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Related Experiment Video

Updated: Apr 26, 2026

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

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Published on: November 28, 2016

Single-Particle Spectroscopic Chromatography Reveals Heterogeneous RNA Loading and Size Correlations in Lipid

Sixuan Li^1,2, Yizong Hu^1,3,4, Jinghan Lin^1,4,5

¹Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, Maryland 21218, United States.

|June 5, 2024

Summary

Researchers developed a new method to analyze lipid nanoparticles (LNPs) for RNA therapeutics. This technique reveals significant heterogeneity in LNP composition and RNA loading, offering crucial insights into LNP assembly.

Keywords:

compositional heterogeneity lipid nanoparticles multiparametric characterization nanoparticle chromatography nanoparticle payload single-particle spectroscopy

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Determining Four Components in a Lipid Nanoparticle RNA Delivery System by Liquid Chromatography Combined with Evaporative Light Scattering Detector

Determining Four Components in a Lipid Nanoparticle RNA Delivery System by Liquid Chromatography Combined with Evaporative Light Scattering Detector

Published on: May 30, 2025

Area of Science:

Biotechnology
Nanotechnology
Pharmaceutical Sciences

Background:

Lipid nanoparticles (LNPs) are key delivery systems for RNA therapeutics.
Previous studies often assumed homogeneity in LNP populations, loading, and composition.
Characterizing LNP heterogeneity at the single-particle level has been challenging due to a lack of suitable tools.

Purpose of the Study:

To develop a generalizable strategy for dissecting multicomponent LNP assembly complexities.
To create a high-throughput platform for characterizing physicochemical properties of LNPs at the single-particle level.
To investigate LNP heterogeneity, including population identity, size, RNA loading, and lipid composition.

Main Methods:

An integrated spectroscopy-chromatography approach was developed.
The platform couples cylindrical illumination confocal spectroscopy (CICS) with single-nanoparticle free solution hydrodynamic separation (SN-FSHS).
This method simultaneously profiles population identity, hydrodynamic size, RNA loading, and lipid distributions in LNPs.

Main Results:

The platform successfully distinguished seven distinct LNP populations in a benchmark siRNA LNP formulation.
Quantitative characterization of size distribution and RNA loading levels across wide ranges was achieved.
Crucially, composition-size correlations were resolved, revealing heterogeneity in RNA packing density and size-dependent loading.

Conclusions:

The developed SN-FSHS-CICS analysis provides critical insights into LNP heterogeneity.
Significant variations in RNA packing density and size-dependent loading were observed.
These findings suggest kinetics-driven assembly mechanisms are responsible for RNA LNP formation.