Facilitation of Gene Transfection with Well-Defined Degradable Comb

Feb 14, 2012 - Further improvement in degradability and transfection efficiency of the PGEA vectors will facilitate their application in gene therapy...
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Facilitation of Gene Transfection with Well-Defined Degradable Comb-Shaped Poly(glycidyl methacrylate) Derivative Vectors X. C. Yang,† M. Y. Chai,† Y. Zhu, W. T. Yang, and F. J. Xu* State Key Laboratory of Chemical Resource Engineering, Key Laboratory of Carbon Fiber and Functional Polymers, Ministry of Education College of Materials Science & Engineering, Beijing University of Chemical Technology, Beijing, China 100029 ABSTRACT: Recently, we reported that ethanolamine (EA)functionalized poly(glycidyl methacrylate) (PGMA) vectors (PGEAs) can produce good transfection efficiency, while exhibiting very low toxicity. Further improvement in degradability and transfection efficiency of the PGEA vectors will facilitate their application in gene therapy. Comb-shaped cationic copolymers have been of interest and importance as nonviral gene carriers. Herein, the degradable high-molecularweight comb-shaped PGEA vectors (c-PGEAs) composed of the low-molecular-weight PGEA backbone and side chains were prepared by a combination of atom transfer radical polymerization (ATRP) and ring-opening reactions. The PGEA side chains were linked with the PGEA backbones via hydrolyzable ester bonds. Such comb-shaped c-PGEA vectors possessed the degradability, good pDNA condensation ability, low cytotoxicity, and good buffering capacity. More importantly, the comb-shaped c-PGEA vectors could enhance the gene expression levels. Moreover, the PGEA side chains of c-PGEA could also be copolymerized with some poly(poly(ethylene glycol)ethyl ether methacrylate) species to further improve the gene delivery system.

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INTRODUCTION Successful gene therapy depends on the design of gene delivery vectors with low cytotoxicity and high transfection efficiency. In comparison with viral vectors and cationic lipids, cationic polymers have been receiving considerable attention, because of their low host immunogenicity, high flexibility, and easy preparation.1,2 Under physiological conditions, polycations can spontaneously condense negatively charged DNA into compact nanocomplexes, reduce the electrostatic repulsion between DNA and cell surfaces, protect plasmid DNA from enzymatic degradation by nucleases, and facilitate cellular transfection. Many kinds of polycations, including polyethylenimine (PEI),3,4 poly(tertiaryamine methacrylate),4 poly(L-lysine),5 and polyamidoamine,6 have been reported to be capable of gene delivery. High transfection efficiency is generally associated with a devastating toxicity. We recently reported that ethanolamine (EA)-functionalized poly(glycidyl methacrylate) (PGMA) or PGEA with plentiful flanking secondary amine and hydroxyl groups can produce good transfection efficiency in different cell lines, while exhibiting low toxicity.7,8 It was noted that such a PGEA vector was nondegradable and its good transfection efficiency was dependent on the high molecular weight. A water-soluble polymer with high molecular weight is not suitable for filtration through human kidney membrane due to the large hydrodynamic radius.9,10 Further improvement in degradability and transfection efficiency of the PGMA derivative vectors will benefit constructing better gene-delivery systems. Comb-shaped cationic copolymers have been of interest and importance as nonviral gene carriers.4,11−13 A comb-shaped © 2012 American Chemical Society

copolymer is a special type of branched polymer composed of one main polymer backbone and some polymer side chains. The side chains could be structurally distinct and extend from the main backbone. The comb-shaped architectures probably could enhance the interaction of the cationic species with pDNA or cellular membranes. It was reported that, in comparison with high-molecular-weight cationic homopolymers, the comb cationic copolymers containing low-molecular-weight polycation side chains exhibited much higher gene transfection efficiency and lower cytotoxicity.12,13 The reactive epoxy groups of PGMA could be converted into initiation sites for forming side chains to produce the comb-like copolymers. In this work, the degradable high-molecular-weight comb-shaped PGMA derivative vectors (c-PGEA), where the low-molecular-weight PGEA side chains were linked with the PGEA backbones via the ester bonds, were proposed via facile atom transfer radical polymerization (ATRP)4,14 (Figure 1). Such degradable combshaped c-PGEA vectors could facilitate the resultant gene delivery and benefit the final removal of the PGEA vectors from the body. Moreover, the PGEA side chains of c-PGEA could also be copolymerized with poly(poly(ethylene glycol)ethyl ether methacrylate) (PPEGEEMA) to further improve the gene transfection efficiency. The proposed degradable comb-shaped c-PGEA vectors would become promising candidates as safe and efficient vectors for future gene therapies. Received: December 6, 2011 Revised: January 16, 2012 Published: February 14, 2012 618

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Figure 1. Preparation processes of different comb-shaped poly(glycidyl methacrylate) (PGMA) derivative vectors.

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EXPERIMENTAL PROCEDURES Materials. Branched polyethylenimine (PEI, Mw ∼25 000 Da), glycidyl methacrylate (GMA, 98%), poly(ethylene glycol)ethyl ether methacrylate) (PEGEEMA, Mn ∼ 246), ethanolamine (EA, 98%), 1,1,4,7,10,10-hexamethyltriethyenetetramine (HMTETA, 99%), α-bromoisobutyric acid (BIBA, 98%), ethyl bromoisobutyrate (99%), and copper(I) bromide (CuBr, 99%) were obtained from Sigma-Aldrich Chemical Co., St. Louis, MO. GMA and PEGEEMA were used after removal of the inhibitors in a ready-to-use disposable inhibitor-removal column (SigmaAldrich). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), penicillin, and streptomycin were also purchased from Sigma-Aldrich Chemical Co., St. Louis, MO. C6 and HEK293 cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Introduction of ATRP Initiation Sites onto PGMA Backbones. The linear PGMA (l-PGMA) backbones were first synthesized using a molar feed ratio [GMA (12 mL)]: [ethyl bromoisobutyrate]:[CuBr]:[HMTETA] of 140:1:1:1.5 at 50 °C in 24 mL of GMA/THF (1/1, v/v). The reaction was performed in a 50 mL flask equipped with a magnetic stirrer and under the typical conditions of ATRP. GMA, THF, ethyl bromoisobutyrate, and HMTETA were introduced into the 50 mL flask equipped with a magnetic stirrer. The reaction mixture was degassed by bubbling argon for 20 min. Then, CuBr was added into the mixture and the flask was then sealed with a rubber stopper under an argon atmosphere. The polymerization was allowed under continuous stirring at 50 °C to produce l-PGMA from 2 h of ATRP (Mn = 9.5 × 103 g/mol,

PDI = 1.32). The reaction was stopped by exposing to air. The l-PGMA was precipitated in excess methanol to remove the catalyst complex. The crude polymer was purified by reprecipitation cycles into methanol to remove the reactant residues, prior to being dried under reduced pressure. As shown in Figure 1, the introduction of the ATRP initiation sites onto l-PGMA was performed via the direct ringopening reactions of the epoxy groups of l-PGMA with αbromoisobutyric acid (BIBA) to produce the bromoisobutylrylterminated l-PGMA (l-PGMA-Br) for the subsequent ATRP. The two l-PGMA-Br macroinitiators with the different distribution of initiation sites were prepared by adjusting the BIBA/l-PGMA feed ratio. With the predetermined molar feed ratio (1/5 or 1/8) of the BIBA/epoxy units, the BIBA (2.11 or 1.32 mmol) in 2 mL of THF was added dropwise at ambient temperature into the flask containing l-PGMA (10.56 mmol) in 10 mL of THF. The reaction was allowed to proceed at 40 °C for 24 h to produce the l-PGMA-Br. The final reaction mixture was precipitated and washed with excess diethyl ether, prior to being dried under reduced pressure. Synthesis of Comb-Shaped Cationic PGMA Derivatives. For the preparation of comb-like PGMA (c-PGMA) polymers via ATRP, the molar feed ratio [GMA (4 mL)]: [CuBr]:[HMTETA] of 100:1:1.5 was used at 50 °C in 8 mL of GMA/THF (1/1, v/v) containing 0.3 g of l-PGMABr. The reaction was conducted in a 25 mL flask equipped with a magnetic stirrer and under the typical conditions for ATRP. GMA, l-PGMABr, and HMTETA were introduced into the flask containing 8 mL of GMA/THF (1/1, v/v), and the 619

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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.12 The particle sizes and zeta potentials of the polymer/pDNA complexes were measured in triplicate using a Zetasizer Nano ZS (Malvern Instruments, Southborough, MA) and procedures similar to those described earlier.12 Cell Viability. The cytotoxicity of the polymer vectors was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay in HEK293 and C6 cell lines. They were cultured in Dulbecco’s modified eagle medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C, under 5% CO2 and 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 or 10 μL polymer/pDNA complex solution (containing 0.5 μg of pDNA) at various N/P ratios, 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. In Vitro Transfection Assay. Transfection assays were performed using plasmid pRL-CMV as the reporter gene in HEK293 and C6 cell lines. In brief, the cells were seeded in 24well plates at a density of 5 × 104 cells in 500 μL of medium/ well and incubated for 24 h. The polymer/pDNA complexes of N/P ratios from 10 to 35 were prepared a priori 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 fresh normal serum medium (supplemented with 10% FBS). The polymer/pDNA complexes were added (20 μL/well containing 1.0 μg of pDNA) into the transfection medium and incubated with the cells for 4 h under the standard incubation 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, giving rise to a total transfection time of 24 h.12 The cultured cells were washed with PBS twice and lysed in 100 μL of the cell culture lysis reagent (Promega Co., Cergy Pontoise, France). 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). All experiments were

reaction mixture was degassed by bubbling argon for 20 min. Then, CuBr was added into the mixture under an argon atmosphere. The flask was then sealed with a rubber stopper under an argon atmosphere. The polymerization was allowed to proceed under continuous stirring at 50 °C for 4 h to produce c-PGMA. The final reaction mixture was precipitated and washed with excess methanol, prior to being dried under reduced pressure. For the preparation of comb-like P(GMA-PEGEEMA) (c-P(GMAPEGEEMA)) polymers via ATRP, the molar feed ratio [GMA (3.4 or 3.7 mL) + PEGEEMA (0.6 or 0.3 mL))]:[CuBr]: [HMTETA] of 100:1:1.5 was used at 50 °C in 8 mL of GMA/ THF (1/1, v/v) containing 0.3 g of l-PGMABr. The polymerization was allowed to proceed for 4 h to produce c-P(GMAPEGEEMA). The ATRP reaction and product purification were performed using the same procedures as those described above. Different EA-functionalized PGMA derivatives were prepared by reacting l-PGMA, c-PGMA, or c-P(GMA-PEGEEMA) with ethanolamine (EA). 0.4 g of l-PGMA, c-PGMA, or c-P(GMAPEGEEMA) was dissolved in 7 mL of DMF. Four mL of EA and 2 mL of triethyleneamine were then added. The reaction mixture was stirred at 50 °C for 72 h to produce l-PGEA, cPGEA, or c-PGEAPEG (Figure 1). The final reaction mixture was precipitated with excess diethyl ether. The crude produce was purified by 24 h dialysis against DDW using a dialysis membrane (MWCO 3500) prior to lyophilization. Polymer Characterization. The molecular weights of polymers were determined by gel permeation chromatography (GPC), chemical structure by nuclear magnetic resonance (NMR) spectroscopy, and chemical composition by X-ray photoelectron spectroscopy (XPS). 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. Monodispersed poly(ethylene glycol) standards were used to obtain a calibration curve. 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 (for PGMA) or D2O (for PGEA) as the solvent. XPS measurements were performed on a Kratos AXIS HSi spectrometer using a monochromatized Al Kα X-ray source (1486.6 eV photons) and procedures similar to those described earlier.7 Degradation Study. The degradation of c-PGEA was analyzed using GPC. c-PGEA was dissolved in PBS solution (pH 7.4), which was constantly shaken in a 37 °C incubator at 100 rpm. The PHPD solution was withdrawn at different time points for GPC analysis to determine the relative molecular mass of degraded products. Characterization of Polymer/pDNA 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 purified pDNA was resuspended in Tris-EDTA (TE) buffer, pH 7.4, 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 of 0.2 μm average pore size and stored at 4 °C. Polymers to DNA ratios are expressed as molar ratios of nitrogen (N) in PGEAs to phosphate (P) in DNA (or as N/P ratios). The average mass weight of 325 per phosphate group of DNA was assumed. All polymer/pDNA complexes were formed by mixing equal 620

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at δ = 3.2 ppm (b) and δ = 2.63 and 2.84 ppm (c) could be assigned to the protons of the epoxide ring. The ratio of peak areas of a, b, and c is about 2:1:2, indicating that the epoxy groups of the PGMA remained intact throughout ATRP. After the ring-opening reactions of some epoxy groups with BIBA, the new peaks of l-PGMABr at δ = 1.85, δ = 3.5, and δ = 4.15 ppm were mainly attributable to the methyl protons ( f, C(Br)−CH3) of the 2-bromoisobutyryl groups, the CH−OH methylidyne protons (e), and the methylene protons (d, CH2− O−CO) from the ring-opening reactions, respectively. When ATRP is carried out from a multifunctional backbone with a high local concentration of initiation sites, the radical−radical 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 l-PGMABr with moderate initiation sites is desired for subsequent comb-shaped copolymers. In this work, the concentration of initiation sites can be controlled by adjusting the feed ratio of the BIBA/epoxy units. With the predetermined molar feed ratio (1/5 or 1/8) of the BIBA/epoxy units, based on the area ratio of peak d and peak a, the l-PGMABr1 (where every 5.2 repeat units contains about one initiation site) and l-PGMABr2 (where every 8.5 repeat units contains about one initiation site) were prepared in this work (Table 1). Such prepared l-PGMA-Br with moderate initiation sites could avoid potential gelation during ATRP. In addition, l-PGMA and l-PGMABr were also characterized by X-ray photoelectron spectroscopy (XPS). Their representative C 1s spectra were shown in Figure 3a,b, respectively. The C 1s core-level spectrum of PGMA could be curve-fitted into three peak components with binding energies (BE’s) at about 284.6, 286.2, and 288.4 eV, attributable to the C−H, C−O, and OC−O species, respectively, in an approximate ratio of 3:3:1, consistent with the chemical structure of PGMA. After the ring-opening reactions, the C 1s spectral line shape of l-PGMABr with the increased compositional ratio of the C−H species was obviously different from that of the original l-PGMA. The corresponding Br 3d core-level spectrum (with BE at about 69 eV) of l-PGMABr was shown in Figure 3b′. The above XPS results also clearly confirmed the successful preparation of l-PGMABr. Synthesis of Comb-Shaped Cationic PGMA Derivatives. Well-defined comb-shaped PGMA (c-PGMA) polymers were subsequently synthesized via ATRP of GMA from l-PGMABr (Figure 1 and Table 1). After 4 h of ATRP, the c-PGMA1 (Mn = 5.5 × 104 g/mol) and c-PGMA2 (Mn = 5.2 × 104 g/mol) were prepared uisng the l-PGMABr1 and l-PGMABr2 macroinitiators, respectively. Based on the assumption of one initiation site out of every 5.2 repeat units for l-PGMABr1 or every 8.5 repeat units for l-PGMABr2, the total numbers of GMA repeat units per side chain were 25 for c-PGMA1 and 38 for c-PGMA2, respectively (Table 1). The typical 1H NMR and C 1s XPS spectra of c-PGMA were shown in Figures 2c and 3c, respectively. As expected, their spectra were very similar to those of l-PGMA (Figure 2a and Figure 3a). In addition, the polydispersity indexes (PDIs) of c-PGMA1 and c-PGMA2 are comparable to that of l-PGMA (Table 1), indicating that ATRP from the starting l-PGMA-Br macroinitiator is well-controlled. It was reported that the introduction of poly(poly(ethylene glycol)ethyl ether methacrylate) (PPEGEEMA) into the cationic gene vectors could probably enhance the gene transfection efficiency.15 In this work, PPEGEEMA was also

repeated at least three times. The data were collected in triplicate and expressed as mean ± standard deviations. Determination of Buffering Capacity. The buffering capacity of cationic polymers in the pH range 2−10 was determined by acid−base titration.7,12 Polymers were dissolved into 20 mL of saline (0.9% NaCl solution) with a 10 mM amino group concentration. The solutions were titrated with a 0.1 N HCl solution with various volume increments. The pH of all the solutions was measured using a TOLEDO 320 pH meter (METTLER).

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RESULTS AND DISCUSSION Preparation of the PGMA Backbones with ATRP Initiation Sites. As shown in Figure 1, the degradable comb-shaped PGMA derivative vectors composed of the PGEA backbones and PGEA-based side chains were prepared by a combination of the ATRP and ring-opening reactions. The starting linear PGMA (l-PGMA) with about 67 GMA units (number average molecular weight (Mn) = 9.5 × 103 g/mol, polydispersity index (PDI) = 1.32, Table 1) was first Table 1. Characterization of the Comb-Shaped PGMA-Based Polymers monomer repeat units per main chain (and side chain) sample a

l-PGMA c-PGMA1c c-PGMA2d c-P(GMAPEGEEMA)1e c-P(GMAPEGEEMA)2g

reaction time (h)

Mn (g/mol)b

2 4 4 4

9.5 5.5 5.2 4.9

× × × ×

10 104 104 104

1.32 1.41 1.52 1.49

67 67 (25)b 67 (38)b 67 (20) f

1.4 f

4

6.4 × 104

1.87

67 (25) f

3.5 f

3

PDIb

GMA

PEGEEMA

b

a

Synthesized using a molar feed ratio [GMA (12 mL)]:[ethyl bromoisobutyrate]:[CuBr]: [1,4,7,10,10,-hexamethyl triethylenetetramine, HMTETA] of 140:1:1:1.5 at 50 °C in 24 mL of GMA/THF (1/1, v/v). bDetermined from GPC results. PDI = weight average molecular weight/number average molecular weight, or Mw/Mn. c Synthesized using a molar feed ratio [GMA (4 mL)]:[CuBr]: [HMTETA] of 100:1:1.5 at 50 °C in 8 mL of GMA/THF (1/1, v/v) containing 0.3 g of l-PGMABr1, where every 5.2 repeat units contains about one initiation site. dSynthesized using a molar feed ratio [GMA (4 mL)]:[CuBr]:[HMTETA] of 100:1:1.5 at 50 °C in 8 mL of GMA/ THF (1/1, v/v) containing 0.3 g of l-PGMABr2, where every 8.5 repeat units contains about one initiation site. eSynthesized using a molar feed ratio [GMA (3.7 mL)+PEGEEMA (0.3 mL)]:[CuBr]: [HMTETA] of 100:1:1.5 at 50 °C in 8 mL of GMA/THF (1/1, v/v) containing 0.3 g of l-PGMABr1. fDetermined from Mn and 1H NMR data. gSynthesized using a molar feed ratio [GMA (3.4 mL)+PEGEEMA (0.6 mL)]:[CuBr]:[HMTETA] of 100:1:1.5 at 50 °C in 8 mL of GMA/THF (1/1, v/v) containing 0.3 g of l-PGMABr1.

synthesized via ATRP. The introduction of the ATRP initiation sites onto l-PGMA was performed via the direct ring-opening reactions of some epoxy groups of l-PGMA with αbromoisobutyric acid (BIBA) to produce the bromoisobutylrylterminated l-PGMA (l-PGMABr). The representative structures of l-PGMA and l-PGMABr were first characterized by 1H NMR spectra as shown in Figure 2a,b, respectively. For l-PGMA, the signals at δ = 3.8 and 4.3 ppm corresponded to the methylene protons adjacent to the oxygen moieties of the ester linkages (a, CH2−O−CO). The peaks 621

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Figure 2. 300 MHz 1H NMR spectra of (a) l-PGMA, (b) l-PGMABr, (c) c-PGMA, (d) c-P(GMA-PEGEEMA), (e) c-PGEA, and (f) c-PGEAPEG.

for c-P(GMA-PEGEEMA)1 and 25 (and 3.5) for c-P(GMAPEGEEMA)2, respectively (Table 1). The ethanolamine (EA)-functionalized PGMA derivatives were prepared by reacting l-PGMA, c-PGMA1, c-PGMA2, c-P(GMA-PEGEEMA)1, or c-P(GMA-PEGEEMA)2 with EA to produce the corresponding l-PGEA, c-PGEA1, c-PGEA2, c-PGEAPEG1, or c-PGEAPEG2 vectors (Figure 1). The typical 1 H NMR spectra of c-PGEA and c-PGEAPEG were shown in Figure 2e and f, respectively. After the ring-opening reactions of PGMA species with EA, the peaks (b,c) associated with the epoxide ring had disappeared completely. The peaks (a, CH2− O−CO) at δ = 3.8 and 4.3 ppm shifted to one new position (d) at δ = 4.05 ppm. The new peak at δ = 2.7 ppm was mainly attributable to the methylene protons (g, NH−CH2). The strong peak at δ = 3.7 ppm was associated with the CH−OH

copolymerized into PGMA side chains to produce the comb-like P(GMA-PEGEEMA) (c-P(GMA-PEGEEMA)) polymers (Figure 1). The c-P(GMA-PEGEEMA)1 (Mn = 4.9 × 104 g/mol) and c-P(GMA-PEGEEMA)1 (Mn = 6.4 × 104 g/mol) polymers were prepared via 4 h of ATRP using different molar feed ratio of GMA and PEGEEMA monomers from the l-PGMABr1 macroinitiators (Table 1). The typical 1H NMR spectrum of c-P(GMA-PEGEEMA) was shown in Figure 2d. In comparison with the spectrum of c-PGMA (Figure 2c), the new peaks of c-P(GMA-PEGEEMA) at δ = 3.7 and δ = 4.15 ppm were mainly attributable to the methylene protons (e, O−CH2 and d, CH2−O−CO) of the P(PEGEEMA) species. Based on the area ratio of peak d and peak a of c-P(GMA-PEGEEMA), the total numbers of GMA (and PEGEEMA) repeat units per side chain were 20 (and 1.4) 622

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Figure 4. Change of molecular weight of c-PGEA1 with degradation time in PBS (pH 7.4) at 37 °C.

various N/P ratios. Figure 5 shows the gel retardation results of various cationic PGMA derivative/DNA complexes with in-

Figure 3. XPS C 1s spectra of (a) l-PGMA, (b) l-PGMABr, (c) cPGMA, (d) c-P(GMA-PEGEEMA), (e) c-PGEA, and (f) c-PGEAPEG, and (b′) Br 3d core-level spectrum of l-PGMABr.

methylidyne and CH2−OH or O−CH2 methylene protons (e). The representative XPS C 1s spectra of c-PGEA and c-PGEAPEG were shown in Figure 3e and f, respectively. They could be curve-fitted into four peak components with BE’s at about 284.6, 285.5, 286.2, and 288.4 eV, attributable to the C−H, C−N, C−O, and OC−O species, respectively. After the ringopening reactions, the C 1s spectral line shapes of c-PGEA and cPGEAPEG were significantly different from their corresponding counterparts (Figure 3c,d). The above results indicated that the oxirane rings of PGMA were opened by EA under the present reaction conditions. Degradation Study. l-PGEA backbone was homopolymer and is nondegradable due its C−C linkages. For the above comb-shaped cationic PGMA derivatives, the low-molecularweight PGEA or PGEAPEG side chains were linked with the PGEA backbones via hydrolyzable ester linkages, which would make the PGEA side chains cleavable from the l-PGEA backbone. Thus, the molecular weight of c-PGEA could be slowly degraded with degradation time as shown in Figure 4, arising from the hydrolysis of ester linkages. Such degradation could benefit the final removal of vectors from the body. Biophysical Characterization of Cationic Vector/pDNA Complexes. A successful gene delivery system requires that plasmid DNA (pDNA) has to be condensed by polycation into nanoparticles small enough to facilitate cellular uptake. The ability to condense DNA is a prerequisite for polymeric gene vectors. In this work, the ability of the PGMA derivative vectors to condense pDNA was confirmed by agarose gel electrophoresis, and particle size and zeta potential measurements. The formation of the polymer/pDNA complexes was first analyzed by their electrophoretic mobility on an agarose gel at

Figure 5. Electrophoretic mobility of plasmid DNA (pDNA) in the complexes of the cationic polymers ((a) l-PGEA, (b) c-PGEA1, (c) c-PGEA2, (d) c-PGEAPEG1, (e) c-PGEAPEG2, and (f) PEI) at various N/P ratios.

creasing nitrogen/phosphate (N/P) ratios in comparison with that of PEI (25 kDa). l-PGEA compacted pDNA completely at the N/P ratios of above 2.5. c-PGEAs exhibited slightly higher condensation capability than l-PGEA and inhibited the migration of pDNA at the N/P ratios of above 2.0, similar to that of the control PEI. The above indicated that the highmolecular-weight c-PGEAs benefited the interaction with pDNA. The c-PGEAPEG1 and c-PGEAPEG2 samples could 623

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completely inhibit the migration of pDNA at the N/P ratios of 2.0 and 3.0, respectively, indicating that excess neutral P(PEGEEMA) species in the c-PGEAPEG vectors would decrease the pDNA condensation capability. The particle size and surface charge of complexes are important factors in modulating their cellular uptake. All the cationic vectors could efficiently compact pDNA into small nanoparticles, as shown in Figure 6a. Generally, the hydrodynamic size

Figure 7. Cell viability assay in (a) HEK293 and (b) C6 cells with various concentrations of l-PGEA, c-PGEA1, c-PGEA2, c-PGEAPEG1, c-PGEAPEG2, and PEI (25 kDa). Cell viability was determined by the MTT assay and expressed as a percentage of the control cell culture.

cytotoxicity. PEI is mainly composed of secondary amine groups. The uniform nonionic hydrophilic hydroxyl groups in PGEAs not only resulted in a lower relative concentration of amino groups, but probably also shielded the harmful charges of the cationic carriers. The above results were consistent with those reported earlier.7,8 It was well-known that the cytotoxicity of polycations increases with the molecular weight.16 However, no obvious difference in cytotoxicity was observed between the l-PGEA and high-molecular-weight c-PGEA vectors, indicating that the introduction of the low-molecular-weight cationic PGEA side chains did not affect substantially the cytotoxicity. The cell viability of the polymer/pDNA complexes as a function of the N/P ratio was also evaluated by using MTT assay (Figure 8). The N/P ratio had a profound impact on the cytotoxicity of complexes. The cell viability of all polymer/ pDNA complexes was observed to decrease with increasing N/P ratios. For the cytotoxicity assay at the given N/P ratio, the cells were incubated in 100 μL of DMEM/well containing 0.5 μg of pDNA. Based on the average mass weights of 325 per phosphate group of DNA and of 203 per nitrogen group of PGEA, the cationic polymer concentrations at the test N/P ratios of 10, 20, and 35 were about 31, 62, and 109 μg/mL, respectively. At higher N/P ratios, the transfection formulation also contains free polymer, besides the compact and positively charged polymer/pDNA complexes. The increased free cationic polymers produced the increasing cytotoxicity. The results of Figure 8 were consistent with those of Figure 7. At the same N/P ratio, no obvious difference was observed among the l-PGEA, c-PGEA, and c-PGEAPEG-mediated complexes, where the control PEI/pDNA complexes exhibited much higher cytotoxicity. In Vitro Transfection Assay. The in vitro gene transfection efficiency of the cationic vector/pDNA complexes was assessed using luciferase as a gene reporter in HEK293 and C6 cell lines in the complete serum media. Figure 9 shows the gene

Figure 6. Particle size (a) and zeta potential (b) of the complexes between the cationic polymers (l-PGEA, c-PGEA1, c-PGEA2, c-PGEAPEG1, c-PGEAPEG2, and PEI) and pDNA at various N/ P ratios.

of the complex decreased with increasing N/P ratio. At the N/P ratio of 2.0, loose large aggregates were formed, due to the lower amount of cationic polymers. At the N/P ratios of above 10, all PGEAs and PGEAPEGs could condense pDNA into nanoparticles of 200 to 300 nm in diameters. The complex sizes of l-PGEA/pDNA were lower than those of c-PGEA/pDNA at most N/P ratios, which was consistent with the relative low condensation ability of l-PGEA/pDNA (Figure 5). Zeta potential is an indicator of surface charges on the polymer/ pDNA nanoparticles. As indicated in Figure 6b, the complex surfaces were positive. A positively charged surface allows electrostatic interaction with anionic cell surfaces and facilitates cellular uptake. Cell Viability Assay. Cytotoxicity is another important factor to be considered in selecting polymeric gene carriers. A successful delivery system should have high transfection efficiency and minimal toxicity. Figure 7 shows the in vitro MTT assay results of cytotoxicity of l-PGEA, c-PGEAs, and c-PGEAPEGs, and PEI in HEK293 and C6 cell lines. All of the cationic polymers exhibit a dose-dependent cytotoxicity effect. l-PGEA and c-PGEAs exhibited substantially lower toxicity than PEI. The slopes of the dose-dependent cytotoxicity curves for PEI were sharply steeper. A high concentration of amino groups is always considered an important factor leading to high 624

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Figure 8. Cell viability of polymer/pDNA complexes at different N/P ratios in (a) HEK293 and (b) C 6 cell line.

Figure 9. In vitro gene transfection efficiency of the cationic polymers (l-PGEA, c-PGEA1, c-PGEA2, c-PGEAPEG1, and c-PGEAPEG2)/ pDNA complexes at various N/P ratios in comparison with that of PEI (25 kDa) at the optimal N/P ratio of 10 in (a) HEK293 and (b) C 6 cells.

transfection efficiency of l-PGEA, c-PGEAs, and c-PGEAPEGs at various N/P ratios in comparison with that of PEI at a N/P ratio of 10, at which branched PEI (25 kDa) usually exhibits the highest transfection efficiency.7,8,12−15 The transfection efficiency generally first increases at lower N/P ratios and then decreases slightly with the increase in N/P ratios. At lower N/P ratios, pDNA cannot be condensed efficiently by the polymers, and the resultant loose polymer/pDNA complex cannot enter the cell easily. In general, after reaching the optimal N/P ratio, the transfection efficiency decreases as the N/P ratios further increase. At higher N/P ratios, the transfection formulation also contains free cationic polymers causing cytotoxicity (Figure 8), besides the compact and positively charged polymer/pDNA complexes. The increasing cytotoxicity may result in a reduction in the transfection efficiency. In both cell lines, the c-PGEA vectors exhibited much higher gene transfection efficiency that mediated by l-PGEA at most N/P ratios. The transfection efficiency of PGEA is dependent on the molecular weights.7 The high-molecular-weight c-PGEA vectors could increase the binding ability and complex stability, probably leading to a much higher transfection efficiency. No obvious difference was observed in the transfection efficiencies mediated by c-PGEA1 (derived from c-PGMA1) and c-PGEA2 (derived from c-PGMA2), probably due to their similar molecular weights, although they possessed different distributions of the ATRP initiation sites (Table 1). As shown in Figure 9, c-PGEAPEG1 (derived from cP(GMA-co-PEGEEMA)1) exhibited significantly higher transfection efficiencies than those mediated by c-PGEA1 (derived from c-PGMA1) at most N/P ratios in both cell lines, although they possessed the similar molecular weights (Table 1). The above results suggested that the introduced suitable amount of P(PEGEEMA) species can further enhance the gene transfection efficiency, which was consistent with the earlier report.15 The good biocompatibility of the P(PEGEEMA), as well as its shielding effect, probably contributed to the enhanced transfection efficiency. However, c-PGEAPEG2 (derived from c-P(GMA-co-PEGEEMA)2)

exhibited significantly lower transfection efficiencies than those mediated by c-PGEA1 at most N/P ratios in both cell lines. c-PGEAPEG2 contained a higher amount of P(PEGEEMA) species. The excess P(PEGEEMA) parts would decrease the pDNA condensation capability (Figure 5), which probably led the lower gene expression levels. Buffering Capacity. After cellular entry via endocytosis, the polymer/plasmid complexes were transported into the lysosome. Efficient escape from endosomes is one of the most important factors to be considered for the design of gene delivery vehicles. This event is associated with the buffering capacity of gene vectors, in which vectors undergo change from extracellular environment to endosomal acid environment.17 By disrupting the endosomal membrane, polycations with high buffering capacity can mediate efficient escape from endosome to cytosol triggered by the acidic environment of endosome. The buffering capacity of the cationic polymers is very useful for the endosomal release of pDNA to the cytoplasm. In this study, acid−base titration under the given 10 mM amino group concentration was performed to evaluate the protonbuffering effects of l-PGEA, c-PGEAs, and c-PGEAPEGs (Figure 10). l-PGEA showed significantly higher buffering capacity than PEI (25 kDa), a well-known transfection agent for its strong proton-sponge effect.18 This is consistent with our earlier report.7 The local environment of the nonionic hydrophilic hydroxyl groups of PGEA as well as its ester species may benefit the improvement the buffering capacity. No obvious difference was observed in the buffering capacities of the l-PGEA, c-PGEA1, c-PGEA2, and c-PGEAPEG1 vectors in the entire test pH range. However, in comparison with c-PGEAPEG1 and PGEA, c-PGEAPEG2 demonstrated a much higher buffering capacity, probably due to its higher amount of P(PEGEEMA) species (Table 1). Unfortunately, as 625

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(2) Niidome, T., and Huang, L. (2002) Gene therapy progress and prospects: nonviral vectors. Gene Ther. 9, 1647−1652. (3) Bisht, H. S., Manickam, D. S., You, Y., and Oupicky, D. (2006) Temperature-controlled properties of DNA complexes with poly(ethylenimine)-graf t-poly(N-isopropylacrylamide). Biomacromolecules 7, 1169−1178. (4) Xu, F. J., and Yang, W. T. (2011) Polymer vectors via controlled/ living radical polymerization for gene delivery. Prog. Polym. Sci. 36, 1099−1131. (5) Wagner, E., Ogris, M., and Zauner, W. (1998) Polylysine-based transfection systems utilizing receptor-mediated delivery. Adv. Drug Delivery Rev. 30, 97−113. (6) Kukowska-Latallo, J. F., Bielinska, A. U., Johnson, J., Spindler, R., Tomalia, D. A., and Baker-Jr, J. R. (1996) Efficient transfer of genetic material into mammalian cells using starburst polyamidoamine dendrimers. Proc. Natl. Acad. Sci. U.S.A. 93, 4897−4902. (7) Xu, F. J., Chai, M. Y., Li, W. B., Ping, Y., Tang, G. P., Yang, W. T., Ma, J., and Liu, F. S. (2010) Well-Defined poly(2-hydroxyl-3-(2hydroxyethlamino)propyl methacylate) vectors with low cytotoxicity and high gene transfection efficiency. Biomacromolecules 11, 1437− 1442. (8) Xu, F. J., Zhu, Y., Chai, M. Y., and Liu, F. S. (2011) Comparison of ethanolamine/ethylenediamine-functionalized poly(glycidyl methacrylate) for efficient gene delivery. Acta Biomater. 7, 3131−3140. (9) Jeong, B., Bae, Y. H., Lee, D. S., and Kim, S. W. (1997) Biodegradable block copolymers as injectable drug-delivery systems. Nature 388, 860−862. (10) Li, J., Li, X., Ni, X., Wang, X., Li, H., and Leong, K. W. (2006) Self-assembled supramolecular hydrogels formed by biodegradable PEO−PHB−PEO triblock copolymers and α-cyclodextrin for controlled drug delivery. Biomaterials 27, 4132−4140. (11) Asayama, S., Maruyama, A., Cho, C. S., and Akaike, T. (2007) Design of comb-type polyamine copolymers for a novel pH-sensitive DNA carrier. Bioconjugate Chem. 8, 833−838. (12) Ping, Y., Liu, C. D., Tang, G. P., Li, J. S., Li, J., Yang, W. T., and Xu, F. J. (2010) Functionalization of chitosan via atom transfer radical polymerization for gene delivery. Adv. Funct. Mater. 20, 3106−3116. (13) Wang, Z. H., Li, W. B., Ma, J., Tang, G. P., Yang, W. T., and Xu, F. J. (2011) Functionalized nonionic dextran backbones by atom transfer radical polymerization for efficient gene delivery. Macromolecules 44, 230−239. (14) Siegwart, D. J., Oh, J. K., and Matyjaszewski, K. (2011) ATRP in the design of functional materials for biomedical applications. Prog. Polym. Sci. 37, 18−37. (15) Xu, F. J., Zhang, Z. X., Ping, Y., Li, J., Kang, E. T., and Neoh, K. G. (2009) Star-shaped cationic polymers by atom transfer radical polymerization from β-cyclodextrin cores for non-viral gene delivery. Biomacromolecules 10, 285−293. (16) Wetering, P. V. D., Moret, E. E., Nieuwenbroek, N. M. E., Steenbergen, M. J. V., and Hennink, W. E. (1999) Structure-activity relationship of water-soluble cationic methacrylate/methacrylmide polymers for nonviral gene delivery. Bioconjugate Chem. 10, 589−597. (17) Boussif, O., Lezoualc’h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297−7301. (18) Wang, D. A., Narang, A. S., Kotb, M., Gaber, A. O., Miller, D. D., Kim, S. W., and Mahato, R. I. (2002) Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery. Biomacromolecules 3, 1197−1207.

Figure 10. Determination of the buffer capacity of l-PGEA, c-PGEA1, c-PGEA2, c-PGEAPEG1, c-PGEAPEG2, PEI, and water by acid−base titration. The cationic polymer solutions with 10 mM amino group concentration were titrated with 0.1 M HCl solution.

shown in Figure 9, c-PGEAPEG2 exhibited much lower gene expression levels, which was probably caused by the decreased pDNA condensation capability of c-PGEAPEG2 (Figure 5). The above results indicated that, in addition to buffering capacity, an efficient gene delivery system still requires other beneficial factors such as good pDNA condensation capability.

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CONCLUSIONS The ATRP initiation sites had been successfully introduced onto PGMA backbones. The degradable high-molecular-weight comb-shaped PGMA derivative (c-PGEA) vectors composed of the low-molecular-weight PGEA backbone and side chains were subsequently prepared by the combination of ATRP and ringopening reactions. Such comb-shaped c-PGEA vectors could possess the good pDNA condensation ability, low cytotoxicity, and good buffering capacity. More importantly, the c-PGEA vectors could improve the gene expression levels and their degradability would benefit the final removal of PGEA from the body. In addition, the PGEA side chains of c-PGEA could also be copolymerized with some PEGEEMA species to further enhance the gene transfection efficiency. Thus, the improvement in degradability and transfection efficiency of PGMA derivative vectors would facilitate the development of effective gene carrier systems.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

X. C. Yang and M. Y. Chai contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (grant numbers 21074007 and 51173014), Research Fund for the Doctoral Program of Higher Education of China (project no. 20090010120007), Program for New Century Excellent Talents in University (NCET-10-0203), SRF for ROCS, SEM and National High Technology Development Program of China (863 Program 2011AA030102).

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REFERENCES

(1) Hunt, K. K., and Vorburger, S. A. (2002) Gene therapy: hurdles and hopes for cancer treatment. Science 297, 415−416. 626

dx.doi.org/10.1021/bc200658r | Bioconjugate Chem. 2012, 23, 618−626