Bates Stephanie M, Munson Michael J, Trovisco Vitor, Pereira Sara, Miller Sophie R, Sabirsh Alan, Betts Catherine J, Blenke Erik Oude, Gay Nicholas J
Immune Safety, Clinical Pharmacology and Safety Sciences, Biopharmaceuticals R&D, AstraZeneca, Cambridge, UK; Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.
Advanced Drug Delivery, Pharmaceutical Sciences, Biopharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
J Control Release. 2025 Jul 18:114047. doi: 10.1016/j.jconrel.2025.114047.
Lipid nanoparticles (LNPs) are widely used to deliver mRNA therapeutics and vaccines; but treatment related inflammation can pose safety issues for applications requiring higher doses or repeat administration. The mechanisms underlying the toxicity of LNPs are not fully understood, so improved understanding of the intracellular pathways that sense LNP entry into the cell will facilitate LNP design for successful deployment across different therapeutic applications. Here we explored how experimental conditions can influence the uptake of LNP-delivered mRNA in vitro and modulate both the expression pattern of the cargo and cytotoxicity. We found that altering serum protein concentrations in culture media influence cellular uptake of LNPs, resulting in expression patterns that were not dose dependent. Additionally, changing or removing the mRNA cargo did not significantly affect cytotoxicity, indicating that this response was driven by the LNP components. Using a fluorescent Galectin 9 reporter system, kinetic experiments with different serum protein concentrations were conducted to measure LNP uptake, endosomal escape and cargo expression to systematically explore how these processes are related to cytotoxicity. We demonstrate that cytotoxicity is driven by both endosomal disruption and lipid chemistry. Our finding that the rate and magnitude of endosomal disruption correlates with cytotoxicity provide a new focus for investigation. Developing LNPs with optimal mRNA release mechanisms, and a reduced rate and duration of endosomal disruption, could alleviate some of the safety concerns associated with LNP/mRNA. Finally, we found that individual cationic lipid components can also influence cytotoxicity that is independent of cargo delivery, implying that at least some of the cytotoxicity can be mitigated with chemical design.
脂质纳米颗粒（LNPs）被广泛用于递送mRNA治疗药物和疫苗；但与治疗相关的炎症可能会给需要更高剂量或重复给药的应用带来安全问题。LNPs毒性的潜在机制尚未完全了解，因此更好地理解感知LNP进入细胞的细胞内途径将有助于设计LNP，以便在不同的治疗应用中成功部署。在这里，我们探讨了实验条件如何在体外影响LNP递送的mRNA的摄取，并调节货物的表达模式和细胞毒性。我们发现，改变培养基中的血清蛋白浓度会影响细胞对LNPs的摄取，导致表达模式不依赖于剂量。此外，改变或去除mRNA货物对细胞毒性没有显著影响，表明这种反应是由LNP成分驱动的。使用荧光半乳糖凝集素9报告系统，对不同血清蛋白浓度进行动力学实验，以测量LNP摄取、内体逃逸和货物表达，从而系统地探索这些过程与细胞毒性的关系。我们证明细胞毒性是由内体破坏和脂质化学共同驱动的。我们发现内体破坏的速率和程度与细胞毒性相关，这为研究提供了一个新的重点。开发具有最佳mRNA释放机制、降低内体破坏速率和持续时间的LNPs，可以减轻与LNP/mRNA相关的一些安全问题。最后，我们发现单个阳离子脂质成分也可以影响与货物递送无关的细胞毒性，这意味着至少部分细胞毒性可以通过化学设计来减轻。
