A Combinatorial Library of Lipid Nanoparticles for Cell Type‐Specific mRNA Delivery

Abstract Ionizable lipid‐based nanoparticles (LNPs) are the most advanced non‐viral drug delivery systems for RNA therapeutics and vaccines. However, cell type‐specific, extrahepatic mRNA delivery is still a major hurdle, hampering the development of novel therapeutic modalities. Herein, a novel ionizable lipid library is synthesized by modifying hydrophobic tail chains and linkers. Combined with other helper lipids and utilizing a microfluidic mixing approach, stable LNPs are formed. Using Luciferase‐mRNA, mCherry mRNA, and Cre mRNA together with a TdTomato animal model, superior lipids forming LNPs for potent cell‐type specific mRNA delivery are identified. In vitro assays concluded that combining branched ester tail chains with hydroxylamine linker negatively affects mRNA delivery efficiency. In vivo studies identify Lipid 23 as a liver‐trophic, superior mRNA delivery lipid and Lipid 16 as a potent cell type‐specific ionizable lipid for the CD11bhi macrophage population without an additional targeting moiety. Finally, in vivo mRNA delivery efficiency and toxicity of these LNPs are compared with SM‐102‐based LNP (Moderna's LNP formulation) and are shown to be cell‐specific compared to SM‐102‐based LNPs. Overall, this study suggests that a structural combination of tail and linker can drive a novel functionality of LNPs in vivo.

After that, sodium triacetoxyborohydride (250 mg, 1.20 mmol, 1.5 equiv.) was added portion wise and stirred for 10 min. Then a solution of linoleyl aldehyde 2 (232 mg, 0.88 mmol, 1.1 equiv.) in dry CH2Cl2 (10 mL) was added, drop wisely, and stirred for another 5 min. Later, the remaining amount of sodium triacetoxyborohydride (250 mg, 1.20 mmol, 1.5 equiv.) was added portion wise and stirred for 8 hr at room temperature under an argon atmosphere. The reaction was quenched with sat.NaHCO3 solution and extracted with CH2Cl2 (3 times). The organic portion was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated, and the residue was purified by column chromatography using 0-10% ethyl acetate in hexane to obtain the desired hydroxylamine 7 (405 mg, 80%) as a colorless liquid.
The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated on a rotary evaporator, and the residue was purified by column chromatography using 0-5% Isopropanol in CHCl3 to obtain the desired ethanolamine 8 (417 mg, 84%) as a colorless liquid.
The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated, and the residue was purified by column chromatography using 0-10% MeOH in CHCl3 to get ethanolamine 11 (450 mg, 45 %) as a colorless liquid.
The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated on a rotary evaporator, and the residue was purified by column chromatography using 0-5% Isopropanol in CHCl3 to obtain the desired ethanolamine 12 (297 mg, 81%) as colorless liquid.
Later, the remaining amount of sodium triacetoxyborohydride (253 mg, 1.2 mmol, 1.5 equiv.) was added portion wise and stirred for 8 hr at room temperature under an argon atmosphere.
The reaction was quenched with sat.NaHCO3 solution and extracted with CH2Cl2 (3 times). The organic portion was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated, and the residue was purified by column chromatography using 0-10% ethyl acetate in hexane to obtain the desired hydroxylamine 13 (460 mg, 76%) as a colorless liquid.

Heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate
The above bromide 15 (1.26 g, 2.73 mmol, 1 equiv.), and ethanolamine (4.95 mL, 82 mmol, 30 equiv.) were dissolved in ethanol (5 mL) and stirred overnight at 65 ºC. After that, the solvent was evaporated under reduced pressure, then the reaction mixture was poured into the water and extracted with ethyl acetate (3 times). The organic portion was washed with brine solution and dried over with anhydrous Na2SO4. The solvent was evaporated, and the residue was
NaHCO3 followed by extract with CH2Cl2 (3 times). Then the organic portion was washed with brine solution and dried over with anhydrous Na2SO4. The solvent was evaporated, and the residue was purified by column chromatography using 0-5% EtOAc in hexane to obtain the desired silyl product (6.1 g, 94%) as a colorless liquid.
The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated on a rotary evaporator, and the residue was purified by column chromatography using 0-5% Isopropanol in CHCl3 to obtain the desired product SM-102 (498 mg, 97%) as a colorless liquid.

RNA encapsulation and quantification.
The Quant-iT RiboGreen RNA assay kit (Life Technologies) was used to measure the mRNA encapsulation in LNPs. In brief, 0.5 µL of LNP was diluted in a final volume of 100 µL of TE buffer (20 mм EDTA, 10 mм Tris-HCL) with or without Triton X-100 (0.5%, Sigma-Aldrich).
Samples were loaded in a 96-well black plate (Costar, Corning). The plate was incubated for 15 min at 37 °C before adding 100 µL of TE buffer (0.5% v/v, RiboGreen reagent) to each well.
The fluorescence was detected using a microplate reader (Biotek Industries) according to the manufacturer's protocol.
In vitro luciferase assay.

TNS assay.
As previously described [2] , the pKa values of LNPs were measured using the 2-(p-toluidino)-6naphthalenesulfonic acid (TNS) assay. In brief, the master buffer was prepared using 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mM 4morpholineethanesulfonic acid (MES), 10 mM ammonium acetate, and 130 mM sodium chloride (NaCl). Sixteen buffers with a pH ranging from 2.5 to 10 were prepared using 1.0 M sodium hydroxide and 1.0 M hydrochloric acid based on the master buffer. 6-(p-toluidino)-2naphthalenesulfonic acid sodium salt (TNS reagent) was prepared as a 0.1 mM stock solution in Milli-Q water. 90 µL of each buffer was added in triplicate to a black 96-well plate, and then