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.

Fig. 1 |. Experimental design for preparation of liver, lung and spleen SORT LNPs.

a, Inclusion of a SORT molecule in traditional four-component LNPs (which consist of ionizable cationic lipids, amphipathic phospholipids, cholesterol and PEG lipids) systematically alters the in vivo delivery profile of the resulting five-component SORT LNPs to enable tissue-specific delivery of mRNA to the liver, lungs and spleen of mice after IV administration. b, Chemical structures of the lipids used in this protocol, including the ionizable cationic lipids 4A3-SC8 and DLin-MC3-DMA (MC3), the phospholipids DOPE and DSPC, cholesterol, DMG-PEG and the SORT molecules DOTAP, 18PA and DODAP. c–e, Three mixing methods are described to prepare SORT LNP formulations introduced in this protocol: pipette mixing (c), vortex mixing (d) and microfluidic mixing methods (e). a adapted from ref., Springer Nature Limited. Images in c–e created with BioRender.com.

Fig. 2 |. Flow chart of the general procedures.

Steps 1–25 outline synthesis and purchase of lipids; Step 26 outlines preparation of SORT LNP formulations; Step 27 outlines characterization; Steps 28–41 outline in vitro/in vivo delivery of Luc-mRNA by SORT LNPs.

Fig. 3 |. Reaction scheme for the synthesis of AEMA (1) and 4A3-SC8 (2).

TEA: triethylamine; CHCl₃: chloroform; BHT: butylated hydroxytoluene; DMPP: dimethylphenylphosphine; RT: room temperature.

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.

a, Formulation details of base four-component 4A3-SC8 mDLNPs and five-component 4A3-SC8 SORT LNPs. b, Encapsulation efficiency of mRNA in 4A3-SC8-based LNPs. c, Representative dynamic light scattering (DLS) analysis of 4A3-SC8-based LNPs, particle sizes and PDIs are shown. d–g, 4A3-SC8-based Luc mRNA SORT LNPs were prepared by three mixing methods and injected intravenously into mice at a dosage of 0.1 mg/kg Luc mRNA. After 6 h, ex vivo images of luminescence (from top to bottom rows: pipette, vortex and microfluidic mixing methods) in major organs (heart, lungs, liver, spleen and kidneys) and quantification data are shown. Data are presented as mean ± s.e.m. (n = 3 biologically independent animals). 4A3-SC8 SORT LNPs with 20% DODAP (e) enhanced liver delivery. Inclusion of 50% DOTAP (f) and 10% 18PA (g) shifted the luciferase protein expression to the lungs and spleen, respectively. Statistical analysis of data in Fig. 4d–g is shown in Supplementary Fig. 4. Please see the Data Availability Statement and source data for more information. All the animal experiments were approved by the IACUC of the UTSW and were consistent with local, state and federal regulations as applicable.

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.

a, Formulation details of base four-component MC3 LNPs and five-component MC3 SORT LNPs. b, Encapsulation efficiency of mRNA in MC3-based LNPs. c, Representative DLS analysis of MC3-based LNPs, particle sizes and PDIs are shown. d–f, MC3-based Luc mRNA SORT LNPs were prepared by three mixing methods and injected intravenously into mice at a dosage of 0.1 mg/kg Luc mRNA. After 6 h, ex vivo images of luminescence (from top to bottom rows: pipette, vortex and microfluidic methods) in major organs (heart, lungs, liver, spleen and kidneys) and quantification data are shown. Data are presented as mean ± s.e.m. (n = 3 biologically independent animals). MC3 LNPs mainly deliver mRNA to the liver. Inclusion of 50% DOTAP (e) and 30% 18PA (f) shifted the luciferase protein expression to the lungs and spleen, respectively. Statistical analysis of data in Fig. 5d–f are shown in Supplementary Fig. 4. Please see the Data Availability Statement and source data for more information. All the animal experiments were approved by the IACUC of the UTSW and were consistent with local, state, and federal regulations as applicable.