Gene Delivery Based On Sustained Releasing Core-shell Structure For Therapy Against Bone Defect

Objective In the past decades, gene therapy has been emerging as a novel approach for treating disease. However, the gene therapy vectors could so far conducted transient tansfection, little cell factors and bone mass express which greatly limits the development of bone tissue engineering. Recently, we are development a derivative of chitason as a novel transfection agent which takes advantages in high transfection efficiency, superior biocompatibility, biodegradability and sustained release of cell factors. Bone morphogenetic protein 4(BMP4) is playing a key role in several stage plays in bone formation by inducing the animal or human’s mesenchymal stem cells to differentiate into bone, cartilage, ligament, tendon and nerve tissue. And now it has become the focus in the area of osteogenic induction. This study is aim to prepare and evaluate the shell-core nanoparticles hydroxybutyl chitosan-thiolated N-alkylated chitosan encapsulated with enhanced green fluorescent protein encoded plasmids as a sustained-release gene carrier.Methods Thiolated N-alkylated chitason and hydroxylbutyl chitason were taken to interact with e GFP-BMP plasmid to form TACS/HBC-p BMP4-EGFP. Then dynamic light scattering, Transmission Electron Microscope, agarose gel electrophoresis were to test whether they could delivery plasmid into cell. The capacity of protect the plasmids from nuclease degradation was shown by gel agarose electrophoresis. And it was tested to wonder the nanoparticles could be used to transfect HEK293 in vitro.Building bilateral 18 mm in length full bone defect model, and then we implanted allogenic acellular bone matrix carrying nanoparticles. At predefined time intervals like0, 4, 8 weeks after operation,.Results With addition of thiolated N-alkylated chitason, the diameter of formed nanoparticle became smaller. When the N/P ratio is 8, the diameter of nanoaprticle is less than 200 nm. After forming a shell out of nanoparticle by addition of hydroxylbutyl chitason, the diameter became a litter larger and the zeta potential came to more neutral.We took advantage of some plasmid which contains both the base pair sequence of e GFP and BMP plasmid. The expression of e GFP was tested by flow cytometry and the expression of BMP was examined by Western blot. X-ray showed that more woven bone-like tissue were visible and trabecular-like structure was formed in the erperiment group.Conclusion TACS/HBC-p BMP4-EGFP owns the ability of sustained release and may be used to repaire bone defect.