Researchers at the University of British Columbia have developed a novel class of liposomal lipid nanoparticles (LNPs) that demonstrate superior mRNA delivery capabilities and enhanced stability compared to conventional formulations. The study, published in Nature Communications, reveals how modifying the ratio of bilayer-forming lipids can dramatically improve both the performance and storage characteristics of mRNA therapeutics.
Enhanced Formulation Design
The research team systematically investigated LNP systems containing varying ratios of bilayer lipids (equimolar egg sphingomyelin and cholesterol) to ionizable lipid, designated as RB/I ratios ranging from 9 to 0.43. Using nor-MC3 as the ionizable lipid component, they formulated four-component systems while maintaining PEG-lipid content at 1.5 mol ratio.
Cryo-transmission electron microscopy studies revealed a remarkable progression in morphology as RB/I ratios decreased. At high RB/I values (9-2.3), the particles exhibited a bilayer liposomal structure containing an aqueous region and an electron-dense solid core. The liposomal LNP system with RB/I = 4 showed particularly promising characteristics, with approximately 84% of particles displaying a bilayer structure containing a solid core occupying roughly 30% of the interior.
Superior In Vitro and Storage Performance
The liposomal LNPs demonstrated potent transfection capabilities in Huh7 cells, with formulations containing RB/I ratios of 4-0.67 exceeding the performance of Onpattro-like compositions. Notably, these systems showed exceptional stability during long-term storage at 4°C for 63 weeks. LNPs with RB/I ratios of 9-2.3 increased in size by less than 20% and maintained mRNA encapsulation levels above 80%, while conventional formulations with lower RB/I ratios experienced size increases up to 300% with encapsulation dropping to as low as 40%.
Dramatic Shift in Biodistribution Profile
In vivo studies using NanoLuc mRNA revealed a markedly different transfection profile for liposomal LNPs compared to Onpattro-like formulations. Rather than predominantly targeting the liver, the liposomal system (RB/I = 4) showed enhanced extrahepatic delivery with 50-fold and 150-fold increases in luminescence in the spleen and inguinal lymph nodes, respectively.
SPECT/CT imaging studies demonstrated that liposomal LNPs exhibited approximately 15-fold longer circulation half-lives compared to conventional formulations. This extended circulation correlated with increased accumulation in heart, thoracic mammary glands, blood, inguinal lymph nodes, pancreas, and bone.
Reduced Protein Corona Formation
Analysis of biomolecular corona formation revealed that liposomal LNPs adsorbed approximately 2-fold lower levels of plasma proteins compared to Onpattro-like systems. Mass spectrometry proteomic analysis identified over 1000 different proteins in both formulations, but liposomal LNPs showed significantly reduced levels of immunoglobulin heavy variable 10-1 and fibrinogen, along with slight decreases in immunoproteins and increases in apolipoproteins.
Novel pH-Responsive Delivery Mechanism
The researchers elucidated a unique mechanism underlying the transfection competence of liposomal LNPs. When exposed to decreasing pH conditions mimicking the endosomal environment, the interior solid core progressively migrated from the LNP interior to present as a protrusion from the particle surface. This pH-responsive behavior creates what the researchers describe as a "localized warhead" containing positively charged ionizable lipids complexed with mRNA cargo that facilitates endosomal escape and cytosolic delivery.
Clinical Translation Potential
The study demonstrates that liposomal LNPs can be rationally designed while maintaining acceptable safety profiles. Although these formulations require higher lipid doses per mRNA dose compared to conventional systems, the researchers note that the lipid doses remain lower than those commonly used for approved liposomal drugs like Doxil, and the bilayer lipid components have established safety profiles.
The enhanced delivery to lymph nodes and pancreatic tissue suggests potential applications for immunotherapies and treatments for pancreatic disorders. The improved stability characteristics also offer advantages for manufacturing, storage, and distribution of mRNA therapeutics.
Broader Implications for mRNA Therapeutics
This research challenges conventional assumptions about optimal LNP design and suggests that incorporating higher proportions of bilayer-forming lipids can unlock new therapeutic possibilities. The ability to achieve potent extrahepatic delivery while maintaining excellent stability profiles could expand the therapeutic applications of mRNA technology beyond the liver-focused treatments that currently dominate the field.
The findings provide a foundation for developing next-generation mRNA delivery systems that could address unmet medical needs in immunotherapy, tissue regeneration, and treatment of diseases affecting organs that have been difficult to target with current LNP formulations.
