Dendronized Chitosan Derivative as a Biocompatible Gene Delivery

Jan 26, 2011 - To improve the transfection efficiency of chitosan as a nonviral gene delivery vector, a dendronized chitosan derivative was prepared b...
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Dendronized Chitosan Derivative as a Biocompatible Gene Delivery Carrier Junjie Deng,†,‡ Yanfang Zhou,†,§,|| Bo Xu,§ Kaijin Mai,‡ Yubin Deng,*,§ and Li-Ming Zhang*,‡ ‡

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DSAPM Lab and PCFM Lab, Institute of Polymer Science, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China § Department of Pathophysiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China Department of Pathophysiology, Guangdong Medical College, Dongguan 523808, China ABSTRACT: To improve the transfection efficiency of chitosan as a nonviral gene delivery vector, a dendronized chitosan derivative was prepared by a copper-catalyzed azide alkyne cyclization reaction of propargyl focal point poly(amidoamine) dendron with 6-azido-6-deoxy-chitosan. Its structure was characterized by 1H NMR and FTIR analyses and its buffering capacity was evaluated by acid-base titration. In particular, its complexation with plasmid DNA was investigated by agarose gel electrophoresis, zeta potential, and particle size analyses as well as transmission electron microscopy observation. Compared to unmodified chitosan, such a chitosan derivative has better water solubility and buffering capacity. Compared to commonly used polyethyleneimine (PEI, 25 kDa), it could exhibit enhanced transfection efficiency in some cases and lower cell toxicity, as confirmed by in vitro transfection and cytotoxicity tests in human kidney 293T and human nasopharyngeal carcinoma CNE2 cell lines. In addition, the effect of serum on its transfection efficiency was also studied.

’ INTRODUCTION Chitosan, a naturally occurring cationic mucopolysaccharide comprised of β-(1-4) linked 2-amino-2-deoxy-β-D-glucose and the N-acetylated analogue, has attracted increasing attention as a nonviral gene delivery vector.1-4 Different from commonly used polyethyleneimine (PEI) with high toxicity,5 such a gene carrier is generally biocompatible, biodegradable, and nontoxic. However, native chitosan usually suffers from poor water solubility and weak buffering capacity at physiological pH, which lead to very low transfection efficiency. To overcome these limitations, some chitosan derivatives with improved physiochemical properties have been synthesized and investigated for gene delivery, including glycosylated chitosan,6-9 pegylated chitosan,10 trimethylated chitosan,11,12 hydrophobized chitosan,13 urocanic acid-modified chitosan,14 and oligoamine-grafted chitosan.15 Although they have relatively higher gene transfection efficiency when compared to unmodified chitosan, the efficiency of gene delivery by these modified chitosan derivatives is still relatively low when compared to commonly used PEI or cationic lipids. Therefore, the development for new chitosan-based gene carriers with high transfection efficiency has always been an endeavor in biomaterials. Dendrimers are monodisperse, synthetic macromolecules with well-defined architectures, precise molecular weights, and multivalent functionalization sites.16,17 They provide tailorable scaffolds for the development of nucleic acid carriers.18 Among them, cationic poly(amidoamine) (PAMAM) dendrimers have been widely used for the delivery of plasmid DNA or siRNA.19-24 Therefore, the r 2011 American Chemical Society

chemical combination of PAMAM dendrimers with chitosan may provide chitosan with more biofunctional characteristics to improve its properties for gene delivery applications. Although Sashiwas et al.25,26 prepared the PAMAM or sialic acid dendrimers having a tetraethylene glycol spacer and attached these dendrimers to chitosan by reductive N-alkylation, they did not explore the gene delivery applications of the resultant dendronized chitosan derivatives. In this work, we use a facile click conjugation strategy to prepare dendronized chitosan derivatives for improved gene delivery. For this purpose, propargyl focal point PAMAM dendron and 6-azido-6deoxy-chitosan were synthesized and then used for the coppercatalyzed azide alkyne cyclization reaction. For resultant dendronized chitosan derivatives, their complexation with plasmid DNA, in vitro cytotoxicity, and transfection efficiency, as well as serum resistance ability, were investigated.

’ EXPERIMENTAL SECTION Materials. Chitosan (Mw = 10 kDa, deacetylation degree = 85.3%) was purchased from Haidebei Marine Bioengineering Co. Ltd. (China) and used as received. Phthalic anhydride, hydrazine monohydrate, N-bromosuccinimide (NBS), triphenylphosphine (TPP), copper sulfate, 1-methy-2pyrrolidinone (NMP) were obtained from Sinopharm Group Chemical Reagent Co., Ltd. (China). Sodium azide (NaN3, 99%) and sodium ascorbate Received: November 1, 2010 Revised: December 19, 2010 Published: January 26, 2011 642

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Scheme 1. Preparation Route to Dendronized Chitosan Derivatives [Cs-g-PAMAM (G = 2, 3)]a

(99%) were purchased from Alfa Aesar. The Dulbecco’s modified Eagle medium (DMEM), trypsin-ethylenediaminetetraaceticacid (TrypsinEDTA), and fetal bovine serum (FBS) were purchased from Gibco-BRL (Canada). Polyethylenimine (PEI, 25 kDa), ethidium bromide (EB), and 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (U.S.A.). Deoxyribonuclease I (DNaseI) was purchased from Fermentas Life Sciences. Plasmid EGFP-N1 (4.7 kb) encoding enhanced green fluorescent protein was purchased from Clontech (Palo Alto, CA). The plasmid DNA (pDNA) was isolated with PureYield (TM) Plasmid Maxiprep System Kits (U.S.A.) according to the manufacturer’s instructions. Its purity was confirmed by spectrophotometry (A260/ A280) and its concentration was determined from the absorbance at 260 nm. The pDNA was stored at -20 °C until the transfection experiments. All other reagents were of analytical grade and were used as received. Preparation of Dendronized Chitosan Derivatives. Dendronized chitosan derivatives [Cs-g-PAMAM (G = 2, 3)] were prepared by copper-catalyzed azide alkyne cyclization reactions of propargyl focal point PAMAM dendrons of second and third generations with 6-azido6-deoxy-chitosan (6-N3-Cs), as illustrated in Scheme 1. First, propargyl focal point PAMAM dendrons (Scheme 1A) were synthesized using a protocol similar to that described by Lee et al.27,28 In brief, a solution of propargylamine (1.5 g, 27.3 mmol) in methanol (5 mL) was added dropwise to the solution of methyl acrylate (9.4 g, 109.2 mmol) in methanol (10 mL). The reaction mixture was stirred for 24 h at room temperature under a nitrogen atmosphere. The reaction solution was evaporated, and then the residue was dried in vacuo at 35 °C to give the methyl esterterminated dendron (G = 0.5). The solution of PAMAM dendron (G = 0.5, 5.36 g, 23.6 mmol) in methanol (20 mL) was added dropwise to the solution of ethylenediamine (21.2 g, 0.35 mol) in methanol (30 mL). The reaction mixture was stirred for 48 h at room temperature under a nitrogen atmosphere. The reaction solution was evaporated, and then the residue was dried in vacuum at 35 °C to give the amino-terminated PAMAM dendron (G = 1). The amino-terminated PAMAM dendrons (G = 2, 3) were synthesized from dendron (G = 1) using the same method as successive Michael addition of primary amines with methyl acrylate and amidation of methyl ester groups with a large molar excess of ethylenediamine. Second, 6-N3-Cs (Scheme 1B) was prepared according to the similar method reported by Satoh et al.29 Chitosan (1) was reacted with phthalic anhydride to obtain N-phthaloyl-chitosan (2). 6-Azido-6-deoxy-Nphthaloyl-chitosan (4) was prepared by converting 2 into 6-bromide6-deoxy-N-phthaloyl-chitosan (3) in the presence of NBS and TPP in NMP. After that, the bromide derivative (3) was substituted with azido groups by reacting with sodium azide to obtaine the product (4). To remove the phthalimide group, 6-azido-6-deoxy-N-phthaloyl-chitosan (4) was added to aqueous hydrazine monohydrate (v/v, 1:1) and stirred at 100 °C for 10 h. After the solvent evaporation, the residue was precipitated with EtOH and then dried under a vacuum, resulting in 6-N3-Cs (5) as a brown powder with a yield of 75%. At last, Cs-g-PAMAM (G = 2, 3) samples (6) were prepared according to Scheme 1C. A mixed solution of the PAMAM dendron (G = 2 or 3; 0.72 mmol), 6-N3-Cs (0.24 mmol) and the catalyst (CuSO4 3 5H2O/sodium ascorbate, 0.12 mmol/0.24 mmol) in 12.0 mL of DMF/water (5:1, v/v) was heated at 50 °C for 24 h in a closed vial. After the reaction, the mixture was precipitated with a large excess of EtOH and then purified by dialysis in water for 2 days with a dialysis tube (MWCO, 3.5 kDa, Sigma). After the dialysis and lyophilization, the dendronized chitosan derivative was obtained as a brown powder with a yield of 92%. 1H NMR (D2O, ppm): δ = 3.30-3.90 (multiplet, D-glucosamine unit, H-3, H-4, H-5, H-6, H-60 ), 2.20-2.30 (multiplet, PAMAM dendron), 2.45 (-CH2CONH-), 3.31 (-CONHCH2-), 2.6-3.0 (protons next to amines), 7.95 (H of triazole). Based on elemental analysis, the degree of substitution (DS) of PAMAM dendron on the chitosan, which is defined as the number of PAMAM dendrons per 100 anhydroglucose units of

a

Reaction conditions: (a) phthalic anhydride, DMF, 120 °C, 8 h; (b) Nbromosuccinimide, triphenylphosphine, NMP, 80 °C, 2 h; (c) sodium azide, NMP, 80 °C, 4 h; (d) hydrazine monohydrate, water, 100 °C, 10 h; (e) propargyl focal point PAMAM dendrons (G = 2, 3), CuSO4 3 5H2O/ sodium ascorbate, DMF/water, 40 °C, 24 h. 643

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chitosan,30 was determined to be 23.3 for Cs-g-PAMAM (G = 2) and 25.1 for Cs-g-PAMAM (G = 3), respectively. Structural Characterization. 1H NMR spectra were measured in D2O by using a Bruker DPX-300 NMR spectrometer (300 MHz) at room temperature. FTIR spectra were recorded on a Perkin-Elmer Paragon 1000 spectrometer at frequencies ranging from 500 to 4000 cm-1. Each sample was thoroughly mixed with KBr and pressed into a pellet form. Buffering Capacity. The buffering capacity of dendronized chitosan derivatives [Cs-g-PAMAM (G = 2, 3)] were determined by acid-base titration assay, as described by Benns et al.31 Briefly, 0.2 mg/mL of each sample solution was prepared in 30 mL of 150 mM NaCl solution. The sample solution was first titrated by 0.1 M NaOH to a pH of 10, and then 0.1 M HCl solution with particular volume was added to the solution to obtained mixtures with different pH values, which were determined using a microprocessor pH meter.

Preparation of Cs-g-PAMAM (G = 2, 3)/pDNA Complexes. Cs-g-PAMAM (G = 2, 3) samples were dissolved in phosphate buffered saline solution (PBS, pH 7.4) with a concentration of 1.0 mg/mL and then filtered using a 0.22 μm filter. pDNA solution (0.24 mg/mL) was added to aqueous Cs-g-PAMAM system and mixed by a vortex mixer at a charge ratio (N/P), which was calculated as a ratio of the number of primary amines in the polymer to the number of anionic phosphate groups in the pDNA. The complex solutions were kept for 30 min at 37 °C. Agarose Gel Electrophoresis. To assess the condensation ability of Cs-g-PAMAM (G = 2, 3) to pDNA, the electrophoresis tests were performed. The Cs-g-PAMAM (G = 2, 3)/pDNA complexes with different N/P ratios ranging from 0.2 to 30 and incubated for 30 min at room temperature to ensure complex formation. The final volume with the 6 agarose gel loading dye mixture was 20 μL. The complexes were loaded onto 0.8% agarose gels with ethidium bromide (0.1 μg/mL) and run with Tris-acetate (TAE) running buffer for 1 h at 90 V. Protection against DNaseI Degradation. A total of 2 μL of DNase I (10 units) was added to naked pDNA or Cs-g-PAMAM/pDNA complexes with various N/P ratios (5/1, 10/1, and 20/1) and incubated at 37 °C under shaking at 100 rpm for 30 min. 4 μL of EDTA (250 mM) and 4 μL of 10% (w/v) sodium dodecyl sulfate (SDS) were added. The resultant mixed solution was incubated at room temperature for 1 h to dissociate the complexes.32 The integrity of DNA was examined using agarose gel electrophoresis. Measurements of Particle Size and Zeta Potentials. The mean particle size and zeta potential were measured in PBS (pH 7.4) using ZetaPALS (Brookhaven Instruments Corporation) at 25 °C. According to the N/P ratios, various concentrations of sample solutions were added to 1 μg pDNA solution in equivalent volume to form Cs-g-PAMAM/pDNA complexes. Then aqueous system containing the complexes was diluted for the measurements of mean particle size and zeta potential. The presented data are the means of three independent measurements. Transmission Electron Microscopy Observation. The morphology of Cs-g-PAMAM (G = 3)/pDNA complexes at the N/P ratios of 5, 10, and 20 were observed using transmission electron microscopy (TEM). Briefly, one drop of Cs-g-PAMAM (G = 3)/pDNA complex solution was placed on a copper grid and incubated for 60 s. Excess liquid was removed by blotting with filter paper. Samples were negatively stained with 2% (w/v) phosphotungstic acid solution for 60s and again blotted dry with filter paper. TEM images were recorded with a transmission electron microscopy (JEM2010) operated at 80 kV. Cell Culture. Two cell lines including 293T from human kidney and CNE2 from human nasopharyngeal carcinoma were cultured in DMEM supplemented with 10% FBS and 1% antibiotics (100 U/mL penicillin G and 0.1 mg/mL streptomycin). All cells were incubated at 37 °C in humidified 5% CO2 atmosphere. Cytotoxicity Assay. Cells were seeded in the 96-well plate at a density of 1  104 cells/well and cultured for 24 h. Growth media were replaced by fresh, serum-free media, which contained different concentrations

Figure 1. FTIR spectra of the PAMAM dendron (G = 3) (a), 6-N3-Cs (b), and Cs-g-PAMAM (G = 3) (c).

Figure 2. 1H NMR spectra of propargyl focal point PAMAM dendron (G = 3; in D2O), 6-N3-Cs (in D2O and 1.0% CF3COOD), and Cs-gPAMAM (G = 3; in D2O). of Cs-g-PAMAM (G = 2, 3). PEI (25 kDa) and chitosan were used as the controls. After an additional incubation for 24 h, the media were changed with DMEM containing 20 μL of MTT solution (0.5 mg/mL). After further incubation for 4 h, the reaction product was solubilized with 150 μL of DMSO. The absorbance value was measured at 570 nm, using plate reader (Bio-Rad, U.S.A.). Cell viability (%) was calculated according to the following equation: cell viability (%) = [A570 (sample)/A570 (control)]  100, where A570 (sample) was obtained in the presence of polymers and A570 (control) was obtained in the absence of polymers. In Vitro Transfection. 293T and CNE2 cells were plated in 24well plates at 1  104 cells/well and were incubated for 12 h. At the time of transfection, the medium in each well was replaced with serum-free media or 10% fetal bovine serum media. The complexes of various polymer samples/ pDNA at different N/P ratios (containing 2 μg pDNA in each N/P ratio) were incubated with the cells for 48 h at 37 °C. After the incubation, the cells were observed with a Olympus IX71 fluorescence microscope (Melville, NY, U.S.A.). PEI (25 kDa) and chitosan were used as the controls. The transfected cells were washed once with PBS and detached with 0.25% trypsin. Transfection efficiency was evaluated by scoring the percentage of cells expressing GFP, using a FACS Aria flow cytometer (Germany). Statistics. All data are summarized as mean ( standard deviation (SD). Statistical analysis of the results was performed by Student’s t test for unpaired samples.

’ RESULTS AND DISCUSSION Characterization of Dendronized Chitosan Derivative. Figure 1 gives FTIR spectra of the PAMAM dendron (G = 3), 644

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Figure 3. Acid-base titration profiles of chitosan, the PAMAM dendrons (G = 2, 3), and Cs-g-PAMAM (G = 2, 3) in aqueous 150 mM NaCl solutions (sample concentration, 0.2 mg/mL).

Figure 5. The particle size (a) and zeta potential (b) of Cs-g-PAMAM (G = 2, 3)/pDNA complexes and chitosan/pDNA complexes at various N/P ratios.

(νN-H), 2934, 2841 (νC-H), 1642 (νCdO), 1556, and 1386 cm-1 (νCO-NH). The characteristic absorption bands of 6-N3-Cs appear at 3444 (νO-H, pyranose), 2107 (azido groups), and 1079 cm-1 (νC-O, pyranose). After the conjugation, the spectrum of the Cs-g-PAMAM did not show the characteristic absorption bands of the azido (2107 cm-1), but exhibited the main characteristic bands of the PAMAM dendron and 6-N3-Cs. These results indicate the success of the click conjugation between the PAMAM dendron and 6-N3-Cs. Figure 2 shows 1H NMR spectra of the PAMAM dendron (G = 3), 6-N3-Cs, and Cs-g-PAMAM (G = 3). Compared to the spectrum of 6-N3-Cs, the spectrum of the Cs-g-PAMAM showed new peaks at 2.20-3.30 ppm, which could be assigned to the PAMAM dendron. In addition, the signal at 7.95 ppm in the spectrum of the Cs-g-PAMAM indicated the presence of the triazole proton, which confirmed further the click conjugation between the PAMAM dendron and 6-N3-Cs. For resultant dendronized chitosan derivatives, their buffering capacity in aqueous NaCl solution was also characterized by acid-base titration in comparison with the buffering capacity of chitosan and the PAMAM dendrons, as shown in Figure 3. In this work, the concentration of each sample was fixed to be 0.2 mg/mL. As seen, Cs-g-PAMAM (G = 2, 3) samples have the buffering capacity stronger than chitosan but weaker than the PAMAM dendrons. The strong buffering capability of the PAMAM dendron is attributed to a high density of amino groups in the macromolecular chains. Therefore, the incorporation of PAMAM dendron into chitosan can enhance its buffering capacity. Compared to Cs-g-PAMAM (G = 2), Cs-g-PAMAM (G = 3) with an equivalent substitution degree of PAMAM dendron has better buffering capacity due to the existence of more amino groups. It is thought that gene vectors with good buffering capacity may help pDNA-containing complexes to escape

Figure 4. (A) Agarose gel electrophoresis retardation assay of Cs-gPAMAM (G = 2, 3)/pDNA complexes and chitosan/pDNA complexes at various N/P ratios. (B) Protection and release assay of pDNA. pDNA was released by adding 10% SDS to Cs-g-PAMAM (G = 2, 3)/pDNA complexes or chitosan/pDNA complexes at various N/P ratios. Lanes 1-2, naked pDNA; lanes 3-5, Cs-g-PAMAM (G = 3)/pDNA complexes (N/P = 5/1, 10/1, and 20/1); lanes 6-8, Cs-g-PAMAM (G = 2)/ pDNA complexes (N/P = 5/1, 10/1, and 20/1); lanes 9-11, chitosan/ pDNA complexes (N/P = 5/1, 10/1, and 20/1).

6-N3-Cs, and Cs-g-PAMAM (G = 3). As seen, the characteristic absorption bands of the PAMAM dendron appear at 3262 645

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Figure 6. TEM images of (a) Cs-g-PAMAM (G = 2)/pDNA complexes at N/P = 5/1, (b) Cs-g-PAMAM (G = 2)/pDNA complexes at N/P = 10/1, (c) Cs-g-PAMAM complexes (G = 3)/pDNA at N/P = 5/1, and (d) Cs-g-PAMAM (G = 3)/pDNA complexes at N/P = 10/1.

Figure 7. Cytotoxicity assay on various polymer samples with different concentrations in 293T cell line (a) and CNE2 cell line (b) (n = 3, means ( SD). Figure 8. Transfection efficiency of Cs-g-PAMAM (G = 2, 3)/pDNA complexes and chitosan/pDNA complexes at various N/P ratios (N/P = 5/1, 10/ 1, 20/1) in comparison with that of PEI/pDNA complexes (N/P = 10/1) in T293 cell line (a) and CNE2 cell line (b) (n = 3, means ( SD, *P < 0.01).

from the endosomes and consequently promote transfection activity.33,34 Complex Formation of Dendronized Chitosan Derivative with pDNA. The formation of Cs-g-PAMAM (G = 2, 3)/pDNA complexes was examined by their electrophoretic mobility on an agarose gel, as shown in Figure 4. Figure 4A gives the gel

retardation results of Cs-g-PAMAM (G = 2, 3)/pDNA and chitosan/pDNA complexes at various N/P ratios. The migration 646

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Figure 9. Typical fluorescence images of (A) 293T and (B) CNE2 cells transfected by chitosan/pDNA complexes (a, e), Cs-g-PAMAM (G = 2)/pDNA complexes (b, f), Cs-g-PAMAM (G = 3)/pDNA complexes (c, g), and PEI (25KDa)/pDNA complexes (d, h). Conditions: N/P ratio, 10/1; AI and BI, fluorescence field images; AII and BII, bright field images.

of pDNA was completely retarded when the N/P ratio of Cs-gPAMAM (G = 2, 3) to pDNA equaled to or was greater than 2/1. In contrast, the migration of pDNA was completely retarded only when the N/P ratio was higher than 5/1. These results indicated that Cs-g-PAMAM (G = 2, 3) have strong pDNA condensation capability due to relatively high density of amino groups. Figure 4B shows the protection effect of Cs-g-PAMAM (G = 2, 3) against pDNA degradation by DNase I. Naked pDNA was completely digested, with no band observed. In contrast, Cs-g-PAMAM (G = 2, 3)/pDNA complexes at all N/P ratios (5/1, 10/1, and 20/1) exhibited a distinct protective effect against DNase I. For resultant Cs-g-PAMAM (G = 2, 3)/pDNA complexes, their mean particle size and zeta potential were investigated, as shown in Figure 5. At the N/P ratio equal to 0.5/1, both of Cs-gPAMAM (G = 2, 3)/pDNA complex and chitosan/pDNA complexes formed large particles (Figure 5a) due to the inadequate positive charge for the condensation of pDNA. The size of the complexes tended to decrease with the increase of N/P ratio until the N/P ratio was 10/1, and remained in the size range from 100 to 150 nm. In contrast, Cs-g-PAMAM (G = 2, 3) samples have stronger condensing ability for pDNA when compared to native chitosan. The polydispersity index of Cs-g-PAMAM (G = 2, 3)/pDNA complexes in Figure 5a was found to range from 0.1 to 0.2. In addition, all complexes were observed to have positive zeta potential values at the N/P ratios above 5:1. The zeta potential of the complexes increased with the increase of N/P ratio, as indicated in Figure 5b. Besides, the zeta potential values of Cs-g-PAMAM (G = 2, 3)/pDNA complexes were higher than those of chitosan/pDNA complex at the same N/P ratio.

The morphology of Cs-g-PAMAM (G = 2, 3)/pDNA complexes was investigated by TEM. As shown in Figure 6, the complexes were found to have a spherical shape and compact structure. Moreover, the TEM images demonstrated that the size of the nanoparticles was roughly in the range of 100-200 nm, which was consistent with the results measured by dynamic light scattering. Therefore, Cs-g-PAMAM (G = 2, 3) could form the nanosize complexes with pDNA under some conditions. Previous studies35,36 had confirmed that the size range of pDNAcontaining complexes from 50 to 400 nm was suitable for cellular endocytosis and in vivo circulation when these complexes were used for gene delivery. In Vitro Cytotoxicity. The in vitro cytotoxicity of Cs-g-PAMAM (G = 2, 3), chitosan, and PEI (25 kDa) in the concentration range from 6.25 to 200 μg/mL were evaluated in 293T and CNE2 cell lines by MTT assays, as shown in Figure 7. For PEI (25 kDa), significant toxicity was observed when its concentration was higher than 25 μg/mL. In this case, the cell viability was found to be lower than 40% in 293T line and 10% in CNE2 cell line, respectively. In contrast, Cs-g-PAMAM (G = 2, 3) and chitosan could exhibit a higher cell viability, even at high concentrations. At a concentration of 50 μg/mL, for example, the cell viability of Cs-g-PAMAM was found to be higher than 80% in 293T line and 45% in CNE2 cell line, respectively. These results could be attributed to good biocompatibity of chitosan. Therefore, Cs-g-PAMAM (G = 2, 3) samples have a low cytotoxicity when compared to PEI (25 kDa), which will become an advantage when they are used for gene delivery. Compared to Cs-g-PAMAM (G = 2), Cs-g-PAMAM (G = 3) has a lower cell viability due to the increase of amino groups from the 647

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Biomacromolecules PAMAM dendron (G = 3).37 In general, a level of 60% cell viability represents nearly nontoxic status for in vivo application.38 In Vitro Transfection. To confirm whether the modification of chitosan by PAMAM dendrons could improve its transfection efficiency, we compared the transfection efficiency of Cs-gPAMAM (G = 2, 3)/pDNA complexes with that of chitosan/ pDNA in 293T and CNE2 cells. As shown in Figure 8, the transfection efficiency of Cs-g-PAMAM (G = 2, 3)/pDNA complexes was significantly higher than that of chitosan/pDNA complexes in two cell lines. Moreover, the transfection efficiency of Cs-g-PAMAM (G = 2, 3)/pDNA complexes increased with the increase of the N/P ratio, and an optimum N/P ratio was found to be 10/1. Compared to Cs-g-PAMAM (G = 2), Cs-gPAMAM (G = 3) was found to have a higher gene transfection efficiency. In addition, the cell type has an effect on the transfection efficiency mediated by Cs-g-PAMAM (G = 2, 3). At the N/P ratio of 10/1, for example, the transfection efficiency of Cs-gPAMAM (G = 3)/pDNA complex in 293T cells was 84% while the transfection efficiency of Cs-g-PAMAM (G = 3)/pDNA complex in CNE2 cells was only 23%. This difference may result from different cellular uptake rate in these cell lines. For a comparison, the transfection efficiency of PEI (25 kDa) / pDNA complexes in 293T or CNE2 cells was also investigated, as indicated in Figure 8. It was found that the transfection efficiency was about 80% in T293 cells and 16% in CNE2 cells, respectively. In contrast, Cs-g-PAMAM (G = 3) has a better ability than PEI (25KDa) at the same N/P ratio (10/1) for in vitro transfection of pDNA on two cell lines (Figure 8). Up to now, PEI has been considered a standard nonviral gene carrier, in spite of high cell toxicity.39 It is a matter of concern to develop and design synthetic nonviral gene carriers with high transfection efficiency and low cytotoxicity in gene therapy. Recently, Ma et al.40 synthesized two different degrees of substitution of PAMAM-triamcinolone acetonide (PAMAM-TA) conjugates for the translocation of pDNA into the nucleus and investigated their in vitro cytotoxicity and transfection efficiency by using PEI (25 kDa) and PAMAM dendrimers (G4) as the controls. Compared to PEI (25 kDa), these conjugates and PAMAM dendrimers were confirmed to have higher cell viability but lower transfection efficiency. It seems that Cs-g-PAMAM (G = 3) with lower cell toxicity and higher transfection efficiency has a great potential as a new nonviral gene carrier. The gene delivery capability of Cs-g-PAMAM (G = 2, 3) was also investigated by fluorescence microscopy. Plasmid pEGFPN1 encoding GFP was used to examine the GFP expression in 293T and CNE2 cell lines. Figure 9 gives typical fluorescence images of 293T and CNE2 cells transfected by chitosan/pDNA complex, Cs-g-PAMAM (G = 2, 3)/pDNA complexes and PEI (25 kDa)/pDNA complex. The GFP expression could not be detected when the transfection was mediated by naked pDNA, which was used as a negative control (data not shown). Strong fluorescence signal could be observed when the transfections were mediated by Cs-g-PAMAM (G = 2, 3) and PEI (25 kDa). In addition, the transfection efficiency of Cs-g-PAMAM (G = 3) was confirmed to be higher than that of Cs-g-PAMAM (G = 2). Serum usually has a negative impact on the transfection efficiency of cationic liposomes.41 The interactions of cationic polymers with serum may serve as a predictive model for the in vivo efficiency of a cationic polymer.42 For this reason, we investigated the effects of serum on the transfection efficiency of Cs-g-PAMAM (G = 2, 3)/pDNA complexes, chitosan/pDNA complexes, and PEI (25 kDa) /pDNA complexes in 293T and CNE2 cell lines, as shown in Figure 10. In all cases, the N/P ratio

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Figure 10. Effects of serum on the transfection efficiency of Cs-gPAMAM(G = 2, 3)/pDNA complexes, chitosan/pDNA complexes, and PEI (25 kDa)/pDNA complexes in 293T (a) and CNE2 (b) cell lines. Condition: N/P ratio, 10/1; serum, 10%.

was fixed to be 10/1. For PEI (25KDa)/pDNA complexes, the presence of serum resulted in an decrease of transfection efficiency, regardless of cell type. In contrast, the serum seems to have a complex effect on the transfection efficiency of Cs-gPAMAM (G = 2, 3)/pDNA complexes or chitosan/pDNA complexes. When the serum was added, a decrease of the transfection efficiency was observed in 293T cell line, while an increase of the transfection efficiency was found in CNE2 cell line. The possible mechanism for such a complex effect is under investigation.

’ CONCLUSIONS Dendronized chitosan derivatives, namely, Cs-g-PAMAM (G = 2, 3) could be prepared by copper-catalyzed azide alkyne cyclization reactions of propargyl focal point PAMAM dendrons with 6-azido-6-deoxy-chitosan. They have better water solubility and buffering capacity when compared to native chitosan. In particular, Cs-g-PAMAM (G = 3) could exhibit enhanced transfection efficiency and lower cytotoxicity in 293T and CNE2 cell lines when compared to commonly used PEI (25 kDa). Such a modified chitosan may be used as a new nonviral gene delivery vector for future gene therapy applications.

’ AUTHOR INFORMATION Corresponding Author

*Tel.: þ86-20-84112354. Fax: þ86-20-84112354. E-mail: ceszhlm@ mail.sysu.edu.cn; [email protected]. 648

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Author Contributions †

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Authors contributed equally to this work.

’ ACKNOWLEDGMENT L.-M.Z. would like to thank the supports from National Natural Science Foundation of China (Grant Nos. 21074152, 20874116 and J0730420) and Natural Science Foundation of Guangdong Province in China (Grant Nos. 8151027501000004 and 9151027501000105) as well as the Doctoral Research Program of Education Ministry in China (20090171110023). ’ REFERENCES (1) Dang, J. M.; Leong, K. W. Adv. Drug Delivery Rev. 2006, 58, 487–499. (2) Ravi Kumar, M. N. V.; Muzzarelli, R. A. A.; Muzzarelli, C.; Sashiwa, H.; Domb, A. J. Chem. Rev. 2004, 104, 6017–6084. (3) Liu, W. G.; Yao, K. D. J. Controlled Release 2002, 83, 1–11.   (4) K€oping-H€oggard, M.; Varum, K. M.; Issa, M.; Danielsen, S.; Christensen, B. E.; Stokke, B. T.; Artursson, P. Gene Ther. 2004, 11, 1441–1452. (5) Neu, M.; Fischer, D.; Kissel, T. J. Gene Med. 2005, 7, 992–1009. (6) Hashimoto, M.; Morimoto, M.; Saimoto, H.; Shigemasa, Y.; Sato, T. Bioconjugate Chem. 2006, 17, 309–316.   (7) Issa, M. M.; K€oping-H€oggard, M.; Tommeraas, K.; Varum, K. M.; Christensen, B. E.; Strand, S. P.; Artursson, P. J. Controlled Release 2006, 115, 103–112. (8) Strand, S. P.; Issa, M. M.; Christensen, B. E.; Varum, K. M.; Artursson, P. Biomacromolecules 2008, 9, 3268–3276. (9) Jiang, H.; Kwon, J.; Kim, E.; Kim, Y.; Arote, R.; Jere, D.; Jeong, H.; Jang, M.; Nah, J.; Xu, C.; Park, I.; Cho, M.; Cho, C. J. Controlled Release 2008, 131, 150–157. (10) Jiang, X.; Dai, H.; Leong, K. W.; Goh, S. H.; Mao, H. Q.; Yang, Y. Y. J. Gene Med. 2006, 8, 477–487. (11) Gao, Y.; Zhang, Z.; Chen, L.; Gu, W.; Li, Y. Biomacromolecules 2009, 10, 2175–2182. (12) Germershaus, O.; Mao, S.; Sitterberg, J.; Bakowsky, U.; Kissel, T. J. Controlled Release 2008, 125, 145–154. (13) Son, S. H.; Chae, S. Y.; Choi, C. Y.; Kim, M. Y.; Ngugen, V. G.; Jang, M. K.; Nah, J. W. Macromol. Res. 2004, 12, 573–580. (14) Kim, T. H.; Ihm, J. E.; Choi, Y. J.; Nah, J. W.; Cho, C. S. J. Controlled Release 2003, 93, 389–402. (15) Lu, B.; Wang, C. F.; Wu, D. Q.; Li, C.; Zhang, X. Z.; Zhuo, R. X. J. Controlled Release 2009, 137, 54–62. (16) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem. Rev. 1999, 99, 1665–1688. (17) Jang, W.; Kamruzzaman Selim, K. M.; Lee, C.; Kang, I. Prog. Polym. Sci. 2009, 34, 1–23. (18) Ma, K.; Hua, M. X.; Qi, Y; Zou, J. H.; Qiu, L. Y.; Jin, Y.; Ying, X. Y.; Sun, H. Y. Biomaterials 2009, 30, 6109–6118. (19) Kim, T.; Seo, H. J.; Choi, J. S.; Jang, H.; Baek, J.; Kim, K.; Park, J. Biomacromolecules 2004, 5, 2487–2492. (20) Yuan, Q.; Yeudall, W. A.; Yang, H. Biomacromolecules 2010, 11, 1940–1947. (21) Santos, J. L.; Oramas, E.; P^ego, A. P.; Granja, P. L.; Tomas, H. J. Controlled Release 2009, 134, 141–148. (22) Nama, H. Y.; Nama, K.; Hahn, H. J.; Kim, B. H.; Lim, H. J.; Kim, H. J.; Choi, J. S.; Park, J. S. Biomaterials 2009, 30, 665–673. (23) Ma, K.; Hua, M. X.; Qi, Y.; Zou, J. H.; Qiu, L. Y.; Jin, Y.; Ying, X. Y.; Sun, H. Y. Biomaterials 2009, 30, 6109–6118. (24) Gillies, E. R.; Frechet, J. M. J. J. Am. Chem. Soc. 2002, 124, 14137–14146. (25) Sashiwa, H.; Shigemasa, Y.; Roy, R. Carbohydr. Polym. 2002, 49, 195–205. (26) Sashiwa, H.; Shigemasa, Y.; Roy, R. Macromolecules 2000, 33, 6913–6915. 649

dx.doi.org/10.1021/bm101303f |Biomacromolecules 2011, 12, 642–649