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Macromolecules 2011, 44, 230–239 DOI: 10.1021/ma102419e
Functionalized Nonionic Dextran Backbones by Atom Transfer Radical Polymerization for Efficient Gene Delivery Z. H. Wang,† W. B. Li,‡ J. Ma,‡ G. P. Tang,§ 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 100029 China, ‡State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences, Beijing 100021 China, and §Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou 310028, China Received October 27, 2010; Revised Manuscript Received December 9, 2010
ABSTRACT: It is of crucial importance to modify dextran-based polysaccharides in the design of novel biomedical materials. A simple one-step method, involving the reaction of hydroxyl groups of dextran with R-bromoisobutyric acid in the presence of 1,10 -carbonyldiimidazole, was first developed to produce bromoisobutyryl-terminated dextran as multifunctional initiators for subsequent atom transfer radical polymerization (ATRP). Well-defined comb-shaped copolymers (DPDs) composed of nonionic hydrophilic dextran backbones and cationic poly((2-dimethyl amino)ethyl methacrylate) (or P(DMAEMA)) side chains were subsequently prepared via ATRP for nonviral gene delivery. The P(DMAEMA) side chains of DPDs can be further partially quaternized to produce the quaternary ammonium DPDs (QDPDs). DPD and QDPDs can condense pDNA into complex nanoparticles of 100 to 150 nm in sizes. QDPDs exhibit stronger ability to complex pDNA, due to increased surface cationic charges. DPDs can exhibit much lower cytotoxicity and better gene transfection yield than high-molecular-weight P(DMAEMA) homopolymers and “gold-standard” polyethylenimine (25 kDa) in HEK293 and L929 cell lines. DPDs also exhibit efficient gene delivery ability in different cancer cell lines, especially in MCF7 cells where the DPD-mediated transfection efficiency is almost 3 times higher than that of the popular Lipfectamine 2000 transfection reagent. This study demonstrated that grafting low-molecular-weight polymer chains from natural dextran backbones via ATRP is an effective means to produce novel polysaccharide-based nanobiomaterials.
1. Introduction Successful gene therapy depends on the design of gene delivery vectors with low cytotoxicity and high transfection efficiency.1,2 Over the last few decades, cationic polymers have been receiving considerable attention as the major type of nonviral gene delivery nanovectors, because of their low host immunogenicity, high flexibility, and easy preparation.3,4 Polycations usually contain primary, secondary, tertiary, and/or quaternary amino groups. 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. A great number of polycations, including polyethylenimine (PEI),5 poly(tertiary amine methacrylate),4,6,7 poly(L-lysine),8 polyamidoamine,9 and polysaccharide-based cationic carriers,2,10-12 have emerged as a leading class of transfection reagents. However, most of them suffer from either low gene transfection efficiency or significant toxicity. New cationic polymeric vectors with high gene transfection efficiency and low cytotoxicity will facilitate their applications in gene therapy. Natural polysaccharides are very suitable candidates for the design of novel biomaterials, because they are renewable, nontoxic, biodegradable and excellent biocompatible materials. Dextran is a highly water-soluble branched polysaccharide composed of glucose units mainly linked by R-1,6-linkages. It is of crucial importance to *To whom correspondence should be addressed. E-mail: xufj@mail. buct.edu.cn. pubs.acs.org/Macromolecules
Published on Web 12/22/2010
modify dextran-based polysaccharides in the designing of biomedical materials. A series of dextran-based cationic polymers including diethylaminoethyl-dextran and dextran-spermine were prepared for the efficient delivery of nucleic acids.2,12-14 Atom transfer radical polymerization (ATRP) is a recently developed ‘controlled’ radical polymerization method, which has been used to prepare graft copolymers from some polysaccharides.11,15-18 Recently, we have reported several types of P(DMAEMA)-based polycations via ATRP as efficient gene nanovectors.6,11,15,16 In particular, comb-shaped copolymers composed of chitosan15 or hydroxypropyl cellulose16 backbones and P(DMAEMA) side chains provide a versatile means for designing advanced nonviral gene carriers from nontoxic polysaccharides. Novel comb-shaped gene delivery vectors using dextran as backbones also will be developed if the hydroxyl groups of dextran could be derivatized to serve as effective initiation sites for growing cationic side chains. Dextran can readily dissolve in dimethyl sulfoxide (DMSO). However, the fixation of alkyl halide initiators on dextran via direct esterification of hydroxyl groups is not successful in DMSO, due to the occurrence of Swern secondary reactions.18 It was reported that such esterification immobilization reaction could be carried out in dimethylformamide (DMF) solvent.17 Unfortunately, the results were not promising, due to the poor solubility of dextran in DMF.18 Considering dextran’s poor solubility in most traditional solvents, the direct fixation of uniform ATRP initiators on dextran is very difficult, usually involving a mutilstep process including protection/deprotection steps.8 In the present study, a simple one-step method was developed to produce bromoisobutyryl-terminated dextran (Dextran-Br) r 2010 American Chemical Society
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Scheme 1. Schematic Diagram Illustrating the Preparation Processes of Partially Quaternized P(DMAEMA)-graft-dextran Comb Copolymers via Atom Transfer Radical Polymerization (ATRP) from 2-Bromoisobutyryl-Terminated Dextran
as multifunctional ATRP initiators (Scheme 1). Well-defined comb-shaped copolymers (DPDs) composed of nonionic hydrophilic dextran backbones and cationic poly((2-dimethyl amino)ethyl methacrylate) (or P(DMAEMA)) side chains were subsequently prepared via ATRP for highly efficient nonviral gene delivery. It was reported that the cationic dextran carriers with quaternary ammonium groups can increase the ability to complex plasmid DNA (pDNA).14,16 The P(DMAEMA) side chains can be further partially quaternized to produce the quaternary ammonium DPDs (QDPDs). DPDs and QDPDs can condense pDNA into complex nanoparticles of 100-150 nm in size. QHPDs exhibit stronger ability to complex pDNA. In comparison with the high-molecular-weight P(DMAEMA) homopolymer and “gold-standard” branched PEI (25 kDa), DPDs exhibit much lower cytotoxicity and better or comparable gene transfection efficiency in HEK293 and L929 cell lines. DPDs also can produce good transfection ability in different cancer cell lines, especially in MCF7 cells where the DPD-mediated transfection efficiency is almost 3 times higher than that of the popular Lipfectamine 2000 transfection reagent. In addition, it was found that DPDs also possess better water solubility and higher transfection efficiency than the hydroxypropyl cellulose-graft-P(DMAEAM) comb carriers reported earlier.16 Thus, the approach grafting low-molecular-weight cationic polymer chains from nonionic hydrophilic dextran backbones can produce a new class of nonviral gene delivery nanovectors with low cytotoxicity and high transfection efficiency for future gene therapy.
2. Experimental Section 2.1. Materials. Dextran from Leuconostoc spp. (mol wt 6000 g/mol), branched polyethylenimine (PEI, weight-average molecular weight (Mw) ∼25000 Da, 99%), R-bromoisobutyric acid (BIBA, 98%), 1,10 -carbonyldiimidazole (CDI, 97%), 1,4,7, 10,10-hexamethyl triethylenetetramine (HMTETA, 99%), copper(I) bromide (CuBr, 99%), 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, 98%), penicillin G potassium (>98%), and streptomycin sulfate salt (>98%) were purchased from Sigma Chemical Co., St. Louis, MO. HEK293, L929, MCF7, and 973 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-2bromoisobutyrate as the initiator.11 2.2. Synthesis of Comb-Shaped Cationic Copolymers via ATRP. The starting bromoisobutyryl-terminated dextran (Dextran-Br) was synthesized via the reaction of hydroxyl groups of dextran with BIBA in the presence of CDI catalyst.19-21 In separate flasks, BIBA (4.2 g, 26.3 mmol), CDI (2.0 g, 12.3 mmol), and dextran (2.0 g, about 37.0 mmol -OH) were independently dissolved in 10 mL of anhydrous DMSO. The CDI solution was added dropwise at ambient temperature into the flask containing BIBA solution. The reaction was allowed to proceed at room temperature (about 28 °C) for 4 h to produce the acid imidazolide. After the
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reaction was complete, the acid imidazolide reaction mixture was added to the dextran solution. The reaction was stirred at 60 °C for 6 h to produce the Dextran-Br macroinitiator. The final reaction mixture was precipitated with 300 mL of diethyl ether. The crude polymer was purified by reprecipitation thrice in 200 mL of diethyl ether/ethanol mixture (20/1, v/v). Finally, the Dextran-Br for the subsequent ATRP was dried under reduced pressure. Yield = 85%. 1H NMR (D2O, δ/ppm): 3.4-4.0 (CH-O and CH2-O), 4.9 (O-CH-O), and 1.92 (C(Br)-CH3). Mn = 5.7 103 g/mol and polydispersity index (PDI) = 1.2 from GPC. The Dextran-g-P(DMAEMA) comb-like polymers (DPDs, Scheme 1) were synthesized using a molar feed ratio [DMAEMA (3 mL, 18.7 mmol)]:[CuBr, 0.31 mmol]:[HMTETA, 0.37 mmol] of 60:1:1.2 at room temperature in 8 mL of methanol/water (3/5, v/v) containing Dextran-Br (0.4 g, about 0.31 mmol Br). The reaction was performed in a 25 mL flask equipped with a magnetic stirrer and under the typical conditions for ATRP.11 DMAEMA, Dextran-Br, and HMTETA were introduced into the flask containing 8 mL of methanol/water (3/5, v/v). After Dextran-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 flask was then sealed with a rubber stopper under an argon atmosphere. The polymerization was allowed to proceed under continuous stirring at room temperature from 5 to 12 h. The reaction was stopped by diluting with tetrahydrofuran (THF). The catalyst complex was removed by passing the blue dilute polymer solution through a short aluminum oxide column. After removal of THF in a rotary evaporator, DPDs were precipitated in 100 mL of n-hexane. The crude polymer was purified by reprecipitation twice in hexane to remove the reactant residues, prior to being dried under reduced pressure. About 0.5 g of DPD1 from 5 h (Mn = 1.49 104 g/mol and PDI = 1.4) and 1.2 g of DPD2 from 12 h (Mn = 3.02 104 g/mol and PDI = 1.6) were obtained. 1H NMR (D2O, δ/ppm): 0.89 (C-CH3), 1.83 (C-CH2), 2.28 (N-CH3), 2.67 (N-CH2), and 4.12 (CH2-O-CdO). The Dextran-g-QP(DMAEMA) (QDPD) with partially quaternized P(DMAEMA) side chains was prepared at 50 °C for 24 h in 6 mL of THF containing 0.25 g (about 1.23 mmol N) of DPD2 and 0.5 (3.5 mmol) or 1.0 (7.0 mol) mL of 1-bromohexane. The QHPD was precipitated in 50 mL of n-hexane and dried under reduced pressure. Yield = 94%. Quaternization extent of P(DMAEMA) side chains determined from XPS: 21% (for DPD2-1 from 0.5 mL) and 35% (for DPD2-1 from 1.0 mL). 1 H NMR (D2O, δ/ppm): 0.89 (C-CH3), 1.83 (C-CH2), 2.28 (N-CH3), 2.67 (N-CH2) 3.17 (Nþ-CH3), 3.42 (Nþ-CH2) and 4.12 (CH2-O-CdO). 2.3. 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 YL9100 GPC system equipped with a UV/vis detector and Waters Ultrahydrogel 250 (packed with crosslinked hydroxylated polymethacrylate-based gels of 250 A˚ pore sizes) and Ultrahydrogel Linear (packed with crosslinked hydroxylated polymethacrylate-based gels of different pore sizes) 7.8 300 mm columns. The Ultrahydrogel 250 and Ultrahydrogel Linear columns allowed the separation of polymers over the molecular weight ranges of 103 to 8 104 and 103 to 7 106, respectively. A pH 3.5 acetic buffer solution was used as the eluent at a low flow rate of 1.0 mL/min at 25 °C. Monodispersed dextran standards were used to obtain a calibration curve. The XPS measurements were performed on a Kratos AXIS HSi spectrometer equipped with a monochromatized AlKR X-ray source (1486.6 eV photons), using the same procedures as those described earlier.11 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 D2O as the solvent. Chemical shifts were referred to the solvent peak, δ = 4.70 ppm.
Wang et al. 2.4. Characterization of Polymer/pDNA Complexes. The plasmid (encoding Renilla luciferase) mainly 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 agrose gel electrophoresis.6 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 DPD and QDPDs to phosphate (P) in DNA (or as N/P ratios). The average mass weight of 325 per phosphate group of DNA was assumed.5 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.6 The polymer/pDNA complexes at various N/P ratios were investigated. Gel electrophoresis was carried out in TAE running buffer (40 mM Tris-acetate, 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 ζ potentials of the polymer/pDNA complexes were measured using a Zetasizer Nano ZS (Malvern Instruments, Southborough, MA) and procedures similar to those described earlier.6 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.6 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 visualizing of the complex in the TEM mode at high spatial resolution. 2.5. Cell Viability and Transfection Assay. The cytotoxicity of the comb-like polymers was evaluated using the MTT assay in HEK293 and L929 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, 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 first using plasmid pRLCMV as the reporter gene in HEK293 and L929 cell lines in the presence of serum. In brief, the cells were seeded in 24-well plates
Article at a density of 5 104 cells in the 500 μL of medium/well and incubated for 24 h. The comb-shaped 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 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.6 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). DPD-mediated transfection was also assessed using the enhanced green fluorescent protein (EGFP) pDNA (BD Biosciences, San Jose, CA) as the reporter gene in human breast cancer MCF7 and human lung adenocarcinoma 973 cell lines, where the commercial Lipfectamine 2000 transfaction reagent was used as the control. The cancer cells were seeded in 24-well plates at a density of 1 105 cells in the 500 μL of RPMI 1640 medium(supplemented with 10% FBS)/well and incubated for 24 h before transfection. The transfection procedures are based on those provided by Lipofectamine 2000 protocol. Lipofectamine 2000 (or DPD at a specific N/P ratio)/pDNA complexes (100 μL/well containing 0.8 μg of pDNA) were formed by mixing equal volumes of polymer and pDNA solutions, followed by incubation for 20 min at room temperature. At the time of transfection, the complexes were added into the medium and incubated with the cells for 6 h under standard incubator conditions. Then, the medium was replaced with the fresh normal medium. The cells were further incubated until a total transfection time of 48 h under the same conditions. To visualize the expression of EGFP in the cancer cells, the evaluation of the EGFP expression was performed using a Leica DMIL Fluorescence Microscope. The percentage of the EGFP positive cells was determined using flow cytometry (FCM). FCM analysis was conducted using Epics Elite ESP (Beckman Coulter, USA). EGFP was excited at 488 nm and measured at 525 nm. EXPO 32 ADC software was used to analyze FCM data.
3. Results and Discussion 3.1. Synthesis and Characterization of Comb-Shaped Cationic Polymers Composed of Dextran Backbones and P(DMAEMA) Side Chains. In order to prepare comb-shaped copolymers using dextran as a backbone via ATRP, it is essential to introduce alkyl halide into dextran. In this work, the bromoisobutyryl-terminated dextran (Dextran-Br) multifunctional initiators were prepared via the direct reaction of hydroxyl groups of dextran (mol wt 6000 g/mol) with R-bromoisobutyric acid in the presence of 1,10 -carbonyldiimidazole (CDI) catalyst in DMSO solvent (Scheme 1). The carboxylic acids can be activated by CDI to produce the reactive acid imidazolide, which can be reacted with hydroxyl groups of polysaccharides without purification.19-21 The C 1s core-level XPS spectra of the pristine dextran and Dextran-Br are shown in Figure 1, parts a and b, respectively. The C 1s core-level spectrum of dextran can be curve-fitted by three peak components with binding energies (BE’s) at about 284.6, 286.2, and 287.6 eV, attributable to the C-H/C-C,
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C-O, and O-C-O species, respectively.6 The C 1s core-level spectrum of Dextran-Br can be curve-fitted by four peak components with BE’s 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 OdC-O species, respectively.6 The new O-CdO peak component is associated with the bromide-capped ester groups of Dextran-Br. The corresponding Br 3d core-level spectrum is shown in Figure 1b0 . From the [Br]/[C] ratio of 0.019 (determined from the sensitivity factor-corrected Br 3d and C 1s core-level spectral area ratio), it was calculated that about 8 glucose units of Dextran-Br possess one ATRP initiation site. The chemical structure of Dextran-Br is also characterized by 1 H NMR spectroscopy in D2O (Figure 2a). The broad chemical shifts in the wide region of 3.4-4.0 ppm are mainly associated with the inner methylidyne and methylene protons (A, CH-O and CH2-O) on glucose units. The chemical shift associated with the unique methylidyne proton (O-CH-O) of glucose units is at about 4.9 ppm. The chemical shift at δ = 1.92 ppm is associated with the methyl protons (b, C(Br)-CH3) of the 2-bromoisobutyryl groups.6 The area ratio of peak b and peak A indicated that very 8.7 glucose units of Dextran-Br possess one initiation site, fairly consistent with that determined by XPS. 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 this work, the prepared Dextran-Br with such moderate initiation sites can avoid potential gelation during ATRP. In addition, the concentration of initiation sites can be controlled by adjusting the BIBA/dextran feed ratio. Well-defined comb-shaped copolymers (DPDs) of dextran and P(DMAEMA), or Dextran-g-P(DMAEMA)s, were subsequently synthesized from Dextran-Br (Scheme 1, Table 1). The DPDs with different lengths of P(DMAEMA) side chains can be synthesized by varying the ATRP time. Table 1 summarizes the GPC results of DPD1 (from 5 h of ATRP) and DPD2 (from 12 h of ATRP). With the increase in reaction time from 5 to 12 h, the Mn of DPD from GPC using linear dextran standards increases from 1.49 104 to 3.42 104 g/mol, and the total number of DMAEMA repeat units per side chain increases accordingly from 12 to 35, based on the assumption of one initiation site out of every 8 glucose units of Dextran-Br (Table 1). This value is lower than those determined by XPS and 1H NMR (Table 1), probably due to the differences in hydrodynamic volumes between comb-like DPDs and dextran standards. The C 1s core-level spectra of DPDs (Figure 1, parts c and d) can be curve-fitted into five peak components with BE’s 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, O-C-O, and OdC-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 Dextran-Br, the increase (and decrease) in intensities of the C-N (and C-O) species is consistent with the presence of P(DMAEMA). The corresponding N 1s core-level spectra of DPDs are shown in Figure 1, parts c0 and d0 . From the [N]/[C] ratios determined by XPS, the number of DMAEMA repeat units per side chain can be calculated (Table 1). In order to increase the surface cationic charges, the P(DMAEMA) side chains of DPD2 was partially quaternized to produce the Dextran-g-QP(DMAEMA)2 (QDPD2) with quaternary ammonium groups. In this work, the QDPD2-1 and QDPD2-2 with different quaternization contents were prepared from DPD2 and 1-bromohexane (Scheme 1 and Table 1). After quaternization, the C 1s core-level spectra of QDPDs (Figure 1, parts e and 1f) can be curve-fitted into five peak components with BE’s at about
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Figure 1. XPS C 1s core-level spectra of (a) Dextran, (b) Dextran-Br, (c) DPD1, (d) DPD2, (e) QDPD2-1, and (f) QDPD2-2, Br 3d core-level spectrum of (b0 ) Dextran-Br, and N 1s core-level spectra of (c0 ) DPD1, (d0 ) DPD2, (e0 ) QDPD2-1, and (f0 ) QDPD2-2.
284.6, 285.5, 286.2, 287.6, and 288.4 eV, attributable to the C-H/C-C, C-N, C-O/C-Nþ, O-C-O, and OdC-O species, respectively. In comparison with the C 1s core-level spectrum (Figure 1d) of the starting DPD2, the decrease (and increase) in intensities of the C-N (and C-O/C-Nþ) species of QDPDs 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 1d0 ) of DPD2, the N 1s core-level spectra (Figures 1(e0 ) and 1(f0 )) of QDPDs can be curve-fitted into two peak components with BE’s at about 399 and 402 eV, attributable to the amino (C-N) and positively charged nitrogen (C-Nþ) species, respectively. The quaternization extent of P(DMAEMA) side chains can be expressed as the [Nþ]/([N] þ [Nþ]) ratio (determined from the corresponding Nþ peak component and total nitrogen spectral area ratio). The average [Nþ]/([N]þ[Nþ]) ratios for QDPD2-1 and QDPD2-2 are about 0.21 and 0.35, respectively (Table 1), indicating that about one out of every 4.8 (for QDPD2-1) or 2.9 (for QDPD2-2) DMAEMA repeat units had been quaternized. Parts b and c of Figure 2 show the respective 1H NMR spectra of DPD2 and QDPDs in D2O. The typical chemical shifts at about 0.89, 1.83, 2.28, and 2.67 ppm are mainly
attributable to the (c0 ) C-CH3 methyl, (d0 ) C-CH2 methylene, (c) N-CH3 methyl, and (d) N-CH2 methylene protons of the P(DMAEMA) side chains, respectively. The chemical shift (at δ = 4.12 ppm) associated with the methylene protons adjacent to the oxygen moieties of the ester linkages (a0 , CH2-O-CdO) overlapped completely with glucose peaks of dextran. The signal intensities associated with the dextran backbone (A) have decreased substantially. From the area ratio of peaks d, d0 , c and peaks A, a0 , the number of DMAEMA repeat units per side chain can be estimated (Table 1). After quaternization, the new signals at about 3.17 and 3.42 ppm are attributable to the (c000 ) Nþ-CH3 methyl and (d000 ) Nþ-CH2 methylene protons of the quaternized P(DMAEMA) side chains, respectively.22 The chemical shift at about 1.36 ppm corresponds to the methylene protons of hexane (d00 , CH2-C). For QDPD2-1 and QDPD2-2, the signal intensities associated with the quaternized P(DMAEMA) units (c000 , d00 , d000 ) increased, which is consistent with their corresponding increase in quanternization extent. Because of 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. 3.2. Polymer/pDNA Complexes. The DNA condensation capability is a prerequisite for polymeric gene vectors. In this
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Figure 2. 300 MHz 1H NMR spectra of the (a) Dextran-Br, (b) DPD2, and (c) QDPDs polymers in D2O. Table 1. Characterization of the Comb-Shaped Cationic Polymers sample
reaction time (h)
Mn (g/mol)c
polydispersity index (PDI)c
[N]/[C]d
[Nþ]/ ([N] þ [Nþ])d
DMAEMA repeat units per side chain
5 1.49 104 1.4 0.092 13c or 17d or 20e Dextran-g-P(DMAEMA)1 or DPD1a Dextran-g-P(DMAEMA)2 or DPD2a 12 3.02 104 1.6 0.108 35c or 40d or 42e Dextran-g-QP(DMAEMA)2-1 or 24 0.21 QDPD2-1b Dextran-g-QP(DMAEMA)2-2 or 24 0.35 QDPD2-2b a Synthesized using a molar feed ratio [DMAEMA (3 mL)]:[CuBr]:[HMTETA] of 60:1:1:1.2 at room temperature in 8 mL of methanol/water (3/5, v/v) containing 0.4 g of Dextran-Br (Mn = 5.7 103 g/mol and PDI = 1.2). About 8 glucose units of Dextran-Br possess one initiation site. b Synthesized at 50 °C in 6 mL of THF containing 0.25 g of DPD2 and 0.5 mL (for QDPD2-1) or 1.0 mL (for QDPD2-2) 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 the 1H NMR spectroscopy data.
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 ζ potential measurements, as well as STEM imaging. The formation of the copolymer/pDNA complexes was first analyzed by their electrophoretic mobility on an agarose gel
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Figure 4. Particle size (a) and ζ potential (b) of the complexes between the cationic polymers (DPD1, DPD2, QDPD2-1, QDPD2-2, P(DMAMEA), and PEI) and pDNA at various N/P ratios.
Figure 3. Electrophoretic mobility of plasmid DNA (pDNA) in the complexes of the cationic polymers ((a) DPD1, (b) DPD2, (c) QDPD2-1, (d) QDPD2-2, (e) P(DMAEMA), and (f) PEI at various N/P ratios.
at various N/P ratios. Figure 3 shows the gel retardation results of the cationic comb copolymer (DPD1, DPD2, QDPD2-1, and QDPD2-2)/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. All the DPDs and QDPDs can compact pDNA completely within the N/P ratio of 1.5. DPD2, QDPD2-1, and QDPD2-2 show increasing capability to condense pDNA, indicating that the increased cationic charges from quaternization could enhance the condensation capability. The control P(DMAEMA) and PEI could inhibit the migration of pDNA at N/P ratios of 1.25 and 2.0, respectively. QDPD2-2 exhibits similar condensation capability to that of the P(DMAEMA) homopolymer. The (a) particle size and (b) ζ potential of the cationic polymer (DPD1, DPD2, QDPD2-1, and QDPD2-2)/pDNA complexes are compared to those of the P(DMAEMA)/pDNA and PEI/pDNA complexes at various N/P ratios in Figure 4. Particle size is a crucial factor for determining the rate of cellular uptake of polymer/DNA complexes. All the cationic comb copolymers can efficiently compact pDNA into small particles. 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, similar to the control P(DMAEMA) and PEI, can condense pDNA into nanoparticles of 100 to 150 nm in diameters. The polymer/pDNA complex within this size range can readily undergo endocytosis.23 ζ potential, an indicator of
surface charges on the polymer/pDNA nanoparticles, is another important factor affecting cellular uptake of the complexes. A positively charged surface allows electrostatic interaction with negatively charged cell surfaces and facilitates cellular uptake. For the pDNA complexes of DPD1, DPD2, QDPD2-1, QDPD2-2, 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 ζ potentials of the pDNA complexes of all the cationic polymers are strongly positive. Because of the increasing quaternization of the P(DMAEMA) side chains, the pDNA complexes of DPD2, QDPD2-1, and QDPD2-2 show increasing surface cationic charges, indicating that they will produce increasing affinity for anionic cell surfaces. In addition, the excess cationic polymers probably do not exert any significant effects on the particle size and ζ potential of the complexes (Figure 4). Figure 5 shows the representative STEM images of the (a) DPD2/pDNA (at a N/P ratio of 1), (b) DPD2/pDNA (at a N/P ratio of 15), and (c) QDPD2-1/pDNA (at a N/P ratio of 15) complexes. The images clearly reveal the morphological difference between the polymer/pDNA complexes at different N/P ratios. For the DPD/pDNA complex at a ratio of 1, loose large aggregates, supercoiled plasmid pDNA, and compact nanoparticles (
