The Preparation And Anti-tumor Activity Of Docetaxel/Chitosan Targeting Nano-drug Delivery System

Gastric cancer(GC)is a common cancer in digestive system.In China,there are approximately 400 thousand cases annually which accounts for 40% of cases of the world and the death rate of GC is in the second place.The common treatment methods for GC are chemotherapy,radiotherapy and surgery.There are many problems in the clinical use of docetaxel,a traditional chemotherapeutic agent for GC.Docetaxel has very poor water-solubility and low targeting ability,which can cause many severe side effects in patients.Chitosan is a kind of macromolecular polymer extracted from shrimp and crab shells and the only alkaline polysaccharide in nature.Chitosan has good biocompatibility,biogradation,non-toxicity and low immunogenicity,which makes it a potential drug carrier.As the nanotechnology develops rapidly,the nanoformulation of docetaxel/chitosan could increase its water solubilty and targeting ability,enhance the bioavailability and anti-tumor effects.This thesis is divided into two parts.Firstly,a new GC-targeted nanoparticle was prepared.Sructurally,GX1 was the GC vascular-targeted molecule and N-deoxycholic acid glycol chitosan was the nano-carrier encapsulating docetaxel,a anti-angiogenesis agent.Different characteristics of nanoparticles were determined and the drug release mechanism was analyzed.Subsequently,the in vitro and in vivo anti-tumor activities were determined.Secondly,three carboxymethyl chitosans with different carboxymethyl sites were synthesized as nano-carriers for docetaxel.The characteristics and in vitro anti-tumor activity of three kinds of nanoparticles were determined.The influence of carboxymethyl sites on nanoparticles was analyzed.The conclusion is as bellows:(1)The GC-targeted nanoparticle(GDD)had encapsulating efficiency of 52.7% and drig loading content of 24.6%.The average size was 150.9 nm and zeta potential was 16.0 mV.GDD was spherical-shaped under TEM and SEM and the surface was smooth and distributed uniformly without accumulation.The release profile of docetaxel from GDD was pH-sensitive controlled release.The release rate under acid pH was significantly faster than that under neutral pH and the release mechanism analysis showed it was non-Fickan diffusion.The in vitro anti-tumor activity study showed that the presence of GX1 could enhance the cellular uptake of GDD in Co-HUVEC cells and GDD had significantly stronger cytotoxicity against Co-HUVEC cells than docetaxel free drug under the docetaxel concentration of 50 ?M.The in vivo study showed that GDD could inhibited the growth of xenografted human gastic tumor in nude mice.The mice had high tolerance for GDD.The TUNEL and HE staining of tumor biopsies showed that GDD induced apoptosis of large amount of tumor cells.(2)Three docetaxel-loaded chitosan with different carboxymethyl sites based(O-carboxymethyl,N-carboxymethyl and N,O-carboxymethyl)nanoparticles were prepared by ionic-crosslinking.The preparing condition of each kind of nanoparticles was optimized by investigated the proportion of carboxymethyl chitosan and CaCl2,the crosslinking agent.The O-carboxymethyl chitosan nanoparticles had the highest encapsulating efficiency and drug loading content and N,O-carboxymethyl chitosan nanoparticles had the largest abosolute zeta potential value.The average sizes of three different nanoparticles were under 200 nm and the size distribution was uniform.The drug release rate and amount of O-carboxymethyl chitosan nanoparticles were the largest among them.The in vitro cytotoxicity showed that the anti-tumor activity against SGC7901 cells of O-carboxymethyl chitosan nanoparticles was strongest,while there was no significant difference between O-carboxymethyl chitosan nanoparticles and N,O-carboxymethyl chitosan nanoparticles.When the concentration of docetaxel was under 50 ?M,the cytotoxicity of three nanoparticles was stronger than the free drug.Furthermore,high cellular uptake of three nanoparticles in SGC 7901 cells was observed under fluorescent microscope.The celluar uptake of O-carboxymethyl chitosan nanoparticles was the most as measured by the flow cytometry,which maybe related to the amount of unsubstituted amine groups.In this thesis,the preparation and characterization of a new GX1-targeting docetaxel/chitosan nanoparticle were described and the in vitro and in vivo anti-tumor activities were determined.GDD exhibited stronger activity than the free drug docetaxel,which provided a new idea for the development of docetaxel nanoformulations with potential clinical use in the future.Besides,after optimizing the preparation condition of carboxymethyl chitosan nanoparticles by ionic-crosslinking,the influence of carboxymethyl sites on physical-chemical characteristics and anti-tumor activity of carboxymethyl chitosan nanoparticles was analyzed.Results showed that O-carboxymethyl chitosan nanoparticles had the strongest anti-tumor activity and the largest cellular uptake,which provided evidence for the development of chitosan-based anti-tumor drugs.