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Jan 10, 2011 - Chitosan grafted with macrocyclic polyamines (Cs-g-MCPA) on the C-2 or the C-6 position was synthesized by a simple method...
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Biomacromolecules 2011, 12, 298–305

Chitosan Grafted with Macrocyclic Polyamines on C-2 and C-6 Positions as Nonviral Gene Vectors: Preparation, Characterization, and In Vitro Transfection Studies Chao Li,† Hua Tian,† Na Rong,† Kun Liu,‡ Feng Liu,‡ Yanjie Zhu,† Renzhong Qiao,*,† and Yuyang Jiang*,‡,§ State Key Laboratory of Chemical Resource Engineering, Department of Pharmaceutical Engineering, Beijing University of Chemical Technology, Beijing 100029, China, State Key Laboratory of Chemical Biology, Guangdong Province, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China, and School of Medicine, Tsinghua University, Beijing 100084, China Received July 19, 2010; Revised Manuscript Received December 14, 2010

Chitosan grafted with macrocyclic polyamines (Cs-g-MCPA) on the C-2 or the C-6 position was synthesized by a simple method. Four copolymers prepared were characterized by 1H NMR, 13C NMR, Fourier transform infrared spectra (FTIR), X-ray diffraction (XRD), and gel permeation chromatography (GPC). Circular dichroism spectra (CD), fluorescence spectra and agarose gel electrophoresis assay showed that Cs-g-MCPA copolymers had good binding ability to DNA. By modification of the MCPAs, the copolymers showed low cytotoxicity and high transfection efficiency as new gene vectors. It was found that Cs-g-MCPA copolymers with different grafted positions showed different properties: copolymers grafted on the C-2 position showed higher cytotoxicity and higher transfection efficiency than those grafted on position C-6.

Introduction Gene therapy is the treatment of human disorders by the introduction of genetic material to specific target cells of a patient, where the production of the encoded protein will occur.1 It is also currently being applied in many different health problems such as cancer, AIDS, and cardiovascular diseases.2 In the field of gene therapy, the development of efficient and safe vector systems able to transfer DNA into cells is a major goal. Although viral vectors are highly efficient in transfecting cells, undesirable complications limit their therapeutic applications. Synthetic nonviral vectors such as cationic liposomes and cationic polymers are being widely sought as alternatives. Among them, chitosan has been considered to be a good gene vector candidate in vitro and in vivo because of its nonallergenicity, biocompatibility, biodegradability, and cationic surface charge in acidic medium. However, as a nonviral gene vector, the transfection efficiency of chitosan is poor. To improve the transfection efficiency and enhance the cell specificity, chemical modification of chitosan using hydrophilic,3-8 hydrophobic,9-16 pH-sensitive,17-20 and thermosensitive21-23 specific ligands,24-27 polymers, and groups has been studied, and the results showed that the modified chitosan vectors exhibited superior transfection efficiency to that of chitosan. As we know, many researchers prefer to introduce a polymer containing an amino/imino group to chitosan as a copolymerization strategy because the protonated amino/imino group can enhance the interaction between the vector and DNA. Representative examples include NMC-g-PEI (PEI: polyethylenimine),28 PLL-g-Chi,29 and PEI-g-chitosan.18 However, the size of the vector is usually increased after conjugation with another * Corresponding author. Tel: 86-010-64413899. Fax: 86-010-82728926. E-mail: [email protected]. † Beijing University of Chemical Technology. ‡ Graduate School at Shenzhen, Tsinghua University. § School of Medicine, Tsinghua University.

polymer, and high molecular weight (MW) is not conductive to achieve efficient intracellular unpacking and reduce cytotoxicity. For these reasons, functional group modification is now becoming a more preferred method for vector derivative synthesis. Quaternized chitosan, 6-amino-6-deoxy chitosan (6ACT), and urocanic acid-modified chitosan (UAC), as functional group modifications of chitosan, have been presented by Thanou,14 Satoh,8 and Kim,17 respectively. Compared with chitosan, the polymers produced by these methods have much higher water solubility and transfection efficiency. In this study, through grafting two kinds of macrocyclic polyamine, 1,4,7,10-tetraazacyclododecane (Cyclen) and 1,4,7triazacyclononane (TACN), to chitosan at one of two different grafting positions (C-2 or C-6), we have prepared a series of new, nonviral gene vectors chitosan grafted with macrocyclic polyamines (Cs-g-MCPA), as shown in Scheme 1. These new copolymers modified with macrocyclic polyamines (MCPAs) not only provide a protonated amino/imino group to improve DNA binding but also decrease the size and MW of the vector. The capability of the resulting copolymers as gene vectors was evaluated with respect to the physiochemical characteristics, morphology, in vitro cytotoxicity, and transfection activity. In a detailed set of experiments, we also found that modifiedchitosan with different grafted positions exhibits dissimilar behavior in terms of physicochemical characterization, interaction with DNA, cytotoxicity, and transfection efficiency. That is to say, the same grafters on different positions of chitosan, such as C-2 or C-6, can contribute distinct results.

Materials and Methods Materials. Chitosan (low MW, 22 kDa) was purchased from SigmaAldrich Chemicals. Deacetylation degree of 88% was determined by 1 H NMR; pUC18 plasmid DNA and DNase I were obtained from Takara Biotechnology (Dalian). Calf thymus DNA (ct-DNA) was purchased from Huamei Biotechnology Company. All other chemicals were of analytical grade.

10.1021/bm100819z  2011 American Chemical Society Published on Web 01/10/2011

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Scheme 1. Structures of Cs-g-MCPA

Synthesis of MCPAs Derivatives. Cyclen and TACN were modified by the same flexible linker, respectively. We dissolved 2.0 g protected Cyclen (3Boc-Cyclen) and 1.2 g potassium carbonate (K2CO3) in 20 mL of acetonitrile, and the solution was stirred at room temperature for 10 min. Then, 483 µL of ethyl bromoacetate was added to the abovementioned reaction. After the reaction finished, we obtained the product (3Boc-Cyclen-CH2COOEt) by removing the solvent. Subsequently, the product with purification was hydrolyzed to the corresponding acid (3Boc-Cyclen-CH2COOH) (2.3 g). The modification of TACN with linker was achieved in the same way. Preparation and Characterization of Cs-g-MCPAs. 6-O-Triphenylmethy1 chitosan (Cs-Tr) was synthesized according to Nishimura et al.,30 and the free amino group of Cs-Tr was coupled to the carboxyl group of 3Boc-Cyclen-CH2COOH (or 2Boc-TACN-CH2COOH), which was converted to its activated ester with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC) to obtain compound Cs-g(2)Cyclen-Tr (or Cs-g(2)-TACN-Tr). Finally, the protective groups were removed simultaneously in the presence of trifluoroacetic acid (TFA), and the crude products were dialyzed with distilled water for 3 days and lyophilized to provide the target products, Cs-g(2)-Cyclen, and Csg(2)-TACN. N-2 of chitosan was protected with phthalic anhydride (NPTh) in DMF to obtain the compound Cs-NPTh. Then, in the presence of diisopropylethylamine (DIEA) and Castro’s reagent (BOP), the free hydroxyl group of Cs-NPTh was coupled to the carboxyl group of 3BocCyclen-CH2COOH (or 2Boc-TACN-CH2COOH) to give compound Cs-g(6)-Cyclen-NPTh (or Cs-g(6)-TACN-NPTh). Subsequently, the protecting groups were removed simultaneously with 25% HCl (l), and the crude product was dialyzed and lyophilized to provide the target products, Cs-g(6)-Cyclen and Cs-g(6)-TACN. 1H NMR spectra were obtained using a Bruker Avance 600 spectrometer (600 MHz) at 298 K in D2O or DMSO-d6. Fourier transform infrared (FTIR) spectra were recorded in KBr pellets with a JASCO FT-IR 410 instrument. The physical form (crystalline or amorphous) of the lyophilized samples was determined by X-ray diffraction (XRD) over the range 2θ from 5 to 40° using a Rigaku D/max 2500 diffractometer with Bragg-Brentano geometry (θ, 2θ) and Ni-filtered Cu-Ka radiation. The degree of substitution (DS) of MCPAs onto C-2 or C-6 of chitosan was calculated on the basis of 1H NMR spectra and elemental analysis. The MW of the polymeric conjugate was measured by gel permeation chromatog-

raphy (GPC) (Waters 515-410, column: Ultrahydrogel 250, solvent: 0.1 M NaCl, 40 °C, flow rate: 1.0 mL/min, standards: PEO). For contrast experiments, copolymers with similar MW and DS were chosen through controllable reaction conditions. MW and DS of Cs-g-MCPAs copolymers are shown in Table S1 of the Supporting Information. Preparation of Cs-g-MCPAs/TPP Nanoparticles. Cs-g-MCPAs/ TPP nanoparticles (N/P 5) were prepared according to the procedure reported by Calvo et al. based on the ionic gelation of chitosan with TPP polyanions.31,32 In brief, 1.0 mg of the synthesized Cs-g-MCPAs was dissolved in 6 mL of 5 mM sodium acetate/acetic acid buffer (pH 5.4). With stirring at room temperature, 1.2 mL of 0.15 mg/mL TPP solution was added dropwise to the above solution and stirred continuously for 1 h to stabilize the nanoparticles through the electrostatic interaction with TPP. (The final concentration of Cs-gMCPAs is 0.14 mg/mL.) The suspension of nanoparticles was then dialyzed against deionized water and passed through a syringe filter (pore size 0.45 µm) prior to lyophilization. Physicochemical Characterization of Nanoparticles. For measurements of particle size, zeta potential, and polydispersity (size distribution) of freshly prepared Cs-g-MCPAs nanoparticles, a Zetaplus (Brookhaven) was used, which is based on dynamic light scattering (DLS) techniques. All DLS measurements were done with a wavelength of 633 nm at 25 °C, with an angle detection of 90°. For zeta potential measurements, samples were diluted with 0.1 mM KCl and measured in the automatic mode. Morphological characteristics of the nanoparticles were observed by Transmission Electron Microscope (TEM, Hitachi, H-800). One drop of freshly made nanoparticles solution was placed on 300 mesh copper grids coated with carbon film and allowed to air-dry. Circular Dichroism (CD). All experiments were performed with a continuous flow of nitrogen purging the Jasco-810 spectropolarimeter with a path length cell of 1 cm at room temperature. Complex solutions of Cs-g-MCPA/ct-DNA with different N/P ratios were prepared. Then, the solutions were diluted with 0.1 M sodium acetate/0.1 M acetic acid buffer to a final DNA concentration of 1.1 × 104 M. The standard scan parameters for all experiments used a wavelength range from 400 to 220 nm. Sensitivity was set at 100 mdeg and scan speed of 200 nm per minute. Three scans were made and computer averaged. Fluorescence Quenching. Fluorescence spectra were recorded on a Hitachi model F-4500 spectrofluorimeter, with excitation and emission

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Scheme 2. Synthetic Routes for Preparation of the Cs-g-MCPA Copolymers

band bass: 10 nm (λex ) 520 nm, λem ) 620 nm). Ethidium bromide (EB) (0.1 mg/mL) was dissolved in 0.1 M sodium acetate/0.1 M acetic acid buffer (pH 5.4). EB solution (20 mL) was added to a 0.1 mg/mL of ct-DNA solution. An aliquot of chitosan or Cs-g-MCPA (1.0 mg/ mL in 0.1 M sodium acetate/0.1 M acetic acid buffer) was then titrated in the ct-DNA/EB solution to various N/P ratios. The mixture was incubated for 30 min at 37 °C, and readings were taken. In the same manner, an aliquot of Cs-g-MCPA (1.0 mg/mL in 0.01 M sodium hydroxide solution) was added dropwise to the EB/DNA solution. Gel Retardation and DNase Resistivity Assay. Cs-g-MCPA and plasmid DNA (pUC18) with varied N/P ratios were placed in 1 mL of 10 mM Tris-HCl buffer containing 10 mM NaCl to obtain the Cs-gMCPA/DNA complexes. Complex formation was carried out for 30 min at room temperature for gel retardation. In the separate setup, 1 or 10 units of DNase I was added to this solution and incubated for 30 min at 37 °C for DNase I resistivity assay. Both of the complex solutions were loaded onto 1.0 wt % agarose gel (85 V), and DNA bands were visualized by EB staining. Cytotoxicity Assay. HepG2 cells (1 × 104 cells/well) were seeded in 96-well plates. The cells were incubated for 24 h with 20 µL of Cs-g-MCPAs at different concentrations. The medium in each well was replaced with 10 µL of fresh complete medium 24 h later. After 20 µL of MTT solution in PBS (5 mg/mL) was added, the cells were incubated for another 4 h. Thereafter, the medium was removed and 150 µL of DMSO was added. Plates were incubated for 30 min at 37 °C. The optical intensity was measured at 570 nm using a microplate reader (Beckman DTX 880). The relative cell viability was calculated as cell viability (%) ) (ODsample/ODcontrol) × 100, where ODcontrol was obtained in the absence of polymers and ODsample was obtained in the presence of polymers. Each value was averaged from four independent experiments. In Vitro Gene Transfection. The HepG2 cells were seeded in 24well plates at a density of 5 × 104 cells/well in 1 mL of complete medium (10% FBS) and incubated for 24 h at 37 °C in 5% CO2. When the cells were at 80-90% confluence, the culture medium was replaced with 200 µL of serum-free medium containing 500 µL of complexes (different pH or N/P ratios Cs-g-MCPAs/DNA complexes). pEGFPCl was used as the reporter plasmid to assay the transfection efficiency. After incubation for 6 h at 37 °C, the cells received 1 mL of complete medium and incubated sequentially until 48 h post transfection. To

assay the expression of luciferase, we removed the medium and gently rinsed the cells with PBS. After thorough lysis of the cells with reporter lysis buffer (Promega; 200 µL/well), the luciferase activity was determined by detecting the light emission from an aliquot of cell lysate incubated with 100 µL of luciferin substrate (Promega) in a luminometer. The protein content of the cell lysate was determined by BCA protein assay kit. All experiments were carried out in triplicate to ascertain reproducibility.

Results and Discussion Synthesis and Characterization of Cs-g-MCPA Copolymers. In this study, macrocyclic polyamines were introduced to the C-2 or C-6 position of chitosan with a flexible linker. We compared the properties of chitosan grafted on the C-2 position with that grafted at C-6 and undertook preliminary studies of the impact of different grafting positions on functions including physicochemical characteristics, interaction with DNA, cytotoxicity, and transfection efficiency. The synthesis route of the Cs-g-MCPAs copolymers is presented in Scheme 2. The amino groups and hydroxyl groups of chitosan were first protected with NPTh and TrCl in that order. The amine groups were deprotected by hydrazinolysis to obtain the product CsTr. Condensation of MCPAs initiated by Cs-Tr was performed in DMA at room temperature. Finally, trityl groups were removed by TFA. We can thereby obtain chitosan grafted with MCPA on the C-2 position. For synthesis of C-6 grafted copolymers, the hydroxyl groups on chitosan directly reacted with MCPAs in the presence of BOP and DIEA after amino groups were protected. Subsequently, amino groups were then deprotected using 25% HCl to obtain the target products. These methods could be used for synthesizing various kinds of C-2 or C-6 modified-chitosans. 1 H NMR spectra of Cs-g-MCPAs are shown in Figure 1. In the case of Cs-g(2)-Cyclen, there still appear some peaks for chitosan and cyclen, the multiplet at 2.65-2.87 ppm being due to the introduction of cyclen and the multiplet at 3.59-3.76 ppm being due to H-3, H-4, H-5, and H-6 of chitosan,

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Figure 1. 1H NMR and D2O exchange).

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13

C NMR spectra of Chitosan (in 5% CF3COOD/D2O), Cs-g(2)-Cyclen (in D2O), and Cs-g(6)-Cyclen (in DMSO-d6 with

respectively. The peak intensity of H-* cannot be clearly defined because of overlap with peaks of chitosan. Small peaks at 7.42-7.66 ppm are assigned to H-7 because CO-NH is unlikely to exchange completely in a short time. Interestingly, in the 1H NMR spectrum of Cs-g(6)-Cyclen, every H signal peak of chitosan and H-* can be almost differentiated in DMSO-d6 solvent. In addition, the 13C NMR spectra of Cs-g-MCPAs were recorded. Chitosan exhibits signals at 100.85, 78.89, 74.71, 72.03, 60.10, and 56.49 ppm, which are assigned to C-1, C-4, C-5, C-3, C-6, and C-2, respectively. The presence of cyclen was shown by signals at 44.18-47.61 ppm. The linker between chitosan and cyclen exhibited an additional signal at 50.61 ppm, belonging to C-*. It should be noted that the C-a signal appeared at 174.21 ppm as evidence of chitosan grafted with cyclen. The FTIR spectra of chitosan, Cs-g(2)-Cyclen, and Cs-g(6)Cyclen are shown in Figure 2. In the chitosan spectrum (Figure 2a), the characteristic absorbance of amide I is ∼1650 cm-1, and the C-O stretching peak of the pyranose ring is at 1029-1071 cm-1. A CdO stretching peak (related to carboxylic ester unit of Cs-g(6)-Cyclen) appears at 1713-1778 cm-1 in Figure 2c and indicates the introduction of the cyclen to the chitosan chain via a carboxylic ester linker.21 The band at 1677 cm-1 is attributed to the formation of imide-linked cyclen and chitosan chain (Cs-g(2)-Cyclen), as shown in Figure 2b.

To identify the physical state and crystallinity of Cs-gMCPAs, the XRD spectra of chitosan, Cs-g(2)-Cyclen, and Csg(6)-Cyclen are presented in Figure 2 (right). As can be seen, the original chitosan powder showed two major broad crystalline peaks at 2θ of around 9.77° and 19.88°, respectively, whereas the diffraction peak of Cs-g(2)-Cyclen was not observed at the same position. The peak at 2θ of ∼9.77° disappeared, and instead, new peaks at 2θ of 21.18 and 26.41° with low intensity were observed. A series of characteristic peaks appear at 2θ of 21.20, 26.30, and 29.20°. These new peaks may be attributed to a polymorph structure transformation when the initial crystal form is not thermodynamically stable and transforms into the most stable form due to the attachment of MCPA to chitosan through a carboxylic ester or imide linker. Combining the results of NMR, FTIR, and XRD, it is reasonable to conclude that MCPAs have been grafted to chitosan at two different positions (C-2 or C-6). Formation of Cs-g-MCPAs/TPP Complexes. The formation of chitosan/TPP and Cs-g-MCPAs/TPP nanoparticles occurs spontaneously upon incorporation of negatively charged TPP into a chitosan derivative solution because of the complexation between oppositely charged species. The process of formation of complexes is very simple and mild, avoiding possible toxicity of chemical reagents and other undesirable effects. As shown

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Figure 2. FTIR and XRD spectra of (a) chitosan, (b) Cs-g(2)-Cyclen, and (c) Cs-g(6)-Cyclen.

Figure 3. TEM images of (a) chitosan, (b) Cs-g(2)-Cyclen, and (c) Cs-g(6)-Cyclen.

in Figure 3a, the chitosan/TPP nanoparticles appear spherical with diameters in the range of 50 to 200 nm. Figure 3b,c illustrates that Cs-g(2)-Cyclen/TPP and Cs-g(6)-Cyclen/TPP nanoparticles formed under the same conditions also have similar spherical form but smaller particle sizes of 40 and 10 nm, respectively. The main interaction is between the negative phosphate groups of the TPP and the positive amino groups of the polymers. The introduction of MCPAs into the chitosan chain with both grafted positions results in the increase in imino groups. Potential protonated groups are greater than those in chitosan and are better accessible for interaction with TPP. Table S2 of the Supporting Information shows the diameter, zeta potential, and polydispersity index of chitosan/TPP and Cs-gMCPA/TPP nanoparticles prepared by ionotropic gelation. Interestingly, the position of derivation in the chitosan molecule seems to have a major impact on size and zeta potential values. Compared with chitosan, surface charges increased remarkably when the amino-containing molecules were grafted, probably because the introduction of amino groups enhances protonation. Zeta potential values of Cs-g-MCPAs rise to 18.7 mV (Cs-g(2)Cyclen/TPP) and 24.3 mV (Cs-g(6)-Cyclen/TPP) from 12.25 mV (chitosan/TPP). We also obtained higher zeta potential values for Cs-g(6)-MCPAs than those for Cs-g(2)-MCPAs. The free 2-NH2 of Cs-g(6)-MCPAs could contribute to more surface charge of polymers. The higher surface charges of chitosan derivatives provide more powerful interaction with TPP, so we observe more compact particles of Cs-g(6)-Cyclen and Cs-g(6)TACN, sized at 122.5 and 132.9 nm, respectively. These results are consistent with the TEM images of chitosan derivatives. The zeta potential is a good indicator of the available surface charge on nanoparticles. Positivite character of nanoparticles is important for their interaction with cellular membrane components and the tight junctions in triggering the paracellular permeation of hydrophilic compounds. The ratio of negative charge (TPP) to positive charge of the chitosan and its derivatives remains in favor of positive charge of the polymers because only a fraction

is neutralized upon binding TPP. It is also noteworthy that the hydrodynamic diameter of nanoparticles measured by DLS was larger than the size estimated from TEM. This was mainly due to the process involved in the preparation of the sample. TEM images depicted the size in the dried state of the sample, whereas DLS determined the size in the hydrated state of the sample. Therefore, size determined by DLS is a hydrodynamic diameter and is larger than size measured by TEM because of solvation effects. Interactions of Cs-g-MCPAs with DNA. Diverse effects of Cs-g-MCPAs on the DNA secondary structure of doublestranded DNA were evaluated by CD measurements. The CD spectra of Cs-g-MCPAs/DNA with varied N/P ratios determined at 20 °C in the case of Cs-g(2)-Cyclen and Cs-g(6)-Cyclen are illustrated in Figure 4. Positive and negative ellipticities centered around 245 and 275 nm arise from the DNA itself, revealing a double-stranded structure for the DNA. Free DNA shows a typical CD spectrum for B-type DNA. Detectable conformational changes of DNA are induced by Cs-g(2)-Cyclen at N/P 1:10. With an increase in the N/P ratio, the molar ellipticities of the DNA are suppressed, and the positive band at 275 nm is clearly red-shifted. Interestingly, one can see two distinct variation trends of DNA conformation in the selected range of N/P ratios. From 1:10 to 2:1, Cs-g(2)-Cyclen shows a decrease in positive CD signal at 275 nm, whereas it shows an increase in signals from 2:1 to 20:1. The change of CD signal observed with Cs-g(2)-Cyclen from 1:10 to 2:1 is typical for B-C-type transformation of DNA duplex, whereas that observed from 2:1 to 20:1 is similar to B-A-type transformation.21 Cs-g(6)-Cyclen shows similar behavior under the same conditions. A competitive displacement assay can characterize the ability of Cs-g-MCPAs to bind to DNA.33 To check the effect of the degree of protonation of chitosan on the interaction between vectors and DNA, fluorescence was determined at pH 5.4, 7.4, and 8.9 (Figure 5). It can be seen that with the increase in the

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Figure 4. CD spectra of (a) Cs-g(2)-Cyclen/ct-DNA and (b) Cs-g(6)-Cyclen/ct-DNA complexes at different N/P ratios.

Figure 5. Fluorescence quenching by chitosan/ct-DNA and Cs-g-MCPAs/ct-DNA with varied N/P ratios at pH 5.4 (0.1 M sodium acetate/acetic acid buffer), pH 7.4, and pH 8.9 (10 mM Tris-HCl buffer containing 10 mM NaCl). 9, pH 8.9; 2, pH 7.4; b, pH 5.4; 1, chitosan, pH 5.4.

amount of polycation, the intensity of fluorescence decreases, indicating that EB is replaced by the added polycation; that is, Cs-g-MCPAs binds selectively to DNA. Importantly, the variation of fluorescence is dependent on the pH of medium. At pH 5.4, the initial fluorescence of chitosan/DNA decreases by up to 90% when the N/P ratio is 20. This is because the amino groups are almost completely protonated at a pH below the pKa of chitosan (6.5), and the charge density of chitosan increases. The electrostatic interaction between chitosan and DNA is enhanced, causing the release of more EB dye.34 However, at both pH 5.4 and 7.4, the fluorescence of Cs-g(6)Cyclen/DNA decreases by up to 80% with an N/P ratio of 20, implying that EB dyes are displaced and the amino groups of Cs-g(6)-Cyclen are significantly protonated at neutrality. Using the Cs-g(2)-Cyclen, we found that a fluorescence decrease by up to 40% as the 2-NH2 function compared with the behavior of Cs-g(6)-Cyclen. Electrophoretic retardation bands on agarose gels can characterize the formation of the two oppositely charged polyelectrolyte partners because the neutralization and/or increase in molecular size of complex results in the strong retardation of anodic DNA migration. Figure 6 shows the agarose gel electrophoresis results for Cs-g(2)-Cyclen/DNA and Cs-g(2)TACN/DNA complexes. One can see that complete retardation occurs at N/P 1.5/1 (lane 5) for the Cs-g(2)-Cyclen/DNA complex, indicating the formation of the complex. For Cs-g(2)TACN/DNA, a partial retardation is observed at the same N/P ratio (lane 12). It is obvious that the presence of imino groups affects the formation of Cs-g-MCPAs/DNA complexes. The resistance of Cs-g(2)-Cyclen/DNA and Cs-g(6)-Cyclen/ DNA complexes to DNA hydrolysis by DNase I was examined to explore their potential as vectors. Although naked DNA was digested completely by the incubation with 1 U/mL of DNase I in 30 min (Figure 6, lane 16), DNA complexed with Cs-g(2)-

Figure 6. Gel retardation and DNase resistivity assay of pUC18 DNA by Cs-g-MCPAs. Lanes 1-7 correspond to different N/P ratios of Csg(2)-Cyclen/DNA as follows: lane 1, 0/1 (DNA only); lane 2, 1/25; lane 3, 1/6; lane 4, 1/1; lane 5, 1.5/1; lane 6, 2/1; lane 7, 3/1. Lanes 8-14 correspond to different N/P ratios of Cs-g(2)-TACN/DNA with N/P ratios the same as for lanes 1-7. Lane 15, naked DNA; lane 16, naked DNA treated with DNase I (1 U/mL); lane 17, Cs-g(2)-Cyclen/ DNA treated with DNase I (1 U/mL); lane 18, Cs-g(2)-Cyclen/DNA treated with DNase I (10 U/mL); lane 19, Cs-g(6)-Cyclen/DNA treated with DNase I (1 U/mL); lane 20, Cs-g(6)-Cyclen/DNA treated with DNase I (10 U/mL).

Cyclen (N/P 2:1) showed resistance to DNase I under different concentrations (lane 17 for 1 U/mL DNase I and lane 18 for 10 U/mL DNase I, respectively). Likewise, DNA and Cs-g(6)Cyclen, at the same N/P ratio, can prevent DNase I from hydrolyzing DNA (lane 19 for 1 U/mL DNase I and lane 20 for 10 U/mL DNase I, respectively). Cytotoxicity of Cs-g-MCPAs. A standard MTT test was introduced to evaluate the cytotoxicity of the Cs-g-MCPAs copolymers against an HepG2 cell line. Chitosan (low MW) was used as a control. Cell viability decreased with increasing polymer concentration (Figure 7). Among the chitosan and four Cs-g-MCPAs copolymers, C-6 grafted copolymers display lower cytotoxicity. After 24 h of exposure to Cs-g(6)-Cyclen and Csg(6)-TACN at a polymer concentration of 50 µg/mL, the viabilities of cells were 79.1 and 80.9%, respectively. At concentrations even at 200 µg/mL, both cell viabilities were ∼80%. The cytotoxicity of both C-2 grafted copolymers was similar to that of chitosan, about 70.4 and 70.5% of the cells

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Figure 7. Cytotoxicity of chitosan and Cs-g-MCPAs at various concentrations against HepG2 cell line.

were viable after exposure to Cs-g(2)-Cyclen and Cs-g(2)-TACN with 100 µg/mL for 24 h. Zhuo28 reported that NMC-g-PEI copolymers showed low cytotoxicity and good transfection activity. However, NMC-gPEI copolymers with various MWs are less toxic than PEI but more toxic than chitosan in the general cell lines tested. Cell viability was ∼40% when the concentration of polymers containing PEI was increased to 100 µg/mL. Cho19 and coworkers obtained ∼70% cell viability at 100 µg/mL concentration of Chi-g-PEI in HepG2 cell line, but this cytotoxicity value is lower than that for a chitosan control. Excessive amino groups in PEI lead to higher cytotoxicity. It is well known that the cytotoxicity of copolymers arises from the introduction of amino/imino group because of the interactions with the plasma membrane or interactions with negatively charged cell components and proteins,35,36 and reduced cytotoxicity of hybrid polymer is achieved by breaking the cationic group into short segments.37 It should be noted, in our case, that although macrocyclic polyamines as modifiers are grafted onto the chitosan backbone, reduced cytotoxicity of C-6 and C-2 grafted copolymers against the HepG2 cell line was observed relative to chitosan (Figure 7). This result demonstrates that small and cyclic amino/imino groups may be good candidates as efficient grafters for chitosan. In Vitro Transfection. To determine whether MCPAs grafted onto the chitosan backbone could improve its in vitro transfection efficiency, we compared the efficiency of Cs-g-MCPAs with that of chitosan. Transfection experiments were performed against HepG2 cells by pEGFP-C1 with different N/P ratios and pH values. As shown in Figure 8, both Cs-g(2)-Cyclen and Cs-g(2)-TACN display higher transfection efficiency compared with chitosan under the same conditions. The increased transfection efficiency of these copolymers can be attributed to the higher amine content from MCPAs. For introduction of MCPAs at the C-2 position of chitosan, the interaction with DNA increased, and the ability of cell uptake increased accordingly compared with that of chitosan. However, the transfection ability of Cs-g(2)-Cyclen was inferior to that of Cs-g(2)-TACN, suggesting that strong binding ability resulting from excess amino groups is not conducive to release of DNA from complexes. The matched affinity for DNA is an important factor for improving the gene transfection efficiency. Interestingly, distinct transfection efficiency was obtained when the transfection experiment was performed using copolymers with two different grafted positions. The transfection efficiencies of Cs-

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Figure 8. Expression of pEGFP-Cl mediated by Cs-g-MCPAs/DNA complexes at various N/P ratios and pH values for HepG2 cell.

g(6)-Cyclen and Cs-g(6)-TACN have advantages compared with those of chitosan at the same N/P ratio and pH value but much lower than that of C-2 grafted copolymers. Although the free amino group in C-2 position could offer more positive charge of its copolymers to improve the binding activity and buffer capacity, a hydroxyl group in the C-6 position may play an important role in favoring cellular uptake and nuclear delivery of DNA. So, chitosan with MCPA groups in the C-2 position maintains the free hydroxyl group in C-6 position so as to exert maximally the function of that hydroxyl group and the MCPA to achieve better transfection.

Conclusions In summary, MCPAs have been grafted onto chitosan. These copolymers have been evaluated as new gene vectors and show suitable physicochemical properties and binding activities with DNA for gene delivery. The introduction of cyclic polyamines does not result in an increase in cytotoxicity compared with chitosan, whereas it can contribute significantly to transfection efficiency. The primary conclusion can be drawn that chitosan grafted at the positions C-2 or C-6 presents distinctly different properties in terms of physicochemical characterization, cytotoxicity, and transfection efficiency. Acknowledgment. Support of this research by the National Nature Science Foundation of China (nos. 20732004, 20872010, 20972014, and 90813013) and Major National Science & Technology Specific projects (no. 2009ZX09501-004) are gratefully acknowledged. Note Added after ASAP Publication. This paper was published ASAP on January 10, 2011. Schemes 1 and 2 have been revised. The correct version was reposted on January 18, 2011. Supporting Information Available. The characterization and physical properties of Cs-g-MCPAs copolymers, such as molecular weight, DS, particle sizes, zeta potential values, and polydispersity indices. This material is available free of charge via the Internet at http://pubs.acs.org.

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