A New Multifunctional Gold Nanorod Mediated Tumor Targeting Gene Silencing Combined With Photothermal Effect Inhibit Liver Cancer Progression

Background:Hepatocellular carcinoma(HCC)is the third leading cause of death in cancer patients in the world.HCC is difficult to be dignosed at the early stage,resulting in most of HCC patients are detected at an advanced stage.Advanced hepatocellular carcinoma has a high degree of malignancy and lacks effective treatment at the end stage,so the overall prognosis of HCC patients is poor.Therefore,novel treatment methods are highly demanding for liver cancer patients in clinic.Objectives:This study was aimed to develop a novel nano system GAL-MUA-PEI-GNR,which can target liver cancer,carry siRNA and retain the photothermal effect of gold nanorods.In order to investigate the effect of GPC-3 gene silencing in hepatocellular carcinoma cells(Hepa1-6)mediated by the nanosystem and to elucidate its biological function of this novel nanosystem.Additionally,we will also study the photothermal effect and targeting of GAL-GNR,and to explore the therapeutic effect of GAL-GNR-siRNA-mediated GPC-3 gene silencing combined with photothermal effect on liver cancer in mice.Methods: 1.Construction of GAL-GNR nanocarriers: Gold nanorods were synthesized using the classic seed solution growth method,and the gold nanorods were functionally modified.The functionally modified gold nanorods were detected by UV-visible spectroscopy,transmission electron microscopy,nuclear magnetic resonance hydrogen spectroscopy,and dynamic light scattering particle size analyzer.2.Gel retard experiments were performed to test the siRNA loading capacity of GAL-GNR and the protective effect of GAL-GNR on siRNA in serum.3.MTT method and Calcein-AM / PI staining method were used to detect the cytotoxicity of gold nanorods before and after functional modification.4.The photothermal conversion ability of GAL-GNR was measured by infrared laser irradiation.5.Cy3-siRNA was used to detect the transfection efficiency of GAL-GNR in Hapa1-6 cells.qPCR was used to detect the silencing effect of GAL-GNR-siGPC-3 on Hapa1-6 cells.6.Lactobionic acid competition assay was used to detect the targeting effect of galactose structure on liver cancer cells.7.MTT experiment,scratch test,transwell,and clone formation test were used to detect the effects of GPC-3 gene silencing effect on Hepa1-6 cell proliferation,invasion,migration and colony forming ability.8.In vivo targeting effect of GAL-GNR-Cy3-siGAPDH was detected by frozen section method.9.C57BL/6 mouse liver cancer tumor-bearing model was to observe the therapeutic effect on mouse liver cancer.10.The liver toxicity of GAL-GNR in mice was evaluated by detecting AST/GOT and ALT/GPT indicators in mouse serum.The serum creatinine and urea indexes were measured to evaluate the renal toxicity of GAL-GNR in mice.The effect of GAL-GNR on the vital signs of mice was evaluated by measuring the weight change of mice.H&E staining was used to evaluate the histopathological changes and bio-distribution of GAL-GNR on the main organs of heart,liver,spleen,lung,and kidney in mice.Results: 1.Compared with the unmodified GNR,the TEM results showed that the size of GAL-GNR had no obvious change and it possessed good dispersion.The modified GNR had obvious red shift under the UV-Vis absorption spectrum;The NMR hydrogen spectrum showed that GAL molecules were successfully connected.2.Gel block experiment showed that the mass ratio of GAL-GNR to siRNA was the best load ratio at 6?1,and GAL-GNR could effectively protect siRNA from being degraded in serum.3.MTT method and Calcein-AM / PI staining results showed that GAL-GNR had lower cytotoxicity and better biocompatibility 4.The infrared laser irradiation results showed that GAL-GNR has excellent light-to-heat conversion ability,and the light-to-heat conversion ability was related to the material concentration and average light density.5.The results of the red fluorescence image showed that the optimal transfection rate could be achieved when the amount of GAL-GNR was 30 ?g / mL,which was similar to that of the positive control group,and the silencing efficiency was about 75%.6.Lactobionic acid competition experiments showed that after the ASGPR receptor was preoccupied by lactobionic acid,the binding ability of GAL-GNR-Cy3-siRNA to liver cancer cells became weaker.7.GAL-GNR-siGPC-3 could effectively silence the GPC-3 gene in hepatoma cells,resulting in inhibiting the proliferation,invasion,migration and clone formation ability of mouse hepatoma cells.8.The results of frozen tissue sections showed that GAL-GNR-Cy3-siGAPDH could be significantly accumulated in tumors after 24 h of tail vein injection of GAL-GNR-Cy3-siGAPDH.9.The results of in vivo treatment showed that GPC-3 gene silencing and photothermal effect could synergistically inhibit tumor growth and tumor volume,and reduce tumor mass.10.No significant weight loss in mice during tumor treatment was observed;GOT,GPT,creatinine,and urea indicators in serum showed that GAL-GNR had lower liver and kidney toxicity;The H&E staining results showed that no significant damage was found to the organs such as heart,liver,spleen,lung,kidney,when GAL-GNR was adminstrated for a long time,indicating good biocompatibility.Conclusion: 1.This study successfully constructed a new nanocarrier GAL-GNR-siRNA,which integrates advantages of tumor targeting,gene silencing and photothermal transcription.2.GAL-GNR can load and effectively protect siRNA,and can transfer siRNA into hepa1-6 cells to silence GPC-3 gene.After GPC-3 gene silencing,the capacity of proliferation,invasion,migration and clone formation of hepatoma cells decreased significantly.3.GAL-GNR-siGPC-3-mediated siRNA gene silencing combined with photothermotherapy can synergistically treat liver cancer tumor-bearing mice.