Study On Dual-targeting Polymeric Drug Carriers Containing Transferrin And Folic Acid

In the drug delivery system,compared with single-targeting polymeric drug carriers,dual-targeting polymeric drug carriers not only have the characteristics of further selectively entering tumor cells and increasing the drug concentration in the pathological parts such as tumors,but also can effectively prevent single-targeting ligand from being interfered to affect the targeting effect.For example,both transferrin receptors(Tf R)and folate receptors(FR)are overexpressed on the surface of mouse glioma cells.Therefore dual-targeting polymeric drug carriers containing transferrin(Tf)and folic acid(FA)are expected to be taken up more effectively by brain tumor cells.In this thesis,both biotinylated and folate-decorated Pluronic F127/poly(lactic acid)block copolymers[Biotin(FA)-F127-PLA]were firstly prepared by physically mixing biotinylated Pluronic F127/poly(lactic acid)block copolymers(Biotin-F127-PLA)and FA-F127-PLA block copolymers through the nano-precipitation method.Afterwards,the biotin-avidin three-step coupling technique was used to realize the dual-targeting behavior of transferrin and folic acid of Biotin(FA)-F127-PLA nanoparticles.The specific steps of three-step coupling technique were as followed:First,the biotinylated transferrin was bonded with overexpressed Tf R on the surface of mice glioma cells;Second,the avidin was bonded with the biotinylated transferrin specifically;Third,Biotin(FA)-F127-PLA nanoparticles were bonded with the avidin.Due to the high intensity of non-covalent binding between biotin and avidin,the dual-targeting behavior of transferrin and folic acid which based on the three steps above was not susceptible by exogenous chemical reactions.Furthermore,the biotin-avidin three-step coupling technique has widely been applied in the linking of other targeted ligands and cell fluorescence imaging.The morphology of Biotin(FA)-F127-PLA nanoparticlesobserved by transmission electron microscope was spherical vesicles with double layers,which were also called polymersomes.Then,the particle size of Biotin(FA)-F127-PLA polymersomes tested by dynamic light scattering was mainly distributed in the range of 30-70nm and the size distribution of these vesicles was relatively uniform.This provided a possibility for the embedment of the anticancer drug paclitaxel(PTX).Besides,it was beneficial that dual-targeting polymersomes were taken in tumor cell.In vitro release study on PLA-F127-PLA/PTX,FA-F127-PLA/PTX,Biotin-F127-PLA/PTX and Biotin(FA)-F127-PLA/PTX polymersomes showed that encapsulated paclitaxel released fast initially followed by a slow release.In the study of the cell compatibility of the materials,the result from MTT assay showed that PLA-F127-PLA,FA-F127-PLA,Tf-F127-PLA and Tf(FA)-F127-PLA were almost nontoxic to C6 cells.Moreover,in the in vitro toxicity study of paclitaxel-loaded polymersomes,we found that the toxicity of these four polymersomes to C6 cells was Tf(FA)-F127-PLA/PTX>FA-F127-PLA/PTX>Tf-F127-PLA/PTX>PLA-F127-PLA/PTX.It is showed that dual-targeting polymeric drug carriers containing transferrin and folic acid have obvious advantages in the anti-tumor toxicity to C6 cells.In the cell fluorescence experiment,C6 cells were used as model cells and coumarin-6(C-6)was used as a fluorescent probe.The qualitative experimental result obtained from fluorescence microscopy showed that the polymeric nanoparticles were mainly absorbed into the cytoplasm of C6 cells.In addition,the quantitative experimental results showed that the order of C6 cells uptake was Tf(FA)-F127-PLA/C-6>FA-F127-PLA/C-6>Tf-F127-PLA/C-6>PLA-F127-PLA/C-6.It is consistent with the result of the cytotoxicity of dual-targeting Tf(FA)-F127-PLA polymersomes on C6 cells.On this basis,we further explored the mechanism of the C6 cells uptake of dual-targeting polymersomes.The results showed that the C6 cell uptake of all polymersomes formulations was active and depended on energy.Their cellular uptakes were also related to the clathrin、caveolin、N~+/H~+exchanger、the transport of endoplasmic reticulum to Golgi body and endosome to lysosome.In addition,the data also demonstrated that the cellular uptake of dual-targeting Tf(FA)-F127-PLA polymersomes was connected with the endocytosis mediated by both FR and Tf R.To sum up,dual-targeting polymeric drug carriers containing transferrin and folic acid have the better application potential and prospect in brain tumor and other complex diseases.