Synthesis Of Anti-CD90 Thermosensitive Magnetoliposomes And Their Targeted Anti-Tumor Effect On Hepatocellular Carcinoma Using MFH

In recent years, it has been assumed by the theory of cancer stem cells that cancer stem cells are derived from a small subset of tumor cells existing in all tumors. They are responsible for tumor formation, maintenance and metastasis. Although the clinical routine therapy targets the tumor cells in multiplying stage, relapse often occur after a period of time because of the cancer stem cells remain. Naturally finding a novel therapeutic approaches focusing on killing cancer stem cells as an effectively supplementary treatment for tumor to reduce the recurrence and metastasis, decrease the mortality rate and improve the curative strategy, is of certain academic significance and potential clinical value.Magnetic fluid hyperthermia(MFH), which delivers magnetic nanoparticles into the target tissue and heated it to 41-46℃ by an alternating magnetic field (AMF), is one of adjuvanticity means to cure tumor. What’s more, thermotherapy, as a cancer treatment by radio sensitization and chemotherapy increase-sensitivity, will result in a need for less chemo and radiation, thereby reducing side effects. Thus, magnetic thermotherapy is a novel technique to heat deep-seated tumors. However, there are certain deficiencies on independent application of hyperthermia alone. It is more desirable to add times of heating to enhance the effect of hyperthermia. Thermotolerance that emerges in tumor cells bearing subsequent heating can result in an invalid apoptosis and less cell kill. It is reported that heat shock proteins (HSPs) have been identified in association with thermoresistance and the induction of the heat shock response, especially the up-regulation of HSP90, would impact on the features or duration of thermotolerance. Inhibition of HSP90 can sensitize tumor cells to hyperthermia and exhibited increased tumor cell apoptosis. 17-allylamino-17-demethoxygeldanamycin (17-AAG) is a HSP90 inhibitor derived from the geldanamycin antibiotic and can kill tumor cells by reversibly associating with HSP90. Meanwhile, as a unique drug targeting on tumor treatment,17-AAG can effectively inhibit many cell signal transduction pathways which maintain the proliferation and survival of tumor cell. What’s more, CD90 is regarded as one of the important makers of liver cancer stem cells (LCSCs). Hence, we prepared an anti-cluster of differentiation 90 monoclonal antibody (anti-CD90 mAb) targeted thermosensitive magnetoliposomes (TMs) encapsulated HSP90 inhibition 17-AAG (CD90@17-AAG/TMs) to target and kill CD90+LCSCs by external magnetic field.In a previous study, we propose a brand-new combined therapy targeting cancer stem cell. In terms of targeting, the biological targeting of anti-CD90 monoclonal antibody was used to ensure the specificity and security of therapy. In terms of treatment, the physical therapy of magnetic nano-particles induction heating is combined with biological therapy of 17-AAG to guarantee the effectiveness, meeting the requirements of specificity, security and effectiveness as far as possible. It is the major task of novel biologic therapies for clinically application.The main work of the thesis could be summarized as follows:1. The relationship between the expression of CD90 and the clinicopathological parameters in hepatocellular carcinoma was explored. The results showed that the expression of CD90 was related to the degree of the differentiation. It indirectly confirmed that CD90 might be the maker of the LCSCs. The expression of CD90 on the surface of hepatocarcinoma cells was assayed by flow cytometry. CD90+ cells were isolated by magnetic-activated cell sorting from Huh7 and BEL-7404 cell lines. Proliferation assay, differentiation assay, colony formation assay, transwell invasion assay, drug resistance assay and tumor formation assay were performed to identify stem cell characteristics. The results suggested that CD90+ cells showed significant stem cell-like properties.2. CD90@17-AAG/TMs was prepared by rotary evaporator-hydration and ’post-insertion method’. It’s physical, chemical and biological properties were characterized by transmission electron microscopy (TEM), particle size analysis, potential detection, saturation magnetization analysis, differential thermal analysis, fourier transform infrared spectroscopy (FTIR) scaning, thermodynamic properties testing, release characteristics detection and immunoagglutination assays and their stability was determined.3. The targeting performance of CD90@17-AAG/TMs in vitro was detected by flow cytometry, Prussian blue staining, confocal laser and 7.0 Tesla MR (magnetic resonance) imaging. Considering the specificity and security of the therapy, the 7.0 Tesla MR imaging and multispectral imaging system were used to detect whether the liposomes could effectively increase the enrichment and accumulation in the tumor.4. In terms of the treatment in vitro, we firstly found that CD90@17-AAG/TMs combining with hyperthemia could increase the sensitivity of CD90+ LCSCs to magnetic hyperthermia-induced apoptosis and cause a significant decrease in drug resistance, invasive potential, colony forming ability and oncogenicity through the experiment of hematoxylin and eosin (MTT), flow cytometer, characterization of stem cell and Western Blot. It might because that 17-AAG could block the signal transduction pathways of Raf-1-MEK-Erk branch and PI3K-AKT pathways in MAPK (mitogen activated protein kinase, MAPK) signaling pathways through combination with HSP90.5. In terms of the treatment in vivo, as the results showed in the analyzation of tumor mass and tumor volume, hematoxylin and eosin (HE) staining, transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining and CD90 immunohistochemistry (IHC) staining assays, the composite liposomes could effectively kill CD90+LCSCs and inhibit the tumor growth.