Anti-Acod1 siRNA-loaded lipid nanoparticles for liver cancer treatment
Abstract
241877
Introduction: Recent studies provide substantial evidence supporting Cis-aconitate decarboxylase 1 (Acod1) as a potential target for cancer immunotherapy. Acod1 is mainly expressed by myeloid cells, particularly tumor-associated macrophages (TAMs), in both human and murine tumors. The Acod1-derived products, namely Itaconate, exhibit inhibitory effects on inflammatory gene expression and impede the effect of CD8+ T cells at tumor sites. This investigation aims at developing a novel Acod1-targeted silence system for the immunotherapy of hepatocellular carcinoma (HCC) using engineered lipid nanoparticles (LNPs) as the delivery system. Furthermore, biological imaging techniques are employed to trace the in vivo distribution of siAcod1-LNPs and monitor their efficacy in tumor treatment.
Methods: Functionalized LNPs were prepared by thin film hydration and subsequently extruded 15 times through a 400 nm porous polycarbonate membrane. Subsequently, Acod1 siRNA was electrostatically combined with LNPs to form siAcod1-LNPs. The hydrodynamic diameter and zeta potential were measured by dynamic light scattering (DLS). The morphology and structure of LNPs and siAcod1-LNPs were characterized by transmission electron microscope. The in vitro efficacy of siAcod1-LNPs was verified in murine bone marrow-derived macrophages (BMDMs) via qPT-PCR analysis. To explore the impact of Acod1 on tumor immunotherapy, Acod1 knockout mice were utilized in conjunction with PD-1 monoclonal antibody treatment to observe the therapeutic effect of the liver tumor. Subsequently, Hepa1-6 hepatoma cells overexpressing luciferase were orthotopically inoculated into the livers of C57BL/6 mice. Tumor growth was monitored through bioluminescence imaging after intravenous injection of siAcod1-LNPs.
Results: DOTAP/cholesterol cationic liposomes were successfully prepared, followed by electrostatic adsorption to generate siAcod1-LNPs. The encapsulation efficiency of siAcod1 was approximately 83%. Dynamic light scattering measurement showed that the hydrodynamic size of LNPs was 281.52 ± 6.09 nm, while that of siAcod1-LNPs was 273.64 ± 9.73 nm. In vitro experiments demonstrated that si-LNPs significantly downregulated the expression of Acod1 in BMDMs (38.35 ± 2.36%). The knockdown of Acod1 expression in mice restrained the growth of liver tumors and enhanced the efficacy of anti-PD-(L)1 immunotherapy. Additionally, the growth of Hepa1-6 tumors was significantly impeded in the livers of C57BL/6 mice by in vivo delivery of siAcod1-LNPs, as evidenced by in vivo small animal bioluminescence imaging.
Conclusions: We successfully developed LNPs vectors for hepatic siAcod1 delivery. siAcod1-LNPs effectively inhibit the expression of the target gene, significantly contributing to the suppression of liver tumor growth. Moreover, employing the therapeutic gene siAcod1 in a synergistic immunotherapy strategy with PD-(L)1 offers significant advantages. Given the absence of reported inhibitors targeting the Acod1, our developed LNPs facilitate efficient and safe in vivo delivery of siAcod1. This approach is anticipated to be employed for anti-tumor therapy either alone or in combination with PD-(L)1 monotherapy.
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