Lipid Nanomedicines Modulate Inflammation-related And Immunosuppressive Factors For Enhanced Tumor Immunotherapy

In recent years,the global burden of cancer has continued to increase,making it a major public health concern.Therefore,it is essential to develop effective strategies for cancer prevention and treatment.The emergence of immunotherapy has presented a glimmer of optimism regarding the potential eradication of cancer.Despite the improved therapeutic effects achieved by small molecule inhibitors or antibody drugs that target immune checkpoints in clinical cancer treatment,the overall response rate remains low,and certain tumor types remain unresponsive to immunotherapy.Additionally,it is associated by a range of significant immune-related adverse responses that further restrict the therapeutic efficacy.Hence,it is imperative to conduct continuous research to improve the therapeutic efficacy and minimize the adverse effects with tumor immunotherapy.Tumor-associated inflammation is recognized as a crucial characteristic of all cancers and plays a significant role in various stages of tumorigenesis,progression,malignancy,invasion,and metastasis.Inflammation-associated molecules play an essential part as mediators that establish a connection between inflammation and cancer,while also governing the progression of cancer through many pathways.For instance,caspase-1 serves as an intermediary connecting the inflammasome and the inflammatory response.Upon activation by classical inflammasome,caspase-1 cleaves cytokine precursors and promotes the secretion of the inflammatory cytokines IL-1βand IL-18.Tumor-associated macrophages（TAMs）represent the primary inflammatory cells in the tumor microenvironment.High levels of TAMs infiltration are typically associated with poor patient prognosis in most solid tumors.Tumor microenvironment stimulates the secretion of IL-1βand IL-18 by TAMs,resulting in tumor immunosuppression.Furthermore,the involvement of caspase-1 in TAMs also encompasses the regulation of cellular metabolic programming and differentiation,hence exerting an influence on immune function.Nevertheless,the existing research on caspase-1 primarily focuses on gene-deficient mice or small molecule inhibitors.The absence of cell-targeted therapy techniques restricts its in vivo application.Hence,it is imperative to devise regulatory approaches that specifically target caspase-1 in macrophages for the purpose of tumor therapy.Immunosuppressive factors are one of the main causes of tumor immune escape.Programmed cell death ligand-1（PD-L1）acts as a critical checkpoint for the immune system.T cell dysfunction is the consequence of the interaction between PD-L1expressed by tumor cells and PD-1 on T cells.Recent studies have demonstrated that the expression of PD-L1 by dendritic cell（DC）is essential in the tumor immune response.Not only does it exhibit an inhibitory impact by binding to PD-1 on the surface of T cells,but it also binds to its own CD80 to impede the activation of DC,hence impeding T cell activation.Currently,numerous studies have been dedicated to investigating therapeutic approaches that specifically target PD-L1 on tumor cells.However,there remains a dearth of publications that explore PD-L1 as a therapeutic target in DC.Furthermore,the therapeutic efficacy of PD-L1 antibodies and inhibitors has been constrained by their harmful side effects.Hence,the development of strategies aimed at efficiently modulatingPD-L1 expression in DC will offer novel insights for enhancing tumor immunotherapy.The potential of nucleic acid drugs in disease treatment lies in their ability to influence gene expression and cellular function.Nevertheless,carriers are necessary for nucleic acid molecules delivery as they have limited stability and face challenges in penetrating cells.Lipid nanoparticle（LNP）is considered the most promising class of nucleic acid drug delivery systems for clinical applications.Optimizing the performance of LNP to enhance the in vivo delivery efficiency and cellular targeting of nucleic acid medicines is essential for enhancing the safety and effectiveness of tumor therapy.This work aims to optimize the LNP for investigating the regulation of inflammation-related factors and immunosuppressive molecules that specifically target immune cells.The objective of this study is to examine the therapeutic efficacy and underlying mechanism of employing LNP as a nucleic acid delivery system for the purpose of alleviating immunosuppression and augmenting immune response in malignancies.The study is divided into two parts:1.BAMPA-O16B lipid was utilized in the construction of lipid nanoparticles for the delivery of caspase-1 siRNA（BAMPA-O16B/siCaspase-1）.In vitro study,we examined uptake capacity of macrophages for BAMPA-O16B/siCaspase-1,evaluated the efficacy of gene silencing,and investigated the impact of reduced caspase-1expression on macrophage polarization,inflammatory protein expression,cellular pyroptosis,and cytokine secretion.The in vivo experiments involved the initial observation of the distribution of BAMPA-O16B/siCaspase-1 and its regulatory effects on macrophages.Subsequently,BAMPA-O16B/siCaspase-1 was administered intravenously to 4T1 tumor-bearing mice to investigate the inhibitory effects on tumor growth.The alterations in the immune system were analyzed using flow cytometry,immunohistochemistry,and immunofluorescence assay to elucidate the effects of the tumor inhibition.These investigations aimed to elucidate the underlying anti-tumor mechanism and evaluate the safety of nanoparticles.In vitro results demonstrated that BAMPA-O16B/siCaspase-1 was taken up by macrophages and successfully suppressed caspase-1 expression.Additionally,it facilitated the polarization of macrophages towards M1 phenotype.The suppression of caspase-1 activity by the reduction of caspase-1 expression in macrophages has the potential to impede macrophage inflammatory death,as well as the production and secretion of inflammatory proteins and cytokines.The in vivo findings demonstrated that the siRNA delivered by BAMPA-O16B/siCspase-1 mostly acted on tumor-associated macrophages,whereas its distribution was comparatively lower in lymphoid lineage cells and tumor cells.BAMPA-O16B/siCaspase-1 exhibited favorable biosafety characteristics and effectively suppressed the breast cancer growth by modulating macrophage activation,inhibiting inflammatory cytokines expression,regulating macrophage repolarization,and enhancing the antitumor activities of effector cells.This study demonstrates the potential of employing LNP to modulate macrophage inflammation-associated factors to enhance tumor therapeutic effects.2.Development of lipid nanoparticles with varying cholesterol densities based on BAMEA-O16B lipid（BLAN）for the purpose of delivering the CRISPR/Cas9 gene editing system.The expression of mRNA delivered by BLAN_mRNA with different cholesterol densities was examined in DC,macrophages,and tumor cells during the in vitro experiments.We also conducted a comparative analysis of the uptake and endosome escape capabilities of BLAN_mRNA with different cholesterol densities in DC.Furthermore,we analyzed the effect of cholesterol density on the knockout of PD-L1within DC by BLAN_mCas9/gPD-L1.The in vivo experiments investigated the distribution of nanoparticles and their regulatory effects on DC using optimized BLAN.The study analyzed changes in the expression level of PD-L1 in DC by intratumoral injection of BLAN_mCas9/gPD-L1.The knockout effects of PD-L1 expression in DC on cell mature,T cell activation,and tumor immune microenvironment were investigated.Finally,the study evaluated the safety and therapeutic effects of in vivo application.In vitro results revealed that the cholesterol content plays a crucial role in the regulation of mRNA expression in DC by BLAN.Decreasing cholesterol density led to an improvement in the efficiency of BLAN uptake by DC,endosomal escape,and sgRNA release.In vivo findings indicated that low cholesterol density BLAN had the ability to accumulate in tumors and draining lymph nodes,as well as facilitate the delivery of Cas9 mRNA and PD-L1 sgRNA to DC.BLAN effectively relieved DC immunosuppression,enhanced cDC1 enrichment,improved DC activation and maturation,and increased the level of T cell-mediated anti-tumor immune response by knockout of PD-L1 expression in cDC1 in draining lymph nodes,ultimately significantly inhibiting colon cancer growth.This study reveals the feasibility and mechanism of using optimized LNP delivery CRISPR/Cas9 system to modulate DC immunosuppressive factors and enhance anti-tumor immune responses.In summary,this study involved the development of two distinct lipid nanomedicines with the aim of facilitating the delivery of nucleic acid drugs to modulate inflammation-related factors and immunosuppressive molecules within immune cells.Reducing the expression of inflammatory molecules in macrophages to promote their differentiation and activation towards the M1 type and mitigat the tumor immunosuppressive microenvironment.Additionally,by knocking down the immunosuppressive molecules in DC,it increases the activation level of DC and enhances the T cell anti-tumor immune response.This study showcases the potential application of performance-optimized lipid nanocarriers for delivering nucleic acid drugs and modulating immune system function,which provides new strategies and approaches for tumor immunotherapy.