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

Area of Science:

• Biotechnology
• Nanotechnology
• Drug Delivery

Background:

• Microfluidics is the standard for producing mRNA-loaded lipid nanoparticles (mRNA-LNPs) due to its reproducibility.
• However, microfluidic methods generate significant material waste, especially for small-scale research and formulation screening.
• The high cost of synthetic mRNA exacerbates the economic limitations of current methods.

Purpose of the Study:

• To develop alternative, cost-effective methods for producing mRNA-LNPs at a research scale.
• To improve the stability of mRNA-LNPs for formulation screening and reduce material waste.
• To investigate post-encapsulation of mRNA into pre-formed vesicles (PFVs) as a viable alternative.

Main Methods:

• Pre-formed vesicles (PFVs) were generated using microfluidic mixing.
• mRNA was encapsulated into PFVs using either a microfluidic or a manual pipetting method.
• The resulting mRNA-LNPs were characterized for physicochemical properties, morphology, and in vitro/in vivo expression levels.

Main Results:

• mRNA-LNPs produced via post-encapsulation showed similar physicochemical properties and morphologies to conventionally produced mRNA-LNPs.
• Both microfluidic and manual pipetting post-encapsulation methods resulted in comparable in vitro and in vivo transfection efficiencies.
• The suitability of these methods was confirmed across different lipids, mRNA types, and microfluidic systems.

Conclusions:

• Post-encapsulation offers robust, simple, and economical alternatives for small-scale mRNA-LNP preparation.
• These methods reduce material waste and enable efficient formulation screening.
• The versatility of post-encapsulation facilitates personalized mRNA therapies by allowing tailored doses and constructs.