Reduction-Degradable Linear Cationic Polymers as Gene Carriers

Dec 2, 2010 - These polymers were studied with a goal of developing a set of new gene carriers. The buffering capacity and DNA binding ability of thes...
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Biomacromolecules 2011, 12, 66–74

Reduction-Degradable Linear Cationic Polymers as Gene Carriers Prepared by Cu(I)-Catalyzed Azide-Alkyne Cycloaddition Yang Wang,† Rui Zhang,† Ning Xu,† Fu-Sheng Du,*,† Ying-Li Wang,‡ Ying-Xia Tan,‡ Shou-Ping Ji,*,‡ De-Hai Liang,† and Zi-Chen Li*,† Beijing National Laboratory for Molecular Sciences, Key Laboratory of Polymer Chemistry and Physics of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, People’s Republic of China, and Department of Molecular Biology, Beijing Institute of Transfusion Medicine, Beijing 100850, People’s Republic of China Received August 27, 2010; Revised Manuscript Received November 9, 2010

Linear reduction-degradable cationic polymers with different secondary amine densities (S2 and S3) and their nonreducible counterparts (C2 and C3) were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) step-growth polymerization of the dialkyne-oligoamine monomers and the diazide monomers. These polymers were studied with a goal of developing a set of new gene carriers. The buffering capacity and DNA binding ability of these polymers were evaluated by acid-base titration, gel retardation, and ethidium bromide (EB) exclusion assay. The polymers with lower amine density exhibit a weaker DNA-binding ability but a stronger buffering capacity in the range of pH 5.1 and 7.4. Particle size and zeta-potential measurements demonstrate that the polymers with higher amine density condense pDNA to form polyplexes with smaller sizes, while the disulfide bond in the backbone shows a negative effect on the condensing capability of the polymers, resulting in the formation of polyplexes with large size and nearly neutral surface. The reduction-sensitive polyplexes formed by polymer S2 or S3 can be disrupted by dithiothreitol (DTT) to release free DNA, which has been proven by the combination of gel retardation, EB exclusion assay, particles sizing, and zeta potential measurements. Cell viability measurements by MTT assay demonstrate that the reduction-degradable polymers (S2 and S3) have little cytotoxicity while the nonreducible polymers (C2 and C3) show obvious cytotoxicity, in particular, at high N/P ratios. In vitro transfection efficiencies of these polymers were evaluated using EGFP and luciferase plasmids as the reporter genes. Polymers S3 and S2 show much higher efficiencies than the nonreducible polymers C3 and C2 in the absence of 10% serum; unexpectedly, the lowest transfection efficiency has been observed for polymer S3 in the presence of serum.

Introduction Gene therapy has attracted a great deal of attention in the past two decades. Although viruses are capable of delivering nucleic acids into cells effectively and most of the present clinical trials of gene therapy are based on viral vectors, the safety issues do limit their applications, thereby spurring the development of nonviral methods for gene delivery.1,2 Among various nonviral carriers, cationic polymers, exemplified by poly(ethylenimine) (PEI), dendritic poly(amido amine) (PAMAM), poly-L-lysine (PLL), chitosan, and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), have been thoroughly studied and widely used for gene delivery because of their advantages such as non- or lower immunogenicity, tunable structures, ease of large scale production, and so on.3-6 Cytotoxicity is an important issue that may hamper the application of cationic polymers. Although the mechanism is not completely clarified, high molecular weight cationic polymers showing high or moderate transfection efficiency are often accompanied by significant cytotoxicity.7,8 To reduce the cytotoxicity while retaining transfection efficiency, various * To whom correspondence should be addressed. Tel.: 86-10-62755543. Fax: 86-10-62751708. E-mail: [email protected] (Z.-C.L.); [email protected] (F.-S.D.); [email protected] (S.-P.J.). † Peking University. ‡ Beijing Institute of Transfusion Medicine.

degradable cationic polymers have been studied.9-12 In addition, because there is a significant redox potential gradient between the intracellular components and the extracellular environments, in the past decade, large pools of reduction-degradable cationic polymers13-18 or reduction-sensitive gene delivery systems19-24 have been developed and summarized in the recent review papers.25-28 The unique point of the reduction-sensitive gene delivery systems is that the active nucleic acids can be selectively released within cells. Recently, Oupicky and colleagues reported that the performance of the reducible cationic polymers was highly dependent on the type of nucleic acids, the innate glutathione level of cells, and the density of disulfide bonds in the polymers.29,30 Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), discovered in 2002,31,32 is one of the most popularly used “click” reactions.33 CuAAC can be performed at ambient conditions in a highly specific manner and is compatible with different functional groups such as amino group, carboxyl group, and hydroxyl group, etc. Therefore, in the past several years, CuAAC has been widely applied in many fields, including the synthesis of polymers with various architectures and functionalities, the conjugation of biomacromolecules, the surface modification of nano/microparticles, and the preparation of cross-linked networks.34-38 However, only minimal work was reported by using CuAAC to construct cationic polymers or molecular clusters as nonviral gene carriers.39-41 Reineke et al. prepared

10.1021/bm101005j  2011 American Chemical Society Published on Web 12/02/2010

Cationic Polymers as Gene Carriers Scheme 1. Synthesis of the Cationic Polymersa

a Reagents and conditions: (a) acryloyl chloride, TEA, r.t., 8 h; (b) ethylenediamine or diethylenetriamine, CHCl3, 25 °C, 72 h; (c) tosyl chloride (TsCl), TEA, trimethylamine, r.t., 2 h; (d) NaN3, DMF, 80 °C, 24 h; (e) CuSO4, L-ascorbic acid sodium salt, t-BuOH/H2O (1:1), 60 °C, 72 h.

a family of cationic glycopolymers containing oligoamine units and trehalose or β-cyclodextrin units by CuAAC polymerization. They systematically studied the effects of chemical structures and molecular weights of these glycopolymers on their biological properties as plasmid DNA carriers, and their results demonstrated that CuAAC polymerization is a useful approach to prepare cationic polymers in which the combination of triazole ring and the adjacent amide group may help to bind and compact DNA.42-44 In this work, we report a new type of reduction-degradable (reducible) linear cationic polymers which were synthesized by CuAAC step-growth polymerization of the disulfide-containing diazides and dialkyne-oligoamines (Scheme 1). The nonreducible counterparts were also prepared to demonstrate the function of disulfide bond in the polymer backbone. As aforementioned, the reduction-degradable cationic polymers are generally lower toxic and may be more efficient compared to their nonreducible counterparts. Positive charge density of the polymers, an important factor for tuning DNA binding/condensing capability as well as the transfection efficiency,45,46 can be simply adjusted by changing the numbers of the amino groups of the dialkyneoligoamines. In addition, the adjacent triazole ring and amide group might be helpful for the polymers to bind and compact DNA.42 Finally, the secondary amino groups in the backbone provide us the opportunity to easily modify these polymers.

Experimental Section Materials. Propargyl amine (99%, Acros), 1,6-hexanediol (99%, Sinopharm Chemical Reagent Co.), diethylenetriamine (98.5%, Acros), 2-mercaptoethanol (99%, Fluka), sodium azide (NaN3, 98%, Zhejiang Dongyang Kaiming Chemicals Co.), dithiothreitol (DTT, 99%, Acros), copper sulfate (CuSO4, 99%, Shanghai Jinshan Tingxin Chemical Reagent), acetic anhydride (97%, Acros), L-ascorbic acid sodium salt (99%, Acros), and linear polyethylenimine (Mw: 25000, Polysciences, Inc.) were used as received. Chloroform, ethylenediamine, and triethylamine (TEA; Beijing Chemical Reagents Co.) were refluxed with CaH2 or sodium and distilled prior to use. Tosyl chloride was purchased from Beijing Chemical Reagents Co. and recrystallized in petroleum ether prior to use. Acryloyl chloride was synthesized by the reaction of acrylic acid and benzoyl chloride.47 2-Hydroxyethyl disulfide was synthesized by the oxidation of 2-mercaptoethanol using DMSO as an oxidant. Agarose, calf thymus DNA (ctDNA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) were purchased from Beijing Huamei Scientific Co. SYBR Green I (10000× in DMSO) was purchased from Beijing Chuanggen Shengtai Co. Other chemical reagents were obtained from Beijing Chemical Reagents Co. and used as received.

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Plasmid DNA. pEGFP-N1 plasmid encoding enhanced green fluorescent protein (EGFP) (BD Biosciences) was amplified in E. coli and harvested according to the protocol (Vigorous Biotechnology Beijing Co., Ltd.). pShuttle-Luc DNA was a gift from Prof. Wenlin Huang (Institute of Microbiology, CAS). pcDNA 3.1 was kindly provided by Nitto Denko Technical Corp. Characterization. Gel permeation chromatography (GPC) was carried out on equipment with a Waters 515 HPLC pump, a Waters 2470 refractive index detector, and two Styragel HR columns (HT4 and HT3). N,N-Dimethylformamide (DMF) containing 20 mM LiBr was used as the eluent at a flow rate of 1.0 mL/min (35 °C). Monodisperse polystyrene standards were used for calibration. NMR spectra were recorded in CDCl3 or D2O on a Bruker ARX-400 spectrometer or a Varian Gemini 300 spectrometer. Elemental analysis was conducted on an Elementar Vario Micro Cube equipment (Germany). Fluorescence measurements were carried using a Hitachi F-4500 fluorescence spectrometer. Synthesis of Dialkyne Monomers (2 and 3 in Scheme 1). Take dialkyne-oligoamine monomer 3 as an example. N-(Prop-2-ynyl)acrylamide (50.7 g, 465 mmol) dissolved in 40 mL of dry chloroform was added dropwise into a solution of diethylenetriamine (8.0 g, 77.5 mmol) in 120 mL of dry chloroform at ambient temperature. The solution was bubbled with nitrogen for ∼20 min and stirred at 25 °C for 72 h. After concentration of the reaction mixture on a rotary evaporator, ethanol was added to dissolve the residue. The insoluble precipitates were removed by filtration, and the filtrate was acidified by adding hydrochloric acid (35%), which afforded a white precipitate. The crude product was purified by dissolving in 35% hydrochloric acid and precipitation in ethanol again, affording the final product (in the form of hydrochloride salt) with a yield of 40%. 1H NMR (400 MHz, D2O, ppm, Figure S7A) δ 2.65 (t, 2H, J ) 2.54 Hz), 2.79 (t, 4H, J ) 6.58 Hz), 3.44 (t, 4H, J ) 6.60 Hz), 3.56-3.61 (m, 8H), 3.99 (d, 4H, J ) 2.52 Hz). 13C NMR (100 MHz, D2O, ppm, Figure S7B) δ 28.9, 30.9, 43.1, 43.5, 44.2, 72.1, 79.6, 171.3. Elem. anal. (C/N, w/w): Calcd, 2.744; found, 2.717. Dialkyne-oligoamine monomer 2 was synthesized following a similar procedure as for monomer 3, yield 33% (HCl salt). 1H NMR (400 MHz, D2O, ppm, Figure S8A) δ 2.70 (t, 2H, J ) 2.50 Hz), 2.82 (t, 4H, J ) 6.62 Hz), 3.47 (t, 4H, J ) 6.62 Hz), 3.58 (s, 4H), 4.02 (d, 4H, J ) 2.52 Hz). 13C NMR (100 MHz, D2O, ppm, Figure S8B) δ 29.0, 31.1, 43.3, 44.2, 72.2, 79.8, 171.4. Elem. anal. (C/N, w/w): Calcd, 3.001; found, 3.018. Synthesis of Diazide Monomers (4 and 5 in Scheme 1). Details of synthesis of these two compounds are provided in the Supporting Information. CuAAC (Click) Step-Growth Polymerization. Take polymer C2 as an example. Diazide monomer 4 (166.2 mg, 0.989 mmol), dialkyneoligoamine monomer 2 (351.2 mg, 0.988 mmol), and L-ascorbic acid sodium salt (158 mg, 0.798 mmol) were charged into a Schlenk tube, to which 7 mL of mixed solvents tert-butanol (t-BuOH)/H2O (1:1, v/v) was added. The reaction mixture was degassed by three freeze-pumpthaw cycles, and the tube was purged with nitrogen gas. After addition of CuSO4 (64 mg, 0.4 mmol), the tube was further treated by three freeze-pump-thaw cycles and sealed under a nitrogen atmosphere. The polymerization was carried out at 60 °C for 72 h. After cooling to room temperature, the reaction mixture was diluted with water, dialyzed against acidic water (pH ) 1) for three days, and then against pure water for an additional 12 h using Spectra/Por Regenerated Cellulose (RC) membrane (MWCO 3500) at room temperature (r.t.). Water was nitrogen-purged prior to the dialysis. After freeze-drying, a yellow powder was obtained in a yield of 60%. The other three polymers (C3, S2, and S3) were prepared by a similar procedure. All the four polymers were characterized by 1H NMR spectra and GPC (in the form of acetylated derivatives). 1H NMR (400 MHz, D2O, ppm, δ, S2, Figure 1A): 2.74 (t, 4H), 3.17 (t, 4H), 3.34 (t, 4H), 3.38 (s, 4H), 4.48 (s, 4H), 4.70 (t, 4H), 7.95 (s, 2H); (S3, Figure 1B): 2.77 (t, 4H), 2.97 (t, 4H), 3.18 (m, 8H), 3.36 (t, 4H), 4.45 (s, 4H), 4.68 (t, 4H), 7.94 (s, 2H);

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Figure 1. 1H NMR (400 MHz) spectra of polymers S2 (A) and S3 (B) in D2O. Table 1. Characterization of the Linear Cationic Polymers polymer

yield (%)

Mna (×103)

Mwa (×103)

PDIa

Nwtb (%)

Neffc (%)

BCd (%)

S2 S3 C2 C3

28 46 60 72

34 38 46 80

46 44 57 180

1.3 1.2 1.2 2.3

22.9 23.8 25.2 25.0

4.6 6.5 5.0 6.8

38.3 22.8 39.9 15.5

a Molecular weight determined by GPC using DMF with 20 mM LiBr as an eluent, 1.0 mL/min, 35 °C, RI detector, narrowly distributed polystyrenes as standards. The value of Mn and Mw was estimated by peakfit software. b Weight percent nitrogen content in the polymer as measured by elemental analysis. c Weight percent content of the secondary amino (protonatable) nitrogen in the polymer as calculated by multiplying Nwt% with a factor of 3/11 for C3 and S3 or 2/10 for C2 and S2. d Buffering capacity calculated from the results of acid-base titration.

(C2, Figure S10): 1.23 (m, 4H), 1.83 (m, 4H), 2.77 (t, 4H), 3.38 (t, 4H), 3.47 (s, 4H), 4.36 (t, 4H), 4.47 (s, 4H), 7.89 (s, 2H); (C3, Figure S11): 1.21 (m, 4H), 1.81 (m, 4H), 2.77 (t, 4H), 2.97 (t, 4H), 3.20 (t, 4H), 3.36 (t, 4H), 4.34 (t, 4H), 4.44 (s, 4H), 7.88 (s, 2H). Acid-Base Titration. Weight percent nitrogen content (Nwt%) in the polymer was determined by elemental analysis. As shown in Scheme 1, for polymers C2 and S2 there are 10 nitrogen atoms in 1 repeating unit, but only 2 of them are protonatable under physiological condition. Similarly, 3 of the 11 nitrogen atoms in 1 repeating unit are protonatable for polymers C3 and S3. Thus, the weight percent content of the secondary (protonatable) amino nitrogen (Neff%) in the polymer was calculated by multiplying Nwt% with a factor of 3/11 for C3 and S3 or 2/10 for C2 and S2, respectively (Table 1). According to the results of elemental analysis, a known amount of polymers was dissolved in 0.1 M NaCl to obtain the sample solution containing 1.0 mM protonatable amino groups. The pH of the polymer solution was adjusted to 2.0 by addition of concentrated hydrochloric acid, and the solution was titrated by 0.1 M NaOH on a thermostat at 25 °C. The solution pH was

measured by a pH meter. A 0.1 M NaCl solution with the same volume was also titrated as the blank. Buffering capacity, defined as the percentage neutralized in the pH range of 5.1-7.4 of the secondary amino groups in the polymers, was calculated by the formula: (NNaOH-polym - NNaOH-blank)/Namine × 100%, where NNaOH-polym and NNaOH-blank represent the molar amount of NaOH consumed by polymer or blank in the pH range of 5.1 to 7.4, respectively. Namine denotes the molar amount of all protonatable amino groups, that is, the secondary amino groups. Preparation of Polyplex. Plasmid DNA (pDNA) was dissolved in 20 mM HEPES (pH 7.4). The solution was centrifuged at 10000 rpm for 2 min and the supernatant was collected as the pDNA stock solution. Calf thymus DNA (ctDNA) was dissolved in 20 mM HEPES and filtered through a 0.22 µm Millipore PVDF (polyvinylidene difluoride) membrane, affording the ctDNA stock solution. DNA concentrations of the stock solutions were determined by measuring the OD at 260 nm assuming one OD260 ) 50 µg/mL of DNA. Polymers were dissolved in 20 mM HEPES and filtered through a 0.22 µm Millipore PVDF membrane, affording the polymer stock solutions. Prior to the polyplex formation, DNA solution was diluted to a predetermined concentration according to different experimental protocols. Then, polymer solution with a calculated concentration was mixed with the DNA solution, followed by pipetting several times or vortexing 30 s at 1200 rpm and incubation for 30 min at r.t. Agarose Gel Electrophoresis. A total of 6 µL of freshly prepared polyplexes solution (0.3 µg pEGFP-N1 DNA) was mixed with 1.2 µL of loading buffer (0.15% bromophenol blue (w/v) dissolved in 40% glycerol (w/v)) and loaded onto agarose gel (0.7% w/v), which was stained by SYBR Green I. The electrophoresis was run in 1× TBE (Tris/Borate/EDTA) buffer at 90 V for 50 min. The UV image was taken by a VDS thermal imaging system (Pharmacia Biotech). Ethidium Bromide (EB) Exclusion Assay. All of the following procedures were carried out in dark. To a ctDNA solution (5 µg/mL), EB was added at a molar ratio of 1:10 (EB:DNA). The mixture was thoroughly mixed and incubated at r.t. for 1 h. Then the required amount of polymers was added into the above-mentioned DNA/EB solution at different N/P ratios and the mixed solution was incubated at r.t. for 30 min. A pure EB solution and the DNA/EB solution without any cationic polymer were used as negative and positive controls, respectively. Fluorescence intensity was measured (λex ) 510 nm, λem ) 590 nm, slitex ) 5 nm, slitem ) 10 nm). EB exclusion efficiency was defined as (F - FEB)/(F0 - FEB), whereas FEB and F0 denote the fluorescence intensity of pure EB solution and the DNA/EB solution without any polymer, respectively. For reduction-degradable properties of the cationic polymers or their polyplexes, two sets of experiments were designed. To explore whether the degraded polymer fragments are capable of binding and condensing ctDNA, the reduction-degradable polymers (S2 and S3) were incubated in the presence of DTT (50 mM) at r.t. for 30 min prior to the polyplex formation. For the second group of experiments, DTT was added into the preformed polyplex solution and the mixture was incubated at r.t. for 30 min prior to the fluorescence measurement. The final DTT concentration was 5.0 mM. Particle Size and Zeta Potential Analysis. Polyplexes were formed in HEPES at a plasmid DNA concentration of 10 µg/mL. Particle size and zeta potential were analyzed on Zeta PALS analyzer (Brookhaven Instruments Corporation, BIC). Plasmid pcDNA 3.1 was used for the particle size and zeta potential measurements unless otherwise mentioned. For the degradation kinetics measurements, known amount of 300 mM DTT was added into the formed polyplexes solution to bring the final DTT concentration to 20 mM, and then the change of particle size and zeta potential versus time was monitored. All the data were measured in triplicate. In Vitro Transfection and Cell Viability. B16F10 and COS 7 cells were obtained from ATCC (American Type Culture Collection, Manassas, VA) and cultured in DMEM (Dulbecco’s Modified Eagle’s Medium, Invitrogen, Carlsbad, CA) growth medium plus 10% FBS

Cationic Polymers as Gene Carriers (fetal bovine serum), penicillin (100 units/mL), and streptomycin (100 mg/mL) at 37 °C with 5% CO2 in a humidified chamber. A total fo 5 × 103 cells were seeded and cultured in each well of a 96-well plate 1 day prior to transfection. To each well, 15 µL polyplexes formed in OPTI-MEM containing 0.3 µg pEGFP-N1 DNA were added. After 4 h incubation, the medium was replaced with 100 µL of supplemented DMEM, and cells were left to grow for another 48 h. The fluorescence image was recorded with a Leica DMI 4000B microscope. These transfected cells were further used for the evaluation of cell viability. To each well, 20 µL of PBS solution with 5 mg/mL MTT was added, and the cells were incubated at 37 °C with 5% CO2 for 4 h. Then the medium was pipetted out carefully and 150 µL of DMSO was added to each well to dissolve the formazan crystals. After incubation for 15 min at r.t., absorbance at 570 nm of the solution was measured on a Molecular Devices Spectra Max M5 Pro spectrophotometer. pShuttleLuc DNA transfection experiments in B16F10 cells were performed following a similar procedure as for pEGFP-N1 DNA. To explore the influence of serum on transfection, the matured polyplexes (15 µL, 0.3 µg pShuttle-Luc DNA) were introduced into the wells containing B16F16 cells cultured in serum-free and 10% FBS supplemented DMEM media, respectively, and incubated for 4 h before changing to the normal supplemented DMEM. For luciferase assay, after 36 h transfection, the cells were lysed with 1× cell culture lysing buffer, and the level of luciferase expression was measured on lumimometer (TD-20120, Turner Biosystems, CA) using a luciferase assay kit (Promega, Madison, WI) according to the manufacturer’s instructions. The amount of protein per well was determined using the Coomassie Bradford Assays kit (MBchem Co., Ltd., Shanghai). Luciferase activity was expressed as relative light units (RLU)/mg protein.

Result and Discussion Syntheses of Dialkyne Monomers and Polymers. The linear reduction-degradable cationic polymers were synthesized by CuAAC step-growth polymerization of diazide monomer 5 with dialkyne monomers 2 and 3, respectively. The nonreducible counterparts were prepared by the same reaction using diazide monomer 4 instead of monomer 5 (Scheme 1). A key step for the preparation of the cationic polymers with a linear structure is to synthesize the expected linear dialkyne monomers through Michael addition reaction of N-propargyl acrylamide with the oligoamines. In recent years, Michael addition reaction between amine and acrylate/acrylamide has been widely used to prepare versatile materials including the well-developed cationic polymers as nonviral gene carriers.12,28 Both primary and secondary amines can react with acrylate/acrylamide, and the reactivity highly depends on the structure of the amines, the temperature, as well as the solvents used.48,49 Kim et al. prepared a family of reducible poly(amido amine)s through Michael addition reactions between cystamine bisacrylamide and different multiamines with both primary and secondary amino groups. Their results demonstrate that a branching structure of the polymers, caused by addition of the secondary amino group to the acrylamide group, is inevitable at 50 °C when using 10% aqueous methanol as the solvent.50 In the case of Michael addition reaction between the aliphatic multiamines (RNH(CH2)2NH2) and a diacrylate, Liu et al. have also elucidated when the R group is larger than methyl group, the reaction rate of the primary and secondary amines can not be distinguished from each other, and the polymers with a hyperbranched structure have been obtained.48 In the present work, Michael addition reactions were carried out in chloroform at ambient temperature to minimize the possible side products with branching structures (3a and 3b in Scheme 2). Even under such a mild condition, an insufficient amount of N-propargyl

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Scheme 2

acrylamide resulted in the formation of byproduct 3b, which was hard to be separated from the dialkyne monomer 3. Increasing feed ratio of the acrylamide to oligoamine could eliminate the byproduct 3b in the reaction mixture (Figure S9). Another byproduct 3a could be removed from monomer 3 by using an acidification-precipitation method because of their different solubilities in organic solvent after protonization. For dialkyne monomer 2, a similar strategy was applied to acquire the pure product. “Click” chemistry has been successfully applied in the stepwise polymerization.42 Reineke et al. synthesized the trehalose-containing cationic polymers through the CuAAC stepgrowth polymerization between a trehalose-derived diazide monomer and a series of dialkyne monomers that contain tertbutoxycarbonyl (Boc)-protected secondary amines, followed by removal of the protective Boc group.42 We used the unprotected dialkyne monomers (2 and 3) to carry out the “click” stepgrowth polymerization, employing the similar condition as in Reineke’s work. The polymers were purified by a sequential dialysis in acidic and neutral water, respectively. Copper ions are reported to cause nucleic acid damage due to the cleavage of the phosphoester bond.51 Residual copper catalyst may cause significant cell cytotoxicity and hamper the biological applications of the polymers. So, the reaction mixtures were dialyzed against acid medium (pH ∼ 1) to reduce the coordination between the amino group and copper ions, as such help to remove copper ions from the polymer. ICP results showed that the residual content of copper in the polymer was lower than 0.003 wt %, ensuring that the downstream measurements of biological properties would not be affected by the trace amount of residual copper in the polymers. Figure 1 shows the 1H NMR spectra of the reducible polymers, S2 and S3. The proton signal (∼7.8 ppm) of the triazole ring was clearly observed. In addition, the chemical shifts of methylene protons adjacent to the secondary amino groups are above 3.0 ppm, indicating that the amino groups were partially protonated. Unlike typical acrylic polymers which have broad and unsplitted proton signals of the backbone, well splitted 1H NMR spectra were obtained for polymers S2 and S3, as well as for their nonreducible counterparts, C2 and C3 (Figures S10 and S11). This is probably due to, the more backbone atoms in each repeating unit of these polymers, there would be fewer coupling between the neighboring repeated units, consistent with that observed for poly(εcaprolactone).52 All four polymers are soluble in water but insoluble in organic solvents such as THF, DMF, and so on. After modification with acetic anhydride, the acetylated polymers became soluble in DMF. The apparent molecular weights of the acetylated polymers were characterized by GPC using DMF as an eluent and the narrowly distributed polystyrenes as the molecular weight standards (Table 1). Because the nitrogen atoms of the amido group and triazole ring are unable to be protonated under physiological condition,42

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Figure 2. Agarose gel electrophoresis of pDNA complexed with various cationic polymers C2 (A), C3 (B), S2 (C), S3 (D). The numbers denote N/P ratios.

Figure 4. Size (A) and zeta potential (B) of the polyplexes at various N/P ratios. Results are presented as the mean ( SD in triplicate.

Figure 3. Relative fluorescence intensity of EB intercalated with ctDNA in the presence of polymers at different N/P ratios.

Neff% is introduced to stand for the percent content of the secondary amino nitrogen atoms in the polymers. Neff% was calculated by multiplying Nwt% with a factor of 3/11 for polymers S3 and C3 or 2/10 for polymers S2 and C2 according to the polymer structures (Scheme 1), where Nwt% (weight percent content of nitrogen in the polymer) was measured by elemental analysis. In this work, Neff% is used to calculate the N/P ratios. DNA Binding. DNA binding capabilities of these cationic polymers were explored by combining both gel retardation and EB exclusion assay. As shown in Figure 2, the nonreducible cationic polymers (C2 and C3) can completely retard the migration of free DNA at and above N/P ratio of 3, while the threshold N/P ratio increased to ∼6 for polymer S2 and ∼4 for polymer S3, respectively. The relatively lower DNA binding capability of the reducible polymers is probably attributed to the more hydrophobic feature of the disulfide bond which might hinder the electrostatic interaction between DNA and the cationic polymers. The results of EB exclusion assay are shown in Figure 3. The relative fluorescence intensities of DNA/polymer solutions greatly decreased with increasing N/P ratio and leveled off at higher polymer concentrations, where only very weak fluorescence was detected. For the polymers with more protonatable nitrogen atoms, that is, C3 and S3, the intensity leveled off at the N/P ratio ∼3, which was lower than that for C2 and S2. These results demonstrate that the polymers with higher secondary amine density have a stronger DNA binding capabil-

ity, which is in accordance with the results in literature.45,53 Above N/P ratio of 4, there was no significant difference among the four polymers. Buffering Capacity. Although the limiting steps in cationic polymer-based gene delivery are not completely clarified, the endosomal escape of polyplexes is widely considered to be a key barrier.25 Branched PEI 25000 has a good reputation in nonviral gene delivery named as “golden standard” partially because of its inherent buffering capacity in the pH range of ∼5.1-7.4, which promotes the endosomal escape process through the well-known “proton sponge” effect.54-56 Furthermore, an appropriate buffering capacity is thought to be essential for a cationic polymer to have high transfection efficiency30,57 even though there are some discrepant opinions.58,59 We estimated the buffering capacity of polymers S2, S3, C2, and C3 by acid-base titration (Figure S12). It is seen that all of these polymers exhibited moderate buffering capacities in the range of pH 5.1-7.4. The polymers with less protonatable nitrogen atoms, that is, C2 and S2, had a larger buffering capacity than C3 and S3 (Table 1). These results are probably explained by the different pKa values of the two dialkyne monomers. The pKa values of ethylenediamine are 9.92 and 6.85, respectively, while for diethylenetriamine they are 10.02, 9.21, and 4.42.60 It is obvious that the ethylenediamine dialkyne monomer would have a pKa close to the pH range of 5.1 to 7.4, enabling the polymers S2 and C2 a higher buffering capacity. Kim et al. reported a similar trend for the reducible poly(amido ethylenimine)s containing different nitrogen atoms in the repeating units.50 Physicochemical Property of the Polyplexes. For efficient transfection, it is critical for the DNA-containing polyplex particles to be internalized efficiently by cells. Although the mechanism is not yet completely understood, it is generally accepted that size and zeta potential of the polyplex particles are key factors that influence internalization and the subsequent intracellular fate of the polyplexes.61 Size and surface charge of the polyplexes may exert significant influence on their transfection efficiency and cytotoxicity.62-64 Figure 4 shows

Cationic Polymers as Gene Carriers

Figure 5. Agarose gel electrophoresis of pDNA complexed with polymers S2 (A) and S3 (B) in the presence of different concentrations of DTT. Left lane on each of the gels denotes intact plasmid, and the other lanes denote the polyplexes with N/P ratio of 16. The numbers denote the concentration (mM) of DTT.

the size and zeta potential of the polyplexes formed by pDNA and the polymers (S2, S3, C2, and C3) at various N/P ratios. It is seen that large particles with sizes more than 800 nm were formed at an N/P ratio of 20 for all the polymers. With the increase of N/P ratio, particle size of the polyplexes decreased gradually, except for polymer S2, which formed polyplexes of ∼1.1 µm at different N/P ratios. In addition, zeta potential of the polyplexes gradually increased with the increase of N/P ratio. However, for the reducible polymers, that is, S2 and S3, the polyplexes formed had almost neutral surface charge with the zeta potential between 0-5 mV even at the highest N/P ratio of 60. Regarding the effect of secondary amine density, it can be seen that the polymers with higher amine density (S3 and C3) formed polyplexes with a smaller size than S2 and C2. This trend is consistent with the results of gel retardation and EB exclusion assay. More protonatable nitrogen atoms in cationic polymers generally lead to a stronger DNA binding capacity and a smaller particle size.42,50 On the other hand, polymers S2 and S3 formed much larger polyplexes with lower surface charges as compared to their counterparts C2 and C3, demonstrating again that the disulfide bond had a negative effect on the condensing capability of the polymers. This result is inconsistent with that reported by Zhong and colleagues. They found that the reducible linear poly(amido amine)s formed a little smaller and more positive polyplexes than the nonreducible counterparts.65 The size of the polyplexes was further revealed by TEM (Figure S13). For the reducible polymers S2 and S3, the larger polyplex particles as overestimated by DLS were aggregated clusters composed of smaller particles less than 300 nm. However, the polyplexes of C3 were mostly observed as individual particles. This difference is probably attributed to the different surface charge of the polyplexes. Polyplexes of S2 and S3 with a nearly neutral surface are expected to have a stronger tendency to aggregate while the polyplex particles of C3 can be stabilized partly by the positive surface charge. Reductive Response of the Polyplexes. It is generally accepted that there is a significant gradient in reduction potential between the extracellular environments and various intracellular organelles. The reduction potential in the cytoplasm or nucleus is much higher than that in blood.26,30,66 In order to study the reductive response of the polyplexes formed by polymers S2 and S3, DTT was applied as a model reducing agent. Figure 5 shows gel retardation results of the reducible polyplexes in the presence of DTT at different concentrations. It can be seen that 0.5 mM of DTT resulted in a significant release of DNA for both S2 and S3 polyplexes, while sufficient dissociation of the

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Figure 6. Relative fluorescence intensity of EB intercalated with ctDNA in the presence of polymers S2 and S3 at different N/P ratios. DTT was added into the solutions before (50 mM) or post (5 mM) the formation of polyplexes.

Figure 7. Change in size (A) and zeta potential (B) of the reductionsensitive polyplexes with incubation time in the presence or absence of 20 mM DTT. Polyplexes were formed at N/P ratio of 60 with pEGFP-N1 DNA. Statistical significance: **p < 0.01.

polyplexes and nearly complete DNA release was observed above 5 mM of DTT. EB exclusion assays were also carried out to confirm the effect of DTT on DNA condensing capability of polymers S2 and S3 (Figure 6). The intensity of EB fluorescence intercalated with DNA did not changed with N/P ratio in the range of 0-6, regardless adding DTT before or after the formation of the polyplexes. This demonstrates that the reducible cationic polymers were degraded by DTT into cationic fragments that were unable to condense DNA and exclude EB. These results of gel retardation and EB exclusion experiments are consistent with those of other reducible cationic polymers.18,50 The effect of DTT on the reduction-sensitive polyplexes was further studied by zeta potential and size measurements (Figure 7). It can be seen that zeta potential of the reduction-sensitive polyplexes remained almost unchanged in the absence of DTT, but dramatically decreased from ∼5 to ∼-20 mV for polymer S2 and ∼-30 mV for polymer S3 in the presence of 20 mM DTT (Figure 7B), demonstrating that the polyplexes were disrupted due to the reduction-cleavage of the polymer backbone. As shown in Figure 7A, in the absence of DTT, size of

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Figure 8. Cell viability after transfection of pEGFP-N1 polyplexes by MTT assay in B16F10 (A) and COS-7 (B) cell lines. Results are presented as the mean ( SD in triplicate. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

the reduction-sensitive polyplexes formed by both polymer S2 and polymer S3 gradually increased with incubation time. It indicates that the freshly formed reduction-sensitive polyplexes were not stable, showing a significant trend of aggregation. By contrast, in the presence of 20 mM of DTT, the size of the reduction-sensitive polyplexes decreased from ∼1000 to ∼600 nm, probably due to the partial dissociation of the polyplexes

Wang et al.

or the presence of loose associates formed by the degraded polymer fragments attached to the DNA. The physicochemical properties of the reduction-sensitive polyplexes were also measured at different DTT concentrations. In the solution with 100 mM DTT, zeta potential and size of the reduction-sensitive polyplexes changed similarly as in 20 mM DTT solution. For the nonreducible C3-formed polyplexes, zeta potential gradually decreased, while the particle size showed a gradual increase with incubation time. However, addition of DTT (100 mM) did not exert significant influence on the C3-formed polyplexes (Figure S14). In Vitro Cell Viability and Transfection. In vitro biological properties of these cationic polymers were evaluated using two cell lines, B16F10 and COS-7 cells. Linear PEI 25000 (lPEI) was used as a control. Figure 8 shows cell viability of the pEGFP-N1/polymer polyplexes evaluated by MTT assay. For B16F10 cells, cytotoxicity of the C3 and C2 polyplexes significantly increased with N/P ratio, while the reductionsensitive polyplexes showed little cytotoxicity only at the highest N/P ratio of 100. In COS-7 cells, cytotoxicity of the polyplexes decreased in the order of C3 > C2, lPEI > S3, S2. Actually, no obvious cytotoxicity was observed for the S2 and S3 polyplexes in the tested range. These results can be attributed to the intracellular reduction-degradability of polymers S2 and S3, consistent with other cationic polymers.30,63 In vitro transfection efficiency of these cationic polymers was first estimated using pEGFP-N1 (Figure 9 and Figure S15). It is seen that polymer S2 exhibited much higher efficiency than the other cationic polymers, even better than lPEI in B16F10 cells. Polymers C3 and C2 failed to transfect B16F10 cells at the higher N/P ratios: 30 or above for C3 and 60 or above for C2, which is probably due to their high cytotoxicity (Figure 8A). In COS-7 cells, the highest efficiency was also observed for polymer S2 (Figure S15). The transfection efficiency of the polymers was further evaluated quantitatively in B16F10 cells using pShuttle-Luc plasmid and the effect of serum (FBS) was

Figure 9. In vitro pEGFP-N1 transfection of various polyplexes in B16F10 cells at different N/P ratios. Fluorescence images were taken after 48 h of transfection. Numbers on the top denote N/P ratio.

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application. Multifunctional modifications of polymers S2 and S3 are in progress in our lab.

Conclusion

Figure 10. In vitro luciferase gene transfection of various polyplexes in B16F10 cells at different N/P ratios in the presence (A) and absence (B) of 10% FBS. Results are presented as the mean ( SD in triplicate. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

also demonstrated (Figure 10). Similar with the EGFP results, in the presence of 10% FBS polymer S2 showed the highest transfection efficiency among the tested cationic polymers, surpassing lPEI (Figure 10A). The optimized efficiency for polymer S2 was observed at N/P ratio of 60. At relatively high N/P ratios, the transfection efficiency of polymer S2 was 30-300 times higher than that of polymers C3 or C2. Unexpectedly, polymer S3 showed the lowest transfection efficiency at each of the tested N/P ratios. As shown in Figure 10B, in the absence of FBS, the transfection efficiency of polymer S3 significantly increased, reaching a similar level of S2, in particular, at the higher N/P ratios. For other cationic polymers, serum was also reported to decrease the in vitro transfection efficiency.67 Both S2 and S3 showed a much greater efficiency than lPEI. By contrast, the transfection efficiency of polymer C2 or C3 decreased with increasing the N/P ratio, being even lower than that in the presence of FBS at the higher N/P ratios, which was probably due to the high cytotoxicity of polymers C2 and C3. For in vitro cell transfection of polyplexes there are generally several key barriers including cellular uptake, endosomal escape, unpacking of the polyplexes, and release of the active pDNA in cytoplasm or in nucleus.4,25 In the present work, the smaller size and larger zeta potential were obtained for C3 or C2 polyplexes compared to those of the S2 polyplexes whose size and zeta potential were similar to S3 polyplexes. Because positively charged polyplexes are generally helpful for enhancing cellular association and uptake,68 we may speculate that cellular uptake is not the rate-limiting step for cell transfection of S2 polyplexes. Furthermore, it was reported that the polyplexes with sizes more than 700 nm63,64 or nearly neutral surface charge69 can efficiently deliver pDNA into cells. Although polymers S2 and S3 (in the absence of serum) show good in vitro transfection efficiency and low cytotoxicity, the instability of their polyplexes against incubation does limit its in vivo

We demonstrated the synthesis of linear reduction-degradable cationic polymers with different secondary amine densities by CuAAC step-growth polymerization of the dialkyne-oligoamine and diazide monomers. These polymers can bind and condense pDNA to form polyplexes. DNA-binding capability of the polymers, particle size, and surface charge of the formed polyplexes are influenced by the amine density and the disulfide bond. In general, a higher amine density and absence of the disulfide bond in the backbone enable the polymers a better DNA binding and condensing capability, resulting in the formation of polyplexes with smaller sizes and more positive surface charges. Both polymers S2 and S3 condense DNA to form reduction-sensitive polyplexes that can be disrupted by DTT, thus, exhibiting much lower cytotoxicity than their nonreducible counterparts, C2 and C3. Both polymers S2 and S3 show high transfection efficacy in the absence of serum, but the lowest efficiency is observed for polymer S3 among the four polymers in the presence of serum, implying that reductive degradability is not the sole factor affecting the transfection efficacy of cationic polymers. High efficiency accompanied with low cytotoxicity make polymer S2 a good carrier for in vitro gene delivery. Unfortunately, S2-formed polyplexes have large particle size in micrometer scale and show poor stability against incubation, which may limit its in vivo application, in particular, for systemic administration. Acknowledgment. This work was supported by the National Natural Science Foundation of China (20874001 and 20474004) and Nitto Denko Technical Corp. We thank Prof. Gu Yuan (College of Chemistry at Peking University), Prof. Wenlin Huang, Dr. Shijuan Gao, and Mr. Chao Zhang (Institute of Microbiology, CAS) for their kind help in gel retardation and luciferase pDNA transfection experiments. Supporting Information Available. Synthesis of intermediate compounds, more 1H NMR and 13C NMR spectra, acid-base titration curves, degradation kinetics under 100 mM DTT, GFP transfection results in COS-7 cells, and TEM micrographs of the polyplexes. This material is available free of charge via the Internet at http://pubs.acs.org.

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