Rational: Tumor immunogenic cell death (ICD), induced by certain chemotherapeutic drugs such as doxorubicin (Dox), is a form of apoptosis potentiating a protective immune response. One of the hallmarks of ICD is the translocation of calreticulin to the cell surface acting as an 'eat me' signal. This manuscript describes the development of a stable nucleic acid-lipid particles (SNALPs) formulation for the simultaneous delivery of ICD inducing drug (Dox) with small interfering RNA (siRNA) knocking down CD47 (siCD47), the dominant 'don't eat me' marker, for synergistic enhancement of ICD. Methods: SNALPs loaded with Dox or siCD47 either mono or combinatory platforms were prepared by ethanol injection method. The proposed systems were characterized for particle size, surface charge, entrapment efficiency and in vitro drug release. The ability of the SNALPs to preserve the siRNA integrity in presence of serum and RNAse were assessed over 48 h. The in vitro cellular uptake and gene silencing of the prepared SNALPs was assessed in CT26 cells. The immunological responses of the SNALPs were defined in vitro in terms of surface calreticulin expression and macrophage-mediated phagocytosis induction. In vivo therapeutic studies were performed in CT26 bearing mice where the therapeutic outcomes were expressed as tumor volume, expression of CD4 and CD8 as well as in vivo silencing. Results: The optimized SNALPs had a particle size 122 ±6 nm and an entrapment efficiency > 65% for both siRNA and Dox with improved serum stability. SNALPs were able to improve siRNA and Dox uptake in CT26 cells with enhanced cytotoxicity. siCD47 SNALPs were able to knockdown CD47 by approximately 70% with no interference from the presence of Dox. The siCD47 and Dox combination SNALPs were able to induce surface calreticulin expression leading to a synergistic effect on macrophage-mediated phagocytosis of treated cells. In a tumor challenge model, 50% of mice receiving siCD47 and Dox containing SNALPs were able to clear the tumor, while the remaining animals showed significantly lower tumor burden as compared to either monotreatment. Conclusion: Therefore, the combination of siCD47 and Dox in a particulate system showed potent anti-tumor activity which merits further investigation in future clinical studies.

中文翻译：

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用于实体瘤全身免疫治疗的“吃我”组合纳米制剂。

理由：肿瘤免疫原性细胞死亡 (ICD) 由某些化疗药物（如阿霉素 (Dox)）诱导，是一种增强保护性免疫反应的细胞凋亡形式。ICD 的标志之一是钙网蛋白易位到细胞表面，充当“吃我”信号。本手稿描述了稳定的核酸脂质颗粒 (SNALP) 制剂的开发，用于同时递送 ICD 诱导药物 (Dox) 和小干扰 RNA (siRNA)，以敲低 CD47 (siCD47)，即“别吃我”的主导药物' 标记，用于 ICD 的协同增强。方法：通过乙醇注射法制备负载Dox或siCD47的单一或组合平台的SNALP。所提出的系统的特征在于粒径、表面电荷、包封效率和体外药物释放。在 48 小时内评估了 SNALP 在血清和 RNAse 存在下保持 siRNA 完整性的能力。在 CT26 细胞中评估了制备的 SNALP 的体外细胞摄取和基因沉默。SNALP 的免疫反应在体外根据表面钙网蛋白表达和巨噬细胞介导的吞噬作用诱导来定义。在携带 CT26 的小鼠中进行体内治疗研究，其中治疗结果表示为肿瘤体积、CD4 和 CD8 的表达以及体内沉默。结果：优化后的 SNALP 粒径为 122 ±6 nm，对 siRNA 和 Dox 的包封率均 > 65%，并且血清稳定性得到改善。SNALP 能够提高 CT26 细胞中 siRNA 和 Dox 的摄取，并增强细胞毒性。siCD47 SNALP 能够将 CD47 敲低约 70%，而不受 Dox 存在的干扰。siCD47 和 Dox 组合 SNALP 能够诱导表面钙网蛋白表达，从而对处理细胞的巨噬细胞介导的吞噬作用产生协同作用。在肿瘤攻击模型中，接受 siCD47 和含有 SNALP 的 Dox 的小鼠中有 50% 能够清除肿瘤，而与任一单一治疗相比，其余动物的肿瘤负荷显着降低。结论：因此，siCD47 和 Dox 在颗粒系统中的组合显示出有效的抗肿瘤活性，值得在未来的临床研究中进一步研究。