Determination of mRNA copy number in degradable lipid nanoparticles via density contrast analytical ultracentrifugation

Abstract

Lipid nanoparticles as delivery system for mRNA have recently attracted attention to a broader audience as COVID-19 mRNA vaccines. Their low immunogenicity and capability to deliver a variety of nucleic acids renders them an interesting and complementary alternative to gene therapy vectors like AAVs. An important quality attribute of LNPs is the copy number of the encapsulated cargo molecule. This work describes how density and molecular weight distributions obtained by density contrast sedimentation velocity can be used to calculate the mRNA copy number of a degradable lipid nanoparticle formulation. The determined average copy number of 5 mRNA molecules per LNP is consistent with the previous studies using other biophysical techniques, such as single particle imaging microscopy and multi-laser cylindrical illumination confocal spectroscopy (CICS). Supplementary Information The online version contains supplementary material available at 10.1007/s00249-023-01663-y.

Analytical Ultracentrifugation to Assess the Quality of LNP-mRNA Therapeutics

“…This is extremely important as it avoids any potential bias in the results that could be introduced by ultrafiltration steps, RNA extraction, or particle disruption that are needed when using other analytical techniques. The simplicity of the sample preparation and the specific detection of the signal from the RNA molecule (using the UV detector at 260 nm) also allow the characterization of LNP-mRNA in physiologically relevant media [ 34 ].…”

Lipid Nanoparticle (LNP) Delivery Carrier-Assisted Targeted Controlled Release mRNA Vaccines in Tumor Immunity

“…LNPs tend to be concentrated in the liver rather than tumor tissue, which limits their precise delivery to tumors [ 256 , 257 , 258 , 259 , 260 ]. In response to this challenge, we can explore improving the targeting of LNPs, designing specific targeting ligands or functionalized molecules, and making them more inclined to be enriched in tumor tissues in order to improve the therapeutic effect [ 261 ]. Lipid nanoparticles (LNP) are a common vaccine delivery system consisting of different lipid components.…”

Analytical Characterization of Heterogeneities in mRNA-Lipid Nanoparticles Using Sucrose Density Gradient Ultracentrifugation

“…19 To the best of our knowledge, reports of in vivo evaluation of different subfractions of mRNA-LNP formulations obtained using DGC are limited. 20 In this research, we synthesized four LNPs encapsulating human erythropoietin (hEPO) mRNA with two novel ionizable lipids, lipid A and lipid B, each formulated with the helper lipid DOPE or DEPE to obtain (i) A-DOPE, (ii) A-DEPE, (iii) B-DOPE, and (iv) B-DEPE. These formulations were subjected to ultracentrifugation on a sucrose density gradient with the following two objectives: (1) to understand the density differences of the subpopulations as a function of ionizable lipid and helper lipid and (2) to evaluate in vivo performance of these variably dense subpopulations when dosed via intravenous (IV) and intramuscular (IM) routes of administration.…”

“…To the best of our knowledge, reports of in vivo evaluation of different subfractions of mRNA-LNP formulations obtained using DGC are limited . In this research, we synthesized four LNPs encapsulating human erythropoietin (hEPO) mRNA with two novel ionizable lipids, lipid A and lipid B, each formulated with the helper lipid DOPE or DEPE to obtain (i) A-DOPE, (ii) A-DEPE, (iii) B-DOPE, and (iv) B-DEPE.…”