Biology subjects

Kamanzi, A.

Publications and source records attributed to Kamanzi, A..

3 recordsLinked to original sources

bioRxiv2026

Direct Counting of mRNA Copies Inside Individual Lipid Nanoparticles Using In Situ Lysis and Labeling

The optimization of mRNA-lipid nanoparticles (mRNA-LNPs) for therapeutic applications is limited in part by the inadequate characterization of mRNA payload heterogeneity. One current challenge is accurately measuring the number of mRNA copies within individual LNPs, where the standard method of intensity-based mRNA number determination is sensitive to fluorescent dye-dye interactions and heterogeneity of mRNA labeling. Here we present a single-particle microscopy method that combines direct counting of the mRNA copies per LNP with LNP size measurements. While confined in microwells, individual mRNA-LNPs are lysed to release their cargo and stained with a dye such that the number of mRNA molecules in each well can be directly counted using fluorescence microscopy. Since the method stains the mRNA cargo in situ, it enables characterization of LNPs formulated with therapeutic grade (e.g., unlabeled) mRNA. We applied this approach to two Onpattro(R)-based LNP formulations prepared using different formulation buffers, where the two formulations had different average mRNA copy number, particle size, and fraction of LNPs lacking mRNA. The ability to directly count the number of mRNA molecules in LNPs establishes a complimentary method to intensity-based mRNA number determination and supports the characterization and screening of clinically relevant LNP formulations.

biophysics↗

bioRxiv2025

Single-particle multi-parametric microscopy reveals structural, size, and payload heterogeneity in mRNA-loaded lipid nanoparticles

Deciphering the heterogeneity of mRNA-containing lipid nanoparticles (LNPs) is essential for understanding the relationship between their microscopic properties and therapeutic function. Here, by combining alternating laser excitation (ALEX) with Convex Lens-induced Confinement (CLiC) microscopy, we simultaneously measure size, multi-color fluorescence, mRNA payload, and Forster resonance energy transfer (FRET) of individual suspended LNPs containing labeled lipid and mRNA molecules. By varying formulation parameters, including ionizable lipids, formulation buffers, and molecular ratios, we investigated and correlated key microscopic properties for relevant vaccine formulations. While the per-particle lipid fluorescence was lower for empty versus mRNA-loaded particles for all formulations, the relative size of empty versus mRNA-loaded particles depended upon the formulation and intraparticle structure. When comparing CLiC-ALEX to cryogenic transmission electron microscopy measurements (Cryo-TEM), for the LNP formulations that display blebs, the fraction of bleb LNPs is in close agreement with the fraction of mRNA-containing LNPs. CLiC-ALEX also enabled quantification of the per-particle mRNA fluorescence and FRET signals, and thus the heterogeneity in the mRNA copy number and mRNA-LNP structural arrangements, where the results were compared with biophysical estimates based on the LNP formulations. These rigorous biophysical insights are critical to inform our understanding of structure-activity relationships and inform rational design of nanomedicines.

biophysics↗

bioRxiv2025

Multiparametric Characterization of Individual Suspended Nanoparticles using Confocal Fluorescence and Interferometric Scattering Microscopy with Microfluidic Confinement

Detailed characterization of the size, mass, payload, and structure of suspended mRNA-lipid nanoparticles (LNPs) is necessary to improve our understanding of how these heterogenous properties influence therapeutic efficacy and potency. Methods currently in use face limitations in reporting ensemble-average particle properties or requiring dedicated home-built microscopes that are beyond the reach of nanoparticle developers. In this work, we overcome these limitations by combining a commercially available confocal microscope and a convex lens-induced confinement (CLiC) instrument to achieve simultaneous characterization and correlation of the size, mass, refractive index, and nucleic acid payload of individual LNPs. We established the accuracy and precision of our method using nanosized beads and used it to investigate the size, payload, and water content of LNPs in different solvent pH. By employing readily available microscopy tools, we open the door to widespread adoption of our quantitative, in-solution nanoparticle characterization method.

biophysics↗