Black Phosphorus Nanosheets Activated By The Second Near-infrared Window Co-delivery Of Sorafenib And PD-L1 M Ab For Combined Photothermal Therapy Of HCC

Primary liver cancer is an important disease that endangers human life.Hepatocellular carcinoma(HCC)is the most common primary liver tumor with a 5-year survival rate of only 18%.Drug therapy is the most common treatment for HCC.However,FDA-approved first-line drug regimens still struggle to meet demand due to the complex tumor growth process.Therefore,exploring new effective strategies to achieve efficient drug delivery to improve the therapeutic effect of HCC is a hot research topic.Photothermal therapy(PTT),as a new type of therapy that has attracted much attention in the field of tumor treatment,can kill tumors while inducing immunogenic cell death(ICD)effect to activate the body’s immune response,making it useful in tumor treatment.has unique advantages.However,PTT tumor therapy alone has limited efficacy.On the one hand,PTT commonly used photothermal agents in the near-infrared first(NIR-Ⅰ)region is not suitable for deep tissue tumors due to their easy absorption and scattering by skin tissue and limited penetration depth.On the other hand,the process of PTT activating the body’s immune response through the ICD effect is affected by the tumor immunosuppressive microenvironment(TIME),which limits the efficacy of PTT in immunity.Immune checkpoint blockade of T cells and suppressive T cell activity on Macrophages(TAMs)with high PD-L1 expression are the two main factors for immunosuppression in HCC.Therefore,adopting an effective combination strategy to alleviate the TIME will further improve the immunotherapy effect of PTT-induced ICD.To solve the above problems,on the one hand,photothermal agents in the near-infrared second(NIR-Ⅱ)region are used for PTT therapy.Due to the longer wavelength,they allow deeper tissue penetration and are suitable for deep tumors.On the other hand,the chemotherapy drug Sorafenib(SF)and the immune checkpoint inhibitor PD-L1 antibody are a very effective combination strategy.PD-L1 antibody can specifically block the immune checkpoint PD-1/PDL1 pathway and restore the immune killing function of T cells.SF exerts the killing effect of chemotherapy.In addition,studies have shown that inhibition of the RAF-MEK-EPK pathway by SF can down-regulate the expression of PD-L1 on tumor cells and TAM cells,further activating T cells.Therefore,the combination of SF and PD-L1 antibody can effectively regulate the TIME,relieve T cell immunosuppression,and enhance the therapeutic effect of PTT-induced ICD.However,the combined application of multiple drug molecules faces a series of challenges,and choosing a reasonable delivery method to achieve the optimal therapeutic effect is an important challenge for combined therapy.Nano-drug delivery systems provide a good solution strategy for co-delivery of HCC drugs.Among them,"Carrier-free" drug delivery systems based on therapeutic carriers have attracted widespread attention due to their advantages of high drug loading,good biocompatibility,and low cytotoxicity.Black phosphorus(BP)has attracted widespread attention in the biomedical community due to its excellent safety,high drug loading,and high photothermal conversion efficiency in the NIR-Ⅰ and NIR-Ⅱ regions.Therefore,BP was selected as NIR-Ⅱ region photosensitizer and drug carrier to construct a BP-based "Carrierfree" drug delivery system,which can not only exert excellent PTT ability,but also fully improve the delivery efficiency and treatment of PD-L1 antibody and SF.Effect.Based on the above design concept,this project uses BP nanosheets(BP nanosheets,BPNSs)as drug carriers and photosensitizers to design a "Carrier-free" drug delivery system(BPSP)that co-loads SF and PD-L1 antibody.In this paper,BPNSs with suitable particle size were first prepared by ultrasonication.Considering that BPNSs are easily oxidized in air,BPNSs were functionalized with amino compound Mal-PEG-NH2 to prepare BP@PEG-Mal.Then,taking advantage of the huge specific surface area on BPNSs,SF was first loaded on BP@PEG-Mal to prepare BP@PEG-Mal/SF.Finally,BP@PEG/SF-PD-L1(BPSP)was prepared by Michael addition reaction between the thiolated PD-L1 antibody and BP@PEGMal/SF.After BPSP co-delivered SF and PD-L1 mAb through PD-L1-mediated active targeting to the HCC site,NIR-Ⅱ region laser was used to irradiate the HCC site,and the temperature of the HCC site increased rapidly,resulting in a corresponding therapeutic effect.Specifically:①NIR-Ⅱ photothermal effect can not only kill tumor cells,but also induce ICD effect and activate the body’s immune response;②Local heating of HCC can partially destroy the deep HCC interstitium and reduce the expression of type I collagenase,Promote the deep penetration of SF and PD-L1;③promote the rapid diffusion of SF and PD-L1 antibodies on BPNSs to kill tumor cells by different mechanisms,relieve T cell immunosuppression,relieve the TIME at HCC sites,and enhance the efficacy of ICD.The main research contents of this subject are as follows:1.Establishment of Methodology for Determination of SorafenibThe sorafenib content determination methodology was established by high performance liquid chromatography.2.Preparation and Evaluation of Co-loaded SF and PD-L1 Multifunctional Black Phosphorus NanosheetsThe physicochemical properties of BPSP were evaluated by analyzing drug loading,morphology,potential,particle size,thickness,X-ray photoelectron spectroscopy and Raman spectroscopy.The stability of in vitro BPSP was evaluated by uv absorption curve.The photothermal properties of BPSP were investigated by NIR-Ⅱ laser and thermometer.The in vitro photothermal release behavior of BPSP was investigated by dialysis.In vitro tumor cytotoxicity of BPSP was detected via MTT assay.The tumor targeting ability of BPFP in vivo and in vitro was evaluated by cell uptake and near infrared imaging of small animals.3.Evaluation of the combined mechanism of co-loaded SF and PD-L1 multifunctional black phosphorus nanosheetsTo evaluate the ability of NIR-Ⅱ laser to promote deep penetration of BPSP,pigskin with different thicknesses was first used to cover the tumor surface to evaluate the penetration depth of BPSP under NIR-Ⅱ laser irradiation;then,the in vitro deep layer of BPCP+L group was evaluated by constructing tumor spheres permeability ability of BPCP was further investigated by laser confocal microscope.Finally,the expression of Collagen I under NIR-Ⅱ laser irradiation was observed by immunofluorescence section.The ability of BPSP to induce ICD effect was evaluated by detecting CRT,HMGB1,ATP and DC maturation.The relationship between BPSP and PD-L1 expression were evaluated by flow cytometry.4.In vivo pharmacodynamic evaluation of multifunctional black phosphorus nanosheets co-loaded with SF and PD-L1In this paper,Hepa1-6-bearing C57BL/6 xenograft mice were selected as the model to verify the combined treatment effect of BPSP in HCC under NIR-Ⅱ laser irradiation,and to investigate its anti-tumor effect in subcutaneous tumors;On this basis,an HCC orthotopic model was established by surgery to investigate the orthotopic tumor inhibition effect of the BPSP+L group.The numbers of CD4+T cells,CD8+T cells,CTL cells and Tregs,the levels of cytokines INF-y,IL-12,TNF-α,IL-10 and TGF-β were examined by flow cytometry and ELISA kits.An investigation was conducted to evaluate the immune-promoting effect of BPSP on photothermal efficacy under NIR-Ⅱ laser irradiation.In addition,the preliminary in vivo safety evaluation of BPSP was carried out by hemolysis test and H&E staining of in vivo organs.In conclusion,this paper enhances the combined treatment effect of HCC from two aspects:increasing the depth of light penetration and enhancing the ICD effect induced by PTT.Using BPNSs as drug carriers and photosensitizers,a "Carrier-free" co-loading SF and PD-L1 antibody was constructed.The nano-delivery system(BPSP)has demonstrated good anti-tumor therapeutic effect in HCC subcutaneous tumor and orthotopic models.This is an innovative tumor combination therapy method,which opens up new possibilities for the comprehensive inhibition of tumor therapy and provides new treatment concepts for various other tumors in the clinic.