Preparation And In Vitro Evaluation Of Long-circulating Folate-conjugated Polymeric Micelles

Polymeric micelles as interests of current drug delivery research have several advantages, such as solubilization for hydrophobic or unsoluble drugs, longevity in blood circulation and active targeting by attaching ligand molecules to the surface of micelles which could specifically initiate ligand-receptor binding-based endocytosis. In this dissertation, folate-conjugated micelles were prepared and evaluated in folate receptor a(FR a)-positive Hela cells. The dissertation consists of five chapters, and is shown as follows.Firstly, folate-PEG-DSPE was synthesized by three-step synthesis method, from PEG-bis-amine,folate and phosphatidylethanolamine, and confirmed structurally by FT-IR and1H NMR.In addition, high performance liquid chromatography (HPLC) was utilized to quantify folate-PEG-DSPE.Secondly, polymeric micelles were prepared by thin-film rehydration method, fluorescent dyes coumarin6as model of hydrophobic drugs.The micelles containing folate-PEG-DSPE were called folate-conjugated micelles(FM) and those containing no folate-PEG-DSPE were called plain micelles(PM).The surface morphology of FM and PM were observed by transmission electron microscope (TEM), the particle size were also examined by dynamic light scattering(DLS), the drug loading and encapsulation efficiency were assayed by HPLC. In TEM pictures, FM and PM were spherical and uniform-sized particles,with diameter about50-60nm.In micelles solution, the mean particle size of FM and PM were about100nm.The drug loading and encapsulation efficiency of FM and PM were about0.8%and15.4%, respectively.Thirdly, polymeric micelles loading mitoxantrone(MTO) were prepared by thin-film rehydration method, effect of DSPE as additive on the MTO loading and encapsulation efficiency was examined. The particle size distribution were examined by dynamic light scattering(DLS), and drug loading and encapsulation efficiency were determined by HPLC.The mean size of MTO-loaded polymeric micelles with or without DSPE was about10nm, but the drug loading and encapsulation efficiency were enhanced by DSPE.Fourthly, FRa positive Hela cells were co-incubated with FM or PM, the endocytosis of micelles were examined by fluorescence microscope and flow cytometer(FCM). The fluorescent images and FCM histogram showed that the endocytosis of FM was more than PM. This results demonstrated the endocytosis of FRa(+) cells could be enhanced by the specific binding of folate on the micelles and FRa on the cells membrane.Finally, Hela cells were treated with dexamethasone(dex,5nM,5OnM,100nM) for96hours in order to upregulating FRa level. And FRa mRNA and membrane FRa level were examined by real-time quantitative polymerase chain reaction (RT-PCR) and FCM. The results showed the mRNA level and FRa level of Hela cells were nearly tripled and doubled by dex treatment, respectively. After Hela cells were treated with dex for96h, folate-conjugated micelles were added to the culture plate and co-incubated for2hours, Hela cells without dex treatment as control, then the endocytosis of micelles were examined by fluorescence microscope and FCM. The results showed that the endocytosis of micelles in Hela cells with dex treatment were more than those without dex treatment.In conclusion, the drug loading and encapsulation efficiency of polymeric micelles for MTO could be increased by DSPE. The endocytosis of foalte-conjugated micelles in Hela cells could be enhanced by upregulating FRa level with dex treatment, and this is a promising strategy for facilitating FRa-based targeting and further research is nessesary.