Payload distribution and capacity of mRNA lipid nanoparticles

Sixuan Li; Yizong Hu; Andrew Li; Jinghan Lin; Kuangwen Hsieh; Zachary Schneiderman; Pengfei Zhang; Yining Zhu; Chenhu Qiu; Efrosini Kokkoli; Tza-Huei Wang; Hai-Quan Mao

doi:10.1038/s41467-022-33157-4

Payload distribution and capacity of mRNA lipid nanoparticles

Nat Commun. 2022 Sep 23;13(1):5561. doi: 10.1038/s41467-022-33157-4.

Authors

Sixuan Li^#¹, Yizong Hu^#^{2

3

4}, Andrew Li⁵, Jinghan Lin^{6

5}, Kuangwen Hsieh¹, Zachary Schneiderman^{6

7}, Pengfei Zhang⁵, Yining Zhu^{6

5

8}, Chenhu Qiu^{6

9}, Efrosini Kokkoli^{6

7}, Tza-Huei Wang^{10

11

12}, Hai-Quan Mao^{13

14

15

16}

Affiliations

¹ Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD, USA.
² Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA. yhu38@jhmi.edu.
³ Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA. yhu38@jhmi.edu.
⁴ Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. yhu38@jhmi.edu.
⁵ Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
⁶ Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA.
⁷ Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.
⁸ Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
⁹ Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD, USA.
¹⁰ Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD, USA. thwang@jhu.edu.
¹¹ Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA. thwang@jhu.edu.
¹² Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA. thwang@jhu.edu.
¹³ Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA. hmao@jhu.edu.
¹⁴ Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA. hmao@jhu.edu.
¹⁵ Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. hmao@jhu.edu.
¹⁶ Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD, USA. hmao@jhu.edu.

^# Contributed equally.

Abstract

Lipid nanoparticles (LNPs) are effective vehicles to deliver mRNA vaccines and therapeutics. It has been challenging to assess mRNA packaging characteristics in LNPs, including payload distribution and capacity, which are critical to understanding structure-property-function relationships for further carrier development. Here, we report a method based on the multi-laser cylindrical illumination confocal spectroscopy (CICS) technique to examine mRNA and lipid contents in LNP formulations at the single-nanoparticle level. By differentiating unencapsulated mRNAs, empty LNPs and mRNA-loaded LNPs via coincidence analysis of fluorescent tags on different LNP components, and quantitatively resolving single-mRNA fluorescence, we reveal that a commonly referenced benchmark formulation using DLin-MC3 as the ionizable lipid contains mostly 2 mRNAs per loaded LNP with a presence of 40%-80% empty LNPs depending on the assembly conditions. Systematic analysis of different formulations with control variables reveals a kinetically controlled assembly mechanism that governs the payload distribution and capacity in LNPs. These results form the foundation for a holistic understanding of the molecular assembly of mRNA LNPs.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Lipids* / chemistry
Liposomes
Nanoparticles* / chemistry
RNA, Messenger / chemistry
RNA, Messenger / genetics
RNA, Small Interfering / genetics

Substances

Lipid Nanoparticles
Lipids
Liposomes
RNA, Messenger
RNA, Small Interfering

Abstract

Publication types

MeSH terms

Substances

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