Preparation And Properties Of Folate/CD44 Dual Receptor Targeted Reduction Responsive PLGA-based Micelles

In this study,poly（lactic-co-glycolic acid）（PLGA）was used as a carrier matrix,hyaluronic acid（HA）and folic acid（FA）were used as target ligands to target tumor sites to express rich CD44 receptors and folate receptors.Disulfide bond was used as a connecting arm to enable micelles to release drugs accurately and rapidly under reductive stimulation.With adriamycin（DOX）as the model drug,a new antitumor drug delivery system with tumor targeting was constructed,which can improve the therapeutic effect of DOX and reduce the side effects,providing experimental basis for the study of PLGA-based micelles.FA-HA-SS-PLGA polymer was synthesized by amidation reaction using EDC and NHS as catalysts.The physicochemical properties of FA-HA-SS-PLGA polymer were characterized and analyzed by Fourier transform infrared spectroscopy（FT-IR）,nuclear magnetic hydrogen spectroscopy,thermogravimetric analysis（TGA）,particle size and Zeta potential analysis,and transmission electron microscopy（TEM）.Then,DOX-loaded micelles were prepared by dialysis method.Response surface methodology（RSM）was used to optimize the preparation process of DOX/FA-HA-SS-PLGA nanomicelles by using Design-Expert software and Box-Behnken method with drug loading ratio,stirring speed and stirring time as three independent variables,drug loading rate and encapsulation efficiency as response values.The size distribution of micelles was analyzed by dynamic light scattering（DLS）and scanning electron microscopy（SEM）.The properties of drug-loaded micelles were analyzed and characterized by hydrophilic experiment,pyrene fluorescence spectroscopy,reduction experiment and hemolysis experiment.The in vitro release of DOX/FA-HA-SS-PLGA in p H 7.4 medium and its stability were investigated.CD44 receptor and folate receptor proteins were screened from PDB database,and the molecular dynamics of synthetic micelles and receptors were studied by Auto Dock Vina software.Finally,MTT method was used to evaluate the cytotoxicity and in vitro antitumor activity of DOX/FA-HA-SS-PLGA.The results of FT-IR,~1H NMR,TGA and Zate potential analysis showed that FA-HA-SS-PLGA polymer was successfully synthesized.The optimization results of the preparation method of DOX/FA-HA-SS-PLGA showed that the preparation process was the best when the drug-loading ratio was 32%,the stirring speed was 612.49 r·min-1,and the stirring time was 4.21 h.At this time,the drug loading rate of DOX/FA-HA-SS-PLGA was 21.02%,and the encapsulation efficiency was 83.61%.The size distribution of nano-drug-loaded micelles DOX/FA-HA-SS-PLGA was uniform,the critical micelle concentration was low,and the micelles were stable.In addition,nano drug-loaded micelles DOX/FA-HA-SS-PLGA also had good hydrophilicity and biocompatibility.In the drug-loaded micelle reduction experiment,it showed sensitivity to the reduction environment.In vitro release experiment was carried out in the medium containing 10m M GSH,and the release amount increased,which further verified that it had the characteristics of reduction stimulus response.More importantly,in vitro cytology studies have shown that compared with free DOX and single receptor micelles,its dual receptor-mediated endocytosis leads to stronger cytotoxicity to tumor cells and more pronounced apoptosis.In this study,DOX/FA-HA-SS-PLGA nano drug delivery system with active targeting effect on tumor was successfully constructed,which improved the antitumor effect of DOX and reduced the side effects of DOX.The experimental results showed that this DOX/FA-HA-SS-PLGA nanocarrier micelles have the potential to be used as chemotherapeutic drugs for precise tumor treatment.