Biodegradable, Endosome Disruptive, and Cationic Network-type

The success of gene therapy is largely dependent on the delivery vector system. Efficient transfection and nontoxicity are two of the most important r...
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Bioconjugate Chem. 2002, 13, 952−957

Biodegradable, Endosome Disruptive, and Cationic Network-type Polymer as a Highly Efficient and Nontoxic Gene Delivery Carrier Yong-beom Lim, Seon-mi Kim, Hearan Suh, and Jong-sang Park* School

of

Chemistry

&

Molecular

Engineering,

Seoul

National

University,

Seoul

151-742,

Korea.

Received April 22, 2002; Revised Manuscript Received July 10, 2002

The success of gene therapy is largely dependent on the delivery vector system. Efficient transfection and nontoxicity are two of the most important requirements of an ideal gene delivery vector. To generate both an efficient and nontoxic vector, we rationally constructed polymeric vectors to have simultaneous multiple functions, i.e., controlled degradation, an endosome disruptive function, and positive charges. Remarkably, the transfection efficiency of network poly(amino ester) (n-PAE) synthesized in this manner was comparable to that of polyethylenimine (PEI), one of the most efficient polymeric gene delivery vectors reported to date. However, there was a marked difference in cytotoxicity between the polymers. The majority of PEI-transfected cells were granulated and dead, whereas most of the cells transfected with n-PAE were viable and healthy. Successive events of efficient endosome escape of n-PAE/DNA polyplex and n-PAE biodegradation should result in high transfection efficiency and favorable cell viability response. The n-PAE-mediated transfection was also very efficient in the presence of serum. These data show that the approach we applied is a very appropriate way of making an ideal gene delivery carrier.

INTRODUCTION

The vector for the delivery of therapeutic DNA has been considered as a major hurdle in achieving successful gene therapy. A number of delivery systems based on viral (1, 2) or nonviral vectors (3-5) have been devised until now; however, none of these have proven to be satisfactory. Nonviral vectors have gained a great deal of interest by their advantages over viral vectors, such as low immunogenicity, limitless DNA incorporation capacity, the ease of large-scale production, and the possibility of fine-tuning the physicochemical and biological properties. However, those nonviral vectors efficient in transfection were toxic because of their nondegradable property (6). On the contrary, the transfection efficiencies of nontoxic nonviral vectors, e.g., biodegradable cationic polymers, were not satisfactory (7-13). Here we demonstrate our approach for generating both a transfection-efficient and nontoxic gene carrier based on a nonviral polymeric vector. To meet these requirements, we rationally designed polymer structure to have simultaneous multiple functions, i.e., biodegradable property, endosome disruptive function, positive charge for DNA condensation, and three-dimensional network-type chain interconnected structure for controlled degradation. n-PAE synthesized in this manner showed very high transfection efficiencies and negligible cytotoxicity in various kinds of mammalian cells. The polymer combined with this type of approach should represent an important step toward the creation of an ideal gene delivery vector. EXPERIMENTAL PROCEDURES

Materials. PEI (average molecular weight 25 kDa) was obtained from Aldrich. Fmoc-6-aminohexanoic acid * To whom correspondence should be addressed. School of Chemistry & Molecular Engineering, Seoul National University, San 56-1, Shinlim-dong, Kwanak-gu, Seoul 151-742, Korea (South), Phone: +82-2-880-6660. Fax: +82-2-877-5110. Email: [email protected].

(Fmoc-eAhx) was purchased from Novabiochem. pGL3Control vector was purchase from Promega. The pCN-LacZ vector encoding β-galactosidase gene was constructed by subcloning E. coli β-galactosidase cDNA to pCN, which contains the 600 bp HCMV IE promoter and its entire 5′ untranslated region consisted of 122 bp exon 1, 827 bp intron 1, and 16 bp exon 2 (14). Synthesis of Network Poly(amino ester). Monomer 1: To a stirred solution of methyl acrylate (150 mL, 1.67 mol) at 45 °C was added a solution of tris(hydroxymethyl)aminomethane (5 g, 41 mmol) in methanol (250 mL). The reaction mixture was stirred for 24 h at 45 °C. Methanol and excess methyl acrylate were evaporated to give 12 g (99%) of monomer 1 as a yellow oil. 1H NMR (300 MHz, DMSO-d6) δ 2.39 (t, 4H, CH2CH2COO), 2.75 (t, 4H, CH2CH2COO), 3.28 (s, 6H, HOCH2), 3.57 (s, 6H, CH3). 13C NMR (75 MHz, DMSO-d6) δ 35.41 (CH2CH2COO), 36.93 (CH2CH2COO), 51.41 (CH3), 59.98 (quaternary carbon), 61.05 (HOCH2), 173.16 (ester carbonyl carbons). MS (MALDI) Calcd for C12H23NO7 (M + H)+ 294.325; found 294.323. Polymer 1: For the polycondensation reaction, monomer 1 (5 g) placed in a glass vial was immersed into a silicon bath. Polymerization was carried out in the bulk at 170 °C under constant argon flow. The temperature of the reaction was increased at a rate of 10 °C/min. After 5 h of reaction, the resulting network-type polymer, which was a solid at room temperature, was pulverized to yield a yellow powder. 1H NMR (300 MHz, DMSO-d6) δ 1.41-2.77 (br m, CH2CH2COO and CH2CH2COO), 3.24-3.42 (br m, HOCH2), 3.50-3.57 (br m, CH3), 4.014.10 (br m, COOCH2). 13C NMR (75 MHz, DMSO-d6) δ 33.63-35.83 (CH2CH2COO), 36.61-37.79 (CH2CH2COO), 51.60 (CH3), 59.32, 62.62 (quaternary carbons), 60.86, 61.44, 64.27 (HOCH2), 165.13-173.53 (ester carbonyl carbons). n-PAE: A mixture polymer 1 (0.73 g) and Fmoc-eAhx (0.91 g, 2.58 mmol) in DMF (7 mL) at room temperature

10.1021/bc025541n CCC: $22.00 © 2002 American Chemical Society Published on Web 08/29/2002

Efficient and Nontoxic Polymeric Gene Carrier

was treated with 1,3-dicyclohexylcarbodiimide (DCC, 1.06 g, 5.15 mmol), 4-(dimethylamino)pyridine (DMAP, 0.16 g, 1.29 mmol), and p-toluenesulfonic acid monohydrate (PTSA, 0.23 g, 1.2 mmol). After 15 h at room temperature, the reaction mixture was filtered to remove DCU. The crude polymer was purified first by precipitating it into a large excess of water three times to remove DMAP and PTSA. The precipitates were collected, dissolved in CHCl3, and precipitated into a large excess of EtOAc/ ether (1:1) three times to remove residual DCC and FmoceAhx. The resulting polymer obtained (0.6 g) was a viscous semisolid. 1H NMR (300 MHz, DMSO-d6) δ 1.211.47 (br m, CH2(CH2)3CH2COO), 2.26 (br, CH2(CH2)3CH2COO), 2.93 (br, CH2(CH2)3CH2COO), 3.21-3.44 (br m, HOCH2), 3.50-3.53 (br m, CH3), 3.91-4.10 (br m, COOCH2), 4.18 (br, CH), 4.26 (br, CHCH2), 7.31-7.94 (br m, aromatic protons). Molecular weight by gel permeation chromatography (GPC): Mn ) 3800, Mw ) 4500, PDI ) 1.18. GPC was carried out using a Waters model 600E Multi Solvent Delivery System, a model 410 refractive index detector, and Autochro-GPC software (Young-In Scientific, Korea) for data acquisition with Styragel 2 and Styragel 4E columns placed in series in order of increasing pore size. THF was used as the eluent at a flow rate of 1 mL/min. The molecular weights were determined relative to narrow molecular weight polystyrene standard. Fmoc protection groups from the Fmoc-eAhx-coupled polymer 1 (0.5 g) was removed by adding 20% piperidine in DMF (v/v). After 5 min of the deprotection reaction, the mixture was added dropwise into a large excess of EtOAc/ether (1:1) three times to give n-PAE (0.21 g) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 1.24-1.47 (br m, CH2(CH2)3CH2COO), 2.27 (br, CH2(CH2)3CH2COO), 2.66 (br, CH2(CH2)3CH2COO), 3.21-3.40 (br m, HOCH2), 3.51-3.55 (br m, CH3), 3.85-4.15 (br m, COOCH2). Quantification of n-PAE Primary Amine Numbers by Using Fluorescamine. A series of known concentrations of n-butylamine (2 mL in 0.2 M sodium borate, pH 9.0) were placed in test tubes. While the tube was vigorously agitated on a Vortex mixer, 700 µL of fluorescamine solution (0.28 mg/mL in acetone) was added (15). Fluorescence measurements were carried out with an SFM 25 spectrofluorometer (Kontron Instruments). Excitation and emission wavelengths were set at 390 and 475 nm, respectively. A standard curve constructed in this way was used to find the amount of primary amines per mg n-PAE. Atomic Force Microscopy (AFM). DNA (pGL3control vector) was dissolved in Hepes-Mg (25 mM Hepes, 10 mM MgCl2, pH 7.6) buffer at a concentration of 1 µg/ mL to obtain a DNA image. Two microliters of the solution was deposited onto a freshly cleaved mica substrate. The solution was allowed to adsorb for 2 min, washed with 1 mL of distilled water, and rapidly dried in a stream of N2 gas. For imaging the polyplex, the polyplex was made by mixing the plasmid solution in water (5 µg/mL) with an equal volume of polymer solution in water. Two microliters of the polyplex solution was deposited onto a freshly cleaved mica substrate. The solution was allowed to dry for 2 min, and then excess fluid was wicked off with filter paper. The solution was dried at room temperature before imaging. AFM was performed using Nanoscope IIIa instrument equipped with an E scanner (Digital Instruments, Santa Barbara, CA). All AFM imaging was conventional ambient tapping mode AFM with scan speeds of about 5 Hz and data collection at 512 × 512 pixels.

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Cytotoxicity Measurement. Cells were seeded at a density of 10000 cells/well in 96-well plate and grown in MEM supplemented with 10% fetal bovine serum (FBS), and 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate to reach 60-70% confluence. Following exposure of the cells with polymer or polyplex solution for 4 h, cells were washed with phosphate-buffered saline (PBS), and 100 µL of the culture medium and 25 µL of 5 mg/mL stock solution of 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were added to each well. After 2 h of incubation at 37 °C, 100 µL of extraction buffer (20% w/v of SDS in a solution of 50% of DMF, pH 4.7) were added. Absorbance was measured at 570 nm after an overnight incubation at 37 °C. Transfection Protocol. Cells were seeded at a density of 10000 cells/well in 96-well plate and grown in MEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate to reach 60-70% confluence prior to transfection. Before transfection, cells were rinsed and serum-free or 10% FBS-containing medium was added to each well. The cells were treated with polyplex solution containing 1 µg of plasmid DNA for 4 h at 37 °C. The concentration of polymers varied depending on the polymer/DNA ratios. Then the transfection mixture was replaced with fresh medium. Cells were further incubated for 48 h at 37 °C. Luciferase gene expression was measured by a luminescence assay. The growth medium was removed, and the cells were rinsed twice with PBS and lysed for 20 min at room temperature in 100 µL of Reporter Lysis Buffer (Promega). The lysate was cleared by centrifugation, and protein content was determined by using Micro BCA Protein Assay Reagent Kit (Pierce). Thirty microliters of the lysate was dispensed into a luminometer tube, and luciferase activity was integrated over 10 s with 2 s measurement delay in a Lumat LB 9507 luminometer (Berthold, Germany) with automatic injection of 100 µL of Luciferase Assay Reagent (Promega). Results were expressed as relative light units per mg of cellular protein. RESULTS AND DISCUSSION

Synthesis and Characterization of n-PAE. The construction of n-PAE commences with the condensation polymerization of monomer 1, which has three hydroxyls, two methyl esters, and one tertiary amine, affording polymer 1 (Scheme 1). Coupling of Fmoc-eAhx acid in the remaining hydroxyl groups of polymer 1 and successive deprotection of Fmoc groups yields n-PAE. Quantification of primary amine numbers by fluorescamine method showed that n-PAE had 1.3 µmol primary amines/mg. GPC analysis with linear polystyrene standard showed that Mn and Mw of Fmoc-eAhx-coupled polymer 1 (n-PAE precursor) were 3800 and 4500, respectively. The unusually low polydispersity index (1.18) as a condensation polymer is likely the result of fractionation during precipitation purification. It is well-known that GPC, based on the hydrodynamic radius (Rh) measurement of polymers, underestimate the true molecular weight distribution (MWD) of branched polymers several times (16). As a result, it is generally accepted that true MWD of branched polymers should be three to five times higher than the values obtained from GPC. In this respect, the true MWD of n-PAE should be much higher than the values obtained by GPC with linear polymer standard. We are currently investigating the absolute MWD of

954 Bioconjugate Chem., Vol. 13, No. 5, 2002 Scheme 1. Synthesis of n-PAEa

a (i) Bulk polycondensation, 170 °C; (ii) Fmoc-6-aminohexanoic acid, DCC, DMAP, PTSA, DMF; (iii) piperidine/DMF (1:4). DCC ) 1,3-dicyclohexylcarbodiimide, DMAP ) 4-(dimethylamino)pyridine, PTSA ) p-toluenesulfonic acid monohydrate.

n-PAE by using a multiangle laser light scattering technique in GPC. The structure of n-PAE was designed to be multifunctional by equipping it with biodegradable ester backbone linkages for nontoxicity, multiple primary

Lim et al.

amines for DNA condensation, multiple tertiary amines for selective protonation according to the pH of surrounding milieu, and network-type chain interconnected structure for controlled degradation. Formation and Stability of n-PAE/DNA Polyplex. The formation and stability of n-PAE/DNA polyplex at pH 7.4, 37 °C were investigated by agarose gel electrophoresis as a function of time at various n-PAE nitrogen/ DNA phosphate ratios (N/P ratios) (Figure 1). Partially formed polyplex was observed at a N/P ratio of 4. When the ratio reached 10, no DNA migrated through the gel, indicating the formation of the polyplex. n-PAE had 1.3 µmol primary amines/mg as describe above. This means that charge neutralization of the polyplex occurs at a N/P ratio of 4.9 on the assumption that only primary amines are protonated at pH 7.4. Therefore, the exact point of charge neutralization should be a N/P ratio of between 4 and 10. As time passed, DNA bands appeared and the polyplex bands at the wells of agarose gel disappeared, indicating that after degradation of n-PAE the condensed DNA was gradually released from the polyplex. The stability of the polyplex increased with increasing N/P ratio where the polyplex remained intact over 3 days at and above a N/P ratio above 20 (Figure 1C and 1D). The appearance of linear DNA, which was not observed in initial plasmid DNA bands, suggests that small parts of nicked circular DNA were cleaved during the incubation period. However, the majority of the released plasmid DNA maintained their initial conformations even after the complete degradation of the polyplex. It has been reported that the linear polyesters having nucleophilic groups (primary amine, secondary amine, and carboxylic acid) in their structure were degraded very rapidly in aqueous solution through a self-destruction mechanism (7, 9). For this reason, the stability of the polyplexes formed with the linear polyesters could not exceed 8 h at pH 7.4, 37 °C. However, there is a need to extend the degradation to a reasonable rate for the polymeric vector to protect DNA from the harsh extracellular and/or cytosolic environment until it enters the cell nucleus. It can be expected that the MWD of the linear polyesters would be halved by a single cleavage of the polymer chain, which explains the rapid degradation kinetics of the polymers. In a network-type chain interconnected structure, however, the MWD of the polymer is not likely to be changed by a single and/or several chain cleavages. For this reason, the stability of n-PAE/DNA polyplex was much extended over that of linear biodegradable cationic polymers. AFM observations showed the formation of polyplex between n-PAE and DNA visually. Supercoiled or nicked circular DNA comprised the majority of the plasmid DNA population (Figure 2A). This result is consistent with the

Figure 1. Formation and stability of n-PAE/DNA (pGL3-control) polyplex. The polyplex formed at n-PAE/DNA ratio (N/P) of (A) 4 and (B) 10 and (C) 20 and (D) 40 and incubated at 37 °C. Aliquots taken at the indicated times were electrophoresed through 0.7% agarose gel and stained with ethidium bromide to visualize DNA. Form I ) supercoiled, form II ) nicked circular, and form III ) linear DNA.

Efficient and Nontoxic Polymeric Gene Carrier

Figure 2. AFM images of (A) plasmid DNA (pGL3-control) and (B) n-PAE/DNA polyplex. The polyplex was formed at a N/P ratio of 10.

electrophoretic observation of the plasmid DNA conformation (vide ante). AFM image of n-PAE/DNA polyplex showed that all of the plasmid DNA was complexed with n-PAE to form a somewhat heterogeneous population of round-shaped particles (Figure 2B). Cytotoxicity Measurement. The cytotoxicity profiles of n-PAE were investigated by using an MTT assay (Figure 3A and 3B). The results indicated that n-PAE was minimally toxic with 93% and 87% of cells remaining viable even at very high polymer concentration (200 µg/ mL) for 293 and HepG2 cells, respectively. The order of cytotoxicity was PEI (branched) > poly[R-(4-aminobutyl)L-glycolic acid] (PAGA) > n-PAE. PAGA is a biodegradable cationic polymer used for gene transfection as reported previously (9, 10). The polyplexes showed similar cytotoxicity profiles (Figure 3C and 3D). The near nontoxic property of n-PAE should be the result of the

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biodegradable property of the polymer, which is in line with several biodegradable cationic polymers investigated in the previous reports (7-13). Transfection Efficiency of n-PAE. The transfection efficiency (TE) evaluated by luciferase reporter gene assay revealed that n-PAE was very efficient in transfection (Figure 4A). Transfection was performed in the absence of chloroquine to show the polymers’ own effect. Chloroquine is known to accumulate in endosomal compartment, buffer endosome acidification, and induce osmotic swelling of the endosome, which eventually results in endosome destabilization and release of internalized polyplex (17). Remarkably, the TE of n-PAE was not less than that of PEI 25 kDa. PEI has been one of the most efficient polymeric gene carriers reported until now. However, the high cytotoxicity of PEI, due to the nondegradable property of the polymer, poses a serious problem for its application in human gene therapy trials (6). In 293 cells, the TE of n-PAE was even about 7-fold higher than that of PEI. In comparison, the TE of PAGA was very low, with values 110 and 320-fold lower than those of n-PAE in 293 and HepG2 cells, respectively. PAGA is a linear biodegradable cationic polymer with multiple primary amines. Similarly, the high TE and low cytotoxicity profile of n-PAE were also observed in other mammalian cell lines (data not shown). In situ X-gal staining of cells transfected with reporter gene expressing β-galactosidase highlighted the differences in cytotoxicity and transfection efficiencies among the polymers more demonstrably (Figure 4B). Far less than 1% of the cells were stained when PAGA was used

Figure 3. Cytotoxicity in 293 and HepG2 cells by MTT assay. (A, B) Cytotoxicity of polymers. (C, D) Cytotoxicity of polyplexes. All polyplexes were formed at polymer/DNA weight ratios of 5. PEI (O), PAGA (3), n-PAE (]), and polymer 1 (0). Relative cell viability was calculated as 100 × [(A570 of polymer treated cells - A570 of blank)/(A570 of control cells - A570 of blank)]. Mean ( SEM (n ) 5).

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Figure 5. Transfection in the presence of serum. Transfection was performed in 293 cells in the absence (open bars) or the presence (filled bars) of 10% FBS. Numbers in parentheses indicate N/P ratios. Mean ( SEM (n ) 5). Transfection experiments were performed without chloroquine and nigericin.

Figure 4. (A) Transfection efficiency (bar graph) and cytotoxicity (filled circle) in 293 and HepG2 cells. The cells were transfected with pGL3 control vector encoding firefly luciferase as a reporter gene. Numbers in parentheses indicate N/P ratios. RLU ) relative light unit. Mean ( SEM (n ) 5) (B) In situ X-gal staining of 293 cells transfected with pCN-LacZ vector encoding β-galactosidase gene. The scale bars represent 50 µm. Transfection experiments were performed without serum, chloroquine, and nigericin.

for transfection. In contrast, nearly all of the cells transfected with PEI and n-PAE were stained blue, which indicated the efficient transfection by both the polymers. However, the cells transfected with PEI are in striking contrast to the cells treated with PAGA and n-PAE. The majority of PEI-transfected cells were granulated and dead, whereas most of the cells transfected with PAGA and n-PAE were viable and looked healthy. If gene delivery carriers are to be used in vivo, the transfection efficiencies of the carriers should be remain relatively unaffected by the presence of serum. As shown in Figure 5, the TE of n-PAE was decrease by 6.3-fold in the presence serum at a N/P ratio of 10. However, n-PAEmediated transfection was even increased by 1.6-fold by the presence of serum at a N/P ratio of 40. The little or lack of serum inhibition on n-PAE transfection implies that n-PAE will be a promising candidate for in vivo gene therapy. Transfection Mechanism of n-PAE/DNA Polyplex. The high TEs of n-PAE and PEI are likely to be the result of the “endosome buffering” or “proton sponge” effect of the polymers (18). Multiple tertiary amines in the polymers, mostly in the form of free bases at slightly basic physiological pH, become protonated at the acidic pH of endosome, which disrupt the endosomal vesicle either by mechanical swelling or osmotic effects. To verify that the high TE of n-PAE is a result of the endosome buffering

Figure 6. Effect of chloroquine or nigericin treatment on the TEs of PEI, PAGA, and n-PAE. (A, B) RLU in the presence of chloroquine (50 µm)/RLU in the absence of chloroquine. (C, D) RLU in the absence of nigericin/RLU in the presence of nigericin (5 µm). Values are indicated above each bar.

effect, transfection experiments were performed in the presence or absence of chloroquine or nigericin (Figure 6). Nigericin is a carboxylic ionophore that mediates exchange of monovalent cations through the membrane and is known to be an inhibitor of endosomal acidification (19). Chloroquine treatment resulted in a decrease or a small increase in the TEs of PEI and n-PAE, respectively, but a large increase in the TE of PAGA in both cell lines tested (Figure 6A and 6B). For example, the TE of PAGA increased by 900-fold upon chloroquine treatment in 293 cells. In contrast, the TEs of PEI and n-PAE increased only by 0.2- and 1.4-fold, respectively. These findings suggest that polyplexes formed with PEI or n-PAE do not need chloroquine assistance for endosome escape, as they are already equipped with endosome buffering function

Efficient and Nontoxic Polymeric Gene Carrier

due to the presence of multiple tertiary amines in their structure. The effect of nigericin treatment on TEs further demonstrated the endosome buffering effect of PEI and n-PAE (Figure 6C and 6D). The efficiencies of PEI- and n-PAE-mediated gene delivery were significantly reduced when nigericin was added in the transfection medium, which are attributed to the inhibitory effect of nigericin on endosomal acidification. However, the TE of PAGA was even enhanced by nigericin treatment. Taken together, these results suggest that transfection of PEI and n-PAE is mediated by endosome acidification followed by release of the polyplex into cytosol, which would eventually increase the amount of DNA entering into the nucleus for the transcription. We have described that through the rational design of polymer structure it was possible to make a nontoxic and efficient gene carrier, which represents a further step in mimicking the cell entry mechanism of viruses, yet maintaining safety profiles. Further elaboration of the structure of n-PAE and/or the analogous polymers based on our approach, such as the attachment of a targeting moiety, a nucleus localization signal, and a stealth function, are expected ultimately to lead to the development of a “safe artificial virus”. ACKNOWLEDGMENT

This work was supported by the Korea Research Foundation (DP-0344) and by the Molecular Therapy Center of KOSEF (R03-2001-00031). LITERATURE CITED (1) Kay, M. A., Liu, D., and Hoogerbrugge, P. M. (1997) Gene therapy. Proc. Natl Acad. Sci. U.S.A. 94, 12744-12746. (2) Lee, H. C., Kim, S., Kim, K., Shin, H., and Yoon, J. (2000) Remission in models of type 1 diabetes by gene therapy using a single-chain insulin analogue. Nature 408, 483-488. (3) Nishikawa, M., and Huang, L. (2001) Nonviral vectors in the new millennium: Delivery barriers in gene transfer. Hum. Gene Ther. 12, 861-870. (4) Wagner, E., Plank, C., Zatloukal, K., Cotton, M., and Birnstiel, M. L. (1992) Transferrin-polycation-DNA complexes: The effect of polycations on the structure of the complex and DNA delivery to cells. Proc. Natl. Acad. Sci. U.S.A. 89, 7934-7938. (5) Vigneron, J., Oudrhiri, N., Fauquet, M., Vergely, L., Bradley, J., Basseville, M., Lehn, P., and Lehn, J. (1996) Guanidiniumcholesterol cationic lipids: Efficient vectors for the transfection of eukaryotic cells. Proc. Natl. Acad. Sci. U.S.A. 93, 9682-9686. (6) Godbey, W. T., Wu, K. K., and Mikos, A. G. (1999) Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc. Natl Acad. Sci. U.S.A. 96, 5177-5181.

Bioconjugate Chem., Vol. 13, No. 5, 2002 957 (7) Lim, Y., Choi, Y. H., and Park, J. (1999) A self-destroying polycationic polymer: Biodegradable poly(4-hydroxy-L-proline ester). J. Am. Chem. Soc. 121, 5633-5639. (8) Putnam, D., and Langer, R. (1999) Poly(4-hydroxy-L-proline ester): Low-temperature polycondensation and plasmid DNA complexation. Macromolecules 32, 3658-3662. (9) Lim, Y., Kim, C., Kim, K., Kim, S. W., and Park, J. (2000) Development of a safe gene delivery system using biodegradable polymer, poly[R-(4-aminobutyl)-L-glycolic acid]. J. Am. Chem. Soc. 122, 6524-6525. (10) Lim, Y., Han, S., Kong, H., Lee, Y., Park, J., Jeong, B., and Kim, S. W. Pharm. Res. (2000) Biodegradable polyester, poly[R-(4-aminobutyl)-L-glycolic acid], as a nontoxic gene carrier. 17, 811-816. (11) Lynn, D. M., and Langer, R. (2000) Degradable poly(βamino esters): Synthesis, characterization, and self-assembly with plasmid DNA. J. Am. Chem. Soc. 122, 10761-10786. (12) Wang, J., Mao, H., and Leong, K. W. (2001) A novel biodegradable gene carrier based on polyphosphoester. J. Am. Chem. Soc. 123, 9480-9481. (13) Lim, Y., Kim, S., Lee, Y., Lee, W., Yang, T., Lee, M., Suh, H., and Park, J. (2001) Cationic hyperbranched poly(amino ester): A novel class of DNA condensing molecule with cationic surface, biodegradable three-dimensional structure, and tertiary amine groups in the interior. J. Am. Chem. Soc. 123, 2460-2461. (14) Lee, Y., Park, E. J., Yu, S. S., Kim, D., and Kim, S. (2000) Improved expression of vascular endothelial growth factor by naked DNA in mouse skeletal muscle: Implication for gene therapy of ischemic disease. Biochem. Biophys. Res. Commun. 272, 230-235. (15) Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., and Weigele, M. (1972) Fluorescamine: A reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 178, 871-872. (16) Feast, W. J., and Stainton, N. M. (1995) Synthesis, structure and properties of some hyperbranched polyesters. J. Mater. Chem. 5, 405-411. (17) Plank, C., Zatloukal, K., Cotton, M., Mechtler, K., and Wagner, E. (1992) Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand. Bioconjugate Chem. 3, 533-539. (18) Boussif, O., Lezoualc’h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc. Natl Acad. Sci. U.S.A. 92, 7297-7301. (19) Uherek, C., Fominaya, J., and Wels, W. (1998) A modular DNA carrier protein based on the structure of diphtheria toxin mediates target cell-specific gene delivery. J. Biol. Chem. 273, 8835-8841.

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