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Updated: Jul 11, 2025

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Published on: February 1, 2019

Investigations into mRNA Lipid Nanoparticles Shelf-Life Stability under Nonfrozen Conditions.

Anne-Gaëlle Reinhart¹, Anja Osterwald², Philippe Ringler³

¹Roche Pharma Research and Early Development, Therapeutic Modalities, pCMC, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, Basel 4070, Switzerland.

Molecular Pharmaceutics

|November 17, 2023

Summary

mRNA lipid nanoparticles (LNPs) can maintain activity without freezing for extended periods. This study found specific LNPs, like C12-200, retain functionality at room temperature, easing storage and transport challenges for mRNA technology.

Keywords:

EGFP LC-MS LNP analytics electron microscopy mRNA shelf-life stability

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Published on: August 9, 2022

Area of Science:

Nanotechnology
Biotechnology
Pharmaceutical Sciences

Background:

mRNA lipid nanoparticles (LNPs) are crucial for drug and vaccine delivery.
Current mRNA LNP formulations require frozen storage, posing logistical and cost challenges.
Developing stable, non-frozen storage conditions is essential for broader mRNA LNP applications.

Purpose of the Study:

To assess the stability and activity of mRNA LNPs under non-frozen storage conditions.
To identify mRNA LNP formulations that maintain functionality at ambient temperatures.
To understand the structure-activity relationships influencing LNP stability.

Main Methods:

Synthesized five structurally diverse mRNA LNPs using microfluidic technology with varying ionizable lipids (C12-200, CKK-E12, MC3, SM-102, lipid 23).
Evaluated shelf-life stability over 9 weeks at 2-8°C, 25°C, and 40°C using analytical techniques (hydrodynamic diameter, zeta potential, encapsulation efficiency, polydispersity).
Assessed in vitro activity via EGFP protein expression in HEK293 cells and analyzed LNP architecture using cryo-EM.

Main Results:

Most LNPs showed minimal changes in physical characteristics (size, charge, encapsulation) during non-frozen storage.
RiboGreen assays suggested consistent mRNA content, but in vitro activity varied significantly.
LNP 1 (C12-200) and LNP 4 (SM-102) showed high initial transfection efficiency.
LNP 1 (C12-200) maintained activity for 11 weeks at 2-8°C and 25°C.
LNP 4 (SM-102) retained activity at 2-8°C but declined at 25°C over 11 weeks.

Conclusions:

Specific mRNA LNP formulations, particularly those with C12-200 lipid, demonstrate potential for non-frozen storage and transport.
In vitro activity assays are crucial for assessing LNP functionality, as physical stability metrics can be misleading.
Understanding LNP architecture and structure-activity relationships is key to overcoming storage limitations and expanding mRNA LNP applications beyond vaccines.