Class I restricted CTL recognition of a soluble protein delivered by liposomes containing lipophilic polylysines

Journal of Immunological Methods, 152 (1992) 237-243

237

© 1992 ElsevierScience Publishers B.V. All rights reserved 0022-[759/92/$05.00

JIM 06377

Class I restricted CTL recognition of a soluble protein delivered by liposomes containing lipophilic polylysines Smita Nair a, Xiaohuai Z h o u b, L e a f H u a n g ba and Barry T. R o u s e ~ Departments of a Microbiology and t, Biochemistry, Unit'ersity of Tennessee, Knoxt'ille, TN 37996, USA

(Received6 January 1992,revised received 17 March 1992.accepted 17 March 1992)

CD8 + cytotoxic lymphocytes ,'ecognize peptides derived from endogenous antigens complexed with class I major histocompatibility complex while CD4 + helper cells recognize peptides from exogenous antigens bound to class II MHC molecules. A soluble protein can be introduced into the class 1 pathway of antigen processing and presentation using an appropriate vehicle to deliver the antigen into the cytosol. Cationic liposomes containing lipophilic polylysine readily form complexes with an anionic, soluble protein ovalbumin. Mouse thymoma EL4 cells incubated with such complexes can be sensitized for killing by OVA-specific CTL effector cells. This method of target sensitization by a soluble antigen is more sensitive than the osmotic loading method previously reported. Key words: Class 1 major histocompatibilitycomplexantigen; Ovalbumin;Cationic liposome;Polylysine;Phorbol ester

Introduction

Antigen presentation to CD8 + cytotoxic T lymphocytes (CTL) requires that the antigen be processed and presented in the context of class 1 molecules (Germain, 1986; Morrison et al., 1986; Bevan, 1987; Davis and Bjorkman, 1989). Peptides derived from endogenous proteins complex with class I molecules in a pre-Golgi compartment, presumably the endoplasmic reticulum (ER), and are transported to the cell surface Correspondence to: B.T. Rouse, Department of Microbiology, Universityof Tennessee, Knoxville,TN 37996-0845, USA. t Present address: Department of Pharmacology,Universityof Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. Abbret'iations: OVA. ovalbumin;CTL, cytotoxicT lymphoo cytes; DPSG, 1,2-dipalmitoyl succinyl glycerol; DOPE, dioleoyl phosphatidylethanolamine; DCC, 1,3-dicyclohexylcarbodiimide; NHS, N-hydroxysuccinimide;LPLL, lipopolyL-lysine;PBS, phosphate-buffered saline; pl, isoelectricpoint.

(Nuchtern et al., 1989; Townsend et al., 1989; Yewdell and Bennink, 1989). Exogenous proteins are processed in the endosomes and the peptides generated associate with class II molecules (Germain, 1986; Bevan, 1987) but not usually with class I molecules. However certain forms of presentation of soluble proteins can result in the class I pathway of antigen processing. Osmotic loading was one of the first well-documented examples (Moore et al., 1988). Another more efficient and less toxic method of sensitizing target cells for class 1 restricted CTL recognition of a soluble protein is to deliver proteins by means of the pH-sensitive iiposomes (Reddy et al., 1991). In the present study we report yet another antigen delivery system, the iipopoly-L-lysine (LPLL) liposome method. This system was chosen since LPLL association has proven to be a convenient and efficient means of introducing DNA into cells, providing an effective transfec-

238 tion reagent (Zhou and Huang, 1991). We demonstrate that the LPLL-liposome approach is a conv,mient and simple to use mild antigen delivery system which effectively sensitizes target cell~ for recognition by CTL. The actual mechanism involved in delivery of a soluble protein t~ a cell for sensitization and class I restricted CTL recognition is essentially unknown. A very likely mechanism is via endocytosis of the LPLL-liposome complexes. Growing evidence suggests the involvement of protein kinase C (PKC) in various cellular functions (Nishizuka, 1986). PKC is a family of calcium/ phospholipid stimulated serine/threonine protein kinases which plays a crucial role in cellular functions like regulation of cell surface receptor function, secretion, cellular metabolism, cell growth and differentiation, modulation of membrane functions and gene expression (Nishizuka, 1984, 1986; Huang, 1989). Tumor promoting phorbol esters, like phorbol 12-myristate 13acetate (PMA) permanently activate PKC and have been widely used to study the effect of PKC in cellular functions. Previous studies suggest that PKC mediated enhanced transfection of exogenous DNA may be due to increased endocytosis of the complexes (Reston et al., 1991). To explore the involvement of PKC activation in antigen delivery by LPLL-liposome-OVA complexes, we examined the sensitization of EL4 cells treated with liposome-OVA complexes in the presence of PMA. Our studies demonstrate that PMA enhances sensitization of EL4 cells which is reflected by increased killing at low OVA concentrations by OVA-specific CTL. This study demonstrates an effective method for delivering soluble proteins to cells to achieve sensitization for class 1 restricted CTL lysis. Studies with PMA suggest that PKC activation enhances sensitization of the ceil, which provides an insight into the mechanism of antigen delivery.

Materials and methods

Animals Female C57BI_./6 mice, 5-6 week old and retired breeders, were obtained from HarlanSprague-Dawley-lndianapolis or from Sasco-

Omaha, Nebraska. In conducting the research described in this report, the investigators adhered to the 'Guide for the Care and Use of Laboratory Animals' as proposed by the Committee on Care of Laboratory Animal Resources Commission on Life Sciences~National Research Council. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care.

Reagents Ovalbumin (OVA batch grade no. 5, Sigma Chemical Company, St. Louis, MO) was used in free form or complexed to LPLL. 1,2-dipaimitoyl succinyl glycerol (DPSG) and dioleoyl phosphatidylethanolamine (DOPE) were obtained from Avanti Polar Lipids, Birmingham, AL. 1,3dicyclohexylcarbodiimide (DCC) and triethylamine were purchased from Aldrich Chemical Company. N-hydroxysuccinimide (NHS), poly-Llysine hydrobromide (PLL) (mol. wt. 3340) and dimethyl sulfoxide were obtained from Sigma Chemical Company. Phorbol 12-myristate 13acetate was obtained from Calbiochem, San Diego, CA.

Preparation of LPLL liposomes containing OVA LPLL was synthesized from DPSG and LPLL as described earlier (Zhou and Huang, 1991a). DPSG was dialyzed against 0.001 N HC! and then lyophilized and redissolved in chloroform. DPSG was activated to NHS ester of DPSG with 1 molar equivalent of each DCC and NHS in ethyl acetate. The reaction proceeded at room temperature for 24 h. Crystals of the reaction by-product, dicyclohexylisourea, was removed by filtration through glass wool. The filtrate was dried in a stream of nitrogen gas and dessicated. NHS ester of DPSG was recrystallized from methanol : ethanol : chloroform (1 : 2 : 2) at 0°C. LPLL was synthesized by mixing PLL with NHS ester of DPSG in dimethyl sulfoxide at 50°C for 10 min in the presence of triethylamine. Triethylamine was removed by evaporation under a stream of N 2 gas. LPLL and OVA were diluted in 1 ml of serum-free RPMI, at the required concentrations, mixed and incubated at room temperature for 15 min. The final volume per tube was 5 ml.

2.39

Target cells The tumor cell lines used were l a - EL4 (C57BL/6, H-2 ~, thymoma), E.GT-OVA (ELA cells stably transfected with eDNA of chicken OVA) (Moore et al., 1988), and YAC-I (H-2d).

OVA-specific cytotoxic effector popul~,tion Splenocytes, obtained from adult mice and depleted of red blood cells, were subjected to osmotic shock as described by Okada and Rechsteiner (Okada et al., 1982). 1.2 × 108 cells were pelleted and resuspended in 1 mi of hypertonic solution containing 10 m g / m i OVA. The hypertonic medium was made up as follows: 0.5 M sucrose, 10% w / v polyethylene glycol 1000, 10 mM Hopes, pH 7.2, in RPMI 1640 medium. The cells were incubated at 37°C for 10 min, then rapidly diluted to 15 ml with warm hypotonic medium and incubated for 2 min at 37°C. The hypotonic solution was made up with 60% PBS and 40% water. The cells were washed, irradiated at 1000 fads, washed twice in PBS and resuspended in PBS at ! × 10~ cells/ml. Young mice were immunized intravenously with 2.5 × 107 splenocytes. Spleen cells (1 × 107) obtained from immunized mice after 7-10 days were restimulated in vitro for 5 days with 1 x 106 E.G7-OVA cells (irradiated 20,000 rads). Cells were maintained in six-well flat bottom plates (Costar, Cambridge, MA) at 37°C and 5% CO z. Each well contained 5 mi of NCTC 109 and RPMI 1640 (1:1 v / v , Gibco), supplemented with 10% heat inactivated fetal calf serum, 10 mM L-glutamine (Gibco), 1 mM oxaloacetic acid (Sigma, St. Louis, MO), 0.2 U / m l bovine insulin (Sigma) and 50 p.M 2mercaptoethanol (Sigma).

Cytotoxicity assay EL4 cells were subjected to osmotic loading, or treated with LPLL liposomes or untreated. 5 x 106 EL4 cells were added to lipopoly-L-lysine with O V A in 5 ml of serum free medium (RPMI supplemented with 50 /tM 2-mercaptoethanol) and incubated in a water bath at 37°C for 3 h. E.G7-OVA, EL4 and EL4 incubated with free, uncomplexed O V A were used as controls. EL4 cells were osmotically loaded with O V A as described earlier. Briefly, 5 × 106 EL4 cells were

suspended in I ml of hypertonic medium containing different concentrations of O V A and incubated for 10 min at 37°C. The cells were rapidly diluted to 15 ml in hypotonic ~lution and further incubated for 2 min at 370C. The cells were washed twice and resuspended in 5 ml RPI0 (RPMI 1640 supplemented with 10% heat inactivated fetal calf serum, 50 /zM 2-mercaptoethanol) and incubated for 3 h at 37"C prior to use in cytotoxicity assay. After 3 h the target cells were washed and labelled with 200 p,Ci of StCr in RPi0 for I h. The labelled targets were washed three times and resuspended in RPI0. Simultaneously, the effector cells were pooled in RP10 and aliquoted in 96-well V-bottomed plates. 104 target cells were added to each well at an effector:target ratio ranging from 50:1 to 3.13:1. The total volume per well was 200 p.I. The plates were centrifuged at 500 x g for 3 rain and incubated for 4 h at 37°C and 5% CO z. 100 p.I of the supernatant fluid was collected to measure radioactivity and specific cytotoxic activity was determined using the formula: % specificrelease

experimental- spontaneous × 100 total - spontaneous

Each assay was performed in triplicate and the spontaneous release was less than 25% of total release by detergent in all assays.

Results

Until recently methods were not available to introduce soluble proteins into the cell such that they would be processed for class ! CTL recognition. As initially reported by Carbone et al. (1990) CTL recognition of a soluble protein OVA can be achieved by osmotically loading EL4 cells with OVA. This approach requires abundant antigen (minimum of 0.5 m g / m l ) and is not without some toxicity (Reddy et ai., 1991). The results in Fig. 1 demonstrate that L P L L / D O P E liposomes complexed to O V A can be used to efficiently sensitize EL4 cells for class i restricted CTL killing. As is evident, sensitization of EL4 cells for class I restricted iysis was demonstrated over a broad range of LPLL lipo-

240

F..GT.0VA 0

tO

ZO

.~0

,~0

SO

PERCENT SPECIFICI.YSIS AT E:T 40:1 Fig. I. The effect of LPLL

liposome

concentration

PERCENTSPECIFIC L¥S15 AT E:T JO:l

on antigen

delivery. The figure represents the amount of lysisobtained at E:T ratio 40:1 with the various target groups. Effectors were generated as described in the materials and methods section, EL4 cells were treated with different concentrations of LPLL liposome ranging from 4.72 to 0.3 p.g/ml with 40 #g/ml OVA in 5 ml of serum-free medium. E.G7-OVA exhibited a significant lysis of 44% and the negative controls EL4 and EL4 treated with free OVA showed insignificant lysis. Antibody depletion studies with anti-CD4 and anti-CD8 monoclonal antibodies were performed to demonstrate that cytotoxictty was mediated by CD8 + T cells (data not shown). some concentrations. However, peak cytotoxicity (41%) was observed at a LPLL liposome concentration of 1.18 p . g / m i declining to 7% specific lysis at a liposome concentration 4.72 / x g / m l . T h e reasons for the decline were not established but !iposome toxicity to target cells s e e m e d not to be the explanation (data not shown). In Fig. 2, the effects of O V A concentration on sensitization for a C T L r e s p o n s e is shown. Sensitization could be achieved at 5 / x g / m l O V A and in all experiments was optimal between 2 0 - 4 0 / ~ g / m l . The efficiency of the cationic liposome m e t h o d c o m p a r e s favorably with the previously r e p o r t e d pH-sensitive liposomal delivery approach (Reddy et al., 1991) and was far m o r e antigen efficient than was osmotic loading. T h e p r e p a r a t i o n of the LPLL liposomes is also less time c o n s u m i n g as c o m p a r e d to pH-sensitive lip o s o m e preparation. Studies have shown that PKC activators can e n h a n c e liposome-mediated D N A transfection ( Z h o u et al., 1992). T h e r e a s o n for this enhancem e n t in the presence of P M A is not known, but P M A could p r e s u m a b l y increase the endocytic

Fig. 2. The effect of varying OVA concentration on antigen presentation. EL4 cells were exposed to LPLL liposomes complexed with OVA, at concentrations ranging from 10 to 80 /~g/ml in 5 ml of serum-free medium and 1.18 p.g/ml LPLL liposomes. Percent specific lysis increased with increasing OVA concentration. Specific lysis of E.G7-OVA was 79%. EL4 treated with free OVA or LPLL liposome alone were used as controls and exhibited no killing. YAC-I cells treated with LPLL liposome (OVA concentration 20/.Lg/ml and 1.18 pg/ml LPLL) showed no lysisldata not shown).

activity of the cell by activating various cellular functions thereby increasing D N A uptake. T o test the uptake hypothesis, EL4 cells were incuso ~ ~ ~ ~

• PMA - . o - .e~ ÷ L ~ ÷p~

4o so-

~ ~

z0-

~

1o0 0

2.5

5

10

20

40

OVA CONCENTRATION (ggl

Fig. 3. Effect of PMA on LPLL liposome-mediated OVA delivery. EL4 cells were treated with liposome-OVA complexes at OVA concentrations of 2.5-40/zg/ml in the presence or absence of PMA (1.5 /zM). E.G7-OVA showed specific lysis of 42%. Negative controls gave no lysis. PMA enhanced the killing obtained at sub-optimal OVA dose of 10 #g/ml to that obtained at optimal OVA dose of 20/zg/ml.

concentrations. PMA had no increased lytic effect on EIA cells subjected to osmotic shock even at OVA concentrations as low as 0.625 m g / m l (Fig. 4). Table I demonstrates the optimum PMA concentration required for enhanced antigen delivery. EL4 cells were treated with liposome-OVA complexes, at varying PMA concentrations with O V A concentration constant at 10 /~g/ml. The optimum concentration of PMA to enhance LPLL liposome-mediated O V A uptake was 1.5/~M.

80' - PMA

i" 0

Discussion o

0.625

1.25

2.5

s.o

Io.o

OVA CnNCE~CTRATInN ling)

Fig. 4. Effect of PMA on deliveryof OVA by osmotic loading. EL4 cells were osmoticallyloaded with OVA at OVA concentrations 0.625-10 mg OVA in the presence or absence of PMA (1.5 pM). Percent specific lysisof E.G7-OVAwas 69%. Negativecontrols showed no lysis. bated with liposome-OVA complexes at varying O V A concentrations and 1.18/zg liposome/ml in the presence or absence of PMA (1.5 g,M PMA). Results in Fig. 3 demonstrate that PMA increases sensitization of EL4 cells, which is evident by the enhanced killing of target cells at lower O V A TABLE ! EFFECT OF VARYING PMA CONCENTRATIONS ON LPLL L1POSOME-MEDIATED OVA DELIVERY Effector cells were generated as described in the materials and methods section. EL4 cells were incubated with 5 ml of serum-free medium containing 20 / ~ g / m l O V A and 1.18 / ~ g / m l L P L L liposome at 37°C in a water-bath. The cells were washed thrice and used in a standard 4 h 5tCr release assay. E . G 7 - O V A and EL4 cells were used as controls. The experiment was repeated thrice with similar results. Groups

% specific lysis at

E:T ratio 30:1 E.G7-OVA

40

EL4 EL4 (500/~g free OVA) EL4/liposome-OVA/PMA(0#M) EL4/liposome-OVA/PMA(0.5/.tM) EL4/liposome-OVA/PMA(1.0/LM) EL4/liposome-OVA/PMA(l.5/zM) EL4/liposome-OVA/PMA(2.0p.M)

0.5 _+1 1 _+1 15 _+I 16 _4=3_ 20 +_2 30 + 2 18 +_l

_+3

This study adds to the approaches that result in sensitizing target cells for class I restricted iysis with soluble non-replicating antigens. The other methods previously described are osmotic loading of cells and the pH-sensitive liposomai delivery approach (Moore et al., 1988; Reddy et al., 1991). The present report demonstrates that positively charged L P L L / D O P E complexed with the negatively charged O V A (pl-4.63) can efficiently deliver the antigen to the cyto~l for class ! restricted processing and presentation. The optimum sensitization occured at LPLL liposome concentration of 1.18 p.g/ml and O V A concentration of above 20 p.g/ml (Figs. 1 and 2). Cationic liposomes are very effective D N A transfection reagents. Recent studies have shown that cationic liposomes composed of L P L L / D O P E and a target-specific ligand can be used as an efficient and simple method of delivering D N A into living cells (Zhou et al., 1991a). Such preparations have been used to deliver DNA, R N A and proteins to cells (Malone et al., 1989; Pinnaduwage et al., 1989; Debs et al., 1990). The advantages of LPLL is the multivalent nature of the cationic compound facilitating the complexing with negatively charged molecules. Anti::en processing for class I recognition may be related to biosynthesis of the antigen within the cell or localization of the antigen in the appropriate cellular compartment for association with the class I molecule. Studies by Moore et al. (1988) and Reddy et al. (1991) have demonstrated that cytoplasmic location of the antigen is a prerequisite for class 1 restricted antigen processing.

242 I n osmotic loading used by Moore et al. 11988). cells were incubated in hypertonic medium and allowed to take up O V A in pinocytic vesicles. Addition of hypotonie medium to the cells caused the pinocytic vesicles to rehydrate and b u l l inside the cell, introducing the antigen directly into the cytosol. Reddy el al. 119911 have de'monstrated sensitization o f cells for class I restricted CTL lysis using pH-scnsitive liposomes, which destabilize in the acidic environment of the endosome thereby releasing part of the antigen into the cytosol. T h e m e c h a n i s m o f c a t i o n i c liposome-mediated antigen delivery is not known, though a likely mechanism is via the cellular endecytosis pathway. The liposomc-OVA complexes may be internalized into the endosome and destabilization of the organelle m e m b r a n e induced by the cationic lipids may result in release of the antigen into the cytosol. Another possibility could be that the lipophilic nature of LPLL perturbs the cell membrane resulting in direct release of O V A into the cytosol. The former mechanism is more likely since cationic complexes are avidly endocytosed by cells (Leonelti el al.. 19901. Studies with P M A were done to shed light on the possible mechanism of L P L L liposomc-mediated antigen delivery. Previous studies have shown that P M A stimulated liposome mediated D N A transfection lZhou et hi.. 19921. The exact mechanism for this effect is not known, though it might be due to increased endocytosis of DNA-liposome complexes (Reston et al.. 1991L Results in Fig. 3 show that liposome-mediated O V A delivery can be significantly enhanced at sub-optimal O V A concentrations of 10 g g / m l in the p r e ~ n c e of PMA. This enhancement was not observed in cells subjected to osmotic loading in the presence of P M A (Fig. 4). T h e increased sensitization with liposome-OVA might be due to P M A induced activation of PKC. M e m b r a n e proteins, contractile and cytoskeletalr proteins and various enz3rmes serve as sabstrates for P K C and activation of these proteins by PKC might cause redistribution and reorientation of cytoskeletal elements, :This may result in modulation of membrane activities thereby increasing endocytosis of the l i p o , s o m e - O V A complex. Preferential effects of PMA on LPLL-liposome may indic.ate that the adsorp-

rive endocytic mechanism is stimulated by elevated PKC activity. Further exploration of the or;act role of P M A will requtre quantitation o f O V A uptake by LPLL-liposome treated cells and osmotically loaded cells with and without P M A . Studies on improved techniques, for measuring protein uptake are currently underway to explore the underlying mechanisms. The main contribution of LPLL-liposome would be as an i~ vitro antigen delivery system for studying antigen prcsentatton. LPLL-liposome method is an efficient means of sensitizing target cells for class I restricted lysls.

Acknowledgements This work was supported by N I H grant AI24762-05. We would like to thank Ms, Paula Keaton and Mrs. Audrey Williams for typing this manuscript.

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