Chitosan As A Preliminary Study Of Gene-based Drug Carriers And Cell Scaffolds

Chitosan (CS), one of natural polysaccharides, have some amidocyanogen and cationic charge after its solubility in dilute acids. Chitosan is abundant in nature and its production is of low cost. Chitosan have favourable biological capability, chitosan is known for being biocompatible. Moreover, chitosan is metabolised by certain human enzymes, and is considered as biodegradable. It possesses antimicrobial property and absorbs toxic metals. In addition, it has good adhesion, coagulation ability, and immunostimulating activity. Chitosan can be produced easily in various forms such as powder, paste, film, fiber, etc. Chitosan is currently receiving a great deal of attention for medical and pharmaceutical applications because of its appealing intrinsic properties.This project carried out an investigation of utilize of chitosan as gene delivery vector and porous scaffold material.We studied the cellular uptake of antisense onligodeoxynucleotides (ODN) mediated by chitosan in vitro. The ODN/CS complexes were prepared by auto-coacervation. The ODN/CS complexes was characterized by particle sizes, agarose electrophoretic retarding. The cellular uptake of ODN/CS in COS7 cell lines were carried out. The effects such as N/P, pH of culture media and serum concentration on cellular uptakes efficiency were investigated. At the same time the chitosan was grafted tetramethyl rhodamine isothiocyanate (TRITC) and complexed with FITC-ODN. The distribution of TRITC-CS and FITC-ODN in cell were determined by fluorescence microscopy. The result showed that ODN/CS complexes could self-assembled through electrostatic effect;Particles size of ODN/CS was under 200nm;ODN in agarose electrophoretic was retarded mostly when N/P was 3, which showed completely and efficiently binding was achieved. It is well known the cell is negative charge and the CS/ODN complex ispositive charge. The CS/ODN complex was adsorbed onto the surfaces of cell and then endocytosed. The positive rate of cell increased with augmentation of N/P. The pH of cultured media could affect the cellular uptake. When the pH was 6. 9, the rate of cellular uptake was highest. There is many components in serum including serum proteins and cationic chitosan by electrostatic absorption. The resulted showed that the low concentration of serum (10%s 20%) had little effect on the cellular uptake almost, and the high concentration of serum (30%) reduced the cellular uptake dramatically. The double marked fluorescence of ODN/CS complex revealed the distribution of CS and ODN. The CS and ODN existed in the cytoplasm and could escaped from endosomes.Although the transfection efficiency of cationic liposome was much higher in non-viral vectors, the toxicity of cationic lipid was relativity great. In contrast to the low toxicity, average liposome which had anionic charge could not be suit for vectors of gene delivery because of its low encapsulate efficiency. Chitosan, one of cationic polymers, was famous for low toxicity, and also well known for its low efficiency of delivering gene. We combined the technology of cationic polymers and liposome. The liposome-DN/CS (L-DN/CS) was formed by mixing the liposome and ODN/CS. The average liposome-ODN/CS (L-DN/CS) was characterized using transmission electron microscope, particle and zeta potential sizer. The hela cell line was cultured in vitro. The toxicity of CSs liposome and mixture of CS and liposome was evaluated by MTT experiment and microscope. The cellular uptake of L-ODN/CS and tranfection mediated by L-ODN/CS was investigated. The liposome was modified by Span-40 and encapsulated Chloroquine for higher transfection. The result showed that the zeta potential of L-DN/CS was declined and the particle sizes was enlarged contrasting to ODN/CS. There was three types of electrostatic coalescence such as (l)adsorption> (2) surroundings (3) encapsulating by the observingof electron microscope. The toxicity experiment showed that CS> liposome and mixture of CS and liposome almost had no toxicity and the cationic liposome had high toxicity. The cellular uptake efficiency of L-DN/CS was much higher than that of chitosan. L-DN/CS had finer transfect efficaciously according to chitosan, especially when the liposome was modified with Span-40 and encapsulated chloroquine.In the study of tissue engineering , it is the primary side to prepare cell scaffold. Chitosan is one of the most important materials. To prepare cells scaffolds with the characteristics of sustained release of proteins. Firstly, Chitosan scaffolds was prepared by freeze-drying. The porosity and water content of scaffolds were determined. Secondly , the bovine serum album was selected as a model protein. The poly (lactic-co- glycolic acid) (PLGA) microspheres were prepared by double emulsion solvent evaporation and encapsulated into chitosan scaffolds. Thirdly, the morphology of PLGA microspheres and various scaffolds were observed using scanning electron microscope. Release behavior of BSA from various chitosan scaffolds was investigated. The results showed that the chitosan scaffold represents porous. At the - 70 "C of quenching temperature, the porosity and water content of chitosan scaffolds were 78.6+1.5% and 85.1+6.2 % , respectively. PLGA microspheres can be uniformly encapsulated into scaffolds without any morphology change. Significant sustained release of BSA from PLGA microspheres encapsulated into scaffolds was obtained. The cumulative release at 168 h was only 33.5%, while that of BSA from chitosan scaffolds at 24 h was above 90%. The release behavior can be controlled by adjusting the amount of chitosan in scaffolds and the type of PLGA. As a conclusion :the novel chitosan scaffolds encapsulating PLGA microspheres proved to be a promising cells scaffolds with controlling the release of growth factors in tissue engineering.To improve the growth of cell on chitosan scaffolds, , porous chitosanscaffold was modified with polyanionic materials such as sodium alginate, gelatin, and sodium heparin . The porosity and water content of scaffolds were determined,, The morphology of blank scaffold and growth situation of Hepatocytes on scaffolds were observed by microscope and scanning electron microscope. The affinity and proliferation rate on different scaffolds were evaluated using MTT. The result showed that polyelectrolyte shaped on the inner surface after the scaffold were modified with polyanion. and then the porosity and water content of scaffolds increased. The porosity and water content of scaffolds modified with sodium alginate and gelatin respectively were 536.7 + 45% , 888.1 ±39%, 457. 8 ±38%, 781. 1+34%;. The Hepatocytes grew onto the inner surface without morphology change in evidence, which was testified by observing the scaffold modified with gelatin using scanning electron microscope. The result also showed the affinity on the unmodified scaffold was much higher than others because of the cationic charge of chitosan . On the contrary the affinity rate became low as the cationic charge was neutralized by polyanion. Sodium alginate and gelatin was physiological factor and could accelerate the proliferation of Hepatocytes. The proliferation rates on the scaffold modified with sodium alginate and gelatin were higher than unmodified scaffold .There was no obviously change of proliferation on the scaffold modified with sodium heparin.