Effects of Side Chain Configuration and Backbone Spacing on the

Cotten, M., Längle-Rouault, F., Kirlappos, H., Wagner, E., Mechtler, K., Zenke, M., Beug, H., and Birnstiel, M. L. (1990) Transferrin-polycation-medi...
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Bioconjugate Chem. 2005, 16, 694−699

694

Effects of Side Chain Configuration and Backbone Spacing on the Gene Delivery Properties of Lysine-Derived Cationic Polymers Sarah E. Eldred,‡ Margaret R. Pancost,‡ Karin M. Otte,‡ David Rozema,§,* Shannon S. Stahl,‡,* and Samuel H. Gellman‡,* Department of Chemistry, University of WisconsinsMadison, 1101 University Ave., Madison, Wisconsin 53706, and Mirus Corporation, 505 S. Rosa Road, Madison, Wisconsin 53719. Received January 25, 2005; Revised Manuscript Received April 3, 2005

A series of lysine-based oligomers (18 residues) that differ in side chain configuration or side chain spacing along the backbone was tested for DNA transfection activity. Although materials constructed from lysine are not the most effective polymeric transfection agents, we have chosen L-lysine-based molecules as a starting point because this system allows us to examine the functional effects of incremental changes in polycation structure. The oligomer constructed from β3-homolysine (β3-hLys) and that from R-D-lysine were superior to an R-L-lysine 18-mer in gene delivery assays. This improved activity is attributed to the fact that the R-L-peptide is a protease substrate while the other 18-mers are not. This conclusion is supported by the effects of chloroquine on transfection activity, based on the protease inhibition activity of chloroquine. To our knowledge, these results represent the first direct comparison of a D-lysine oligomer with an L-lysine oligomer in the context of gene delivery. Poly(β3-hLys) was synthesized from the ring opening polymerization of the corresponding lactam. The DNA transfection ability of this polymer was compared with that of commercially available poly(Llysine) (PLL). In each case the polymer was more active than the corresponding oligomer.

INTRODUCTION

Techniques for efficient delivery of foreign DNA into cells could lead to gene therapies for many human disorders, but realizing this potential will require improvement relative to current gene delivery methods (1). Injection of naked DNA into the body results in its rapid clearance and degradation, which precludes therapeutic efficacy (2-3). A further problem is that naked DNA is only poorly taken up by cells because of the large persistent length (stiffness) of this anionic biopolymer. Therefore, a delivery vehicle or “vector” is required. Viral vectors are among the most efficient gene delivery vehicles, but concerns about their safety have curtailed their use in clinical trials (1, 3). Nonviral vectors are safer, but they suffer from lower transfection efficiencies (4). Two classes of nonviral vectors are being investigated for gene delivery: cationic lipids and cationic polymers. Neutral polymers have been used in transfections but only when coupled to adenoviruses (5). The lipids can be effective, but inconsistencies in their preparation and difficulties in their purification have led to irreproducible transfection results in some cases (4, 6). Additionally, minor lipid modifications, desirable in terms of analyzing structure-activity relationships, can be difficult to achieve. A further problem is that lipid-DNA interactions are relatively weak and unstable in the presence of serum proteins (2). Polycations, on the other hand, are relatively easy to prepare, modify, and formulate with DNA. Thus, polycationic gene delivery vectors have received wide* To whom correspondence should be addressed. E-mail addresses: [email protected], [email protected], [email protected]. ‡ University of WisconsinsMadison. § Mirus Corporation.

Figure 1. Amino acid building blocks.

spread attention (7-14). Much research has focused on increasing the uptake of vector-DNA complexes (“polyplexes”) by attaching cell-adhesive ligands to the vectors in order to take advantage of receptor-mediated endocytosis. Another common transfection strategy has been to enhance endosomolysis (following polyplex uptake) by the incorporation of pH buffering or membrane-lytic agents into the vector. Less studied are the factors that control the intracellular dissociation of DNA from the cationic polymer, which is ultimately required for transcription and which may be important for transfection (15-22). We have undertaken a fundamental study of the effects of cationic polymer structure on transfection efficiency, using several lysine-based oligomers and analogous polymers. Poly(L-lysine) (PLL) and L-lysine oligomers are well-known to condense DNA, and to provide modest gene delivery to cells in culture (23-25). Although the activity of L-lysine-based polycations is inferior to that of other cationic polymers, we have chosen L-lysine-based molecules as a starting point because this system allows us to examine incremental changes in polycation structure. We have examined both the effect of changing the configuration of the side chain attachment point (use of D-lysine in place of L-lysine) and the effect of altering the backbone (use of β3-homolysine (β3-hLys) in place of lysine) on gene delivery activity (Figure 1).

10.1021/bc050017c CCC: $30.25 © 2005 American Chemical Society Published on Web 05/03/2005

Lysine-Derived Cationic Polymers

Oligomers and polymers of L-Lys are substrates for proteolytic enzymes, while polycations comprising D-Lys or β3-hLys are not (26-28). Therefore, comparisons among these three polycations should indicate whether proteolytic degradation is favorable or unfavorable with regard to delivery efficacy. (Comparisons among the Lysand β3-hLys-derived cations probe more subtle effects of polymer structure on delivery.) Polymer degradation too early in the uptake process would presumably be detrimental to transfection efficiency, but such degradation in later stages might enhance the necessary DNA unpacking. The effect of delivery vehicle degradation has received relatively little attention, even though poly(Dlysine) is commonly used in gene delivery studies instead of poly(L-lysine) (29). Side chain configuration effects have been examined elsewhere to track the intracellular fate of plasmid DNA during transfection (30), but to our knowledge the present study is unique in comparing the transfection efficacy of well-defined enantiomeric lysine oligomers and in extending the comparison from side chain configuration to side chain spacing along the backbone. EXPERIMENTAL PROCEDURES

General. All commercially available compounds were purchased from Aldrich and used as received unless otherwise noted. Fmoc-Lys(Boc)-OH (D and L versions) was purchased from Nova Biochem. Anhydrous CH2Cl2 was obtained from Fisher Scientific in FisherPak containers. It was passed through a solvent purification system prior to use (31). 1H and 13C NMR spectra were recorded on Bruker AC-300 MHz (75 MHz for 13C) spectrometers, and CDCl3 was purchased from both Cambridge Isotope Laboratories, Inc., and Aldrich. The chemical shifts (δ) are given in parts per million relative to internal TMS (0 ppm for 1H) and CDCl3 (77.23 ppm for 13C). IR spectra were recorded on a Bruker Tensor 27 IR instrument with an ATR attachment (Pike Technologies). MALDI-TOF-MS was performed using a Bruker REFLEX II spectrometer with a 337 nm laser using the R-cyano-4-hydroxycinnamic acid matrix. HPLC purification was performed using a Shimadzu instrument equipped with a reverse phase C4-silica column. Flash chromatography was performed on silica gel 60 (particle size 0.040-0.063 nm, 200-400 mesh ASTM, purchased from Sorbent Technology). Polymer molecular weight was determined by Dr. Michelle Chen at Wyatt Technologies using GPC with Shodex SB guard columns along with a Wyatt DAWN EOS multiangle light scattering detector and a Wyatt Optilab rEX concentration detector. Oligomer Synthesis. β-Amino acid residues were synthesized according to literature procedures (32-34). Peptide oligomers were synthesized using standard Fmoc solid-phase peptide synthesis techniques with HBTU activation on Rink amide resin (Applied BioSystems) on a Synergy automated synthesizer (Applied Biosystems). The resin-bound peptides were cleaved from the solid support and deprotected simultaneously by using trifluoroacetic acid. The peptides were precipitated by evaporation of the deprotection solution and addition of excess cold anhydrous diethyl ether. The peptide products were purified by reverse-phase HPLC and analyzed using MALDI-TOF-MS. Lactam 7. β3-hLys (32-33) 5 (1 equiv) was cyclized following the general procedure of Huang and co-workers (35). Briefly, 5 was placed in a round-bottomed flask with 2-chloro-1-methylpyridinium iodide (1.1 equiv), and CH3CN was added to make the reaction 0.01 M in substrate.

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Triethylamine (2.2 equiv) was then added, and the reaction was refluxed under nitrogen for 24 h. The solvent was then removed by rotary evaporation, and the residue was purified by column chromatography in ethyl acetate/hexanes. 1H NMR (CDCl3) δ 1.33-1.81, m, 15H; 2.57, dq J ) 14.7, 1.2 Hz, 1H; 3.06, ddd J ) 15.0, 5.1, 2.4 Hz, 1H; 3.13, q J ) 6.6 Hz, 2H; 3.61, qd J ) 4.8, 2.4 Hz, 1H; 4.51, br s, 1H; 6.0, br s, 1H. 13C NMR (CDCl3) δ 23.62, 28.63, 30.08, 35.23, 43.72, 48.21, 77.66, 156.24, 168.14. FTIR (ATR): 1518 cm-1 (amide II); 1689 cm-1 (carbamate); 1791 cm-1 (amide I); 3366 cm-1 (N-H). MS-ESI: m/z ) 265.1 [M + Na]+ (Calculated m/z ) 242.2 [M]+.) Poly(β3-hLys). In the glovebox, lactam 7 (1 equiv) was placed in a round-bottomed flask with Sc(NTMS2)3(THF)3 (2.5%) and anhydrous CH2Cl2 to make the reaction 0.02 M in substrate. The flasks were sealed, removed from the glovebox, and stirred at RT for 24 h. After 24 h the flasks were opened, and the contents were poured into diethyl ether to precipitate the polymer. The polymer was collected by filtration. Deprotection was accomplished by stirring in trifluoroacetic acid until the polymer dissolved (usually 2-3 days). The deprotected polymer was dialyzed against DI H2O with MW 1000 cutoff membranes and then lyophilized. 1H NMR (D2O) δ 1.33-1.66, br m, 6H; 2.38, br s, 2H; 2.96, t J ) 7.5 Hz, 2H; 4.10, br s, 1H. FTIR (ATR): 1561 cm-1 (amide II); 1646 cm-1 (amide I); 1676 cm-1 (TFA); 3280 cm-1 (N-H). Molecular weight was determined using GPC. Mn ) 62 000, Mw ) 93 000, PDI ) 1.5. Transfections. Plasmid DNA pCIluc (10 µg/mL, pCIluc is prepared according to a published procedure (36) where the luciferase reporter gene was under the cytomegalovirus promoter) in 0.25 mL of 5 mM HEPES pH 7.5 was complexed with polymers at a weight ratio of 1:2.66 DNA:polymer (N:P ratio of 1:3.5 using the TFA salt molecular weight for the polycation). Other weight ratios were tested and gave similar results. The DNA complexes were then added (100 µL) to wells containing cultured cells (mouse hepatoma Hepa-1clc7 cells or monkey kidney COS-7 cells, obtained from the American Type Culture Collection (ATCC)) in Dulbecco’s modified Eagle Media containing 10% fetal bovine serum. The cells were allowed to incubate for 48 h. The percentages of well surface area covered with cells were then determined using an Olympus CK2 inverted microscope. We have previously found (37) that assays of cell toxicity using confluency and methylthiazolyldiphenyltetrazolium bromide (MTT) give, within error, equivalent results. Due to the ease of measurement and the ability to assay toxicity and transfection using the same sample, confluency was used as a measure of toxicity in our studies. Cells in each well were 85-100% confluent at the time of the assay, indicating that the oligomers and polymers tested were noncytotoxic at the levels used for transfection. The cells were harvested and assayed for luciferase expression as previously reported (38) using a Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany) luminometer. Reported data is an average of at least six trials. Chloroquine Studies. Transfection studies with chloroquine were performed in a manner identical to that used in the non-chloroquine studies, save for the addition of 80 µM chloroquine to the wells along with the polyplexes. In these cases, the medium was changed after 2 h, and the cells were allowed to incubate for 46 h more. DNA Titration Assays. The collapse of TMR-labeled DNA was assessed using a quantitative assay based on condensation-induced quenching of a fluorophore covalently attached to DNA (39). Briefly, tetramethyl rhodamine-labeled DNA (TMR-DNA) was synthesized

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using tetramethyl rhodamine LabelIT (Mirusbio Corp) according to manufacturer’s protocol at a 1:1 DNA:TMR weight ratio. TMR-DNA (10 µg) was mixed with increasing quantities of polymers in 0.5 mL of 10 mM HEPES, pH 7.5. After addition of the polymer, TMR fluorescence of the samples was measured using a Cary spectrofluorometer (excitation wavelength (λex) of 546 nm; emission wavelength (λem) of 576 nm) at room temperature. A similar method was used to carry out the NaCl titration study. Polyplexes were generated using a 1:2.66 TMRDNA:oligomer ratio, and the TMR fluorescence was measured following NaCl additions. Particle Sizing. Particles comprised of DNA and polycations were formulated in 20 mM HEPES buffer pH 7.5 at a DNA:polycation ratio of 1:2.66 at a DNA concentration of 10 µg/mL. The size of the particles was assayed by light scattering at 532 nm using a Brookhaven Instruments Corporation, ZetaPlus Particle Sizer, I90. RESULTS AND DISCUSSION

Activity Comparisons among 18-Mers. Our studies compared (R-L-Lys)18 (1), which is known to be modestly active in gene delivery (23-25), with (R-D-Lys)18 (2), and the homologue of (1), (β3-hLys)18 (3). We also examined β-peptide 4, the analogue of 3, in which β3-hLys at onethird of the positions is replaced with a more rigid but still cationic residue, trans-4-aminopiperidine-3-carboxylic acid (APiC (34)). Transfection assays with hepa cells indicated that the β-oligomers (3 and 4) and (R-D-Lys)18 (2) outperformed (R-L-Lys)18, but that the two β-peptides were comparable to one another (Table 1). Because the APiC monomer is more difficult to prepare than is β3hLys (34), subsequent β-peptide studies were limited to molecules containing exclusively β3-hLys.

Transfections of both hepa and COS cells showed enhanced activity for both 2 and 3 relative to 1 (Table 1). The hepa cell line was chosen because of long-term interest in gene delivery to hepatocytes, while the COS cell line was selected to demonstrate generality (COS cells are not hepatocytes) and because the transfection properties of COS cells are well-known. In the transfection assays, cells were plated into wells at 40-50% confluency, as determined by light microscopy. At the conclusion of the 48-h incubation period, the confluency of the cells was again determined by light microscopy. Previous work (37) has shown that any toxic effects of transfection agents will be manifested as low confluency after the incubation period, and that confluency is comparable to an MTT assay as an indicator of toxicity (or lack thereof). In our studies, all cells were between

Eldred et al. Table 1. DNA Transfection Activity of Oligomers 2-4 Relative to 1a transfection transfection relative to 1 relative to 1 entry oligomer cell line (no chloroquine) (with 80 µM chloroquine) 1 2 3 4 5

2 3 4 2 3

hepa hepa hepa COS COS

2.3 ( 0.6 4.1 ( 1.5 4.2 ( 1.9 24 ( 10 32 ( 10

0.5 ( 0.2 0.6 ( 0.5 1.1 ( 0.2 1.3 ( 0.04

a Transfections were performed at a DNA:oligomer ratio of 1:2.66. Data reported is an average of at least six trials. Errors are one standard deviation from the mean.

85 and 100% confluent at the end of the 48-h incubation period, indicating that the oligomers used were not cytotoxic at the levels employed for transfection. Our primary goal was to determine the relative transfection efficiencies of a set of related lysine-based oligocations. This information is intended to illuminate the relationship between polycation structure and transfection efficacy and thereby provide a basis for developing more effective transfection agents in the future. Since the crucial observations in this case are the relative transfection efficiencies among oligomers 1-4, and not the absolute transfection results, the data have been normalized to the transfection efficiency of oligomer 1. The results in Table 1 show that 2 and 3 are superior to 1 for both hepa cells (entries 1 and 2) and COS cells (entries 4 and 5), but that the advantage is larger for COS cells. We cannot explain this difference. The most important point, however, is that for both cell types (R-D-Lys)18 and (β3-hLys)18 display enhanced activity relative to (R-L-Lys)18. Chloroquine Effects. Chloroquine is a well-known transfection-boosting reagent that has been used to promote endosomal escape by DNA-cation polyplexes (40). It is hypothesized that chloroquine’s favorable effect derives from its buffering activity, which prevents the normal endosomal acidification process and causes release of the endosomal contents into the cytoplasm (4, 40, 41). Not surprisingly, transfection assays of 18-mers 1-3 conducted in the presence of chloroquine led to overall activity enhancements relative to assays conducted without chloroquine. More interesting is the observation that the transfection ability of proteasesensitive L-Lys 18-mer 1 has a significantly greater enhancement relative to those of 2 or 3 in the presence of chloroquine. The activity of 1 is greater than or equal to the activity of enantiomer 2 or β-peptide 3 when chloroquine is used with either hepa cells (Table 1, entries 1-2) or COS cells (Table 1, entries 4-5). As in the absence of chloroquine, cells were 85-100% confluent at the completion of the 48 h incubation period; thus, the chloroquine concentration we used does not exert significant toxic effects. Chloroquine acts as a protease inhibitor in addition to its buffering ability (16, 42, 43). We attribute chloroquine’s greater enhancement of transfection by 1, relative to the effects on 2 or 3, to the protease inhibition activity of chloroquine. L-Lys 18-mer 1 should be readily degraded by cellular proteases while D-Lys 18-mer 2 and β3-hLys 18-mer 3 should be intrinsically resistant to proteolysis. Previous research has shown that L-Lys oligomers shorter than 18 units are not effective for DNA transfection (44); therefore, it is reasonable to assume that the fragments of oligomer 1 would not be effective for transfection. Our observations concerning chloroquine’s enhancement suggest that in the case of protease-labile delivery vectors,

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Lysine-Derived Cationic Polymers

Figure 2. Quenching of fluorescent DNA as a function of amount of added oligomer for 1 and 3. Error bars are one standard deviation from the mean. Full condensation was reached at a DNA:oligomer weight ratio of 1:1.

Figure 3. Effect of NaCl concentration on polyplex stability for polyplexes formed between fluorescently labeled DNA and 1 or 3. Error bars are one standard deviation from the mean. Scheme 1

pH-buffering is not the only activity of chloroquine responsible for increased transfection activity. The differential effect of chloroquine within polycation series 1-3 is consistent with our hypothesis that the superior gene delivery activity of 2 and 3 relative to 1 in the absence of chloroquine stems from the chemical instability of 1 in cellular fluids. Since the polycation must dissociate from the DNA at some point within the cell to allow transcription, and since cleavage of the polycation should encourage dissociation from the DNA, it is not obvious a priori whether proteolytic degradation of the cationic vector would enhance or diminish delivery efficiency. Our results suggest that breakdown of a polycationic delivery vector can impede transfection. Evaluation of DNA-Oligocation Interactions. In an effort to elucidate the origin of the difference in gene delivery activity among 18-mers 1-3, we compared DNA polyplex formation with these oligomers. Complexes formed at a 1:2.66 DNA:oligomer ratio (effective for transfection) were found to be indistinguishable in size. Polyplexes generated from 1 had an average diameter of 48 nm, polyplexes from 2 had an average diameter of 60 nm, and polyplexes from 3 had an average diameter of 58 nm. These differences are not significant. We used titration experiments to probe the efficiency of DNA-polycation complexation (39). In these experiments, varying amounts of polycation are added to fluorescently labeled DNA. Polycation-induced condensation of the DNA is detected as a decrease in fluorescence. When the fluorescence reaches a steady minimum, we assume that the DNA is fully condensed. Figure 2 shows that the results for 1 and 3 are identical; results for 2 (not shown) were also indistinguishable. These findings indicate that the DNA-polycation ratios in these polyplexes are not altered by backbone spacing or side chain configuration. In addition, the observation that the same level of quenching is reached for both polycations indicates that the average distance between flourophores is the same, which implies that the condensed-DNA particles have very similar packing and structure. A third experiment was performed in order to compare the stabilities of the polyplexes. Polyplexes formed between fluorescently labeled DNA and oligocation 1, 2, or 3 were exposed to increasing concentrations of NaCl in order to determine the salt concentration required to “unpack” the polyplex (as indicated by an increase in fluorescence). In the absence of polycation, high salt concentrations result in a nominal increase in fluorescence; however, this increase is gradual as a function of salt concentration, unlike the sharp rise seen in Figure 3 at approximately 550 mM NaCl. The magnitude of the

Scheme 2

change is much smaller in the absence of polycation than in its presence. Figure 3 shows that the results for 1 and 3 are identical; results for 2 (not shown) were indistinguishable. Thus, the strength of DNA-oligocation association is not influenced by the variation in backbone spacing of side chains or side chain configuration. Overall, we find no significant differences among polyplexes formed between DNA and 18-mers 1-3 can be detected by three distinct measurements. Cationic Polymers. The transfection efficacy for lysine polymers depends on molecular weight, and an oligomer containing only 18 residues is expected to be less effective on a weight basis than are longer polymers (23-25). We examined higher molecular weight polymers of β3-hLys in order to determine whether a similar trend would be observed in these cases as well. Commercially available poly(L-lysine) (PLL) was used as a positive control for comparison with poly(β3-hLys) prepared in our laboratory. We selected 70 and 188 K PLL as our control polymers in order to bracket the molecular weight of the poly(β3-hLys) (93 K). The poly-β-peptide was synthesized by metal-catalyzed ring-opening polymerization of a protected β-lactam (45). Synthesis of this β-lactam begins with a commercially available protected form of amino acid L-lysine (5), which is homologated using the Arndt-Eistert sequence to provide β-amino acid 6 (Scheme 1) (32-33).

698 Bioconjugate Chem., Vol. 16, No. 3, 2005 Table 2. DNA Transfection Activity of Poly(β3-hLys) Relative to PLLa entry

polycation

cell line

relative transfection

1 2 3 4

poly(β3-hLys) poly(β3-hLys) poly(β3-hLys) poly(β3-hLys)

hepa hepa COS COS

13 ( 8 (relative to 70 K PLL) 18 ( 5 (relative to 188 K PLL) 11 ( 3 (relative to 70 K PLL) 9 ( 7 (relative to 188 K PLL)

a Transfections performed at a DNA: oligomer ratio of 1:2.66. Data reported is an average of at least six trials. Errors are one standard deviation from the mean.

Deprotection and cyclization then yield β-lactam 7 (35). Polymerization of 7 leads to the side chain-protected polymer (Scheme 2). Deprotection with trifluoroacetic acid followed by dialysis provides poly(β3-hLys) with Mn ) 62 000 and Mw ) 93 000 (PDI ) 1.5). Transfection assays with hepa cells (Table 2, entries 1-2) show that poly(β3-hLys) outperforms both PLL controls. Studies with COS cells (Table 2, entries 3-4) indicate superiority of poly(β3-hLys) over both 70 and 188 K PLL. CONCLUSIONS

We have shown that oligomers or polymers of D-Lys or β3-hLys are more effective gene delivery agents than are analogous oligomers or polymers of L-Lys for cultured cells. This difference in activity appears to result from the fact that proteases can degrade vectors composed of L-Lys, while vectors composed of D-Lys or β3-hLys are not susceptible to proteolysis. By several measures, the polyplexes formed between DNA and 18-mers of L-Lys, D-Lys or β3-hLys do not differ significantly from one another. Our results indicate that premature breakdown of a cationic oligomer or polymer used to package DNA for delivery can diminish gene transfer activity. ACKNOWLEDGMENT

This work was supported in part by the NSF Collaborative Research in Chemistry Program (CHE0404704). Initial phases of the research were supported by seed funding (UW-Madison MRSEC) and by an Industrial and Economic Development Research grant from UW-Madison. We thank Dr. Michelle Chen of Wyatt Technologies for polymer molecular weight characterization. LITERATURE CITED (1) Mulligan, R. C. (1993) The basic science of gene therapy. Science 260, 926-932. (2) Langner, M. (2000) The intracellular fate of non-viral DNA carriers. Cell. Mol. Biol. Lett. 5, 295-313. (3) Piskin, E., Dinc¸ er, S., and Tu¨rk, M. (2004) Gene delivery: intelligent but just at the beginning. J. Biomater. Sci. Polym. Ed. 15, 1181-1202. (4) Pouton, C. W., Lucas, P., Thomas, B. J., Uduehi, A. N., Milroy, D. A., and Moss, S. H. (1998) Polycation-DNA complexes for gene delivery: A comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids. J. Controlled Release 53, 289-299. (5) Bellocq, N. C., Kang, D. W., Wang, X., Jensen, G. S., Pun, S. H., Schluep, T., Zepeda, M. L., and Davis, M. E. (2004) Synthetic biocompatible cyclodextrin-based constructs for local gene delivery to improve cutaneous wound healing. Bioconjugate Chem. 15, 1201-1211. (6) Guo, X., and Szoka, F. C., Jr. (2003) Chemical approaches to triggerable lipid vesicles for drug and gene delivery. Acc. Chem. Res. 36, 335-341. (7) Wolfert, M. A., Dash, P. R., Nazarova, O., Oupicky, D., Seymour, L. W., Smart, S., Strohalm, J., and Ulbrich, K. (1999) Polyelectrolyte vectors for gene delivery: Influence of

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