Construction Of FA-BSP-SA/TPGS Micelles Drug Delivery System And Its Anti-tumor Effect Of Loading With Zinc Phthalocyanine And Doxorubicin

In the early stage of our research group,we have constructed a p H-sensitive FA-BSP-SA and TPGS mixed micellar delivery system.In the study,we found that the addition of TPGS can enhance the inhibitory effect on breast cancer 4T1 cells.This study combined in vitro quality evaluation parameters（encapsulation efficiency and loading capacity and particle size,etc.）,biocompatibility and comprehensive evaluation of in vitro anti-tumor experiments to further explore FA-BSP-SA and TPGS as the best quality ratio of the drug delivery system,and used doxorubicin（Dox）and Zinc phthalocyanine（ZnPc）as model drugs to investigate their synergistic anti-tumor effects.This research consists of three parts:（1）FA-BSP-SA copolymer and ZnPc were prepared by chemical synthesis and characterized by ¹H-NMR.The critical micelle aggregation concentration was determined by pyrene fluorescence spectrometry.FA-BSP-SA/TPGS micelles,Dox@FA-BSP-SA/TPGS micelles and ZnPc@FA-BSP-SA/TPGS micelles were prepared,and the particle size,Zeta potential,encapsulation efficiency（EE）and loading capacity（LC）were measured.The drug release behavior in vitro of free drugs and drug-loaded micelles at different p H values and temperatures was studied by dialysis bag method.The safety evaluation of blank micelles was done through hemolysis test.MTT method was used to determine the cytotoxicity of FA-BSP-SA/TPGS micelles on HCM cells,and the in vitro anti-tumor effects of FA-BSP-SA/TPGS micelles,Dox@FA-BSP-SA/TPGS micelles and ZnPc@FA-BSP-SA/TPGS micelles on 4T1 cells.Based on the above experimental results,determined the best quality ratio of FA-BSP-SA and TPGS.（2）The MTT method was used to determine the in vitro synergistic anti-tumor effects of Dox@FA-BSP-SA/TPGS₂₅ micelles+ZnPc@FA-BSP-SA/TPGS₂₅ micelles.The level of ROS,mitochondrial membrane potential,apoptosis and intracellular uptake were observed and photographed by fluorescence inverted microscope.（3）The distribution of micelles in tumor sites and main organs of tumor-bearing BALB/c mice were observed by targeted distribution experiment in vivo.And the anti-tumor effect of drug-loaded micelles in vivo had been studied.The critical micelle aggregation concentrations were 5.88,13.18,12.88,12.59and 35.48μg/m L when the mass ratio of TPGS was 0%,15%,20%,25%and 100%,respectively.The LC and EE of hydrophobic doxorubicin by electrostatic adsorption method were higher than those by alkali neutralization method.It was determined that the best quality ratio of TPGS was 25%by investigating the particle size,Zeta potential,LC,EE,hemolysis rate and anti-tumor experiments in vitro.The particle sizes of FA-BSP-SA/TPGS₂₅,Dox@FA-BSP-SA/TPGS₂₅ and ZnPc@FA-BSP-SA/TPGS₂₅micelles were 110.2 nm,107.9 nm and 114.47 nm,respectively.Zeta potentials of FA-BSP-SA/TPGS₂₅,Dox@FA-BSP-SA/TPGS₂₅ and ZnPc@FA-BSP-SA/TPGS₂₅micelles were-18.9 m V,-13.20 m V and-20.63 m V,respectively.The LC and EE of Dox@FA-BSP-SA/TPGS₂₅ micelles were 7.24%and 92.84%.The LC and EE of ZnPc@FA-BSP-SA/TPGS₂₅ micelles were 2.07%and 26.48%.The micelles were p H-sensitive,and as the p H decreased,the particle size tends to increased.In vitro release experiments showed that p H value had no effect on the release of free Dox,while the release rate of micelles was p H-sensitive,and the drug release rate increased with the decrease of p H value.The drug release rate increased with the increase of temperature.When the concentration of FA-BSP-SA/TPGS₂₅ micelles was 0.25mg/m L,the hemolysis rate was 3.50%,indicating that FA-BSP-SA/TPGS₂₅ had good biocompatibility.Cytotoxicity experiments show that when the concentration of FA-BSP-SA/TPGS₂₅ micelles is 50μg/m L,the survival rate of HCM cells is still above 85%,which further proves that its biocompatibility is good.In vitro anti-tumor experiments showed that FA-BSP-SA/TPGS₂₅ micelles,Dox@FA-BSP-SA/TPGS₂₅micelles and ZnPc@FA-BSP-SA/TPGS₂₅ micelles had the best anti-tumor effects.Based on the above experimental results,FA-BSP-SA/TPGS₂₅ micelles were selected as the carrier for further research.The results of the in vitro anti-tumor experiment showed that when the drug concentrations of Dox@FA-BSP-SA/TPGS₂₅ micelles and ZnPc@FA-BSP-SA/TPGS₂₅micelles were 1.10 and 0.10μg/m L,respectively,the survival rate of 4T1 cells was25%.Cell experiments showed that TPGS,Dox and ZnPc could induce tumor cell apoptosis by decreasing mitochondrial membrane potential and increasing the level of intracellular ROS.In vivo anti-tumor experiments showed that the Dox@FA-BSP-SA/TPGS₂₅+ZnPc@FA-BSP-SA/TPGS₂₅ micelle group was treated with a combination of chemotherapy and photodynamic therapy,showing a strong tumor treatment effect.In conclusion,FA-BSP-SA/TPGS₂₅ micelles can target anti-tumor drugs to th In vivo targeting distribution experiment in tumor-bearing BALB/c mice showed that FA-BSP-SA/TPGS₂₅ micelles could make the drug more enriched to the tumor site,so as to reduce the side effects of other organs and tissues,and enhance the anti-tumor effect.Therefore,FA-BSP-SA/TPGS₂₅ micelles are expected to become nano-carrier materials for the targeted delivery of insoluble antitumor drugs.