Reactive Oxygen Species-Responsive Pyroptosis Nanoinitiators Promote Immune Cell Infiltration and Activate Anti-Tumor Immune Response

Background: Immunotherapy, particularly immune checkpoint inhibitors, has become the standard treatment strategy for diverse malignant tumors. However, the inadequate infiltration of immune cells in tumors coupled with the immunosuppressive tumor microenvironment severely hinders the efficacy of immunotherapy. Methods: A poly(ethylene glycol)- block -poly(lysine) copolymer (mPEG- b -PLL) was prepared through ring-opening polymerization and deprotection, and thioketal (TK) was attached to the amino group of mPEG- b -PLL via the condensation reaction to obtain mPEG- b -PLL-TK. Doxorubicin (DOX) and decitabine (DAC) were encapsulated in mPEG- b -PLL-TK to prepare the pyroptosis nanoinitiator (NP/(DAC+DOX)). The drug release behavior, cellular uptake, pyroptosis-triggering performance, and cytotoxicity of NP/(DAC+DOX) were evaluated in vitro experiments. The in vivo pharmacokinetics and biodistribution of NP/(DAC+DOX) were assessed through fluorescence imaging and high-performance liquid chromatography analysis. CT26 and 4T1 tumor-bearing mouse models were established to evaluate the anti-tumor efficacy, pyroptosis-triggering performance, and immune activation effects of NP/(DAC+DOX). Results: NP/(DAC+DOX) exhibited excellent reactive oxygen species (ROS)-responsive drug release behavior and could be effectively taken up by tumor cells. Experiments both in vitro and in vivo demonstrated that NP/(DAC+DOX) effectively triggered pyroptosis in tumor cells, which was attributed to the DOX-induced activation of caspase-3 and the upregulation of GSDME expression caused by DAC. Following intravenous administration, NP/(DAC+DOX) specifically aggregated in tumor tissues. NP/(DAC+DOX) significantly suppressed tumor growth and extended the survival time of tumor-bearing mice. Furthermore, NP/(DAC+DOX) promoted dendritic cell maturation, enhanced the infiltration of cytotoxic T lymphocytes within the tumor, and decreased the proportion of myeloid-derived suppressor cells. Conclusion: This study developed a ROS-responsive pyroptosis nanoinitiator to precisely induce the pyroptosis of tumor cells, thereby enhancing intratumoral immune cell infiltration and activating anti-tumor immune responses. Keywords: pyroptosis, reactive oxygen species-responsive nanomicelles, immune cell infiltration, immunotherapy