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

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.

Recent Advances in the Lipid Nanoparticle-Mediated Delivery of mRNA Vaccines

“…Although the predictable and rational design of LNPs for nucleic acid delivery to extrahepatic tissues remains difficult (LNPs resemble very-low-density lipoprotein and adsorb apolipoprotein E in blood plasma, resulting in preferential accumulation in the liver and uptake into hepatocytes), this challenge must be overcome in order to realize the full potential of mRNA LNP technology for broad therapeutic developments [ 55 ]. However, such mechanisms are effective in treating liver diseases, including cancer.…”

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“…The shift in target from the liver to the spleen to the lungs was observed as an expression of luciferase protein, as shown in Figure 12. Another SORT molecule, 4A3-SC8, was used with 20% DODAP for liver targeting, 50% DOTAP for lung targeting, and 10% 18 PA for spleen targeting (Wang et al, 2022). Effects of incorporation of SORT molecules in LNPs formulations visible from luminescence profile for tested mice models.…”

“…The added SORT molecule controls the biodistribution, apparent pKa, and interaction with serum protein. The change in tissue tropism depends on surface charges of SORT molecules and their amount, which may help to predict the targeting of the LNPs for expression of load in particular organs ( Wang et al, 2022 ). Compared to other targeting strategies such as aptamers or antibodies, SORT molecules offer innate targeting without any surface modifications.…”

Biodrug Delivery Systems: Do mRNA Lipid Nanoparticles Come of Age?

“…Nanoparticles can be engineered to target specific cells and organs in the body via 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 [19].…”