| Nowadays,the incidence of tumor is increasing,that has become the first killer to human health and life.The research and treatment of tumor is vastly studied by physicians and pharmacy industry.The treatments of tumor normally include surgery,radiotherapy and chemotherapy.Chemotherapy was one of the most common methods.At present,chemotherapy in clinical has obviously toxic side effects,which could damage the patients' immune function.Photodynamic therapy?PDT?is a new mean of treating tumor in recent years.It has a great deal of excellent characteristics such as noninvasive,local direct treatment.It can be used for the selective elimination of local tumors without injuring the normal tissue.However,there are some the disadvantages,for example,the poor selectivity of photosensitizer,small distribution in tumor site,low laser penetration which leads to inadequate depth of treatment and other shortcomings,affect its further application.Therefore,this research constructed a delivery system which containing chemotherapeutic drugs,two-photon compounds and photosensitizers,and it can deliver the nano-drug to tumor targetly,which could increase the organ selectivity of chemotherapeutic agents and photosensitizers.The two-photon conveyor extended the tissue penetration depth of drugs.This nano-drug delivery systems combined the chemotherapy and the photodynamic therapy together against cancer.The main research contents are as follows.In this study,a paclitaxel?PTX?conjugated cathepsin B sensitive dendritic polymer prodrug was synthesized,carrying two-photon compounds of?T1?and pyropheophorbide a?PPa?,combining chemotherapy and photodynamic therapy to inhibit the tumor growth.In the first part,dendrimeric prodrug was synthesized and drug-loaded nanoparticles were prepared.The POEGMA-GFLG-PTX was synthesized via a one-step reversible addition-fragmentation chain transfer reaction?RAFT?.The content of PTX in dendrimeric was determined by high performance liquid chromatography.The molecular weight of the polymeric prodrug was determined by size exclusion chromatography.The thin-film hydration method was used to prepare the drug-loaded nanoparticles.The characterization and in vitro study of drug-loaded nanoparticles includes: concentrations of T1 and PPa loaded nanoparticles were measured by UV spectrophotometry and fluorescence spectrophotometry;The morphology,particle size and stability of drug-loaded nanoparticles were determined by transmission electron microscopy?TEM?and Zetasizer Nano ZS;The critical nanoparticles concentration?CMC?of the polymer was determined via pyrene fluorescence probe method;The enzymatic sensitivity and in vitro release of drug-loaded nanoparticles were investigated by dialysis method.Two-photon induced fluorescence method was applied to determine the cross-sectional area of two-photon absorption.Conclusion: dendrimeric prodrug POEGMA-GFLG-PTX was successfully synthesized and the mass fraction of PTX in the polymer was 5%?w/w?,the molecular weight of the polymer prodrug is 264 kDa.Meanwhile,the drug-loaded nanoparticles were successfully prepared.According to the characteristic absorption peak,T1 and PPa are loaded together into the nanoparticles;The drug-loaded nanoparticles were spherical-shaped,the average particle size was 164.2 nm ± 2.1 nm,and the nanoparticles size was stable in 36 h;the CMC of polymer prodrug was 4.9×10-3 mg/m L;the release of drug-loaded nanoparticles were related to the presence of enzymes,and the cumulative release rates in 36 h were above 90%.The two-photon absorption cross-sectional area of T1 at 808 nm in TPNps is 293.7?37?/GM.The second part was in vitro pharmacodynamics study of drug-loaded nanoparticles.Photobleaching method was used to detect the production of singlet oxygen.Two-photon confocal microscopy was used to capture the cellular uptaking and endocytosis mechanisms after adding the endocytosis inhibitors;the CCK-8 method was used to determine the dark/light toxicity of drug-loaded nanoparticles.The apoptotic population of 4T1 cells was detected by flow cytometry.The intracellular fluorescence intensity of DLNps was measured by Lambda Stack.The reactive oxygen species level was detected by reactive oxygen species assay Kit?DCHF-DA?.The cell morphology was measured by two-photon confocal microscopy.Conclusion: Drug-loaded nanoparticles in PBS solution can achieve long-wavelength excitation induced singlet oxygen generation.4T1 cells were able to uptake drug-loaded nanoparticles which were uptaken through the clathrin and caveolin-mediated internalization pathways;the concentration of T1?10.70 mg/mL?and PPa?8.30 mg/m L?had no significant effect on the survival rate of 4T1 cells;The IC50 values of BNps,TPNps,PSNps and DLNps were: 12.8 mg/mL,14.1 mg/m L,11.4 mg/m L,and 11.1 mg/m L respectively;after light exposure,the cell survival rate decreased while the illumination time and intensity increased;the absorption spectra of T1 and PPa overlaps a large part area;the apoptosis cell population of PDT group was 92.7% in 3 minutes by flow cytometry;Under the two-photon confocal microscope,the intracellular ROS levels were significantly increased and the cell morphology was collapsed while scanning by 808 nm laser beam.The third part was the establishment of the subcutaneous breast cancer mice model and the investigation of the antitumor effect of DLNps.The model of mouse breast cancer was constructed by transplanting 4T1 cells;the targeting of tumor tissue and the distribution of major organs were observed in 36 hours post injection and experiment was perfromed by using small animal living body imaging instrument;in order to investigate the anticancer effect of DLNps,mice were divided into the administration group and the control groups.The tumor volume and the body weight were recorded;hematoxylin-eosin staining was used to examine the tissue morphology.Conclusion: the maximum accumulation of drug-loaded nanoparticles in tumor site was 24 hours post injection.After combined with PDT,the tumor growth in administration group was inhibited,meanwhile there was no significant effect on the body weight of mice;there was no obvious necrosis for normal organization in the administration group.In summary,combined with two-photon photodynamic therapy,the paclitaxel conjugated cathepsin B sensitive dendrimeric prodrug has a significant inhibitory effect on 4T1 breast cancer mice,and it is expected to become a safe and effective anti-tumor nano-medicine. |
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