Preparation And Evaluation Of Redox-sensitive Paclitaxel Mixed Micelles

Cancer is still one of the leading causes of human death worldwide,and among women,breast cancer is the most common type of cancer.At present,the treatment methods for cancer mainly include surgery,chemotherapy,radiation therapy and Chinese medicine treatment.In the actual treatment plan,two or more treatments are often combined to achieve better therapeutic effect,and chemotherapy is the most widely used cancer treatment.However,simple chemotherapy drugs often have poor effects or serious side effects.In recent years,the development of nanotechnology has provided an effective method for the delivery of chemotherapy drugs.At present,the researches of nanotechnology for drug delivery mainly include three types:prodrugs,nanomaterial-encapsulated drug substances,and drug-material linking complexes Considering the targeted delivery ability and the drug controlled release performance,the last option was chosen for the study.Briefly,paclitaxel was connected with mPEG-NH2 and FA-PEG-NH2 by disulfide bond to form mPEG-SS-PTX and FA-PEG-SS-PTX drug-material bonding complexes,which have an amphiphilic structure characteristic with hydrophobic core and hydrophilic shell,they can also self-assemble in aqueous solution to form amphiphilic mixed micelles.The micelles were redox sensitivity and tumor targeting.When circulating to the tumor site,aggregation occurs at the tumor site due to enhanced permeability and retention effect(EPR effect),followed by specific binding of folic acid(FA)on the micelle and folate receptor(FR)on the tumor cell membrane,which cause receptor-mediated endocytosis to endocytose the micelle into the tumor cells.The glutathione(GSH)content in the tumor cells is abnormally higher than that in the nonnal tissues,so it can enter the micelle and break the disulfide bond,thereby release paclitaxel to kill cancer cells.The researches of this subject mainly include four parts:(1)synthesis and characterization of mPEG-SS-PTX and FA-PEG-SS-PTX;(2)preparation and physicochemical properties study of mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE mixed micelles;(3)in vitro cell experiments of mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE mixed micelles;(4)in vivo distribution and antitumor effects of mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE mixed micelles.The main research processes and results are as follows:(1)Synthesis and characterization of mPEG-SS-PTX and FA-PEG-SS-PTX:Firstly,the highly reactive dithiodipropionicanhydride(DTDPA)was synthesized,and its structure and melting point were confirmed by nuclear magnetic resonance(H-NMR)analysis and melting point determination;Subsequently,DTDPA was linked to the 2'hydroxyl group of paclitaxel to obtain PTX-SS-COOH.The structure of the product was verified by high performance liquid chromatography(HPLC),fourier transform infrared spectroscopy(FT-IR)analysis and 1H-NMR.Finally,PTX-SS-COOH was linked with mPEG-NH2 and FA-PEG-NH2 to obtain mPEG-SS-PTX and FA-PEG-SS-PTX.The structure of the product was verified by FT-IR and 1H-NMR.The graft ratios of paclitaxel in the obtained mPEG-SS-PTX and FA-PEG-SS-PTX were 39%and 74%,respectively.(2)Preparation and physicochemical properties study of mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE mixed micelles:Mixed micelles were prepared by emulsification dialysis method,the particle size,zeta potential and morphology of micelles were investigated by dynamic light scattering(DLS)and transmission microscopy(TEM).The influences of oil-water phase volume ratio,FA-PEG-SS-PTX/mPEG-SS-PTX mass ratio and FITC-PEG-DSPE/FA-PEG-SS-PTX+mPEG-SS-PTX mass ratio on the particle size,zeta potential and morphology of micelles were screened by single factor method.The oil-water phase volume ratio was selected as 2:15,FA-PEG-SS-PTX/mPEG-SS-PTX mass ratio,according to the results.50%and 10%of FITC-PEG-DSPE/FA-PEG-SS-PTX+mPEG-SS-PTX mass ratio were used for the preparation of the subsequent micelles.The obtained micelles had a particle size of 96.42±6.38 nm and zeta potential of-2.53±0.51.The critical micelle concentration(CMC)of the mixed micelles was then examined using a ruthenium-fluorescent probe method and found to be 0.0163 mg/ml.The in vitro release behavior of the mixed micelles was investigated by dialysis bag method.In order to study the redox sensitivity of the micelles,10 mM dithiothreitol(DTT)was added to the experimental group to simulate the high reducing environment of the tumor site.The results showed that the experimental group possess significant increase in release rate and cumulative release amount compare with control group,indicating that the micelle has significant redox sensitivity,(3)In vitro cell experiments of mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE mixed micelles:Firstly,human breast cancer cell(MCF-7 cells)were used as model cells to re-examined FA-PEG-SS-PTX/mPEG-SS-PTX mass ratio and FITC-PEG-DSPE/FA-PEG-SS-PTX+mPEG-SS-PTX mass ratio by fluorescence microscopy method.The results showed that when FA-PEG-SS-PTX/mPEG-SS-PTX mass ratio is 50%,the difference in fluorescence brightness between the folate-positive group and the negative group was the largest,indicating that the FA had the best active targeting at this ratio,so FA-PEG-SS-PTX/mPEG-SS-PTX mass ratio 50%was chosen.When the mass ratio of FITC-PEG-DSPE/FA-PEG-SS-PTX+mPEG-SS-PTX is 10%,the fluorescence intensity is stronger than that at 20%,so the chosen ratio of FITC-PEG-DSPE/FA-PEG-SS-PTX+mPEG-SS-PTX is 10%.The cytotoxicity was investigated by murine melanoma cells(B16 cells)and MCF-7 cells.The cell viability of material group was always above 80%,indicating that the material has good safety.The cell viability of mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE micelle group show significantly time-dependence and dose-dependence.And as dose increased,the anti-tumor effect of mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE micelle was better than that of the paclitaxel.Human lung cancer cell(A549 cells)and MCF-7 cells were used as modal cell to investigate the targeting ability of FA by flow cytometry method(FCM).The results showed that the fluorescence intensity in MCF-7 cells was 10 times stronger than that in A549 cells,indicating that the micelles have good FA targeting ability.(4)In vivo distribution and anti-tumor effect of mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE mixed micelles:Tumor model was established by female Kunming mouse bearing B16 cells,and treatment was started when the tumor volume reached about 150 mm3.The tumor volume and body weight were measured once every two days.After 14 days' treatment,the mice were sacrificed,and the major organs include heart,liver,spleen,lung and kidney as well as tumor tissues were taken for hematoxylin-eosin staining,(H&E staining)to investigate the biosafety of micelles.In vivo distribution and tumor targeting effect of the micelles were examined using the same tumor modal by optical in vivo imaging(OI).The tumor volume curve shows that the mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE micelles have a good anti-tumor effect compare with Taxol(?).The body weight of Taxol(?)group showed significantly decreased in the late treatment period,but there was no significant change in the mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE micelle group and the saline group.HE staining showed that the mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE micelle group had a larger area of pink inflammation than the Taxol(?)group and the saline group,indicating a good antitumor effect of the mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE micelle.The HE staining results of major organs showed that the tissue of the mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE micelle group had no obvious side effects compared with the saline group.In summary,the prepared mPEG-SS-PTX/FA-PEG-SS-PTX/FITC-PEG-DSPE micelles have high-efficiency,as well as low-toxic anti-tumor effects compared to the Taxol(?).