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Bioconjugate Chem. 2009, 20, 322–332
A Biodegradable Low Molecular Weight Polyethylenimine Derivative as Low Toxicity and Efficient Gene Vector Yuting Wen,†,‡ Shirong Pan,‡,* Xin Luo,† Xuan Zhang,‡ Wei Zhang,‡ and Min Feng† School of Pharmaceutical Sciences and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China. Received October 8, 2008; Revised Manuscript Received December 15, 2008
Polyethylenimine (PEI) is a class of cationic polymers proven to be effective for gene delivery. However, PEI is nondegradable and the molecular weight of PEI affects the cytotoxicity and gene transfer activity. Aiming to prepare a biodegradable gene vector with high transfection efficiency and low cytotoxicity, we conjugated low molecular weight (LMW) PEIs to the biodegradable backbone polyglutamic acids derivative (PEG-b-PBLG) by aminolysis to form PEIs combined PEG-b-PLG-g-PEIs (GGI). Two copolymers, GGI 30 and GGI 40, were synthesized. The chemistry of GGI was characterized using IR, 1H NMR and 13C NMR, GPC, and CD, respectively. The degradation behaviors of copolymer GGI in papain solution were investigated. GGIs showed good DNA condensation ability and high protection of DNA from nuclease degradation. The zeta potential of the GGI/ pDNA polyplexes was ∼15 mV, and the particle size was in the range 102-138 nm at N/P ratios between 10 and 30. The particle size and the morphology of the polyplex was further confirmed by transmission electron microscope (TEM). In cytotoxicity assay, GGIs were significantly less toxic than PEI 25k. The degradation product of GGI exhibited negligible effects on cells even at high copolymer concentration. The results of GFP flow cytometry and fluorescence imaging showed that the trasnfection efficiencies of GGIs were all markedly higher than PEI 25k in Hela, HepG2, Bel 7402, and 293 cell lines. Importantly, the presence of serum had a lower inhibitive effect on the transfection activity of GGI in comparison to PEI 25k and Lipofectamine 2000. Therefore, PEGb-PLG-g-PEI copolymers may be attractive cationic polymers for nonviral gene therapy.
INTRODUCTION In the past decade, cationic polymers have become the major type of nonviral gene delivery carriers, due to their advantages over viral vectors, including the ability to deliver larger DNA molecules, low immunogenicity, and relative safety (1-4). Among the cationic polymers, polyethylenimine (PEI) is regarded to be one of the most successful and widely studied gene delivery polymers. PEI has been proven to be effective in gene delivery due to its condensation of DNA, which facilitates endocytosis, as well as its “proton sponge effect”, which can prevent the DNA from endosomal disruption (5, 6). Many studies have shown that the molecular weight of PEI is the most effective parameter for the gene transfection efficiency and cytotoxicity (7-11). It was observed that PEI gene transfer activity increased with an increase in molecular weight, whereas the cytotoxicity appears to increase with increasing polymer size. For example, low molecular weight (LMW) PEI ( Bel7402 > Hela > HepG2.
CONCLUSION In this study, we demonstrated that a novel degradable PEGb-PLG-g-PEIs copolymer, a kind of combined LMW PEIs, was synthesized and evaluated as a new gene vector. The obtained polymers GGI 30 and GGI 40 showed degradability, a great ability to condense DNA and to protect pDNA against DNase I. The physicochemical properties, particle size, morphology, and zeta potential of GGI/pDNA polyplexes were suitable for gene delivery. The in vitro cell viability as well as gene transfection suggested that GGI had great potential as a gene carrier, especially GGI 40, which had significantly lower cytotoxicity and higher transfection efficiency, as compared to PEI 25k. In particular, the transfection efficiency of GGI was not markedly decreased in the presence of serum in comparison with PEI 25k and Lipofectamine 2000. Therefore, GGI can be a promising new nonviral gene delivery vector for future gene therapy applications, due to its degradability, low cytotoxicity, and high transfection efficiency.
ACKNOWLEDGMENT The authors greatly acknowledge the financial support from the National Natural Science Foundation of the People’s Republic of China (30570500). Figure 11. (A) Comparison of the in vitro gene transfection efficiency between GGI 40 and HMW PEI 25k at their optimal N/P ratios in Hela, HepG2, Bel 7402, and 293 cells. Significant differences between GGI and PEI 25k polyplexes are marked with an asterisk: *p < 0.05, **p < 0.01. (n ) 3, error bars represented standard deviation.) (B) Comparison of the fluorescence images of (1) Hela, (2) HepG2, (3) Bel 7402, and (4) 293 cells transfected with (A) HMW PEI 25k at its optimal N/P ratio of 10 and (B) GGI 40 at its optimal N/P ratio of 20 (magnification 200×).
fluorescent spots of Lipofectamine 2000 were much dimmer than that of GGI 40, indicating that GGI 40 expressed more fluorescent proteins than those with Lipofectamine. The results were in accordance with the flow cytometry results, suggesting that LMW PEI combined copolymer GGI was more effective as a gene carrier, compared with HMW PEI 25k and Lipofectamine 2000. In addition, we studied the effect of serum on the transfection efficiency of GGI/pDNA complexes. Since therapeutic gene transfection is in vivo, it is crucial to investigate the influence of serum on the transfection efficiency. As shown in Figure 10B, for GGI 30 and GGI 40 at N/P ratio of 10 and 20, the transfection efficiencies were both slightly decreased in the serum-supplemented medium. At
Supporting Information Available: Figure S1. The aggregation tendency of GGI/DNA complexes prepared in 10 mM NaCl, in comparison with that of PEI 25k. Polyplexes were prepared at N/P ratio of 20. Figure S2. Cytotoxicity of GGI degradation products at the concentration of 1 mg/mL and 2 mg/mL. The GGI degradation products were prepared by inbubating GGI with papain for 24 and 96 h at 37 °C. This material is available free of charge via the Internet at http:// pubs.acs.org.
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