Transfection Potency of Lipid Nanoparticles Containing MRNA Depends on Relative Loading Levels

Overview

Journal ACS Appl Mater Interfaces

Publisher American Chemical Society

Specialties Biomedical Engineering
Biotechnology

Date 2024 Dec 31

PMID 39737597

Authors

Suiyang Liao

Shuangyu Wang

Abishek Wadhwa

Alexandra Birkenshaw

Kevin Fox

Miffy Hok Yan Cheng

Irafasha C Casmil

Armando Alcazar Magana

Nuthan Vikas Bathula

Chia Hao Ho

Jin-Yu Cheng

Leonard J Foster

Kenneth W Harder

Colin J D Ross

Pieter R Cullis

Anna K Blakney

Affiliations

Soon will be listed here.

Abstract

When formulating mRNA into lipid nanoparticles (LNP), various copy numbers of mRNA are encapsulated, leading to a distribution of mRNA loading levels within the LNPs. It is unclear whether the mRNA loading level affects the functional delivery of the message. Here we show that depending on the mRNA loading level, LNPs exhibit distinct mass densities and can be fractionated via ultracentrifugation. Upon fractionation, we investigated if mRNA loading levels influence LNP sizing, lipid composition, and morphology. We further conducted and functional delivery of mRNA and found that the LNP fraction with the highest mRNA loading levels was the least transfection competent.

References

Kloczewiak M, Banks J, Jin L, Brader M . A Biopharmaceutical Perspective on Higher-Order Structure and Thermal Stability of mRNA Vaccines. Mol Pharm. 2022; 19(7):2022-2031. PMC: 9257798. DOI: 10.1021/acs.molpharmaceut.2c00092. View

Guerrini G, Mehn D, Scaccabarozzi D, Gioria S, Calzolai L . Analytical Ultracentrifugation to Assess the Quality of LNP-mRNA Therapeutics. Int J Mol Sci. 2024; 25(11). PMC: 11171944. DOI: 10.3390/ijms25115718. View

Buschmann M, Carrasco M, Alishetty S, Paige M, Alameh M, Weissman D . Nanomaterial Delivery Systems for mRNA Vaccines. Vaccines (Basel). 2021; 9(1). PMC: 7836001. DOI: 10.3390/vaccines9010065. View

Brader M, Williams S, Banks J, Hui W, Zhou Z, Jin L . Encapsulation state of messenger RNA inside lipid nanoparticles. Biophys J. 2021; 120(14):2766-2770. PMC: 8390897. DOI: 10.1016/j.bpj.2021.03.012. View

Henrickson A, Kulkarni J, Zaifman J, Gorbet G, Cullis P, Demeler B . Density Matching Multi-wavelength Analytical Ultracentrifugation to Measure Drug Loading of Lipid Nanoparticle Formulations. ACS Nano. 2021; 15(3):5068-5076. DOI: 10.1021/acsnano.0c10069. View

Zhao H, Sousa A, Schuck P . Flotation Coefficient Distributions of Lipid Nanoparticles by Sedimentation Velocity Analytical Ultracentrifugation. ACS Nano. 2024; 18(28):18663-18672. PMC: 11256894. DOI: 10.1021/acsnano.4c05322. View

Leung A, Hafez I, Baoukina S, Belliveau N, Zhigaltsev I, Afshinmanesh E . Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core. J Phys Chem C Nanomater Interfaces. 2012; 116(34):18440-18450. PMC: 3434764. DOI: 10.1021/jp303267y. View

Baden L, El Sahly H, Essink B, Kotloff K, Frey S, Novak R . Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N Engl J Med. 2020; 384(5):403-416. PMC: 7787219. DOI: 10.1056/NEJMoa2035389. View

Bepperling A, Richter G . Determination of mRNA copy number in degradable lipid nanoparticles via density contrast analytical ultracentrifugation. Eur Biophys J. 2023; 52(4-5):393-400. PMC: 10248324. DOI: 10.1007/s00249-023-01663-y. View

10.

Whitehead K, Matthews J, Chang P, Niroui F, Dorkin J, Severgnini M . In vitro-in vivo translation of lipid nanoparticles for hepatocellular siRNA delivery. ACS Nano. 2012; 6(8):6922-9. PMC: 3429655. DOI: 10.1021/nn301922x. View

11.

Polack F, Thomas S, Kitchin N, Absalon J, Gurtman A, Lockhart S . Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med. 2020; 383(27):2603-2615. PMC: 7745181. DOI: 10.1056/NEJMoa2034577. View

12.

Kulkarni J, Witzigmann D, Leung J, van der Meel R, Zaifman J, Darjuan M . Fusion-dependent formation of lipid nanoparticles containing macromolecular payloads. Nanoscale. 2019; 11(18):9023-9031. DOI: 10.1039/c9nr02004g. View

13.

Heinz F, Stiasny K . Distinguishing features of current COVID-19 vaccines: knowns and unknowns of antigen presentation and modes of action. NPJ Vaccines. 2021; 6(1):104. PMC: 8368295. DOI: 10.1038/s41541-021-00369-6. View

14.

Li S, Hu Y, Li A, Lin J, Hsieh K, Schneiderman Z . Payload distribution and capacity of mRNA lipid nanoparticles. Nat Commun. 2022; 13(1):5561. PMC: 9508184. DOI: 10.1038/s41467-022-33157-4. View

15.

Wilson E, Goswami J, Baqui A, Doreski P, Perez-Marc G, Zaman K . Efficacy and Safety of an mRNA-Based RSV PreF Vaccine in Older Adults. N Engl J Med. 2023; 389(24):2233-2244. DOI: 10.1056/NEJMoa2307079. View

16.

Kim J, Eygeris Y, Gupta M, Sahay G . Self-assembled mRNA vaccines. Adv Drug Deliv Rev. 2021; 170:83-112. PMC: 7837307. DOI: 10.1016/j.addr.2020.12.014. View

17.

Cheng M, Leung J, Zhang Y, Strong C, Basha G, Momeni A . Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency. Adv Mater. 2023; 35(31):e2303370. DOI: 10.1002/adma.202303370. View

18.

Szebeni J, Kiss B, Bozo T, Turjeman K, Levi-Kalisman Y, Barenholz Y . Insights into the Structure of Comirnaty Covid-19 Vaccine: A Theory on Soft, Partially Bilayer-Covered Nanoparticles with Hydrogen Bond-Stabilized mRNA-Lipid Complexes. ACS Nano. 2023; 17(14):13147-13157. PMC: 10373524. DOI: 10.1021/acsnano.2c11904. View

19.

Li S, Hu Y, Lin J, Schneiderman Z, Shao F, Wei L . Single-Particle Spectroscopic Chromatography Reveals Heterogeneous RNA Loading and Size Correlations in Lipid Nanoparticles. ACS Nano. 2024; 18(24):15729-15743. PMC: 11191693. DOI: 10.1021/acsnano.4c02341. View

20.

Paunovska K, Sago C, Monaco C, Hudson W, Gamboa Castro M, Rudoltz T . A Direct Comparison of in Vitro and in Vivo Nucleic Acid Delivery Mediated by Hundreds of Nanoparticles Reveals a Weak Correlation. Nano Lett. 2018; 18(3):2148-2157. PMC: 6054134. DOI: 10.1021/acs.nanolett.8b00432. View