Cisplatin-loaded Gold Nanoparticle For Anti-melanoma Therapy In Vitro And In Vivo

Melanoma is one of the most aggressive types of skin cancer developing from melanocytes (pigment-producing cells). The5-year overall survival rate of patients with metastatic melanoma is less than10%. Convention treatments of late-stage metastatic melanoma mainly include chemotherapy, radiation, immunotherapy and targeted therapy. However, the effectiveness of these therapies has not been satisfactory because of advanced side effects, difficulty to reach tumor site and tolerance. Cisplatin was found to be able to inhibit the growth of cancer cells in the1970s. It is extensively used for chemotherapy of patients with advanced malignant tumor including metastatic melanoma, but has significant side effects. It is well known that "Enhanced Permeability and Retention (EPR) effect" and acidic microenvironment exist in tumor tissues. As an attractive inorganic nanocarrier for successfully deliverying small drug molecule, gold nanoparticles (AuNP) attract a lot of attention. Selective accumulation of drug-loaded AuNPs within tumor occurs by the EPR effect and improves the efficacy of drug. At the same time, the issue of acidic environment may provide the base for the control release of the drug and also reduce the side effect of the drug. Based on the above condition, pH-sensitive cisplatin-loaded AuNPs is designed and synthesized by our group and used for the study of effects on melanoma therapy in vitro and in vivo.Objective:The aim of our research is to synthesize pH-sensitive cisplatin-loaded AuNPs, followed by the investigation of its cytotoxicity to Ml4human melanoma cell line in vitro and inhibition of melanoma in M14tumor-bearing mice in vivo.Methods:1. Synthesize Au-PNIPAm-PEG-cisplatin nanoparticle:AuNP was fabricated by seeding growth and ligand exchange method with PNIPAm-PEG at the particle surface and conjugated cisplatin. The size and morphology of AuNPs and Au-PNIPAm-PEG-cisplatin NPs were observed by transmission electron microscopy (TEM). Absorption spectra of AuNP in deionized water were recorded by UV spectrophotometer. The concentration of platinum in the Au-PNIPAm-PEG-cisplatin NPs was estimated by ICP-MS. 2. In vitro test:FITC-labeled Au-PNIPAm-PEG-cisplatin NPs were investigated to determine the cellular uptake by fluorescence microscopy. TEM was applied to further investigate the intracellular distribution of Au-PNIPAm-PEG-cisplatin. The in vitro cytotoxic effects of Au-PNIPAm-PEG-cisplatin were determined by the MTT assay.3. In vivo test:Human melanoma implant model was established by using nude mice. The tumors in mice were locally injected with Au-PNIPAm-PEG-cisplatin NPs for20days. The tumor morphologies of the mice were recorded and tumor sizes were measured. The weight and survival time of mice were recorded. The histological characteristics of organs and tumors stained with hematoxylin eosin in mice were examined by optical microscopy. Intracellular localization of Au-PNIPAm-PEG-cisplatin in transplanted tumor cell was determined by TEM.Results:1. AuNP and Au-PNIPAm-PEG-cisplatin NPs could disperse well. The diameter of AuNP was8.0±0.8nm. The concentration of cisplatin in Au-PNIPAm-PEG-cisplatin was100.55μM.2. Au-PNIPAm-PEG-cisplatin NP could be observed in the cytoplasm, endosome and lysosome in M14cells. The IC50results indicated that Au-pNIPAM-peg-cisplatin was almost1.7fold (48h) and1.5fold (72h) more efficient than cisplatin. Au-PNIPAm-PEG without cisplatin did not exhibit apparent cytotoxicity against M14cells.3. In vivo, the anti-tumor rate of Au-PNIPAm-PEG-cisplatin NPs and free cisplatin were76.29%and66.41%respectively (20d). Mice had longer survival time than normal saline and Au-PNIPAm-PEG without cisplatin groups and the specimen of tissue revealed transplanted tumor without distant metastasis in Au-PNIPAm-PEG-cisplatin NP group. Au-PNIPAm-PEG-cisplatin was aggregated in the endosome of tumor cells.Conclusion:1. Cisplatin is successfully loaded in Au-PNIPAm-PEG-cisplatin NP.2. Au-PNIPAm-PEG-cisplatin has more effect of inhibiting M14cells than free cisplatin.3. Au-PNIPAm-PEG-cisplatin can significantly enhance the survival time of nude mice bearing melanoma and inhibit tumor growth and metastasis.