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Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery

Nature Protocols volume 18, pages 265–291 (2023)Cite this article

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Abstract

A new methodology termed selective organ targeting (SORT) was recently developed that enables controllable delivery of nucleic acids to target tissues. SORT lipid nanoparticles (LNPs) involve the inclusion of SORT molecules that accurately tune delivery to the liver, lungs and spleen of mice after intravenous administration. Nanoparticles can be engineered to target specific cells and organs in the body by passive, active and endogenous targeting mechanisms that require distinct design criteria. SORT LNPs are modular and can be prepared using scalable, synthetic chemistry and established engineering formulation methods. This protocol provides detailed procedures, including the synthesis of a representative ionizable cationic lipid, preparation of multiple classes of SORT LNPs by pipette, vortex and microfluidic mixing methods, physical characterization, and in vitro/in vivo mRNA delivery evaluation. Depending on the scale of the experiments, the synthesis of the ionizable lipid requires 4–6 d; LNPs can be formulated within several hours; LNP characterization can be completed in 2–4 h; and in vitro/in vivo evaluation studies require 1–14 d, depending on the design and application. Our strategy offers a versatile and practical method for rationally designing nanoparticles that accurately target specific organs. The SORT LNPs generated as described in this protocol can therefore be applied to multiple classes of LNP systems for therapeutic nucleic acid delivery and facilitate the development of protein replacement and genetic medicines in target tissues. This protocol does not require specific expertise, is modular to various lipids within defined physicochemical classes, and should be accomplishable by researchers from various backgrounds.

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Fig. 1: Experimental design for preparation of liver, lung and spleen SORT LNPs.
Fig. 2: Flow chart of the general procedures.
Fig. 3: Reaction scheme for the synthesis of AEMA (1) and 4A3-SC8 (2).
Fig. 4: LNP characterization and tissue-specific Luc mRNA delivery results for 4A3-SC8-based SORT LNPs prepared by pipette mixing, vortex mixing and microfluidic mixing methods.
Fig. 5: LNP characterization and tissue-specific Luc mRNA delivery results for DLin-MC3-DMA (MC3)-based SORT LNPs prepared by pipette mixing, vortex mixing and microfluidic mixing methods.

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Data availability

All data supporting this protocol are available within the article and its Supplementary Information files and from the corresponding author upon reasonable request.

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Acknowledgements

D.J.S. acknowledges financial support from the National Institutes of Health (NIH) National Institute of Biomedical Imaging and Bioengineering (NIBIB) (R01 EB025192-01A1) and National Cancer Institute (R01 CA269787-01), the Cystic Fibrosis Foundation (CFF) (SIEGWA18XX0 and SIEGWA21XX0) and the Cancer Prevention and Research Institute of Texas (CPRIT) (RP190251). S.M.L. and J.R. acknowledge support from the NIH (T32GM127216). T.W. acknowledges support from a CPRIT Training Grant (RP160157). The authors acknowledge the UTSW Small Animal Imaging Shared Resource, which is supported in part by the NIH NCI Support Grant (P30 CA142543).

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Authors and Affiliations

  1. Department of Biochemistry, Simmons Comprehensive Cancer Center, The University of Texas Southwestern Medical Center, Dallas, TX, USA

    Xu Wang, Shuai Liu, Yehui Sun, Xueliang Yu, Sang M. Lee, Qiang Cheng, Tuo Wei, Junyu Gong, Joshua Robinson, Di Zhang, Xizhen Lian, Pratima Basak & Daniel J. Siegwart

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  1. Xu Wang
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Contributions

X.W. and D.J.S. conceived and designed the experiments. X.W. performed the majority of experiments, analyzed the data and wrote the first draft of the manuscript. S.L. assisted with the design of the experiments, chemical synthesis, in vitro and in vivo studies. Y.S. and X.Y. assisted in improving mixing methods for the preparation of LNP formulations. X.Y., S.M.L., J.G. and J.R. assisted with the chemical synthesis. Q.C. and T.W. assisted with the design of the experiments. D.Z. assisted with the vortex mixing method. X.L. and P.B. assisted with the preparation of complete lipid mix solutions and general experiments. D.J.S. supervised all the experiments, wrote and edited the manuscript, and managed the project. All authors contributed to the development of the protocol and improved the manuscript.

Corresponding author

Correspondence to Daniel J. Siegwart.

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Competing interests

The University of Texas System has filed patent applications related to the SORT technology, with some authors listed as co-inventors. D.J.S. is a co-founder/consultant to ReCode Therapeutics, which has licensed intellectual property from UT Southwestern. D.J.S. is on the Scientific Advisory Board of Tome Biosciences.

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Nature Protocols thanks Michael Mitchell, Dan Peer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Cheng, Q. et al. Nat. Nanotechnol. 15, 313–320 (2020): https://doi.org/10.1038/s41565-020-0669-6

Wei, T. et al. Nat. Commun. 11, 3232 (2020): https://doi.org/10.1038/s41467-020-17029-3

Dilliard, S. A. et al. Proc. Natl. Acad. Sci. USA 118, e2109256118 (2021): https://doi.org/10.1073/pnas.2109256118

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Wang, X., Liu, S., Sun, Y. et al. Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery. Nat Protoc 18, 265–291 (2023). https://doi.org/10.1038/s41596-022-00755-x

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