A Study On The Application Of Nanomedicines Based On Removing Physical Barriers To Enhance Immunotherapy In HCC

[Background]In recent years,tumour immunotherapy,a rapidly developing novel therapy,with immune checkpoint inhibitors(PD-1/PD-L1,CTLA-4 antibodies,etc.)has achieved encouraging results in the treatment of melanoma,lung cancer,lymphoma and other tumours,but has had limited efficacy in HCC.According to recent studies,T-cell infiltration within tumors is closely related to the efficacy of immune checkpoint inhibitor therapy.Unlike melanoma and lung cancer,where T-cell infiltration is high,HCC has a low infiltration of T-cells.It has been suggested that the cause of inadequate T-cell infiltration within hepatocellular carcinoma is related to the production of excess extracellular matrix(ECM)during HCC development.Aimed at the typical feature of high hyaluronic acid expression in ECM,HAase is used to clear the tumour stromal barrier and promote T-cell infiltration.At the same time,interleukin-12(IL-12)can polarise macrophages from M0 to M1 which clears tumour cells.Thus,HAase combined with IL-12 may effectively promote CTL infiltration and proliferation,thereby enhancing the antitumour effect of immune checkpoint blockade(ICB)in HCC.However,systemic administration of IL-12 is likely to cause cytokine syndrome and systemic administration of HAase degrades hyaluronic acid in non-tumour organs,causing a range of adverse effects.Additionally,systemic administration of anti-PD-L1 antibodies can result in T cells attacking their own organs,causing a broad range of immune-related adverse events(ir AEs),as normal tissues express this receptor.[Objective]To address the above problems,this study proposes to prepare a tumour-accurate nanomedicine delivery to address the challenge of ineffective immunotherapy for HCC.For one thing,nanomedicine delivery removes the physical barrier of the tumour and promotes T-cell infiltration and proliferation,thus achieving efficient HCC immune cell infiltration.For the other thing,the sensitivity design of nanomedicines enables one-stop precise delivery of pro-T-cell infiltrating drugs and immune checkpoint inhibitors,preventing adverse drug reactions while achieving efficient immunotherapy for liver cancer.The purpose of this study is to improve the effectiveness of immunotherapy in HCC and to provide a new strategy for formulating clinical treatment protocols for this disease.[Methods]Design and synthesis of biosafety-friendly block polymers,characterisation and optimisation.Preparation of nanomedicines and characterisation of physicochemical properties.Preparation of nanomedicines loaded with HAase,IL-12 and αPD-L1 antibodies.Characterisation of the particle size,potential and phase profile of the nanodrugs by means of different experimental techniques.Detection of the drug release behaviour of nanodrugs.Quantitative assessment of the release of IL-12,HAase andαPD-L1 antibodies under different conditions(simulated blood and tumour).Study of the safety and delivery efficiency of nanodrugs in vivo.To examine the safe injectable dose and biosafety of nanomedicines.A mouse subcutaneous tumour model for hepatocellular carcinoma was established.The nanocarriers were labelled with fluorescent molecules and the distribution of the prepared nanodrugs in the subcutaneous tumour and major organs was examined by a small animal live imager.Study on the effect of nanomedicine in HCC treatment.A mouse subcutaneous tumour model of hepatocellular carcinoma was established,and after the drug was administered in the tail vein,tumour growth was recorded regularly,and the infiltration of relevant immune cells and the expression level of cellular molecules in the tumour were detected by histology and molecular biology to elucidate the therapeutic mechanism.[Result]In this study,a tumour microenvironment-responsive(p H and MMP-2 enzyme dual-responsive)nanocarrier was prepared for one-stop delivery of HAase,IL-12 andαPD-L1 antibodies into hepatocellular carcinoma.For one thing,Calcium phosphate(Cap)nanoparticles are broken down in the acidic tumour microenvironment and release HAase and IL-12,eliminating the physical barrier of the tumour and promoting T-cell infiltration and proliferation.For the other thing,the release of αPD-L1 triggered by MMP-2alleviated the immunosuppression of T cells by tumour cells.This combined strategy effectively increased the number and enhanced the activation of CTLs,thus exerting a powerful synergistic anti-tumour effect,inhibiting tumour growth and prolonging the survival of the animals.In addition,IL-12 released by the nanodrug induced M1 polarization and inhibited M2 polarization of Tumour‐associated macrophages(TAMs),which would facilitate macrophage-mediated antitumor effects.[Conclusion]The dual-sensitive nanodrug prepared in this study has demonstrated to be an effective immunotherapeutic modality for the treatment of HCC with the potential to be applied to other solid tumors with intensive ECM features.