Functional Graphene Oxide As A Plasmid-based Stat3SiRNA Carrier Inhibits Mouse Malignant Melanoma Growth

Background: Malignant Melanoma is a common cancer, which has high aggressivemalignancy. Patients are effectively treated by surgery at early stages. However, theincidence rates of malignant melanoma in many countries are increasing evidently inrecent years. There are some important biological characteristics in malignantmelanoma, such as high aggressive malignancy, early metastasis, poor prognosis,easily recrudescence and large mortality. Most patients are resistant to chemotherapyand radiotherapy at present. With increasing improvement of researching tumormethods, the molecular mechanisms of cancer are investigated clearly. It is hoped thatutilization of the RNAi method will help to inhibit target gene expression and inducetumor apoptosis. However, it is a serious challenge to researchers how to deliver geneinto tumor tissues safely and effectively. In recent years, nanotechnology has provideda novel approach to researching gene transmission and also new hope in treatingcancer. Thus, it is important to develop better gene delivery system for treatmentcancer.Graphene, a novel and promising nanomaterial, has attracted tremendousattention due to its remarkable electronic, mechanical, and thermal properties.Functional graphene oxide （GO）was often applied in biomedical field. More andmore researchers had demonstrated that modified graphene oxide could improve genetransfection efficiency and biocompatibility in physiological environments.Polythyleneimine（PEI）was widely used for gene delivery, due to binding DNAability, protection of DNA against enzymatic degradation and high endosomal escape.GO-PEI functionalized with polyethylene glycol (PEG) had exhibited high solubilityand stability in physiological solutions. In this work, we applied GO-PEI-PEG asStat3siRNA gene delivery.Signal transducer and activator of transcription3（Stat3）has a crucial role insignal transduction. Stat3is closely related to cell proliferation, differentiation andapoptosis. Constitutive activation of Stat3is the most intimately linked to tumourgenesis. Stat3has been found in an activated state in a wide variety of solidtumors, including malignant melanoma, liver cancer, and prostate cancer. Aberrantlyactive Stat3promotes proliferation, inhibits apoptosis, and influences cell cycleprogression. In previous work, we used a DNA vector-based Stat3-specific RNAiapproach to block Stat3signaling, and successfully inhibited of Stat3overexpressionin liver cancer and prostate cancer. But RNAi approach can not completely blocktarget gene expression in mammalian cells. Photothermal therapy has advantage overchemotherapy and radiotherapy, which has no toxicity or inhibits tumor growthinduction of cell apoptosis. Thus, we show much better therapeutic effficacy usingtargeted delivery in cancer treatment. Photothermal therapy and gene therapy havecombined effect on induction of tumor cell apoptosis.We apply GO-PEI-PEG as a nanovehicle for Stat3-specific RNAi gene delivery tobetter kill tumor cells. We then utilize the strong optical absorbance of GO in theinfrared region for in vivo photothermal therapy, achieving ultraefficient tumorablation after intratumor administration of GO. Furthermore, in this work, we explorethe antitumor effect and therapeutic mechanism of functional GO carriedStat3-specific RNAi in malignant melanoma mouse model.Objective: To evaluate the antitumor effect on Stat3-specific RNAi delivered byGO-PEI-PEG in mouse malignant melanoma and explore the antitumor underlyingmechanism in vitro and in vivo.Method: We chemically synthetize GO-PEI-PEG nanovehicle. Nansheet sizes andzeta potential were determined by employing dynamic light scattering methodology.The morphology and size of nanosheets were imaged tapping-mode atomic forcemicroscopy (AFM). To determine stability of GO and GO-PEI-PEG nanosheets, thetwo samples were separately put into PBS, DMEM medium and serum at roomtemperature. To study Stat3-specific siRNA plasmid loading onto GO-PEI–PEGcomplexes, a gel electrophoresis assay was performed after incubation ofGO-PEI-PEG with si-Stat3plasmid at different mass ratios. The cytotoxicity ofGO-PEI-PEG to B16cells was evaluated by a CCK8assay. B16cells were transfectedwith si-Stat3plasmid delivered by GO-PEI-PEG. Tumor cell cycle was detected by PIassay. RT-PCR and Western blot analysis to detect the expression of Stat3relatedgenes and proteins changes were performed. To study the effects of si-Stat3on malignant melanoma growth in vivo, we developed a C57BL/6mouse tumorxenograft model. The morphology of apoptosis cells and GO-PEI-PEG was observedby transmission electron microscopy. Immunohistochemical analyses for CD34andPCNA protein expressions were carried out. TUNEL analyses were carried out toobserve cell apoptosis of tumor. BUN, ALT and AST in the serum were measured byauto-chemistry analyzer. The morphology of organs was examined in every group byhematoxylin and eosin (H&E) method.Result: The nanosheets of GO-PEI-PEG were successfully synthesized. The zetapotential and effective diameter of the GO-PEI-PEG were determined to be＋21.02mV and298.9nm, respectively. After binding si-Stat3plasmid, the zeta potential andeffective diameter of complexes were determined to be＋7.52mV and339.2nm.Significant stability of GO-PEI-PEG was observed in physiological solutions afterextensive incubation.GO-PEI-PEG complexes are a safe and effective nanocarrier for delivery ofplasmid Stat3siRNA in vitro. Transfection with GO-PEI-PEG/si-Stat3-GFP to B16cells were visualized green fluorescent protein (GFP). The Stat3mRNA, proteinexpression and Stat3related protein decreased significantly in the transfected withGO-si-Stat3by RT-PCR and Western blot. Cell cycle was blocked S cycle of tumor.The results in vivo indicated significant regression in tumor growth and tumorweight after plasmid-based Stat3siRNA delivered by GO-PEI-PEG with simultaneousinfrared irradiation on the tumor. The Stat3mRNA and protein expression decreasedsignificantly in the mice transfected with GO-si-Stat3. Moreover, Stat3related proteinexpressions had significant alteration. Apoptosis of tumor cells in the GO-si-Stat3group were observed though H&E staining and transmission electron microscopy.Proliferating cells and tumor microvessels decreased in the GO-si-Stat3group viaimmunohistochemistry assay. The BUN, AST and ALT levels in the blood were alsonormal. No noticeable toxic effect on organs was found and no GO-PEI-PEG wasdistributed in heart, liver, spleen, lung and kidney in each group.Conclusion: GO-PEI-PEG is an excellent nanocarrier for effective delivery ofplasmid Stat3siRNA. We then utilize the strong optical absorbance of GO in theinfrared region for in vivo photothermal therapy, achieving ultraefficient tumorablation after intratumor administration of GO. Plasmid Stat3siRNA delivered by GO-PEI-PEG with simultaneous infrared irradiation on mouse malignant melanomasuppressed tumor growth.