A Research Of Hybrid Membrane Coated Metal-organic Framework For Photodynamic Immunotherapy

Cancer immunotherapy(IT)inhibits tumor metastasis and recurrence by activating systemic immune response,but its efficacy is limited by the immunosuppressive state of tumor microenvironment(TME)and insufficient systemic immune response.Photodynamic therapy(PDT)can induce immunogenic cell death(ICD)and immune inflammatory infiltration at the tumor site while killing cancer cells by reactive oxygen species(ROS)generated by photosensitizer under light.As a local therapy model with immune activation,PDT has the inherent advantage of forming a combined treatment platform with IT.In order to enhance the ICD effect induced by PDT and activate the systemic antitumor immune response,we synthesized a metal-organic framework(MOF)material with photosensitizer meso-tetra(4-carboxyphenyl)porphine(TCPP)as an organic linker,and modified its surface with a biomimetic hybrid membrane(B16-NK)obtained by coextrusion of mouse melanoma(B16F10)cell membrane and mouse primary natural killer(NK)cell membrane to get a photoimmunotherapy(PIT)nanoplatform MOFB16NK.Firstly,we prepared MOF by a solvothermal method.The size of MOF was 57.8±1.2 nm and Zeta potential was 33.1±0.4 m V measured by DLS;TEM showed that MOF particles were uniform in size and dispersed evenly;The BET surface area of MOF measured by nitrogen adsorption and desorption method was 799.2994 m2/g;the structure of MOF was characterized by FT-IR and PXRD;the successful introduction of TCPP was proved by UV-vis and fluorescence scanning spectra.Then,we coextruded the extracted B16F10 cell membrane and NK cell membrane into B16-NK hybrid membrane vesicles through a liposome extruder.The morphology of B16-NK was characterized by DLS and fluorescence microscope.We used the mass ratio3:1 of hybrid membrane proteins to MOF to obtain MOFB16NK.The hybrid membrane structure about 20 nm in the outer layer of MOFB16NK was observed by TEM;the colocalization relationship between B16-NK and MOF was verified by fluorescence microscope;the stability of MOFB16NK under physiological conditions was verified by DLS;the optical properties of MOFB16NK was verified by UV-vis and fluorescence spectroscopy;the retention of proteins on B16F10 membrane and NK membrane in MOFB16NK was verified by SDS-PAGE and WB respectively.In vitro study,CLSM and flow cytometry showed that uncoated MOF,single B16F10 membrane coated MOF and hybrid membrane coated MOF had significant incremental material internalization ability in B16F10 cells,and MOFB16NK had a great intracellular ROS production.MTT assay showed that MOFB16NK had low dark toxicity and high phototoxicity,and had ideal photoselective cytotoxicity in vitro.At the same time,MOFB16NK can promote the exposure of calreticulin(CRT)and the release of extracellular adenosine triphosphate(ATP)in B16F10 cells under light irradiation,indicating that the nanosystem can enhance ICD induced by PDT.Flow cytometry showed that the culture supernatant of B16F10 cells treated with MOFB16NK under light could promote the maturation of mouse bone marrow-derived dendritic cells(BMDCs)and activate the downstream cytotoxic T lymphocytes(CD8~+T).TNF-αand IL-12 in BMDCs culture supernatant was increased while IL-10 decreased measured by ELISA.And IFN-γsecretion was increased in the supernatant of the coincubation system of BMDCs and spleen lymphocytes.In vivo study,we used living fluorescence imaging to assess the in vivo distribution of MOFB16NK in mouse melanoma model,and measured the content of Zr in tumor site by ICP-MS to further verify the tumor targeting effect of nanosystem in vivo.After four cycles of treatment,the relative tumor volume(RTV)of mice in MOFB16NK group with light was the lowest and the tumor tissue damage was the most serious.To evaluate the local immune stimulation effect of MOFB16NK,we performed immunofluorescence staining on tumor tissue sections.The results showed that MOFB16NK could promote the expression of CRT and high mobility group protein B1(HMGB1)in TME,as well as the infiltration of CD8~+T cells and M1-like macrophages;ELISA results showed that MOFB16NK could increase the secretion of IL-12 and reduce the secretion of IL-10 in tumor tissue lysate.To evaluate the anti-tumor metastasis and systemic immunostimulatory effects of nanoparticles,we constructed a bilateral metastasis model of mouse melanoma and only the primary tumor was treated with drugs and light.After multiple courses of treatment,flow cytometry was used to detect T cells and macrophages in mouse spleen,and IFN-γin mouse serum.The results showed that MOFB16NK could inhibit the growth of both primary and distal tumors,and significantly prolong the survival time of mice,increasing the proportion of CD8~+T cells and M1-like macrophages in mouse spleen and IFN-γin serum.In conclusion,MOFB16NK can increase the in vitro cell internalization and the in vivo tumor targeting ability of the material through the dual targeting effect of B16F10membrane component and NK membrane component,so as to promote ICD induced by PDT and activate local immune response;NK membrane also plays an immunomodulatory role by cooperating with the ICD effect at the tumor site to cause systemic immune response and finally inhibit the growth and metastasis of melanoma.