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Low Molecular Weight Polyethylenimine Grafted N-Maleated Chitosan for Gene Delivery: Properties and In Vitro Transfection Studies Bo Lu, Xiao-Ding Xu, Xian-Zheng Zhang,* Si-Xue Cheng, and Ren-Xi Zhuo* Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China Received April 29, 2008; Revised Manuscript Received June 22, 2008
To develop chitosan-based efficient gene vectors, chitosans with different molecular weights were chemically modified with low molecular weight polyethylenimine. The molecular weight and composition of polyethylenimine grafted N-maleated chitosan (NMC-g-PEI) copolymers were characterized using gel permeation chromatography (GPC) and 1H NMR, respectively. Agarose gel electrophoresis assay showed that NMC-g-PEI had good binding ability with DNA, and the particle size of the NMC-g-PEI/DNA complexes was 200-400 nm, as determined by a Zeta sizer. The nanosized complexes observed by scanning electron microscopy (SEM) exhibited a compact and spherical morphology. The NMC-g-PEI copolymers showed low cytotoxicity and good transfection activity, comparable to PEI (25 KDa) in both 293T and HeLa cell lines, except for NMC50K-g-PEI. The results indicated that the molecular weight of NMC-g-PEI has an important effect on cytotoxicity and transfection activity, and low molecular weight NMC-g-PEI has a good potential as efficient nonviral gene vectors.
Introduction The success of gene therapy greatly depends on the availability of suitable delivery vectors, which can be generally categorized into viral vectors and nonviral vectors. Although viral vectors display rather good transfection properties, several drawbacks, including the lack of specificity toward target cells, limitation in the size of inserted DNA, high cost of production, and safety concerns such as risk of potential immunogenecity and chromosomal insertion of viral genome, greatly limit the practical applications of viral vectors.1-3 Synthetic nonviral vectors such as cationic liposomes and cationic polymers are being sought as alternatives, which have several advantages over viral counterparts, including ease of production, low immune response, the ability to transfer large DNA molecules, and a potential cell targeting property.4,5 Cationic natural polysaccharides as gene vectors have several attractive advantages over synthetic polymer-based gene carriers. Because the polysaccharides are derived from natural sources, they are expected to be nontoxic, biocompatible, and biodegradable. Chitosan is a linear, natural cationic polysaccharide comprising β 1f4-linked glucosamine partly containing Nacetyl-D-glucosamine. It is considered that chitosan is a biodegradable polysaccharide and has been shown to have low toxicity in experimental animals and humans.6-8 Moreover, chitosan is recognized by tumor cells and therefore can be used for tumor targeting drug delivery.9 Due to its favorable properties, chitosan is one of the most widely reported nonviral naturally derived polymeric gene vectors10,11 with a low cytotoxicity and biodegradability. However, as a nonviral gene vector, the transfection efficiency of chitosan is poor. To improve transfection efficiency and enhance cell specificity, chemical modifications of chitosan using hydrophilic,12,13 hydrophobic,14,15 pH-sensitive,16 thermosensitive,17,18 and cell-specificity19-21 polymers and groups were studied, and the * To whom correspondence should be addressed. Fax: 86-27-68754509. E-mail:
[email protected] (X.Z.Z.);
[email protected] (R.X.Z.).
results showed that the transfection efficiency of the modified chitosan vectors exhibited superior to that of chitosan vector. As we know, efficient gene transfection involves multiple steps including DNA complexation, cellular uptake of the complexes, release of DNA from the vectors, and transfer into the nucleus.22 The high stability of chitosan/DNA complexes is suggested to be a major barrier for intracellular release of DNA and, therefore, is a major drawback for efficient transfection.23 It had been reported that the ability of polycation/ DNA complexes to escape the endolysosomal compartment could be correlated to the buffering capacity of the polycation in the pH range of 5-7.24 To overcome this major barrier, some effective chemical modifications of chitosan were carried out. Leong et al. reported PEI-g-chitosan prepared by cationic polymerization of aziridine initiated by the free amino groups of water-soluble chitosan,25 Cho et al. synthesized the chitosangraft-PEI copolymer through the reaction between periodateoxidized chitosan and PEI.26 The results showed that those polymers have an increased solubility at physiologic pH and superior transfection efficiency compared to chitosan. However, the grafting degree of PEI is not easy to be well controlled in these reactions. In addition, it is also difficult to synthesize high molecular weight chitosan with grafted polyethylenimine (PEI). It is well-known that low molecular weight PEI (800 Da) is not competent in transfection due to the lack of DNA condensing property, but has much lower cytotoxicity compared to the high molecular weight PEI (25 KDa). In this paper, through grafting low molecular weight PEI (800 Da) to N-maleated chitosans with different molecular weights by Michael addition, we prepared a serial of novel gene vectors and the capability of resulted copolymers as the gene vectors was evaluated in vitro with respect to the physiochemical characteristics, morphology, in vitro cytotoxicity and transfection activity.
Materials and Methods Materials. Branched polyethylenimines (Mw ) 25 KDa and Mw ) 800 Da) were purchased from Sigma-Aldrich. Chitosans (Mw ) 5, 10,
10.1021/bm8004676 CCC: $40.75 2008 American Chemical Society Published on Web 08/13/2008
Polyethylenimine Grafted N-Maleated Chitosan 50 KDa, deacetylation degree ) 85.5, 85.3, 85.3%, respectively) were purchased from Haidebei Marine Bioengineering Co. Ltd., Jinan, China. Dimethyl sulfoxide (DMSO) was obtained from Shanghai Chemical Reagent Co., China, which was refluxed with anhydrous MgSO4 overnight and was then distilled under reduced pressure. Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin-streptomycin, trypsin, fetal bovine serum (FBS), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and Dulbecco’s phosphate-buffered saline (PBS) were purchased from Invitrogen Corp. The reporter plasmids, pEGFP-C1 and pGL3-Luc, were purchased from Invitrogen and Promega, respectively. The plasmid DNA was stored at -20 °C until the transfection experiments. All other reagents were of analytical grade and were used as received. Synthesis of N-Maleated Chitosan (NMC) and Polyethylenimine Grafted N-Maleated Chitosan (NMC-g-PEI). A total 2 g chitosan was dissolved in 50 mL of 0.1 M acetic acid, precipitated with 40 mL of 0.2 M NaOH, collected by filtration, and then washed with water to pH 7. The chitosan obtained from above was dispersed in 150 mL of DMSO with stirring. DMSO solution containing 3.5 g maleic anhydride was added into above solution, and then the mixture was placed in a 60 °C oil bath for 8 h. The product was precipitated in 500 mL of acetone, filtered, washed with acetone and ether (3×), and then dried to obtain NMC. Chitosans with molecular weights of 5, 10, and 50 KDa were used to synthesize N-maleated chitosan (NMC) and the products were designated as NMC5K, NMC10K, NMC50K, respectively. To graft PEI onto NMC, 0.2 g NMC was dissolved in 20 mL of 0.25% sodium hydroxide solution. The aqueous solution with 2.0 g PEI (800 Da) was then added into the above solution. The mixture was stirred and the reaction was carried out at 60 °C for 24 h. Then hydrochloric acid was added in the mixture with stirring until pH value reaches 7.0. The product was dialysized (MWCO: 3500 Da) against distilled water for 3 days and then lyophilized for 3 days. Polymer Characterization. 1HNMR spectra of chitosan, NMC, and NMC-g-PEI were recorded on a Mercury VX-300 spectrometer at 300 MHz (Varian, U.S.A.) by using D2O as a solvent and TMS as an internal. The molecular weight of the NMC-g-PEI copolymers was measured by a gel permeation chromatography (GPC) equipped with a Waters 2690 separation module and a Waters 2410 refractive index detector. Waters millennium module software was used to calculate the molecular weight based on a universal calibration curve generated by PEG standards of narrow molecular weight distribution. Acetic acid/ ammonium acetate (pH 5.3) buffer solution was used as elute at flow rate of 1.0 mL/min for NMC5K-g-PEI and NMC10K-g-PEI, and acetic acid/sodium acetate (pH 2.7) buffer solution was used as elute at flow rate of 1.0 mL/min for NMC50K-g-PEI. The column temperature was maintained as 35 °C. Cell Culture. 293T and HeLa cells were incubated in DMEM supplemented with 10% (v/v) FBS and 1% antibiotics (penicillinstreptomycin, 10000 U/mL) at 37 °C in a humidified atmosphere containing 5% CO2. Cells were subcultured prior to confluence using trypsin-EDTA. Cytotoxicity Assay. Cells were seeded in the 96-well plate at a density of 6000 cells/well and cultured 24 h in 200 µL of DMEM containing 10% FBS. After the polymer was added for 48 h, the medium was replaced with 200 µL of fresh medium. Then 20 µL of MTT (5 mg/mL) solution was added for 4 h. Thereafter, the medium was removed and 150 µL of DMSO was added. Plates were incubated for 5 min at 37 °C, and then absorbance was measured at 570 nm using a microplate reader (BIO-RAD, Model 550, U.S.A.). 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. Preparation of NMC-g-PEI/DNA Nanoparticles. NMC-g-PEI was dissolved in NaCl solution (150 mM) with a concentration of 2 mg/ mL and the solution was filtered by a 0.22 µm filter. A plasmid DNA stock solution (120 ng/µL) was prepared in 40 mM Tris-HCl buffer
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solution. Nanoparticles were prepared by adding copolymer solution to equal volumes of DNA solution (contain 1 µg DNA) at various weight ratios, with gentle vortexing and incubated at 37 °C for 30 min before use. Agarose Gel Electrophoresis. To assess DNA condensation ability of the copolymers, electrophoresis was performed. The NMC-g-PEI/ DNA complexes with different weight ratios ranging from 0 to 7 were formed according to the conditions described above. Then 6 µL of complex suspension containing 0.1 µg DNA was electrophoresed on the 0.7% (w/v) agarose gel containing GelRed and with Tris-acetate (TAE) running buffer at 80 V for 80 min. DNA retardation was observed by irradiation with UV light and assayed with Cam2com software. Measurement of Particle Size and ζ-Potential. The particle size and ζ-potential measurements were performed at pH ) 7.0 in triplicates by Nano-ZS ZEN3600 (MALVERN Instrument) at 25 °C. The NMCg-PEI/DNA complexes were prepared by adding appropriate volume of copolymer solution (150 mM NaCl solution) to 1 µg of DNA (in 40 mM Tris-HCl buffer solution) at weight ratios ranging from 0 to 30. Then the solution containing complexes was diluted by 150 mM NaCl solution for particle size measurement or diluted by distilled water for ζ-potential measurement to 1 mL and then incubated at 37 °C for 30 min. Scanning Electron Microscopy (SEM). The polymer/DNA complexes were prepared according to the conditions described above. A total of 100 µL of complex suspension was deposited onto a glass slide. After drying at room temperature, the morphology of the sample was observed by a scanning electron microscope (SEM, FEI-QUANTA 200, Holland). Before the SEM observation, the samples were fixed on an aluminum stub and coated with gold for 7 min. Determination of Buffering Capacity of Polymers. The buffering capacity of PEI and NMC-g-PEI copolymers was determined by acid-base titration assay over the pH of 10 to 2, as described by Benns et al.27,28 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 with 0.1 M NaOH to a pH of 10, and then 0.1 M HCl solutions with particular volumes were added to the solution to obtained mixtures with different pH values that were measured using a microprocessor pH meter. Because of the poor solubility of chitosan in base solution, the buffering range between pH 7.0 and 2.0 was measured. In Vitro Transfection. Cells were seeded in 24-well plates at an initial density of 6 × 104 cells/well with 1 mL of DMEM containing 10% FBS and incubated at 37 °C for 24 h in 5% CO2 (to reach 70% confluence at the time of transfection). The polymer/DNA complexes were formed at different weight ratios ranging from 5 to 40, according to the conditions described above (containing 1 µg DNA in every weight ratio). Before transfection, the cells were washed with phosphate buffered saline (PBS, 0.1 M, pH 7.4), and the cells were incubated with the complexes in serum-free or 10% serum-containing culture medium for 4 h at 37 °C. Then the medium was replaced with fresh medium containing serum and incubated for 48 h. To assay the expression of luciferase, the medium was removed and the cells were rinsed gently by 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 (Lumat LB9507, Berthold). The protein content of the cell lysate was determined by BCA protein assay kit (Pierce).29 All the experiments were carried out in triplicate to ascertain the reproducibility.
Results and Discussion Synthesis of NMC-g-PEI Copolymers. In this study, we used a new method to synthesize a serial of NMC-g-PEI copolymers and the synthesis route of NMC-g-PEI is shown in Scheme 1. The amine groups of chitosan were first reacted with maleic anhydride. Then, the product was obtained by the
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Scheme 1. Synthesis of Polyethylenimine Grafted N-Maleated Chitosan (NMC-g-PEI)
reaction between amino groups of PEI (800 Da) with the double bonds of N-maleated chitosan. The synthesis route is simple and can be conveniently controlled for synthesizing graft copolymers with different molecular weights. The 1H NMR spectra of chitosan, NMC, and NMC-g-PEI are shown in Figure 1. After the maleated reaction, new peak appears at 6.2 and 5.7 ppm in the spectrum of NMC (Figure 1a), which is assigned to -CHdCH-. By comparing the intensity of peak at 3.4∼3.6 ppm (multiplet, assigned to glucosamine unit, H-3, H-4, H-5, H-6, H-6′), the maleated chitosan degrees are calculated as 76.9, 68.4, and 69.1% per glucosamine unit in NMC5K, NMC10K, and NMC50K, respectively. The decreased degree from NMC5K to NMC10K or NMC50K was probably attributed to the reduced reaction activity of the increased molecular weight of chitosan. NMC-g-PEI copolymers were synthesized through Michael addition of NMC with PEI. In this study, we used excessive PEI to avoid gelation. As shown in Figure 1, in the spectrum of NMC-g-PEI, the peak ascribed to -CHdCH- disappears, and the proton peaks of PEI (-NHCH2CH2-) appear at 2.5∼3.0 ppm (Figure 1b), indicating that PEI was grafted to the chitosan chain. The PEI grafting degree (GD, grafting degree was defined as the percentage of PEI grafted to NMC per glucosamine unit,including nonacetylated and acetylated glucosamine units) was determined by comparing the 1H NMR signal integrals from -CH2 protons of PEI with integrals of the polysaccharide backbone proton signals.30 The PEI GD and molecular weight of NMC-g-PEI are shown in Table 1. The average numbers of ethylenimine units per saccharide unit are 3.4, 2.6, and 2.4 for NMC5K-gPEI, NMC10K-g-PEI, and NMC50K-g-PEI, respectively. Cytotoxicity of NMC-g-PEI. The cytotoxicity of NMC-gPEI copolymers on 293T and HeLa cell lines was evaluated by
Figure 1. 1H NMR spectra of (A) CHI (10 KDa), (B) NMC10K, and (C) NMC10K-g-PEI in D2O.
Table 1. Molecular Weight and PEI Grafting Degree (GD) of NMC-g-PEI Copolymers NMC5K-g-PEI
NMC10K-g-PEI
NMC50K-g-PEI
Mwa 14600 24000 114000 Mwb 16500 (PDI ) 1.37) 23100 (PDI ) 1.48) 97570 (PDI ) 1.74) GD 27.2% 21.0% 19.0% a
Calculated from 1H NMR.
b
Determined by GPC measurement.
MTT assay.31 From Figure 2 we found that high molecular weight PEI was significantly more toxic than low molecular weight PEI. The results are in agreement with previous reports.32,33 As we expected, NMC-g-PEI copolymers are less toxic than PEI (25 KDa) but more toxic than PEI (800 Da) in both cell lines tested. It is reported that the cytotoxicity of cationic polymers is probably caused by the interactions with the plasma membrane or interactions with negatively charged cell components and proteins.34,35 When a number of PEI (800 Da) molecules were grafted to chitosan, the molecular weight and charge density of resulted copolymers increased correspondingly. Therefore, copolymers are more toxic than PEI (800 Da). However, in comparison to PEI (25 KDa), the cytotoxicity of copolymers is significantly reduced because of the presence of chitosan backbone. Similar finding was also reported that reduced cytotoxicity of hybrid polymer was achieved by breaking the cationic polymer into short segments with monosaccharide spacers.36 Among three NMC-g-PEI copolymers, NMC50K-g-PEI displayed the highest cytotoxicity. However, NMC10K-g-PEI is less toxic than NMC5K-g-PEI, which was ascribed to the fact that the charge density of NMC5K-g-PEI molecule is higher than that of NMC10K-g-PEI because the GD of PEI for NMC5K is higher than the one of NMC10K. Characterization of NMC-g-PEI/DNA Complexes. The ability of NMC-g-PEI to form complexes with DNA was confirmed by gel retardation assay. In this study, weight ratio instead of N/P ratio (N/P refer to the ratio of moles of the amine groups of copolymer to moles of phosphates of DNA), was used to represent the properties of the copolymer. The relationship between the weight ratio and N/P ratio is 2.40, 2.22, and 2.15, respectively, for corresponding NMC5K-g-PEI/DNA, NMC10Kg-PEI/DNA, and NMC50K-g-PEI/DNA. Complexes containing 0.1 µg DNA were prepared at various weight ratios in 150 mM NaCl solution. As shown in Figure 3, migration of DNA in agarose gel was completely retarded when the NMC-g-PEI/DNA weight ratio is higher than 3, indicating that all copolymers could bind DNA strongly. Moreover, the binding ability increased from NMC5K-g-PEI to NMC50K-g-PEI, which was in agreement
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Figure 2. Cytotoxicity of copolymers at various concentrations for (A) 293T and (B) HeLa cells.
Figure 4. Particle size of the NMC-g-PEI/DNA complexes at various w/w ratios.
Figure 3. Agarose gel electrophoresis retardation assay of the copolymers at various weight ratios of NMC-g-PEI/DNA complexes. (A) NMC5K-g-PEI, (B) NMC10K-g-PEI, (C) NMC50K-g-PEI.
with the literature37 reporting that the binding ability to DNA increased with the increasing of length and charge of polymer. Appropriate size and surface charge are important to ensure the uptake of the polymer/DNA complexes by cells. It is reported that cells typically uptake particles ranging from about 50 to several hundred nanometers.38 The diameters of the complexes formed at various weight ratios were measured by dynamic light scattering. In Figure 4, the diameters of the NMC5K-g-PEI/DNA and NMC10K-g-PEI/DNA complexes were in the range of 200-300 nm and the diameter of NMC50K-gPEI/DNA complexes was in the range of 300-400 nm. The size of the complexes decreased with the increasing polymer/ DNA weight ratio. The morphology of NMC-g-PEI/DNA complexes was observed by SEM. As shown in Figure 5, the typical images showed that
the complexes had a spherical shape and a compacted structure. Moreover, the SEM images demonstrated the sizes of the nanoparticles were roughly 200-400 nm, which was consistent with the results measured by DLS. Therefore, we have confirmed that our copolymers could form nanoparticles with DNA and the average diameters of the complexes were within the size requirements for efficient cellular endocytosis.10,38,39 The surface charge of the complexes is known to be one of the major factors influencing transfection efficiency.40 The zeta potential of the complexes at various weight ratios is shown in Figure 6. All complexes are positively charged and the zeta potential of NMC-g-PEI/DNA complexes was found to diminish with a decrease of molecular weight of polymers. It is agreement with the report that higher Mw chitosan had a higher ζ-potential than lower Mw chitosan.41 In addition, the ζ-potential increased with the polymer/DNA weight ratio and gradually approached a plateau due to the saturation of polycations complexed with DNA. Buffer Capacity. In the current study, to overcome the barrier for intracellular release of DNA and improve the transfection efficiency, NMC-g-PEI copolymers were designed using PEI to act as a proton sponge to enhance the release of the complexes into the cytoplasm following endocytosis. The buffering capacities of the NMC-g-PEI copolymers, PEI (25 KDa), PEI (800 Da), and chitosan (10 KDa) were determined by an acid-base titration method. The polymer with a high buffering ability would undergo a small change in pH when the same amount of HCl was added into the polymer solution during titration.42 From the Figure 7, we found that PEI (25 KDa) and PEI (800 Da) had a similar and strong buffer capacity, but chitosan (10 KDa) displayed a very limited buffer capacity. When chitosan grafted with PEI (800 Da), the buffer capacity of NMC-g-PEI was improved but still lower than that of PEI. The buffer capacity of copolymers decreased with increasing molecular weight of
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Figure 5. SEM images of (a) NMC5K-g-PEI/DNA (w/w ) 20/1), (b) NMC10K-g-PEI/DNA (w/w ) 20/1), and (c) NMC50K-g-PEI/DNA (w/w ) 20/1) complexes.
Figure 6. ζ-Potential of NMC-g-PEI/DNA complexes at various w/w ratios.
Figure 7. Buffering capacity of PEI (25 KDa), PEI (800 Da), and the NMC-g-PEI copolymers in 150 mM NaCl solutions.
NMC chain. The possible reason is as follows. The buffer capability of polycations depends on the presence of primary, secondary, and tertiary amines groups, when chitosan grafted with PEI (800 Da), the amine group of per copolymer molecule increased and resulted the improved buffer capacity as compared with chitosan. Nevertheless, compared with PEI, the charge densities of copolymers was lower, so the buffer capacity of copolymers was lower than that of PEI. In addition, with the grafting degree of PEI decreasing, the charge densities of the copolymer decreased also, therefore the buffer capacity of NMCg-PEI decreased with increasing molecular weight of NMC chain. In Vitro Transfection. To investigate in vitro gene transfer capability of NMC-g-PEI copolymers, transfections of 293T and
HeLa cells by pEGFP-C1 and pGL3-Luc were carried out, respectively. Figure 8 showed the typical fluorescence images of the transfected 293T and HeLa cells. It can be seen that NMC5K-g-PEI and NMC10K-g-PEI copolymers have relatively higher gene transfection ability, whereas the gene transfection ability of NMC50K-g-PEI is lower. The gene transfection activity of the copolymers was also evaluated by luciferase assay, using PEI (25 KDa) at its optimal ratio (N/P ) 10) as the control. From Figure 9 we can find that transfection activity of the copolymers was dependent on cell lines, and the higher transfection activity was observed in 293T cell line. In addition, the gene expression increased with increasing NMC-g-PEI/DNA weight ratio and gradually approached saturation values. The optimum weight ratios for the copolymers are in the range of 30 to 35. Among the three gene vectors, NMC10K-g-PEI showed the highest transfection activity. In its optimum value, NMC10Kg-PEI has higher gene transfer ability than PEI (25 KDa), which has been considered to be the highly effective cationic gene vector. Interestingly, NMC5K-g-PEI has a slight lower gene transfer ability compared with NMC10K-g-PEI, while NMC50Kg-PEI has the lowest gene transfection activity. It is reported that the molecular weight of chitosan has a major influence on its biological and physicochemical properties,43,44 and chitosan with low molecular weight (Mw < 5000) had difficulty in forming a complex with DNA. When low molecular weight chitosan was grafted with PEI (800 Da), the binding ability and buffer capacity were improved; therefore, the transfection activity was enhanced. The low gene activity of NMC50K-gPEI may be attributed to the following reason. NMC50K-g-PEI with the high molecular weight could much easily entangle free DNA through electrostatic interactions, and the strong interactions between DNA and NMC50K-g-PEI resulted in highly stable complexes, thereby preventing dissociation within the cell and ultimately precluding the translation of DNA. Similar findings were also reported that chitosan of high molecular weight could form extremely stable complexes with DNA and the release of DNA from the chitosan/DNA complexes was affected, resulting in low gene expression.11,45 The development of gene vectors that are stable in serum is very important for the practical application of gene therapy in vivo. One of the practical problems for in vivo gene delivery mediated by PEI is that the transfection is inhibited by serum.46 Thus, in the current study, we investigated the effect of serum on the transfection activity of the NMC-g-PEI copolymers. As show in Figure 10, the gene expression of NMC5K-g-PEI and NMC10K-g-PEI were not affected obviously in the presence of
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Figure 8. Typical fluorescence images of (A) 293T and (B) HeLa cells transfected by NMC-g-PEI/DNA complexes at the optimized polymer/ DNA weight ratios and PEI/DNA complexes at optimized N/P ratio. (Aa and Ba: fluorescence field images; Ab and Bb: bright field images).
Figure 9. Expression of pGL3-Luc mediated by NMC-g-PEI/DNA complexes at various NMC-g-PEI/DNA weight ratios for (A) 293T and (B) HeLa cells.
serum, while the transfection activity of NMC50K-g-PEI was improved greatly than that without serum. Sato et al. reported that the transfection efficiency of chitosan increased about 2-3 times higher than that without serum.23 Erbacher et al. also showed that the transfection efficiency of chitosan was higher in the presence of 10% serum.47 The increased transfection ability of NMC50K-g-PEI in presence of serum may be attributed to the possibility that the serum components contributed to the DNA release from the compact complexes. In addition, the cell function was raised by the addition of serum,23 and the gene transfer ability was enhanced. However, the detailed mechanism still needs further investigations.
Conclusion In this study, three NMC-g-PEI copolymers were synthesized by using chitosans with different molecular weights. After the introduction of low molecular weight PEI, all the copolymers exhibit improved solubility and buffering capacity. These copolymers were evaluated as new gene vectors and they showed suitable physicochemical properties for gene delivery. NMC5K-g-PEI and NMC10K-g-PEI showed comparable transfection activity and lower cytotoxicity as compared with PEI (25 KDa) in both 293T and HeLa cells, whereas NMC50K-gPEI showed higher cytotoxicity and lower transfection activity
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Figure 10. Effect of serum on gene transfection. 293T cells were incubated with copolymer/DNA (pGL-3) complexes in the presence of 10% serum or in the absence of serum.
compared with NMC5K-g-PEI and NMC10K-g-PEI. The presence of fetal bovine serum did not affect the transfection activity of NMC5K-g-PEI and NMC10K-g-PEI remarkably but resulted in increased transfection ability of NMC50K-g-PEI. Acknowledgment. This work was financially supported by National Key Basic Research Program of China (2005CB623903) and Ministry of Education of China (Cultivation Fund of Key Scientific and Technical Innovation Project 707043).
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