Peng Shasha, Zhang Yifan, Zhao Xin, Wang Yibin, Zhang Zihan, Zhang Xin, Li Jiali, Zheng Huiwen, Zhang Ying, Shi Haijing, Li Heng, Liu Longding
Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650118, China.
Key Laboratory of Systemic Innovative Research on Virus Vaccine, Chinese Academy of Medical Sciences, Kunming, Yunnan 650118, China.
Bioconjug Chem. 2024 Dec 18;35(12):2015-2026. doi: 10.1021/acs.bioconjchem.4c00536. Epub 2024 Dec 10.
Ionizable cationic lipids have been developed to mitigate the toxicity of quaternary ammonium lipids, such as DOTAP. Despite its toxicity, DOTAP can promote localization of lipid nanoparticles (LNPs) in target tissues, serving as one of the ionizable cationic helper lipids. Notably, DOTAP-based nanoadjuvants prepared via microfluidic methods showed a better T-cell response. Previous studies showed that DOTAP-based LNPs prepared by the lipid-film method resulted in obvious adverse events. Therefore, our research focused on evaluating the tissue localization and adverse toxicity of a DOTAP-based delivery system prepared through microfluidic techniques. We assessed the delivery efficacy, biodistribution, inflammatory response, and pathological injury in various tissues. In our study, the plasmid DNA encoding the receptor-binding domain (RBD) of SARS-CoV-2 was encapsulated using a mixture of lipids that included DOTAP, DOPE, cholesterol, and DMG-PEG2000 via microfluidic mixing. The LNP-RBDs were smaller than those prepared via the traditional lipid membrane system. We found that LNP-DNA complexes can be effectively delivered and expressed in muscle tissue, with specific antibodies in serum induced postimmunization. Initial distribution of the liposomes was observed in the muscle and liver. Interestingly, both LNPs and DNA showed sustained presence in the lungs and spleen in the group immunized with DNA-encapsulated DOTAP-based LNPs, whereas lower amounts of DNA were detected in the group immunized with dissociative DNA. We detected obvious inflammatory responses and pathological injuries in the muscle, heart, and liver, and the side effects decreased when the immunization dose decreased. These findings suggest that DOTAP-based LNPs have obvious advantages for targeting the lungs and spleen. Additionally, inflammatory responses and pathological injuries occur in a dose-dependent manner in the muscles, heart, and liver. In conclusion, these findings contribute to the development of an LNP delivery system with DOTAP, highlighting its potential to enhance tissue localization and promote high levels of expression when coordinated with ionizable lipids.
可电离阳离子脂质已被开发出来以减轻季铵盐脂质（如DOTAP）的毒性。尽管DOTAP具有毒性，但它可以促进脂质纳米颗粒（LNP）在靶组织中的定位，作为可电离阳离子辅助脂质之一。值得注意的是，通过微流控方法制备的基于DOTAP的纳米佐剂显示出更好的T细胞反应。先前的研究表明，通过脂质膜法制备的基于DOTAP的LNP会导致明显的不良事件。因此，我们的研究重点是评估通过微流控技术制备的基于DOTAP的递送系统的组织定位和不良毒性。我们评估了各种组织中的递送效果、生物分布、炎症反应和病理损伤。在我们的研究中，通过微流控混合，使用包括DOTAP、DOPE、胆固醇和DMG-PEG2000的脂质混合物包裹编码严重急性呼吸综合征冠状病毒2（SARS-CoV-2）受体结合域（RBD）的质粒DNA。基于LNP的RBD比通过传统脂质膜系统制备的更小。我们发现LNP-DNA复合物可以在肌肉组织中有效递送并表达，免疫后血清中诱导产生特异性抗体。观察到脂质体最初分布在肌肉和肝脏中。有趣的是，在用基于DOTAP的LNP包裹DNA免疫的组中，LNP和DNA在肺和脾脏中均持续存在，而在用解离DNA免疫的组中检测到的DNA量较低。我们在肌肉、心脏和肝脏中检测到明显的炎症反应和病理损伤，当免疫剂量降低时副作用减少。这些发现表明基于DOTAP的LNP在靶向肺和脾脏方面具有明显优势。此外，炎症反应和病理损伤在肌肉、心脏和肝脏中呈剂量依赖性发生。总之，这些发现有助于开发基于DOTAP的LNP递送系统，突出了其与可电离脂质协同作用时增强组织定位和促进高水平表达的潜力。
