Comb-Shaped Copolymers Composed of Hydroxypropyl Cellulose

Jul 31, 2009 - The reactive hydroxyl groups on HPC can be used as initiation sites for forming side chains to produce the comb-like copolymers via liv...
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Bioconjugate Chem. 2009, 20, 1449–1458

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Comb-Shaped Copolymers Composed of Hydroxypropyl Cellulose Backbones and Cationic Poly((2-dimethyl amino)ethyl methacrylate) Side Chains for Gene Delivery F. J. Xu,*,† Y. Ping,‡ J. Ma,§ G. P. Tang,| W. T. Yang,*,† J. Li,‡ E. T. Kang,⊥ and K. G. Neoh⊥ State Key Laboratory of Chemical Resource Engineering, College of Materials Science & Engineering, Beijing University of Chemical Technology, Beijing 100029, China, Division of Bioengineering, National University of Singapore, 7 Engineering Drive 1, 117574 Singapore, State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences, Beijing 100021, China, Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou, 310028, People’s Republic of China, and Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, 119260 Singapore. Received January 30, 2009; Revised Manuscript Received July 10, 2009

Cationic polymers have been of interest and importance as nonviral gene delivery carriers. Herein, well-defined comb-shaped cationic copolymers (HPDs) composed of long biocompatible hydroxypropyl cellulose (or HPC) backbones and short poly((2-dimethyl amino)ethyl methacrylate) (or P(DMAEMA)) side chains were prepared as gene vectors via atom transfer radical polymerization (ATRP) from the bromoisobutyryl-terminated HPC biopolymers. The P(DMAEMA) side chains of HPDs can be further partially quaternized to produce the quaternary ammonium HPDs (QHPDs). HPDs and QHPDs were assessed in vitro for nonviral gene delivery. HPDs exhibit much lower cytotoxicity and better gene transfection yield than high-molecular-weight P(DMAEMA) homopolymers. QHPDs exhibit a stronger ability to complex pDNA, due to increased surface cationic charges. Thus, the approach to well-defined comb-shaped cationic copolymers provides a versatile means for tailoring the functional structure of nonviral gene vectors to meet the requirements of strong DNA-condensing ability and high transfection capability.

INTRODUCTION Gene therapy shows much promise in tackling various genetic diseases and cancers, viral infection, and cardiovascular disorders (1-3). Successful gene therapy depends on the design of gene delivery vectors with low cytotoxicity and high transfection efficiency (4, 5). In comparison with viral vectors and cationic lipids, cationic polymers have been receiving considerable attention as the major type of nonviral gene delivery vectors, because of their low host immunogenicity, high flexibility, and easy preparation (6, 7). Polycations can spontaneously condense negatively charged DNA by electrostatic interaction into compact nanocomplexes and reduce the electrostatic repulsion between DNA and cell surfaces. Polycationic vectors can also protect plasmid DNA from enzymatic degradation by nucleases and facilitate cellular transfection. A great number of polycations, including polyethylenimine (PEI) (8), poly((2-dimethyl amino)ethyl methacrylate) (6, 9, 10), poly(Llysine) (11), polyamidoamine (12), and polysaccharide-based cationic carriers (5, 13), have been reported to deliver nucleic acids. Among these cationic polymers, PEI with a molecular weight of 25 kDa is considered the gold standard for polymeric nonviral gene delivery due to its high transfection efficiency (7). However, most of them suffer from either low gene transfection efficiency or significant toxicity. * To whom correspondence should be addressed. E-mail: xufj@ mail.buct.edu.cn (Xu FJ), [email protected] (Yang WT). † Beijing University of Chemical Technology. ‡ Division of Bioengineering, National University of Singapore. § Chinese Academy of Medical Sciences. | Zhejiang University. ⊥ Department of Chemical and Biomolecular Engineering, National University of Singapore.

Natural polysaccharides are very suitable candidates for gene delivery, because they are renewable, nontoxic, biodegradable, and excellent biocompatible materials. Hydroxypropyl cellulose (HPC) is a derivative of natural polysaccharide cellulose with both water and organic solubility, where some of the hydroxyl groups of cellulose have been hydroxypropylated to form propylene oxide groups (14-17). HPC materials have been approved by the United States Food and Drug Administration (FDA) and widely used in food and drug formulations (15, 18, 19). Comb-shaped cationic copolymers have been of interest and importance as nonviral gene carriers (4, 20-22). The reactive hydroxyl groups on HPC can be used as initiation sites for forming side chains to produce the comb-like copolymers via living radical polymerizations (16, 23). In the present work, welldefined comb-shaped cationic copolymers (HPDs) of long, biocompatible HPC backbones and short poly((2-dimethyl amino)ethyl methacrylate) (P(DMAEMA)) side chains are prepared via atom transfer radical polymerization (ATRP) (24) from the bromoisobutyryl-terminated HPC (HPC-Br) backbones (Scheme 1). The P(DMAEMA) side chains can be further partially quaternized to produce the quaternary ammonium HPDs (QHPDs). HPDs and QHPDs can condense plasmid DNA (pDNA) into complex nanoparticles of 150 to 200 nm in size at nitrogen/phosphate (N/P) ratios of 5 or higher. In comparison with the long P(DMAEMA) homopolymer, HPDs show much lower cytotoxicity and higher gene transfection efficiency. QHPDs exhibit a stronger ability to complex pDNA, due to increased surface cationic charges. The novel HPDs and QHPDs may have potential application as nonviral gene vectors for future gene therapy.

EXPERIMENTAL PROCEDURES Materials. Hydroxypropyl cellulose powder (HPC, SigmaAldrich cat #435007: Mn ) 10 000 g/mol and Mw ) 80 000

10.1021/bc900044h CCC: $40.75  2009 American Chemical Society Published on Web 07/31/2009

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Scheme 1. Schematic Diagram Illustrating the Preparation Processes of Cationic Comb-Shaped Copolymers Composed of HPC Backbones and Short Partially Quaternized P(DMAEMA) Side Chains

g/mol; from gel permeation chromatography (GPC) with THF as mobile phase: Mn ) 28 015 g/mol and Mw ) 64 655 g/mol; molar substitution of propylene oxide groups (MS) of about 3 per glucose unit 16, 17), branched polyethylenimine (PEI, Mw ∼25 000 Da), 1,4,7,10,10,-hexamethyl triethylenetetramine (HMTETA, 99%), copper(I) bromide (CuBr, 99%), 2-bromoisobutyryl bromide (BIBB, 98%), and (2-dimethylamino)ethyl methacrylate (DMAEMA, >98%) were obtained from Sigma-Aldrich Chemical Co., St. Louis, MO. DMAEMA was used after removal of the inhibitors in a ready-to-use disposable inhibitor-removal column (Sigma-Aldrich). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), penicillin, and streptomycin were purchased from Sigma Chemical Co., St. Louis, MO. HEK293 cell lines were purchased from the American type Culture Collection (ATCC, Rockville, MD). The control P(DMAEMA) homopolymer with about 180 repeat units of DMAEMA was prepared via ATRP, using the reported ethylene glycol di-2-bromoisobutyrate as the initiator (24). Synthesis of Comb-Shaped Cationic Copolymers via ATRP. The starting bromoisobutyryl-terminated HPC (HPC-Br) was synthesized following the procedures. About 2.0 g of HPC was dissolved completely in 100 mL of anhydrous methylene chloride with stirring and then kept in an ice bath. About 0.3 mL of BIBB in 5 mL of methylene chloride was added dropwise into the flask through an equalizing funnel for a period of 10 min at 0 °C. After this addition, the flask was sealed. The reaction was allowed to proceed at room temperature for another 5 h to produce the HPC-Br macroinitiator. The final reaction mixture was precipitated with 1000 mL of diethyl ether. The crude polymer was purified by reprecipitation thrice in diethyl ether. Finally, the HPC-Br for the subsequent ATRP was dried under reduced pressure.

The HPC-g-P(DMAEMA) comb-like polymers (HPDs, Scheme 1) were synthesized using a molar feed ratio (DMAEMA (about 1 mL))/(CuBr)/(HMTETA) of 60:1:1.2 at room temperature in 8 mL of isopropanol/water (20/1, v/v) containing 0.2 g of HPC-Br. The reaction was performed in a 25 mL flask equipped with a magnetic stirrer and under the typical conditions for ATRP (24). DMAEMA, HPC-Br, and HMTETA were introduced into the flask containing 8 mL of isopropanol/water (20/1, v/v). After HPC-Br had dissolved completely, the reaction mixture was degassed by bubbling argon for 30 min. Then, CuBr was added into the mixture under an argon atmosphere. The reaction mixture was purged with argon for another 10 min. The flask was then sealed with a rubber stopper under an argon atmosphere. The polymerization was allowed to proceed under continuous stirring at room temperature for 2 to 8 h. The reaction was stopped by diluting with THF. The catalyst complex was removed by passing the blue dilute polymer solution through a short aluminum oxide column. A colorless solution was obtained. After removal of THF in a rotary evaporator, the HPDs were precipitated in excess n-hexane. The crude polymer was purified by reprecipitation twice in hexane to remove the reactant residues, prior to being dried under reduced pressure. The HPD1 (from ATRP time of 2 h) and HPD2 (from ATRP time of 8 h) yields are about 0.4 and 0.72 g, respectively. The HPC-g-QP(DMAEMA) (QHPDs) with partially quaternized P(DMAEMA) side chains were prepared at 50 °C for 24 h in 5 mL of THF containing 0.4 g of HPD2 and 200 or 400 µL of 1-bromohexane. The QHPDs were precipitated in excess n-hexane and dried under reduced pressure.

Comb-Shaped Copolymers

Polymer Characterization. The molecular weights of polymers were determined by gel permeation chromatography (GPC), chemical composition by X-ray photoelectron spectroscopy (XPS), and chemical structure by nuclear magnetic resonance (NMR) spectroscopy. GPC measurements were performed on a Waters GPC system equipped with Waters Styragel columns, a Waters-2487 dual wavelength (λ) UV detector, and a Waters-2414 refractive index detector. THF containing 2 vol % triethylamine was used as the eluent at a low flow rate of 1.0 mL/min. Monodispersed polystyrene standards were used to generate the calibration curve. The XPS measurements were performed on a Kratos AXIS HSi spectrometer equipped with a monochromatized Al KR X-ray source (1486.6 eV photons), using the same procedures as those described earlier (24).1H NMR spectra were measured by accumulation of 1000 scans at a relaxation time of 2 s on a Bruker ARX 300 MHz spectrometer, using CDCl3 or D2O as the solvent. Characterization of Polymer/Plasmid DNA Complexes. The plasmid (encoding Renilla luciferase) used in this work was pRL-CMV (Promega Co., Cergy Pontoise, France), which was cloned originally from the marine organism Renilla reniformis. The plasmid DNA (pDNA) was amplified in Escherichia coli and purified according to the supplier’s protocol (Qiagen GmbH, Hilden, Germany). The purity and concentration of the purified DNA were determined by absorption at 260 and 280 nm and by agarose gel electrophoresis (9). The purified pDNA was resuspended in tris-EDTA (TE) buffer and kept in aliquots of 0.5 mg/mL in concentration. All polymer stock solutions were prepared at a nitrogen concentration of 10 mM in distilled water. Solutions were filtered via sterile membranes (0.2 µm) of average pore size and stored at 4 °C. Comb-like polymers to DNA ratios are expressed as molar ratios of nitrogen (N) in the HPDs and QHPDs to phosphate (P) in DNA (or as N/P ratios). The average mass weight of 325 per phosphate group of DNA was assumed (8). All polymer/pDNA complexes were formed by mixing equal volumes of polymer and pDNA solutions to achieve the desired N/P ratio. Each mixture was vortexed and incubated for 30 min at room temperature. Each cationic polymer was examined for its ability to bind pDNA through agarose gel electrophoresis using the similar procedures as those described earlier (9). The polymer/pDNA complexes at various N/P ratios were investigated. Gel electrophoresis was carried out in TAE running buffer (40 mM Trisacetate, 1 mM EDTA) with a voltage of 110 V for 30 min in a Sub-Cell system (Bio-Rad Lab, Hercules, CA). DNA bands were visualized and photographed by a UV transilluminator and BioDco-It imaging system (UVP Inc., Upland, CA). The particle sizes and zeta potentials of the polymer/pDNA complexes were measured using a Zetasizer Nano ZS (Malvern Instruments, Southborough, MA) and procedures similar to those described earlier (9). The polymer/pDNA complexes were also observed with a field-emission scanning electron microscope equipped with a scanning transmission electron microscopy (STEM) detector (JEOL JSM-6700F) and procedures similar to those described earlier (9). The complexes were visualized by transmitted electrons (STEM-in-SEM mode) at a low voltage of 15 kV, in comparison to about 200 kV for the conventional TEM. Such low voltage can minimize damage to the polymer/ pDNA complex and allow visualization of the complex in the TEM mode at high spatial resolution. Cell Viability and in Vitro Transfection Assay. The cytotoxicity of the comb-like polymers was evaluated using the MTT assay in HEK293 cell lines. They were cultured in Dulbecco’s modified eagle medium (DMEM), supplemented with 10% heatinactivated fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 µg/mL of streptomycin at 37 °C, under 5% CO2 and

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95% relative humidity atmosphere. The cells were seeded in a 96-well microtiter plate at a density of 104 cells/well and incubated in 100 µL of DMEM/well for 24 h. The culture media were replaced with fresh culture media containing serial dilutions of polymers, and the cells were incubated for 24 h. Then, 10 µL of sterile-filtered MTT stock solution in PBS (5 mg/mL) was added to each well, reaching a final MTT concentration of 0.5 mg/mL. After 5 h, the unreacted dye was removed by aspiration. The produced formazan crystals were dissolved in DMSO (100 µL/well). The absorbance was measured using a microplate reader (Spectra Plus, Tecan, Zurich, Switzerland) at a wavelength of 570 nm. The cell viability (%) relative to control cells cultured in media without polymers was calculated from (A)test/(A)control × 100%, where (A)test and (A)control are the absorbance values of the wells (with the polymers) and control wells (without the polymers), respectively. For each sample, the final absorbance was the average of those measured from six wells in parallel. Transfection assays were performed using plasmid pRL-CMV as the reporter gene in HEK293 cell lines. In brief, the cells were seeded in 24-well plates at a density of 5 × 104 cells in the 500 µL of medium/well and incubated for 24 h. The combshaped polymer/pDNA complexes (20 µL/well containing 1.0 µg of pDNA) at various N/P ratios from 7 to 22 were prepared by adding the polymer into the DNA solutions, followed by vortexing and incubation for 30 min at room temperature. At the time of transfection, the medium in each well was replaced with 300 µL of serum-free medium or fresh normal medium (supplemented with 10% FBS). The complexes were added into the transfection medium and incubated with the cells for 4 h under standard incubator conditions. Then, the medium was replaced with 500 µL of the fresh normal medium (supplemented with 10% FBS). The cells were further incubated for an additional 20 h under the same conditions, resulting in a total transfection time of 24 h. The cultured cells were washed with PBS twice and lysed in 100 µL of the cell culture lysis reagent (Promega Co., Cergy Pontoise, France) using the similar procedures as those described earlier (9). Luciferase gene expression was quantified using a commercial kit (Promega Co., Cergy Pontoise, France) and a luminometer (Berthold Lumat LB 9507, Berthold Technologies GmbH. KG, Bad Wildbad, Germany). Protein concentration in the cell samples was analyzed using a bicinchoninic acid assay (Biorad Lab, Hercules, CA). Gene expression results were expressed as relative light units (RLUs) per milligram of cell protein lysate (RLU/mg protein). Statistical Analysis. All experiments were repeated at least three times. The data were collected in triplicate and expressed as mean ( standard deviation. Error bars represent the standard deviation. The statistical assay was performed using t-test, and the differences were considered statistically significant with p < 0.05.

RESULTS AND DISCUSSION Synthesis and Characterization of Comb-Shaped Cationic Copolymers Composed of HPC Backbones and P(DMAEMA) Side Chains. The preparation of functional polymers is usually carried out in organic solvent. Because of its unique characterisitcs of both water and organic solubility, HPC is an ideal substrate for developing novel polysaccharide biopolymer-based cationic carriers. The molecular weight (Mn) of the HPC used in this work from gel permeation chromatography (GPC) is about 2.80 × 104 g/mol, and its corresponding polydispersity index (PDI) is about 2.3. The C 1s core-level X-ray photoelectron spectrum (XPS) (Figure 1a) of the pristine HPC can be curve-fitted by three peak components with binding energies (BEs) at about 284.6, 286.2, and 287.6 eV, attributable to the

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Figure 1. XPS C 1s core-level spectra of (a) HPC, (b) HPC-Br, (c) HPD1, (d) HPD2, (e) QHPD2-1, and (f) QHPD2-2, Br 3d core-level spectrum of (b′) HPC-Br, and N 1s core-level spectra of (c′) HPD1, (d′) HPD2, (e′) QHPD2-1, and (f′) QHPD2-2.

C-H/C-C, C-O, and O-C-O species, respectively (9, 24). From the (C-O)/(O-C-O) ratio of 11, the average number of propylene oxide groups (-OCH2CH(OH)CH3) per anhydroglucose unit of HPC is about 3, consistent with that (2.9) determined using 1 H NMR (16, 17). In order to prepare comb-shaped copolymers using HPC as a backbone via ATRP, it is essential to introduce alkyl halide into HPC. In this work, the bromoisobutyryl-terminated HPC (HPC-Br) was prepared via the reaction of hydroxyl groups of HPC with 2-bromoisobutyryl bromide (BIBB) (Scheme 1). When ATRP is carried out from a multifunctional backbone with a high local concentration of initiation sites, the radicalradical coupling of the propagating chains will occur and result in a gel. In order to avoid potential gelation and introduce some flexibility onto the comb-like cationic copolymers, the starting HPC-Br with moderate initiation sites is desired for preparing well-defined comb-shaped copolymers. The C 1s core-level XPS spectrum (Figure 1b) of HPC-Br can be curve-fitted by four peak components with BEs at about 284.6, 286.2, 287.6, and 288.4 eV, attributable to the C-H/C-C, C-O/C-Br, O-C-O, and O)C-O species, respectively (9, 24). The new O-C)O peak component is associated with the bromide-capped ester groups of HPC-Br. The corresponding Br 3d core-level spectrum is shown in Figure 1b′. From the (Br)/(C) ratio of 0.011 (determined from the sensitivity factor-corrected Br 3d and C 1s corelevel spectral area ratio), it was calculated that about six glucose units of HPC-Br possess one ATRP initiation site. The chemical

structure of HPC-Br is also characterized by 1H NMR spectroscopy (Figure 2a). The chemical shift at δ ) 1.12 ppm is attributable to the methyl protons of propylene oxide groups (a, CH3). The chemical shift at δ ) 1.92 ppm is associated with the methyl protons (b, C(Br)-CH3) of the 2-bromoisobutyryl groups (24). The broad chemical shifts in the wide region 2.9-4.3 ppm are mainly associated with the inner methylidyne and methylene protons (c, c′, CH and CH2) on glucose units and propylene oxide groups. From the area ratio of peak a and peak b, the initiator density of HPC-Br is that every 6.8 glucose units possess one initiation site, fairly consistent with that determined by XPS. Well-defined comb-shaped copolymers (HPDs) of HPC and P(DMAEMA), or HPC-g-P(DMAEMA), were subsequently synthesized via ATRP of DMAEMA from HPC-Br (Scheme 1). The HPDs with different lengths of P(DMAEMA) side chains can be synthesized by varying the ATRP time. Table 1 summarizes the GPC results of HPD1 (from 2 h of ATRP) and HPD2 (from 8 h of ATRP). With the increase in reaction time from 2 to 8 h, the Mn of HPD from GPC using linear polystyrene standards increases from 5.42 × 104 to 8.86 × 104 g/mol, and the total number of DMAEMA repeat units per side chain increases accordingly from 13 to 30. These values are lower than those determined by XPS and 1H NMR (Table 1), probably due to the differences in hydrodynamic volumes between comblike and linear polymers. In addition, the PDIs of HPDs are comparable to that of the pristine HPC, indicating that the ATRP

Comb-Shaped Copolymers

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Figure 2. 300 MHz 1H NMR spectra of the (a) HPC-Br, (b) HPD2, and (c) QHPD2-1 polymers.

of DMAEMA is well-controlled. In order to increase the surface cationic charges, the P(DMAEMA) side chains of HPD can be partially quaternized to produce the HPC-g-QP(DMAEMA) (QHPD) with quaternary ammonium groups. In this work, the QHPD2-1 and QHPD2-2 with different quaternization contents were prepared by using HPD2 and 1-bromohexane (Scheme 1 and Table 1). The C 1s core-level spectra of HPD1 and HPD2 (Figure 1c and d) can be curve-fitted into five peak components with BEs

of about 284.6, 285.5, 286.2, 287.6, and 288.4 eV, attributable to the C-H/C-C, C-N, C-O, O-C-O, and O)C-O species, respectively. The C-N peak is associated with the P(DMAEMA) side chains. In comparison with the C 1s core-level spectrum (Figure 1b) of HPC-Br, the increase (and decrease) in intensities of the C-N (and C-O) species with ATRP time is consistent with the increase in P(DMAEMA) contents. The corresponding N 1s core-level spectra of HPD1 and HPD2 are shown in Figure 1c′ and d′, respectively. From the (N)/(C) ratios determined by

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Table 1. Characterization of the Comb-Shaped Cationic Polymers sample HPC HPC-g-P(DMAEMA)1 or HPD1a HPC-g-P(DMAEMA)2 or HPD2a HPC-g-QP(DMAEMA)2-1 or QHPD2-1b HPC-g-QP(DMAEMA)2-2 or QHPD2-2b

reaction time (h) Mn (g/mol)c PDIc (N)/(C)d (N+)/(N)+(N+)d DMAEMA repeat units (per side chain) 2 8 24 24

2.80 × 104 5.42 × 104 8.86 × 104 -

2.3 2.2 1.8 -

0.073 0.095 -

0.22 0.43

13e 30e

16f 35f -

15g 37g

a Synthesized using a molar feed ratio (DMAEMA (1 mL)):(CuBr):(HMTETA) of 60:1:1:1.2 at room temperature in 8 mL of isopropanol/water (20/ 1, v/v) containing 0.2 g of HPC-Br. The calculated molecular format of glucose units of HPC is C15H28O8, and about six glucose units of HPC-Br possess one initiation site. b Synthesized at 50 °C in 5 mL of THF containing 0.4 g of HPD2 and 200 (for QHPD2-1) or 400 µL (for QHPD2-2) of 1-bromohexane. c Determined from GPC results. PDI ) Weight average molecular weight/Number average molecular weight, or Mw/Mn. d Determined from XPS. e Determined from Mn and the molecular weights of DMAEMA (157 g/mol). f Determined from (N)/(C) ratio, where (N)/(C) ≈ n(DMAEMA)/((6 × 15 + 4) + 8n(DMAEMA)). g Determined from the 1H NMR spectroscopy data.

XPS, the P(DMAEMA) contents of HPDs can be calculated (Table 1). After quaternization, the C 1s core-level spectra of QHPD2-1 and QHPD2-2 (Figure 1e and f) can be curve-fitted into five peak components with BEs at about 284.6, 285.5, 286.2, 287.6, and 288.4 eV, attributable to the C-H/C-C, C-N, C-O/C-N+, O-C-O, and O)C-O species, respectively (25). In comparison with the C 1s core-level spectrum (Figure 1d) of the starting HPD2, the increase in intensities of the C-H/C-C and C-O/C-N+ species of QHPD2-1 and QHPD2-2 indicates that the P(DMAEMA) side chains have been successfully quaternized by 1-bromhexane. In comparison with the monopeak component of the N 1s core-level spectrum (Figure 1d′) of HPD2, the N 1s core-level spectra (Figure 1e′ and f′) of QHPD2-1 and QHPD2-2 can be curve-fitted into two peak components with BEs at about 399 and 402 eV, attributable to the amino (C-N) and positively charged nitrogen (C-N+) species, respectively (25). The quaternization extent of P(DMAEMA) side chains can be expressed as the (N+)/((N) + (N+)) ratio (determined from the corresponding N+ peak component and total nitrogen spectral area ratio). The average (N+)/((N) + (N+)) ratios for QHPD2-1 and QHPD2-2 are about 0.22 and 0.43, respectively (Table 1), indicating that about 1 out of every 4.5 (for QHPD2-1) or 2.3 (for QHPD2-2) DMAEMA repeat units had been quaternized. Figure 2b and c shows the respective 1H NMR spectra of HPD2 and QHPD2-1 in D2O. The typical chemical shifts at about 0.89, 1.81, 2.28, and 2.69 ppm are mainly attributable to the (a′) C-CH3 methyl, (d′′) C-CH2 methylene, (d) N-CH3 methyl, and (d′) N-CH2 methylene protons of the P(DMAEMA) side chains, respectively. The chemical shift associated with the methylene protons adjacent to the oxygen moieties of the ester linkages (c′′, CH2-O-CdO) overlapped completely with glucose peaks of HPC. The signal intensities associated with the HPC backbone (a, c, c′′) have decreased substantially. From the area ratio of peaks d, d′, d′′ and peaks a, a′, the P(DMAEMA) contents of HPDs can be calculated, and the results are comparable to those obtained from XPS (Table 1). After quaternization, the new signals at about 3.17 and 3.41 ppm are attributable to the (d′′′) N+-CH3 methyl and (d′′′′) N+CH2 methylene protons of the quaternized P(DMAEMA) side chains, respectively (26). The chemical shift at about 1.36 ppm corresponds to the methylene protons of hexane (a′′, CH2-C). Due to the serious overlaps in the characteristic shifts of the components, it is very difficult to quantify the P(DMAEMA) and QP(DMAEMA) segments by 1H NMR. Polymer/Plasmid DNA Complexes. For cellular transfection, DNA has to be first condensed by cationic polymers into polymer/plasmid nanoparticles suitable for cellular entry. Thus, the DNA condensation capability is a prerequisite for polymeric gene vectors. In this work, the ability of the comb-shaped cationic copolymers to condense plasmid DNA (pDNA) into particulate structures was confirmed by agarose gel electrophoresis, particle size, and zeta potential measurements, as well as STEM imaging. Figure 3 shows the gel retardation results

Figure 3. Electrophoretic mobility of plasmid DNA (pDNA) in the complexes of the cationic polymers ((a) HPD1, (b) HPD2, (c) QHPD2-1, (d) QHPD2-1, (e) P(DMAEMA), and (f) PEI at various N/P ratios.

of the cationic comb copolymer (HPD1, HPD2, QHPD2-1, and QHPD2-2)/pDNA complexes with increasing nitrogen (N)/ phosphate (P) (or N/P) ratios, in comparison with those of the P(DMAEMA) homepolymer (with about 180 DMAEMA repeat units)/pDNA and branched PEI (25 kDa)/pDNA complexes. HPD1 and HPD2 can compact pDNA effectively at N/P ratios of 1.75 and 1.5, respectively, due to the increasing density of amino groups in the comb-like copolymers (Table 1). The migration of pDNA was completely retarded by QHPD2-1 or QHPD2-2 at an N/P ratio of 1.25, indicating that the increased surface cationic charges from quaternization could enhance the condensation capability of the comb copolymers. The control P(DMAEMA) and PEI could inhibit the migration of pDNA at N/P ratios of 1.25 and 2.0, respectively, indicating that the prepared comb copolymers have good condensation capability.

Comb-Shaped Copolymers

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Figure 4. Particle size (a) and zeta potential (b) of the complexes between the cationic polymers (HPD1, HPD2, QHPD2-1, QHPD2-2, P(DMAEMA), and PEI) and pDNA at various N/P ratios.

The (a) particle size and (b) zeta potential of the cationic polymer (HPD1, HPD2, QHPD2-1, and QHPD2-2)/pDNA complexes are compared to those of the P(DMAEMA)/pDNA and PEI/pDNA complexes at various N/P ratios in Figure 4. All the cationic comb copolymers can efficiently compact pDNA into nanoparticles. Generally, the hydrodynamic sizes of the complexes decrease with increasing N/P ratios. After reaching the N/P ratio of 5, all the comb copolymers can condense pDNA into nanoparticles of 150 to 200 nm diameter. The polymer/ pDNA complex within this size range can readily undergo endocytosis (27). Zeta potential is an indicator of surface charges on the polymer/pDNA naoparticles. A positively charged surface allows electrostatic interaction with negatively charged cell surfaces and facilitates cellular uptake. For the pDNA complexes of HPD1, HPD2, QHPD2-1, QHPD2-2, P(DMAEMA), and PEI, the net surface charge increases dramatically as the N/P ratio increases from 1 to 5, and stabilizes at N/P ratios of 10 and above (Figure 4b). After reaching the N/P ratio of 10, the zeta potentials of the pDNA complexes of all the cationic polymers are strongly positive. Due to the increasing quaternization of the P(DMAEMA) side chains, the pDNA complexes of HPD2, QHPD2-1, and QHPD2-2 show increasing surface cationic charges, indicating that they will produce increasing affinity for anionic cell surfaces. On the other hand, the excess cationic polymers probably do not exert any significant effect on the particle size and zeta potential of the complexes (Figure 4). In addition, the observed N/P ratios for completely retarding the migration of pDNA in the agarose gel electrophoresis (Figure 3) represented the starting points where the polymer/pDNA complexes become positively charged. At such N/P ratios, pDNA cannot be packed tightly. With increasing N/P ratios, the hydrodynamic sizes (or net surface charges) of the complexes decrease (or increase) and stabilize at higher N/P ratios (Figure 4). Therefore, the results from electrophoresis are consistent with those from Figure 4. Figure 5 shows the representative STEM images of the (a) HPD2/pDNA (at a N/P ratio of 1), (b) HPD2/pDNA (at a N/P ratio of 15), and (c) QHPD2-1/pDNA (at a N/P ratio

Figure 5. STEM images of the (a) HPD2/pDNA (at a ratio of 1), (b) HPD2/pDNA (at a ratio of 15), and (c) QHPD2-1/pDNA (at a ratio of 15) complexes.

of 15) complexes. The FESEM equipped with a STEM detector allows the visualization of polymer complexes in the TEM mode at a low voltage of 15 kV to avoid destruction of the polymer/pDNA complex. The images clearly reveal the morphological difference between the polymer/pDNA complexes at different N/P ratios. For the HPD2/pDNA complex at a ratio of 1, loose aggregates (>400 nm), supercoiled pDNA, and compact nanoparticles (