Study Of Calcium-crosslinked And Microenvironment Responsive Nanoparticle Deliver Cytokine For Cancer Immunotherapy

Background and objective:In recent years,cancer has become one of the major diseases that risk human health and effective therapeutic strategies are urgently needed.Immunotherapy shows many outstanding advantages compared with traditional surgery,chemotherapy and radiotherapy.However,there are still significant challenges in cancer immunotherapy including the powerful immunosuppressive tumor microenvironment,immune evasion and adverse off-target side effects.M-CSF is a member of the colony-stimulating factor family,by binding to receptor to regulate mononuclear phagocytic cells and macrophages proliferation,differentiation and survival.It can enhance the anti-tumor effect of the immune system by improving the tumor immune microenvironment.However,the side effects of M-CSF on the systemic immune system limit its application in tumor immunotherapy.With the development of nanotechnology,nano-drug delivery systems can carry drug molecules of different nature,improve the metabolic kinetics and biodistribution and enhance the enrichment and controlled release of drugs in tumor tissues,and improve the antitumor effect and increase security.The large amount of lactic acid and hydrogen ions produced by tumor cell metabolism make the tumor cells have a weak acidic microenvironment?pH=6.5-6.8?,which provides feasibility for designing a targeted nano-delivery system with tumor acidity response.The purpose of this study is to prepare a calcium-crosslinked tumor microenvironment-responsive nanoparticle delivering M-CSF.The nanoparticle improves the tumor immune microenvironment,and enhances the anti-tumor effect of the immune system.Methods:We prepared pH-responsive nanoparticles by calcium-crosslinked for tumor targeting deliver cytokine?M-CSF?.First,we characterized the particle sizes and zeta potentials of CaCO₃ nanoparticles and observed the morphology of the nanoparticles by scanning and transmission electron microscopy.Then,we evaluated the pH responsiveness of CaCO₃ nanoparticles in vitro by measuring the size and morphology after incubating in different pH solutions.We observed the accumulation of nanoparticles in tumor tissues and extracellular drug release by in vivo small animal imaging system and laser confocal imaging macroscopy.Finally,we evaluated the antitumor therapeutic effects of M-CSF encapsulated CaCO₃ nanoparticles and explored its mechanism by B16 tumor-bearing mice.Results:1.The NP/M-CSF/CaCO₃ by CaCO₃ as a crosslinker were 512.4±7.1 nm and the zeta potential were-19.7±0.3 mV.The nanoparticles were regular spherical structure.2.Under the acidic condition?pH 6.5?,As the incubation time increases,the NP/M-CSF/CaCO₃ nanoparticles size gradually decreased.The destruction of nanoparticle structure was observed by scanning electron microscopy.3.In vivo small animal images indicate that CaCO₃ nanoparticles could accumulate in the tumor tissue,and the released payloads were mainly distributed at the tumor stroma.4.Intravenous injection of NP/M-CSF/CaCO₃ could significantly increase the ratio of CD8⁺/CD4⁺and M1/M2 in tumor tissues,and inhibit tumor growth.At the same time,NP/M-CSF/CaCO3 had good safety and no obvious side effects.Conclusion:In this research,we established a“smart”nanoparticle delivery cytokine M-CSF by calcium cross-linker.The nanoparticle had good pH response characteristics.CaCO₃nanoparticles could be rapidly accumulated in the tumor site,and the released cytokine M-CSF were mainly distributed in the tumor stroma,which ensured that M-CSF could play a role in tumor.By evaluating the tumor therapeutic effects of CaCO₃ nanoparticles,it was observed that CaCO₃ nanoparticles inhibited tumor growth by enhancing T cell function and affecting the polarization balance of macrophages in the tumor microenvironment.This study provides a new approach to cascading amplification of anti-tumor effects and regulation of the tumor microenvironment.