| Osteosarcoma is the most common malignant tumor in the human skeletal system,ranking sixth in incidence among childhood and adolescent tumors.The 5-year survival rate for patients with limited osteosarcoma is approximately 60%,and only 20%for patients with metastasis or recurrence.The current primary treatment is a combination of neoadjuvant chemotherapy and surgery.However,the survival rate of patients with osteosarcoma has not improved over the past 30 years.The reasons for this are mainly toxic side effects caused by high-dose chemotherapeutic drugs and drug resistance of primary or secondary tumor cells.The results of clinical trials of immune checkpoint inhibitors in osteosarcoma to date have also been unsatisfactory,mainly related to the presence of multiple immune cells that suppress the immune response in the immune microenvironment of osteosarcoma,including M2-type tumor-associated macrophages and TIM3+T lymphocytes.Secondly,the high expression of TIM molecules,an immune checkpoint negative molecule,by osteosarcoma cells and the high expression of EZH2 transcription factors by their stem cells constitute an unfavorable microenvironment leading to treatment failure in osteosarcoma.tazemetostat(EPZ643 8)is a potentially specific oral EZH2 inhibitor that has been approved by the FDA for the treatment of metastatic or locally advanced epithelioid sarcoma that is not amenable to surgery.The aim of this study was to construct a microsphere-vesicle drug delivery system loaded with EPZ6438 and anti-TIM3 antibody(αTIM3)to improve the immune microenvironment,and to investigate the killing effect and mechanism of action of this drug delivery system on osteosarcoma in mice.Part Ⅰ Preparation and characterization of microsphere-vesicle compound drug delivery systemMethods:(1)GelMA hydrogel microspheres were prepared by microfluidics,and the morphology of the microspheres was observed by inverted microscopy and scanning electron microscopy(SEM).(2)Vesicles were prepared using a vesicle preparation device,and the morphology of the vesicles was observed by transmission electron microscopy(TEM).and the particle size distribution and zeta potential were measured by nanometer and zeta potential meter.(3)Prepare the linkage structure between microspheres and vesicles.DSPE-PEG2000-GPLGLAGC.and detect the structure by nuclear magnetic hydrogen spectroscopy.(4)Assemble the microsphere-vesicle complex drug delivery system and observe the morphology by inverted fluorescence microscopy.(5)Verify the MMP2 responsiveness of the composite drug delivery system by adding matrix metalloproteinase 2(MMP2)to the composite drug delivery system and observing its supernatant fluorescence intensity under an inverted microscope.(6)Verify that the MMP2-responsive vesicles released by the compound delivery system can be phagocytosed by co-culturing the supernatant of method 5 with K7M2 cells and observing them under an inverted fluorescence microscope.(7)The composite delivery system loaded with αTIM3 was immersed in ultrapure water,and the liquid was aspirated at fixed time points to detect the protein concentration in the liquid using high performance liquid chromatography.Results:(1)GelMA microspheres showed regular spherical structure,the diameter of microspheres was mostly 120-130 μm surface with pores of different sizes,the pores were evenly distributed on the surface of microspheres,the pore size was mostly 3 μm.(2)The vesicles showed regular spherical shape,the surface was visible as a double layer structure,the outer circular structure was translucent,the inner layer structure was lighter in color.The nanoparticle size is about 185.53 ±7.19 nm,and the zeta potential is-47.03 ± 2.17 mV.(3)The characteristic peaks of each part of DSPE-PEG2000-GPLGLAGC can be seen by NMR hydrogen spectroscopy.(4)The fluorogram of the composite drug delivery system can be observed by the uniform distribution of vesicles on the surface of GelMA microspheres,and the assembly of the composite drug delivery system is completed.(5)The supernatant of the compound drug delivery system without the addition of MMP2 had some free vesicles,and its total fluorescence intensity was about 6× 105 AU.The supernatant of the compound drug delivery system with the addition of MMP2 was full of vesicles,and its total fluorescence intensity was about 6.5×108 AU,and its responsive release of vesicles was about 1000 times that of the control.(6)The vesicles responsively released by the compound drug delivery system could be endocytosed by K7M2 cells.(7)The encapsulation rate of aTIM3 by this compound drug delivery system was 85.92%,and its release reached a plateau at 120h.Conclusion:The GelMA hydrogel microspheres,extracellular vesicles,and the above three parts of DSPE-PEG2000-GPLGLAGC of the composite drug delivery system were prepared,and the assembly and responsiveness of the composite drug delivery system were verified,confirming that the responsively released extracellular vesicles of the drug delivery system can be used as excellent drug delivery carriers.In this part,GelMA microspheres were selected as the antibody release carrier,extracellular vesicles as the drug delivery carrier,and MMP2 was used as the tumor microenvironment responsive release condition to synthesize the critical structure connecting the two parts of the carrier,and the above three parts were integrated into an injectable composite drug delivery system,which can be used as a tumor microenvironment responsive therapy.Part Ⅱ Study of microsphere-vesicle compound drug delivery system to improve tumor immune microenvironment to inhibit osteosarcoma growthMethods:(1)Exploring the biocompatibility of material extracts by live-dead cell staining and CCK8.(2)To explore the effect of different concentrations of EPZ6438 on the inhibition of K7M2 proliferation by CCK8.(3)To explore the blockage of TIM3 on the surface of K7M2 cells by aTIM3 using flow cytometry.(4)Explore the effect of compound drug delivery system on K7M2 apoptosis using flow cytometry and real-time fluorescence quantitative PCR(RT-PCR).(5)Explore the effect of the combination drug delivery system on the polarization of bone marrow-derived macrophages using flow cytometry and RT-PCR.(6)To explore the effect of the combination drug delivery system on subcutaneous tumor-bearing mice by monitoring body weight and survival time of subcutaneous tumor-bearing mice.(7)Measurement of tumor size in mice with subcutaneous tumors to monitor changes in tumor volume after treatment with the combination drug delivery system in mice with subcutaneous tumors.(8)Serological analysis of inflammatory factor secretion in mice with subcutaneous tumors after treatment with the drug delivery system.(9)HE staining analysis of tumor tissues in mice with subcutaneous tumors after treatment with the drug delivery system.(10)Immunofluorescence staining analysis of tumor infiltrating macrophage polarization and T-cell activation in tumor tissues of mice treated with the combination drug delivery system.(11)HE staining analysis of heart,liver.lung and kidney tissues after the treatment of subcutaneous tumor-bearing mice with the drug delivery system.Results:(1)The K7M2 cells treated with the material extract showed similar cell survival and proliferation viability as the control group at 1.3 and 5 days.(2)The 5-day inhibition rate of K7M2 cells by 10 μM concentration of EPZ6438 was close to 100%.(3)10 μg/mL of αTIM3 on K7M2 cells treated for 12h showed a surface TIM3 blockade rate of 52.57±1.75%.(4)The compound drug delivery system could induce K7M2 apoptosis and thus inhibit osteosarcoma cell proliferation by upregulating Caspase3 gene ex pression.(5)The combination drug delivery system promoted the up-regulation of CD86 expression and inflammatory factor gene expression in M1 macrophages,and down-regulated CD206 expression and inflammatory factor gene expression in M2 macrophages.(6)The effect of compound drug delivery system on the body weight of mice was not obvious,but it could prolong the survival time.(7)The compound drug delivery system can inhibit tumor growth.(8)The compound drug delivery system can elevate the level of inflammatory factor secretion.(9)The tumor tissue structure was loosened after treatment with the combination drug delivery system.(10)After treatment with the combination drug delivery system,subcutaneous tumor-bearing mice had the largest area of TUNEL+ region.CD11b+iNOS+ region and CD3+CD25+ region in frozen sections,and the lowest area of CD11b+ARG1+ region.(11)No significant abnormalities were observed in HE staining of paraffin sections of heart,liver,lung and kidney in the compound drug delivery system group.Conclusion:The compound drug delivery system can achieve the killing function by inducing apoptosis of K7M2 cells;its released αTIM3 can achieve the blockage of TIM3 on the surface of K7M2 cells;it can interfere with the polarization direction of bone marrow-derived macrophages,enhance the inflammatory function of M1-type macrophages and inhibit the anti-inflammatory function of M2-type macrophages.The compound drug delivery system can kill subcutaneous tumor-bearing mice tumor cells.delay tumor growth and enhance survival time;increase the secretion of inflammatory cytokines such as serum IL-6 and TNF-α,increase the number of Ml-type macrophages and decrease the number of M2-type macrophages in the tumor microenvironment,and enhance the functional activation of T cells;and have no significant toxicity to heart,liver,lung and kidney.In summary,the compound drug delivery system constructed in this thesis can kill osteosarcoma cells while improving the tumor immune microenvironment.After its injection into tumor tissues,it can not only kill osteosarcoma cells by releasing EPZ6438(EZH2 inhibitor)contained in vesicles in response to high MMP2 in the tumor microenvironment,but also promote TAMs to M1 The system also promotes the polarization of TAMs to M1-type macrophages by releasing EPZ6438,releasing inflammatory factors,phagocytosing presenting antigens,reactivating the immune process,activating T-cell killing,and blocking the suppressive effect of TIM3 antibodies in the immune microenvironment by slowly releasing them through GelMA microspheres.The system mimics the "chain reaction" of the normal immune system in its pre-killing function to utilize better the above triple action of the drug delivery system and at the same time "To take its strength " to reshape the immunosuppressive microenvironment to play a killing role as well. |
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