 pl S o v a b l e a n d d i g e s t e d for 2 h at 6 0 ° C . S o l u b l i z e d s a m p l e s w e r e s u b j e c t e d to scinti l lat ion c o u n t i n g in a B e c k m a n L S 3 8 0 1 scinti l lat ion c o u n t e r . 2.2.8 Dot blot and Southern blot analyses E x p e r i m e n t a l p ro toco ls w e r e the s a m e a s the 1 4 C - l a b e l e d lipid u p t a k e e x p e r i m e n t s , e x c e p t that all ce l l s w e r e t ryps in ized after incuba t ion in the t rans fec t ion mix tures for 4 h. T h e cent r i fuged pel lets w e r e w a s h e d with i so ton ic buf fer a n d s u b s e q u e n t l y l y s e d by us ing the lysis buffer (10 m M T r i s , p H 7.5, 0 . 5 % S D S , 1 m M E D T A ) c o n t a i n i n g P r o n a s e E at 1 m g / m l ( S i g m a ) overn ight at 3 7 ° C . G e n o m i c D N A a n d d e l i v e r e d p l a s m i d D N A w e r e iso la ted a c c o r d i n g to a p u b l i s h e d p ro toco l ( S a m b r o o k et a l . , 1989) . Ex t rac ted D N A w a s r e s u s p e n d e d in T E buffer. D N A r e c o v e r y w a s d e t e r m i n e d by m e a s u r i n g the a b s o r b a n c e at 2 6 0 n m . 2 p g of total D N A f r o m e a c h s a m p l e w a s ei ther dot blotted onto a ny lon t ransfer m e m b r a n e ( A m e r s h a m ) with a set of p C M V p s t a n d a r d s (0 to 10 pg) or l o a d e d into a 1% a g a r o s e ge l a n d s i z e f ract ionated at 6 0 V for 2 h for the S o u t h e r n a n a l y s i s . B o t h b lots w e r e hybr id i zed overn ight at 6 8 ° C to a 3 2 P - l a b e l e d p l a s m i d D N A p r o b e , w h i c h w a s p r e p a r e d with B a m H I c u t - p C M V p p l a s m i d u s i n g the T 7 Q u i c k P r i m e ™ Kit ( P h a r m a c i a B io tech ) . B lo ts w e r e w a s h e d 3 t i m e s at 6 8 ° C with 2x S S C c o n t a i n i n g 0 . 1 % S D S , a n d w e r e then e x p o s e d o n a P h o s p h o r i m a g e r s c r e e n w h i c h w a s s u b s e q u e n t l y s c a n n e d ( M o l e c u l a r D y n a m i c s - P h o s p h o r i m a g e r ™ S I ) . 46 2.3 R E S U L T S 2.3.1 The transfection potency of plasmid DNA-lipid complexes is increased in the presence of C a 2 + In o r d e r to e x a m i n e the effect of C a 2 + o n c o m p l e x - m e d i a t e d t rans fec t ion , B H K ce l ls w e r e t r a n s f e c t e d with p l a s m i d D N A - c a t i o n i c lipid c o m p l e x e s p r e p a r e d in the p r e s e n c e of i n c r e a s i n g c o n c e n t r a t i o n s of c a l c i u m (0 to 100 m M ) . P l a s m i d D N A ( p C M V p ) w a s c o m p l e x e d with D O D A C / D O P E l i p o s o m e s at a ca t ion ic lipid to p l a s m i d D N A c h a r g e ratio of 1.5 (mol of posi t ive c h a r g e s / m o l of n e g a t i v e c h a r g e s ) . T h e s e c o m p l e x e s w e r e immed ia te ly m i x e d with the a p p r o p r i a t e c o n c e n t r a t i o n of C a 2 + , i n c u b a t e d for 3 0 min b e f o r e diluting to the f inal v o l u m e with D M E M cul ture m e d i a , a n d then a p p l i e d to ce l ls . A s s h o w n in F i g u r e 2 .1 , n o p-gal activity w a s d e t e c t e d w h e n ce l ls w e r e t rans fec ted with c a l c i u m a n d p l a s m i d D N A a l o n e . H o w e v e r , p-gal act ivi t ies w e r e c lear ly e n h a n c e d in ce l l s that w e r e t r a n s f e c t e d with c o m p l e x e s i n c u b a t e d in the p r e s e n c e of C a 2 + . In part icular, up to 20 - fo ld i n c r e a s e s in t r a n s g e n e e x p r e s s i o n w e r e d e t e c t e d at C a 2 + c o n c e n t r a t i o n s b e t w e e n 5 a n d 2 5 m M . T r a n s f e c t i o n p o t e n c i e s d e c r e a s e d for C a 2 + c o n c e n t r a t i o n s at 5 0 m M or greater , w h e r e m o r e than 2 0 % r e d u c t i o n s in total cel lu lar protein leve ls w e r e o b s e r v e d , indicat ing cytotoxici ty (data not s h o w n ) . C e l l u l a r protein l eve ls at C a 2 + c o n c e n t r a t i o n s b e l o w 5 0 m M w e r e s imi lar to t h o s e of ce l ls t r a n s f e c t e d with l i p i d - D N A c o m p l e x e s p r e p a r e d without c a l c i u m . In o r d e r to a v o i d cel l d a m a g e , C a 2 + c o n c e n t r a t i o n s of 10 m M or l e s s w e r e e m p l o y e d in s u b s e q u e n t e x p e r i m e n t s . 4 7 1 2 0 0 0 0.0 0.1 0 .2 0.4 0 .8 1.6 3.1 6 .3 12 .5 2 5 . 0 5 0 . 0 100 .0 C a l c i u m c o n c e n t r a t i o n s ( m M ) F i g u r e 2.1 E f fec t of i n c r e a s i n g C a c o n c e n t r a t i o n s o n the t rans fec t ion act ivi t ies of p l a s m i d D N A - c a t i o n i c l i p o s o m e c o m p l e x e s . p C M V p (0.25 pg) in the p r e s e n c e (• ) or a b s e n c e (• ) of D O D A C / D O P E l i p o s o m e s w a s a d d e d to C a 2 + a n d i n c u b a t e d for 2 0 min at r o o m t e m p e r a t u r e prior to t ransfect ing B H K c e l l s . D O D A C / D O P E w a s c o m p l e x e d to p C M V p at a c h a r g e ratio of 1.5 (mol /mol ) . C e l l s w e r e e x p o s e d to the c o m p l e x e s for 4 h a n d then the t ransfect ion mix tures w e r e r e p l a c e d with c o m p l e t e D M E M m e d i a for a further 2 0 h incubat ion be fo re a s s a y i n g for p-gal e x p r e s s i o n , a s out l ined in Mate r ia ls a n d M e t h o d s . T h e error b a r s r e p r e s e n t the s t a n d a r d dev ia t ion of th ree e x p e r i m e n t s . 48 2.3.2 The proportion of cells transfected is increased in the presence of C a 2 * I n c r e a s e d t ransfec t ion in the p r e s e n c e of C a 2 + c o u l d b e d u e to ce l l s e x p r e s s i n g a h i g h e r level of the d e l i v e r e d g e n e or a g r e a t e r propor t ion of ce l l s b e i n g t r a n s f e c t e d , or a c o m b i n a t i o n of both . In o r d e r to e x a m i n e the t rans fec t ion e f f i c iency in a p o p u l a t i o n of ce l ls , t ransfect ion w a s m o n i t o r e d e m p l o y i n g f l u o r e s c e n t m i c r o s c o p y . B H K ce l ls w e r e t r a n s f e c t e d with p C M V G F P at a 0 .75 c a t i o n i c l ipid-to-p l a s m i d D N A c h a r g e ratio in the p r e s e n c e or a b s e n c e of 2 .5 m M C a 2 + . C o m p l e t e D M E M m e d i u m w a s r e p l a c e d at the e n d of the 4 h t ransfec t ion p e r i o d a n d ce l ls w e r e i n c u b a t e d in n o r m a l g r o w i n g c o n d i t i o n s for ind ica ted p e r i o d s of t ime. G F P e x p r e s s i o n w a s v i s u a l i z e d at 24 , 4 8 , a n d 72 h post - t rans fec t ion (F ig . 2.2). S i n c e the p l a s m i d D N A - c a t i o n i c l i p o s o m e c o m p l e x e s w e r e p r e p a r e d at a 0 .75 c h a r g e ratio, o n l y a s m a l l popu la t ion of ce l l s w a s e x p e c t e d to s h o w e x p r e s s i o n of the G F P t r a n s g e n e (F ig . 2 .2 , C o l . A ) . W h e n t r a n s f e c t e d with c o m p l e x e s i n c u b a t e d in the p r e s e n c e of C a 2 + , a m u c h h igher proport ion of ce l ls e x p r e s s i n g G F P c o u l d b e d e t e c t e d (F ig 2.2 , C o l . B) . A s the d e l i v e r e d p l a s m i d resu l ted in t rans ient e x p r e s s i o n , the n u m b e r of ce l ls e x p r e s s i n g G F P d e c r e a s e d a s ce l ls c o n t i n u e d to g r o w a n d d iv ide o v e r the 7 2 - h t ime p e r i o d . N e v e r t h e l e s s , a s igni f icant n u m b e r of c e l l s t r a n s f e c t e d with the C a 2 + - e n h a n c e d c o m p l e x e s c o n t i n u e d to e x p r e s s the t r a n s g e n e o v e r the 7 2 -h t ime p e r i o d . T h e s e d a t a d e m o n s t r a t e that the i n c r e a s e in t rans fec t ion e f f i c iency is d u e , at l eas t in part, to a h igher propor t ion of ce l l s b e i n g t r a n s f e c t e d w h e n C a 2 + is p r e s e n t in the t ransfec t ion c o m p l e x mixtures . 49 A B 72 h Figure 2.2. Fluorescence microscopy of BHK cells transfected with p C M V G F P using complexes in the presence of Ca 2 + . p C M V G F P (1 ng) was complexed to liposomes at a charge ratio of 0.75 and incubated on cells for 4 h in the absence (column A) or presence (column B) of 5 mM Ca 2 + . Cells were grown continuously on tissue cultured dishes with supplement of fresh DMEM media and splitting as required. Photographs of cells expressing G F P were taken at 24,48, and 72 h post-transfection using a Zeiss fluorescence microscope with an XF100 filter set. 50 2.3.3 Cation-dependent transfection enhancement is C a 2 * specific In o r d e r to d e t e r m i n e w h e t h e r C a 2 + spec i f i ca l ly e n h a n c e s t rans fec t ion , the ef fect of C a C b , M g C I 2 , a n d N a C l w e r e c o m p a r e d . Increas ing c o n c e n t r a t i o n s (0 to 50 m M ) of C a C I 2 , M g C I 2 , a n d N a C l w e r e i n c u b a t e d with p C M V p - D O P E / D O D A C c o m p l e x e s , w h i c h w e r e s u b s e q u e n t l y u s e d to t ransfect B H K ce l ls . A s s h o w n in F i g u r e 2 . 3 A , p-gal e x p r e s s i o n w a s e n h a n c e d on ly in the p r e s e n c e of C a 2 + . N o s igni f icant i n c r e a s e in t r a n s g e n e e x p r e s s i o n w a s o b s e r v e d w h e n t rans fec t ion w a s p e r f o r m e d in the p r e s e n c e of M g 2 + , a n d on ly a sl ight i n c r e a s e w a s d e t e c t e d at h i g h e r c o n c e n t r a t i o n s of N a + . T o further conf i rm the C a 2 + s p e c i f i c nature of this s t imulatory effect , the ability of E G T A to inhibit C a 2 + - e n h a n c e d t ransfec t ion w a s e x a m i n e d . E G T A w a s e i ther p r e - i n c u b a t e d with C a 2 + for 5 min prior to the addi t ion of ca t ion ic l i p o s o m e s a n d p l a s m i d D N A , or it w a s a d d e d to the t ransfec t ion c o m p l e x e s after the th ree c o m p o n e n t s h a d b e e n i n c u b a t e d for 30 min at r o o m t e m p e r a t u r e . F i g u r e 2 . 3 B s h o w s that the C a 2 + - e n h a n c e d t ransfect ion w a s signi f icant ly r e d u c e d in the p r e s e n c e of E G T A , w h e t h e r it w a s a d d e d be fore or after the c o m p l e x f o r m a t i o n . T a k e n together , t h e s e resul ts ind icate that t ransfect ion w a s e n h a n c e d spec i f i ca l l y by C a 2 + . 2.3.4 C a 2 + increases the rate of transfection by complexes T h e ef fect of C a 2 + o n t ransfect ion a s a funct ion of the t ime c o m p l e x e s w e r e i n c u b a t e d with ce l l s w a s invest iga ted . C o m p l e x e s p r e p a r e d in the p r e s e n c e or a b s e n c e of C a 2 + w e r e i n c u b a t e d with ce l ls for 30 min to 4 h. A t the a p p r o p r i a t e t ime po in ts , t ransfec t ion m e d i u m w a s r e m o v e d a n d r e p l a c e d with c o m p l e t e m e d i u m . 51 2000 0.0 0.8 1.6 3.1 6.3 12.5 2 5 . 0 50 .0 C a l c i u m c o n c e n t r a t i o n (mM) >. 2 & Q. 3 7000 6000 5000 4000 3000 2000 1000 0 B J L I I 1 1 I 1 2 3 4 5 F i g u r e 2 .3 . Spec i f i c i ty of C a 2 + for e n h a n c i n g t ransfec t ion . (A) p C M V p (0.25 ug) w a s c o m p l e x e d with D O D A C / D O P E ( c h a r g e ratio 1.5) in the p r e s e n c e of i n c r e a s i n g c o n c e n t r a t i o n s (0 to 50 m M ) of C a C I 2 ( • ) , M g C I 2 ( • ) , or N a C l (^ ) . (B) E f fec t of E G T A o n C a 2 + - e n h a n c e d t ransfec t ion . A n e q u i m o l a r a m o u n t o f E G T A w a s a d d e d to C a 2 + (5 m M ) e i ther prior to the addi t ion of l i p o s o m e s a n d D N A , o r after the fo rmat ion of the C a 2 + - e n h a n c e d D N A - l i p i d c o m p l e x e s . 1. u n t r a n s f e c t e d ce l ls ; 2. D N A - l i p i d c o m p l e x e s ; 3. C a 2 + - e n h a n c e d D N A - l i p i d c o m p l e x e s ; 4. E G T A i n c u b a t e d with C a 2 + prior to addi t ion of l i p o s o m e s a n d p l a s m i d D N A ; 5. E G T A a d d e d after fo rmat ion of C a 2 + - e n h a n c e d D N A - l i p i d c o m p l e x e s . T h e error b a r s r e p r e s e n t the s t a n d a r d dev ia t ion of th ree e x p e r i m e n t s . 52 C e l l s w e r e further i n c u b a t e d overn ight b e f o r e a s s a y i n g for p-gal e x p r e s s i o n . A t the 1 h t ime point, on ly min ima l t r a n s g e n e e x p r e s s i o n w a s d e t e c t e d in c e l l s t r a n s f e c t e d with the contro l p l a s m i d D N A - c a t i o n i c lipid c o m p l e x e s (F ig . 2.4). H i g h e r g e n e e x p r e s s i o n w a s o b s e r v e d at longer incubat ion p e r i o d s , w h e r e m a x i m u m p-gal activity w a s a c h i e v e d w h e n the t ransfect ing mixtures w e r e i n c u b a t e d o n c e l l s for 3 to 4 h. O n the o ther h a n d , e x p o s u r e of ce l ls for just 3 0 min to t rans fec t ion mix tures p r e p a r e d in the p r e s e n c e of C a 2 + resu l ted in t r a n s g e n e e x p r e s s i o n a s h igh a s the m a x i m u m t ransfec t ion level a c h i e v e d after a 4 h incubat ion of c o m p l e x e s p r e p a r e d in the a b s e n c e of C a 2 + . 2.3.5 Increased intracellular delivery of lipid is observed in the presence of C a 2 + T h e shor te r incubat ion per iod requi red for g e n e e x p r e s s i o n e m p l o y i n g p l a s m i d D N A - c a t i o n i c lipid c o m p l e x e s p r e p a r e d in the p r e s e n c e of C a 2 + s u g g e s t s i n c r e a s e d ra tes of intracel lular de l ivery of c o m p l e x e s . T h e k inet ics of ca t ion ic lipid u p t a k e w e r e there fore e x a m i n e d . D O D A C / D O P E l i p o s o m e s l a b e l e d with a t race a m o u n t of 1 4 C - r a d i o l a b e l e d D O D A C w e r e u s e d to p r e p a r e c o m p l e x e s in the p r e s e n c e or a b s e n c e of C a 2 + . L ipid u p t a k e w a s m o n i t o r e d o v e r a 2 4 - h t ime p e r i o d . In the a b s e n c e of C a 2 + , ce l lu lar lipid u p t a k e i n c r e a s e d up to the 4 h t ime point a n d r e m a i n e d s t a b l e for the rema in ing t ime (F ig . 2.5). E n h a n c e d lipid u p t a k e w a s o b s e r v e d for ce l ls t r a n s f e c t e d with c o m p l e x e s p r e p a r e d in the p r e s e n c e of C a 2 + . A t the 1 h t ime point, ce l lu lar lipid u p t a k e s imi lar to the m a x i m a l level of c o m p l e x e s p r e p a r e d without C a 2 + w a s a c h i e v e d . T h i s w a s e q u i v a l e n t to a m o r e t h a n 2- fo ld 53 1 2 0 0 0 1 0 0 0 0 - \ 0 1 2 3 T i m e (h) F i g u r e 2.4. E f fec t of incubat ion t ime o n t ransfect ion e m p l o y i n g contro l D N A - l i p i d a n d C a 2 + - e n h a n c e d D N A - l i p i d c o m p l e x e s . T r a n s f e c t i o n mix tures p r e p a r e d in the p r e s e n c e ( • ) o r a b s e n c e (• ) of C a 2 + w e r e i n c u b a t e d with B H K ce l ls for 0 .5, 1, 2, 3, a n d 4 h. A t the appropr ia te t ime points , c o m p l e t e m e d i a w a s r e p l a c e d a n d ce l ls w e r e a l l o w e d to r e c o v e r overn ight a n d p-gal activity w a s a s s a y e d . L i p o s o m e s w e r e c o m p l e x e d to 0 .25 p g p C M V p at a 1.5 c h a r g e ratio. T h e er ror b a r s r e p r e s e n t the s t a n d a r d dev ia t ion of three e x p e r i m e n t s . 54 CO 0 •E If ~ o ° CO , O (1) 03 Q. •g C L TO 15 O < Q O Q O E c 2.5 2.0 1.5 1.0 -A 0.5 0.0 1.00 0.75 0.50 0.25 0.00 CO CD c ^ iz Q3 * i "O CD i O CD 31 CO » C L •g Q. CD _<D O C O < o Q j) o E c F i g u r e 2 .5 . K ine t ic a n a l y s i s of the intracel lular de l ivery of l ipids in the p r e s e n c e of C a 2 + . U p t a k e of 1 4 C - r a d i o l a b e l e d D O D A C w a s e x a m i n e d o v e r a 2 4 - h t ime p e r i o d for both w h o l e cel l (solid l ines) or n u c l e a r (dotted l ines) f rac t ions , p r e p a r e d a s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . C e l l s t rans fec ted with D N A - l i p i d c o m p l e x e s p r e p a r e d in the p r e s e n c e ( • ) o r a b s e n c e (• ) of 5 m M C a 2 + w e r e s o l u b i l i z e d at the ind ica ted t ime po in ts (1, 2, 4, a n d 24 h) a n d a s s a y e d by scinti l lat ion c o u n t i n g to d e t e r m i n e lipid u p t a k e . T h e er ror b a r s r e p r e s e n t the s t a n d a r d dev ia t ion of th ree e x p e r i m e n t s . 55 i n c r e a s e in intracel lular lipid up take w h e n c o m p a r e d to the contro l c o m p l e x e s at the s a m e t ime point. T h i s g rea te r than 2-fold i n c r e a s e w a s m a i n t a i n e d a s the t rans fec t ion p r o c e s s c o n t i n u e d th rough the 4 -h incubat ion p e r i o d . It is interest ing to note that lipid u p t a k e c o n t i n u e d to i n c r e a s e o v e r the 2 4 - h t ime p e r i o d (after r e m o v a l of the c o m p l e x e s at 4 h), s u g g e s t i n g c o n t i n u e d u p t a k e of c o m p l e x e s a d s o r b e d to the cel l s u r f a c e . E x a m i n a t i o n of the n u c l e a r f ract ion s h o w e d that the level of n u c l e a r a s s o c i a t e d lipid r e m a i n e d low e v e n in the p r e s e n c e of C a 2 + (F ig . 2.5). 2.3.6 Increased intracellular delivery of intact plasmid DNA is observed in the presence of C a 2 + Intracellular de l ivery of p l a s m i d D N A w a s d e t e r m i n e d u s i n g the dot blot a s s a y , a n d the integrity of the p l a s m i d w a s e x a m i n e d e m p l o y i n g the S o u t h e r n blot a n a l y s i s . C e l l s w e r e e x p o s e d to c o m p l e x e s p r e p a r e d in the p r e s e n c e or a b s e n c e of c a l c i u m . T h e leve ls of p l a s m i d D N A up take for the different s y s t e m s w e r e c o m p a r e d after isolat ion of D N A f rom ce l ls that h a d b e e n t rans fec ted for 4 h, a s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e resul ts of the dot blot a n d S o u t h e r n blot a n a l y s e s a r e s h o w n in F i g u r e 2.6 . O n a v e r a g e e a c h cel l took up ~ 8 , 0 0 0 c o p i e s of p l a s m i d D N A w h e n ce l ls w e r e t r a n s f e c t e d with c o m p l e x e s p r e p a r e d in the a b s e n c e of C a 2 + . T h i s is c o n s i s t e n t with p r e v i o u s work indicat ing that readi ly t rans fec tab le ce l l s t a k e up ~ 1 0 5 p l a s m i d s w h e n t rans fec ted by c o m p l e x e s ( T s e n g et a l . , 1997; Z a b n e r et a l . , 1995) . A s s h o w n in F i g u r e 2 . 6 A , w h e n ce l ls w e r e t r a n s f e c t e d with the c o m p l e x e s p r e p a r e d in the p r e s e n c e of C a 2 + the n u m b e r of p l a s m i d s taken up by e a c h cel l i n c r e a s e d by m o r e t h a n 2 .5 - fo ld . A S o u t h e r n a n a l y s i s of the d e l i v e r e d D N A s h o w e d s o m e 56 I? V > 3 u . 0 co C o o !*• eg rt 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 B 4 5 6 7 8 9 10 11 F i g u r e 2 .6 . A n a l y s i s of p l a s m i d D N A up take us ing dot blot a n d intracel lular integrity u s i n g S o u t h e r n blot. (A) Intracellular de l ivery of p l a s m i d D N A d e t e r m i n e d by the dot blot a s s a y . P l a s m i d D N A d e l i v e r e d by: 1. D O D A C / D O P E with C a 2 + ; 2. D O D A C / D O P E a n d 3. u n t r a n s f e c t e d ce l ls . (B) S o u t h e r n blot a n a l y s i s of intracel lular p l a s m i d D N A . L a n e s 1: p C M V B s t a n d a r d ; 2: T E buffer; 3-5: u n t r a n s f e c t e d ce l ls ; L a n e s 6-8: p l a s m i d d e l i v e r e d by D O D A C / D O P E ; a n d 9-11: p l a s m i d d e l i v e r e d by D O D A C / D O P E p r e p a r e d with C a 2 + . 2 p g p C M V B c o m p l e x e d to l i p o s o m e s at a 1.5 c h a r g e ratio. C e l l s w e r e t r a n s f e c t e d for 4 h, in the p r e s e n c e or a b s e n c e of 5 m M C a 2 + . G e n o m i c a n d p l a s m i d D N A (total D N A ) w e r e purif ied f rom the cel l l y s a t e s a s d e s c r i b e d in Mater ia ls a n d M e t h o d s . 2 (ig of total D N A w e r e dot ted onto a n i t roce l lu lose m e m b r a n e or l o a d e d into a 1% a g a r o s e g e l , w h i c h w a s run at 6 0 V for 2 h. B lo ts w e r e hybr id i zed to a 3 2 P - l a b e l e d p r o b e overn ight for de tec t ion of p l a s m i d u s i n g a p h o s p h o r i m a g e r . T h e n u m b e r of p l a s m i d c o p i e s w a s quant i ta ted a c c o r d i n g to a p l a s m i d s t a n d a r d a n d the resul ts n o r m a l i z e d by d iv id ing by the n u m b e r of c e l l s . T h e er ror b a r s r e p r e s e n t the s t a n d a r d dev ia t ion of three e x p e r i m e n t s . 57 d e g r a d a t i o n of the d e l i v e r e d p l a s m i d for both the control a n d c a l c i u m - e n h a n c e d l ipid-D N A c o m p l e x e s , a s ind ica ted in F i g u r e 2 . 6 B by the s m e a r i n g of the D N A b a n d s a n d t h e a p p e a r a n c e o f add i t iona l D N A c o n f o r m a t i o n s a s c o m p a r e d to t h e cont ro l p l a s m i d . H o w e v e r , the resul ts d e m o n s t r a t e that s igni f icant ly m o r e intact p l a s m i d D N A is p r e s e n t in ce l ls t r a n s f e c t e d with c o m p l e x e s p r e p a r e d in the p r e s e n c e of 2.3.7 The level of transfection enhancement is dependent on the time of Ca addition E x p e r i m e n t s w e r e p e r f o r m e d to d e t e r m i n e if the point at w h i c h C a 2 + is a d d e d dur ing p repara t ion of the c o m p l e x e s af fects the level of t ransfec t ion e n h a n c e m e n t . T h r e e s i tua t ions w e r e invest iga ted u s i n g equ iva len t c o n c e n t r a t i o n s of C a 2 + (5 m M ) for e a c h . In the first s i tuat ion, C a 2 + w a s i n c u b a t e d with e i ther the D O D A C / D O P E l i p o s o m e s or the p C M V p for 15 min prior to a d d i n g the o ther c o m p o n e n t , after w h i c h the mix tures w e r e a l l o w e d to i n c u b a t e for a n o t h e r 15 min b e f o r e b e i n g u s i n g for t rans fec t ion . In the s e c o n d c a s e , l i p o s o m e s a n d D N A w e r e i n c u b a t e d for 3 0 min a n d C a 2 + w a s a d d e d just prior to a p p l y i n g the t ransfect ion mix tures to c e l l s . In the last c a s e , l i p o s o m e s a n d p l a s m i d D N A w e r e m i x e d toge ther a n d i m m e d i a t e l y a d d e d to C a 2 + a s d e s c r i b e d in Mater ia ls a n d M e t h o d s , a n d the th ree c o m p o n e n t s w e r e i n c u b a t e d for 3 0 min b e f o r e app ly ing to ce l ls . A s s h o w n in F i g u r e 2.7 , the g rea tes t e n h a n c e m e n t o c c u r r e d w h e n C a 2 + , p l a s m i d D N A , a n d l i p o s o m e s w e r e m i x e d a n d i n c u b a t e d together . Incubat ion of p l a s m i d D N A with C a 2 + b e f o r e the addi t ion of l i p o s o m e s resu l ted in s imi lar 5 8 12000 loooo A c •> °-o g> co c "ro "ro c o c o i i co. ca. E 8000 6000 4000 2000 0 F i g u r e s 2 .7 . Ef fect of the o r d e r o f C a ^ + addi t ion o n t ransfec t ion p o t e n c y . C e l l s w e r e t r a n s f e c t e d with D N A - c a t i o n i c lipid c o m p l e x e s (0.25 u.g D N A ) that w e r e m i x e d with 5 m M C a 2 + at different s t a g e s . 1. l i p o s o m e s a n d p l a s m i d D N A mix tures a d d e d to C a 2 + s i m u l t a n e o u s l y ; 2. C a 2 + m i x e d with ca t ion ic l i p o s o m e s first; 3. C a 2 + m i x e d with p l a s m i d D N A first; 4. C a 2 + a d d e d just b e f o r e t ransfec t ion a n d after fo rmat ion of D N A -lipid c o m p l e x e s . T h e error b a r s r e p r e s e n t the s t a n d a r d dev ia t ion of th ree e x p e r i m e n t s . 59 t rans fec t ion leve ls . H o w e v e r , a m o r e than 5 0 % reduct ion in t ransfec t ion p o t e n c y w a s o b s e r v e d w h e n C a 2 + w a s i n c u b a t e d first with the ca t ion ic l i p o s o m e s a n d t h e n with p l a s m i d D N A . M o s t signif icant ly , t ransfect ion leve ls w e r e c o n s i d e r a b l y r e d u c e d (~5-fold) w h e n C a 2 + w a s a d d e d to p r e - f o r m e d p l a s m i d D N A - c a t i o n i c lipid c o m p l e x e s i m m e d i a t e l y prior to the app l ica t ion to ce l l s . T h e s e d a t a s u g g e s t that the s t imula tory e f fec ts of C a 2 + d e p e n d to s o m e extent o n the ability of C a 2 + to interact d i rect ly with the p l a s m i d D N A in the c o m p l e x . 2.3.8 Ca 2 + -enhanced transfection for complexes is observed in a variety of cell lines In o r d e r to il lustrate the genera l i ty of the t ransfect ion e n h a n c i n g ef fect of C a 2 + , the in f luence of C a 2 + o n c o m p l e x - m e d i a t e d t ransfec t ion of a n u m b e r of di f ferent cel l l ines w a s e x a m i n e d . P r e v i o u s work h a s s h o w n that the type of cel l e m p l o y e d c a n c a u s e large var ia t ions in ca t ion ic l i p o s o m e - m e d i a t e d t rans fec t ion e f f i c i e n c i e s ( H a r r i s o n et a l . , 1995) . T h i s var iat ion w a s a l s o o b s e r v e d for the cel l l ines tes ted h e r e . T r a n s f e c t i o n e m p l o y i n g p C M V B - D O D A C / D O P E c o m p l e x e s p r e p a r e d in the p r e s e n c e or a b s e n c e of C a 2 + w a s p e r f o r m e d o n B H K , C V - 1 , 2 9 3 , S K O V - 3 , a n d H S 5 7 8 T c e l l s . In the a b s e n c e of C a 2 + , B H K ce l ls s h o w e d the h i g h e s t t rans fec t ion e f f i c i e n c i e s a m o n g the cel l l ines e x a m i n e d (F ig . 2.8). I n c r e a s e d t rans fec t ion w a s o b s e r v e d in the p r e s e n c e of C a 2 + for all cel l l ines, a l t h o u g h dif ferent ce l l l ines d e m o n s t r a t e d different d e g r e e s of e n h a n c e m e n t (from 2 to 80-fold) . 60 c cu •+-» o : f I 5 o £ CD CD — CO c/> 2 0 0 0 0 1 6 0 0 0 « 1 2 0 0 0 8 0 0 0 CO I CO. c ZJ E Cell types F i g u r e 2.8 . E f fec t of C a 2 + o n t ransfect ion for different ce l l l ines . C e l l s w e r e t r a n s f e c t e d with 0 .25 fxg p C M V p c o m p l e x e d to D O D A C / D O P E in the p r e s e n c e ( d a s h e d bars ) or a b s e n c e ( o p e n bars) of C a 2 + ( 5 m M ) . C e l l s w e r e e x p o s e d to the c o m p l e x e s for 4 h a n d then the t ransfect ion mix tures w e r e r e p l a c e d with the a p p r o p r i a t e cul ture m e d i a for a further 2 0 h b e f o r e a s s a y i n g for p-gal e x p r e s s i o n . T h e er ror b a r s r e p r e s e n t the s t a n d a r d dev ia t ion of three e x p e r i m e n t s . 61 2.3.9 C a 2 * enhances the transfection potency of complexes containing a variety of cationic lipids T h e st imulatory effect of C a 2 + o n the t ransfect ion proper t ies of p l a s m i d D N A -ca t ion ic lipid c o m p l e x e s conta in ing a var iety of ca t ion ic l ipids w e r e e x a m i n e d . Pr ior to app l ica t ion to ce l ls , p l a s m i d D N A w a s c o m p l e x e d to l i p o s o m e s c o n t a i n i n g e i ther 50 m o l % of D O D A C , D O T M A , D S D A C or D D A B a n d 50 m o l % D O P E . E n h a n c e d p-g a l e x p r e s s i o n w a s o b s e r v e d for all the cat ion ic l i p o s o m e fo rmula t ions e x a m i n e d in the p r e s e n c e of C a 2 + , with D N A - l i p i d c o m p l e x e s con ta in ing D O T M A s h o w i n g the h i g h e s t t rans fec t ion e f f ic iency, fo l lowed by D O D A C , D S D A C , a n d D D A B (F ig . 2 . 9A ) . It w a s of interest to inc lude D O D A C / D O P C l i p o s o m e s in o r d e r to s e e if C a 2 + c o u l d e n h a n c e t ransfec t ion for a " n o n f u s o g e n i c " formula t ion . It h a s b e e n d e m o n s t r a t e d that on ly the f u s o g e n i c lipid D O P E is a b l e to s h o w h e l p e r activity in facil i tating ca t ion ic l i p o s o m e - m e d i a t e d g e n e t ransfer ( F a r h o o d et a l . , 1995; F e i g n e r et a l . , 1987; M o k & C u l l i s , 1997) . R e s u l t s s h o w e d that ce l ls t rans fec ted with the D O D A C / D O P C formula t ion exh ib i ted v e r y low leve ls of p-gal activity. T r a n s f e c t i o n e f f i c ienc ies i n c r e a s e d m o r e t h a n 5-fold w h e n C a 2 + w a s i n c l u d e d , a l t h o u g h the e n h a n c e d t rans fec t ion w a s still s igni f icant ly lower than for the D O P E - c o n t a i n i n g fo rmula t ion in the a b s e n c e of C a 2 + . T h e ability o f C a 2 + to e n h a n c e t ransfect ion for the D O D A C / D O P C s y s t e m w a s further inves t iga ted for i n c r e a s i n g c o n c e n t r a t i o n s of C a 2 + in c o m p l e x e s p r e p a r e d at a 1.5 c h a r g e ratio. A s s h o w n in F i g u r e 2 . 9 B , s igni f icant e n h a n c e m e n t s in t rans fec t ion w e r e o b s e r v e d in the p r e s e n c e of 6 to 2 5 m M C a 2 + , w h e r e i n c r e a s e s in p-gal act ivi t ies f r o m 0 to 6 0 0 milliunits p e r m g of total ce l lu lar protein w e r e o b s e r v e d . A s 62 c CD 7000 6000 >.| 5 0 0 0 | S 4000 co E To ro 3000 CO CO 2000 — 1000 0 CD ^2 > Q. ro E "ro "co co co C Q . C O . ZJ E 1000 1 2 3 4 5 6 cat ion ic l i p o s o m a l fo rmula t ions 0.0 0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5 25.0 50.0100.0 c a l c i u m c o n c e n t r a t i o n (mM) F i g u r e 2.9. E f fec t of C a o n t ransfect ion proper t ies of dif ferent l i p o s o m a l f o r m u l a t i o n s . (A) T r a n s f e c t i o n proper t ies of c o m p l e x e s c o n t a i n i n g D O D A C , D O T M A , D S D A C , or D D A B p r e p a r e d at a 1:1 m o l a r ratio with D O P E , a s wel l a s a " n o n f u s o g e n i c " formulat ion ( D O D A C / D O P C ) , in the p r e s e n c e ( d a s h bars ) o r a b s e n c e (sol id bars ) o f 10 m M C a 2 + . D N A w a s m i x e d with 1. C a 2 + only; 2. D O D A C / D O P C ; 3. D O D A C / D O P E ; 4. D O T M A / D O P E ; 5. D S D A C / D O P E ; o r 6. D D A B / D O P E . (B) Ef fect of i n c r e a s i n g C a 2 + c o n c e n t r a t i o n o n t rans fec t ion p roper t i es of " n o n f u s o g e n i c " D N A - l i p i d c o m p l e x e s . D N A w a s c o m p l e x e d to D O D A C / D O P C a n d a d d e d to 0 to 100 m M C a 2 + . A l l l i p o s o m e fo rmula t ions w e r e c o m p l e x e d to 0.25 jag p C M V p at 1.5 c h a r g e ratio. T h e error b a r s r e p r e s e n t the s t a n d a r d dev ia t ion of th ree e x p e r i m e n t s . 63 w a s o b s e r v e d for the D O D A C / D O P E s y s t e m , D O D A C / D O P C t ransfec t ion d i m i n i s h e d w h e n 5 0 m M or h igher C a 2 + c o n c e n t r a t i o n s w e r e u s e d . 64 2.4 DISCUSSION T h e s t u d i e s repor ted h e r e s h o w that the p r e s e n c e of C a 2 + dur ing fo rmat ion of D N A - c a t i o n i c lipid c o m p l e x e s c a n result in s igni f icant i m p r o v e m e n t s in t rans fec t ion p roper t i es in vitro. T h r e e a r e a s warrant further d i s c u s s i o n . First , it is of interest to c o m p a r e the i m p r o v e m e n t in t ransfect ion e f f ic iency a c h i e v e d in the p r e s e n c e of C a 2 + with that a c h i e v e d by other p ro toco ls . A s e c o n d a r e a c o n c e r n s the in teract ions of C a 2 + with the c o m p o n e n t s of the c o m p l e x e s . F inal ly , w e d i s c u s s p o s s i b l e m e c h a n i s m s w h e r e b y C a 2 + resul ts in e n h a n c e d t ransfec t ion p o t e n c y of p l a s m i d D N A - c a t i o n i c lipid c o m p l e x e s . C o m p a r e d to viral v e c t o r s , p l a s m i d D N A - c a t i o n i c l i p o s o m e c o m p l e x e s a r e relat ively ineff icient g e n e t ransfer a g e n t s ( Z a b n e r et a l . , 1995) . T r a n s f e c t i o n e f f i c iency m a y b e limited by the ability of c o m p l e x e s to en te r the ce l l , r e l e a s e of p l a s m i d f r o m the e n d o s o m a l c o m p a r t m e n t , the stability of p l a s m i d in the c y t o p l a s m , a n d entry of p l a s m i d into the n u c l e u s . A var iety of a g e n t s h a v e b e e n p r o p o s e d to e n h a n c e the t ransfec t ion p o t e n c y of c o m p l e x e s by r e m o v i n g o n e or m o r e of t h e s e l imitat ions. L y s o s o m o t r o p i c a g e n t s s u c h a s c h l o r o q u i n e h a v e b e e n s u g g e s t e d to p r o m o t e e s c a p e of e n d o c y t o s e d mater ia ls f rom the e n d o s o m e ( F a r h o o d et a l . , 1995; F e i g n e r et a l . , 1994) . H o w e v e r , a l t h o u g h s o m e i n c r e a s e in t rans fec t ion h a s b e e n d e t e c t e d (~ 4- fold) , the effect of c h l o r o q u i n e h a s b e e n f o u n d to va ry for different t y p e s of c o m p l e x e s to the extent that it c o u l d inhibit t rans fec t ion ( F e i g n e r et a l . , 1994) . D N A - b i n d i n g m o l e c u l e s s u c h a s po ly lys ine h a v e b e e n p r o p o s e d to inhibit b r e a k d o w n of intracel lular D N A a n d thus i n c r e a s e t ransfec t ion , resul t ing in a 3 to 9-fold i n c r e a s e in t ransfec t ion e f f ic iency ( B o u l i k a s & Mar t in , 1997; C h i o u et a l . , 1994; 65 G a o & H u a n g , 1996) . T h e resul ts p r e s e n t e d h e r e d e m o n s t r a t e that the p r e s e n c e of C a 2 + c a n e n h a n c e the t ransfect ion p o t e n c y of c o m p l e x e s by fac tors r a n g i n g f r o m 3 to 20 - fo ld d e p e n d i n g o n C a 2 + c o n c e n t r a t i o n s e m p l o y e d . O p t i m a l C a 2 + c o n c e n t r a t i o n s to s t imulate D N A - c a t i o n i c lipid c o m p l e x e s t rans fec t ion r a n g e d b e t w e e n 5 a n d 2 5 m M , w h i c h w a s a h igher ext race l lu lar C a 2 + c o n c e n t r a t i o n w h e n c o m p a r e d to the level f o u n d in D M E M cul ture m e d i a , w h i c h norma l ly c o n t a i n e d ~1 .8 m M C a 2 + . T h e genera l i ty of this st imulatory effect w a s c o n f i r m e d by t rans fec t ing a n u m b e r of different cel l l ines a s wel l a s e m p l o y i n g v a r i o u s ca t ion ic l i p o s o m a l s y s t e m s , w h i c h further e s t a b l i s h e d C a 2 + a s a potent st imulatory a g e n t for e n h a n c i n g ca t ion ic l i p o s o m e - m e d i a t e d cel l t ransfec t ion . In this regard a recen t report h a s s h o w n that C a 2 + i ons c a n e n h a n c e n u c l e a r p r o t e i n - m e d i a t e d t rans fec t ion but not ca t ion ic l i p o s o m e - m e d i a t e d t ransfec t ion ( H a b e r l a n d et a l . , 1999) . T h i s d i s c r e p a n c y likely a r i s e s f r o m prepara t ion of the c o m p l e x e s in the a b s e n c e of C a 2 + , with s u b s e q u e n t addi t ion of C a 2 + immed ia te ly prior to app l ica t ion to c e l l s . T h e resul ts p r e s e n t e d h e r e s h o w that the t ransfect ion p o t e n c y is s igni f icant ly r e d u c e d u n d e r s u c h s i tuat ions . It is likely, but not yet p r o v e n , that the ability of C a 2 + to e n h a n c e the t rans fec t ion proper t ies of c o m p l e x e s i n v o l v e s a direct a s s o c i a t i o n with the p l a s m i d D N A in the c o m p l e x . T w o l ines of e v i d e n c e s u p p o r t this c o n c l u s i o n . First , the o b s e r v a t i o n that eff icient t ransfect ion in the p r e s e n c e of c a l c i u m is o b s e r v e d at lower ca t ion ic l ip id - to -p lasmid D N A c h a r g e ratios than in the a b s e n c e of c a l c i u m s u g g e s t s that c a l c i u m m a y subst i tute to s o m e extent for ca t ion ic lipid in the c o m p l e x . S e c o n d , e n h a n c e d t rans fec t ion p o t e n c i e s w e r e o b s e r v e d if c o m p l e x e s w e r e i n c u b a t e d with 66 C a for 30 min immed ia te ly after c o m p l e x fo rmat ion , but t ransfec t ion leve ls w e r e m u c h r e d u c e d if C a 2 + w a s a d d e d immed ia te ly b e f o r e app l ica t ion to c e l l s . It h a s b e e n s h o w n that p l a s m i d in p l a s m i d D N A - c a t i o n i c lipid c o m p l e x e s is at least partially p ro tec ted f r o m the externa l a q u e o u s e n v i r o n m e n t ( W h e e l e r et a l . , 1999) , w h i c h w o u l d p r e c l u d e rapid interact ion with external C a 2 + . T h e m e c h a n i s m w h e r e b y C a 2 + e n h a n c e s the t ransfect ion p o t e n c y of c o m p l e x e s a p p e a r s to b e directly re lated to a n ability to i n c r e a s e up take of c o m p l e x e s into c e l l s , resul t ing in i n c r e a s e d leve ls of intact intracel lular p l a s m i d D N A . T h e m e c h a n i s m w h e r e b y C a 2 + c o u l d s t imulate up take is not yet c lear , but likely a r i s e s f r o m i n c r e a s e d intracel lular l eve ls of C a 2 + . It h a s b e e n s h o w n that i n c r e a s e d intracel lular C a 2 + l eve ls c o u l d l e a d to i n c r e a s e s in the rates of e n d o c y t o s i s ( E l i a s s o n et a l . , 1996; E p s t e i n et a l . , 1992) a n d thus c o u l d p r o m o t e the u p t a k e of c o m p l e x e s . T h e d e p e n d e n c e of t rans fec t ion e n h a n c e m e n t o n the t ime a n d o r d e r of C a 2 + addi t ion s u g g e s t s that c o m p l e x - a s s o c i a t e d C a 2 + , a s o p p o s e d to f ree C a 2 + , p l a y s a pr imary role in s t imulat ing u p t a k e . It is p o s s i b l e that the c o m p l e x - a s s o c i a t e d C a 2 + c a n g a i n a c c e s s to the intracel lular c o m p a r t m e n t m o r e readi ly , o r that the c o m p l e x - a s s o c i a t e d C a 2 + s i m p l y r e p r e s e n t s the largest poo l of C a 2 + in the e n d o s o m e fo l lowing u p t a k e of the c o m p l e x . In s u m m a r y , w e h a v e s h o w n that C a 2 + c a n act a s a potent s t imulatory a g e n t to e n h a n c e t ransfec t ion proper t ies of ca t ion ic D N A - l i p i d c o m p l e x e s . T h e p ro toco l is s i m p l e a n d a p p e a r s to b e of utility in a var iety of cel l l ines a n d for a var ie ty of ca t ion ic l ipids. T h e m e c h a n i s m w h e r e b y C a 2 + s t imula tes t ransfect ion a p p e a r s to invo lve 6 7 e n h a n c e d u p t a k e of p l a s m i d D N A . It is ant ic ipa ted that this p ro toco l will f ind g e n e r a l appl icabi l i ty a s a m e a n s to e n h a n c e ca t ion ic l i p o s o m e m e d i a t e d t rans fec t ion . 68 C H A P T E R 3 CALCIUM ENHANCES THE TRANSFECTION P O T E N C Y OF STABILIZED PLASMID-LIPID PARTICLES 3.1 INTRODUCTION In o r d e r for g e n e t h e r a p y to b e cl inical ly use fu l , a n ef fect ive a n d s a f e g e n e de l ive ry s y s t e m is requ i red . V i ra l v e c t o r s a r e relat ively eff icient g e n e de l ive ry v e h i c l e s , but suf fer f rom a var iety of l imitations s u c h a s the potent ial for r e v e r s i o n to the wild t y p e a n d i m m u n e r e s p o n s e c o n c e r n s . A s a result, c o n s i d e r a b l e efforts h a v e b e e n m a d e to d e v e l o p non-v i ra l g e n e de l ivery s y s t e m s ( H o p e et a l . , 1998; P e e t e r s et a l . , 1996; W o r g a l l et a l . , 1997; Y e i et a l . , 1994) . P l a s m i d D N A - c a t i o n i c l i p o s o m e c o m p l e x e s ( l ipoplexes) a r e current ly the m o s t c o m m o n l y e m p l o y e d non-v i ra l g e n e de l ive ry v e h i c l e s ( C h o n n a n d Cu l l i s , 1995; F e i g n e r , 1997) . L i p o p l e x e s c a n exhibi t g o o d t rans fec t ion proper t ies in vitro but h a v e cer ta in l imitations in v ivo . F o r e x a m p l e , l i p o p l e x e s a r e large , h ighly c h a r g e d s y s t e m s that a r e rapidly c l e a r e d f r o m the c i rcula t ion fo l lowing s y s t e m i c admin is t ra t ion a n d c a n a l s o elicit tox ic s i d e e f fec ts (Har r i son et a l . , 1995; H o f l a n d et a l . , 1997; H u a n g a n d L i , 1997; T e m p l e t o n et a l . , 1997) . R e c e n t work f rom this laboratory h a s s h o w n that p l a s m i d D N A c a n b e e n c a p s u l a t e d in s m a l l (~ 7 0 n m d iameter ) "stabi l ized p lasmid - l ip id par t ic les" ( S P L P ) that c o n s i s t of a s i n g l e p l a s m i d e n c a p s u l a t e d within a b i layer lipid v e s i c l e ( W h e e l e r et a l . , 1999) . T h e s e S P L P conta in the " f u s o g e n i c " lipid d i o l e o y l p h o s p h a t i d y l e t h a n o l a m i n e ( D O P E ) , low leve ls of ca t ion ic lipid a n d a r e 69 s tab i l i zed in a q u e o u s m e d i a by the p r e s e n c e of a p o l y e t h y l e n e g l y c o l ( P E G ) c o a t i n g . S P L P h a v e s y s t e m i c app l ica t ion a s they exhibit e x t e n d e d c i rculat ion l i fet imes fo l lowing i n t r a v e n o u s (i.v.) inject ion, a c c u m u l a t e preferent ia l ly at distal t u m o u r s i tes d u e to the e n h a n c e d v a s c u l a r permeabi l i ty in s u c h r e g i o n s a n d c a n m e d i a t e t r a n s g e n e e x p r e s s i o n at t h e s e t u m o u r s i tes (Tarn et a l . , 2000 ) . T h e leve ls of t r a n s g e n e e x p r e s s i o n in v ivo a r e g rea te r than c a n b e a c h i e v e d with n a k e d D N A or c o m p l e x e s but a r e lower than m a y b e requi red for t h e r a p e u t i c benef i t ( Z h a n g et a l . , 1999 ; M o k et a l . , 1999) . Efforts h a v e therefore b e e n f o c u s e d o n d e v e l o p i n g S P L P that h a v e m o r e potent t ransfect ion proper t ies . In p r e v i o u s work w e h a v e s h o w n that C a 2 + c a n e n h a n c e the t rans fec t ion p o t e n c i e s of p l a s m i d D N A - c a t i o n i c lipid c o m p l e x e s by 20- fo ld or m o r e ( L a m a n d C u l l i s , 2000 ) . T h i s e n h a n c e d p o t e n c y w a s a s c r i b e d to i n c r e a s e d cel l u p t a k e of the c o m p l e x e s w h e n C a 2 + is p resent . In the p r e s e n t work w e e x a m i n e d w h e t h e r C a 2 + c o u l d a l s o e n h a n c e the t ransfect ion activity of S P L P . It is s h o w n that C a 2 + c a n result in up to 600 - fo ld e n h a n c e m e n t s in S P L P t ransfect ion p o t e n c y . S u r p r i s i n g l y , it w a s f o u n d that t h e s e e n h a n c e d t ransfect ion activit ies d id not result f r o m e n h a n c e d cel l u p t a k e , but a p p e a r to b e re lated to a n ability of C a 2 + to d e s t a b i l i z e the e n d o s o m a l m e m b r a n e fo l lowing e n d o c y t o s i s . F inal ly , the effect of C a 2 + o n the t rans fec t ion p o t e n c y of S P L P fo rmula t ions conta in ing ei ther a ca t ion ic P E G lipid ( C P L ) or h i g h e r D O D A C c o n t e n t w a s e x a m i n e d . 70 3.2 MATERIALS AND METHODS 3.2.1 Materials A/ , /V -d io ley l -A / ,A / -d imethy lammonium ch lo r ide ( D O D A C ) w a s o b t a i n e d f r o m Dr. S . A n s e l l a n d 1-0- (2 - (co -methoxyethyleneglycol)succinoyl) -2-A/-a r a c h i d o y l s p h i n g o s i n e ( P E G - C e r C 2 o ) w a s s y n t h e s i z e d by Dr. Z . W a n g at Inex P h a r m a c e u t i c a l s C o r p o r a t i o n ( B u r n a b y , B C ) . 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 e t h a n o l a m i n e ( D O P E ) , a n d 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 c h o l i n e ( D O P C ) w e r e o b t a i n e d f rom Nor thern L ip ids ( V a n c o u v e r , B C ) . 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 - L - s e r i n e ] ( D O P S ) a n d 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 e t h a n o l a m i n e - A / - ( L i s s a m i n e R h o d a m i n e B Sul fony l ) ( R h - D O P E ) w e r e p u r c h a s e d f r o m A v a n t i P o l a r L ip ids (A labas te r , A L ) . C h o l e s t e r o l , o c t y l g l u c o p y r a n o s i d e ( O G P ) , H E P E S , M g C b . , a n d N a C l w e r e o b t a i n e d f r o m S i g m a C h e m i c a l C o . (St. L o u i s , M O ) . D E A E S e p h a r o s e C L - 6 B a n i o n i c - e x c h a n g e c o l u m n mater ia ls w e r e o b t a i n e d f rom S i g m a C h e m i c a l C o . (St. L o u i s , M O ) . T h e l u c i f e r a s e a s s a y kit w a s p u r c h a s e d f rom P r o m e g a C o r p . ( M a d i s o n , W l ) . P i c o g r e e n d s D N A de tec t ion r e a g e n t w a s o b t a i n e d f rom M o l e c u l a r P r o b e s ( E u g e n e , O R ) . P l a s m i d D N A ( p C M V L u c ) c o d i n g for the luc i fe rase reporter g e n e u n d e r the contro l of the h u m a n C M V i m m e d i a t e ear ly p r o m o t e r - e n h a n c e r e l e m e n t w a s o b t a i n e d f r o m Inex P h a r m a c e u t i c a l s C o r p o r a t i o n ( B u r n a b y , B C ) . B o v i n e h a m s t e r k i d n e y ( B H K ) ce l l s w e r e o b t a i n e d f rom the A m e r i c a n T i s s u e C u l t u r e C o l l e c t i o n ( A T C C C C L - 1 0 , R o c k v i l l e , M D ) a n d cul tured in D u l b e c c o modi f ied E a g l e m e d i u m ( D M E M ) s u p p l e m e n t with 10 % fetal b o v i n e s e r u m ( F B S ) , 100 U /ml of penici l l in a n d 100 71 f ig /ml of s t r e p t o m y c i n . B H K ce l ls w e r e m a i n t a i n e d a s a m o n o l a y e r at 3 7 ° C in a humid i f ied a t m o s p h e r e conta in ing 5.0 % CO2. 3.2.2 Preparation of S P L P S P L P w e r e p r e p a r e d a s d e s c r i b e d by W h e e l e r et a l . ( W h e e l e r et a l . , 1999) with s o m e modi f i ca t ions . Briefly, a total of 10 u m o l e s of D O D A C , D O P E , P E G -CerC2o (7:83:10; m o l / m o l / m o l ) w e r e d i s s o l v e d in c h l o r o f o r m a n d dr ied u n d e r a s t r e a m of n i t rogen g a s . R e s i d u a l s o l v e n t w a s r e m o v e d u n d e r h igh v a c u u m for 2 h. T h e resul t ing lipid film w a s hydra ted in 1 ml of H B S buffer (20 m M H E P E S a n d 150 m M N a C l , p H 7.5) conta in ing 0.2 M O G P with c o n t i n u o u s vor tex ing . P l a s m i d D N A (400 ug/ml ) w a s a d d e d to the hydra ted l ipids a n d the mix tures w e r e d i a l y s e d a g a i n s t H B S buffer for 36 to 4 8 h with 2 buffer c h a n g e s . N o n - e n c a p s u l a t e d p l a s m i d w a s r e m o v e d by D E A E a n i o n e x c h a n g e c h r o m a t o g r a p h y a n d e m p t y lipid v e s i c l e s w e r e r e m o v e d by e m p l o y i n g a s u c r o s e dens i ty grad ient a s p r e v i o u s l y d e s c r i b e d ( M o k et a l . , 1999) . F o r the h igh D O D A C conten t formulat ion ( D O D A C / D O P E / P E G - C e r C 2 o , 14:76:10, m o l / m o l / m o l ) , S P L P w e r e initially p r e p a r e d in H B S buffer c o n t a i n i n g 30 m M s o d i u m citrate a s d e s c r i b e d p rev ious ly ( Z h a n g et a l . , 1999) . S P L P w e r e c h a r a c t e r i z e d with r e s p e c t to p l a s m i d e n t r a p m e n t u s i n g a p r e v i o u s l y d e s c r i b e d P i c o g r e e n a s s a y ( Z h a n g et a l . , 1999) a n d s i z e d u s i n g q u a s i e l a s t i c light sca t te r ing . 3.2.3 Transfection in the presence of C a 2 + Pr ior to t ransfec t ion , B H K ce l ls w e r e p la ted at a d e n s i t y of 1 x 1 0 4 ce l l s p e r wel l in a 96-wel l p late overn ight . 2 0 0 m M C a C I 2 s t o c k so lut ion w a s p r e p a r e d in 72 d H 2 0 a n d ster i l ized by filtering. 0.5 p g p l a s m i d D N A e n c a p s u l a t e d in S P L P w a s u s e d p e r wel l of t ransfec t ion . S P L P w e r e first a d d e d to a p p r o p r i a t e c o n c e n t r a t i o n s of C a 2 + a s requ i red by the e x p e r i m e n t , after w h i c h cul ture m e d i a w a s a d d e d to the mix tures to obta in the final t ransfect ion v o l u m e of 100 uJ/well. C a 2 + c o n c e n t r a t i o n s a d d e d to the S P L P w e r e c a l c u l a t e d with r e s p e c t to the f inal v o l u m e of the t rans fec t ion m e d i u m a p p l i e d to ce l ls . T h e final v o l u m e c o n t a i n e d 2 0 % vo l C a 2 + a n d S P L P mix tures a n d 8 0 % vol cul ture m e d i a . C e l l s w e r e i n c u b a t e d with the t rans fec t ion c o m p l e x e s for the appropr ia te t ime p e r i o d s b e f o r e a s s a y i n g for g e n e e x p r e s s i o n a s d e s c r i b e d p rev ious ly ( W h e e l e r et a l . , 1999) . R e l a t i v e l u c i f e r a s e activity w a s n o r m a l i z e d a g a i n s t total ce l lu lar protein d e t e r m i n e d by u s i n g the M i c r o B C A prote in a s s a y r e a g e n t kit (P ie rce , Illinois). 3.2.4 Determination of S P L P uptake into cells B H K ce l ls w e r e p la ted at 1 x 1 0 5 ce l ls p e r wel l o f 12-wel l p la tes the d a y prior to the e x p e r i m e n t . S P L P cel lu lar up take de te rmina t ion w a s p e r f o r m e d by incorpora t ing 0.5 m o l % R h - D O P E into the lipid fo rmula t ions ( D O D A C / D O P E / P E G -C e r C 2 o / R h - D O P E ; 7:82.5:10:0 .5 m o l / m o l / m o l / m o l ) a n d S P L P w e r e p r e p a r e d u s i n g the d e t e r g e n t d ia lys is m e t h o d a s d e s c r i b e d prev ious ly . S P L P m i x e d with i n c r e a s i n g c o n c e n t r a t i o n s of C a 2 + (0 to 14 m M ) w e r e a d d e d to ce l ls at a lipid d o s e of 8 0 n m o l e s in c o m p l e t e m e d i a (1 ml final v o l u m e ) . Af ter incubat ion at 3 7 ° C for 4 , 8, a n d 24 h, ce l l s w e r e w a s h e d with P B S a n d l y s e d by the addi t ion of buffer c o n t a i n i n g 0.1 % T X -100 in 2 5 0 m M p h o s p h a t e buffer (pH 8.0). R h o d a m i n e f l u o r e s c e n c e of the lysa te w a s m e a s u r e d o n a Perk in E l m e r L u m i n e s c e n c e S p e c t r o p h o t o m e t e r u s i n g A, e x of 5 6 0 73 n m a n d Xem o f 5 9 0 n m with slit w id ths of 10 a n d 10 n m , respec t i ve ly . L ip id u p t a k e w a s d e t e r m i n e d by c o m p a r i n g lysate f l u o r e s c e n c e to that of a lipid s t a n d a r d n o r m a l i z i n g it to the total ce l lu lar prote in. 3.2.5 Fluorescence microscopy studies F l u o r e s c e n c e m i c r o s c o p y w a s e m p l o y e d to v i s u a l i z e the intracel lu lar lipid distr ibut ion fo l lowing u p t a k e of la rge un i lamel la r v e s i c l e s ( L U V ) . C e l l s w e r e i n c u b a t e d with L U V with the s a m e lipid c o m p o s i t i o n a s the S P L P formula t ion e x c e p t that 4 m o l % R h - D O P E w a s incorpora ted to s e r v e a s the f l u o r e s c e n c e m a r k e r ( L U V c o m p o s i t i o n : D O D A C / D O P E / P E G - C 2 0 / R h - D O P E , 7:79:10:4). L U V w e r e p r e p a r e d by u s i n g the ex t rus ion m e t h o d a s d e s c r i b e d p rev ious ly ( L a m a n d C u l l i s , 2000 ) . T h e t rans fec t ion m e d i a w a s r e p l a c e d with c o m p l e t e m e d i a prior to a n a l y s i s u n d e r the f l u o r e s c e n c e m i c r o s c o p e . F l u o r e s c e n c e m i c r o g r a p h s w e r e t a k e n o n a n A x i o v e r t 100 Z e i s s F l u o r e s c e n t m i c r o s c o p e (Car l Z e i s s J e n a G m b H ) u s i n g a r h o d a m i n e filter f r o m O m e g a O p t i c a l s (Brat t leboro, V T ) with the fol lowing s p e c i f i c a t i o n s , Xex=560 ± 2 0 n m , 6 0 0 n m L P , a n d D C 5 9 0 n m . 3.2.6 Intracellular processing of plasmid DNA B H K ce l ls w e r e p la ted at 3 x 1 0 5 ce l ls p e r wel l o f 6-wel l p la tes the d a y pr ior to the e x p e r i m e n t . 2 .5 jag p l a s m i d D N A e n c a p s u l a t e d in S P L P w e r e i n c u b a t e d with ce l ls for 2, 4, a n d 8 h, in the a b s e n c e or p r e s e n c e (8 m M ) of C a 2 + . A t the a p p r o p r i a t e t ime points , ce l l s w e r e w a s h e d with P B S a n d ex terna l S P L P w e r e r e m o v e d by t ryps in iza t ion . T r y p s i n i z e d ce l l s w e r e pe l le ted by centr i fugat ion a n d 74 c e l l s w e r e r e s u s p e n d e d a n d w a s h e d with isoton ic buffer (250 m M s u c r o s e , 3 m M M g C I 2 , 5 0 m M H E P E S , p H 7.2). S u b s e q u e n t l y , pe l le ted ce l l s w e r e l y s e d by incuba t ing with 2 5 0 pl of lysis buffer (10 m M T r i s , p H 7.5, 0 . 5 % S D S , 1 m M E D T A ) c o n t a i n i n g P r o n a s e E at 1 m g / m l ( S i g m a ) overn ight at 3 7 ° C . D N A ( g e n o m i c D N A a n d d e l i v e r e d p l a s m i d D N A ) w e r e ex t rac ted a s d e s c r i b e d p r e v i o u s l y ( S a m b r o o k et a l . , 1989) . D N A r e c o v e r y w a s d e t e r m i n e d by m e a s u r i n g the a b s o r b a n c e at 2 6 0 n m . 6 |a,g of total D N A f r o m e a c h s a m p l e w a s ei ther dot blotted on to a ny lon t rans fe r m e m b r a n e ( A m e r s h a m ) with a se t of p C M V L u c s t a n d a r d s (0 to 5 pg) or l o a d e d into a 1% a g a r o s e ge l a n d s i z e f ract ionated at 6 0 V for 2 h for the S o u t h e r n a n a l y s i s . B o t h blots w e r e hybr id i zed overn ight at 6 8 ° C to a 3 2 P - l a b e l e d p l a s m i d D N A p r o b e , w h i c h w a s p r e p a r e d with Pst\ c u t - p C M V L u c p l a s m i d u s i n g the T 7 Q u i c k P r i m e ™ Kit ( P h a r m a c i a B io tech ) . B lo ts w e r e w a s h e d 3 t imes with 2x S S C c o n t a i n i n g 0 . 1 % S D S , a n d w e r e t h e n e x p o s e d o n a P h o s p h o r i m a g e r s c r e e n w h i c h w a s s u b s e q u e n t l y s c a n n e d ( M o l e c u l a r D y n a m i c s - P h o s p h o r i m a g e r ™ S I ) . 3.2.7 3 1 P NMR spectroscopy S o l i d - s t a t e 3 1 P N M R s p e c t r a w e r e r e c o r d e d with b r o a d - b a n d d e c o u p l i n g at 81 .02 M H z o n a B r u k e r M S L 2 0 0 s p e c t r o m e t e r , us ing a 3.8-(j,s 6 0 ° p u l s e a n d a 1.5-s r e p e a t t ime. T h e f ree induct ion d e c a y (FID) w a s a c c u m u l a t e d o v e r 2 5 0 0 - 3 0 0 0 s c a n s a n d w a s F o u r i e r t r a n s f o r m e d with 5 0 - H z line b r o a d e n i n g . P h o s p h o l i p i d mix tures (25 u m o l of total p h o s p h o l i p i d ) w e r e d i s p e r s e d to f o r m mul t i lamel lar v e s i c l e s ( M L V ) by vor tex mix ing in 2 ml of buffer (20 m M H E P E S buffer, p H 7.4). I nc reas ing c o n c e n t r a t i o n s of C a 2 + w e r e titrated into the v e s i c l e s by a d d i n g a l iquots of 75 a 2 0 0 m M C a C ^ s t o c k so lu t ion . C a equi l ibrat ion w a s e n s u r e d by p e r f o r m i n g th ree c y c l e s of f r e e z e - t h a w i n g . T h e t e m p e r a t u r e w a s m a i n t a i n e d at 2 5 ° C with a B r u k e r v a r i a b l e t e m p e r a t u r e unit. A mixture of p h o s p h o r i c a c i d / D 2 0 w a s u s e d a s the r e f e r e n c e for c h e m i c a l shifts in a l l 3 1 P N M R s p e c t r a . 3.2.8 Entrapment of C a 2 + inside S P L P S P L P ( D O D A C / D O P E / P E G - C e r C 2 0 / R h - D O P E , 10:79 .5:10:0 .5 , m o l / m o l / m o l ) w e r e p r e p a r e d a s d e s c r i b e d in citrate buffer (150 m M s o d i u m citrate a n d 150 m M citric a c i d , p H 4). N o n - e n c a p s u l a t e d p l a s m i d w a s r e m o v e d by D E A E a n i o n e x c h a n g e c h r o m a t o g r a p h y equi l ibrated in H B S buffer ( p H 7.5) a n d e m p t y lipid v e s i c l e s w e r e r e m o v e d by e m p l o y i n g a s u c r o s e d e n s i t y grad ient a s p r e v i o u s l y d e s c r i b e d ( M o k et a l . , 1999) . C a 2 + load ing w a s p e r f o r m e d by incuba t ion of the D N A -l o a d e d v e s i c l e s with 2 .5 m M C a C b a n d the i o n o p h o r e s A 2 3 1 8 7 ( 0 . 1 u g / u r n o l e lipids) for 3 0 min at r o o m t e m p e r a t u r e . U n l o a d e d C a 2 + a n d i o n o p h o r e s w e r e r e m o v e d by d ia lys is in H B S buffer with 2 buffer c h a n g e s . Internal C a 2 + c o n c e n t r a t i o n s w e r e d e t e r m i n e d in the a b s e n c e a n d p r e s e n c e of T X - 1 0 0 (0.2%) by e m p l o y i n g the m e m b r a n e n o n - p e r m e a n t a b s o r b a n t indicator A s e n a z o III (0.1 m M in 10 m M H E P E S buffer, p H 7), a g a i n s t a s t a n d a r d c u r v e g e n e r a t e d e m p l o y i n g C a C b . C a 2 + l eve ls w e r e m e a s u r e d by m e a s u r i n g a b s o r b a n c e at 6 5 0 n m . Internal c o n c e n t r a t i o n s of C a 2 + w e r e f o u n d to b e ~ 1 7 5 m M . S P L P w e r e c h a r a c t e r i z e d with r e s p e c t to p l a s m i d e n t r a p m e n t u s i n g a p rev ious ly d e s c r i b e d P i c o g r e e n a s s a y ( Z h a n g et a l . , 1999) a n d s i z e d u s i n g q u a s i e l a s t i c light sca t te r ing . 7 6 3.2.9 Insertion of CPL Prior to CPL insertion, SPLP were prepared as described in the previous section with some modification. SPLP containing a total of 10 umoles of DODAC, DOPE, PEG-CerC 2 0 , and Rh-DOPE (7:82.5:10:0.5; mol/mol/mol/mol) were hydrated in 1 ml of HBS buffer (20 mM HEPES and 150 mM NaCl, pH 7.5) containing 0.2 M OGP with continuous vortexing. Plasmid DNA (400 |ag/ml) was added to the hydrated lipids and the mixtures were dialysed against HBS buffer for 36 to 48 h with 2 buffer changes. Non-encapsulated plasmid was removed by DEAE anion exchange chromatography. CPL insertion was performed after the DEAE column as described (Fenske et al., submitted). Briefly, CPL stocks in methanol labeled with a dansyl fluorescence marker were added to the vesicles to give the desired molar ratio (4 mol % CPL relative to vesicle lipid). CPL and SPLP were incubated for up to 3 h at 60 °C, and cooled on ice to room temperature. Both empty lipid vesicles and non-inserted CPL were removed by using sucrose density gradient centrifugation (at step gradients of 1%, 2.5%, and 10% sucrose). The insertion levels of CPL were quantitated by using the Perkin Elmer Luminescence Spectrophotometer as described previously (Fenske et al., submitted). Briefly, the initial dansyl/rhodamine (D/Rj) fluorescence ratio prior to sucrose density gradient and the final D/R (D/Rf) ratio of the isolated CPL-SPLP were measured. Rhodamine flouresence was assayed at A, e x = 560 nm and A, e m = 590 nm, while dansyl fluorescence was assayed at Xex= 340 nm and Xem = 510 nm, with slit widths of 10 and 10 nm. The %-insertion was calculated as follows: 77 %-insertion = ([D/R]f)*100/(D/R)j CPL-SPLP were further characterized with respect to plasmid entrapment the Picogreen assay and sized using quasielastic light scattering. 78 3.3 R E S U L T S 3.3.1 The transfection potencies of S P L P are dramatically enhanced in the presence of C a 2 + P r e v i o u s work h a s s h o w n that S P L P , part icular ly S P L P s tab i l i zed by P E G -CerC2o, exhibi t v e r y low leve ls of t ransfect ion in vitro ( W h e e l e r et a l . , 1999; M o k et a l . , 1999) . W e h a v e a l s o s h o w n that the t ransfec t ion p o t e n c i e s of p l a s m i d D N A -c a t i o n i c lipid c o m p l e x e s c a n b e e n h a n c e d up to 20- fo ld w h e n C a 2 + is p r e s e n t in the t rans fec t ion m e d i u m ( L a m a n d Cu l l i s , 2000) . H e r e w e e x t e n d t h e s e s t u d i e s to moni to r the e f fec ts of C a 2 + o n the t ransfect ion p o t e n c y of S P L P . S P L P p r e p a r e d f r o m D O P E / D O D A C / P E G - C e r C 2 o (84:6:10; mol :mol :mol ) lipid mixture a n d p C M V L u c e m p l o y i n g the de te rgen t d ia lys is m e t h o d w e r e purif ied f r o m e m p t y v e s i c l e s a n d u n e n c a p s u l a t e d p l a s m i d a s d e s c r i b e d in Mater ia ls a n d M e t h o d s . A p p r o p r i a t e a m o u n t s of C a C b w e r e then a d d e d to the S P L P prepara t ion to g ive r ise to the d e s i r e d C a 2 + c o n c e n t r a t i o n s fo l lowing dilution into the m e d i a b e f o r e a p p l y i n g to the B H K ce l ls . T h e B H K ce l ls a n d the S P L P w e r e i n c u b a t e d t o g e t h e r for 24 h, after w h i c h the t r a n s f e c t e d ce l ls w e r e a s s a y e d for luc i fe rase e x p r e s s i o n . A s s h o w n in F i g u r e 3.1 , the p r e s e n c e of C a 2 + resu l ted in d r a m a t i c e n h a n c e m e n t s in l u c i f e r a s e e x p r e s s i o n leve ls , with a ~600 - fo ld i n c r e a s e in S P L P t ransfec t ion p o t e n c y o b s e r v e d at the opt ima l C a 2 + c o n c e n t r a t i o n s . T h i s C a 2 + - m e d i a t e d i n c r e a s e in t rans fec t ion is s igni f icant ly g rea te r for the S P L P s y s t e m than p r e v i o u s l y o b s e r v e d for p l a s m i d D N A -ca t ion ic lipid c o m p l e x e s . T h e opt imal c o n c e n t r a t i o n s of C a 2 + r equ i red for s t imulat ing S P L P t rans fec t ion p o t e n c i e s w e r e in the r a n g e of 8 to 10 m M , s o m e w h a t lower t h a n 79 6 0 0 0 5 0 0 0 - \ 0 2 4 6 8 10 12 14 ca t ion c o n c e n t r a t i o n (mM) F i g u r e 3 .1 . Caz+ e n h a n c e s the t ransfect ion p o t e n c y of S P L P in a s p e c i f i c m a n n e r . I n c r e a s i n g c o n c e n t r a t i o n s o f C a C I 2 ( • ) , M g C I 2 ( • ) , o r N a C l (^ ) (0 to 14 m M - f inal c o n c e n t r a t i o n s ) w e r e titrated into S P L P prior to their add i t ion to ce l l s . 0.5 (j,g of p C M V L u c p l a s m i d e n c a p s u l a t e d in S P L P ( D O D A C / D O P E / P E G - C e r C 2 0 ; 7:83:10 m o l / m o l / m o l ) v e s i c l e s w a s u s e d to t ransfect ce l l s p la ted at 1 x 1 0 4 ce l ls /we l l o f 96 -wel l p la tes . C e l l s w e r e i n c u b a t e d with S P L P for 2 4 h, a n d L u c activity w a s m e a s u r e d a s d e s c r i b e d in Mate r ia ls a n d M e t h o d s . T h e er ror b a r s r e p r e s e n t the s t a n d a r d dev ia t ion of three e x p e r i m e n t s . 80 that requ i red (5-25 m M ) for opt imal st imulat ion of the t rans fec t ion p o t e n c i e s of p l a s m i d D N A - c a t i o n i c lipid c o m p l e x e s . F inal ly , the ability of C a 2 + to s t imula te the t rans fec t ion p o t e n c y of S P L P w a s spec i f i c . A s s h o w n in F i g u r e 3.1 , if M g C I 2 o r N a C l w a s subst i tu ted for C a C I 2 n o e n h a n c e m e n t in t ransfect ion p o t e n c y w a s o b s e r v e d . 3.3.2 S P L P are stable in the presence of C a 2 + S P L P with the P E G - C e r C 2 o coa t ing a r e highly s tab le s y s t e m s that exhibi t e x t e n d e d c i rculat ion t imes in v ivo , protect e n c a p s u l a t e d p l a s m i d f r o m ex te rna l n u c l e a s e s a n d d o not interact readi ly with ce l l s ( M o n c k et a l . , 2 0 0 0 ; M o k et a l . , 1999 ; W h e e l e r et a l . , 1999) . It is there fore important to d e m o n s t r a t e that the e n h a n c e d t rans fec t ion proper t ies of S P L P in the p r e s e n c e of C a 2 + w e r e not d u e to des tab i l i za t ion of the S P L P lead ing to e n h a n c e d cel l u p t a k e . T h e stabil ity of the S P L P in the p r e s e n c e of C a 2 + w a s e x a m i n e d e m p l o y i n g q u a s i - e l a s t i c light sca t te r ing ( Q E L S ) to de tec t c h a n g e s in s i z e a n d the P i c o g r e e n f l u o r o p h o r e a s s a y to d e t e c t D N A l e a k a g e . F o r the Q E L S e x p e r i m e n t s , C a C I 2 w a s a d d e d to the S P L P s u s p e n s i o n to a c h i e v e c o n c e n t r a t i o n s a s high a s 5 0 m M . N o c h a n g e in the S P L P s i z e or s i z e distr ibut ion w a s o b s e r v e d (F ig . 3 .2A) . F o r the p l a s m i d r e l e a s e e x p e r i m e n t s , S P L P w e r e i n c u b a t e d at 37 ° C in H B S buffer conta in ing 10 % F B S in the p r e s e n c e or a b s e n c e of 8 m M C a 2 + . P l a s m i d r e l e a s e w a s a s s a y e d o v e r 2 4 h e m p l o y i n g the P i c o g r e e n a s s a y . N o p l a s m i d r e l e a s e w a s o b s e r v e d (F ig . 3 .2B) . 3.3.3 C a 2 + does not influence the cellular uptake of S P L P T h e ability of C a 2 + to e n h a n c e the t ransfect ion activity of p l a s m i d D N A -81 E c 120 100 -A 80 60 2 40 -A co i— d) - 4 — » E co •a a) o CO cu > 20 - J 4 6 8 10 12 14 20 50 calcium concentrations (mM) 120 Figure 3.2. Stability of S P L P in the presence of C a 2 + . (A) Effect of C a ^ + on ves ic le s i ze mean diameters. Increasing concentrations of C a 2 + (0 to 50 mM) were titrated into S P L P , and vesic le mean diameters were determined using quasie last ic light scattering. (B) Percent plasmid D N A retention in the presence of C a 2 + . S P L P were incubated at 37°C in H B S (1), 1 0 % F B S (2), or 8 m M C a 2 + w / 1 0 % F B S (3). A n aliquot w a s taken from each sample and levels of Rd f luorescence were determined in the a b s e n c e and presence of T X - 1 0 0 (0.2%) as descr ibed in Materials and Methods. 82 ca t ion ic lipid c o m p l e x e s h a s b e e n attr ibuted to a n i n c r e a s e in the u p t a k e of the c o m p l e x e s into ce l l s in the p r e s e n c e of C a 2 + ( L a m a n d Cu l l i s , 2000 ) . In this r e g a r d , the low t rans fec t ion p o t e n c i e s of S P L P a s c o m p a r e d to c o m p l e x e s a r ise , at least in part, f r o m v e r y low leve ls of ce l lu lar u p t a k e of S P L P ( M o k et a l . , 1999) . It w a s there fore important to d e t e r m i n e w h e t h e r C a 2 + s t imula ted S P L P t rans fec t ion p o t e n c i e s by i n c r e a s i n g S P L P u p t a k e into ce l ls . S P L P c o n t a i n i n g 0.5 m o l % R h -D O P E w e r e e m p l o y e d to d e t e r m i n e S P L P u p t a k e into B H K ce l ls in the p r e s e n c e of up to 14 m M C a 2 + a s d e s c r i b e d in Mater ia ls a n d M e t h o d s . T h e S P L P w e r e i n c u b a t e d with ce l l s for 4, 8, a n d 24 h a n d the leve ls of intracel lular lipid d e t e r m i n e d . L ip id u p t a k e at e a c h t ime-point w a s n o r m a l i z e d a g a i n s t total cel l prote in in o r d e r to a c c o u n t for cel l g rowth . A s s h o w n in F i g u r e 3.3, the resul ts ind icate that C a 2 + d id not s igni f icant ly i n c r e a s e the ce l lu lar up take of S P L P e v e n t h o u g h the t rans fec t ion p o t e n c i e s of the S P L P v a r i e d by s e v e r a l h u n d r e d - f o l d o v e r the r a n g e of C a 2 + c o n c e n t r a t i o n s t e s t e d . 3.3.4 Fluorescence studies indicate enhanced endosomal destabilization following uptake of LUV in the presence of C a 2 + T h e fact that u p t a k e of S P L P is not s t imula ted by addi t ion of C a 2 + s u g g e s t s that the C a 2 + - d e p e n d e n t e n h a n c e m e n t of t ransfect ion m u s t a r i s e f r o m m o r e eff icient uti l ization of S P L P that a r e a c c u m u l a t e d . O n e possibi l i ty is that C a 2 + s o m e h o w faci l i tates destab i l i za t ion of e n d o s o m e s fol lowing up take of S P L P , t h u s e n h a n c i n g intracel lular de l ivery of p l a s m i d . P r e v i o u s work h a s s h o w n that e n d o s o m a l des tab i l i za t ion fo l lowing up take of v e s i c l e s con ta in ing f l u o r e s c e n t l y - l a b e l e d l ipids c a n 83 5 1 I I I I I 1 I 0 2 4 6 8 10 12 14 calcium concentration (mM) Figure 3.3. Influence of C a on the cellular uptake of S P L P . S P L P containing 0.5 mol % Rh- labeled D O P E ( D O D A C / D O P E / P E G - C e r C 2 0 / R h - D O P E ; 7:82.5:10:0.5 mol /mol /mol /mol) were employed to monitor cellular lipid uptake. 80 nmoles of lipid ves ic les prepared in the presence of C a 2 + (0 to 14 m M - final concentrations) were incubated on cel ls for the appropriate time periods. Levels of lipid uptake were determined by measuring Rh f luorescence at 4 h ( • ) , 8 h ( • ) , or 24 h (^) as descr ibed in Materials and Methods. The error bars represent the standard deviation of three experiments. 84 b e d e t e c t e d by f l u o r e s c e n c e m i c r o s c o p y a s a d i f fuse intracel lular f l u o r e s c e n c e , w h e r e a s u p t a k e into s tab le e n d o s o m e s g i v e s r ise to a l o c a l i z e d "punctate" a p p e a r a n c e ( F e i g n e r et a l . , 1987) . In o r d e r to b e a b l e to v i s u a l i z e the e f fec ts of C a 2 + , L U V with the s a m e lipid c o m p o s i t i o n a s the S P L P but with a h igh level of the f l u o r e s c e n t lipid R h - D O P E w e r e c o n s t r u c t e d ( D O D A C / D O P E / P E G - C 2 0 / R h - D O P E , 7:79:10:4, m o l / m o l / m o l / m o l ) . T h e s e R h - l a b e l e d L U V w e r e i n c u b a t e d with B H K c e l l s in the p r e s e n c e a n d a b s e n c e of 10 m M C a 2 + a n d the cel l m o r p h o l o g y w a s e x a m i n e d at 8 h by f l u o r e s c e n c e m i c r o s c o p y . S imi la r leve ls of r h o d a m i n e f l u o r e s c e n c e w e r e d e t e c t e d in the a b s e n c e or p r e s e n c e of C a 2 + , in a g r e e m e n t with the quant i ta t ive m e a s u r e m e n t s of S P L P up take noted in the p r e v i o u s s e c t i o n . H o w e v e r , a s s h o w n in F i g u r e 3.4, the a p p e a r a n c e of the ce l ls a s d e t e c t e d by f l u o r e s c e n c e m i c r o s c o p y w a s qui te dif ferent in the p r e s e n c e or a b s e n c e of C a 2 + . A l t h o u g h s o m e p u n c t a t e s t ruc tu res a r e o b s e r v e d , B H K ce l ls conta in ing the f l u o r e s c e n t l y - l a b e l e d L U V exh ib i ted a m o r e d i f fuse pattern w h e n C a 2 + w a s i n c l u d e d . In the a b s e n c e o f C a 2 + , the f l u o r e s c e n c e pattern w a s largely puncta te , c o n s i s t e n t with retent ion of the L U V in the e n d o s o m a l c o m p a r t m e n t s . 3.3.5 Intracellular processing of delivered plasmid DNA T h e p r e c e d i n g f l u o r e s c e n t m i c r o s c o p y resul ts s u g g e s t that C a 2 + e n h a n c e s t rans fec t ion by destab i l i z ing the e n d o s o m a l c o m p a r t m e n t s , t h u s e n h a n c i n g c y t o p l a s m i c de l ivery of the S P L P - a s s o c i a t e d p l a s m i d . If S P L P p l a s m i d c a n e s c a p e f r o m the e n d o s o m e m o r e readi ly in the p r e s e n c e of C a 2 + , it s h o u l d a v o i d b r e a k d o w n in the l y s o s o m a l p a t h w a y a n d m o r e intact intracel lular p l a s m i d D N A s h o u l d b e 8 5 Figure 3.4. Influence of C a 2 + on cell morphology following uptake of LUV labelled with R h - D O P E as detected using f luorescence microscopy. LUV (DODAC/DOPE/PEG-C 2 0 /Rh -DOPE, 7:79:10:4 mol/mol/mol/mol) were prepared as indicated in Materials and Methods and amounts corresponding to100 nmoles lipid were incubated with cells (plated at 1x10 5 cells per well of a 12-well plate) in the absence (A) or presence (B) of calcium (10 mM). At the 8 h time point, transfecting media was replaced with complete DMEM media and cells were examined using fluorescence microscopy. Fluorescence micrographs were taken as indicated in Materials and Methods. 81 p r e s e n t . A dot blot a s s a y w a s e m p l o y e d to m e a s u r e intracel lular de l ivery of p l a s m i d D N A , a n d the integrity of the p l a s m i d w a s e x a m i n e d by u s i n g the S o u t h e r n blot a n a l y s i s . C e l l s w e r e i n c u b a t e d with S P L P in the a b s e n c e or p r e s e n c e of 8 m M C a 2 + for 2, 4, a n d 8 h. T h e leve ls of intact, intracel lular p l a s m i d D N A for the dif ferent s y s t e m s w e r e c o m p a r e d after isolat ion of D N A f r o m the ce l l s a s d e s c r i b e d in Mate r ia ls a n d M e t h o d s , a n d the resul ts a r e s h o w n in F i g u r e 3.5. A s s h o w n in F i g u r e 3 . 5 A , w h e n ce l ls w e r e t r a n s f e c t e d with the S P L P in the p r e s e n c e of C a 2 + the a m o u n t of intact p l a s m i d in the B H K ce l ls w a s i n c r e a s e d by a p p r o x i m a t e l y 10- fold after a n 8 h incubat ion pe r iod . T h i s is a l s o ref lected by a S o u t h e r n a n a l y s i s w h i c h s h o w e d that m o r e intact p l a s m i d D N A w a s p r e s e n t in ce l l s t r a n s f e c t e d with S P L P p r e p a r e d in the p r e s e n c e of C a 2 + (F ig . 3 .5B) . S u c h e n h a n c e d leve ls of intact p l a s m i d D N A w e r e not o b s e r v e d w h e n M g 2 + w a s subst i tu ted for C a 2 + , d e m o n s t r a t i n g the speci f ic i ty of C a 2 + (F ig . 3 .5B) . 3.3.6 C a 2 + destabilizes bilayer lipid structures in a manner consistent with an ability to destabilize endosomal membranes R e c e n t work s u g g e s t s that ca t ion ic l ipids s t imulate intracel lular de l ive ry of m a c r o m o l e c u l e s s u c h a s p l a s m i d D N A by c o m b i n i n g with a n i o n i c l ipids a n d fo rming ion pa i rs that d e s t a b i l i z e b i layer m e m b r a n e s by i n d u c i n g n o n - b i l a y e r (Hn p h a s e ) s t ructure ( H a f e z et a l . , 2 0 0 0 ; H a f e z a n d Cu l l i s , submi t ted) . In this r e g a r d , it is wel l k n o w n that C a 2 + c a n des tab i l i ze lipid b i layers con ta in ing a c i d i c l ipids s u c h a s p h o s p h a t i d y l s e r i n e ( P S ) in c o m b i n a t i o n with u n s a t u r a t e d P E ' s by i n d u c i n g the n o n -87 F i g u r e 3.5 . Inf luence of C a 2 + o n the integrity of S P L P p l a s m i d fo l lowing u p t a k e of S P L P into B H K ce l ls . S P L P ( D O D A C / D O P E / P E G - C 2 0 , 7 :83:10 m o l / m o l / m o l ) c o n t a i n i n g 2 .5 p.g p l a s m i d D N A w e r e u s e d to t ransfect B H K ce l ls a s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . A t appr opr ia te t ime points (2 h, 4 h, or 8 h), D N A w a s ex t rac ted f r o m the ce l ls a n d intracel lular p l a s m i d D N A w a s d e t e c t e d by hybr id izat ion to a s p e c i f i c 3 2 P - l a b e l e d p l a s m i d D N A p r o b e , (a) L e v e l s of p l a s m i d D N A u p t a k e d e t e r m i n e d by dot blot a n a l y s i s in the p r e s e n c e ( • ) or a b s e n c e ( • ) of 8 m M C a 2 + . (b) Integrity of intracel lular p l a s m i d D N A u s i n g the S o u t h e r n blot a n a l y s i s . L a n e s 1 a n d 11: p C M V L u c contro l ; l a n e s 2, 5, 8 a n d 12: u n t r a n s f e c t e d contro l ; l a n e s 3, 6, 9 a n d 13: c e l l s t r a n s f e c t e d with S P L P ; l a n e s 4 , 7, 10 a n d 14: ce l l s t r a n s f e c t e d with S P L P a n d 8 m M C a 2 + ; a n d l ane 15: ce l ls t rans fec ted with S P L P a n d 8 m M M g 2 + . T h e er ror b a r s r e p r e s e n t the s t a n d a r d dev ia t ion of th ree e x p e r i m e n t s . 88 bi layer h e x a g o n a l H M p h a s e structure ( H o p e a n d C u l l i s , 1979 ; T i l c o c k a n d C u l l i s , 1981 ; Ba l ly et a l . , 1983) . It h a s a l s o b e e n s h o w n that C a 2 + c a n i n d u c e H M p h a s e s t ructure in re lated s y s t e m s conta in ing p h o s p h a t i d y l c h o l i n e ( P C ) a n d c h o l e s t e r o l . F o r e x a m p l e , addi t ion of C a 2 + to mixtures of D O P C / D O P E / D O P S / C h o l e s t e r o l (1:1:1:3; m o l a r ratios) a l s o t r iggers b i layer to h e x a g o n a l Hu p h a s e t rans i t ions (T i l cock et a l . , 1984) . It m a y there fore b e p r o p o s e d that C a 2 + s t imu la tes S P L P t rans fec t ion by ac t ing in c o n c e r t with the ca t ion ic lipid in the S P L P to d e s t a b i l i z e the lipid b i layer of e n d o s o m a l m e m b r a n e s . In o r d e r to illustrate this potential w e invest iga ted the C a 2 + - d e p e n d e n t p o l y m o r p h i s m of M L V c o m p o s e d of D O P C / D O P E / D O P S / C h o l e s t e r o l (1:1:1:3; m o l a r ratios) in the a b s e n c e a n d p r e s e n c e of s m a l l a m o u n t s of D O D A C e m p l o y i n g 3 1 P N M R . C o n s i d e r a b l e p r e v i o u s work h a s s h o w n that M L V c o m p o s e d of p h o s p h o l i p i d s in the b i layer o r g a n i z a t i o n g ive r ise to a s y m m e t r i c 3 1 P N M R line s h a p e s with a low field s h o u l d e r a n d h igh field p e a k , w h e r e a s p h o s p h o l i p i d s in the h e x a g o n a l H M p h a s e g ive r ise to a line s h a p e with r e v e r s e d a s y m m e t r y that is a fac tor of two n a r r o w e r (Cul l is a n d d e Kruijff, 1979) . A s s h o w n in F i g u r e 3 . 6 A , in the a b s e n c e of D O D A C , C a 2 + is a b l e to s t imula te a transit ion f rom the b i layer to the h e x a g o n a l Hu p h a s e a s repor ted by 3 1 P N M R at the C a 2 + - t o - D O P S ratio of 0 .5:1. A l ternat ive ly , in M L V c o n t a i n i n g s m a l l a m o u n t s of D O D A C ( D O P C / D O P E / D O P S / C h o l e s t e r o l / D O D A C ; 1:1:1:3:0.25; m o l a r ratios), C a 2 + - t o - D O P S ratios of on ly 0.25:1 a r e requ i red to i n d u c e p r e d o m i n a n t l y H M p h a s e structure (F ig . 3 .6B) . T h e nar row cent ra l p e a k m a y a r i s e f r o m s m a l l lamel la r s t ruc tures or lipid in n o n - b i l a y e r s t ruc tures s u c h the a s c u b i c p h a s e in w h i c h c o m p o n e n t p h o s p h o l i p i d s e x p e r i e n c e isot rop ic mot iona l a v e r a g i n g . 89 C a ^ / D O P S C a / + / D O P S ' - - t , i , i—,—i—i—i—i—J 40 20 0 -20 -40 PPM (A) (B) F i g u r e 3.6. P N M R s p e c t r a of v a r i o u s m o d e l m e m b r a n e s y s t e m s in the p r e s e n c e of C a 2 + . C a 2 + w a s titrated into M L V c o m p o s e d of (A) D O P E / D O P S / D O P C / C h o l , 1:1:1:3; or (B) D O P E / D O P S / D O P C / C h o l / D O D A C , 1:1:1:3:0.25, to a c h i e v e C a 2 + / D O P S rat ios r a n g i n g f r o m 0:1 to 0.5:1 (molar rat ios). Equi l ibra t ion of the c a t i o n s w a s e n s u r e d by th ree c y c l e s of f r e e z e - t h a w i n g . S p e c t r a h a v e b e e n n o r m a l i z e d to the s a m e p e a k height . E x p e r i m e n t s w e r e car r ied out a s d e s c r i b e d in Mater ia ls a n d M e t h o d s . 90 3.3.7 External C a 2 * is required to enhance S P L P transfection potency It w a s of interest to d e t e r m i n e w h e t h e r C a 2 + e n c a p s u l a t e d within the S P L P c o u l d s t imula te t r a n s g e n e e x p r e s s i o n . A s deta i led e l s e w h e r e ( W h e e l e r et a l . , 1994) , C a 2 + c a n b e l o a d e d into large uni lamel lar v e s i c l e s ( L U V ) in r e s p o n s e to a p H g rad ien t ( ins ide ac id ic ) w h e n the C a 2 + i o n o p h o r e A 2 3 1 8 7 is p r e s e n t . Internal C a 2 + c o n c e n t r a t i o n s a s h igh a s 2 5 0 m M c a n b e a c h i e v e d . A s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s , S P L P c o u l d b e readi ly p r e p a r e d at p H 4 in the p r e s e n c e of a citrate buffer a n d t h e n the externa l p H c o u l d b e ra ised to p H 7.5 fo l lowing the d e t e r g e n t d ia lys is p r o c e d u r e . A d d i t i o n of externa l C a C I 2 a n d i o n o p h o r e then resu l ted in l o a d i n g of C a 2 + into the S P L P to a c h i e v e internal c o n c e n t r a t i o n s of ~ 175 m M . H o w e v e r , a s s h o w n in F i g u r e 3.7, the p r e s e n c e of e n c a p s u l a t e d C a 2 + d id not result in s ign i f icant e n h a n c e m e n t of S P L P t ransfect ion p o t e n c y . 3.3.8 Effect of C a 2 * on S P L P systems containing cationic P E G lipids and higher levels of DODAC O n e of the l imitations with S P L P is that the s y s t e m is not readi ly t a k e n up by ce l l s a s a result of l imited ca t ion ic lipid a n d p r e s e n c e of P E G o n the v e s i c l e s ( M o k et a l . , 1999) . R e c e n t l y , a n e w c l a s s of ca t ion ic lipid k n o w n a s ca t ion ic p o l y ( e t h y l e n e g lycol ) lipid c o n j u g a t e s ( C P L ) h a s b e e n s y n t h e s i z e d in o u r laboratory ( C h e n et a l . , 2000 ) . C P L e m p l o y e d in this s t u d y c o n t a i n s a h y d r o p h o b i c c e r a m i d e a n c h o r , w h i c h is a t t a c h e d to a hydroph i l ic P E G s p a c e r that is l inked to a ca t ion ic h e a d g r o u p m a d e of four lys ine r e s i d u e s . It h a s b e e n s h o w n that C P L e n h a n c e in teract ions b e t w e e n the l i p o s o m e s a n d cel l p l a s m a m e m b r a n e , thus lead ing to h igher ce l lu lar b ind ing a n d 91 2 5 0 0 H 0 2 4 6 8 10 12 14 16 Ex te rna l C a c o n c e n t r a t i o n (mM) F i g u r e 3.7. E x t e r n a l C a 2 + w a s r e s p o n s i b l e for s t imulat ing t rans fec t ion . C a 2 + w a s l o a d e d e m p l o y i n g A 2 3 1 8 7 in the p r e s e n c e of a p H grad ien t a s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . Increas ing c o n c e n t r a t i o n s of C a 2 + (0 to 14 m M ) w e r e a d d e d to both S P L P (•) a n d C a 2 + - c o n t a i n i n g S P L P (•) prior to D M E M di lut ion. 0 .5 p g of p C M V L u c p l a s m i d e n c a p s u l a t e d in S P L P w e r e u s e d to t ransfect c e l l s p la ted at 1 x 1 0 4 ce l ls /we l l o f 96-wel l p la tes . L u c i f e r a s e activity w a s m e a s u r e d a s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e error b a r s r e p r e s e n t the s t a n d a r d d e v i a t i o n of th ree e x p e r i m e n t s . 92 u p t a k e ( C h e n et a l . , 2000) . E x p e r i m e n t s w e r e car r ied out to d e t e r m i n e the in f luence of C a 2 + o n the C P L - S P L P s y s t e m . In addi t ion , S P L P c o n t a i n i n g h i g h e r D O D A C c o n t e n t (14 m o l %) w e r e a l s o inves t iga ted , a s e n h a n c e d t rans fec t ion p o t e n c i e s h a v e b e e n o b s e r v e d for s u c h s y s t e m s ( Z h a n g et a l . , 1999) . F o r the C P L - S P L P p r e p a r a t i o n , p l a s m i d D N A w a s l o a d e d into l i p o s o m e s u s i n g the p r e v i o u s l y d e s c r i b e d d e t e r g e n t d ia lys is m e t h o d a n d C P L w e r e inser ted into the p r e f o r m e d S P L P u s i n g a wel l c h a r a c t e r i z e d insert ion m e t h o d ( F e n s k e et a l . , submi t ted) . C P L w e r e inser ted to obta in a f inal 4 m o l % insert ion e f f ic iency, a s this level h a s b e e n s h o w n to p r o v i d e g o o d ce l lu lar b ind ing a n d up take ( F e n s k e et a l . , submi t ted) . C a 2 + at 8 m M w a s a d d e d to the S P L P a n d C P L - S P L P p r e p a r a t i o n s , di luted into the m e d i a b e f o r e a p p l y i n g to the B H K ce l ls . G e n e e x p r e s s i o n w a s d e t e r m i n e d by a s s a y i n g for l u c i f e r a s e 2 4 h after incubat ion of the B H K ce l ls toge ther with the t ransfec t ing l i p o s o m e s . A s s h o w n in F i g u r e 3.8, C a 2 + w a s a l s o requ i red for the i m p r o v e d S P L P s y s t e m s . In part icular , a - 2 0 0 0 - f o l d i n c r e a s e a n d a 10 5 - fo ld i n c r e a s e in t rans fec t ion w e r e d e t e c t e d for the S P L P conta in ing ei ther h igher D O D A C c o n t e n t or C P L , r e s p e c t i v e l y . 93 D O D A C (14mol%) C P L (4 m o l % ) F i g u r e 3.8. E f fec t of C a o n S P L P s y s t e m s conta in ing ca t ion ic P E G l ipids or h i g h e r a m o u n t s of D O D A C . S P L P conta in ing h igher D O D A C c o n t e n t (14 m o l %) or C P L (4 m o l %) w e r e u s e d to t ransfect ce l ls in the p r e s e n c e ( d a s h e d bars ) or a b s e n c e ( o p e n bars ) of 8 m M C a 2 + . 0 .5 n g of p C M V L u c w a s u s e d in e a c h formula t ion in e a c h t rans fec t ion e x p e r i m e n t . C e l l s w e r e e x p o s e d to the v e s i c l e s for 2 4 h b e f o r e a s s a y i n g for L u c e x p r e s s i o n , a s out l ined in Mater ia ls a n d M e t h o d s . T h e er ror b a r s r e p r e s e n t the s t a n d a r d dev ia t ion of three e x p e r i m e n t s . 94 3.4 DISCUSSION T h i s s t u d y d e m o n s t r a t e s that C a 2 + g i v e s r ise to a la rge e n h a n c e m e n t of S P L P t rans fec t ion p o t e n c y in vitro. T h r e e a r e a s warrant further d i s c u s s i o n . T h e s e i n c l u d e the p o s s i b l e m e c h a n i s m s w h e r e b y C a 2 + resul ts in a n e n h a n c e d t rans fec t ion p o t e n c y of S P L P , the relat ion b e t w e e n the resul ts p r e s e n t e d h e r e a n d p r e v i o u s d e m o n s t r a t i o n s that C a 2 + c a n i m p r o v e the t ransfect ion p o t e n c y of p l a s m i d D N A -c a t i o n i c lipid c o m p l e x e s ( L a m a n d Cu l l i s , 2000) a n d the impl ica t ions for the d e s i g n of S P L P that exhibi t e n h a n c e d t ransfect ion p o t e n c y in v ivo . I will d i s c u s s t h e s e t o p i c s in turn. T h e m e c h a n i s m w h e r e b y C a 2 + s t imu la tes the t ransfec t ion p o t e n c y of S P L P m u s t a c c o u n t for s e v e r a l o b s e r v a t i o n s . First , the e n h a n c e d t rans fec t ion a p p e a r s to result f r o m h igher intracel lular leve ls of intact p l a s m i d in the p r e s e n c e of C a 2 + ; h o w e v e r t h e s e h igher leve ls of p l a s m i d d o not a r i se f rom i n c r e a s e d u p t a k e of S P L P into c e l l s . S e c o n d , the p r o c e s s is a s s o c i a t e d with a reduc t ion in the "punctate" a p p e a r a n c e of ce l l s fol lowing up take of f luorescent ly l a b e l e d S P L P . F ina l ly , the ef fect is C a 2 + s p e c i f i c . T h e first two o b s e r v a t i o n s a r e c lear ly c o n s i s t e n t with e n h a n c e d e n d o s o m a l destab i l i za t ion of the B H K ce l ls fo l lowing e n d o c y t o s i s of S P L P a n d the q u e s t i o n that r e m a i n s is h o w C a 2 + c o u l d p r o m o t e this des tab i l i za t ion in a s p e c i f i c m a n n e r . In this r e g a r d , there is p resent ly n o c o n s e n s u s a s to h o w e n d o s o m e s c a n b e d e s t a b i l i z e d to e n h a n c e r e l e a s e of their c o n t e n t s , h o w e v e r a n u m b e r of l e a d i n g o b s e r v a t i o n s h a v e b e e n m a d e . C h i e f a m o n g s t t h e s e is the o b s e r v a t i o n that ca t ion ic l ipids c a n dramat ica l ly e n h a n c e the intracel lular de l i ve ry of m a c r o m o l e c u l e s s u c h a s p l a s m i d s a n d a n t i s e n s e o l i g o n u c l e o t i d e s ( B a r r o n et a l . , 95 1999; B e n n e t t et a l . , 1992) a n d that this p r o c e s s a p p e a r s to rely o n a n ability of ca t ion ic l ipids to d e s t a b i l i z e e n d o s o m a l m e m b r a n e s , t h u s facil itating intracel lular r e l e a s e of e n d o s o m a l c o n t e n t s (Watt iaux et a l . , 1997; X u a n d S z o k a , 1996) . R e c e n t w o r k h a s s h o w n that ca t ion ic l ipids exhibit a s a g e n e r a l proper ty the ability to c o m b i n e with a n i o n i c l ipids to fo rm n o n - b i l a y e r h e x a g o n a l Hn p h a s e s t ructure ( H a f e z et a l . , 2 0 0 0 ; H a f e z a n d Cu l l i s , submi t ted) , l ead ing to the p r o p o s a l that the m e c h a n i s m w h e r e b y ca t ion ic l ipids des tab i l i ze e n d o s o m e s re l ies o n a n ability to d is rupt the b i layer o rgan iza t ion of the e n d o s o m a l m e m b r a n e . In the s a m e v e i n , if C a 2 + c o u l d d isrupt b i layer o r g a n i z a t i o n a n d i n d u c e Hu p h a s e s t ructure s imi la r e n h a n c e m e n t s in intracel lular de l ivery w o u l d b e e x p e c t e d . T h e r e is c o n s i d e r a b l e e v i d e n c e that C a 2 + c a n i n d u c e Hu p h a s e s t ructure in p r e v i o u s l y b i layer lipid s y s t e m s conta in ing a n i o n i c l ipids a n d that this ef fect is C a 2 + -s p e c i f i c in that o ther c a t i o n s s u c h a s M g 2 + e i ther c a n n o t i n d u c e Hu s t ructure or requi re h igher c o n c e n t r a t i o n s to p r o d u c e s imi lar e f fects (Bal ly et a l . , 1983; T i l c o c k et a l . , 1984) . A s s h o w n in this work , C a 2 + c a n i n d u c e Hu p h a s e st ructure in b i l ayers c o m p o s e d of D O P C : D O P E : D O P S : c h o l e s t e r o l a n d c a n act in s y n e r g y with low leve ls of the ca t ion ic lipid D O D A C to tr igger Hn p h a s e fo rmat ion . It is difficult to direct ly re late the m o d e l m e m b r a n e b e h a v i o u r to the b e h a v i o u r ins ide the e n d o s o m e , h o w e v e r it s h o u l d b e noted that up take of a n S P L P resul ts in de l ivery of on ly low leve ls of ca t ion ic lipid. A s s u m i n g the e n d o s o m e h a s a d i a m e t e r of 2 0 0 n m a n d the lipid c o m p o s i t i o n d e t e r m i n e d e l s e w h e r e ( K o b a y a s h i et a l . , 1998) it is s t ra ight forward to s h o w that u p t a k e of a 7 0 n m d i a m e t e r S P L P conta in ing 7 m o l % D O D A C p r o v i d e s o n l y 5 % of the ca t ion ic lipid n e c e s s a r y to fo rm ion pa i rs with all of the a n i o n i c lipid in 96 the e n d o s o m a l m e m b r a n e . T h i s con t ras ts with the si tuat ion for l i p o p l e x e s , a s u p t a k e of a l ipoplex s y s t e m of ~ 9 0 n m d i a m e t e r c o r r e s p o n d s to u p t a k e of suff ic ient ca t ion ic lipid a n d "helper" lipid to i n d u c e H M p h a s e o r g a n i z a t i o n for the ent ire e n d o s o m a l m e m b r a n e ( H a f e z a n d Cu l l i s , submit ted) . It is p o s s i b l e that the e n h a n c e d sensi t iv i ty of S P L P t rans fec t ion p o t e n c y to C a 2 + a s c o m p a r e d to l i p o p l e x e s m a y reflect this d i f f e rence . In part icular it m a y b e s u g g e s t e d that the u p t a k e of S P L P with low leve ls of ca t ion ic lipid resul ts in a r e q u i r e m e n t for C a 2 + in o r d e r to a c h i e v e m a x i m u m des tab i l i za t ion of the e n d o s o m a l m e m b r a n e , w h e r e a s u p t a k e of a l ipoplex resul ts in de l ive ry of suff ic ient ca t ion ic lipid a n d h e l p e r lipid to d e s t a b i l i z e the e n d o s o m a l m e m b r a n e i n d e p e n d e n t of C a 2 + l eve ls . In s u m m a r y , the resul ts repor ted h e r e s u p p o r t the possibi l i ty that C a 2 + e n h a n c e s t ransfect ion of S P L P by p r o m o t i n g e n d o s o m a l destab i l i za t ion in s y n e r g y with ca t ion ic lipid. S u c h a p r o p o s a l is a l s o in a g r e e m e n t with the o b s e r v a t i o n that the addi t ion of C a 2 + to l y s o b i s p h o s p h a t i d i c a c i d ( L B P A ) , a ma jor e n d o s o m a l a n i o n i c lipid ( K o b a y a s h i et a l . , 1998) , resul ts in fo rmat ion of the Hu p h a s e (I. H a f e z , u n p u b l i s h e d results) . A s u r p r i s i n g a s p e c t of this s t u d y c o n c e r n s the d i s c r e p a n c y b e t w e e n the in f luence of C a 2 + o n the t ransfect ion proper t ies of p l a s m i d D N A - c a t i o n i c lipid c o m p l e x e s p r e v i o u s l y repor ted ( L a m a n d Cu l l i s , 2000 ) a n d the resul ts repor ted h e r e for S P L P . In part icular , the p r e v i o u s work d e m o n s t r a t e d that C a 2 + c o u l d e n h a n c e the t rans fec t ion p o t e n c y of c o m p l e x e s by up to 20- fo ld a n d that this c o u l d b e attr ibuted to e n h a n c e d u p t a k e of the c o m p l e x e s into the ce l l s , rather t h a n e n h a n c e d e n d o s o m a l r e l e a s e . T h e surpr is ing a s p e c t is that the i n c r e a s e d t rans fec t ion p o t e n c y of S P L P in the p r e s e n c e of C a 2 + c o u l d not b e re lated to i n c r e a s e d u p t a k e of S P L P 9 7 by the c e l l s , w h e r e a s C a c a u s e d at least a 2-fold i n c r e a s e in u p t a k e of c o m p l e x e s a s e v i d e n c e d by u p t a k e of both lipid a n d p l a s m i d ( L a m a n d C u l l i s , 2000 ) . It is likely that this d i s c r e p a n c y is re lated to the m u c h different p h y s i c a l p roper t ies of S P L P a s c o m p a r e d to c o m p l e x e s . C o m p l e x e s a re large, posi t ive ly c h a r g e d s y s t e m s c o n t a i n i n g h igh (equimolar ) leve ls of ca t ion ic lipid w h e r e a s S P L P a r e s m a l l , s t a b l e , e s s e n t i a l l y neutra l v e s i c l e s with a P E G coa t ing that conta in low leve ls of ca t ion ic l ipid. T h e f inal top ic of d i s c u s s i o n c o n c e r n s e x t e n s i o n of the resul ts p r e s e n t e d h e r e to g e n e r a t e S P L P that exhibit e n h a n c e d t ransfect ion p o t e n c i e s in v ivo . A r e m a r k a b l e a s p e c t of the resul ts p r e s e n t e d h e r e is that the C a 2 + s t imulat ion of S P L P t rans fec t ion p o t e n c y is e v e n m o r e p r o n o u n c e d for s y s t e m s conta in ing C P L , w h i c h resul ts in e n h a n c e d u p t a k e into ce l l s . T h e s e resul ts c lear ly indicate that S P L P s h o u l d b e u s e d in c o n j u n c t i o n with C a 2 + w h e n e v e r p o s s i b l e . A s e m p h a s i z e d e l s e w h e r e ( W h e e l e r et a l . , 1999 , Z h a n g et a l . , 1999) , S P L P h a v e b e e n d e s i g n e d for s y s t e m i c a p p l i c a t i o n s w h e r e long c i rculat ion l i fet imes a n d a c c u m u l a t i o n at d i s e a s e s i tes s u c h a s t u m o u r s i tes is r e q u i r e d . A c h i e v e m e n t of e n h a n c e d t ransfect ion p o t e n c y by i n c r e a s i n g the local c o n c e n t r a t i o n of C a 2 + in v ivo is not a stra ightforward p r o p o s i t i o n . A s ind ica ted h e r e , e n c a p s u l a t i o n of C a 2 + within the S P L P did not result in i n c r e a s e d t rans fec t ion p o t e n c y , indicat ing that C a 2 + is requ i red o u t s i d e the S P L P in o r d e r to g ive r ise to e n h a n c e d t rans fec t ion . It is p o s s i b l e that s t ra teg ies a i m e d at i n c r e a s i n g s u r f a c e C a 2 + c o n c e n t r a t i o n s by a t t a c h m e n t of C a 2 + - c h e l a t i n g a g e n t s to S P L P m a y g i v e r ise to e n h a n c e d in v ivo t ransfect ion a n d t h e s e a n d re lated s t ra teg ies a r e u n d e r ac t i ve invest iga t ion . 98 In s u m m a r y the resul ts p r e s e n t e d h e r e d e m o n s t r a t e that C a d ramat ica l l y e n h a n c e s the t ransfec t ion p o t e n c y of S P L P in vitro. T h e effect is C a 2 + - s p e c i f i c a n d a p p e a r s to ref lect i n c r e a s e d destab i l i za t ion of the e n d o s o m a l m e m b r a n e with a s s o c i a t e d r e l e a s e of S P L P - a s s o c i a t e d p l a s m i d into the c y t o p l a s m rather t h a n i n c r e a s e d ce l lu lar up take of S P L P . F inal ly , the ability of C a 2 + to d e s t a b i l i z e the e n d o s o m a l m e m b r a n e w o u l d b e c o n s i s t e n t with its ability to i n d u c e n o n - b i l a y e r Hu p h a s e s t ructure in m o d e l m e m b r a n e s y s t e m s in s y n e r g y with ca t ion ic l ipids. 99 C H A P T E R 4 SUMMARY AND FUTURE DIRECTIONS 4.1 SUMMARY T h e s t u d i e s p e r f o r m e d in this work w e r e a i m e d at e x a m i n i n g the ef fect of C a 2 + o n two l i p i d - b a s e d g e n e t ransfer s y s t e m s . W e first e x a m i n e d a n d c h a r a c t e r i z e d the ef fect of C a 2 + o n the p l a s m i d D N A - l i p i d c o m p l e x e s . T h e n the in f luence C a 2 + h a s o n the S P L P s y s t e m a n d its m e c h a n i s m of st imulat ing t ransfec t ion w a s inves t iga ted a n d c o m p a r e d with the larger c o m p l e x e s . C h a p t e r 2 d e s c r i b e s the o b s e r v a t i o n that inc lus ion of C a 2 + s igni f icant ly i n c r e a s e d the t ransfect ion p o t e n c y of p l a s m i d D N A - l i p i d c o m p l e x e s a n d the s u b s e q u e n t e x p e r i m e n t s d e s i g n e d to c h a r a c t e r i z e the C a 2 + effect. E v i d e n c e of C a 2 + act ing a s a c o f a c t o r for ca t ion ic l i p o s o m e - m e d i a t e d t ransfec t ion c o m e s f r o m the o b s e r v a t i o n that ce l l s t r a n s f e c t e d with C a 2 + a n d p l a s m i d D N A a l o n e d id not l e a d to a n y t r a n s g e n e activity. T h e speci f ic i ty of C a 2 + w a s ver i f ied by c o m p a r i s o n s with o ther c a t i o n s s u c h a s M g 2 + a n d N a + , a s wel l a s by the d e m o n s t r a t i o n of the inhibitory ef fect of E G T A . F u r t h e r m o r e , s i n c e the other c a t i o n s did not s t imula te s imi lar i n c r e a s e s in t ransfec t ion , the C a 2 + - m e d i a t e d e n h a n c e m e n t w a s likely not a s s o c i a t e d with a n e lect rosta t ic ef fect b e t w e e n the c a t i o n s a n d the p l a s m i d D N A - l i p i d c o m p l e x e s . S t imula t ion of t ransfect ion w a s d e p e n d e n t o n the C a 2 + c o n c e n t r a t i o n , with op t ima l t ransfec t ion rang ing f rom 6 to 2 5 m M . Cytotoxic i ty w a s o b s e r v e d at 5 0 m M C a 2 + o r h igher , indicat ing that ce l ls c o u l d on ly to lerate ext r ins ic C a 2 + c o n c e n t r a t i o n b e l o w 5 0 m M in the p r e s e n c e of ca t ion ic l i p o s o m e s . A s e v i d e n c e d 100 f r o m f l u o r e s c e n c e a n a l y s i s of ce l ls t rans fec ted with the p l a s m i d e n c o d i n g G F P , the i n c r e a s e in the c o m p l e x t ransfect ion p o t e n c y w a s re lated to a h igher p o p u l a t i o n of ce l l s e x p r e s s i n g the t r a n s g e n e . T h e rate of t ransfect ion w a s f o u n d to b e faster , s u g g e s t i n g that C a 2 + faci l i tated the ce l lu lar de l ivery of p l a s m i d D N A - l i p i d c o m p l e x e s . T h i s w a s c o n f i r m e d b y resul ts f r o m kinet ic a n a l y s i s of r a d i o l a b e l e d - l i p i d s a n d d o t blot a s s a y s of p l a s m i d D N A , in w h i c h the de l ivery of p l a s m i d D N A - l i p i d c o m p l e x e s w a s e n h a n c e d f r o m 2- to 4 - fo ld . M o r e signif icant ly , a h igher level of intact p l a s m i d D N A w a s o b s e r v e d ins ide the ce l ls w h e n C a 2 + w a s i n c l u d e d . F ina l ly , it w a s the C a 2 + a d d e d s i m u l t a n e o u s l y with p l a s m i d D N A a n d ca t ion ic l i p o s o m e s that g a v e r ise to the h i g h e s t t ransfec t ion e n h a n c e m e n t . T h e s e resul ts s u g g e s t that it w a s likely the c o m p l e x - a s s o c i a t e d C a 2 + , rather than f ree C a 2 + , that w a s ult imately r e s p o n s i b l e for facil i tating t rans fec t ion . T h e g e n e r a l app l ica t ion of C a 2 + a s a n eff icient c o f a c t o r w a s c o n f i r m e d by eva lua t ing its in f luence in a n u m b e r of different ca t ion ic l i p o s o m a l f o r m u l a t i o n s a s wel l a s by t ransfect ing a n u m b e r of different cel l l ines . T h e f inding that C a 2 + e n h a n c e d p l a s m i d D N A - l i p i d c o m p l e x t rans fec t ion p o t e n c y s u g g e s t e d that it c o u l d exhibit s imi lar s t imulatory e f fects for S P L P , a s y s t e m with p h y s i c a l charac te r is t i cs m o r e su i tab le for in v ivo app l ica t ion but w h i c h su f fe rs f r o m limited t ransfec t ion activit ies. C h a p t e r 3 p r e s e n t e d the resul ts of the e x p e r i m e n t s d e s i g n e d to e x a m i n e the in f luence of C a 2 + o n S P L P . T r a n s f e c t i o n d a t a s h o w e d that a >600-fold i n c r e a s e in S P L P t ransfect ion e f f i c ienc ies w a s a c h i e v e d in the p r e s e n c e of C a 2 + , indicat ing that C a 2 + exh ib i ted a m o r e potent ef fect for S P L P t h a n for the c o m p l e x e s . Q E L s h o w e d that the v e s i c l e d i a m e t e r a n d s i z e distr ibut ion r e m a i n e d u n a l t e r e d , a n d p i c o g r e e n D N A l e a k a g e a s s a y s by incuba t ing S P L P at 101 3 7 ° C a n d 1 0 % F B S s h o w e d that p l a s m i d D N A w a s still e n c a p s u l a t e d ins ide v e s i c l e b i l ayers in the p r e s e n c e of C a 2 + . T h e s e resul ts c lear ly d e m o n s t r a t e d that S P L P m a i n t a i n e d their integrity, at least prior to ce l lu lar entry. S imi la r to the c o m p l e x e s , the C a 2 + ef fect o n S P L P w a s f o u n d to b e qui te spec i f i c . O n the o ther h a n d , the op t ima l C a 2 + r a n g e w a s lower a n d nar rower (8 to 10 m M ) with S P L P w h e n c o m p a r e d to the c o m p l e x e s . It w a s a l s o u n e x p e c t e d that, d e s p i t e the d r a m a t i c e n h a n c e m e n t in t rans fec t ion activity, n o s igni f icant i n c r e a s e in S P L P de l ivery w a s d e t e c t e d . W e h y p o t h e s i z e d that C a 2 + c o u l d a s s i s t in o v e r c o m i n g a n intracel lular barr ier that c o u l d potent ia l ly l e a d to a n i n c r e a s e in t ransfec t ion . O n e o b v i o u s barr ier for S P L P -m e d i a t e d g e n e t ransfer w a s the e s c a p e f rom e n d o s o m e s a n d r e l e a s e of the p l a s m i d D N A b e i n g re ta ined ins ide the v e s i c l e s . E v i d e n c e s u g g e s t i n g that e n d o s o m a l des tab i l i za t ion o c c u r r e d c a m e initially f rom f l u o r e s c e n c e s t u d i e s of ce l l s t r a n s f e c t e d with R h - l a b e l e d L U V with a lipid c o m p o s i t i o n s imi lar to S P L P , w h i c h exh ib i ted a m o r e d i f fuse rather than p u n c t a t e c y t o p l a s m i c distr ibution in the p r e s e n c e of C a 2 + . N o r m a l l y , e n d o s o m e s m a t u r e into l y s o s o m e s a n d their internal conten t is s u b j e c t e d to d e g r a d a t i o n by l y s o s o m a l e n z y m e s . B y destab i l i z ing e n d o s o m e s , the d e l i v e r e d p l a s m i d D N A c o u l d better e s c a p e the d e g r a d a t i v e p a t h w a y . Indeed , both dot blot a n d S o u t h e r n blot a n a l y s i s s h o w e d that i n c r e a s e d leve ls (2- to 10-fold) of intact p l a s m i d D N A w a s d e t e c t e d in the p r e s e n c e of C a 2 + . F u r t h e r m o r e , n o s u c h i n c r e a s e in intact p l a s m i d w a s d e t e c t e d w h e n M g 2 + w a s e m p l o y e d , indicat ing that e n d o s o m a l des tab i l i za t ion is faci l i tated spec i f ica l ly by C a 2 + . U s i n g 3 1 P N M R a n d a m o d e l m e m b r a n e s y s t e m , w e d e m o n s t r a t e d that C a 2 + h a d the ability to m e d i a t e lipid p h a s e t rans i t ions f r o m the b i layer to Hu p h a s e . In addi t ion , the N M R resul ts s h o w e d that 102 C a w o r k s in s y n e r g y with the ca t ion ic lipid. Lipid reorgan iza t ion f rom b i layer to Hn h a s b e e n s u g g e s t e d to s igni fy m e m b r a n e f u s i o n , a n d recent f ind ings ind ica te that ca t ion ic lipid p l a y s a n important role in p romot ing H M p h a s e s t ructure ( H a f e z a n d C u l l i s , submi t ted ) . T h e s y n e r g i s t i c effect b e t w e e n C a 2 + a n d ca t ion ic lipid is further d e m o n s t r a t e d w h e n e v e n h igher t ransfect ion e n h a n c e m e n t w a s o b s e r v e d w h e n C a 2 + w a s e m p l o y e d in S P L P s y s t e m s conta in ing h igher ca t ion ic lipid conten t . A ~ 2 0 0 0 - f o l d i n c r e a s e in t ransfect ion activity w a s a c h i e v e d u s i n g a S P L P formula t ion c o n t a i n i n g h igher D O D A C conten t (14 m o l %) a n d a 10 5 - fo ld i n c r e a s e in t rans fec t ion with the C P L - S P L P s y s t e m , w h i c h c o n t a i n s a n o v e l ca t ion ic lipid c o a t e d o n the v e s i c l e s u r f a c e . 103 4.2 FUTURE DIRECTIONS T h e invest igat ions d e s c r i b e d in this s t u d y in t roduced a n e w a r e a in the d e v e l o p m e n t of l i p o s o m a l - m e d i a t e d t ransfect ion s y s t e m s , at the s a m e t ime n e w q u e s t i o n s a r e b rought forth. A l t h o u g h our o b s e r v a t i o n s a l low u s to p r o p o s e s o m e p o s s i b l e p a t h w a y s in w h i c h C a 2 + faci l i tates e n h a n c e d t ransfec t ion , the u n d e r l y i n g m e c h a n i s m of h o w C a 2 + s t imula tes t ransfect ion is still unc lea r . It is not a p p a r e n t w h y C a 2 + is on ly ef fect ive within a cer ta in c o n c e n t r a t i o n r a n g e a n d w h y the work ing r a n g e w o u l d vary b e t w e e n the c o m p l e x e s a n d S P L P . B y c o m p a r i s o n , the S P L P w a s m o r e sens i t i ve to the C a 2 + c o n c e n t r a t i o n than the la rger c o m p l e x e s , in that t ransfect ion e f f ic iency r ises dramat ica l ly at 8 to 10 m M a n d w a s a l m o s t a s d ramat ica l l y inhibited at h igher c o n c e n t r a t i o n s . Un l ike the c o m p l e x e s , in w h i c h cel l toxicity w a s cor re la ted with l e s s g e n e e x p r e s s i o n at h igher C a 2 + , n o tox ic ef fect w a s o b s e r v e d with S P L P . In addi t ion to dr iving e n d o s o m e destab i l i za t ion , it is p o s s i b l e that the influx of C a 2 + with the l i p o s o m e s c o u l d affect the C a 2 + g rad ient a c r o s s the cel l p l a s m a m e m b r a n e . B e i n g a potent intracel lular m e s s e n g e r , C a 2 + is k n o w n for its ef fect in m o d u l a t i n g v a r i o u s ce l lu lar func t ions . A s a result , ce l lu lar act ivi t ies that c o u l d potent ia l ly affect g e n e e x p r e s s i o n might b e s t imula ted with the influx of C a 2 + . It w o u l d b e use fu l to d e t e r m i n e the leve ls of C a 2 + influx dur ing t rans fec t ion m e d i a t e d by l i p o s o m e s . E x a m i n a t i o n of s p e c i f i c prote ins that c o u l d b e tu rned o n by C a 2 + c o u l d a l s o b e a n indicator to d e t e r m i n e w h e t h e r o ther ce l lu lar activity w a s a f fec ted by the C a 2 + influx. T h e pre l iminary o b s e r v a t i o n s noted in C h a p t e r 3 that C a 2 + exhib i ts e v e n 104 g r e a t e r s t imulat ion for S P L P conta in ing C P L or m o r e ca t ion ic lipid a l s o n e e d to b e c h a r a c t e r i z e d further to d e t e r m i n e the m a x i m u m leve ls of t rans fec t ion that m a y b e a c h i e v a b l e . A l s o , it is of part icular i m p o r t a n c e to try to e x t e n d t h e s e resu l ts to in v ivo s i tua t ions . It is p o s s i b l e that incorporat ion of C a 2 + c h e l a t o r s or c o - d e l i v e r y of C a 2 + in l i p o s o m e s m a y p r o v e use fu l . T h e interest in l i p o s o m e s a s carr iers of m a c r o m o l e c u l e s is b a s e d o n their potent ia l to e n c l o s e a n d protect d i v e r s e mater ia ls of b io log ica l interest a n d to de l i ve r t h e m , funct ional ly intact a n d in s igni f icant quant i t ies , to the interior of m a n y ce l l t y p e s . In o r d e r to g e n e r a t e the m o s t eff icient l i p o s o m e s a s g e n e car r ie rs , both the ex t race l lu la r a n d intracel lular barr iers m u s t b e o v e r c o m e . T h e p r e s e n c e of P E G c o a t i n g o n the S P L P v e s i c l e s a p p e a r s to o v e r c o m e the ext race l lu lar barr iers , w h i c h i n c l u d e o p s o n i n s , p h a g o c y t e s , ext race l lu lar matrix, a n d d e g r a d a t i v e e n z y m e s . T h e cur rent i m p r o v e m e n t in the t ransfect ion p o t e n c i e s of S P L P u s i n g C a 2 + a p p e a r s to b e a s s o c i a t e d with a n intracel lular effect. It w o u l d b e use fu l to e x a m i n e o ther e n d o s o m a l des tab i l i z ing a g e n t s , e s p e c i a l l y o n e s that c o u l d b e e n c a p s u l a t e d with the p l a s m i d D N A . F ina l ly , s i n c e the addi t ion of posi t ive c h a r g e s o n the S P L P is b a s e d o n n o n - s p e c i f i c target ing of e n g i n e e r e d l i p o s o m e s , it w o u l d b e interest ing to c o m b i n e the C a 2 + e f fect with the a t t a c h m e n t of a s p e c i f i c target ing e l e m e n t onto the S P L P in o r d e r to i m p r o v e the speci f ic i ty of g e n e e x p r e s s i o n . 105 R E F E R E N C E S A l l e n , T . M . (1994) . Long-c i rcu la t ing (sterical ly s tabi l ized) l i p o s o m e s for t a rge ted d r u g de l ivery . Trends in Pharmacological Sciences, 15 :215 -220 . A l m o g , S . , K u s h n i r , T . , Nir, S . , a n d L i c h t e n b e r g , D. (1986). K inet ic a n d structura l a s p e c t s of reconst i tut ion of 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 by di lut ion of p h o s p h a t i d y l c h o l i n e - s o d i u m c h o l a t e m i x e d m i c e l l e s . Biochemistry, 2 5 : 2 5 9 7 - 2 6 0 5 . A l t o n , E . W . a n d G e d d e s , D . M . (1995). G e n e t h e r a p y for c y s t i c f ibros is : a c l in ical p e r s p e c t i v e . Gene Therapy, 2 :88 -95 . A n d e r s o n , W . F . (1992) H u m a n g e n e therapy . Science, 2 5 6 : 8 0 8 - 8 1 3 . B a d i n g , H . , H a r d i n g h a m , G . E . , J o h n s o n , C M . , a n d C h a w l a , S . (1997) . G e n e regula t ion by n u c l e a r a n d c y t o p l a s m i c c a l c i u m s i g n a l s . Biochem Biophys Res Commun, 2 3 6 : 5 4 1 - 5 4 3 . B a k e r , D, W u e s t e h u b e , L., S c h e k m a n , R., a n d S e g e v , N . (1990) . G T P - b i n d i n g Ypt1 prote in a n d C a 2 + funct ion i n d e p e n d e n t l y in a ce l l - f ree protein t ranspor t reac t ion . Proc. Natl. Acad. Sci. USA, 8 7 : 3 5 5 - 3 5 9 . B a k e r , P . F . a n d Knight , D . E . (1984). C h e m i o s m o t i c h y p o t h e s e s of e x o c y t o s i s : a cr i t ique. R e v i e w . Bioscience Reports, 4 :285 -298 . Ba l ly , M . B . , T i l c o c k , C P . , H o p e , M . J . , a n d Cu l l i s , P . R . (1983) . P o l y m o r p h i s m of p h o s p h a t i d y l e t h a n o l a m i n e - p h o s p h a t i d y l s e r i n e m o d e l s y s t e m s : i n f l u e n c e of c h o l e s t e r o l a n d M g 2 + o n C a ^ - t r i g g e r e d b i layer to h e x a g o n a l ( H „ ) t ransi t ion. Canadian Journal of Biochemistry & Cell Biology 61 , 3 4 6 - 3 5 2 . B a n g h a m , A . D . , S t a n d i s h , M . M . , a n d W a t k i n s , J . C . (1965). D i f fus ion of un iva lent i o n s a c r o s s the l a m e l l a e of s w o l l e n p h o s p h o l i p i d s . Journal of Molecular Biology, 13 :238 -252 . B a r e n h o l z , Y . , A m s e l e m , S . , a n d L i c h t e n b e r g , D. (1979). A n e w m e t h o d for p r e p a 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 ( l i p o s o m e s ) - F r e n c h p r e s s . FEBS Letters, 9 9 : 2 1 0 - 2 1 4 . B a r r o n , L . G . , U y e c h i , L . S . , a n d S z o k a , F . C . J . (1999). C a t i o n i c l ipids a r e e s s e n t i a l for g e n e de l ive ry m e d i a t e d by i n t r a v e n o u s admin is t ra t ion of l i p o p l e x e s . Gene Ther, 6 : 1 1 7 9 - 1 1 8 3 . B a r u , M . , A x e l r o d , J . H . , a n d Nur , I. (1995). L i p o s o m e - e n c a p s u l a t e d D N A - m e d i a t e d g e n e t ransfer a n d s y n t h e s i s o f h u m a n factor IX in m i c e . Gene, 1 6 1 : 1 4 3 - 1 5 0 . 106 B e c k e r s , C . J . M . a n d B a l c h W . E . (1989). C a 2 + a n d G T P : e s s e n t i a l c o m p o n e n t s in v e s i c u l a r traff icking b e t w e e n the e n d o p l a s m i c ret iculum a n d G o l g i a p p a r a t u s . J. Cell Biol., 1 0 8 : 1 2 4 5 - 1 2 5 6 . B e n n e t t , C . F . , C h i a n g , M . Y . , C h a n , H . , S h o e m a k e r , J . E . , a n d Mirabel l i , C . K . (1992) . C a t i o n i c l ipids e n h a n c e cel lu lar up take a n d activity of p h o s p h o r o t h i o a t e a n t i s e n s e o l i g o n u c l e o t i d e s . Mol Pharmacol, 4 1 : 1 0 2 3 - 1 0 3 3 . B e r r i d g e , M . J . (1993). Inositol t r i s p h o s p h a t e a n d c a l c i u m s igna l l ing . Nature, 3 6 1 : 3 1 5 - 3 2 5 . B h a t t a c h a r y a , S . a n d M a n d a l , S . S . (1998). E v i d e n c e of interl ipidic ion -pa i r ing in a n i o n - i n d u c e d D N A r e l e a s e f rom ca t ion ic a m p h i p h i l e - D N A c o m p l e x e s . M e c h a n i s t i c imp l ica t ions in t ransfec t ion . Biochemistry, 3 7 : 7 7 6 4 - 7 7 7 7 . Bot tger , M . , Z a i t s e v , S . V . , Ot to , A . , H a b e r l a n d , A . , a n d V o r o b ' e v , V.I . (1998) . A c i d n u c l e a r ex t rac ts a s m e d i a t o r s of g e n e t ransfer a n d e x p r e s s i o n . Biochimica et Biophysica Acta, 1395 :78 -87 . B o u l i k a s , T a n d Mart in , F. (1997). H i s t o n e s , p r o t a m i n e , a n d p o l y l y s i n e but not p o l y ( E : K ) e n h a n c e t ransfect ion e f f ic iency. International J o u r n a l o f O n c o l o g y 10, 3 1 7 -3 2 2 . C h e n , T . , W o n g , K . F . , F e n s k e , D . B . , P a l m e r , L . R . , a n d C u l l i s , P . R . (2000) . F l u o r e s c e n t - l a b e l e d po ly (e thy lene glycol ) lipid c o n j u g a t e s with distal ca t ion ic h e a d g r o u p s . Bioconjugate Chem., 11 :433 -437 . C h e r n o m o r d i k , L . V . a n d Z i m m e r b e r g , J . (1995). B e n d i n g m e m b r a n e s to the task: structural in te rmed ia tes in b i layer f u s i o n . Current Opinion in Structural Biology, 5 :541 -547 . C h e r n o m o r d i k , L .V . , K o z l o v , M . M . , M e l i k y a n , G . B . , A b i d o r , I.G., M a r k i n , V . S . , a n d C h i z m a d z h e v , Y . A . (1985). T h e s h a p e of lipid m o l e c u l e s a n d m o n o l a y e r m e m b r a n e f u s i o n . Biochim Biophys Acta, 8 1 2 : 6 4 3 - 6 5 5 . C h e r n o m o r d i k , L . V . , L e i k i n a , E . , F r o l o v , V . , B r o n k , P. , a n d Z i m m e r b e r g , J . (1997) . A n ear ly s t a g e of m e m b r a n e f u s i o n m e d i a t e d by the low p H c o n f o r m a t i o n of i n f l u e n z a h e m a g g l u t i n i n d e p e n d s u p o n m e m b r a n e l ipids. J. Cell Biol., 136 :81 -93 . C h i o u , H . C . , T a n g c o , M . V . , L e v i n e , S . M . , R o b e r t s o n , D., K o r m i s , K., W u , C . H . , a n d W u , G . Y . (1994) . E n h a n c e d r e s i s t a n c e to n u c l e a s e d e g r a d a t i o n of n u c l e i c a c i d s c o m p l e x e d to a s i a l o g l y c o p r o t e i n - p o l y l y s i n e carr iers . Nucleic Acids Research, 2 2 : 5 4 3 9 - 5 4 4 6 . C h o n n , A . a n d Cu l l i s , P . R . (1995). R e c e n t a d v a n c e s in l i p o s o m a l d r u g - d e l i v e r y s y s t e m s . Current Opinion in Biotechnology, 6 :698 -708 . C l a p h a m , D . E . (1995) . C a l c i u m s igna l ing . Cell, 80 :259 -268 . 107 C o l o m b o , M.I., B e r o n W . , a n d S t a h l , P . D . (1997). C a l m o d u l i n r e g u l a t e s e n d o s o m e f u s i o n . J. Biol. Chem., 2 7 2 : 7 7 0 7 - 7 7 1 2 . C u l l i s , P . R . a n d d e Kruijff, B. (1978). P o l y m o r p h i c p h a s e b e h a v i o u r of lipid mix tures a s d e t e c t e d by 3 1 P N M R . E v i d e n c e that c h o l e s t e r o l m a y d e s t a b i l i z e b i layer s t ructure in m e m b r a n e s y s t e m s conta in ing p h o s p h a t i d y l e t h a n o l a m i n e . Biochimica et Biophysica Acta, 5 0 7 : 2 0 7 - 2 1 8 . C u l l i s , P . R . a n d d e Kruijff, B . (1979). Lipid p o l y m o r p h i s m a n d the funct iona l ro les of l ipids in b io log ica l m e m b r a n e s . Biochimica et Biophysica Acta, 5 5 9 : 3 9 9 - 4 2 0 . C u l l i s , P . R . a n d H o p e , M . J . (1985). P h y s i c a l p roper t ies a n d funct iona l ro les of l ipids in m e m b r a n e s . Biochemistry of lipids and membranes, J . E . V a n c e a n d D . E . V a n c e , ed i tors . B e n j a m i n / C u m m i n g s , M e n l o Park , C A . , 2 5 - 7 2 . C u l l i s , P . R . , H o p e , M . J . , a n d T i l c o c k , C P . (1986). L ip id p o l y m o r p h i s m a n d the ro les of l ipids in m e m b r a n e s . Chemistry & Physics of Lipids, 4 0 : 1 2 7 - 1 4 4 . C u l l i s , P . R . , H o p e , M . J . , d e Kruijff, B . , Verk le i j , A . , a n d T i l c o c k , C P . S . (1985) . St ructura l p roper t ies a n d funct iona l ro les of p h o s p h o l i p i d s in m e m b r n e s . Phospholipids and Cellular Regulation, J . F . K u o (Ed. ) C R C P r e s s , B o c a R a t o n , F l o r i d a , C h a p t e r 1:1-60. D e n c h e r , N A . a n d H e y n , M . P . (1978). F o r m a t i o n a n d p roper t i es of b a c t e r i o r h o d o p s i n m o n o m e r s in the n o n - i o n i c d e t e r g e n t s o c t y l - b e t a - D - g l u c o s i d e a n d Tr i ton X - 1 0 0 . FEBS Letters, 9 6 : 3 2 2 - 3 2 6 . D z a u , V . J . , M a n n , M . J . , Mor ish i ta , R., & K a n e d a , Y . (1996) . F u s i g e n i c viral l i p o s o m e for g e n e t h e r a p y in c a r d i o v a s c u l a r d i s e a s e s . Proceedings of the National Academy of Sciences of the United States of America 93, 1 1 4 2 1 - 1 1 4 2 5 . E d w a r d s o n , J . M . , A n , S . , a n d J a h n , R. (1997). T h e s e c r e t o r y g r a n u l e prote in s y n c o l l i n b i n d s to syntax in in a C a 2 + - s e n s i t i v e m a n n e r . Cell, 9 0 : 3 2 5 - 3 3 3 . E i b l , H . a n d W o o l e y , P . (1979). E lec t ros ta t ic in teract ions at c h a r g e d lipid m e m b r a n e s . H y d r o g e n b o n d s in lipid m e m b r a n e s u r f a c e s . Biophys.Chem., 10 :261 -2 7 1 . E l O u a h a b i , A . , Th i ry , M . , P e c t o r , V . , F u k s , R., R u y s s c h a e r t , J . M . , a n d V a n d e n b r a n d e n , M . (1997). T h e role of e n d o s o m e destab i l i z ing activity in the g e n e t rans fe r p r o c e s s m e d i a t e d by ca t ion ic l ipids. FEBS Letters, 4 1 4 : 1 8 7 - 1 9 2 . E l i a s s o n , L. , P r o k s , P . , A m m a l a , C , A s h c r o f t , F . M . , Bokv is t , K., R e n s t r o m , E . , R o r s m a n , P . , a n d S m i t h , P A . (1996). E n d o c y t o s i s of s e c r e t o r y g r a n u l e s in m o u s e p a n c r e a t i c b e t a - c e l l s e v o k e d by t ransient e leva t ion of c y t o s o l i c c a l c i u m . Journal of Physiology, 4 9 3 : 7 5 5 - 7 6 7 . 108 E p s t e i n , R . J . , Druker , B . J . , Irminger, J . C , J o n e s , S . D . , R o b e r t s , T . M . , a n d St i les , C D . (1992) . Ex t race l lu la r c a l c i u m m i m i c s the a c t i o n s of p la te le t -der ived growth fac tor o n m o u s e f ibroblasts . Cell Growth & Differentiation, 3 :157 -164 . E r x l e b e n , C , K l a u k e , N. , F l o t e n m e y e r , M . , B l a n c h a r d , M - P . , B r a u n , C , a n d Plattner , H. (1997) . Microdomain Ca2+ activation during exocytosis in Paramecium cells. Superimposition of local subplasmalemmal Ca2+ store activation by local Ca2+ influx. J. Cell. Biol., 1 3 6 : 5 9 7 - 6 0 7 . F a r h o o d , H . , S e r b i n a , N. , a n d H u a n g , L. (1995). T h e role of d io leoy l p h o s p h a t i d y l e t h a n o l a m i n e in ca t ion ic l i p o s o m e m e d i a t e d g e n e t ransfer . Biochimica et Biophysica Acta, 1 2 3 5 : 2 8 9 - 2 9 5 . F a s b e n d e r , A . J . , Z a b n e r , J . , a n d W e l s h , M . J . (1995). Op t im iza t ion of ca t ion ic l ipid-m e d i a t e d g e n e t ransfer to a i rway epi the l ia . American Journal of Physiology, 2 6 9 : L 4 5 - L 5 1 . F e i g n e r , J . H . , K u m a r , R., S r idhar , C . N . , W h e e l e r , C . J . , T s a i , Y . J . , B o r d e r , R., R a m s e y , P . , Mar t in , M . , a n d F e i g n e r , P . L . (1994). E n h a n c e d g e n e de l ive ry a n d m e c h a n i s m s t u d i e s with a n o v e l s e r i e s of ca t ion ic lipid fo rmula t ions . Journal of Biological Chemistry, 2 6 9 : 2 5 5 0 - 2 5 6 1 . F e i g n e r , P . L . (1997) . Nonv i ra l s t ra teg ies for g e n e therapy . Scientific American, 2 7 6 : 1 0 2 - 1 0 6 . F e i g n e r , P . L . a n d R i n g o l d , G . M . (1989). C a t i o n i c l i p o s o m e - m e d i a t e d t rans fec t ion . Nature, 3 3 7 : 3 8 7 - 3 8 8 . F e i g n e r , P . L . , G a d e k , T . R . , H o l m , M . , R o m a n , R., C h a n , H . W . , W e n z , M . , Nor th rop , J . P . , R i n g o l d , G . M . , a n d D a n i e l s e n , M . (1987). L ipo fec t ion : a h ighly eff icient, l ipid-m e d i a t e d D N A - t r a n s f e c t i o n p r o c e d u r e . Proceedings of the National Academy of Sciences of the United States of America, 8 4 : 7 4 1 3 - 7 4 1 7 . F e i g n e r , P . L . , T s a i , Y . J . , S u k h u , L., W h e e l e r , C . J . , M a n t h o r p e , M . , M a r s h a l l , J . , a n d C h e n g , S . H . (1995). Improved ca t ion ic lipid fo rmula t ions for in v ivo g e n e t h e r a p y . Annals of the New York Academy of Sciences, 7 7 2 : 1 2 6 - 1 3 9 . F e n s k e , D . B . , P a l m e r , L .R . , C h e n , T . , W o n g , K . F . , a n d C u l l i s , P . R . , (2000) C a t i o n i c P E G - l i p i d s i n c o r p o r a t e d into p r e - f o r m e d v e s i c l e s e n h a n c e b ind ing a n d u p t a k e to B H K c e l l s . S u b m i t t e d to publ icat ion in Biochimica et Biophysica Acta. Fewtre l l , C . (1993) . C a 2 + osc i l la t ions in n o n - e x c i t a b l e ce l l s . Annual Review of Physiology, 5 5 : 4 2 7 - 4 5 4 . F i s k e , C . H . a n d S u b b a r o w , Y . (1925). T h e co lor imetr ic d e t e r m i n a t i o n of p h o s p h o r u s . Journal of Biological Chemistry 66 , 3 7 5 - 3 7 9 . 109 F r a l e y , R., S u b r a m a n i , S . , B e r g , P. , a n d P a p a h a d j o p o u l o s , D. (1980) . Introduction of l i p o s o m e - e n c a p s u l a t e d S V 4 0 D N A into ce l ls . Journal of Biological Chemistry, 2 5 5 : 1 0 4 3 1 - 1 0 4 3 5 . F r a l e y , R . T . , F o r n a r i , C . S . , a n d K a p l a n , S . (1979). E n t r a p m e n t of a bacter ia l p l a s m i d in p h o s p h o l i p i d v e s i c l e s : potential for g e n e t ransfer . Proceedings of the National Academy of Sciences of the United States of America, 7 6 : 3 3 4 8 - 3 3 5 2 . F r i e n d , D . S . , P a p a h a d j o p o u l o s , D., a n d D e b s , R . J . (1996) . E n d o c y t o s i s a n d intracel lular p r o c e s s i n g a c c o m p a n y i n g t ransfect ion m e d i a t e d by ca t ion ic l i p o s o m e s . Biochimica et Biophysica Acta, 1278 :41 -50 . Fur th , A . J . (1980) . R e m o v i n g u n b o u n d de te rgent f rom h y d r o p h o b i c p ro te ins . Analytical Biochemistry, 109 :207 -215 . G a o , X . a n d H u a n g , L. (1991). A nove l ca t ion ic l i p o s o m e r e a g e n t for eff icient t rans fec t ion of m a m m a l i a n ce l ls . Biochemical & Biophysical Research Communications, 179 :280 -285 . G a o , X . a n d H u a n g , L. (1996). Potent iat ion of ca t ion ic l i p o s o m e - m e d i a t e d g e n e de l ive ry by p o l y c a t i o n s . Biochemistry, 3 5 : 1 0 2 7 - 1 0 3 6 . G e r s h o n , H . , G h i r l a n d o , R., G u t t m a n , S . B . , a n d M i n s k y , A . (1993) . M o d e of fo rmat ion a n d structural f e a t u r e s of D N A - c a t i o n i c l i p o s o m e c o m p l e x e s u s e d for t r ans fec t ion . Biochemistry, 3 2 : 7 1 4 3 - 7 1 5 1 . G l o r i o s o , J . C , G o i n s , W . F . , M e a n e y , C A . , F ink , D . J . , a n d D e L u c a , N . A . (1994) . G e n e t rans fe r to bra in u s i n g h e r p e s s i m p l e x v i rus v e c t o r s . Ann. Neurol, 3 5 : S 2 8 - 3 4 . G r a h a m , F . L . a n d V a n d e r E b , A . J . (1973). A n e w t e c h n i q u e for the a s s a y of infectivity of h u m a n a d e n o v i r u s 5 D N A . Virology, 5 2 : 4 5 6 - 4 6 7 . G r e g o r i a d i s , G . (1993). L i p o s o m e s , a tale of d r u g target ing. Journal of Drug Targeting, 1:3-6. G r u n e r , S . M . , Cu l l i s , P . R . , H o p e , M . J . , a n d T i l c o c k , C P . (1985) . L ip id p o l y m o r p h i s m : the m o l e c u l a r b a s i s of n o n b i l a y e r p h a s e s . Annual Review of Biophysics & Biophysical Chemistry, 14 :211 -238 . G r u n h a u s , A . a n d Horwitz , M . S . (1992). A d e n o v i r u s e s a s c lon ing v e c t o r s . Seminars in virology, 3 :237 -252 . G u s t a f s s o n , J . , A r v i d s o n , G . , K a r l s s o n , G . , a n d A l m g r e n , M . (1995) . C o m p l e x e s b e t w e e n ca t ion ic l i p o s o m e s a n d D N A v i s u a l i z e d by c r y o - T E M . Biochimica et Biophysica Acta, 1235 :305 -312 . no H a b e r l a n d , A . , K n a u s , T . , Z a i t s e v , S . V . , S t a h n , R., Mistry, A . R . , C o u t e l l e , C , Hal ler , H . , a n d Bot tger , M . (1999). C a l c i u m ions a s eff icient c o f a c t o r o f p o l y c a t i o n - m e d i a t e d g e n e t ransfer . Biochimica et Biophysica Acta, 1445 :21 -30 . H a e n s l e r , J . a n d S z o k a , F . C . , Jr . (1993). P o l y a m i d o a m i n e c a s c a d e p o l y m e r s m e d i a t e eff icient t ransfect ion of ce l l s in cul ture . Bioconjugate Chemistry, 4 : 3 7 2 - 3 7 9 . H a f e z , I.M., A n s e l l , S . , a n d Cu l l i s , P . R . (2000). T u n a b l e p H - s e n s i t i v e l i p o s o m e s c o m p o s e d of mix tures of ca t ion ic a n d a n i o n i c l ipids. Biophys J, 7 9 : 1 4 3 8 - 1 4 4 6 . H a f e z , I.M. a n d Cu l l i s , P . R . (2000). O n the m e c h a n i s m w h e r e b y c a t i o n i c l ipids p r o m o t e intracel lular de l ivery of p o l y n u c l e i c a c i d s . S u b m i t t e d for pub l ica t ion in Gene Therapy. H a g s t r o m , J . E . , S e b e s t y e n , M . G . , B u d k e r , V . , L u d t k e , J . J . , Fri tz , J D , a n d Wolf f , J . A . (1996) . C o m p l e x e s of n o n - c a t i o n i c l i p o s o m e s a n d h is tone H1 m e d i a t e eff icient t rans fec t ion of D N A without e n c a p s u l a t i o n . Biochimica et Biophysica Acta, 1 2 8 4 : 4 7 -5 5 . H a r d i n g h a m , G . E . , C r u z a l e g u i , F . H . , C h a w l a , S . , a n d B a d i n g , H . (1998) . M e c h a n i s m s control l ing g e n e e x p r e s s i o n by n u c l e a r c a l c i u m s i g n a l s . Cell Calcium, 2 3 : 1 3 1 - 1 3 4 . H a r r i s o n , G . S . , W a n g , Y . , T o m c z a k , J . , H o g a n , C , S h p a l l , E . J . , C u r i e l , T . J . , F e i g n e r , & P L (1995) . O p t i m i z a t i o n of g e n e t ransfer u s i n g ca t ion ic l ipids in cel l l ines a n d pr imary h u m a n C D 4 + a n d C D 3 4 + h e m a t o p o i e t i c ce l l s . Biotechniques 19, 8 1 6 - 8 2 3 . H a y , J . C . a n d S c h e l l e r , R . H . (1997). S N A R E s a n d N S F in ta rge ted m e m b r a n e f u s i o n . Curr. Opin. Cell. Biol., 9 :505-512 . H o f l a n d , H . E . , N a g y , D., L i u , J . J . , Sprat t , K., L e e , Y . L . , D a n o s , O . , a n d S u l l i v a n , S . M . (1997) . In v i v o g e n e t ransfer by i n t r a v e n o u s admin is t ra t ion of s t a b l e ca t ion ic l i p i d / D N A c o m p l e x . Pharmaceutical Research, 14 :742 -749 . H o l l a n d , J . W . , C u l l i s , P . R . , a n d M a d d e n , T . D . (1996b) . P o l y ( e t h y l e n e glycol ) - l ip id c o n j u g a t e s p r o m o t e b i layer format ion in mix tures of n o n - b i l a y e r - f o r m i n g l ipids. Biochemistry, 3 5 : 2 6 1 0 - 2 6 1 7 . H o l l a n d , J . W . , H u i , C , Cu l l i s , P . R . , a n d M a d d e n , T . D . (1996). P o l y e t h y l e n e g lyco l ) - -lipid c o n j u g a t e s regula te the c a l c i u m - i n d u c e d f u s i o n of l i p o s o m e s c o m p o s e d of p h o s p h a t i d y l e t h a n o l a m i n e a n d p h o s p h a t i d y l s e r i n e . Biochemistry, 3 5 : 2 6 1 8 - 2 6 2 4 . H o n g , K., Z h e n g , W . , B a k e r , A . , a n d P a p a h a d j o p o u l o s , D. (1997) . S tab i l i za t ion of ca t ion ic l i p o s o m e - p l a s m i d D N A c o m p l e x e s by p o l y a m i n e s a n d p o l y ( e t h y l e n e g l y c o l -p h o s p h o l i p i d c o n j u g a t e s for eff icient in v ivo g e n e de l ivery . FEBS Letters, 4 0 0 : 2 3 3 -2 3 7 . i n H o p e , M . J . a n d C u l l i s , P . R . (1979). T h e b i layer stability of inner m o n o l a y e r l ipids f r o m the h u m a n ery throcyte . FEBS Letters, 107 :323 -326 . H o p e , M . J . , M u i , B . , A n s e l l , S . , a n d A h k o n g , Q . F . (1998). C a t i o n i c l ipids, p h o s p h a t i d y l e t h a n o l a m i n e a n d the intracel lular de l ivery of p o l y m e r i c , n u c l e i c a c i d -b a s e d d r u g s . Molecular Membrane Biology, 15:1-14. H u a n g , C . (1969). S t u d i e s o n 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 . F o r m a t i o n a n d p h y s i c a l c h a r a c t e r i s t i c s . Biochemistry, 8 :344-352 . H u a n g , L. a n d L i , S . (1997). L i p o s o m a l g e n e del ivery: a c o m p l e x p a c k a g e . Nature Biotechnology, 15 :620 -621 . H u i , S . W . , L a n g n e r , M . , Z h a o , Y . L . , R o s s , P. , Hur ley , E . , a n d C h a n , K. (1996) . T h e role of h e l p e r l ipids in ca t ion ic l i p o s o m e - m e d i a t e d g e n e t ransfer . Biophysical Journal, 7 1 : 5 9 0 - 5 9 9 . I banez , M . , G a r i g l i o , P. , C h a v e z , P. , S a n t i a g o , R., W o n g , C , a n d B a e z a , I. (1996) . S p e r m i d i n e - c o n d e n s e d D N A a n d c o n e - s h a p e d l ipids i m p r o v e de l ivery a n d e x p r e s s i o n of e x o g e n o u s D N A t ransfer b y l i p o s o m e s . Biochemistry & Cell Biology, 7 4 : 6 3 3 - 6 4 3 . K a n a s e k i , T . , K a w a s a k i , K., M u r a t a , M . , Ikeuchi , Y . , a n d O h n i s h i , S.I. (1997) . St ructura l f e a t u r e s of m e m b r a n e f u s i o n b e t w e e n in f luenza v i rus a n d l i p o s o m e a s r e v e a l e d by q u i c k - f r e e z i n g e lec t ron m i c r o s c o p y . J . Cell. Biol., 1 3 7 : 1 0 4 1 - 1 0 5 6 . Ki l l ian, J . A . a n d d e Kruijff, B . (1986) . T h e in f luence of p ro te ins a n d p e p t i d e s o n the p h a s e proper t ies of l ipids. Chemistry & Physics of Lipids, 4 0 : 2 5 9 - 2 8 4 . K i w a d a , H . , N i i m u r a , H . , Fu j isak i , Y . , Y a m a d a , S . , a n d K a t o , Y . (1985) . A p p l i c a t i o n of syn the t ic alkyl g l y c o s i d e v e s i c l e s a s d rug carr iers . I. P r e p a r a t i o n a n d p h y s i c a l proper t ies . Chemical & Pharmaceutical Bulletin, 3 3 : 7 5 3 - 7 5 9 . K o b a y a s h i , T . , S t a n g , E . , F a n g , K . S . , d e M o e r l o o s e , P . , P a r t o n , R . G . , a n d G r u e n b e r g , J . (1998). A lipid a s s o c i a t e d with the a n t i p h o s p h o l i p i d s y n d r o m e r e g u l a t e s e n d o s o m e structure a n d funct ion . Nature, 3 9 2 : 1 9 3 - 1 9 7 . K u n i t a k e , K. a n d O k a h a t a , Y . (1977). A totally synthet ic b i layer m e m b r a n e s . Journal of American Chemical Society, 9 9 : 3 8 6 0 - 3 6 8 1 . L a m , A . M . I , a n d Cu l l i s , P . R . (2000). C a l c i u m e n h a n c e s the t rans fec t ion p o t e n c y of p l a s m i d D N A - c a t i o n i c l i p o s o m e c o m p l e x e s . Biochim Biophys Acta, 1 4 6 3 : 2 7 9 - 2 9 0 . L a s c h , J . , B e r d i c h e v s k y , V . R . , Torch i l in , V . P . , K o e l s c h , R., a n d K r e t s c h m e r , K. (1983) . A m e t h o d to m e a s u r e critical de te rgen t p a r a m e t e r s . P r e p a r a t i o n of l i p o s o m e s . Analytical Biochemistry, 133 :486 -491 . 112 L a s i c , D . D . , Mart in , F . J . , G a b i z o n , A . , H u a n g , S K , a n d P a p a h a d j o p o u l o s , D. (1991) . S ter ica l ly s tab i l i zed l i p o s o m e s : a h y p o t h e s i s o n the m o l e c u l a r or igin of the e x t e n d e d c i rcu la t ion t i m e s . Biochimica et Biophysica Acta, 1070 :187 -192 . L e c h a r d e u r , D., S o h n , K . J . , Haard t , M . , J o s h i , P . B . , M o n c k , M . , G r a h a m , R . W . , Beat ty , B . , S q u i r e , J . , O ' B r o d o v i c h , H . , a n d L u k a c s , G . L . (1999). M e t a b o l i c instabil ity of p l a s m i d D N A in the c y t o s o l : a potential barr ier to g e n e t ransfer . Gene Therapy, 6 :482 -497 . L e g e n d r e , J . Y . a n d S z o k a , F . C . , Jr . (1992). De l ivery of p l a s m i d D N A into m a m m a l i a n cel l l ines us ing p H - s e n s i t i v e l i p o s o m e s : c o m p a r i s o n with ca t ion ic l i p o s o m e s . Pharmaceutical Research, 9 :1235 -1242 . Li , S . a n d H u a n g , L. (1997). In v ivo g e n e t ransfer v i a i n t r a v e n o u s admin is t ra t ion of c a t i o n i c l i p i d - p r o t a m i n e - D N A ( L P D ) c o m p l e x e s . Gene Therapy, 4 : 8 9 1 - 9 0 0 . Li , S . , T s e n g , W . C . , S t o l z , D . B . , W u , S . P . , W a t k i n s , S . C . , a n d H u a n g , L. (1999) . D y n a m i c c h a n g e s in the charac te r is t i cs of ca t ion ic lipidic v e c t o r s after e x p o s u r e to m o u s e s e r u m : impl ica t ions for i n t r a v e n o u s l ipofect ion. Gene Therapy, 6 :585 -594 . L i n d m a n , B . a n d W e n n e r s t r o m , H . (1980). M i c e l e s . A m p h i p h i l e a g g r e g a t i o n in a q u e o u s so lu t ion . Topics in Current Chemistry, 87 :1 -87 . L i tz inger , D . C . a n d H u a n g , L. (1992). P h o s p h a t i d y l e t h a n o l a m i n e l i p o s o m e s : d r u g de l ivery , g e n e t ransfer a n d i m m u n o d i a g n o s t i c app l i ca t ions . Biochimica et Biophysica Acta, 1 1 1 3 : 2 0 1 - 2 2 7 . L i u , Y . , Liggitt, D., Z h o n g , W . , T u , G . , G a e n s l e r , K., a n d D e b s , R. (1995) . C a t i o n i c l i p o s o m e - m e d i a t e d i n t r a v e n o u s g e n e del ivery . Journal of Biological Chemistry, 2 7 0 : 2 4 8 6 4 - 2 4 8 7 0 . L i u , Y . , M o u n k e s , L . C . , Liggitt, H . D . , B r o w n , C . S . , S o l o d i n , I., H e a t h , T . D . , a n d D e b s , R . J . (1997) . F a c t o r s in f luencing the e f f ic iency of ca t ion ic l i p o s o m e - m e d i a t e d i n t r a v e n o u s g e n e de l ivery . Nature Biotechnology, 15 :167 -173 . L l i n a s , R., S u g i m o r i , M . , a n d Si lver , R . B . (1992). M i c r o d o m a i n s of h igh C a 2 + c o n c e n t r a t i o n in a p r e s y n a p t i c termina l . Science, 2 5 6 : 6 7 7 - 6 7 9 . Loyter , A . , S c a n g o s , G . A . , a n d R u d d l e , F . H . (1982). M e c h a n i s m s of D N A u p t a k e by m a m m a l i a n ce l ls : fate of e x o g e n o u s l y a d d e d D N A m o n i t o r e d by the u s e of f l u o r e s c e n t d y e s . Proceedings of the National Academy of Sciences of the United States of America, 7 9 : 4 2 2 - 4 2 6 . L u r q u i n , P . F . (1979). E n t r a p m e n t o f p l a s m i d D N A by l i p o s o m e s a n d their in teract ions with plant pro top las ts . Nucleic Acids Research, 6 : 3 7 7 3 - 3 7 8 4 . M a r k i n , V . S . , K o z l o v , M . M . , a n d Borov jag in , V . L . (1984). O n the theory of m e m b r a n e f u s i o n . T h e stalk m e c h a n i s m . General Physiology & Biophysics, 3 :361 -377 . 113 M a y e r , A . (1999) . Intracellular m e m b r a n e f u s i o n : S N A R E s o n l y ? Curr. Opin. Cell Biol., 11 :447 -452 . M a y e r , L . D . , H o p e , M . J . , a n d Cu l l i s , P . R . (1986). V e s i c l e s of v a r i a b l e s i z e s p r o d u c e d by a rapid ex t rus ion p r o c e d u r e . Biochimica et Biophysica Acta, 8 5 8 : 1 6 1 - 1 6 8 . M a y e r , L . D . , H o p e , M . J . , Cu l l i s , P . R . , a n d Janof f , A . S . (1985) . . S o l u t e d ist r ibut ions a n d t rapp ing e f f i c ienc ies o b s e r v e d in f r e e z e - t h a w e d mul t i lamel lar v e s i c l e s . Biochimica et Biophysica Acta, 8 1 7 : 1 9 3 - 1 9 6 . M c K u s i c k , V . A . (1992) . H u m a n g e n e t i c s : the last 35 y e a r s , the p r e s e n t , a n d the future. American Journal of Human Genetics, 5 0 : 6 6 3 - 6 7 0 . M i m m s , L . T . , Z a m p i g h i , G . , N o z a k i , Y . , T a n f o r d , C , a n d R e y n o l d s , J . A . (1981) . P h o s p h o l i p i d v e s i c l e format ion a n d t r a n s m e m b r a n e protein incorpora t ion u s i n g octy l g l u c o s i d e . Biochemistry, 2 0 : 8 3 3 - 8 4 0 . M i z u g u c h i , H . , N a k a g a w a , T . , N a k a n i s h i , M . , Imazu , S . , N a k a g a w a , a n d M a y u m i , T . (1996) . Ef f ic ient g e n e t ransfer into m a m m a l i a n ce l ls u s i n g f u s o g e n i c l i p o s o m e . Biochemical & Biophysical Research Communications, 2 1 8 : 4 0 2 - 4 0 7 . M o k , K . W . a n d Cu l l i s , P . R . (1997). St ructura l a n d f u s o g e n i c p roper t i es of ca t ion ic l i p o s o m e s in the p r e s e n c e of p l a s m i d D N A . Biophysical Journal, 7 3 : 2 5 3 4 - 2 5 4 5 . M o k , K . W . , L a m , A . M . , a n d Cu l l i s , P . R . (1999). S tab i l i zed p lasmid - l ip id par t ic les: f ac tors in f luenc ing p l a s m i d e n t r a p m e n t a n d t ransfect ion proper t i es . Biochimica et Biophysica Acta, 1419 :137 -150 . M o n c k , J . R . a n d F e r n a n d e z , J . M . (1994). T h e e x o c y t o t i c f u s i o n p o r e a n d neuro t ransmi t te r r e l e a s e . Neuron, 12 :707-716 . M o n c k , M . A . , M o r i , A . , L e e , D., T a r n , P. , W h e e l e r , J . J . , C u l i i s , P . R . a n d S c h e r r e r , P. (2000) . S t a b i l i z e d p lasmid- l ip id par t ic les: p h a r m a c o k i n e t i c s a n d p l a s m i d de l ivery to distal t u m o r s fo l lowing i n t r a v e n o u s inject ion. Journal of Drug Targeting, 7 :439 -452 . M o n t a l , M . (1999) . E lec t ros ta t ic attraction at the c o r e of m e m b r a n e f u s i o n . FEBS Letters 447, 129 -130 . M u z y c z k a , N . (1992) . U s e of a d e n o - a s s o c i a t e d v i rus a s a g e n e r a l t r a n s d u c t i o n v e c t o r for m a m m a l i a n ce l l s . Curr. Top. Microbiol. Immunol., 158 :97 -129 . N a b e l , G . J . , N a b e l , E . G . , Y a n g , Z . Y . , F o x , B . A . , P lau tz , G . E . , G a o , X . , H u a n g , L , S h u , S . , G o r d o n , D., a n d C h a n g , A . E . (1993). D i rect g e n e t ransfer with D N A -l i p o s o m e c o m p l e x e s in m e l a n o m a : e x p r e s s i o n , b io log ic activity, a n d lack of toxicity in h u m a n s . Proceedings of the National Academy of Sciences of the United States of America, 9 0 : 1 1 3 0 7 - 1 1 3 1 1 . 114 N a k a n i s h i , M . , U c h i d a , T . , S u g a w a , H. , Ishiura, M . , a n d O k a d a , Y . (1985) . Ef f ic ient in t roduct ion of c o n t e n t s of l i p o s o m e s into ce l ls u s i n g H V J ( S e n d a i v i rus) . Experimental Cell Research, 159 :399 -409 . N i c o l a u , C . a n d R o t t e m , S . (1982). E x p r e s s i o n of a b e t a - l a c t a m a s e activity in M y c o p l a s m a c a p r i c o l u m t r a n s f e c t e d with the l i p o s o m e - e n c a p s u l a t e d E .co l i p B R 3 2 2 p l a s m i d . Biochemical & Biophysical Research Communications, 1 0 8 : 9 8 2 - 9 8 6 . O l n e s , M . J . a n d Kur l , R . N . (1994). Isolation of n u c l e a r ex t racts f r o m fragi le ce l l s : a s impl i f ied p r o c e d u r e a p p l i e d to t h y m o c y t e s . Biotechniques, 17 :828 -829 . O r r a n t i a , E . a n d C h a n g , P . L . (1990). Intracellular distr ibution of D N A in terna l ized t h r o u g h c a l c i u m p h o s p h a t e precipi tat ion. Experimental Cell Research, 1 9 0 : 1 7 0 - 1 7 4 . P a l m e r , L . R . , C h e n , T . , L a m , A .M . I . , F e n s k e , D . B . , W o n g , K . F . , a n d C u l l i s , P . R . (2000) . T r a n s f e c t i o n proper t ies of s tab i l i zed p lasmid- l ip id par t ic les c o n t a i n i n g ca t ion ic P E G l ipids. S u b m i t t e d for publ icat ion in Gene Therapy. P a p a h a d j o p o u l o s , D., V a i l , W . J . , N e w t o n , C , Nir, S . , J a c o b s o n , K., P o s t e , G . , a n d L a z o , R. (1977) . S t u d i e s o n m e m b r a n e f u s i o n . III. T h e role of c a l c i u m - i n d u c e d p h a s e c h a n g e s . Biochimica et Biophysica Acta 465, 5 7 9 - 5 9 8 . P a r e n t e , R . A . a n d L e n t z , B . R . (1984). P h a s e b e h a v i o r of la rge un i lamel la r v e s i c l e s c o m p o s e d of synthe t ic p h o s p h o l i p i d s . Biochemistry, 2 3 : 2 3 5 3 - 2 3 6 2 . P a u k k u , T . , L a u r a e u s , S . , H u h t a n i e m i , I., a n d K i n n u n e n , P . K . (1997) . N o v e l ca t ion ic l i p o s o m e s for D N A - t r a n s f e c t i o n with h igh e f f ic iency a n d low toxicity. Chemistry & Physics of Lipids, 87 :23 -29 . P e e t e r s , M . J . , Pat i jn, G . A . , L ieber , A . , M e u s e , L., a n d K a y , M . A . (1996) . A d e n o v i r u s -m e d i a t e d h e p a t i c g e n e t ransfer in m i c e : c o m p a r i s o n of in t ravascu la r a n d biliary admin is t ra t ion . Human Gene Therapy, 7 :1693 -1699 . P e r e z - T e r z i c , C , P y l e , J . , J a c o n i , M . , S tehno-B i t t e l , L , a n d C l a p h a m , D . E . (1996) . C o n f o r m a t i o n a l s ta tes of the n u c l e a r p o r e c o m p l e x i n d u c e d by d e p l e t i o n of n u c l e a r C a 2 + s t o r e s . Science, 2 7 3 : 1 8 7 5 - 1 8 7 7 . P e t e r s , C . a n d M a y e r , A . (1998). C a 2 + / c a l m o d u l i n s i g n a l s the c o m p l e t i o n of d o c k i n g a n d t r iggers a late s t e p of v a c u o l e f u s i o n . Nature, 3 9 6 : 5 7 5 - 5 7 9 . P e t e r s e n , O . H . , G e r a s i m e n k o , O . V . , G e r a s i m e n k o , J . V . , M o g a m i , H . , a n d T e p i k i n , A . V . (1998) . T h e c a l c i u m s tore in the n u c l e a r e n v e l o p e . Cell Calcium, 2 3 : 8 7 - 9 0 . Por t is , A . , N e w t o n , C , P a n g b o r n , W . , a n d P a p a h a d j o p o u l o s , D. (1979) . S t u d i e s o n the m e c h a n i s m of m e m b r a n e f u s i o n : e v i d e n c e for a n i n t e r m e m b r a n e C a 2 + -p h o s p h o l i p i d c o m p l e x , s y n e r g i s m with M g 2 + , a n d inhibition by s p e c t r i n . Biochemistry, 18 :780 -790 . 115 R a d l e r , J . O . , Ko l tover , I., Salditt , T . , a n d S a f i n y a , C R . (1997) . S t ruc ture of D N A -ca t ion ic l i p o s o m e c o m p l e x e s : D N A intercalat ion in mul t i lamel lar m e m b r a n e s in dist inct interhel ical p a c k i n g r e g i m e s . Science, 2 7 5 : 8 1 0 - 8 1 4 . Ri l fors , L , L i n d b l o m , G . , W i e s l a n d e r , A . , a n d C h r i s t i a n s s o n , A . (1984) . L ip id B i l a y e r Stabi l i ty in B i o l o g i c a l M e m b r a n e s . Membrane Fluidity, eds., Mor r is K a t e s a n d L i o n e l M a n s o n , P l e n u m P u b l i s h i n g C o r p o r a t i o n : 2 0 5 - 2 4 5 . R o i z m a n , B . a n d S e a r s , A . E . (1990). H e r p e s s i m p l e x v i r u s e s a n d their rep l ica t ion . In B . N . F i e l d s a n d D . M . K n i p e ( E d s . ) , Fields Virology ( 2 n d e d . , p p . 1 7 9 5 - 1 8 4 2 ) . N e w Y o r k : R a v e n P r e s s . R o t h m a n , J . E . (1994) . M e c h a n i s m s of intracel lular protein t ransport . Nature, 3 7 2 : 5 5 - 6 3 . S a m b r o o k , J . , F r i t s c h , E . F . , a n d Man ia t is , T . (1989). Ext rac t ion a n d puri f icat ion of p l a s m i d D N A . In M o l e c u l a r C l o n i n g : A L a b o r a t o r y M a n u a l I, 1 .21-1 .52. C o l d S p r i n g H a r b o r , N e w Y o r k , C . N o l a n , editor. C o l d S p r i n g H a r b o r L a b o r a t o r y . S a n t e l l a , L. a n d C a r a f o l i , E . (1997). C a l c i u m s igna l ing in the cel l n u c l e u s FASEB Journal, 1 1 : 1 0 9 1 - 1 1 0 9 . S c h u h , J . R . , B a n e r j e e , U . , Mul ler , L., a n d C h a n , S.I. (1982) . T h e p h o s p h o l i p i d p a c k i n g a r r a n g e m e n t in s m a l l b i layer v e s i c l e s a s r e v e a l e d by proton m a g n e t i c r e s o n a n c e s t u d i e s at 5 0 0 M H z . Biochimica et Biophysica Acta, 6 8 7 : 2 1 9 - 2 2 5 . S i e g e l , D . P . (1993) . E n e r g e t i c s of in te rmedia tes in m e m b r a n e f u s i o n : c o m p a r i s o n of stalk a n d inver ted mice l la r in termedia te m e c h a n i s m s . Biophysical Journal, 6 5 : 2 1 2 4 -2 1 4 0 . S i e g e l , D . P . a n d E p a n d , R . M . (1997). T h e m e c h a n i s m of lamel la r - to - inver ted h e x a g o n a l p h a s e t ransi t ions in p h o s p h a t i d y l e t h a n o l a m i n e : imp l ica t ions for m e m b r a n e f u s i o n m e c h a n i s m s . Biophysical Journal, 7 3 : 3 0 8 9 - 3 1 1 1 . S i e g e l , D . P . , B u r n s , J . L . , C h e s t n u t , M . H . , a n d T a l m o n , Y . (1989). In termedia tes in m e m b r a n e f u s i o n a n d b i l ayer /nonb i layer p h a s e t ransi t ions i m a g e d by t i m e - r e s o l v e d c r y o - t r a n s m i s s i o n e lec t ron m i c r o s c o p y . Biophysical Journal, 5 6 : 1 6 1 - 1 6 9 . S i e g e l , D . P . , G r e e n , W . J . , a n d T a l m o n , Y . (1994). T h e m e c h a n i s m of lamel lar - to -inver ted h e x a g o n a l p h a s e t ransi t ions: a s t u d y u s i n g t e m p e r a t u r e - j u m p c r y o - e l e c t r o n m i c r o s c o p y . Biophys.J., 6 6 : 4 0 2 - 4 1 4 . S i n g e r , S . J . a n d N i c o l s o n , G . L . (1972). T h e fluid m o s a i c m o d e l of the s t ructure of cel l m e m b r a n e s . Science, 175 :720 -731 . S o n g , Y . K . , L i u , F . , C h u , S . , a n d L iu , D. (1997). C h a r a c t e r i z a t i o n of ca t ion ic l i p o s o m e - m e d i a t e d g e n e t ransfer in v ivo by i n t r a v e n o u s admin is t ra t ion . Human Gene Therapy, 8 :1585 -1594 . 116 S p e c t o r , M . S . a n d S c h n u r , J . M . (1997). D N A order ing o n a lipid m e m b r a n e . Science, 2 7 5 : 7 9 1 - 7 9 2 . S t e g m a n n , T . a n d L e g e n d r e , J . Y . (1997). G e n e t ransfer m e d i a t e d by ca t ion ic l ipids: lack of a corre la t ion b e t w e e n lipid mix ing a n d t ransfec t ion . Biochimica et Biophysica Acta, 1 3 2 5 : 7 1 - 7 9 . S t e r n b e r g , B . , S o r g i , F . L . , a n d H u a n g , L. (1994). N e w s t ruc tures in c o m p l e x fo rmat ion b e t w e e n D N A a n d ca t ion ic l i p o s o m e s v i s u a l i z e d by f r e e z e - f r a c t u r e e l e c t r o n m i c r o s c o p y . FEBS Letters, 3 5 6 : 3 6 1 - 3 6 6 . S u d h o f , T . C . a n d R i z o , J . (1996). S y n a t o t a g m i n s : C 2 - d o m a i n pro te ins that regu la te m e m b r a n e traffic. Neuron, 17 :379 -388 . S z o k a , F . J . a n d P a p a h a d j o p o u l o s , D. (1978). P r o c e d u r e for p r e p a r a t i o n of l i p o s o m e s with large internal a q u e o u s s p a c e a n d high c a p t u r e by r e v e r s e - p h a s e e v a p o r a t i o n . Proceedings of the National Academy of Sciences of the United States of America, 7 5 : 4 1 9 4 - 4 1 9 8 . S z o k a , F . J . a n d P a p a h a d j o p o u l o s , D. (1980). C o m p a r a t i v e p roper t ies a n d m e t h o d s of p repara t ion of lipid v e s i c l e s ( l i p o s o m e s ) . Annual Review of Biophysics & Bioengineering, 9 :467 -508 . T a r n , P . , M a c L a c h l a n , I., M o n c k , M . , L e e , D., L u d k o v s k i , O , L e n g , E . C . , C l o w , K., B u c h k o w s k y , S . , S tark , H . , S c h e r r e r , P. , a n d Cu l l i s , P . R . (2000) . S t a b i l i z e d p l a s m i d -lipid par t ic les for s y s t e m i c g e n e therapy . Gene Therapy, in p r e s s . T e m p l e t o n , N . S . , L a s i c , D . D . , F reder ik , P . M . , S t rey , H . H . , R o b e r t s , D D , a n d P a v l a k i s , G . N . (1997). Improved D N A : l i p o s o m e c o m p l e x e s for i n c r e a s e d s y s t e m i c de l ive ry a n d g e n e e x p r e s s i o n . Nature Biotechnology, 15 :647 -652 . Th ie r ry , A . R . , L u n a r d i - l s k a n d a r , Y . , Bryant , J . L . , R a b i n o v i c h , P . , G a l l o , R . C . , a n d M a h a n , L . C . (1995). S y s t e m i c g e n e therapy: biodistr ibut ion a n d long - te rm e x p r e s s i o n of a t r a n s g e n e in m i c e . Proceedings of the National Academy of Sciences of the United States of America, 9 2 : 9 7 4 2 - 9 7 4 6 . T i l c o c k , C P . (1986) . L ip id p o l y m o r p h i s m . Chemistry and Physics of lipids, 4 0 : 1 0 9 -125 . T i l c o c k , C P . a n d Cu l l i s , P . R . (1981). T h e p o l y m o r p h i c p h a s e b e h a v i o u r of m i x e d p h o s p h a t i d y l s e r i n e - p h o s p h a t i d y l e t h a n o l a m i n e m o d e l s y s t e m s a s d e t e c t e d by 3 1 P -N M R . Biochimica et Biophysica Acta, 6 4 1 : 1 8 9 - 2 0 1 . T i l c o c k , C P . , Ba l ly , M . B . , F a r r e n , S . B . , a n d C u l l i s , P . R . (1982) . In f luence of c h o l e s t e r o l o n the structural p r e f e r e n c e s of d i o l e o y l p h o s p h a t i d y l e t h a n o l a m i n e -d i o l e o y l p h o s p h a t i d y l c h o l i n e s y s t e m s : a p h o s p h o r u s - 3 1 a n d d e u t e r i u m n u c l e a r m a g n e t i c r e s o n a n c e s tudy . Biochemistry, 2 1 : 4 5 9 6 - 4 6 0 1 . 117 T i l c o c k , C P . , Ba l ly , M . B . , F a r r e n , S . B . , Cu l l i s , P . R . , a n d G r u n e r , S . M . (1984) . C a t i o n - d e p e n d e n t s e g r e g a t i o n p h e n o m e n a a n d p h a s e b e h a v i o r in m o d e l m e m b r a n e s y s t e m s c o n t a i n i n g p h o s p h a t i d y l s e r i n e : in f luence of c h o l e s t e r o l a n d a c y l c h a i n c o m p o s i t i o n . Biochemistry, 2 3 : 2 6 9 6 - 2 7 0 3 . T i l c o c k , C P . , Cu l l i s , P . R . , a n d G r u n e r , S . M . (1988) . C a l c i u m - i n d u c e d p h a s e s e p a r a t i o n p h e n o m e n a in m u l t i c o m p o n e n t u n s a t u r a t e d lipid mix tures . Biochemistry, 2 7 : 1 4 1 5 - 1 4 2 0 . T s e , F . W . , T s e , A . , Hil le, B . , H o r s t m a n n , H. , a n d A i m e r s , W . (1997) . L o c a l C a 2 + r e l e a s e f r o m internal s t o r e s contro ls e x o c y t o s i s in pituitary g o n a d o t r o p h s . Neuron, 18 :121 -132 . T s e n g , W . C . , H a s e l t o n , F . R . , a n d G i o r g i o , T . D . (1997). T r a n s f e c t i o n by ca t ion ic l i p o s o m e s u s i n g s i m u l t a n e o u s s ing le cel l m e a s u r e m e n t s of p l a s m i d de l i ve ry a n d t r a n s g e n e e x p r e s s i o n . Journal of Biological Chemistry, 2 7 2 : 2 5 6 4 1 - 2 5 6 4 7 . T s i e n , R . W . a n d T s i e n , R . Y . (1990). C a l c i u m c h a n n e l s , s t o r e s , a n d osc i l l a t ions . Annual Review of Cell Biology, 6 :715 -760 . V a h e r i , A . a n d P a g a n o , J . S . (1965). Infectious pol iov i rus R N A : a s e n s i t i v e m e t h o d of a s s a y . Virology, 2 7 : 4 3 4 - 4 3 6 . V a n d e r W o u d e , I., V i s s e r , H W , ter B e e s t , M . B . , W a g e n a a r , A . , Ru i te rs , M . H . , E n g b e r t s , J . B . , a n d H o e k s t r a , D. (1995). P a r a m e t e r s in f luenc ing the int roduct ion of p l a s m i d D N A into ce l l s by the u s e of synthet ic a m p h i p h i l e s a s a carr ier s y s t e m . Biochimica et Biophysica Acta, 1240:34 -40 . V a r m u s , H . (1988). R e t r o v i r u s e s . Science, 2 4 0 : 1 4 2 7 - 1 4 3 5 . Verk le i j , A . J . (1984). L ip id ic i n t r a m e m b r a n o u s par t ic les . Biochimica et Biophysica Acta , 7 7 9 : 4 3 - 6 3 . Wat t iaux , R., J a d o t , M . , Warn ie r -P i ro t te , M .T . , a n d W a t t i a u x - D e , C S . (1997) . C a t i o n i c l ipids d e s t a b i l i z e l y s o s o m a l m e m b r a n e in vitro. FEBS Letters, 4 1 7 : 1 9 9 - 2 0 2 . W h e e l e r , J . J . , P a l m e r , L , O s s a n l o u , M . , M a c L a c h l a n , I., G r a h a m , R W , Z h a n g , Y . P . , H o p e , M . J . , S c h e r r e r , P. , a n d C u l l i s , P . R . (1999). S t a b i l i z e d p lasmid - l ip id par t ic les: c o n s t r u c t i o n a n d charac te r i za t ion . Gene Therapy, 6 :271 -281 . W h e e l e r . J . J . , V e i r o . J . A . , C u l l i s , P . R . (1994). l o n o p h o r e - m e d i a t e d l o a d i n g of C a 2 + into large un i lamel la r v e s i c l e s in r e s p o n s e to t r a n s m e m b r a n e p H g r a d i e n t s . Molecular Membrane Biology, 11: 151 -157 . W h i t e , E . M . , A l l is , C D . , G o l d f a r b , D . S . , S r i v a s t v a , A . , W e i r , J . W . , a n d G o r o v s k y , M . A . (1989) . N u c l e u s - s p e c i f i c a n d tempora l ly restr icted loca l iza t ion of p ro te ins in T e t r a h y m e n a m a c r o n u c l e i a n d m i c r o n u c l e i . Journal of Cell Biology, 1 0 9 : 1 9 8 3 - 1 9 9 2 . 118 W i l s c h u t , J . , H o e k s t r a , D. e d s . (1991). Membrane Fusion, N e w Y o r k : D e k k e r . W o o d l e , M . C . a n d L a s i c , D . D . (1992). Ster ica l ly s tab i l i zed l i p o s o m e s . Biochimica et Biophysica Acta, 1113:171 -199 . W o r g a l l , S . , Wolf f , G . , F a l c k - P e d e r s e n , E . , a n d C r y s t a l , R . G . (1997) . Innate i m m u n e m e c h a n i s m s d o m i n a t e e l iminat ion of a d e n o v i r a l v e c t o r s fo l lowing in v ivo admin is t ra t ion . Human Gene Therapy, 8 :37-44. W r o b e l , I. a n d C o l l i n s , D. (1995). F u s i o n of ca t ion ic l i p o s o m e s with m a m m a l i a n ce l l s o c c u r s after e n d o c y t o s i s . Biochimica et Biophysica Acta, 1 2 3 5 : 2 9 6 - 3 0 4 . X u , Y . a n d S z o k a , F . C . , Jr . (1996). M e c h a n i s m of D N A r e l e a s e f r o m ca t ion ic l i p o s o m e / D N A c o m p l e x e s u s e d in cel l t ransfec t ion . Biochemistry, 3 5 : 5 6 1 6 - 5 6 2 3 . Y e i , S . , Mi t tereder , N. , T a n g , K., O ' S u l l i v a n , C , a n d T r a p n e l l , B . C . (1994) . A d e n o v i r u s - m e d i a t e d g e n e t ransfer for cys t i c f ibrosis: quant i tat ive e v a l u a t i o n of r e p e a t e d in v i v o v e c t o r admin is t ra t ion to the lung . Gene Therapy, 1 :192-200 . Y o s h i m u r a , K., R o s e n f e l d , M . A . , N a k a m u r a , H . , S c h e r e r , E . M . , P a v i r a n i , A . , L e c o c q , J . P . , a n d C r y s t a l , R . G . (1992). E x p r e s s i o n of the h u m a n c y s t i c f ib ros is t r a n s m e m b r a n e c o n d u c t a n c e regulator g e n e in the m o u s e lung after in v ivo in t ra t racheal p l a s m i d - m e d i a t e d g e n e transfer . Nucleic Acids Research, 2 0 : 3 2 3 3 -3 2 4 0 . Z a b n e r , J . , F a s b e n d e r , A . J . , M o n i n g e r , T . , Poe l l inger , K . A . , W e l s h , a n d M J (1995) . C e l l u l a r a n d m o l e c u l a r barr iers to g e n e t ransfer by a ca t ion ic l ipid. Journal of Biological Chemistry, 2 7 0 : 1 8 9 9 7 - 1 9 0 0 7 . Z a i t s e v , S . V . , H a b e r l a n d , A . , Ot to , A . , V o r o b ' e v , V.I., Hal ler , H . , a n d Bot tger , M . (1997) . H1 a n d H M G 1 7 ex t rac ted f rom cal f t h y m u s nuc le i a r e eff icient D N A carr iers in g e n e t ransfer . Gene Therapy, 4 :586 -592 . Z h a n g , Y . P . , S e k i r o v , L., S a r a v o l a c , E . G . , W h e e l e r , J . J . , T a r d i , P. , C l o w , K., L e n g , E . , S u n , R., C u l l i s , P . R . , a n d S c h e r r e r , P. (1999). S tab i l i zed p lasmid - l ip id par t ic les for reg iona l g e n e therapy: formulat ion a n d t ransfec t ion proper t i es . Gene Therapy, 6 : 1 4 3 8 - 1 4 4 7 . Z h o u , X . a n d H u a n g , L. (1994). D N A t ransfect ion m e d i a t e d by ca t ion ic l i p o s o m e s c o n t a i n i n g l ipopoly lys ine: charac te r i za t ion a n d m e c h a n i s m of a c t i o n . Biochimica et Biophysica Acta, 1 1 8 9 : 1 9 5 - 2 0 3 . Z h u , N . , Liggitt, D. , L iu , Y . , a n d D e b s , R. (1993). S y s t e m i c g e n e e x p r e s s i o n after i n t r a v e n o u s D N A de l ivery into adult m i c e . Science, 2 6 1 : 2 0 9 - 2 1 1 . Z i m m e r b e r g , J . , C u r r a n , M . , a n d C o h e n , F . S . (1991) A l ipid/protein c o m p l e x h y p o t h e s i s for exocy to t i c f u s i o n p o r e fo rmat ion . Annals N.Y. Acad. Sci. USA, 6 3 5 : 3 0 7 - 3 1 7 . 119 

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