Synthesis and Characterizations of New Lysine-Based Biodegradable

Mar 26, 2009 - The results indicate that PMMD (6) is an attractive cationic poly(urethane-co-ester) for gene delivery and an interesting candidate for...
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Bioconjugate Chem. 2009, 20, 774–779

Synthesis and Characterizations of New Lysine-Based Biodegradable Cationic Poly(urethane-co-ester) and Study on Self-Assembled Nanoparticles with DNA Zheng-Yan Jian,† Jiunn-Kae Chang,‡ and Min-Da Shau*,‡ Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan, and Department of Biotechnology, Chia-Nan University of Pharmacy and Science, Tainan 71710, Taiwan. Received November 19, 2008; Revised Manuscript Received February 16, 2009

To improve the self-assembly efficiency of nanoparticles with DNA, we synthesized lysine-based poly(urethaneco-ester) PMMD (6) and polyester PDMA (8) bearing ester linkages in the backbone and tertiary amines in the side chain. Both poly(urethane-co-ester) PMMD (6) and polyester PDMA (8), readily self-assembled with plasmid DNA (pCMV-β-gal) in HEPES buffer, were characterized by dynamic light scattering and zeta-potential. The results reveal that PMMD (6) and PDMA (8) were able to self-assemble particles with DNA and yield complexes with positive charges of 80-115 nm and 170-180 nm in size at mass ratios (W/W) of 2/1 and 20/1, respectively. The degradation studies indicate that the half-life of PMMD (6) in the HEPES buffer was 20 h at pH 7.4. Titration studies were performed to determine the buffering capacities of the polymers. In addition, the COS-7 cell viabilities in the presence of PMMD/DNA, PDMA/DNA, and PEI/DNA were studied. The results indicate that PMMD (6) is an attractive cationic poly(urethane-co-ester) for gene delivery and an interesting candidate for further study.

INTRODUCTION Plasmid DNA has great potential as a therapeutic agent and in new vaccines (1). DNA-based pharmaceuticals largely depend on the characteristics of the delivery systems (2). To achieve controlled delivery of DNA to the target, vectors have been substantially developed. Because viral vectors have drawbacks such as the random insertion of viral sequences into the host chromosomes, indication of immunological responses, and limitations associated with DNA size, considerable attention has been given to nonviral vectors. Polycations (3-9) have been widely investigated in nonviral gene delivery in a variety of materials. Cationic polymers condense DNA into nanoparticles small enough to enter a cell, and protect negatively charged strands of DNA from nuclease degradation. Cytotoxicity and nonbiodegradability are concerns for long-term safety. Several amine-containing polymers, such as poly(urethane) (4, 10), poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) (11), poly(amido-amine) (12, 13), poly(ethylene imine) (PEI) (14), poly(N-methyldietheneamine sebacate) (15), poly(amido ethylenimine) (16), PAMAM-PEG (17), and poly(ethylene glycol)s (18), have been synthesized. Release of the delivery vehicle from these sections into the cytoplasm is believed to be the limiting step in the transfection mediated by many cationic polymers (19). Molecular weights of polymers and the polymer/ DNA ratio have been shown to be important for transfection efficiency. The molecular weight of a polymer affects the affinity between the cation-chain of the polymer and the anion-strand of DNA (20, 21). Polyesters are biodegradable biomaterials that are used in a wide range of applications, including the controlled release of DNA-based therapeutic agents. These include the controlled degradation of the polymer and a great diversity of side groups and backbones. In this study, amine-containing polyester (PDMA) and poly(urethane-co-ester) (PMMD) were * Corresponding author. Tel: 886-6-2664911ext. 2505. Fax: +8866-266-2132. E-mail: [email protected]. † National Cheng Kung University. ‡ Chia-Nan University of Pharmacy and Science.

designed and synthesized. Their structures in correlation to DNA condensation capacity, biodegradability, and cytotoxicity are discussed in this article.

MATERIALS AND METHODS Materials. 1,4-Butanediol diacrylate was purchased from Lancaster. L-Lysine methyl ester diisocyanate was from LDI, Kyowa Hakko Kogyo, Japan. 2-Hydroxyethyl acrylate (HEA), n-hexane, 2-dimethyaminoethylamine (DMAE), and glutaraldehyde were obtained from Fluka Co. (Switzerland). The solvent of N,N-dimethylformamide (DMF, Tedia co., USA) was dried over calcium hydride and distilled just before use. N-[2Hydroxyethyl] piperazine-N′-[2-ethanesulfonic acid] (HEPES) was obtained from Sigma Co. (USA). N-Methyl dibenzopyrazine methyl sulfate (electron-coupling reagent) and sodium (2,3bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) (XTT) were purchased from Roche Co. (USA). The plasmid pCMV-LacZ (pCMV-βgal) (4, 22) contained a CMV promotertodriveβ-galactosidase(LacZ)geneexpression(4,14,23). The plasmid DNA was amplified in Escherichia coli (DH5R strain) and purified using column chromatography (Qiagen Plasmid Mega kit, Germany). The purified plasmid DNA was dissolved in a tris(hydroxymethyl) methylamine-ethyldiaminetetraacetic acid (Tris-EDTA) buffer (pH 8.0) and determined using the ratio of UV absorbance at 260 nm/280 nm. Monkey SV40 transformed kidney fibroblast COS-7 cells were obtained from American type Culture Collection (ATCC, CRL-1651). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GibcoBRL Co., Ltd.) supplemented with 10% FBS, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 4 mM L-glutamine, and maintained at 37 °C in a humidified 5% CO2containing atmosphere. Polymer Characterizations. The structures of the polymers were characterized by nuclear magnetic resonance (NMR, Bruker AMX-400 spectrometer) and Fourier transform infrared (FT-IR, Mattson Galaxy Series 5000 spectroscope). The molecular weight and distribution of the polymer was determined

10.1021/bc800499w CCC: $40.75  2009 American Chemical Society Published on Web 03/26/2009

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Scheme 1. Synthesis Scheme of PMMD

by gel permeation chromatography analysis (GPC, Waters Model LC-2410) based on polystyrene standards in THF. Synthesis of PMMD (6). PMMD (6) was synthesized as shown in Scheme 1. HEA (2) and DMAE (1) with a molar ratio of 2/1 (-CdC/-NH2) were mixed in THF in a three-necked reaction flask under a dry nitrogen purge and then heated to 45 °C to react for 24 h. The product was precipitated in anhydrous ethyl ether and vacuum-dried at 40 °C. The purity of diol (3) was about 98%. The diol (3) and polymers were characterized by FT-IR, 1H NMR, and 13C NMR. Diol (3). 1H NMR (400 MHz, d6-DMSO, ppm) δ: 4.6 (1H, -CH2CH2OH), 2.3-2.5 (6H, -CH2CH2-, -CH2CH2COO-), 3.1 (2H, -CH2COO-), 4.1 (2H, -COOCH2-), 3.7 (2H, -CH2OH), 2.2 (6H, -N(CH3)2). 13C NMR (400 MHz, d6-DMSO, ppm) δ: 57.5 (-COOCH2CH2-), 60.2 (-COOCH2CH2-), 53.9 (-CH2COO-), 59.3 (NCH2CH2-), 58.9 (NCH2CH2N(CH3)2), 59.1 (NCH2CH2N(CH3)2), 44.2 (-N(CH3)2), 174.5 (-COO-). The monomers of diol (3) and LDI (4) with an -OH/-NCO molar ratio of 2/1 were mixed and then heated to 70 °C to react using 0.5 wt % dibutyltin dilaurate as a catalyst. The chain extension was carried out by slowly adding MDEA (Nmethyldiethanolamine) (5) to the isocyanate-terminated prepolymer until the -NCO/-OH molar ratio was 1/1. PMMD (6) was synthesized through the aminolysis reaction as previously reported (4). PMMD (6). 1H NMR (400 MHz, d6-DMSO, ppm) δ: 2.0-2.2 (3H, NCH3), 2.3-2.5 (4H, -CH2CH2N), 3.1 (2H, -CH2COO-), 3.7-4.2 (4H, -OCH2CH2O-), 3.0 (1H, -CHCONH-), 1.3-1.8 (6H, -CH2CH2CH2-), 3.6 (2H, -CH2NHCOO-), 3.7-4.2 (2H, -NHCOOCH2-), 3.6 (2H, -CONHCH2-), 5.3 (1H, -CH2NHCOO-), 5.7 (1H, -COONHCH-), 7.0 (1H, -CONHCH2-). 13C NMR (400 MHz, d6-DMSO, ppm) δ: 43.7 (NCH3), 56.1 (-CH2CH2N), 40.3 (-CH2NHCOO-), 22.3 (-CH2CH2CH2CH2NH-

COO-), 29.2 (-CH2CH2CH2CH2NHCOO-), 31.8 (-CH2CH2CH2CH2NHCOO-), 53.6 (-OOCNHCH), 36.5 (-CONHCH2CH2N), 58.6 (-CONHCH2CH2N(CH3)2), 44.6 (-CONHCH2CH2N(CH3)2), 57.5 (-CH2COOCH2CH2OOCNH-), 60.2 (-CH2COOCH2CH2OOCNH-), 53.9 (NCH2CH2COO-), 59.3 (NCH2CH2COO-), 58.9 (NCH2CH2N(CH3)2), 59.1 (NCH2CH2N(CH3)2), 44.2 (NCH2CH2N(CH3)2), 156.1 (-NHCOO-), 156.7 (-OOCNH-), 162.5 (-CONH-),174.5 (-COO-). Synthesis of PDMA (8). PDMA (8) was synthesized via standard steps as shown in Scheme 2. 1,4-Butanediol diacrylate was added to (DMAE) (1) according to the method of Ferruti et al. (3, 24, 25). PDMA (8). 1H NMR (400 MHz, d6-DMSO, ppm) δ: 3.7 (2H, -COOCH2CH2-), 4.1 (2H, -COOCH2CH2-), 3.1 (2H, -CH2COO-), 2.3-2.5 (6H, -CH2CH2-, -CH2CH2COO-), 2.2 (6H, -N(CH3)2). 13C NMR (400 MHz, d6-DMSO, ppm) δ: 57.5 (-COOCH2CH2-), 60.2 (-COOCH2CH2-), 53.9 (-CH2COO-), 59.3 (NCH2CH2-), 58.9 (NCH2CH2N(CH3)2), 59.1 (NCH2CH2N(CH3)2), 44.2 (-N(CH3)2), 174.5 (-COO-). Acid-Base Titration Assay of Polymers. Acid-base titration was used to evaluate the buffering capacity of synthesized cationic polymers. In this assay, 10 mg of PMMD (6) was dissolved in 10 mL of 150 mM NaCl. One hundred microliters of 1 N NaOH was then added to the solution to adjust the PH to 11.6 before it was titrated with acid. The solution was titrated with increasing volumes of 0.1 N HCl solutions, and the results were measured using a pH meter. The pH range of PDMA (8) was determined using the same procedure. Hydrolytic Degradation of Polymers. PDMA (8) and PMMD (6) were dissolved in a buffer solution (pH 7.4 and pH 5.1) with a concentration of 10 mg/mL and then incubated in a water bath at 37 °C for various durations. After hydrolysis for various durations, the solution was dried in vacuum for several

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Scheme 2. Synthesis Scheme of PDMA

hours to remove water. The molecular weight of the polymer was determined using gel permeation chromatography (GPC). XTT Assay. The influence of the polymer concentration on cell viability was evaluated in a cell culture for various polymers. The cytotoxicities of PDMA (8)/DNA and PMMD (6)/DNA for comparison with that of PEI/DNA were evaluated using the XTT assay (4, 22). In a 96-well plate, COS-7 cells were cultured in complete DMEM and then seeded at a density of 1.0 × 104 cells/well. The cells were incubated at 37 °C and 5% CO2 in a humidified atmosphere for 24 h. Subsequently, the cells were incubated for 1 h in 200 µL of FBS-free DMEM containing polymer with various concentrations. The cells were incubated in DMEM as a negative control. After 1 h, the cells were washed with 200 µL of PBS solution and replaced by complete DMEM for a further 48 h of incubation. Then, 50 µL of XTT labeling mixture was added to each well, and the cells were further incubated at 37 °C for 1 h. Results are expressed as the relative cell viability (%) with respect to control wells containing culture medium. Formation of Polymer/DNA Complexes. The polymer (10.0 mg/mL) was dissolved in 20 mM HEPES buffer (pH7.4), and its serial dilutions were made with the mass ratio of PMMD (6)/DNA (w/w) from 1/2 to 50/1. The complexes were then allowed to self-assemble in the HEPES buffer. They were incubated at room temperature for 30 min before measurements. Characterizations of Polymer/DNA Complexes. The particle sizes and surface charges of the polymer/DNA complexes were determined by dynamic light scattering (Nicomp 380 system, USA) and electrophoretic mobility at 25 °C with a Zetapotential system (Nicomp Instrument, USA). DNA Gel Retardation Assay of Polymer/DNA Complexes. PDMA (8)/DNA complexes and PMMD (6)/DNA complexes were loaded into a 0.7% agarose gel containing ethidium bromide (0.3 µg/mL) in a tris-acetate-EDTA (TAE) buffer and electrophoresis performed at 100 V for 45 min. After electrophoresis, the DNA bands were visualized using UV-irradiation. PDMA (8)/DNA complexes and PMMD (6)/DNA complexes with ratios of 0.5/1, 1/1, 2/1, 3/1, 4/1, 5/1, 6/1, 20/1, and 50/1 (w/w) were prepared.

cm-1 (N-H, bending, amide), and 3340 cm-1 (N-H, stretching, urethane) represent the absorptions of urethane groups in PMMD (6), as shown at the top of Figure 2. The peak at 1740 cm-1 (CdO, stretching, ester) represents the absorption of ester induced from PDMA (8), as shown at the bottom of Figure 2. The chemical shifts of characterized protons and carbons of diol, PMMD, and PDMA are shown in the Materials and Methods section. In addition, the GPC data of PMMD (6) and PDMA (8) show that the weight-averaged molecular weights were 17600 and 16800 with polydispersities of 1.8 and 1.7, respectively, relative to polystyrene standards in THF. Size and Zeta-Potential analysis of Polymer/DNA Complexes. Figures 3 and 4 show the size and zeta-potential of PMMD (6)/DNA and PDMA (8)/DNA complexes at various mass ratios, determined using dynamic light scattering (DLS) and electrophoretic mobility at 25 °C with zeta-potential analysis. The size of complexes decreased with an increase of the mass content of the polymers until the mass ratio of PMMD (6)/DNA reached 2/1 and that of PDMA (8)/DNA reached 20/ 1. The results show that the average diameters (80-115 nm) of PMMD (6)/DNA and the average diameters (170-180 nm) of PDMA (8)/DNA fall within the optimal range (40nm-200nm) required for cellular endocytosis (26). The above data demon-

Figure 1. FT-IR spectra of (a) DMAE, (b) HEA, and (c) the synthesized diol.

RESULTS AND DISCUSSION Structural Characterizations of Diol (3), PMMD (6), and PDMA (8). Diol (C14H28O6N2), PMMD ((C26H48O9N6)mC5H11N1)n, and PDMA (C14H26O4N2)n were synthesized as shown in Schemes 1 and 2. The synthesis of diol (3) containing tertiary amine in the backbone, in accordance with the synthesis of poly(amido amine), was reported by Ferruti et al. (3, 24, 25). Figure 1 shows the FT-IR spectra of the synthesized diol (3) and starting materials. The peaks at 3400 cm-1 (-OH, stretching, hydrogen bond), 1650 cm-1 (-C ) C, stretching, alkene), and 1640 cm-1 (-NH2, amide) disappeared from the starting materials, and a peak at 1740 cm-1 (CdO, stretching, ester) of diol (3) was observed. Figure 2 shows the FT-IR spectra of the synthesized PMMD and PDMA. The peaks at 1720 cm-1 (CdO, stretching, urethane), 1668 cm-1 (CdO, stretching, amide), 1537

Figure 2. FT-IR spectra of (a) PMMD and (b) PDMA.

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Figure 3. Sizes of PMMD/DNA and PDMA/DNA complexes prepared at various mass ratios. Results are presented as the mean ( SD (n ) 3).

Figure 6. Acid-base titration profile of various polymers with 1 N HCl. Ten milligrams of various polymers was dissolved in 10 mL of 150 mM NaCl, the polymeric solution was added to 100 µL of 1 N NaOH, and the pH was recorded.

Figure 4. Zeta-potential of PMMD/DNA and PDMA/DNA complexes prepared at various mass ratios. Results are presented as the mean ( SD (n ) 3).

Figure 7. Cytotoxicity of polymer/DNA complexes in COS-7 cells. Results are presented as the mean ( SD (n ) 3).

Figure 5. DNA gel retardation assay of PMMD and PDMA. Lanes: (1) 400 ng DNA; (2) PMMD/DNA and PDMA/DNA (w/w) 0.5/1; (3) PMMD/DNA and PDMA/DNA (w/w) 1/1; (4) PMMD/DNA and PDMA/DNA (w/w) 2/1; (5) PMMD/DNA and PDMA/DNA (w/w) 3/1; (6) PMMD/DNA and PDMA/DNA (w/w) 4/1; (7) PMMD/DNA and PDMA/DNA (w/w) 5/1; (8) PMMD/DNA and PDMA/DNA (w/w) 6/1; (9) PMMD/DNA and PDMA /DNA (w/w) 20/1; (9) PMMD/DNA and PDMA/DNA (w/w) 50/1.

strates that PMMD (6) bearing urethane groups in the backbone condenses DNA better than PDMA (8) without the urethane group. The zeta-potential of the resulting complex changed from negative charge to positive charge when the amounts of PMMD (6) and PDMA (8) increased. When the mass ratio of PMMD (6)/DNA complexes was higher than 2/1, the surface of pCMVβ-gal was fully occupied with the PMMD (6) molecules, forming positive charge complexes. When the mass ratio of PDMA (8)/DNA complexes was higher than 10/1, positive zetapotentials of the complexes formed. Complexes with extra

Figure 8. Hydrolytic degradation of PDMA (8) and PMMD (6), which were dissolved in a buffer solution (pH 7.4 and pH 5.1) with a concentration of 10 mg/mL and then incubated in a water bath at 37 °C for various durations. Degradation is expressed over time based on GPC data. (Molecular weight ratio: Mw after degradation/Mw before degradation).

positive charges on their surfaces had better interaction with the target cell membrane, resulting in an enhanced uptake. DNA Gel Retardation Assay. The electrophoretic mobility behavior of free DNA, PMMD (6)/DNA, and PDMA (8)/DNA is shown in Figure 5. Increasing amounts of PMMD (6) and PDMA (8) led to the neutralization of DNA negative charges, as shown by gel retardation. The DNA mobility on agarose gel was influenced by the presence of PMMD (6) and PDMA (8). With a low mass ratio of PMMD (6) (2/1), plasmid DNA was totally retained, as indicated in lane 4. Plasmid DNA was partially retained by the presence of PDMA (8) at a mass ratio of 6/1 (lane 8) and totally retained at a mass ratio of 20/1 (lane 9). These results suggest that DNA was fully completed with PMMD (6) and PDMA (8) to form complexes, showing that amine groups of PMMD (6) and PDMA (8) with positive charges could interact with the charge of negative phosphate groups of DNA strands to form complexes.

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Figure 9. AFM (atomic force microscopy) of PMMD/DNA complexes at a mass ratio of 20/1.

Buffering Capacity of Polymers. Titration studies were performed to determine the buffering capacities of the various polymers regarding a proton buffering effect within the endosomal/lysosomal compartments of the cell (Figure 6). All of the polycation solutions had a pH of 11.5 to 11.8 after the addition of 1.0 N NaOH. The nonviral vector PEI showed a buffering capacity over a wide pH range, which is probably due to the high amount of amine functions present in the polymeric chain. The incorporation of tertiary amine content changed the buffering region of the polycation. PMMD (6) has more tertiary amine than PDMA (8) in the backbone and side chain. The data show the pH range of PMMD (6) to be between 11.5 and 5.4 and that of PDMA (8) to be between 11.5 and 7.5. It was found that the polymers with high tertiary amine content have a high buffering capacity. Cytotoxicity of Polymer/DNA. A critical element for the gene delivery system is cytotoxicity. Cell damage resulting from a cytotoxic delivery system is deleterious because cells must be capable of supporting translation and transcription. To determine the cytotoxicity of PMMD (6)/DNA and PDMA (8)/ DNA for comparison with that of PEI/DNA, we performed an XTT assay using the COS-7 cell line. Cells were incubated with increasing amounts of PMMD (6)/DNA and PDMA (8)/DNA (ranging from 5 µg/mL to 800 µg/mL) and PEI/DNA (ranging from 5 µg/mL to 20 µg/mL). The results show that PMMD (6)/ DNA and PDMA (8)/DNA exhibited substantially lower toxicity on COS-7 cells than did PEI/DNA, as shown in Figure 7. IC50 (defined as the concentration resulting in 50% inhibitory activity (cell death)) for PEI/DNA was around 15 µg/mL, whereas PMMD (6)/DNA and PDMA (8)/DNA had more than 75% viable cells even at a higher dose (800 µg/mL) with no significant change in cell morphology and proliferation relative to controls. We infer that poly(urethane-co-ester)/DNA can provide better cytotoxicity profiles than these presently used for PEI/DNA due to the introduction of amino groups and urethane units in the polycation backbone, which reduces the high charge density of the polycation. In Vitro Hydrolysis of Polymers. Figure 8 shows the degradation profiles of polymers, which were monitored at 37 °C at buffer pH values of 5.1 and 7.4 in order to approximate the environments within endosmal vesicles and lysosome, respectively. The polymers generally degraded faster at pH 7.4

than at pH 5.1. PMMD (6) at pH 5.1 had a half-life of 32-33 h. In contrast, PDMA (8) was completely degraded in less than 16 h at pH 5.1. These results show that PDMA (8) exhibited a higher hydrolytic degradation rate than did PMMD (6) at pH 7.4 and 5.1. AFM (Atomic Force Microscopy) Images of PMMD/ DNA Nanoparticles. Figure 9 shows the atomic force microscopy (AFM) images of PMMD/DNA complexes at a mass ratio of 20/1. At a mass ratio of PMMD/DNA (20/1), nearly all complexes condensed to a roughly spherical shape. The discretely spherical images verify the very efficient DNA condensation obtained using PMMD. The AFM images of complexes were captured after the complexes were coated on mica and dried with compressed air. The observed dimensions, therefore, are different from the ones determined in aqueous media using DLS.

CONCLUSIONS Poly(urethane-co-ester) (PMMD) with low cytotoxicity and high biodegradability was synthesized and then characterized using FT-IR, NMR, and GPC in this study. PMMD and DNA had a strong electrostatic interaction to self-assemble nanoparticles. The positive charges in the backbone and side chain of PMMD condense DNA to form DNA-polycation complexes. The complex efficiency was better than that of the polyester of PDMA, which resulted in numerous amine groups of PMMD with two tertiary amines in the backbone and two tertiary amines in the side chain. Biodegradable PMMD could potentially be used in a nonviral gene delivery system.

LITERATURE CITED (1) Shroff, K. E., Smith, L. R., Bain, T., and Higgins, T. T. (1999) Potential for plasmid DNAs as vaccines for the new millennium. Pharm. Sci. Technol. Today 5, 205–212. (2) Friedman, T. (1997) Overcoming obstacles in gene therapy. Sci. Am. 6, 96–101. (3) Ferruti, P., Manzoni, S., Richardson, S. C. W., Duncan, R., Pattrick, N. G., Mendichi, R., and Casolaro, M. (2000) Amphoteric linear poly(amido-amine)s as endosomolytic polymers: Correlation between physicochemical and biological properties. Macromolecules 33, 7793–7800.

Biodegradable Cationic Poly(urethane-co-ester) (4) Shau, M. D., Tseng, S. J., Yang, T. F., Cherng, J. Y., and Chin, W. J. (2006) Effect of molecular weight on the transfection efficiency of novel polyurethane as a biodegradable gene vector. J. Biomed. Mater. Res., Part A 77A, 736–746. (5) Akinc, A., Lynn, D. M., Anderson, D. G., and Langer, R. (2003) Parallel synthesis and biophysical characterization of a degradable polymer library for gene delivery. J. Am. Chem. Soc. 125, 5316– 5323. (6) Buchanan, S. A., Balogh, N. L. P., and Meyerhoff, M. E. (2004) Potentiometric response characteristics of polycation-sensitive membrane electrodes toward poly(amidoamine) and poly(propylenimine) dendrimers. Anal. Chem. 76, 1474–1482. (7) Zugates, G. T., Anderson, G. D., Little, S. R., Lawhorn, I. E. B., and Langer, R. (2006) Synthesis of poly(β-amino ester)s with thiol-reactive side chains for DNA delivery. J. Am. Chem. Soc. 128, 12726–12734. (8) Choi, M. K., Arote, R., Kim, S. Y., Chung, S. J., Shim, C. K., Cho, C. S., and Kim, D. D. (2007) Transfection of primary human nasal epithelial cells using a biodegradable poly (ester amine) based on polycaprolactone and polyethylenimine as a gene carrier. J. Drug Targeting 15, 684–690. (9) Wu, C. B., Hao, J. Y., Deng, X. M., and Li, X. H. (2008) Copolymer of tetraethylenepentamine and ethylene glycol diacrylate: Synthesis and degradation. Polym. Degrad. Stab. 93, 932–940. (10) Boussif, O., Lezoualc’h, F., Zanta, M. A., Mergny, M. D., Scheman, D., Demeneix, B., and Behr, J. P. (1995) A versatile vector for gene and oligouncleotide transfer into cells in culture and in-vivo-polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297–7301. (11) Cherng, J. Y., Wetering, P. V., Talsma, H., Crommelin, D. J. A., and Hennink, W. E. (1996) Effect of size and serum proteins on transfection efficiency of poly((2-dimethylamino)ethyl methacrylate)-plasmid nanoparticles. Pharm. Res. 13, 1038– 1042. (12) Anderson, W. F. (1998) Human gene therapy. Nature (London) 392, 25–30. (13) Friedman, J. M. (2004) Modern science versus the stigma of obesity. Nature Med. 10, 116. (14) Crystal, R. G. (1995) Transfer of genes to humans: early lessons and obstacles to success. Science 270, 404–410. (15) Wang, Y., Ke, C. Y., Beh, C. W., Liu, S. Q., Goh, S. H., and Yang, Y. Y. (2007) The self-assembly of biodegradable cationic

Bioconjugate Chem., Vol. 20, No. 4, 2009 779 polymer micelles as vectors for gene transfection. Biomaterials 28, 5358–5368. (16) Christensen, L. V., Chang, C. W., Kim, W. J., and Kim, S. W. (2006) Reducible poly(amido ethylenimine)s designed for triggered intracellular gene delivery. Bioconjugate Chem. 17, 1233– 1240. (17) Kim, Y., Klutz, A. M., and Jacobson, K. A. (2008) Systematic investigation of polyamidoamine dendrimers surface-modified with poly(ethylene glycol) for drug delivery applications: Synthesis, characterization, and evaluation of cytotoxicity. Bioconjugate Chem. 19, 1660–1672. (18) Braunova, A., Pechar, M., Laga, R., and Ulbrich, K. (2007) Hydrolytically and reductively degradable high-molecular-weight poly (ethylene glycol) s. Macromol. Chem. Phys. 208, 2642– 2653. (19) Deshmukh, H. M., and Huang, L. (1997) Cationic liposomemediated DNA delivery to the lung endothelium. New J. Chem. 21, 113–124. (20) Sanford, J. C. (1988) The biolistic process. Trends Biotechnol. 6, 299–302. (21) Bieber, H., Meissner, W., and Kostin, S. (2002) Non-viral gene therapy: polycation-mediated DNA delivery. J. Controlled Release 82, 441–454. (22) Cherng, J. Y., van de Wetering, P., Talsma, H., Crom-melin, D. J. A., and Hennink, W. E. (1996) Effect of size and serum proteins on transfection efficiency of poly((2-dimethylamino)ethyl methacrylate)-plasmid nanoparticles. Pharm. Res. 13, 1038– 1042. (23) Lim, K., and Chae, C. B. (1989) A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase. Biotechnol. 7, 576–579. (24) Barbucci, R., Ferruti, P., Improta, C., Delfini, M., Segre, A. L., and Conti, F. (1978) Protonation studies of multifunctional polymers with a poly(amido-amine) structure. Polymer 19, 1329– 1334. (25) Ranucci, E., and Ferruti, P. (1991) Block copolymers containing poly(ethylene glycol) and poly(amido- amine) or poly(amido thioether amine) segments. Macromolecules 24, 3747–3752. (26) Kabanov, A. V., and Kabanov, V. A. (1995) DNA complexes with polycations for the delivery of genetic material into cells. Bioconjugate Chem. 6, 7–20. BC800499W