Combining Amphiphilic Chitosan And Bioglass For Mediating Cellular Osteogenic Growth Peptide Gene

Gene therapy has already begun to be used in the diagnosis and treatment of primary disease. Gene transfection is the key step, and the research of the vector is the hot spot of research. In this paper, the research object of the composite of biological glass and the amphiphilic chitosan was used to explore the function and mechanism of gene transfection.In this paper, a sacrificial liquid template method was used to prepare the bioglass. Its particle size was measured in the range of 100-150 nm by SEM and Zeta. The morphology of the particles was similar to that of the sea hedgehog-like morphology, and the interior was rich in nanopore structure. In addition, the modification of chitosan has improved the solubility of chitosan in aqueous solution. mPEG as the hydrophilic segment and PCL as hydrophobic segment were introduced into the side chain of chitosan, which made it become the amphiphilic chitosan. The value of CMC was characterized by UV. The average molecular weight of CMC was measured by GPC. The chemical structure was characterized by FT-IR and NMR.A full-length OGP sequence was found in the universal GenBank database and was purchased from Thermo Company. The resulting products were digested with Xho I/Hind III and then cloned into the mammalian cell expression vector, pAc-EGFPN1. Plasmids were verified by PCR, enzyme digestion and sequence analysis until the results showed that the gene sequence was correct. The recombinant plasmid included OGP, GFP, and Kanamycin resistance gene. We named the recombinant plasmid as pOGP.In our present study, on the one hand, the smaller size of MBG, which was about 140 nm,has been found to be suitable for gene transfection; on the other hand, the amphiphilic chitosan(CS), including PEG as a hydrophilic segment and polycaprolactone(PCL) as a hydrophobic one, has been investigated as a gene vector due to its self-assembly to form micelles in aqueous solutions. The aim of our present study is to evaluate the influence of MBG for the gene transfection of CS-PCL-mPEG/pOGP complexes in different types of cell lines. Osteogenic growth peptide(OGP) was chosen as a model gene, which can induce differentiation of osteoblasts. The designed model gene has 4.7 kb, which is longer than siRNA or EGFP gene and more difficult to be delivered. The MBG/CS-PCL-mPEG/pOGPcomplexes were investigated in terms of zeta potential, particle size, pOGP binding ability,and in vitro pOGP release. The in vitro gene transfection efficiency of the complexes was monitored in two cell lines(HeLa and MG63).