Gold Nanorod-mesoporous Silica Core Shell Nanocomposites For NIR-Ⅱ Photothermal Ablation And Dual PD-L1/VEGF Blockade Therapy In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma（HCC）is the sixth most common cancer and listed as the third leading cause of cancer-related deaths,with an increasing incidence worldwide.Although traditional treatments such as surgery,radiofrequency ablation,transcatheter arterial embolization,and antiangiogenic drugs can benefit patients with early HCC,most patients are already in advanced stages at their initial diagnosis,and the above strategies show little effect.Immune checkpoint blockade（ICB）therapy can significantly improve the anti-tumor immune response by blocking the binding of inhibitory checkpoints,such as programmed cell death 1（PD-1）,cytotoxic T lymphocyte associated protein 4（CTLA-4）and their ligands between tumors and immune cells,and relieving the inhibition of immune effector cells such as T cells.At present,a variety of immune checkpoint inhibitors（ICIs）have shown considerable efficacy in clinical practice,greatly improving the median survival of patients.However,the heterogeneity of tumors and rapid occurrence of drug resistance make the objective response rate rather low,and most patients cannot benefit from them.A large number of studies have shown that antiangiogenic drugs such as sorafenib can significantly increase the number of infiltrating cytotoxic CD8⁺T cells by normalizing tortuous blood vessels in tumors,and alleviate the immunosuppressive state caused by hypoxia.Therefore,the combination of the two different drugs has made some progress in clinical efficacy.However,due to the lack of tumor specificity,the incidence of toxic side effects caused by"off-target effect"is still high.With the continuous development of nanomedicine in recent years,nano drug delivery system（NDDS）has shown multiple advantages in drug transport and synergistic treatment strategies,thereby attracting widespread attention in the field of cancer treatment.Photothermal therapy（PTT）mediated by nanomaterials is a noninvasive treatment modality that can target a variety of solid tumors.Photothermal agents（PTAs）ablate local tumor tissues by converting light energy into heat,while inducing immunogenic cell death（ICD）,promoting the maturation and activation of antigen presenting cells,and further enhancing the body’s anti-tumor immune response.Therefore,PTT can synergistically enhance ICB therapy by improve the response rate of the latter.The tumor targeting ability of NDDS can further enchances therapeutic effect and alleviates side effects.Gold nanorod（GNR）possesses excellent optical properties,high photothermal conversion efficiency and proper bioinertness,and its surface plasmon resonance wavelength can be adjusted according to the length-width ratio,which makes it one of the critically acclaimed PTAs.At the same time,the pore structure inside the mesoporous silicon dioxide（Si O₂）nanoparticles provides sufficient space for drug loading,and shows good biological safety and stability.Purpose:In this study,we designed and synthesized a gold-silicon nanocomposite（named GSBVVP）loaded with BMS-1166 small molecule drug and the surface was further modified with polyethylene glycol（PEG）and angiostatic peptide vaccine against VEGF（VVP）to mediate near infrared（NIR）Ⅱ photothermal therapy combined with PD-L1/VEGF dual blockade for HCC treatment.We explored the therapeutic effect of GSBVVP in HCC and the underlying molecular mechanism through studying physicochemical properties,in vitro function and in vivo therapeutic effect.This study provides a new strategy for the clinical treatment of HCC based on nanomaterials,and provides a research basis for early clinical transformation of nanomedicine.Method:（1）GSBVVP was prepared,and its morphology,Zeta potential,UV-visible light absorption spectrum,crystal structure,element composition,photothermal conversion ability and stability,dispersion,drug loading efficiency and releasing ability were studied.（2）Through cell experiments,including cellular uptake,cell proliferation-toxicity assay,LIVE/DEAD cell staining,and enzyme linked immunosorbent assay,we studied the targeting ability,cytotoxicity,in vitro therapeutic effect,and the underlying mechanisms of GSBVVP against HCC.（3）Through the establishment of H22 tumor-bearing mouse double tumor models,we studied the biosafety,tissue distribution,in vivo therapeutic effect,immune-activating ability,and anti-angiogenesis effect of GSBVVP.（4）Through the establishment of H22 tumor-bearing mouse recurrence models（reimplantation）,we further explored the in vivo therapeutic effect of GSBVVP and its ability to induce long-term immune memory.Result:（1）In this study,GSBVVP was successfully synthesized.GSBVVP had a unique core-shell structure and high aspect ratio,where the horizontal length of GNR was 51±4 nm and the vertical length was 8±3 nm.The thickness of the lateral Si O₂ was 27±4 nm,and the mesoporous shell had proper pore sizes and large internal drug loading space.When the mass ratio of BMS drug to GSBVVP was 2:1,the maximum loading capacity was 86%,and the actual loading of BMS-1166 inhibitor in GSBVVP was 25.6ng/mg.The absorption peak of GNR in GSBVVP was about 1200nm in the NIR Ⅱ region,which had better penetration and photothermal conversion ability than most NIR I PTAs.The photothermal conversion efficiency（PCE）of 50μg/m L GSBVVP under the irradiation of 0.5W/cm² 1208nm laser was 31.1±1.4%.In H22 tumor-bearing mouse models,50μg/m L GSBVVP could rise to 53.3℃within 1 minute under external laser irradiation,showing promising photothermal therapeutic potentials in vivo.（2）GSBVVP showed a high targeting ability towards HCC in vitro,while it represented almost no toxicity to normal cells and did not exhibit cellular uptake.In addition,GSBVVP irradiated by 1208nm laser showed good photothermal ablating ability and was able to induce the generation of ICD.（3）In H22 tumor-bearing mouse double tumor models,GSBVVP showed promising biosafety and HCC targeting ability.GSBVVP also exhibited significant therapeutic effect under 1208nm laser irradiation.The primary tumor growth inhibition rate was 95.1%and the distant one was 97.3%（P<0.01）,the 60-day survival rate of mice in GSBVVP（+）group reached 100%.By blocking the PD-1/PD-L1 pathway,the release of BMS-1166 in response to irradiation significantly increased the expression levels of CD8⁺T cells and CD4⁺T cells to 22.5%and 23.8%,respectively.The expression level of immunosuppression-related regulatory T cells（Tregs）was also decreased to 10.3%,which could enchance the function of immune effector cells such as CD8⁺T cells;By inducing the body to produce VEGF antibodies,VVP peptides significantly reduce the expression levels of angiogenesis-related proteins p-AKT and p-ERK.Meanwhile,the expression level of memory T cells in GSBVVP（+）group was55.8%,1.7 times and 1.9 times to that of GS（+）group and BMS group,respectively,showing effective ability to induce immune memory.（4）In the recurrence models,the immune memory function mediated by GSBVVP（+）group inhibited the regrowth of H22 cells,and the tumors was almost completely eliminated.During the 60-day survival observation period,all mice were survived,which once again proved that GSBVVP could prevent tumor recurrence.Summary:In this study,we constructed a gold-silicon nanocomposite that could mediate NIR Ⅱ PTT synergizing with PD-L1/VEGF dual signaling pathway blockade,achieving good targeting ability and controllable drug release against HCC.In addition,ICD produced by PTT could synergically enhance the therapeutic effect of ICIs and anti-angiogenic drugs,which could significantly enhance the anti-tumor immune response function while eliminating both the primary and distant tumors in the body,induced the generation of long-term immune memory,and reduced the risk of recurrence.Meanwhile,the GSBVVP nanocomposite exhibited good biocompatibility and represented great potential for clinical transformation.The construction of the GSBVVP nanosystem provided new ideas and experimental support for the combined treatment strategy of advanced HCC and the clinical transformation of gold-silicon nanocomposite system.