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Updated: May 23, 2025

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Multiparametric functional characterization of individual lipid nanoparticles using surface-sensitive

Mattias Sjöberg1,2, Erik Olsén1, Mokhtar Mapar1

  • 1Division of Nano and Biophysics, Department of Physics, Chalmers University of Technology, Gothenburg 412 96, Sweden.

Proceedings of the National Academy of Sciences of the United States of America
|May 22, 2025
PubMed
Summary

Improving lipid nanoparticle (LNP) gene therapeutics requires understanding how particle properties affect delivery. This study uses advanced microscopy to link LNP structure and fusogenicity to functional mRNA delivery, revealing limitations in current characterization methods.

Keywords:
fluorescencelight scatteringlipid nanoparticlesmRNAoptical microscopy

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Area of Science:

  • Biotechnology and Nanomedicine
  • Gene Therapy Delivery Systems
  • Advanced Microscopy Techniques

Background:

  • Lipid nanoparticles (LNPs) are crucial for gene therapeutics, protecting genetic material and facilitating cellular entry.
  • Current LNP formulations have limited efficacy, necessitating a deeper understanding of factors influencing their performance.
  • Key factors include LNP size, lipid composition, cargo loading, and pH-dependent structural changes.

Purpose of the Study:

  • To develop and apply a single-particle-resolved multiparametric characterization method for LNPs.
  • To investigate how LNP properties correlate with functional mRNA delivery efficiency.
  • To identify limitations in current in situ LNP characterization techniques.

Main Methods:

  • Combined surface-sensitive fluorescence and label-free scattering microscopy for single LNP analysis.
  • Microfluidics for controlled liquid exchange and varying refractive index measurements.
  • Quantification of LNP size, refractive index, and Cy5-labeled mRNA cargo content.

Main Results:

  • Multiparametric characterization quantified LNP size, refractive index, and cargo content, showing cargo scales with LNP volume.
  • Two LNP formulations with similar size and mRNA content showed different functional delivery efficiencies.
  • Differences in LNP fusogenicity under early endosomal conditions (pH 6.0) correlated with observed in vitro cellular data.

Conclusions:

  • Single-particle multiparametric characterization provides detailed LNP insights but may not fully predict functional performance.
  • LNP fusogenicity, particularly under endosomal conditions, is a critical factor for successful gene delivery.
  • Current in situ LNP characterization tools focusing on structural properties need enhancement to capture functional aspects.