ACS applied materials & interfacesAug 13, 2026

Cholesterol-like and Vitamin-linked Lipids Improve Lipid Nanoparticle Delivery in Lab and Animal Studies

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗

Updated Aug 14, 2026

Abstract

In vitro-oriented LNPs using β-sitosterol achieved a two orders of magnitude increase in protein expression.

LNPs optimized for in vitro applications showed enhanced protein expression, but did not perform well in vivo.
Modification of LNPs with β-sitosterol led to reduced stability for in vivo use.
Eight vitamin-conjugated ionizable lipids were synthesized for improved in vivo delivery.
Two of these lipids resulted in higher protein expression in animals compared to commercial LNPs.
The optimized in vivo LNP demonstrated good stability and superior immunogenicity with lower doses of mRNA.
In vivo safety studies indicated that the new LNPs had a better safety profile than existing commercial options.

Simplified

LNP-mediated mRNA delivery encounters challenges with insufficient efficacy, poor safety, and unstable performance. Moreover, the distinct requirements of in vitro and in vivo applications call for specific LNP strategies. To address this, we developed two complementary LNP strategies tailored to different application scenarios: one optimized for in vitro transfection efficiency and the other enhanced for in vivo delivery performance. For the in vitro-oriented strategy, β-sitosterol was used to replace cholesterol in LNPs. The resulting β-sitosterol-based LNPs displayed distinct internal fingerprint structures visualized by Cryo-TEM and markedly enhanced in vitro protein expression by two orders of magnitude, making it a high-efficiency tool for in vitro mRNA delivery. However, this modification did not enhance in vivo transfection efficiency and led to a significant reduction in LNP stability, confirming its suitability for in vitro use only. For the in vivo-oriented strategy, eight vitamin-conjugated ionizable lipids were synthesized. Six of these lipids were selected for animal screening, and two exhibited higher in vivo protein expression levels compared with the commercial LNP control. Accelerated stability studies demonstrated that LNPs containing these vitamin-conjugated ionizable lipids had good stability. Furthermore, the optimized in vivo-oriented LNP was used to encapsulate varicella-zoster virus (VZV) mRNA and respiratory syncytial virus (RSV) mRNA, and animal immunization experiments showed that it induced superior immunogenicity at a lower dose than the commercial delivery system. In vivo safety studies further confirmed that the in vivo-oriented LNPs had a better safety profile than commercial LNPs, with no obvious cytotoxicity or adverse reactions. Herein, we developed two complementary LNP strategies based on cholesterol analogs and vitamin-conjugated ionizable lipids, which address the limitations of traditional LNP systems, providing targeted solutions for both in vitro and in vivo translational applications.