Preparation And Properties Of Hyaluronic Acid Oligosaccharide Modified Chitosan Microspheres As Drug Carrier

As the world's second-largest killer,cancer has seriously threatened human health and has become one of the difficult problems that humanity needs to overcome.At present,there are three main methods for cancer treatment,including chemical drug therapy(chemotherapy),radiation therapy(radiotherapy),and surgical treatment(surgery).Among them,radiotherapy and surgery can not eliminate the metastatic lesions.Chemotherapy drugs into the blood,in theory,can reach all parts of the body to kill the metastatic tumor cell,therefore,chemotherapy is still one of the most promising methods of cancer treatment.However,the toxicity of chemical drugs is relatively large,and normal cells are killed in the course of chemotherapy.In addition,some drugs are poorly soluble in water and are easily metabolized by the body,failing to exert efficacy Therefore,further improvement in the medication mthods and drug routes is needed.Drug carriers prepared from natural polymeric materials can serve as sustained release and targeting,and are therefore widely used in the treatment of tumors.Such as hyaluronic acid(HA)and chitosan(CTS),in which HA can recognize the CD44 receptor on the surface of tumor cells,playing a targeted role,CTS can improve drug stability,playing a sustained release role.However,the stability of HA alone as a drug carrier is poor,and it is easily degraded,which severely limits its application prospects as a drug carrier.The carrier rate of CTS alone as a drug carrier is low.Therefore,crosslinking HA and CTS can make up for the inadequacies of each other.,increasing in vivo stability,increasing drug loading,and can preserve the targeting of HA to tumor cells,as well as the role of sustained release of CTSAt present,many studies have focused on the use of HA as a drug carrier targeting material,but the use of HA is a large molecule,the targeting is weak.The reason is that the cell surface CD44 receptor is multivalently bound to a relatively high molecular weight HA,which masks the binding site of the CD44 receptor and leads to the inactivation of the CD44 receptor.whereas the small molecule HA can not only fully bind to the cell surface CD44 receptor,but also competes with the endogenous macromolecule HA for binding to the receptor CD44,and thus can improve the targeting of tumor cells.In this thesis,CTS was used as a carrier,low molecular weight HA was used as a CD44 receptor on tumor cell surface,and docetaxel(DTX)was used as an antitumor drug to prepare microspheres.The main work is as follows:(1)Different molecular weights of HA are linked to CTS by EDC/NHS cross-linking method.HA includes HA with a molecular weight of 5K(5K HA),hyaluronan oligosaccharides(OHAS),and HA with a molecular weight of 870K(870K HA).5K HA-CTS,OHAS-CTS,and 870K HA-CTS were prepared,and the HA content in the 5K HA-CTS was measured to be about 12%to 14%.The content of HA in OHAS-CTS is similar to that of 5K HA-CTS,which is about 13%.The content of HA in 870K HA-CTS is about 20%.FTIR proved that HA-CTS was successfully prepared.After that,the fluorescent dye FITC was respectively connected with the carrier material CTS and HA-CTS to obtain CTS-FITC and HA-CTS-FITC,which prepared for subsequent microspheres into the cell experiment.(2)CTS microspheres,5K HA-CTS microspheres,OHAS-CTS microspheres,DTX-5K HA-CTS microspheres,CTS-FITC fluorescent microspheres,5K HA-CTS-FITC fluorescent microspheres,and OHAS-CTS-FITC fluorescent microsphereswere successfully prepared by emulsification cross-linking method.The SEM morphology observation showed that the microspheres had regular morphology,smooth surface,and no adhesion between the microspheres.(3)The HPLC method was used to determine the drug loading,encapsulation efficiency and drug release of the microspheres.The drug loading rate and entrapment rate results are as follows:The drug/carrier is 1/7 microsphere encapsulation rate is about 77.95%,the drug loading rate is about 9.74%;the drug/carrier is 1/5 microsphere encapsulation rate is about 70.51%,the drug loading rate is about 11.75%;drug/carrier is 1/3 of the encapsulation rate of microspheres is about 57.31%,the drug loading rate is about 14.33%;For microsphere of drug/carrier is 1/7,encapsulation rate is about 77.95%,drug loading rate is about 9.74%;For microsphere of drug/carrier is 1/5,encapsulation rate is about 70.51%,drug loading rate is about 11.75%;For microsphere of drug/carrier is 1/3,encapsulation rate is about 57.31%,drug loading rate is about 14.33%.The drug release experiment results are as follows:We measured that for drug/carrier is 1/7 microspheres,drug/carrier is 1/5 microspheres and drug/carrier is 1/3 microspheres,Drug release is complete in approximately 4 days in PBS with pH is 7.4.The release rate reached about 94%;In the PBS with pH is 7.0,6.7,and 6.4,the release rate of microspheres accelerated in turn.(4)Cell proliferation inhibition test:the thiazolyl blue colorimetric assay(MTT method)was used to determine.the selected cells were human lung cancer cell line A549,human breast cancer cell MCF-7,pig endothelial cell PIEC,human normal breast cell MCF-10A,Drug-loaded microspheres have a significant inhibitory effect on cancer cells.(5)Microspheres entering the cells:The microspheres can be taken up by the cells by laser scanning confocal microscope(LSCM observation,and the uptake of HA-CTS microspheres by A549 cells and MCF-7 cells is greater than the uptake of PIEC cells and MCF-10A cells,indicating that HA-bound CTS microspheres have selective or potential targeting to tumor cells.The results of laser confocal microscopy were further confirmed by the results of flow cytometry.It was found that OHAS was more likely to enter tumor cells than 5K HA-attached CTS microspheres,indicating that the targeting of OHAS was better.Through the research of this subject,it was found that the prepared vector has a sustained release and targeting effect on tumor cells.