A Study Of The Novel Nanometer DNA Transferring System And Its Application To Cartilage Repairing And So On

Objective To study the preparation of low molecular weight chitosan (LMWC) by controllable degradation of high molecular weight chitosan (HMWC). To construct a novel nanometer DNA transferring system to deliver gene in vitro and in vivo safely and efficiently with DNA-loaded low molecular weight chitosan nanoparticles. Physico-chemical property and biological characteristics were observed for the further application in the articular cartilage injury. To investigate the transferring mechanism of this nanometer local delivery system (NLDS). To evaluate the quantity and location of target gene expression with the premise of safe, controllable, persistent and validity transfection. And to study the separation, cultivation, freezing and preservation, resuscitation and passage of chondrocytes from rabbit's cartilage articular. To evaluate the activities of transfection to chondrocytes in vitro and in vivo. To explore the necessary conditions and methods of developing this system into a clinically applicable approach to enhance natural repair mechanisms by in vivo transfection using a non-viral gene delivery system targeting at chondrocytes. And according to the experiment results, some modification should be made. At the same time, to investigated the effect of this system in other fields of joint surgery by combination of ASO: prosthesis failure and rheumatoid arthritis.Methods High molecular weight chitosan was chemically treated with NaNO2 in 0.1mol/L acetic acid at 25°C for 10h to produce low molecular weight chitosan. After marking LMWC with FITC, the average molecular weight of HMWC and LMWC were determined by HPLC. The average amino group content of chitosan was determined by metachromatic titration using Xylidine Tonceau 2R (C.I. Acid Red 26). The plasmids of EGFP,β-Galactosidase and TGF-β1 were used as target gene to generate DNA-loaded low molecular weight chitosan nanoparticles by molecular self-assembly. Transmission electron microscope (TEM) and atomic force microscope (AFM) were employed to observe these particles and a laser particle analyzer were used to survey the particle sizes and Zeta potentials. And DNase I assay was performed to test the gene protential of the nanopaticles. The cytotoxity of the nanopaticles was examined by the MTT assay. The transfection activities to the chondrocytes of the nanopaticles were valued by in vitro and in vivo gene transfection test. Then these nano-complexes then were employed to animal models with full-thick cartilage defect to demonstrate the feasibility of delivering the growth factor gene in vivo, with the aim to promoting cartilage healing by intra-articular injections. At the same time the quality and location of target gene expression also been observed, and their fluctuation changed with time as well. Before a in vivo transfection was performed, a safety assessment was done. Besides, the murine calvaria osteolysis model in ICR mice and type II collagen induced RA model in DBA1 mice also were employed to evaluate this system in wide rang.Results LMWC had far lower viscosity than HMWC, and could dissolve in the 0.1mol/L acetic acid quite easily. After being treated with NaNO2, the average molecular weight of HMWC decreased from 145 KDa to 18 KDa. Although the average amino group content of chitosan also decreased from 71% to 51% as one of the results of oxidation reaction. From the ultrastructure observation, we can find that LMWC can form stable complexes with DNA effectively, and the LMWC/ DNA complexes are found to be near spherical of 70 nm in diameter with homogeneous structure. Preliminary studies were also performed to identify the experimental conditions for the formation of LMWC/DNA complexes. Results show that LMWC can condense plasmid DNA effectively. When the N/P ratio of LMWC/DNA complex increased from 1 to 6, the zeta potential increased from -7 mV to 13 mV, and the effective diameter of complexes decreased from 340nm to 250nm. At the condition of N/P ratio of 5 , the zeta potential was 7.35mV±0.22mV and the average effective diameter of complexes was 258nm±2.5nm. Nanopaticles with positive charges could be formed by the interaction of chitosan and plasmid, that can protect the DNA from degradation by DNase I in certain degree. There is almost no toxicity to chondrocytes. Gene could be effectively expressed in the chondrocytes, when the latter were transfected by the gene-loaded low molecular weight chitosan nanoparticles. When used in vivo, LMWC/ TGF-β1 gene nano-complexes could enhance the transfection efficiency and prolong the expression of TGF-β1 gene. In the animal models of articular osteochondral defect of rabbits, much better healing and much gentler degeneration could be observed comparing with the controls. And when carrying ASO, this system also can suppress particle-induced osteolysis after the implantation of Co-Cr-Mo alloy particles. The expression of TNF-αmRNA and protein descented dramatically, and at the same time, the quantity of osteoclast, the quality of TRAP, and the area of resorption pit decented too. And this suppression can reestablish by the addition of TNF-α. When applied in the treatment of rheumatoid arthritis, this system with anti-TNF-αASO could suppress the body weight loosing in the type II collagen induced rheumatoid arthritis model mice. And arthrocele could be also suppressed by this system. X ray and functional test verified this suppression.Conclusion LMWC from HMWC has very good dissolubility and very low toxicity, and can carry DNA to form a nanometer local delivery system. Gene-Loaded Low molecular weight chitosan nanoparticles can delivery DNA effectively as a vectors to chondrocytes in vitro and in vivo. During this course, some superiority such as DNA protection, delayed release and passive targeting were observed. When carrying TGF-β1 plasmids, this system can delivery the therapeutic factor gene efficiently, and harvest very good result in rabbit's articular cartilage defect model. When carrying anti- TNF-αASO, this system also can delivery oligonucleotide to cells, suppress the TNF-αdramatically, and provide a new treatment strategy for particle induced osteolysis and rheumatoid arthritis. Gene-Loaded Low molecular weight chitosan nanoparticles is a novel nanometer DNA transferring system and has a very nice prospect.