Exploration Of Using Thiolated Chitosan As Carrier To Load Gene Via Oral Drug Administration

With the property of minimal immunogenicity for Thiolated chitosan(TCS) non-viral vectors, the efficacy of nano gene via oral drug administration has been demonstrated. In this paper, TCS nanoparticles with different particle size and potential were manufactured based on ionic crosslinking method. A new TCS nano-microsphere with a shell-core structure was built simultaneously. The stability of TCS-pDNA nanoparticle and TCS-pDNA nano-microsphere loading gene drug via oral drug administration were studied systematically, absorption and expression of TSC as carrier were discussed primarily.The preparation method of TCS nanoparticle was optimized by the use of the response surface method. Results indicated that a good shape and well-dispersed TCS nanoparticles with the particle from 100-500 nm and potential of +20-50 mV can be manufactured, with the condition that the concentration of TCS and sodium polyphosphate was 1-4 mg·mL-1, 1-2 mg·mL-1, the speed and pH was 1000 rpm, 3.5-5.5, respectively. Further study showed that the hollow TCS-pDNA nano-microsphere had spherical particles and smooth surface can be prepared by the use of 1-5% of PCL as shell and 10-60 mg TCS as core, with the speed of 3000-5000 rpm.Based on the pDNA, the TCS-pDNA nanoparticle and nano-microsphere were prepared. The stability of TCS-pDNA nanoparticle in the DNA-I enzyme environment and the stability of TCS-pDNA nanoparticle and nano-microsphere in simulated gastric fluid and intestinal fluid were studied. Results indicated that good stability was obtained for the different potential TCS-pDNA nanoparticle in the pDNA-I enzyme and simulated gastric fluid and intestinal fluid, while the stability increased with the increasing of the particle size. The stability of pDNA was good, for the protection by TCS-pDNA nano-microsphere with the respect to the destruction and degradation from enzyme and pH.The transfection efficiency of TCS-pDNA nanoparticles with different particles in Caco-2 cell was evaluated by establishing the Caco-2 cell model. Results indicated that all the TCS-pDNA nanoparticles have the transfection ability, the transfection efficiency decreased with the increasing of the particle size. Time dependence was observed in the test.The transfection efficiency of TCS-pDNA nanoparticles and nano-microspheres with different particles in small intestine was evaluated by establishing the mouse intestinal loop model. After dosing for 2 d, the expression of transgene EGFP can be detected, and the transfection efficiency decreased with the increasing of the particle size. It should be noted that the transfection efficiency of TCS-pDNA nano-microsphere was makly higher than it of TCS-pDNA nanoparticle.This paper presents results that the use of PCL as the out shell to prepare the shell-core structural TCS-pDNA nano-microspheres can improve the efficiency of gene expression of oarl absorption. Strengthening the quality of the biopharmaceutical of nano gene drug research is essential, for which can lay the foundation for the designation and clinical application of oral nano gene drug dosage form...