溶瘤纳米颗粒CVA21的制备及其抗肿瘤活性分析

Preparation and antitumor activity characterization of oncolytic nanoparticles encapsulating CVA21

摘要:

为了通过包封柯萨奇病毒A21型(Coxsackievirus A21,CVA21)病毒全基因组mRNA制备溶瘤纳米颗粒CVA21(CVA21@ONP)，探究其复活CVA21病毒诱导宿主细胞凋亡的能力以及其在免疫正常的BALB/c荷瘤小鼠中的抗肿瘤免疫效应，本研究采用脂质体纳米颗粒(lipid nanoparticle,LNP)包封CVA21病毒全基因组mRNA制备CVA21@ONP，通过空斑实验和细胞计数试剂盒(cell counting kit-8,CCK-8)确定杀伤效果，通过流式细胞术检测HT29、CT26-iRFP细胞凋亡情况。小鼠经瘤内给药CVA21@ONP，检测近红外荧光蛋白(infra-red fluorescent protein,iRFP)表达的肿瘤生长状况，流式细胞术检测脾脏中免疫细胞的种类与变化。结果表明CVA21@ONP能在HT29与U87MG细胞中成功组装CVA21病毒。空斑实验显示CVA21@ONP对人源和鼠源细胞均具有良好的杀伤效果，流式检测结果显示实验组凋亡细胞显著增加，瘤内检测到CVA21@ONP组iRFP蛋白表达量显著下降。通过流式检测发现CVA21@ONP治疗有效降低了脾脏中包括髓源性抑制细胞(myeloid-derived suppressor cells,MDSCs)和调节性T细胞(regulatory T cells,Tregs)在内的免疫抑制细胞的水平，同时增强了T细胞依赖性的抗肿瘤免疫效应。本研究经瘤内给药验证了CVA21@ONP的溶瘤性能，可进一步挖掘其治疗潜力并推动肿瘤治疗领域的发展。

Abstract:

This study aims to investigate the potential of oncolytic nanoparticles encapsulating Coxsackievirus A21 (CVA21) full-genome mRNA (CVA21@ONP) to resurrect CVA21 and induce apoptosis in host cells, as well as the antitumor immune effects of CVA21@ONP in immunocompetent tumor-bearing BALB/c mice. We used lipid nanoparticles (LNPs) to encapsulate CVA21 full-genome mRNA, thus preparing CVA21@ONP. The killing efficacy of CVA21@ONP was determined by the plaque assay and cell counting kit-8 (CCK-8), and the apoptosis in HT29 and CT26-iRFP cells was evaluated by flow cytometry. Mice were administrated with CVA21@ONP at high and low doses intratumorally, and the growth of tumors expressing infra-red fluorescent protein (iRFP) was monitored. Additionally, the types and changes of immune cells in the spleen were analyzed by flow cytometry. The results demonstrated that CVA21@ONP successfully resurrected CVA21 in both HT29 and U87MG cells. The plaque assay revealed robust killing effects of CVA21@ONP against both human and murine cell lines, and flow cytometry results showed increased early and late apoptotic cells. Notably, intratumoral detection revealed significantly down-regulated expression of iRFP in both high- and low-dose CVA21@ONP groups. Flow cytometry results further indicated that CVA21@ONP treatment effectively reduced the levels of immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), in the spleen, while enhancing T cell-dependent antitumor immune responses. These findings suggest that CVA21@ONP can replicate and survive extensively both in vitro and in vivo, activating the immune system of mice administrated with CVA21@ONP to target cells at the tumor site, thereby remodeling the tumor immune microenvironment and accelerating the suppression or even complete regression of tumors. The oncolytic performance of CVA21@ONP has been verified through intratumoral injection administration in this study, aimed at further exploring its therapeutic potential and promoting the development of the field of tumor treatment.