Construction Of Chitosan-based Drug Delivery System And Study On The Controlled Release Of CCCH-ZAP

ALV-J is a tumorigenic retrovirus,which could cause severe immunosuppression by inhibiting T lymphocytes,especially CD⁴⁺cell proliferation.ALV-J could induce the occurrence of bone marrow cell tumor and many other tumors in chickens,and cause severe immunosuppression in infected chickens,resulting in huge economic losses to poultry industry.Studies have shown that CCCH-ZAP exerts an antiviral role by activating T lymphocytes,indirectly promoting the production of antiviral antibodies,and thus inhibiting the replication of ALV-J.However,traditional transfection in vivo is not practical for large-scale and intensive poultry industry.Besides,due to the poor stability of protein drugs and stress response caused by injection,it is particularly important to find reasonable non-invasive methods of CCCH-ZAP administration.Oral administration is the most convenient way,but the oral utilization rate of protein drugs is low,so designing an appropriate oral administration system for protein drugs is the focus of this study.Chitosan-based drug delivery system is safe,non-toxic,cheap and easy to get.It is widely used in the drug-delivery system.Chitosan could be protonated under acidic conditions and the deprotonated under neutral conditions.Based on this characteristic,it is widely used in the field of drug loading,but mainly in the biomedical field.In this paper,we intend to build a p H-responsive chitosan drug delivery system through ion cross-linking or covalent cross-linking,so as to realize the loading and targeted and controlled release of CCCH-ZAP,and solve the problem of low utilization rate of protein drugs through oral administration.Main research contents and results are as follows:（1）Through ion cross-linking,COS-TPP drug loading system was constructed,and its morphology was characterized,the factors affecting its particle size and loading rate were discussed.With BSA as the model protein,the Zeta potential and particle size of nanoparticles before and after loading BSA were measured,and the loading mechanism and release in vitro were discussed.Results showed that the loading rate of BSA reached 66.5%when p H was 7.4.After 48 h,the release rates of BSA at p H=7.4,5 and 2 were 89.2%,81.5%and 18.6%,respectively.The antiviral activity,cytotoxicity and transmembrane transport of CCCH-ZAP were investigated.Results showed that COS-TPP system could realize the loading of CCCH-ZAP,with a loading rate of 58.3%when p H was 7.4.After loading,the inhibition rates of CCCH-ZAP on ALV-J virus were enhanced significantly,and the inhibition rate reached 46.0%at 72 h.The system above could protect protein drugs from the degradation of gastric acid conditions,achieve sustained release in intestinal epithelium,and is suitable for sustained release in the weakly acidic environment of cancer cells.It is a potential carrier of protein drugs and anticancer drugs.（2）Through ion cross-linking,CSH/SBE-β-CD loading system was constructed,and BSA was used as the model protein.The results showed that the loading rate of CSH/SBE-β-CD on BSA was 71.9%when p H was 7.4.At p H 7.4,5 and 2,the 48 h release rates of BSA were84.2%,89.5%and 26.0%,respectively.CCCH-ZAP could be loaded by CSH/SBE-β-CD system,and the load rate is 61.5%when p H is 7.4.After loading,the inhibitory rate of CCCH-ZAP on ALV-J was significantly enhanced,and the inhibitory rate reached 55.0%after 72 h.With the increase of time,the gap between the inhibitory activity of CCCH-ZAP and that before loading increased.The system could be loaded with protein drugs and anticancer drugs.（3）Through covalent cross-linking,COS-PAMK and COS-PGPS nanodrug-carrying systems were constructed,and their morphologies were characterized.The influencing factors on the preparation of nanoparticles were also discussed.With BSA as the model protein,the loading ability of COS-PAMK and COS-PGPS nanoparticles were investigated.The particle size and Zeta potential of the nanoparticles were measured before and after loading.The results show that both systems could realize the load of BSA,with load rates of 36.7%and 31.3%.At p H 7.4,5 and 2,the release rates of BSA in COS-PAMK system were 62.5%,92.8%and 22.1%,respectively.In the COS-PGPS system,the load rates of BSA were 52.6%,80.8%and 20.2%,respectively.COS-PAMK and COS-PGPS systems could load CCCH-ZAP,and the loading rates are 21.2%and 18.9%when p H is 7.4.Follow-up studies are underway.