Construction And In Vitro And In Vivo Evaluation Of Gene Delivery System With Composite Function

Magnetotactic bacteria synthesized intracellular magnetic nanoparticles by biomineralization, also called magnetosomes or bacterial magnetic particles (BMPs), which were consisted of magnetic core enveloped by stable lipid membrane which contained some lipids and proteins. Magnetosomes with the special characteristics possessed the broad applicable prospects in many fields. After being modified, the BMPs were constructed to a novel gene delivery system with the composite function of magnetic targeting and translocational capability which were used to delivery gene to glioma and investigated inhibition effect in vitro and in vivo in this article.In this article, the crystalline, morphology, crystal-size distributions, iron element content and magnetic properties of the magnetosomes were researched by XRD, TEM, HRTEM, ICP-MS and SQUID. Furthermore, in order to connect magnetosomes with small biomolecule to serve as a new magnetic delivery system, we characterized the functional groups on its surface by FTIR and XPS analysis and quantified the primary amino groups on its surface by fluorescamine assay for the first time, the primary amino groups in the membrane of magnetosomes is 58.58 nmol/mg and its saturation magnetization was 74.33 emu/g. Iron element content in the magnetosomes was 0.605mg/mg, which meaned one milligram of magnetosomes contained 3.3549×1012 magnetosomes nanoparticles. Finally, the cytotoxicity of magnetosome tested with MTT method showed that lipid biomembrane on magnetosome surface endowed them with better biocompatibility.BMPs-PAMAM gene delivery system was synthesized by linking PAMAM to the surface of BMPs using EDC·HCl. On this basis, Tat-BMPs-PAMAM was constructed by connecting tat peptide to the surface of BMPs-PAMAM using Sulfo-LC-SPDP. The configuration, components, magnetic properties and morphology of BMPs-PAMAM and Tat- BMPs-PAMAM were characterized and reaesrched by FTIR,XPS,Zeta Potential Analyzer,TEM,SQUID. The mean size of Tat-BMPs-PAMAM nanoparticle was 50nm and its saturation magnetization was 62.46 emu/g. One milligram of magnetosomes contained 0.056mg PAMAM and 0.027 mg Tat peptides that meaned every magnetosome contained 1472 PAMAM and 3190 Tat peptides.The stable complexes of vectors (BMPs-PAMAM or Tat-BMPs-PAMAM) and plasmid (pEGFP-N1 and pGL-3) at different mass ratios were investigated via agarose gel electrophoresis. The complexes could protect plasmid from digested by DNase-I, and the MTT assay showed that vectors had better biocompatibility which suggested their advantages for gene delivery in vitro. Maximal transfection efficiency of the vectors was obtained at BMPs-PAMAM to plasmid mass ratio of 20:1 and at Tat-BMPs-PAMAM to plasmid mass ratio of 12.5:1. Maximal transfection efficiency of BMPs-PAMAM and Tat-BMPs-PAMAM were 16.65% and 20.56% respectively. In addition, orientation experiment in vitro with the magnetic field, transfection efficiency of BMPs-PAMAM and Tat-BMPs-PAMAM, which were 11.56% and 13.28% respectively would be higher than that of Lipofectamine 2000 which was 9.86%.The biodistribution of 99mTc-labeled complexes of BMPs-PAMAM/pGL-3 and Tat-BMPs-PAMAM/pGL-3 in SD rat was investigated using SPECT. The effections of injection approaches, tat peptide and magnetic field on the biodistribution had also been further investigated in detail.The gene transfection and tumor inhibition assay in vitro were investigated using U251 glioma cell line as cell model. Cell immunofluorescence, cell immuno- histochemistry and western blot were used to analyze the expression of target proteins.And the cell experiments in vitro showed the lowered cell proliferation activity, attenuated cell invasive ability and induced apoptosis of glioma cell after the psiRNA delivery to the U251 cell by BMPs-PAMAM, Tat-BMPs-PAMAM and Lipofectamine 2000. However, Tat-BMPs-PAMAM showed more significant tumor inhibition effect than BMPs-PAMAM and with no difference with that of Lipofectamine 2000.The gene transfection and tumor inhibition assay in vivo were investigated using subcutaneous U251 glioma model established in nude mice. The tumor growth was measured every other day and cell apoptosis and expression of target proteins after gene therapy were detected by TUNEL method and tissue immunochemistry. The results of research in vivo showed Tat-BMPs-PAMAM owned more significant tumor inhibition effect than BMPs-PAMAM and with no difference with that of Lipofectamine 2000, which were in accordance with those in vitro.In this study, the newly nanotechnology and molecular biology were combined to develop a novel kind of non-viral gene delivery system, BMPs-PAMAM and Tat-BMPs-PAMAM. This novel non-viral gene delivery system with high transfection efficiency, safety, magnetic targeting and translocational capability was developed to provide foundation for gene therapy, especially for gene therapy of CNS disease.