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Sequencing of mRNA from Whole Blood using Nanopore Sequencing
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Screening lipid nanoparticles using DNA barcoding and qPCR.

Dawei Liu1, Xing Wang1, Letao Xu2

  • 1School of Chemical Engineering, Faculty of Science, Engineering and Technology, The University of Adelaide, Adelaide, SA 5005, Australia.

Colloids and Surfaces. B, Biointerfaces
|March 1, 2025
PubMed
Summary
This summary is machine-generated.

This study introduces a cost-effective DNA barcoding method to track lipid nanoparticle (LNP) biodistribution for mRNA delivery. The method accurately reflects LNP localization and functional mRNA delivery in vivo.

Keywords:
BiodistributionDNA barcodesIn vivo trackingLipid nanoparticles (LNPs)MRNA delivery

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Pharmacology

Background:

  • Accurate quantification of lipid nanoparticle (LNP) biodistribution is essential for optimizing messenger RNA (mRNA) delivery systems.
  • Current methods for assessing LNP biodistribution have limitations, hindering the development of effective mRNA therapeutics.

Purpose of the Study:

  • To develop and validate a cost-effective method using double-stranded DNA (dsDNA) barcodes and quantitative polymerase chain reaction (qPCR) for rapid analysis of mRNA-LNP biodistribution.
  • To evaluate the functional delivery of mRNA by LNPs in vivo using this novel barcoding approach.

Main Methods:

  • Designed three unique 100-bp dsDNA barcodes to represent three FDA-approved LNP formulations.
  • Mixed three LNP formulations carrying luciferase mRNA with DNA-barcoding LNPs into a pooled formulation.
  • Administered the pooled LNPs intravenously to mice and analyzed biodistribution using qPCR and bioluminescence imaging.

Main Results:

  • qPCR analysis showed the highest abundance of DNA barcodes and luciferase mRNA in the spleen.
  • A positive correlation was observed between DNA barcode presence and mRNA localization across organs, validating DNA barcodes as reliable indicators of mRNA-LNP biodistribution.
  • Bioluminescence imaging confirmed successful delivery and protein translation of luciferase mRNA mediated by the LNPs.

Conclusions:

  • The integrated DNA barcoding and luciferase mRNA assay provides a robust methodology for comprehensive evaluation of LNP performance.
  • This approach offers valuable insights into the localization patterns and mRNA delivery capabilities of different LNP formulations.
  • The developed method facilitates the advancement of more effective and targeted mRNA-based therapeutics.