| The current situation of tumor treatment is becoming increasingly rigorous, with morbidity and mortality increasing year by year. The treatment effect of the existing anti-tumor drugs is not satisfactory, mainly because of their low solubility, poor stability, toxicity, as well as the severe drug resistance. In this thesis, novel folate-modified polymeric micelles were designed and prepared in order to solve these problems. These novel micelles are composed of three moieties:biodegradable block poly (ε-caprolactone)(PCL) serves as drug storage to improve the stability of the drug; poly(2-ethyl-2-oxazoline)(PEOz) plays the "stealth" role to extend blood circulation and suppress unwanted uptake by liver and spleen; and folate, a commonly used small-molecule ligand, is conjugated on the surface of the micelle to realize active targeting. The applications of the micelles in chemotherapy and photothermal therapy were studied in detail. The main research content is as follows:In the first part, an amphiphilic block copolymer, Boc-NH-PEOz-PCL, was synthesized by2-step ring-opening reaction with PEOz as hydrophilic chain and PCL as hydrophobic chain. Boc-NH-PEOz-PCL was further covalently modified with folate to form FA-PEOz-PCL for active tumor targeting. The structure of FA-PEOz-PCL was characterized by FT-IR, H-NMR, GPC, DSC and X-ray diffaction. The micellar behavior of this polymer in water was studied by pyrene fluorescence probe method. The shape and particle size of blank micelles were measured by SEM and dynamic light scatter. The safety of the polymers was also evaluated by hemolysis tests. The results showed the composition and construction of the polymer were well identified and the synthesis was controllable. Both Boc-NH-PEOz-PCL and FA-PEOz-PCL could self-assemble in water to form well dispersed spherical or near-spherical nanoparticles with averge particle size at around100nm and CMC at0.012g·L-1and0.007g·L-1separately. Good biocompatibility was demonstrated by the MTT assay, and the hemolysis ratios for both polymers were lower than5%.In the second part, DOX-loaded micelles (DM) and folate modified DOX-loaded micelles (FDM) were prepared by dialysis method using Boc-NH-PEOz-PCL and FA-PEOz-PCL as carrier materials. The drug loading was12.9%for DM and10.2%for FDM. The average particle size of DM and FDM was162nm and187nm with Zeta potentials close to zero. DOX was shown in amorphous state in both DM and FDM based on X-ray diffraction results. The in vitro release behavior of DOX from DM and FDM was found in a sustained and pH sensitive manner. DOX was released very slowly at physiological pH while much faster in weak acidic media. Cell uptake of DM and FDM was investigated by laser scanning confocal microscope and flow cytometry, and the cytoxicity was evaluated by MTT assay. The cellular uptake and cytoxicity of FDM were significantly higher than DM in folate acceptor (FR) positive Hela and KB cells while showing no significant differences in FR negative A549cells. Further investigation showed cell uptake and cytoxicity of FDM was significantly inhibited by adding free folic acid and was enhanced with increasing ratio of folate modification. MCF7/ADR cell line was chosen as drug-resistant cell models to investigate the reversion of multidrug resistance of the DOX-micelles. Results showed that the cellular uptakes of DM and FDM were3.1and5.1times higher than that of free DOX after3h incubation, and the cytotoxicity were5.0and7.2times higher than that of free DOX after48h incubation, demonstrating that DM and FDM strongly reversed the drug resistance of MCF7/ADR cells. In vivo tumor inhibition was conducted using KB tumor bearing nude mice as animal model. On the35th day after intravenous administration, FDM showed an average tumor inhibition ratio of53.3%, better than DM (37.8%) and free doxorubicin (45.1%). Body weight data showed free DOX induced obvious body weight decrease during administration, while no abnormal body weight change was observed in the mice from DM and FDM group, which demonstrated DM and FDM can effectively reduce systemic toxicity of the drug.In the third part, indocyanine green (ICG)-loaded micelles were prepared by organic solvent evaporation method, termed as IM and FIM for non-modified micelles and folate-modified micelles, respectively. IM and FIM showed maximum absorption wavelength and fluorescence emission wavelength at796nm and832nm, average particle size of28.2nm and43.8nm. TEM photos showed IM and FIM were spherical and well dispersed. The near infrared absorption and fluorescence was much more stable for FIM and IM than free ICQ due to the protection from the micelles.The in vitro and in vivo targeting of FIM was evaluated by cellular uptake experiments and NIR imaging. The results showed that, for Hela and KB cells, cellular uptake of FIM was significantly greater than IM; while for A549cells, cellular uptake of FIM and IM were comparable. In vivo living imaging showed that, FIM could specifically accumulate in KB tumors within24h and emit stable fluorescence signals for at least7days. Semi-quantitative analysis results show that the signal intensity of FIM in tumor was2.5times and5times stronger than that in the liver and in the muscle, respectively, which suggested FIM has excellent targeting ability and is perfectly suitable for NIR imaging. Then, the photothermal cytotoxicity was evaluated by MTT assay. In the absence of laser irradiation, FM showed no obvious cytotoxicity, even when high concentrations were used, suggesting that FM has good biocompatibility. Laser irradiation alone (1.95W·cm-2,5min) was also found to have little effect on cell viability. However, when laser irradiation and ICG treatment were combined, concentration-depending cytotoxicity was observed. For FIM, over40%of the cells were killed when ICG concentration was1.25μg·mL-1, and almost all cells were killed when the ICG concentration was increased to20μg·mL-1. Hoechst/PI dual-staining experiments further confirmed the FIM cytotoxicity, and reveals the mechanism of PTT depends on the extent of hyperthermia. At a relatively low temperature, cells would be killed in an apoptotic manner; while when the concentration increases to a certain amount, necrosis is more likely to occur.Finally, the in vivo PTT was investigated on mice bearing KB tumors. Intratumor injection with FIM plus laser irradiation at1.95W·cm-2for10min completely destroyed the tumors. Intravenous injection with2mg·Kg-1FIM plus laser irradiation resulted in a tumor inhibition ratio of72.6%, significantly better than IM group (44.8%) and free ICG group (25.0%). Further, common characteristics for cell apoptosis and necrosis such as loss of membrane structure, cell shrinkage, vacuolization, and coagulation were found in the tumor sections from the FIM group, demonstrating the PTT effect induced by FIM. |
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