An In Vitro Experimental Study Of Targeted Tumor Therapy Using Magnetic Nanoparticles

Background:The anthracycline antibiotic adriamycin or doxorubicin is a highly efficient antineoplastic agent commonly used in the therapy of a variety of cancers like osteosarcoma, leukaemia, lymphomas, ovarian cancer and late stage breast cancer. During chemotherapy, however, pharmacologically active doxorubicin reaches the tumor tissue with poor specificity and can induce dose-limiting toxicity. Moreover, the cancer cells may eventually develop resistance to multiple chemotherapeutics. These problems have long been primary hindrances for the clinical application of doxorubicin. To tackle these difficulties, decades of research have focused on developing cancer-specific drugs or delivery systems that can selectively localise chemotherapeutics to the tumor site. Recent advances in nanotechnology promise further developments in tumor targeted drug delivery systems. Nanoparticle-based targeted drug delivery may use passive or active strategies. Passive targeting occurs as a result of extravasation of the nanoparticles at the tumor site where the microvasculature is hyperpermeable and leaky, a process aided by tumor-limited lymphatic drainage. Combined, these factors lead to the selective accumulation of nanoparticles in tumor tissue, a phenomenon known as enhanced permeability and retention (EPR) effect. Active targeting is based on the over or exclusive expression of different epitopes or receptors in tumor cells, and on specific physical characteristics (e.g. sensitivity to temperature, pH, electric charge, light, sound or magnetism). The potential of nanoparticle-based drug delivery systems stems from significant advantages such as:(1) the ability to achieve multiple target access allowing for even better specificity and selectivity to the tumor mass; (2) the reduction of the quantity of drug needed to attain a particular concentration in the vicinity of the target; (3) the reduction of the drug concentration at normal tissue, minimizing severe side effects; (4) the ability to act at the cellular level through endocytosis or phagocytosis; (5) the capability to creat multifunctional nanoparticle formulations combining tumor imaging, drug targeting, guided hyperthermia and radiation in an all-in-one system.Objective:The purpose of the present study is to construct a multiple targeted drug delivery system-doxorubicin loaded magnetic Fe3O4 nanoparticles with monoclonal antibody conjugated to the surface, which can selectively target nanoparticles to the tumor mass under the co-ordination of magnetic field, antibody and nanoparticle-based targeting mechanisms, thus reducing overall dosage and side effects. At the present stage, the study consists of two parts:(1) in vitro study of the cytotoxicity of two kinds of doxorubicin loaded magnetic nanoparticles with different particle sizes; (2) preparation, purification and identification of the monoclonal antibody against human osteosarcoma OS-732 cell line.Methods:(1)Two kinds of doxorubicin loaded magnetic Fe3O4 nanoparticles (Fe3O4-DEX-DOX and Fe3O4-PLGA-DOX) with different sizes were prepared and utilized in the present study to compare their cytotoxic effects on cancer cells. The celluar uptake and distribution of doxorubicin loaded magnetic nanoparticles were observed by fluorescence microscopy; The inhibitory effects on the proliferation of cancer cells were evaluated in vitro by MTT assay; The cytotoxic effects on the membrane damage of cancer cells were evaluated in vitro by lactate dehydrogenase (LDH) assay; The apoptotic and necrotic rates of cancer cells exposed to doxorubicin loaded magnetic nanoparticles were determined by flow cytometry using the Annexin V-FITC/PI staining method. (2)The monoclonal antibody against human osteosarcoma OS-732 cell line was prepared through ascites induced by hybridoma cells, and purified by Protein A Sepharose CL-4B affinity chromatography and its properties were evaluated by SDS-PAGE,Immunohistochemistry and ELISA.Results:(1)The observation of fluorescence microscopy demonstrated that doxorubicin loaded magnetic nanoparticles faciliated internalization of doxorubicin to cancer cells with selectivity, and particularly may penentrate the nuclear membrane, however with much less uptake by human embryonic lung cells. (2)Doxorubicin loaded magnetic nanoparticles showed great potential to reverse multidrug resistance of cancer cells. (3)The FCM results illustrated that magnetic nanoparticles loaded with doxorubicin induced higher necrotic rates than free doxorubicin, and the necrotic rates increased in a dose and time dependent manner. (4) The MTT results indicated that the inhibitory effects of magnetic nanoparticles loaded with doxorubicin on cell proliferation were higher than that of free doxorubicin, with statistical significance (p<0.05). (5)The LDH leakages in groups exposed to magnetic nanoparticles loaded with doxorubicin were significantly higher than that of free doxorubicin group(p<0.05). (6) Magnetic nanoparticles loaded with doxorubicin were preferrentially ready for uptake by the mononuclear macrophages and human umbilical vascular endothelial cells, suggesting that the administration routes should be separated from circulation system. (7)With immunohistochemical staining, the monoclonal antibody showed positive reactions on formaldehyde-fixed sections from human osteosarcoma, chondrosarcoma, liposarcoma, Ewing's sarcoma, malignant fibrous histiotoma and so on; the purity of monoclonal antibody was identified about 93%with SDS-PAGE (10%); the immunoactivity was 1 X 10-7 by ELISA.Conclusions:(1)Magnetic nanoparticles loaded with doxorubicin can faciliate penetration and retention in cancer cells selectively, enhance cytotoxicity of doxorubicin, and induce necrosis instead of apoptosis. (2)The monoclonal antibodies produced are of high purity and immunoactivity with specificity to malignant bone tumors.