Post-encapsulation methods for the preparation of mRNA-LNPs.

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.