Depleting Tumor Infiltrating B Cells To Boost Antitumor Immunity With Tumor Immune-microenvironment Reshaped Hybrid Nanocage

The effect of tumor tissue resident and recruited immune cells to the development of tumor has gained notable attention in clinic.Tumor infiltrating B cells(TIB)have been regarded as one of the most important immune cells in tumor tissues.TIB were commonly found in various solid tumors such as breast and melanoma.Based on recent research,TIB could secrete suppressive cytokines including TGF-β and IL-10,increase skewing of T-helper cells toward inhibitory Th2 cells,suppress Th1 cells,support expansion of regulatory T cells(Tregs),and/or inhibit CD4+ and CD8+T cell directly to suppress antitumor immunity.Therefore,as important regulatory cells in the tumor immune-microenvironment,the relationship between TIB and tumor development cannot be ignored.Otherwise,its immunosuppression will affect the effects of tumor therapy,which mean that TIB-dependent immunotherapy could effectively boost antitumor immunity through systemic regulation of tumor immune-microenvironment.Ibrutinib(Ib)as a small molecule inhibitor can bind to BTK at residue C481,causing the downstream of BTK in B-cell receptor(BCR)signaling to be blocked,resulting in the depletion of B cells.Ib is currently approved agent that has become attractive treatment for patients with certain B cell lymphoma in clinics.Otherwise,it has certain effect in the treatment of solid tumors such as colon cancer and pancreatic ductal adenocarcinoma.Therefore,TIB depletion to reshape the tumor immune-microenvironment could achieved with the application of Ib in tumor treatment.Docetaxel(DTX)directly kills tumor cells by strengthening tubulin polymerization and inhibiting microtubule depolymerization,which was widely used in clinical tumor treatment including melanoma.Herein,A new strategy that combines chemotherapy and TIB depletion related immunotherapy was introduced,we combined lb and DTX to regulate immune response and kill tumor cells directly,the applied Ib would effectively reshape the immune-microenvironment of melanoma by depletion of TIB in tumors,which could enhance the antitumor effect of chemoimmunotherapy.Co-delivery system of drugs to their target sites was extremely needed due to separately targeting delivery of Ib and DTX to TIB and tumor cells.An ideal co-delivery system could increase the co-location of differ-targeted drugs in tumor tissues,and deliver drugs to targeted cells respectively to facilitate cellular uptake for synergistic antitumor effects.Herein,a tumor immune-microenvironment reshaped hybrid nanocage consisted of biomacromolecule shell and differ-targeted lipid nanoparticles cores was designed to co-delivery Ib and DTX to stepwise targeting TIB and tumor cells in tumor tissues separately,which realized co-delivery of differ-targeted drugs and enhanced treatment effects of chemoimmunotherapy.We synthesized N-acetylneuraminic acid(Neu5Ac)modified phospholipid material that can specifically bind to the CD22 receptor on the TIB surface,Cys-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val(α-MSH)modified phospholipid material that can specifically bind to the melanocortin-1 receptor on the surface of melanoma cells,and asparagines-glycine-arginine(NGR)modified tumor microenvironment pH-responsive charge reversal material carboxymethyl chitosan-polyethylene glyco-NGR(CMCS-PEG-NGR,CPN)that can specifically bind to the CD 13 receptor on the surface of tumor neovascular endothelial cells,respectively.Ib and DTX were loaded into cationic lipid nanoparticles(cLnp)by thin film dispersion method to form different target lipid nanoparticles cores(NLI and MLD),and self-assemble with CPN through electrostatic interaction to form hybrid nanocage(CPN-NLI/MLD).After the accumulation of CPN-NLI/MLD in tumor tissues through NGR-mediated active targeting,CPN could be disassembled from nanocage in response to the tumor acidic microenvironment,CPN-NLI/MLD changed from negative to positive,differ-targeted NLI and MLD were released,and Neu5Ac and a-MSH mediated TIB and tumor cells targeting respectively as expected.It was beneficial to increase the uptake of Ib by TIB and DTX by tumor cells,respectively.DTX directly killed tumor cells,and Ib could effectively reshape the immune-microenvironment of melanoma by depleting TIB,which could improve the chemoimmunotherapy effects.Main contents and results are listed as follows in this study:1.Determination method for Ibrutinib and DocetaxelMethod for the determination of Ib and DTX was established by HPLC,respectively.The results showed that determination methods were specific and linear relationships were good.The precisions and recoveries could meet the test requirements.2.Preparation and characterization of co-loaded with Ibrutinib and Docetaxel tumor immune-microenvironment reshaped hybrid nanocageFirstly,materials modified by different targeting ligands were synthesized,respectively.In order to achieve tumor tissue accumulation and tumor acid microenvironment responsive depolymerization,the charge reversal materials CPN modified by targeting molecule NGR was synthesized.In order to differ-targeting delivery Ib and DTX,Neu5Ac and a-MSH modified phospholipid materials were synthesized respectively.Targeted loaded with Ib and DTX cLnp(NLI and MLD)were prepared by thin film dispersion respectively,and CPN-NLI/MLD was prepared by electrostatic adsorption.The particle size,zeta potentials,morphology and the charge reversal properties were evaluated.The results showed that CPN-NLI/MLD were spheres with average diameters of 117.5±14.9 nm and zeta potentials of-21.5±1.05 mV.The particle sizes of CPN-NLI/MLD changed from 121.2±16.2 nm to 49.4±4.9 nm in pH 6.5,and the zeta potentials changed from-8.1±1.2 mV to 2.4±0.3 mV.The cumulative release of Ib and DTX at pH 6.5 was 58.0%and 54.9%,respectively,which were higher(p<0.001,p<0.001)than that of pH 7.4(43.3%and 36.3%)due to the responsive disassembly of CPN-NLI/MLD in mimic acid tumor microenvironment.3.Evaluation of stepwise targeting delivery for co-loaded with Ibrutinib and Docetaxel tumor immune-microenvironment reshaped hybrid nanocageMelanoma was selected as the tumor model.The active targeting behavior and differ-targeting delivery properties of CPN-NLI/MLD were investigated.The results of cellular uptake and real-time imaging assays showed the active targeting of CPN-NLI/MLD mediated by NGR.The results of cellular uptake experiments showed that Ib and DTX could be taken up by TIB and tumor cells,respectively.And the uptake under pH 6.5 was significantly higher than that under pH 7.4(p<0.05,p<0.05).The results of co-culture experiments showed that CPN-NLI/MLD has better differ-targeting delivery ability.These results indicate that NLI and MLD could target B cells and tumor cells separately after disassembly of CPN-NLI/MLD under acidic tumor microenvironment.4.Study on antitumor efficacy and safety for co-loaded with Ibrutinib and Docetaxel tumor immune-microenvironment reshaped hybrid nanocageThe cytotoxic effects of CPN-NLI/MLD to WEHI 231 B cells and B16 cells were investigated through cytotoxicity assay and Western-blot experiments.The results showed that the enhanced TIB depletion of CPN-NLI were achieved by inhibition of BTK activation(p<0.05)compared with 1b solution.CPN-MLD has higher cytotoxicity to B16(p<0.01)compared with DTX solution.The outcomes of the immunofluorescence staining showed that CPN-NLI/MLD group decreased the activation of BTK,which depleted TIB,inhibited Treg proliferation,promoted CD4+and CD8+T cells infiltration,improved immunogenic cytokines IFN-γ and IL-2,reduced related immunosuppressive cytokines IL-4,IL-10 and TGF-β,and increased the proportion of M1/M2 tumor-associated macrophages,dendritic cells and natural killer cells,thus reshaped the immune microenvironment and enhanced antitumor immunity.In vivo antitumor experiments showed that CPN-NLI/MLD could significantly improve therapeutic efficacy compared with CPN-NLI and CPN-MLD groups(p<0.001,p<0.01).And the results of H&E,Ki67 and TUNEL stain demonstrated that CPN-NLI/MLD could enhance the tumor therapeutic effect.No obvious lesions were observed in H&E staining of major organs in vivo.In summary,this study constructed a tumor microenvironment pH responsive tumor immune-microenvironment reshaped hybrid nanocage based on the TIB depletion immunotherapy,which was used to differ-targeting delivery Ib and DTX to TIB and tumor cells,respectively.The hybrid nanocage could deplete TIB,reshape the tumor immune microenvironment,and improve the effect of chemoimmunotherapy.CPN-NLI/MLD represents a promising method for advanced design of co-delivery drug carriers of differ-targeted drugs and effective combination therapy based on the TIB related immunotherapy.