| The malignant melanoma (MM), a most aggressive form of skin cacer , which is easy to hematogenous metastasis and has high mortality. Despite a greater understanding of the risk factors as well as the genetic and epigenetic causes of melanoma, the death rate from melanoma continues to increase fast including original low incidence area in recent years. To date, no agent has promoted a clinically meaningful prolongation of overall survival. The current clinical treatment of patients with metastatic melanoma is mainly by systemic chemotherapy after adequate surgery. Although DTIC is so far the most active single agent for treatment of metastatic melanoma, its efficiency is only 20%, the targeting effect of the chemotherapeutic agents is poor and toxic side effect of it is serious, otherwise, malignant melanoma cell is generally refractory to conventional chemotherapy agents. Thus, novel therapy strategy is still to be proposed and there is a critical need for the development of therapeutic agents that could target aberrant survival pathways in metastatic melanomas to improve the treatment of this disease. As DR4 or DR5 is abundantly expressed on the surface of MM cells, it can be expected that the TRAIL-R1 or TRAIL-R2 monoclonal antibodies (DR4 or DR5 mAb) will be very promising immunotherapy bioactive molecules to MM. The purpose of this thesis is to study how to constructe a active targeting drug delivery system with high therapeutic effect and low-toxic, which can best combine chemotherapy and active speci?c immunotherapy for MM therapy.In our study, to overcome the limitations of the current therapy strategies, we take the DR5 monoclonal antibody (TRAIL-receptor 2 mAb, DR5 mAb) which can efficiently induce the apoptosis of tumor cells without any toxic side effects on normal cells as the targeting functional group, MPEG-PLA nanoparticles and dacarbazine (DTIC) are selected as drug-loaded systems and model drug , respectively, to build a new active targeting drug delivery system (DTIC-NPs-DR5 mAb) that combining of chemotherapy and active specific immunotherapy. This thesis was carried out as follow four parts:On the first part, Dacarbazine (DTIC) loaded methoxy polyethylene glycol– polylactide (MPEG-PLA) nanoparticles (NPs) were prepared by modified w/o/w double emulsion-solvent evaporation method through ultrasonic processor without any additional additives. The prescription and preparation technology of DTIC-NPs were optimizd by single-factor test and orthogonal design. Then the DTIC-NPs was prepared by the optimizd prescription and preparation technology, and its pharmaceutical characterization, such as appearance, strutcture, particle size ,zeta potential, surface element, drug loading, encapsulation efficiency as well as in vitro release characters were evaluated and tested by dynamic light-scattering detector (DLS), transmission electron microscope (TEM), x-ray photoelectron ppectroscopy (XPS) and HPLC. The small sized nanoparticles (NPs) with a particle size of 148.6±9.8 nm in diameter and drug encapsulation ef?ciency of 70.1±2.3% are easy to be dispersed in water. The results showed that the nanoparticles were uniformly spherical and the average diameter of DTIC-NPs-DR5 mAb was 148.6±9.8 nm, DL and EE were 15.2±0.4μg/mg and 71.06±2.63 %, respectively. The behavior of drug release in vitro indicated that nanoparticles took on sustained-release character, within 7 days, up to 91% drug release was observed from DTIC-NPs.On the second part, to obtain the nano targeted drug delivery system DTIC-NPs-DR5 mAb, TRAIL-receptor 2 monoclonal antibody (DR5 mAb) which can efficiently induce the apoptosis of tumor cells without any toxic side effects on normal cells were covalently conjugated to the surface of DTIC-NPs by a two-step carbodiimide method. Then the characterization of the DTIC-NPs-DR5 mAb, such as particle size, zeta potential and morphology, were evaluated by DLS and TEM. The resulting DTIC-NPs-DR5 mAb were sized at 166.0±8.1 nm with the zeta potential of -36.8±3.2 mV. The dual fluorescent marker method and Micro BCA protein content determination method were utilized to determine whether the DR5 mAb conjugated to the DTIC-NPs and the quantity of DR5 mAb on the DTIC-NPs, the conjugation quantity and efficiency were about 12.8±2.4μg DR5 mAb/mg NPs and 43%. The DTIC-NPs-DR5 mAb can keep the activity of DR5 mAb and increase the therapeutic efficiency due to its double activity of antiangiogenesis and local enrichment.On the third part, the cellular uptake and the targeted effect of DTIC-NPs-DR5 mAb on human malignant melanoma cell A375 were investigated by flow cytometry and laser-scanning confocal microscopy. The cytotoxic activity of DTIC-NPs-DR5 mAb against A375 or NIH cells was evaluated by MTT assay, and the in vitro cytotoxicity of blank NPs (MPEG-PLA-NPs and MPEG-PLA-NPs-DR5 mAb which did not encapsulate DTIC) was also tested by MTT assay. The FCM results showed that DTIC-NPs-DR5 mAb could recognize and specificity bind to the targeted cell, the biological activities of DR5 mAb on the NPs were well protected during the preparation process. The binding and internalization of targeted NP were detected on the A375cell membrane only after 5 min incubation and in the cytoplasm of cells after 30 min incubation by LSCM. The MTT tests results indicated that the DTIC-NPs-DR5 mAb was more cytotoxic against A375 (cells viability of 19.3±3.6%) and can induce remarkable quantity of A375 cell apoptosis (58.5±5.0%) when compared with DTIC. The blank NPs were found to be non-toxic at each of the tested concentrations. We did not observe a signi?cant difference in the toxicity of MPEG-PLA NPs and MPEG-PLA-DR5 mAb NPs, although there was a slight reduction in cell viability at higher concentrations (2.2 mg/ml), average cell viability was keeped above 80.1±3.9%, suggesting that MPEG-PLA-NPs-DR5 mAb may be used as a safety target delivery carrier.On the fourth part, we inspected the anti-tumor effect and internal security of targeted nanoparticles though human malignant melanoma tumors xenograft model. Compared with various therapeutic agents, we investigated and evaluated the in vivo nonspecific toxicity and anti-tumor effect of DTIC-NPs-DR5 mAb on malignant melanoma with high expression of DR5. The nude routine blood test,liver and kidney function test results indicate that the toxicity DTIC-NPs-DR5 mAb is much lower than DTIC, DTIC embedding in MPEG-PLA and oriented functions of the target head of antibody can effectively reduce the nonspecific intake of DTIC in normal tissue. The results of in vivo treatment on nude demonstrated that DTIC-NPs-DR5 mAb has a much better therapeutic efficacy in inhibiting tumor growth compared with DTIC and other controls, the final mean tumor volume was 241.86±84.74 mm3 (the final mean tumor volume of PBS blank control group was 1308.12±182.73 mm3).These results suggested that the nano active targeting drug delivery system (DTIC-NPs-DR5 mAb) were successfully, mediated by DR5 mAb which can efficiently induce the apoptosis of tumor cells without any toxic side effects on normal cells. In this approach, antibody is programmed through chemistry to bind a target of biological interest, the programmed antibody maintains advantages characteristic of both antibodies and the targeting agent. It is a combination of chemotherapy and immunotherapy, DTIC and DR5 mAb in the active targeting delivery system (DTIC-NPs-DR5 mAb) induce cancer cell apoptosis through intrinsic pathway and extrinsic pathway, respectively. Such chemoimmunotherapy could be resulted in enhanced anti-tumor immunity and improved therapeutic outcome with decrease side-effect. The in vitro and in vivo experiments demonstrated that DTIC-NPs-DR5 mAb is a safe and effective noval antitumor targeted formulation, which has high targeted efficiency,significant antitumor activity as well as lower side-effect. This study could provid a new research route for the therapy of metastatic malignant melanoma as well as the chemotherapy and targeting immunotherapy for other cancers.
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