Duffrène Joanna, Muzard Chloé, Seguin Johanne, Izabelle Charlotte, Vrai Thibaut, Ejlalmanesh Tina, Bombled Marianne, Hamdi Samir, Lemdani Katia, Alhareth Khair, Mignet Nathalie
Université Paris Cité, CNRS, INSERM, Unité des Technologies Chimiques et Biologiques pour la Santé (UTCBS), Paris, 75006, France.
Neovacs SA, Suresnes, France.
Drug Deliv Transl Res. 2025 May 6. doi: 10.1007/s13346-025-01866-0.
Microfluidics mixing is the current lab-scale method used for producing mRNA-loaded lipid nanoparticles (mRNA-LNPs) thanks to reproducibility and robustness of microfluidic mixing. Despite these advantages, the production of small LNP volumes is associated with significant material waste. Given the high cost of synthetic mRNA, this waste can be a major limitation, particularly for early-stage screening of formulations. This study proposes alternative methods for mRNA-LNP formulation aiming to improve their stability for both formulation and mRNA screening, while reducing material waste on a research scale. Specifically, we investigated post-encapsulation of mRNA into pre-formed vesicles (PFVs) obtained by microfluidic mixing. These PFVs were complexed with mRNA by: (1) a microfluidic or (2) a manual pipetting method. The resulting mRNA-LNPs produced using these two post-encapsulation methods exhibit similar physicochemical properties and morphologies to those obtained by conventional microfluidic protocol. These mRNA-LNPs were assessed on in vitro and in vivo expression. mRNA-LNPs prepared by our alternative methods showed a similar transfection level compared to the conventional formulation taken as a control. The suitability of post-encapsulation methods to other lipids, mRNAs and microfluidic systems was also confirmed. This work offers robust, simple and economic alternative methods for preparing small volumes of mRNA-LNPs. The versatility of post-encapsulation methods allows to screen mRNA formulations in a wide range of laboratories. These methods could be applied to encapsulate tailored doses of mRNA and various mRNA constructs to achieve an optimal and personalized therapy.
微流控混合是目前用于生产载有mRNA的脂质纳米颗粒（mRNA-LNPs）的实验室规模方法，这得益于微流控混合的可重复性和稳健性。尽管有这些优点，但小体积LNP的生产会产生大量材料浪费。鉴于合成mRNA成本高昂，这种浪费可能是一个主要限制因素，特别是在制剂的早期筛选方面。本研究提出了mRNA-LNP制剂的替代方法，旨在提高其在制剂和mRNA筛选方面的稳定性，同时在研究规模上减少材料浪费。具体而言，我们研究了将mRNA后包封到通过微流控混合获得的预形成囊泡（PFVs）中的方法。这些PFVs通过以下两种方法与mRNA复合：（1）微流控法或（2）手动移液法。使用这两种后包封方法生产的mRNA-LNPs在物理化学性质和形态上与通过传统微流控方案获得的相似。对这些mRNA-LNPs进行了体外和体内表达评估。与作为对照的传统制剂相比，通过我们的替代方法制备的mRNA-LNPs显示出相似的转染水平。还证实了后包封方法对其他脂质、mRNA和微流控系统的适用性。这项工作为制备小体积mRNA-LNPs提供了稳健、简单且经济的替代方法。后包封方法的多功能性使得可以在广泛的实验室中筛选mRNA制剂。这些方法可用于包封定制剂量的mRNA和各种mRNA构建体，以实现最佳的个性化治疗。
