Formulations Insights: Using Natural Sterol Alternatives to Replace Cholesterol in LNP Drug-Delivery Formulations

Posted on December 03, 2021

Sterol Lnp Formulations

If you’re trying to package, protect, and deliver genetic material to the inside of a cell, it is now common practice to use a lipid nanoparticle (LNP) delivery system. And who better to trust with your lipid delivery systems research than Avanti? With a quality standard that accepts nothing less than the best, Avanti’s lipid products are the best choice for preparing lipid delivery systems used in vaccine research.

The importance of cholesterol in LNP formulations.

Cholesterol is typically one of the four major components, other than genetic material, used in LNPs. LNP formulations usually have between 35-45% composition with respect to cholesterol. There are two primary roles that cholesterol plays in these formulations: (1) Cholesterol enhances the stability of the LNP, and (2) cholesterol assists the transfection of RNA. A recent study evaluated the structure of LNPs containing natural phytosterols in the place of cholesterol. The study showed that changing the sterol component of the LNP significantly affects its morphology, as well as its ability to deliver the mRNA. So, why does changing the structure of the LNP result in such an improvement in its transfection efficacy? Let’s see what researchers at Oregon State University discovered.

How changing the sterol component of an LNP increases its transfection efficiency.

The researchers prepared LNPs with the following composition: cholesterol analogs, DLin-MC3-DMA, DSPC, DMG-PEG-2000 (38.5:50:10:1.5 lipid molar ratios) and Fluc-mRNA. The prepared LNPs were studied to determine their size and polydispersity index (PDI), as well as their ability to transfect HeLa cells. Cholesterol LNPs had the smallest size and PDI; whereas, sitosterol containing LNPs showed the greatest transfection rate.

Cryo-electron microscopy was used to determine the structure of the prepared LNPs. LNPs prepared with sitosterol and stigmasterol were found to exhibit a faceted surface. LNPs prepared with cholesterol and campesterol were observed to form a spherical structure. Furthermore, the LNPs prepared with sitosterol, stigmasterol, and campesterol formed multilamellar structures, while LNPs prepared with cholesterol and fucosterol formed single bilayers.

The membrane rigidity of each LNP formulation was studied using a fluorescent TMA-DPH probe. A lower anisotropy associated with this test means that the bilayer structure is less organized. In previous reports, higher membrane rigidity has been associated with higher transfection efficiency. Cholesterol and campesterol LNPs exhibited the highest anisotropy, followed by sitosterol and stigmasterol. Fucosterol had the lowest anisotropy of the LNPs tested.

The structures above are the proposed structures of the various phytosterol LNPs based on the aforementioned test results. (A) this LNP has a unilamellar perimeter with smooth particle curvature and a homogenous core (cholesterol); (B) this structure has a multilamellar perimeter and a homogeneous core (campesterol); (C) this structure has a bilamellar perimeter, but lipid phase separation creates pockets (fucosterol); (D) this structure is polymorphic (sitosterol or stigmasterol); (E) this is a polymorphic and multilamellar structure (sitosterol and stigmasterol).

LNP morphology is an important area of research. Changes associated with replacing phospholipids, PEG-conjugated lipids, and cationic lipids have been reported, but until recently the morphological changes in LNP structure related to cholesterol replacement were not well understood. This study revealed that using a phytosterol replacement for cholesterol can result in various size distributions, and in the case of sitosterol, a ten-fold increase in transfection. A future study that would be interesting is what effect using a combination of cholesterol and phytosterols might have on the morphology and transfection of LNPs containing mRNA.

Avanti offers a wide variety of Sterols to facilitate your LNP research needs.

Full Research Article in Nano Letters:

Deconvoluting Lipid Nanoparticle Structure for Messenger RNA Delivery

Image Credit: Original Research Publication