Dextran-spermine Polycation As A Vector For Gene Transfection In Vitro

Background: Gene therapy is an approach for human disease treatment by inserting therapeutic gene into cells in order to produce desired proteins with specific functions and to inhibit expression of abnormal genes. The principle problem encountered in gene therapy is lack of efficient, safe and targeted gene delivery system, the gene vector capable of delivery of gene into targeted cells and achieving high level of gene expression with minimum toxicity. Gene delivery systems are classified into viral and nonviral systmes. Although in vitro and in vivo transfection efficiency of the nonviral gene vectors are lower than those of viral vectors and the gene expression is transient, the nonviral systems offer several advantages including low immunogenicity and increased biological safety. Cationic polymers, polycations, are the most potential class of nonviral DNA deliver system because their physiochemical and biological properties could be easily varied by structural modification.Objective: To synthesize water-soluble dextran-spermine polycation (DSP) as a gene vector, and investigate the methods of preparation of gene gun bullets based on DSP as gluing agent and the biophysical properties of DSP/DNA complexes as well as gene gun bullets, and preliminarily discuss how the biophysical properties of polyplexes contribute to transfection activity.Methods: 40 KDa Dextran was oxidized under room temperature using potassium periodate to obtain oxidized dextran, which was then reacted with spermine to form imine-based polycation. The Schiff base polycation was reduced to the stable amine-based dextran-spermine polycation by sodium borohydride. The total nitrogen and primary amine content were determined by elemental analysis and TNBS, respectively, The chemical structure of DSP was elucidated by ~1H-NMR and FT-IR. By incubating with pEGFP-C1, DSP was able to condense the plasmid to form polyplexes by electrostatic interactions. The particle size and zeta potential were measured by laser particle size analyzer; the physical shape of DSP was observed by TEM; the ability of DSP to complex with plasmid DNA was observed by agarose gel electrophoresis; the protective ability of DSP against DNase I was also demonstrated by agarose gel electrophoresis; the buffering capacity of DSP was examined by acid titration. Polyplexes were introduced to SMMC-7721 and BHK-21 cells in vitro and were visualized with inverted fluorescent microscopy for transfection efficiency assessment. The cytotoxicity of polyplexes was evaluated by MTT assay. The preparation of gene gun bullets base on DSP as gluing agent was also investigated. The stability, ability of protect DNA from DNase I and cytotoxicity of gene gun bullets was individually evaluated by agarose gel electrophoresis, DNase I degradation and MTT assay.Results: The aldehyde content of oxidized dextran was 8.47+0.15 mmol/g and the degree of grafting of DSP was 38.9%0 DSP formed stable polyplexeswhen the weight ratio (DSP/DNA) varied from 4:1 to 20:1. The particle size and zeta potential of polyplexes were in the range of 162.6-187.9 nm and increased from +8.45mV to +39.6mV, respectively. DSP was able to tightly condense and interact with DNA and to effectively protect condensed DNA from DNase I degradation, and it showed strong buffering capacity in a certain pH range. The highest yields of transfection efficiency were 27.0±4.42% and 22.2±4.63%, which were found to be as high as Lipofectamine 2000 when the polyplexes were transfected to SMMC-7721 and BHK-21 cells at the weight ratio of 8:1. The cytotoxicity of polyplexes was dependent on DNA concentration and DSP/DNA weight ratios. The gene gun bullets prepared by DSP were able to firmly glue DNA and to protect DNA from DNase degradation at different DSP/DNA and DNA/gold ratios. The higher the gold content and DSP/DNA ratio, the greater the cytotoxicity.Conclusion: This research indicates particle size, Zeta potential, stability, ability to protect DNA against DNase and cytotoxicity of DSP/DNA complexes influence their transfection activity in certain degree. The synthesized DSP is a highly efficient and easily prepared gene vector with low toxicity, and it is hoped to be applied to clinical gene therapy in the future. The gene gun bullets prepared by DSP showed strong ability to protect DNA against DNase and are expected to enhance transfection efficiency of particle-mediated gene transfer in vivo.