Ionizable Lipids And Liposomal Nanoparticle Compositions

Abstract

20 26 20 22 00 20 M ar 2 02 6 A B S T R A C T 2 0 2 6 2 0 2 2 0 0 2 0 M a r 2 0 2 6 A B S T R A C T 2 0 2 6 2 0 2 2 0 0 2 0 M a r 2 0 2 6

Description

RELATED APPLICATIONS This patent application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 775,688, filed on March 21, 2025, U.S. Provisional Patent Application No. 63 / 869,090, filed August 22, 2025, U.S. Provisional Patent Application No. 63 / 944,616, filed December 19, 2025, and U.S. Provisional Patent Application No. 64 / 007,071, filed March 16, 2026, the entire content of each of which are hereby incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 18, 2026, is titled 191016-011602_AU_SL.xml and is 237,272 bytes in size. FIELD Aspects of the present disclosure relate to ionizable lipids useful for the preparation of lipid nanoparticles (LNP) encapsulating a nucleic acid. BACKGROUND Lipid nanoparticles (LNP) are used for the delivery of therapeutic nucleic acids to cells, including vaccines to deliver mRNA therapeutics. LNP formulations typically include an ionizable lipid, a sterol, and a phospholipid. Ionizable lipids in LNP compositions facilitate delivery of encapsulated nucleic acid by remaining mostly neutral at physiological pH, but becoming protonated in the acidic environment of endosomes after cellular uptake. Ionizable lipids that are hydrolysable or bioreducible offer desirable in vivo clearance properties for vaccine-focused lipid nanoparticle (LNP) delivery systems by enhancing both efficacy and safety. Hydrolysable lipids, which break down through controlled chemical or enzymatic cleavage, and bioreducible lipids, which degrade in response to intracellular reducing conditions, promote timely LNP disassembly after delivering the vaccine payload. There remains a need for ionizable lipids that are hydrolysable and / or bioreducible for use in the preparation of LNP formulations for therapeutic delivery of nucleic acids. 2026202200   20 Mar 2026 SUMMARY In some embodiments, this disclosure provides ionizable cationic lipid compounds with significantly improved hydrolysis (Example 4) with significantly improved mRNA transfection efficiency in dendritic cells from LNPs encapsulating the mRNA (Example 5). In some embodiments, an ionizable lipid compound consists of a dioxolane ring covalently bound to both (x) an alkylamine head group having a pKa of 6-7.5, and (y) a pair of identical geminal functionalized aliphatic chains at the 2-position of the dioxolane ring, each having 18-25 total carbon atoms and each consisting of (i) a 7-10 carbon linear aliphatic group covalently bound to the dioxolane ring and to (ii) an ester functional group, and (iii) a 10-20 carbon aliphatic chain covalently bound to the ester functional group. In some embodiments, the dioxolane ring is a 1,3-dioxolane ring and each of the functionalized aliphatic chains comprises two or more branched C5 hydrocarbon monomer units such as isopentane (IPN) or 2-methylbut-2-ene (2MB). For example, in some embodiments, the ionizable lipid is selected from the group consisting of: (KC3-X1-PH12),                                         (KC3-X2-PH12), or a salt thereof. In some embodiments, a lipid nanoparticle (LNP) composition comprises a mRNA nucleic acid encapsulated in lipidic nanoparticles formed from the ionizable lipid disclosed herein at a N / P ratio of 3.5 to 8 relative to the mRNA nucleic acid in a total amount of 40-65 mol% of the total lipid content of the LNP composition; a sterol in a total amount of 25-45 mol% of the total lipid content of the LNP composition; one or more phospholipids in a total amount of phospholipids of 5-25 mol% of the total lipid content of the LNP composition; and a PEGcontaining conjugated lipid in a total amount of 0.5-5.0 mol% of the total lipid content of the LNP composition. In some embodiments, the two or more phospholipids in the LNP 2026202200   20 Mar 2026 composition comprise a total of 5 mol% of a phosphatidyl-L-serine (PS). In some embodiments, the LNP composition comprises the conjugated lipid in a total amount of 0.5-2.5 mol% of the total lipid content of the LNP composition. In some embodiments, the phospholipids consist of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a total amount of 5 mol% of the total lipid content of the LNP composition, and a phosphatidyl-L-serine (DPPS), such as dipalmitoylphosphatidyl-L-serine (DPPS) or distearoylphosphatidyl-L-serine (DSPS), in a total amount of 5 mol% of the total lipid content of the LNP composition. In some embodiments, the PEG-containing conjugated lipid in the LNP is PEG(2000)-dimyristoylglycerol (PEG-DMG) or PEG(2000)-dilauroylglycerol (PEG-DLG). In some embodiments, a lipid nanoparticle (LNP) composition comprises a mRNA nucleic acid encoding an antigenic protein encapsulated at a N / P ratio of 3.25-8 in lipidic nanoparticles consisting of: a KCn-X1 or KCn-X2 ionizable cationic lipid disclosed herein and cholesterol in a molar ratio of the ionizable cationic lipid to the cholesterol of from 65:35 to 45:55, X mol% of an anionic phospholipid such as DPPS (wherein X is 2.5-7.5 mol%) , (10-X) mol% phosphocholine phospholipid such as DSPC, and 1.5 - 4 mol% of a PEG conjugated lipid such as PEG-DMG. In some embodiments, a lipid nanoparticle (LNP) composition comprises a mRNA nucleic acid encapsulated at a N / P ratio of 5.25 in lipidic nanoparticles consisting of: 56 mol% of the KC3-X1-PH12 ionizable cationic lipid, 5 mol% DPPS, 5 mol% DSPC, 1.5 mol% PEG-DMG and 32.5 mol% cholesterol. In some embodiments, a lipid nanoparticle (LNP) composition comprises a mRNA nucleic acid encapsulated at a N / P ratio of 5.25 in lipidic nanoparticles consisting of: 52 mol% of the KC3-X1-PH12 ionizable cationic lipid, 5 mol% DPPS, 5 mol% DSPC, 1.5 mol% PEG-DMG and 36.5 mol% cholesterol. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A is a synthetic scheme for preparing certain ionizable cationic lipids. Figure 1B is a synthetic scheme for preparing an exemplary ionizable cationic lipid designated KC3-X1-PH12. Figure 1C is a table of ionizable cationic lipids. Figure 2A is a synthetic scheme for preparing certain ionizable cationic lipids. Figure 2B is a synthetic scheme for preparing an exemplary ionizable cationic lipid designated KC3-X2-PH12. 2026202200   20 Mar 2026 Figure 2C is a table of ionizable cationic lipids. Figure 3A is a table of various ionizable cationic lipids of Formula (I). Figure 3B is a table of various ionizable cationic lipids of Formula (I). Figure 4A is a graph of the biodegradability of ionizable cationic lipids (ICLs) measured after 15 minutes in the presence of pancreatin enzymes (including trypsin, amylase and lipase) measured according to the assay of Example 4. Figure 4B is a graph of the biodegradability of ionizable cationic lipids (ICLs) measured after 3 hours in the presence of pancreatin enzymes (including trypsin, amylase and lipase) measured according to the assay of Example 4. Figure 4C is a graph showing the enzymatic degradation of various ionizable lipids. Figure 5 is a schematic of a mRNA construct comprising a 5’-cap, a 5’-UTR, a cassette comprising a signal peptide, an open reading frame and a terminal domain encoding a synthetic polypeptide, a 3’-UTR and a poly-A region. Figure 6A-6E are figures describing an experiment comparing B cell responses in BALB / c after prime / boost vaccination with mRNA encoding an influenza A subtypes H1, H3, and B / Victoria and RSV preF mRNA (Example 10). The mRNA was formulated with LNPs comprised with different ionizable lipids. Figure 6A is a table summarizing the experimental design. Figure 6B is a graph showing the anti-H1 IgG titers 21 days after vaccination with a high (0.03 mcg) dose. Figure 6C is a graph showing the anti-H1 IgG titers 21 days after vaccination with a low (0.01 mcg) dose. Figure 6D is a graph showing the anti-H3 IgG titers 21 days after vaccination with a high (0.045 mcg) dose; Figure 6E is a graph showing the anti-H3 IgG titers 21 days after vaccination with a low (0.015 mcg) dose. DETAILED DESCRIPTION In some embodiments, the present disclosure provides ionizable cationic lipid compounds of Formula (I): 2026202200   20 Mar 2026 ,R1 (I) or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are each independently C1-4 alkyl optionally substituted with hydroxyl; Z is *-(CH2)n-**, wherein * is a covalent bond to the 1,3-dioxolane ring and ** is a covalent bond to -NR1R2, and n is 2, 3 or 4; -X1- and -X2- are the same of different and are each independently -(CH2)(X)-, wherein (x) is 7-10; -A1- and -A2- are the same or different and are each independently ester groups; -Y1- and -Y2- are the same or different and are each an aliphatic chain having (y) total carbon atoms, optionally comprising two or more branched C5 hydrocarbon monomer units, wherein (y) is 10-17; and the sum of (x)+(y) is 18-25. In some embodiments, ionizable lipid is a compound of Formula (I-A): (I-A) or a pharmaceutically acceptable salt thereof, wherein R1, R2, Z, X1, X2, A1, A2, Y1 and Y2 are as defined with respect to Formula (I). In some embodiments, R1 and R2 are each methyl and n is 2, 3 or 4. In some embodiments, n is 2 and R1 and R2 are each methyl. In some embodiments, n is 3 and R1 and R2 are each methyl. In some embodiments, n is 4 and R1 and R2 are each methyl. In some embodiments, *-Z-N(R1)(R2) is selected from -(CH2)2-4-N(CH3)2, and -(CH2)2—4-N(CH3)-(CH2)2—4-OH, wherein * is a covalent bond to the 1,3-dioxolane ring in Formula (I). 2026202200   20 Mar 2026 In some embodiments, -Xi- and -X2- are both -(CH2)(x)-, wherein (x) is 7, 8, 9 or 10; Ai and A2 are the same and are both independently *-C(O)-O-** or *-O-C(O) -**, wherein * is a covalent bond to Yi and Y2, respectively, and ** is a covalent bond to Xi and X2, respectively; and -Y1- and -Y2- are each an aliphatic chain having (y) total carbon atoms, comprising two or more branched C5 hydrocarbon monomer units, wherein (y) is 12, 13, 14, or 15; and wherein the sum of (x)+(y) is 22. In some embodiments, -Yi and -Y2 are each independently *-(CH2)o-2-(Ry)2-4-H wherein each Ry is independently an isopentane (IPN) or a 2-methylbut-2-ene (2MB) hydrocarbon monomer unit, and * is a covalent bond to A1 or A2. In some embodiments, Ry is -CH2-CH(-CH3)-CH2-CH2- or -CH2-C(-CH3)=CH-CH2-. In some embodiments, the ionizable lipid is a compound of Formula (I), wherein Xi-Ai-Yi and -X2-A2-Y2 are identical. In some embodiments, the compound is a compound of Formula (I), wherein Ri and R2 are each methyl and n is 2, 3 or 4. In some embodiments, the compound is a compound of Formula (I), wherein n is 2 and Ri and R2 are each methyl. In some embodiments, the compound is a compound of Formula (I), wherein n is 3 and Ri and R2 are each methyl. In some embodiments, the compound is a compound of Formula (I), wherein n is 4 and Ri and R2 are each methyl. In some embodiments, the compound is a compound of Formula (I), wherein n is 2, 3 or 4, Ri is -(CH2)2-OH and R2 is methyl. In some embodiments, the compound is a compound of Formula (I), wherein n is 2, Ri is -(CH2)2-OH and R2 is methyl. In some embodiments, the compound is a compound of Formula (I), wherein n is 3, Ri is -(CH2)2-OH and R2 is methyl. In some embodiments, the compound is a compound of Formula (I), wherein n is 4, Ri is -(CH2)2-OH and R2 is methyl. In some embodiments, the compound is a compound of Formula (I), wherein Ri and R2 are each Ci-4 alkyl optionally substituted with hydroxyl. In some embodiments, the compound is a compound of Formula (I), wherein Ri is Ci-4 alkyl and R2 is Ci-4 alkyl optionally substituted with hydroxyl. In some embodiments, the compound is a compound of Formula (I), wherein Ri is methyl and R2 is -(CH2)2-4-OH. In some embodiments, the compound is a compound of Formula (I), wherein Ri is methyl and R2 is -(CH2)2-OH. In some embodiments, the compound is a compound of Formula (I), wherein Ri is methyl and R2 is -(CH2)3-OH. In some embodiments, the compound is a compound of Formula (I), wherein Ri is methyl and R2 is -(CH2)4-OH. In some embodiments, the compound is a compound of Formula (I), wherein -X1- and -X2-are each independently -(CH2)(x)-, wherein (x) is 7, 8, 9 or 10; -Ai- and -A2- are the same or 2026202200   20 Mar 2026 different and are each independently hydrolysable or bioreducible groups; -Y1- and -Y2- are the same or different and are each an aliphatic chain comprising two or more branched C5 hydrocarbon monomer units and having (y) total carbon atoms, wherein (y) is 10, 11, 12, 13, 14, 15, 16 or 17; and the sum of (x)+(y) is 18-25. In some embodiments, the compound is a compound of Formula (I), wherein each of A1 and A2 is a hydrolysable group such as an ester, carbonate, carbamate, acetal, ketal, orthoester, acid-labile dioxolane, phosphoester or phosphoramidate. In some embodiments, the compound is a compound of Formula (I), wherein -Y1 and -Y2 comprise one or more branched C5 hydrocarbon monomer units, such as n-pentane, isopentane, neopentane, 1-pentene, cis-2-pentene, trans-2-pentene, 3-methyl-1-butene, 2-methylbut-2-ene, 2-methylbut-1-ene, 3-methylbut-1-ene (isopentane), isoprene, 1,4-pentadiene, 1,5-pentadiene, 1,3-pentadiene (cis, trans or cis / trans), 2-methyl-1,3-pentadiene, 3-methyl-1,3-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne, or 3-methyl-2-butyne (isopentyne). In some embodiments, the compound is a compound of Formula (I), wherein -Y1 and -Y2 comprise two, three or four C5 hydrocarbon monomer units in a configuration forming a covalent bond from the C1 carbon of a C5 hydrocarbon monomer to the C4 carbon of an adjacent C5 hydrocarbon monomer, such as IPN (-CH2-CH(-CH3)-CH2-CH2-) and / or 2MB (-CH2-C(-CH3)=CH-CH2-). In some embodiments, the compound is a compound of Formula (I), wherein -X1-A1-Y1 and -X2-A2-Y2 are identical; -X1- and -X2- are each independently -(CH2)(X)-, wherein (x) is 7, 8, 9 or 10; each of Ai and A2 is independently *-C(O)-O-** or *-O-C(O)-**, wherein * is a covalent bond to Y1 and Y2, respectively, and ** is a covalent bond to Xi and X2, respectively; -Yi and -Y2 are each *-(CH2)o-2-(Ry)2-4-(CH2)o-2-H wherein each Ry is independently a C5 hydrocarbon monomer unit such as an IPN or a 2MB hydrocarbon monomer unit, and * is a covalent bond to A1 or A2; (y) is 12, 13, 14 or 15; and the sum of (x) + (y) is 22. Some aspects of the disclosure relate to bioreducible ionizable cationic lipids of Formula (I) or (I-A), wherein each Ri is independently linear hydrocarbon alkyl chains each comprising a hydrolysable or bioreducible group, such as an ester group (e.g, -O-CO- or -CO-O-); and R2 and R3 are each independently (Ci-C4) alkyl optionally substituted with hydroxyl; and n is an integer equal to 2, 3 or 4. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R2 and R3 is methyl, -Z- is -(CH2)n- 2026202200   20 Mar 2026 and n is 3 or 4 and each R1 is the same. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R2 and R3 is methyl and each R1 is a hydrocarbon chain comprising a single ester group with a total 15-22 atoms, and optionally comprising one or two olefin groups. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R2 and R3 is methyl and each R1 is a hydrocarbon chain comprising a single ester group with a total 15-22 atoms, and optionally comprising one olefin group. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R2 and R3 is methyl and each R1 is a hydrocarbon chain comprising a single ester group with a total 15-22 atoms, and optionally comprising two olefin groups separated by at least two methylene groups. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R1 and R2 is methyl, -Z- is -(CH2)n-, n is 3 or 4 and each of -X1-A1-Y1 and -X2-A2-Y2 is the same or different and each comprises an ester joining a proximal saturated C7-8 hydrocarbon at -X1- and -X2- and a distal C12-15 hydrocarbon at -Y1 and -Y2 each optionally comprising one or more olefins and one or more methyl substituents. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of Ri and R2 is methyl, Z is -(CH2)n- and n is 3 or 4 and each of -X1-A1-Y1 and -X2-A2-Y2 is the same or different and each comprises an ester at -A1- and -A2- joining a proximal saturated C4-10 hydrocarbon at -X1- and -X2- and a distal C10-20 hydrocarbon at -Y1 and -Y2 optionally comprising one or more olefins and one or more methyl substituents. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R1 and R2 is methyl, -Z- is -(CH2)n-, n is 3 or 4 and each of -X1-A1-Y1 and -X2-A2-Y2 is the same and each comprises a proximal C7-10 hydrocarbon at -X1- and -X2- and a distal saturated isoprenoid alkane group at -Y1 and -Y2 joined by an ester at -A1- and -A2-. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R1 and R2 is methyl, -Z- is -(CH2)n-, n is 3 or 4 and each of -X1-A1-Y1 and -X2-A2-Y2 is the same and each comprises a proximal C7-10 hydrocarbon at -X1- and -X2- and a distal saturated phytane group at -Y1 and -Y2 joined by an ester at -A1- and -A2-. In some embodiments, a bioreducible 2026202200   20 Mar 2026 or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R1 and R2 is methyl, -Z- is -(CH2)n-, n is 3 or 4 and each of -X1-A1-Y1 and -X2-A2-Y2 is the same and each comprises a proximal C7-10 hydrocarbon at -X1- and -X2- and a distal unsaturated phytane group at -Y1 and -Y2 comprising one or more olefins joined by an ester at -A1- and -A2-. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R1 and R2 is methyl, -Z- is -(CH2)n-, n is 3 and each of -X1-A1-Y1 and -X2-A2-Y2 is the same and each comprises a proximal C7-10 hydrocarbon at -X1- and -X2- joined by an ester at -A1- and -A2- to a distal branched hydrocarbon chain comprising two or three isoprene or isopentane monomer groups at -Y1 and -Y2 . In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R1 and R2 is methyl, Z- is -(CH2)n-, n is 3 or 4 and each -X1-A1-Y1 and -X2-A2-Y2 are each -(CH2)7-io-A-Y, wherein A is -C(=O)-O- or -O-C(=O)-, and Y is -(CH2-CH2-CH(-CH3)-CH2)3-H or -(CH2-CH2-CH(-CH3)-CH2)3-H or - CH2-CH2 -(CH2-CH2-CH(-CH3)-CH2)2-H. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R1 and R2 is methyl, Z- is -(CH2)n-, n is 3 or 4 and each -X1-A1-Y1 and -X2-A2-Y2 is -(CH2)7-A-Y, wherein A is -C(=O)-O- or -O-C(=O)-, and Y is -(CH2-CH2-CH(-CH3)-CH2)3-H. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R2 and R3 is methyl, Z- is -(CH2)n-, n is 3 or 4 and each -X1-A1-Y1 and -X2-A2-Y2 is -(CH2)s-A-Y, wherein A is -C(=O)-O- or -O-C(=O)-, and Y is -(CH2-CH2-CH(-CH3)-CH2)3-H or -(CH2)2-CH=CH-(CH2)8-H or -(CH2)12-H. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R2 and R3 is methyl, Z- is -(CH2)n-, n is 3 or 4 and each -X1-A1-Y1 and -X2-A2-Y2 is -(CH2)io-A-Y, wherein A is -C(=O)-O- or -O-C(=O)-, and Y is - CH2-CH2 -(CH2-CH2-CH(-CH3)-CH2)2-H. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R1 and R2 is methyl, Z- is -(CH2)n-, n is 3 and each -X1-A1-Y1 and -X2-A2-Y2 are each -(CH2)7-io-A-Y, wherein A is -C(=O)-O- or -O-C(=O)-, and Y is -(CH2-CH2-CH(-CH3)-CH2)3-H or -(CH2-CH2-CH(-CH3)-CH2)3-H or - CH2-CH2 -(CH2-CH2-CH(-CH3)-CH2)2-H. In some embodiments, a bioreducible or hydrolysable 2026202200   20 Mar 2026 ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of Ri and R2 is methyl, Z- is -(CH2)n-, n is 3 and each -X1-A1-Y1 and -X2-A2-Y2 is -(CH2)7-A-Y, wherein A is -C(=O)-O- or -O-C(=O)-, and Y is -(CH2-CH2-CH(-CH3)-CH2)3-H. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R2 and R3 is methyl, Z- is -(CH2)n-, n is 3 and each -X1-A1-Y1 and -X2-A2-Y2 is -(CH2)8-A-Y, wherein A is -C(=O)-O- or -O-C(=O)-, and Y is -(CH2-CH2-CH(-CH3)-CH2)3-H or -(CH2)2-CH=CH-(CH2)8-H or -(CH2)12-H. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) wherein each of R2 and R3 is methyl, Z- is -(CH2)n-, n is 3 and each -X1-A1-Y1 and -X2-A2-Y2 is -(CH2)10-A-Y, wherein A is -C(=O)-O- or -O-C(=O)-, and Y is - CH2-CH2 -(CH2-CH2-CH(-CH3)-CH2)2-H. In some embodiments, a bioreducible or hydrolysable ionizable cationic lipid comprises a compound of Formula (I) or Formula (I-A) as described in the table in Figure 3A or the table in Figure 3B. In some embodiments, the disclosure provides certain compounds useful as ionizable lipids in the preparation of lipid nanoparticles encapsulating a nucleic acid such as mRNA. Aspects of the disclosure are based in part on the discovery of ionizable lipids containing one or more functionalized aliphatic tails interrupted by ester linkages with certain defined ratios of aliphatic chain length on either side of the ester emerged from systematic structure-activity relationship studies aimed at improving nucleic acid delivery while enhancing biodegradability. Applicants discovered that positioning an ester within the lipid tail, rather than only at the headgroup-tail junction, allowed control over certain properties of the ionizable lipids. The ionizable lipids with ester-containing, asymmetrically segmented lipid tails are useful in preparing lipid nanoparticle formulations for the delivery of nucleic acids, such as in mRNA vaccine and therapeutic applications. An August 2025 study shows the biodistribution patterns of ionizable cationic lipid components of FDA-approved vaccines, including ALC-0315 and SM-102 (Yuxiang R, et al., “Impact of ionizable lipid type on the pharmacokinetics and biodistribution of mRNA-lipid nanoparticles after intravenous and subcutaneous injection,” Journal of Controlled Release, v. 384, 2025, 113945, ISSN 0168-3659). The publication WO2022 / 115645 (PCT / US2021 / 060877) discloses ionizable cationic lipids including KC3-OA. 2026202200   20 Mar 2026 The hydrolysis of various ionizable lipids were evaluated in the pancreatin enzyme assay of Example 4. Pancreatin contains a mixture of digestive enzymes, including lipases and esterases, that mimic the enzymatic environment encountered by LNPs after administration and cellular uptake. Figure 4A is a graph of the biodegradability of ionizable cationic lipids (ICLs) measured after 15 minutes in the presence of pancreatin enzymes (including trypsin, amylase and lipase) measured according to the assay of Example 4. In Figure 4A, the compounds of Formula (I) were hydrolyzed about 29%-98% more than ALC-0315, about 27%-98% more than SM-102 and about 35%-98% faster than KC3-OA. Figure 4B is a graph of the biodegradability of ionizable cationic lipids (ICLs) measured after 3 hours in the presence of pancreatin enzymes (including trypsin, amylase and lipase) measured according to the assay of Example 4. In Figure 2B, the compounds of Formula (I) were hydrolyzed about 77-99% more than ALC-0315, about 58-99% more than SM-102 and about 81-99% faster than KC3-OA. Figure 4C is a graph showing the enzymatic degradation of various ionizable cationic lipids. The ionizable lipids of Formula (I) were hydrolyzed significantly faster than ALC-0315 and SM-102 in the assay of Example 4. The hydrolysis of the ionizable lipids of Formula (I) was significantly greater and more rapid than comparator ionizable lipids ALC-0315, SM102 and KC3-OA, when evaluated in an assay incorporating pancreatin enzymes in Example 4. In some embodiments, the disclosure provides certain LNP compositions comprising one or more ionizable lipids disclosed herein, such as the ionizable lipids of Formula (I). In some aspects, the LNP compositions comprise a nucleic acid encapsulated in lipid nanoparticles formed from: (x) an ionizable lipid of Formula (I) at a N / P ratio of 3 to 8 relative to the nucleic acid in a total amount of 40-65 mol% of the total lipid content of the LNP composition; (y) a sterol in a total amount of 0.5-50 mol% of the total lipid content of the LNP composition; (c) one or more phospholipids in a total amount of phospholipids of 5-50 mol% of the total lipid content of the LNP composition; and (d) a conjugated lipid in a total amount of 0.5-2.5 mol% of the total lipid content of the LNP composition. Unless explicitly indicated otherwise, the isomer form of the phosphatidylserine (PS) lipids used in the Examples is phosphatidyl-L-serine. In some aspects, the LNP composition is further characterized in that: the nucleic acid is mRNA; the ionizable cationic lipid is present in the LNP composition at a N / P ratio of 4 to 7 relative to the nucleic acid; the one or more phospholipids in the LNP comprise a phosphatidylserine (PS) lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition; and the conjugated lipid is a PEG- 2026202200   20 Mar 2026 containing conjugated lipid. In some embodiments, the LNP is a vaccine composition comprising lipid nanoparticles encapsulating mRNA encoding a vaccine antigen (e.g., Tables 5A and 5B), and formed from (x) the ionizable lipid of Formula (I) (e.g., 45-55 mol% KC3-X1-PH5, KC3-X1-PH12, or KC3-X2-PH12), (y) the sterol (e.g., 25-45 mol% cholesterol), (c) a phosphocholine phospholipid (e.g.,   1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), or egg sphingomyelin (SM)), (d) a phosphatidyl-L-serine or phosphatidylglycerol phospholipid selected to target dendritic cells (e.g., 2.5-10 mol%, 2.5-7.5 mol%, or 5 mol% dipalmitoylphosphatidyl-L-serine (DPPS), distearoylphosphatidyl-L-serine (DSPS) or (1,2-Distearoyl-sn-glycero-3-phosphoglycerol (DSPG)), and (e) a PEG-conjugated lipid (e.g., 0.5-4.0 mol%, 1.0-4.0 mol%, 1.5-4.0 mol%, 1.5-2.5 mol%, 0.5 mol%, 1.0 mol%, 1.5 mol%, 2.0 mol%, or 2.5 mol% PEG(2000)-dimyristoylglycerol (PEG-DMG) or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DLG)). In some embodiments, the LNP comprises a phospholipid. A phospholipid is a lipid that comprises a phosphate group and at least one alkyl, alkenyl, or heteroalkyl chain. A phospholipid may be naturally occurring or non-naturally occurring (e.g., a synthetic phospholipid). A phospholipid may comprise an amine, amide, ester, carboxyl, choline, hydroxyl, acetal, ether, carbohydrate, sterol, or a glycerol. In some embodiments, a phospholipid may comprise a phosphocholine, phosphosphingolipid, or a plasmalogen. Exemplary phospholipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dipalmitoylphosphatidyl-L-serine (DPPS), distearoylphosphatidyl-L-serine (DSPS), hydrogenated soy phosphatidylcholine (HSPC), and / or sphingomyelin (SM). Additional phospholipids that may be included in an LNP described herein are disclosed in Li, J. et al. (Asian J. Pharm. Sci. 10:81-98 (2015)), which is incorporated herein by reference in its entirety. In some embodiments, LNP compositions comprising lipid nanoparticles comprising the ionizable lipid(s) disclosed herein and encapsulating mRNA. Figure 5 is a schematic of a mRNA construct comprising a 5’-cap, a 5’-UTR, a cassette comprising a signal peptide, an open reading frame and a terminal domain encoding a synthetic polypeptide, a 3’-UTR and a poly-A region. In some embodiments, the mRNA (100) is a single mRNA comprising a 5’ region (110) and a 3’ region (190) flanking a cassette (150) comprising one or more open reading frames (ORFs). The 5’ region of the mRNA (110) can comprise a 5’ cap and a 5’ UTR region. The 3’ region of the 2026202200   20 Mar 2026 mRNA (190) can comprise a 3’UTR (182) and a polyA sequence (184). The mRNA cassette (150) comprises (from 5’ to 3’) a signal peptide, one or more open reading frame regions encoding one or more proteins optionally separated by mRNA spacer region(s) and a terminal domain comprising a transmembrane and cytoplasmic domain. The mRNA cassette (150) can be configured to encode one or more antigen proteins and comprises one or more spacer sequences between mRNA encoding each antigen protein. For example, a first cassette (151) comprises a signal peptide (SP), an ORF encoding a first antigen protein (P.1) and a second antigen protein (P.2) separated by a spacer mRNA sequence (S1). A concatenated mRNA cassette of various lengths (152) can be constructed with a signal peptide (SP), a ORF encoding a first antigen protein (P.1), a spacer sequence (Si) between the first antigen protein (P.1) encoding region and each subsequent antigen protein encoding regions (Pi), and a third or subsequent antigen protein encoding region (Pn+2), where each consecutive antigen encoding region is separated by a spacer mRNA sequence (Sn). Another cassette (153) comprises a signal peptide (SP), a ORF encoding a first antigen protein (P.1), a second antigen protein (P.2), a third antigen protein encoding region (P.3), and a fourth antigen protein encoding region (P.4), wherein each antigen protein encoding region is separated by a spacer mRNA sequence (S1), (S2) and (S3) respectively. The mRNA spacer sequences can comprise a G / P (gly-pro), G / S (gly-ser) or AAY spacer polynucleotide sequence between mRNA polynucleotide ORF regions encoding the therpeutic antigens. In some embodiments, the mRNA polynucleotide sequence of the cassette (150) further encodes (x) a LAMP-1 signal peptide and a LAMP-1 transmembrane and cytoplasmic domain; or (y) a human HLA Class I signal peptide and a LAMP-1 transmembrane and cytoplasmic domain. SEQ ID NO:35 is the N-terminal signal peptide of human LAMP-1 (Uniprot ID P11279, amino acids 128); SEQ ID NO: 36 is the C-terminal domain of human LAMP-1, including the transmembrane and cytoplasmic domains. The RNA according to aspects of the disclosure may have modified ribonucleotides in order to increase its stability and / or decrease cytotoxicity. For example, in some embodiments, 5-methylcytidine in the RNA is substituted partially or completely, for cytidine. In some embodiments, 5-methylcytidine in the RNA is substituted completely for cytidine. Alternatively or additionally, in some embodiments, pseudouridine in the RNA used is substituted partially or completely, for uridine. In some embodiments, pseudouridine in the RNA used is substituted completely for uridine. In some aspects, the LNP comprises nucleic acid comprising a 5’ 2026202200   20 Mar 2026 untranslated region (UTR) and 3’ UTR, polyA tail of about 80 to about 140 nucleotides in length, and (i) a 5’ enzymatic or (ii) a 5’ clean cap. In some aspects, the LNP comprises a chemically modified mRNA, wherein the chemically modified mRNA comprises N1-methylpseudouridine. In other embodiments, the mRNA does not include N1-methyl-pseudouridine. Exemplary LNP formulations can deliver mRNA encoding any protein antigen derived from an infectious pathogen or disease that can be targeted with the immune system. Listed in Tables 5A and 5B is a non-exhaustive list of vaccine antigens that could be encoded by mRNA and encapsulated in LNPs to comprise a vaccine using the lipids, methods, and formulations described in the Examples. Table 5A. Examples of antigens that could be encoded by mRNA and encapsulated in LNPs to comprise a novel vaccine. Infectious pathogen / disease vaccine antigen description Reference (PMID) respiratory syncytial virus (RSV) Prefusion stabilized F (DS-Cav1) and subsequent iterative structural improvements. E.g. sc9-10 DS-Cav1 A149C Y458C, DS-Cav1-I53-50A • SEQ ID NO:9 RSV-G antigen •  SEQ ID NO:509 •  SEQ ID NO:510 •  SEQ ID NO:511 •  SEQ ID NO:512 •  SEQ ID NO:513 •  SEQ ID NO:515 24179220, 27478931, 30849373, 37689061 Influenza virus (IAV, IBV) HA (e.g., SEQ ID NO: 5, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 and / or SEQ ID NO:18); HA stabilized stem (Group 1 and 2), neuraminidase (NA) (e.g. SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39); M2; stabilized H5 (e.g., SEQ ID NO:34), 26301691, 26303961, 30808695, 36356572, 35869085, 35383176, A Dosey et al. bioRxiv 2025.07.30.667762 Lassa virus (LASV) stabilized LASV GPC; nucleoprotein; NP-GPC chimeric fusion protein (e.g., SEQ ID NO:801 or SEQ ID NO:803) 38177144, 32269122, 32142546 2026202200   20 Mar 2026 human immunodeficiency virus (HIV) gp120 eOD-GT-based immunogens (e.g. eOD-GT8), stabilized Env trimers (e.g. BG505 DS-SOSPI), conserved protease cleavage sites, gp41 fusion peptide, HLA-E-restricted epitopes 23539181, 31515132, 27174988, 37098956, 38997302, 33766848, 26687358 individual Mtb proteins (e.g. ESAT-6, CFP10, Ag85B, Ag85A Esx family proteins), •  SEQ ID NO:444 •  SEQ ID NO:445 •  SEQ ID NO:447 •  SEQ ID NO:449 •  SEQ ID NO:450 Mycobacterium tuberculosis (tuberculosis) synthetic fusion proteins (e.g. M72, H4, H56, H65, H107, CysVac2, BNT164B1 / 2) •  SEQ ID NO:401 •  SEQ ID NO:403 •  SEQ ID NO:405 •  SEQ ID NO:407 •  SEQ ID NO:409 •  SEQ ID NO:411 •  SEQ ID NO:413 •  SEQ ID NO:415 •  SEQ ID NO:417 •  SEQ ID NO:419 •  SEQ ID NO:421 •  SEQ ID NO:423 •  SEQ ID NO:425 •  SEQ ID NO:427 •  SEQ ID NO:429 •  SEQ ID NO:431 •  SEQ ID NO:437 •  SEQ ID NO:438 •  SEQ ID NO:440 •  SEQ ID NO:442 •  SEQ ID NO:451 31114037, 36604540, 15187142, 24349004, 22133873, 34795205, 29263854, 29996082; N Agrawal et al. 2025, bioRxiv 2025.09.30.679428 Plasmodium falciparim (malaria) circumsporozoite protein (CPS), RTS,S, R21, CSP-HBsAg fusion (hepatitis B surface Ag); Pfs25; PfGARP; CelTOS, RH5.1, RH5.2 27355532,33964223, 37168860, 35822551, 27457156, 39019011, 39025972, Table 5B. Examples of antigens that could be encoded by mRNA and encapsulated in LNPs to comprise a novel vaccine. 2026202200   20 Mar 2026 Infectious pathogen / disease vaccine antigen description Reference (PMID) respiratory syncytial virus (RSV) RSVpreF (DS-Cav1) and subsequent iterative structural improvements. E.g. sc9-10 DS-Cav1 A149C Y458C, DS-Cav1-I53-50A 24179220, 27478931, 30849373, 37689061 Influenza virus (IAV, IBV) HA, HA stabilized stem, NA, M2 26301691, 26303961, 30808695, 36356572, 35869085, 35383176, 3480945 Lassa virus (LASV) stabilized LASV GPC, nucleoprotein 38177144, 32269122, 32142546 SARS-CoV-2, betacoronaviruses spike (S) protein, nucleocapsid, membrane, ORF3a, ORF1ab 33128877 Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) glycoprotein 28807998 Ebola virus (EBOV) glycoprotein (GP), nucleoprotein (NP) 32418798, 31589088, 32234486 Marburg virus (MARV) glycoprotein (GP), nucleoprotein (NP) 31589088,32234486 human metapneumovirus (HMPV) F glycoprotein 34551978 parainfluenza virus Type 1 and type 4. Prefusion stabilized fusion proteins (e.g. prefusion PIV3 F) 30420505 human immunodeficiency virus (HIV) gp120 eOD-GT-based immunogens (e.g. eOD-GT8), stabilized Env trimers (e.g. BG505 DS-SOSPI), conserved protease cleavage sites, gp41 fusion peptide 23539181, 31515132, 27174988 human papilloma viruses (HPV) L1 capsid protein, E7, glycoprotein D, gDE7 fusion 38462683, 36867683 Zika virus (ZIKV) prM-E 28222903 Powassan virus (POWV) prM, prE 30566864 MPox virus A35, B6, M1, H3 38366591 norovirus capsid protein VP1 38811605 human cytomegalovirus (HCMV) glycoprotein B (gB), gH, gL, UL128, UL130, UL131 38324766, 29456015 Ebstein Barr virus (EBV) glycoprotein gp350, gp42, gH / gL (gp42 / gp350) 38479361, 38926438 human varicella-zoster virus (VZV) envelope glycoprotein E (gE) 25916341 Rabies virus (RABV) glycoprotein (G) 37349310 West Nile virus (WNV) premembrane (prM), envelope (E) 37125778 2026202200   20 Mar 2026 Dengue virus (DENV) envelope protein (E), premembrane (prM), NS1, NS3, NS5, NS4B 35608749 Chikungunya virus (CHIKV). other Alphaviruses of the Togaviridae family (RRV, ONNV, BFV, MAYV, SINV, EEEV, WEEV, VEEV) E1, E2, E3 glycoproteins 23670192, 38005854, 36474012 Nipah virus (NiV) glycoprotein (G) 38726013 Mycobacterium tuberculosis (tuberculosis) individual Mtb proteins (e.g. ESAT-6, CFP10, Ag85B, Esx family proteins), synthetic fusion proteins (e.g. M72) 31114037, 36604540 Plasmodium falciparim (malaria) circumsporozoite protein (CPS); RTS,S; CSP-HBsAg fusion (hepatitus B surface Ag); Pfs25; PfGARP; CelTOS 27355532, 33964223, 37168860 Borrelia burgdorferi (Lyme disease) outer surface protein A (OspA) 37533256 cancer cancer neoantigens 37165196, 28678784, 35970920, 38538867 A. americanum (alpha-gal hypersensitivity), I. scapularis Salivary proteins (e.g. Salpfamily proteins, TSLPI) 34788080, 37407406 Mumps (MuV) Fusion glycoprotein F, prefusion F variants, HN 39527740, PCT / US2020 / 064619, WO2021119497A1 Measles (MeV) Fusion glycoprotein F, prefusion F PCT / US2020 / 064619, WO2021119497A1 mRNAs encoding antigens for influenza A subtypes H1 (SEQ ID NO:13 encoding SEQ ID NO:12), H3 (SEQ ID NO:15 encoding SEQ ID NO:14), influenza B / Victoria (SEQ ID NO:17 encoding SEQ ID NO:16), and RSVpreF (SEQ ID NO:10 encoding SEQ ID NO:9) were combined at equal weight ratios and encapsulated into LNPs comprising ionizable lipids of Formula (I) as described in Example 2. To measure the ability of these mRNA-LNPs to induce antibodies, BALB / c mice were immunized at a high and 3-fold lower dose in the caudal thigh in a volume of 50 uL. The experimental design and dosing schema is shown in Figure 6A. Twenty-one days post vaccination, serum was collected and stored at -80°C until analysis. Mouse IgG endpoint antibody titers against the matching H1, H3, B-Victora, RSVpreF or H5 antigens were 2026202200   20 Mar 2026 determined by standard ELISA. For H1-specific Ab titers, we found for KC3-X1-PH12 that immunogenicity of freeze-dried material was not deleterious for biological activity (Figure 6B and Figure 6C). This was true for 52 and 56 mol% ICL with N / P = 5.25. Second, Ab titers were comparable between LNPs comprised of KC3-X1-PH12 and KC3-X2-PH12, showing that either orientation of the biodegradable ester bond can be used for PH12 with a KC3 headgroup. Third, we found for KC3-X1-PH12 that either 52 or 56 mol% with N / P = 5.25 elicited similar immune responses; the same was true for X2-PH12. For H3-specific Ab titers (Figure 6D and Figure 6E), results were similar to H1 titers: lyophilization of the mRNA-LPN was not deleterious to biological activity (H3 titers elicited by lyophilized material trended higher than for nonlyophilized, X1-PH12 and X2-PH12 were comparably immunogenic, and these ICLs can be used with either 52 or 56 mol% without impacting antibody titers. The method of making a LNP can comprise mixing a first solution with a second solution. Mixing can be achieved using standard liquid mixing techniques, such as propellor mixing, vortexing solutions or preferably through microfluidic mixing or high efficiency T-mixing. In some embodiments, the first solution comprises a lipid or a plurality of lipids and a nucleic acid, where all components are solubilized, in water / solvent system. The solvent may be any water miscible solvent (e.g., ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethylsulfoxide, dioxane or tetrahydrofuran). In some embodiments, the first solution comprises a small percentage of water or pH buffered water. In some embodiments, the first solution comprises a single type of lipid, for example, an ionizable lipid, a phospholipid, a sterol, or a PEG-containing lipid. In some embodiments, the first solution comprises a plurality of lipids. In some embodiments, the plurality comprises an ionizable lipid, a phospholipid, a sterol, or a PEG-containing lipid. In some embodiments, the second solution is water. In some embodiments, the second solution is an aqueous buffer with a pH between 3-6 (e.g., a pH of about 3, about 4, about 5, or about 6). The second solution may comprise a load component, e.g., a nucleic acid (e.g., mRNA). The second solution may comprise a small percentage of water-miscible organic solvent. In some embodiments, the solution comprising a mixture of the first and second solutions comprising the LNP suspension can be diluted. In some embodiments, the pH of the solution comprising a mixture of the first and second solutions comprising the LNP suspension can be adjusted. Dilution or adjustment of the pH of the LNP suspension can be achieved with the addition of water, acid, base or aqueous buffer. In some embodiments, no dilution or adjustment of the pH 2026202200   20 Mar 2026 of the LNP suspension is carried out. In some embodiments, both dilution and adjustment of the pH of the LNP suspension is carried out. In some embodiments, excess reagents, solvents, unencapsulated nucleic acid maybe removed from the LNP suspension by tangential flow filtration (TFF) (e.g., diafiltration). The organic solvent (e.g., ethanol) and buffer may also be removed from the LNP suspension with TFF. In some embodiments, the LNP suspension is subjected to dialysis and not TFF. In some embodiments, the LNP suspension is subjected to TFF and not dialysis. In some embodiments, the LNP suspension is subjected to both dialysis and TFF. In one aspect, the present disclosure features a method comprising treating a sample of LNPs comprising nucleic acid, with a fluid comprising a detergent (e.g., Triton X-100, or anionic detergents (such as, but not limited to, sodium dodecyl sulfate (SDS), or non-ionic detergent, such as but not limited to 0-octylglucoside, or Zwittergent 3-14) for a period of time suitable to degrade the lipid layer and thereby release the encapsulated and / or entrapped nucleic acid(s). In an embodiment, the method further comprises analyzing the sample for the presence, absence, and / or amount of the released nucleic acid(s). Methods of making lipid nanoparticle (LNP) compositions encapsulating mRNA are provided in Example 7 (Preparing ligand-targeted LNPs) and Example 9 (Preparation of lipidic nanoparticles) of Patent Cooperation Treaty Patent Application PCT / US2021 / 060877 (WO2022 / 115645), are incorporated herein by reference in their entirety. Methods of making lipid nanoparticle (LNP) compositions encapsulating mRNA are provided in Example 7 (Preparing ligand-targeted LNPs) and Example 9 (Preparation of lipidic nanoparticles) of Patent Cooperation Treaty Patent Application PCT / US2023 / 067517 (WO2023 / 230587), are incorporated herein by reference in their entirety. Methods of characterizing lipid nanoparticle (LNP) compositions encapsulating mRNA are provided in Example 10 (LNP Characterization) and Example 11 (Determination of transfection efficiency in murine dendritic cells of LNPs using mCherry mRNA) of Patent Cooperation Treaty Patent Application PCT / US2021 / 060877 (WO2022 / 115645), are incorporated herein by reference in their entirety. Methods of making lipid nanoparticle (LNP) compositions encapsulating mRNA are provided in Example 10 (LNP Characterization) and Example 11 (Determination of transfection efficiency in murine dendritic cells of LNPs using mCherry mRNA) of Patent Cooperation Treaty Patent Application PCT / US2023 / 067517 (WO2023 / 230587), are incorporated herein by reference in their entirety. These methods can be modified by using a microfluidics chip from ChipShop (187) to make 2026202200   20 Mar 2026 LNPs at flow rates of 18 mL / min instead of the Nanoprecision Systems microfluidics cassette at 6 ml / min whilst keeping downstream processing and analytical procedures the same as described in the published patents referenced above. Additional disclosure of methods for the manufacture of mRNA lipid nanoparticle compositions are provided in: S.J. Shepherd, X. Han, A.J. Mukalel, R. El-Mayta, A.S. Thatte, J. Wu, M.S. Padilla, M. Alameh, N. Srikumar, D. Lee, D. Weissman, D. Issadore, & M.J. Mitchell, Throughput-scalable manufacturing of SARS-CoV-2 mRNA lipid nanoparticle vaccines, Proc. Natl. Acad. Sci. U.S.A. 120 (33) e2303567120, https: / / doi.org / 10.1073 / pnas.2303567120 (2023), incorporated by reference herein in its entirety. EXAMPLES Certain examples are provided below to illustrate various embodiments of the embodiments disclosed herein. One of ordinary skill in the art will recognize that the various embodiments disclosed herein are not limited to these specific illustrative examples. Example 1: Synthesis of Ionizable Cationic Lipids Synthesis of KC3-X1Ionizable Cationic Lipids Figure 1A is a general synthesis for compounds of Formula (I) wherein W1 and W2 are each *-O-C(O)-**, * is a covalent bond to Y1 or Y2 and ** is a covalent bond to X1 or X2 (respectively), Xi or X2 are each -(CH2)x-, and Yi or Y2 are each identical linear or branched hydrocarbon chains with (y) total carbons (i.e., “KCn-X1” ionizable cationic lipids). A general synthesis for compounds of formula (I) shown in Scheme i involves i) esterification of the ketone dicarboxylic acid, ii) acid catalyzed trans ketal formation, iii) Mesylation of the primary alcohol, and finally iv) displacement of the mesyl group using dialkylamine resulting in a tertiary amine. A general synthesis of ketone dicarboxylic acid starting material in Scheme i is presented below in Scheme 2. A general synthesis of ketone dicarboxylic acid starting material shown in Scheme 2 involves i) treatment of suitable bromo alkyl carboxylate with toluenesulfonylmethyl isocyanide in the presence of a base, ii) conversion of the TosMIC adduct to ketone in the presence of HCl, and finally iv) hydrolysis of the ethyl carboxylate to ketone dicarboxylic acid. Figure 1B is a chemical synthesis for preparing an ionizable cationic lipid of Formula (I) designated KC3-X1-PH12, as described below. 2026202200   20 Mar 2026 To a solution of KC3-X1-AA-C23 acid (800 mg, 2.01 mmol, 1 eq) and KC3-X1-PH7_4 (1.12 g, 6.02 mmol, 3 eq) in DMF (10 mL) was added NMI (1.65 g, 20.07 mmol, 1.60 mL, 10 eq) and MNBA (2.07 g, 6.02 mmol, 3 eq). The mixture was stirred at 70 °C for 12hr. The reaction mixture was quenched by addition aq. NaHCOs (sat. 30 mL) at 25°C, and extracted with EtOAc (10 mL*3). The combined organic layers were washed with brine (10 mL* 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0~3%Ethylacetate / Petroleum ethergradient @ 60 mL / min) to give compound 1 (1.5 g, 1.43 mmol, 71.2% yield, 70.1% purity) as a white solid. 1H NMR (400 MHz, CHLOROFORM-d, EW58311-330-P1A1) 8 4.00 (t, J = 6.8 Hz, 4H), 2.31 (t, J = 7.4 Hz, 4H), 2.22 (t, J = 7.6 Hz, 4H), 1.58 - 1.42 (m, 16H), 1.32 - 1.19 (m, 36H), 1.09 - 1.00 (m, 8H), 0.81 - 0.78 (m, 18H). A mixture of compound 1 (1.5 g, 2.04 mmol, 1 eq), PPTS (256.36 mg, 1.02 mmol, 0.5 eq) and compound 2 (588.36 mg, 3.67 mmol, 1.8 eq) in Tol. (20 mL) was heated to reflux and remove water by Dean-Stark trap. The mixture was stirred at 135 °C for 12hr. The reaction mixture was quenched by addition aq.NaHCO3 (sat. 60 mL) at 25°C, and extracted with EtOAc (30 mL*3). The combined organic layers were washed with brine (20 mL* 3), dried overNa2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0~10% Ethyl acetate / Petroleum ether gradient @ 60 mL / min) to give compound 3 (1 g, 1.19 mmol, 58.5% yield) as a yellow oil. 1H NMR (400 MHz, CHLOROFORM-d, EW58311-334-P1A1) 8 4.13 - 4.05 (m, 6H), 3.75 - 3.66 (m, 2H), 3.52 - 3.45 (m, 1H), 2.31 (t, J = 7.6 Hz, 4H), 1.74 - 1.48 (m, 20H), 1.41 - 1.26 (m, 41H), 1.18 - 1.08 (m, 8H), 0.90 - 0.85 (m, 18H). To a solution of compound 3 (1 g, 1.19 mmol, 1 eq) in DCM (10 mL) was added TEA (604.23 mg, 5.97 mmol, 831.13 pL, 5 eq) and MsCl (630 mg, 5.50 mmol, 425.68 pL, 4.61 eq). The mixture was stirred at 0 °C for 1hr. The reaction mixture was quenched by addition ice-water (30 mL) at 0°C, and extracted with EtOAc (10 mL*3). The combined organic layers were washed with brine (10 mL* 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0~15% Ethyl acetate / Petroleum ether gradient @ 40 mL / min) to give compound 4 (1 g, 1.09 mmol, 91.5% yield) as a yellow oil. 1H NMR (400 MHz, CHLOROFORM-d, EW58311-335-P1A1) 8 4.36 - 4.24 (m, 2H), 4.12 - 4.03 (m, 6H), 3.54 - 3.44 2026202200   20 Mar 2026 (m, 1H), 3.03 (s, 3H), 2.31 (t, J = 7.6 Hz, 4H), 1.99 - 1.79 (m, 2H), 1.69 - 1.52 (m, 20H), 1.40 -1.27 (m, 38H), 1.19 - 1.06 (m, 8H), 0.94 - 0.83 (m, 18H). A solution of compound 4 (900 mg, 983.14 gmol, 1 eq) in Me2NH / THF (2 M, 20 mL, 40.69 eq) was stirred at 55 °C for 12hr. The mixture was concentrated in vacuo to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0~8% Ethyl acetate / (Petroleum ether / Dichloromethane=3 / 1, 0.1% TEA as eluant) @ 40 mL / min) and followed by reversed-phase HPLC(column: PHS XP Phenyl-Hexyl 150 * 25 mm * 7 um; mobile phase: [H2O (0.225% FA)-EtOH]; gradient: 65%-95% B over 15.0 min). KC3-X1-PH12 (0.094 eq. FA salt, 435.5 mg, 495.75 pmol, 53.6% yield, 98.4% purity) was obtained as a colorless oil. LCMS (EW58311-340-P1C1): CAD, m / z, M+H=865.0. HRMS (ESI) m / z: [M + H]+ Calcd for C54H106NO6+ 864.8015, found 864.8044. 1H NMR (400 MHz, CHLOROFORM-d, EW58311-340-P1A1) 8 8.55 (s, 0.094H, FA salt), 4.15 - 3.98 (m, 6H), 3.52 - 3.40 (m, 1H), 2.36 (br t, J = 7.2 Hz, 2H), 2.32 - 2.26 (m, 10H), 1.67 - 1.47 (m, 18H), 1.41 - 1.19 (m, 39H), 1.16 - 1.00 (m, 8H), 0.93 - 0.76 (m, 18H). Figure 1C is a table of additional examples of KCn-X1 compounds that were prepared. Synthesis of KC3-X2 Ionizable Cationic Lipids Figure 2A is a chemical synthesis (Scheme 3 and Scheme 4) for preparing ionizable cationic lipids of Formula (I) wherein -A1- and -A2- are each *-C(O)-O-** wherein * is a covalent bond to -Y1 and -Y2, respectively, and ** is a covalent bond to -Xi- and -X2-, respectively, wherein (n) is 2, 3 or 4. (designated “KCn-X2 ICLs”). Scheme 3 is a general synthesis for compounds of Formula (I) wherein W1 and W2 are each *-C(O)-**, * is a covalent bond to Y1 or Y2 and ** is a covalent bond to Xi or X2 (respectively), Xi or X2 are each -(CH2)x-, and Y1 or Y2 are each identical linear or branched hydrocarbon chains with (y) total carbons (i.e., “KCn-X2” ionizable cationic lipids). A general synthesis for compounds of formula (I) wherein W is -C(O)O- shown in Scheme 3 involves i) esterification of the dihydroxy ketone with a suitable carboxylic acid , ii) acid catalyzed trans ketal formation, iii) Mesylation of the primary alcohol, and finally iv) displacement of the mesyl group using dialkylamine resulting in a tertiary amine. A general synthesis of dihydroxy ketone starting material in Scheme 3 is presented below in Scheme 4. A general synthesis of dihydroxy ketone shown in Scheme 4 involves i) a suitable hydroxy alkyl 2026202200   20 Mar 2026 bromide is treated with toluenesulfonylmethyl isocyanide in the presence of sodium hydride in DMSO, and ii) TosMIC adduct is then converted to a ketone in the presence of HCl. Figure 2B is a chemical synthesis for preparing an ionizable cationic lipid of Formula (I) designated KC3-X2-PH12, as described below. A solution of NaH (5.90 g, 147.51 mmol, 60% purity, 2.4 eq) in DMSO (300 mL) was stirred at 25°C for 1hr.Then TBAI (2.27 g, 6.15 mmol, 0.1 eq) and Tosmic (12 g, 61.46 mmol, 1 eq) was added. The mixture was stirred at 25°C for 15min. After the addition of compound 1 (30.61 g, 129.07 mmol, 2.1 eq), the mixture was stirred at 25°C for 1hr. The reaction mixture was quenched by addition sat-NH4Cl (aq.200 mL) at 25°C, and then diluted with ice-water (100mL) and extracted with EtOAc (200 mL *3). The combined organic layers were washed with brine (200 mL *3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The crude product was used directly for the next step without further purification. Compound 2 (32 g, crude) was obtained as a brown oil. To a solution of compound 2 (32 g, 63.02 mmol, 1 eq) in DCM (210 mL) was added con.HCl (70 mL). The mixture was stirred at 25 °C for 2hr. The reaction mixture was concentrated under reduced pressure to remove the solvent and then lyophilized. The crude product was triturated with (PE: EtOAc =1:1) at 0 oC for 30 min to give compound 3 (18.9 g, crude) as a yellow solid. 1H NMR (400 MHz, CHLOROFORM-d, EW58397-468-P1A1) 8 3.63 (t, J = 6.8 Hz, 4H), 3.38 (t, J = 6.8 Hz, 2H), 2.38 (t, J = 7.2 Hz, 4H), 1.58-1.52 (m, 8H), 1.36-1.25 (m, 24H). To a solution of compound 3 (1.6 g, 4.67 mmol, 1 eq) in DCM (20 mL) was added EDCI (3.58 g, 18.68 mmol, 4 eq), DIEA (6.04 g, 46.71 mmol, 8.14 mL, 10 eq), KC3-X2-PH11_4 (2.00 g, 9.34 mmol, 2 eq) and DMAP (1.43 g, 11.68 mmol, 2.5 eq). The mixture was stirred at 40 °C for 16hr. The reaction mixture was concentrated in vacuum. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=1 / 0 to 94 / 6) to give compound 4 (1.8 g, 2.41 mmol, 51% yield) as a yellow oil. 1H NMR (400 MHz, CHLOROFORM-d, EW58397-474-P1A1) 8 4.05 (t, J = 6.8 Hz, 4H), 2.38 (t, J = 7.2 Hz, 4H), 2.30 (t, J = 7.4 Hz, 4H), 1.65 - 1.50 (m, 14H), 1.41 - 1.21 (m, 38H), 1.17 - 1.02 (m, 8H), 0.90 - 0.80 (m, 18H). A solution of compound 4 (2 g, 2.72 mmol, 1 eq), PPTS (205.08 mg, 816.09 pmol, 0.3 eq) and compound 5 (871.64 mg, 5.44 mmol, 2 eq) in Tol. (30 mL) was heated to reflux and the water was removed by Dean-Stark trap. The mixture was stirred at 135 °C for 16hr. The reaction mixture was quenched by addition sat-NaHCO3 (aq. 80 mL) at 25°C, and extracted with EtOAc (30 mL*3). 2026202200   20 Mar 2026 The combined organic layers were washed with brine (20 mL* 3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0~10% Ethyl acetate / Petroleum ether gradient @ 60 mL / min) to give compound 6 (850 mg, 832.40 gmol, 31% yield) as a yellow oil. 1H NMR (400 MHz, CHLOROFORM-d, EW58311-476-P1A1) 8 4.12 - 4.03 (m, 6H), 3.72 - 3.64 (m, 2H), 3.52 - 3.43 (m, 1H), 2.32 - 2.28 (m, 4H), 1.64 - 1.56 (m, 18H), 1.35 - 1.24 (m, 42H), 1.16 - 1.05 (m, 8H), 0.88 - 0.83 (m, 18H). To a solution of compound 6 (840 mg, 1.00 mmol, 1 eq) in DCM (10 mL) was added TEA (507.56 mg, 5.02 mmol, 698.15 gL, 5 eq) and MsCl (270.00 mg, 2.36 mmol, 182.43 gL, 2.35 eq) at 0°C. The mixture was stirred at 0 °C for 1hr. The reaction mixture was quenched by addition ice-water (30 mL) at 0°C, and extracted with DCM (10 mL * 3). The combined organic layers were washed with brine (10 mL * 3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0~10% Ethylacetate / Petroleum ethergradient @ 40 mL / min) to give compound 7 (760 mg, 788.70 gmol, 79% yield,) as a yellow oil. 1H NMR (400 MHz, CHLOROFORM-d, EW58311-477-P1A1) 8 4.33 - 4.22 (m, 2H), 4.09 - 4.01 (m, 6H), 3.53 - 3.42 (m, 1H), 3.02 (s, 3H), 2.30 (t, J = 7.6 Hz, 4H), 1.64 - 1.55 (m, 20H), 1.36 - 1.23 (m, 40H), 1.16 -1.04 (m, 8H), 0.89 - 0.83 (m, 18H). A solution of compound 7 (760 mg, 830.21 gmol, 1 eq) in Me2NH / THF (2 M,10 mL) was stirred at 50 °C for 10hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0~8% Ethyl acetate / (Petroleum ether / Dichloromethane=3 / 1, 0.1% TEA as eluant)@ 40 mL / min) and followed by reversed-phase HPLC (column: PHS XP PhenylHexyl 150 * 25 mm * 7 um; mobile phase: [H2O (0.225% FA) -ACN]; gradient: 65%-95% B over 15.0 min) to give AKG-KC3-X2-PH12 (371.1 mg, 422.87 gmol, 48% yield, 98.5% purity) as a yellow oil. LCMS (EW58311-481-P1C1): CAD, m / z, M+H=865.0. HRMS (ESI) m / z: [M + H]+ Calcd for C54H106NO6+ 864.8015, found 864.8032. 1H NMR (400 MHz, CHLOROFORM-d, EW58311-481-P1A1) 8 4.13 - 3.99 (m, 6H), 3.52 - 3.40 (m, 1H), 2.44 - 2.21 (m, 12H), 1.67 - 1.48 (m, 18H), 1.39 - 1.19 (m, 42H), 1.17 - 1.04 (m, 8H), 0.92 - 0.82 (m, 18H). Figure 2C is a table of additional examples of KCn-X2 compounds that were prepared. 2026202200   20 Mar 2026 Example 2. LNP Preparation Lipid nanoparticle (LNP) compositions encapsulating mRNA can be prepared as described in Example 9 (Preparation of lipidic nanoparticles) of Patent Cooperation Treaty Patent Application PCT / US2021 / 060877 (WO2022 / 115645), and Example 9 (Preparation of lipidic nanoparticles) of Patent Cooperation Treaty Patent Application PCT / US2023 / 067517 (WO2023 / 230587), are incorporated herein by reference in their entirety, modified by using a microfluidics chip from ChipShop (187) to make LNPs at flow rates of 18 mL / min instead of the Nanoprecision Systems microfluidics cassette at 6 ml / min whilst keeping downstream processing and analytical procedures the same as described in the published patents referenced above. Stock solutions of each lipid are prepared. Ionizable lipids are weighed out in 4 mL glass vials (Thermo B7999-2) and dissolved in ethanol (Sigma-Aldrich 200 proof, RNase free) to a final concentration of 10 mM. Other lipids such as DSPC, DPPC-NH4, DPPS-NH4 and PEG-DMG are weighed out and dissolved in ethanol to a concentration of 1 mM, and cholesterol to concentration of 10 mM. Lipid mixtures for each individual LNP are prepared by adding the desired volume of each lipid stock solution to a new vial, adding ethanol if needed to achieve a final volume of 1.2 mL. mRNA solutions are prepared by thawing frozen mRNA (e.g. FFluc-mRNA) vials and diluting mRNA in 6.25 mM sodium acetate (pH 5.0) to a final concentration of 0.033 mg / mL To prepare LNPs, a NanoAssemblr Benchtop microfluidic device (from Precision Nanosystems) is used. 3 mL of mRNA solution is loaded into a 3 mL disposable syringe (BD 309656) and 1 ml of lipid mixture in a 1 ml syringe (BD309659) and placed in the NanoAssemblr heating block for 4 min prior to mixing. LNP formation is achieved by pumping the liquid streams through a disposable microfluidics cassette at 3:1 aqueous: alcohol volume ratio at 6 mL / min mixing speed, or through a ChipShop microfluidics (Jena, Germany) chip (#187) at 18 ml / min. After mixing, 3.6 mL of LNP mixture is collected, while the initial mixed volume of 0.35 mL and last 0.05 mL of mix is discarded. Ethanol is removed by buffer exchange using SpectraPor dialysis tubing (12-14k MWCO) in PBS (Cytiva, SH30256.01) or by sequential concentration and dilution using Amicon Ultra-4 centrifugal concentrators (10k MWCO, at 500 g). LNPs are typically exchanged into PBS, pH 7.4 and then 10 mM Tris, pH 7.4, 10% sucrose, concentrated to 20-50 ug / mL mRNA using an Amicon-Ultra 4 (100,000 MWCO) spin 2026202200   20 Mar 2026 column, sterile filtered (Thermo Nalgene 0.2 pm #720-1320) prior to freezing by immersion in liquid nitrogen for 5 min and long-term storage at - 80 °C. Example 3: Characterization of lipidic nanoparticles (LNPs) containing mRNA. A. mRNA Encapsulation efficiency Materials: Quant-iT™ RiboGreen™ RNA reagent (1 mL solution in DMSO), 20X TE (25 mL in DEPC-treated water), DNase / RNase-Free Distilled Water (Invitrogen Cat # 10977023, or equivalent), 96-Well Clear-Bottom Microplate MED BINDING (Greiner Bio-One Cat # 655096), Detergent (Triton™): Surfact-AmpsTM X-100 Cat # 28314, RNase / DNase-free 1.5 mL Microcentrifuge tubes (ThermoFisher, Invitrogen Cat # AM12400) or equivalent sterile, nuclease-free tubes. A portion of 1X TE buffer prepared for the experiment was allotted for the dilution of RiboGreen dye. This dye was diluted 200X in 1X TE buffer. As the dye is light sensitive, allow to come to RT protected from light. Ensure the dye has melted (become liquid) as it will remain crystalline in temperatures below RT. Before pipetting the dye vortex mix for one second, three times. Prepare the volume of 1X TE buffer needed in a 15mL conical tube. Vortex or invert the diluted dye and store protected from light. Standards corresponding to 1500, 1200, 1000, 800, 500, 250, 50, 25 and 0 ng / mL mRNA were prepared in 1X TE buffer from the mRNA used in the preparation of LNPs. The blank contains no mRNA. LNPs are diluted in 1X TE buffer to approximately 2 pg / mL. 100 pL of blank, standards and samples were added to the wells of a 96-well plate in triplicate to which 100 uL of diluted Ribogreen is added and mixed well by pipetting up and down 4-5 times. The plate is covered with foil to protect from light and after 5 min incubation at room temperature. The standards and samples were read using a microplate fluorometer (e.g., SpectraMax M2) using excitation at 485 nm, emission at 530 nm, and instrument default acquisition parameters. A 2% Triton solution is prepared from the 10% stock solution of Surfact-AmpsTM X-100 by performing a 1:5 dilution in 1X TE buffer. Invert 5X to mix. Once the pre-Triton readings have been taken and saved, empty prepared Triton (diluted to 2% in 1X TE buffer) into a clean reagent reservoir. A multichannel pipettor was used to add 20 pL of 2% Triton to each plate well and slowly pipette mix 5X. The plate can be read 5 minutes after mixing. The pre-Triton mRNA concentrations are determined by extrapolation to the pre-Triton standard curves and the post- 2026202200   20 Mar 2026 Triton mRNA concentrations are determined by extrapolation to the Triton containing standard using a linear curve fit. % Encapsulation Efficiency (% E.E) expresses the concentration of encapsulated mRNA as a percent of the total, post-Triton concentration. The formula can be expressed as: % Encapsulation Efficiency = 100 - ((Pre-Triton concentration / Post-Triton concentration)*100) B. LNP Particle size 30 uL of LNP was mixed with 1.5 mL of 0.1um filtered PBS in a polystyrene cuvette (Sarstedt, #67.754) and analyzed for particle size using a ZetaSizer Pro (Malvern) The Z-average size and polydispersity index value were obtained by the method of cumulants using ZS Xplorer software, version number 1.4.0.105. Typically, size measurements of LNPs were taken post LNP mixing, post buffer exchange, post sterile filtering and after freezing for 2 min in liquid nitrogen and thawing at 37 °C. C. LNP Zeta Potential 30 uL of LNP is mixed with 1.5 mL 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 15 mM sodium chloride, pH 5 or 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 15 mM sodium chloride, pH 7 and injected into a disposable folded capillary cell (Malvern Nanoseries DTS1070) and zeta potential measured on a ZetaSizer Pro at 25 °C. D. LNP Apparent pKa Determination 1.0L of buffer consisting of 10 mM sodium phosphate, 10 mM ammonium acetate, 10 mM sodium citrate, and 150 mM sodium chloride solution in WFI was prepared. The solution is divided into 50 mL aliquots and the pH of each adjusted with 5M HCl or 10N NaOH to span the pH range of 2-12 in approximately 0.5-unit increments. The actual pH of each aliquot was measured with a pH meter (Thermo Orion 4 Star with a glass combination micro pH electrode (8220BNWP) calibrated with Thermo Orion pH 4.01, 7.00, 10.01 standards). A 10 mM solution of 6-p-Toluidino-2-naphthalenesulfonic acid (TNS, Abcam ab275049) in 10 mL of DMSO is prepared from which a 1 mM solution in water is prepared. LNPs were diluted to 1 ugmL mRNA in a well of a 96-well plate in a final volume of 2026202200   20 Mar 2026 200 uL for each pH prepared above and TNS added to a final concentration of 10 uM after which each well is mixed by pipetting up and down 10 times with a multichannel pipette. The plate was incubated at room temperature for 3 min, then read on a plate reader at Ex.331 / Em.445 nm. A plot of pH versus TNS fluorescence signal was prepared in Graph Pad Prism. The pH value at the half maximum value of fluorescence represents the pKa of nanoparticles and is calculated by performing a non-linear regression analysis for a half maximal fluorescence. This value is the pH at which the numbers of ionized (protonated) and deionized groups are equal. Example 4. In Vitro Assessment of Biodegradability of Ionizable Lipids Using Pancreatin Enzymes. An in vitro assay was developed to evaluate the biodegradability of ionizable cationic lipids (ICLs) in the presence of pancreatin enzymes (including trypsin, amylase and lipase). Individual ICL stock solutions were prepared in ethanol at a concentration of 50 mg / mL and stored at -80 °C until use. Bile salts (Millipore, Cat. No. 48305) were dissolved in Dulbecco’s phosphate-buffered saline (DPBS; Sigma, Cat. No. 59300C) at 2 mg / mL, and pancreatin (Sigma, Cat. No. P7545) was prepared at 10 mg / mL in the same buffer. The pH of the DPBS was adjusted to 8.0 using 1 M NaOH. All solutions were maintained at 37 °C under continuous mixing. For each reaction, 50 nmol of ICL (from the 50 mg / mL ethanol stock) was added to 2 mL of pancreatin solution. Reactions were performed in duplicate across six time points. The mixture was vortexed for 30 seconds, after which 100 uL aliquots were transferred into separate 2 mL tubes (two replicates per time point). Samples were incubated at 37 °C with shaking. To terminate the reaction, 10 volumes of isopropanol / acetonitrile (IPA / MeCN, 70:30 v / v) were added. The resulting mixture was sonicated for 5 minutes to facilitate phase separation of lipids and enzymes, followed by centrifugation at 14,000 x g for 15 minutes. The supernatant was collected and transferred to HPLC vials for analysis. Lipid degradation was quantified using a Thermo Scientific Vanquish Flex UHPLC system equipped with a Charged Aerosol Detector (CAD) and a C18 column (Phenomenex Kinetex 1.7 pm C18 100 A 150 x 2.1 mm). Operating conditions for UHPLC are detailed in Table 6. The extent of lipid degradation was expressed as the percentage of the main ICL peak remaining at each time point relative to the peak area at 2026202200   20 Mar 2026 time zero. Quantification was performed using ICL standards prepared in IPA at concentrations ranging from 5 to 40 ugmL. Table 6. Chromatographic Conditions HPLC Instrument HPLC Column HPLC guard column Column Temperature Flow Rate, ml / min Injection Volume CAD Run Time Sample Solvent Mobile Phase A Mobile Phase B Thermo Scientific Vanquish Flex UHPLC C18 column (Phenomenex Kinetex 1.7 um C18 100 A 150 x 2.1 mm) None 50 °C 0.35 5 uL 10H 12 min 70% IPA / 30% MeCN 0.1% TFA in water 0.05 % TFA in MeCN Gradient Time, min      Mobile Phase A%   Mobile Phase B% -0.5                   15                    85 0                   15                   85 0.5                   15                    85 5                    5                   95 11                   5                   95 11.5                   15                    85 12                   15                   85 Results are shown in the graphs in Figure 2A and Figure 2B showing biodegradability of ionizable cationic lipids (ICLs), measured after 15 minutes (Figure 2A) and 3 hours (Figure 2B) in the presence of pancreatin enzymes (including trypsin, amylase and lipase) measured according to the assay of Example 4. Figure 4A and Figure 4B are graphs of the biodegradability of ionizable cationic lipids (ICLs) over time in the presence of pancreatin enzymes (including trypsin, amylase and lipase) measured according to the assay of Example 4. The ionizable cationic lipids of Formula (I) that were tested (Table 7 with structures in Table 8) showed significantly greater degradation compared to the ALC-0315, SM-102 or KC3-OA ionizable lipid comparator compounds. 2026202200   20 Mar 2026 Table 7. Results Obtained from Ionizable Lipids Tested in Example 4. ICL t=15 min. t =3 hr KC3-OA 0.98 0.94 ALC-0315 0.92 0.94 SM-102 0.89 0.53 KC3-X1-PH5 0.67 0.20 KC3-X1-PH12 0.29 0.03 KC3-X1-AA 0.23 0.01 KC3-X2-PH12 0.08 0.02 Table 8. Chemical structures of Ionizable Lipids Tested in Example 4. KC3-X1-PH5 ALC-0315 KC3-X1-PH12 KC3-X2-PH12 SM-102 2026202200   20 Mar 2026 KC3-X1-AA Example 5. Determination of transfection efficiency in murine dendritic cells of LNPs using firefly luciferase mRNA. Immortalized mouse dendritic cells (MutuDC 1940, Fuertes Marraco et al., Front. Immunol. 2012, 3:331) are cultivated in Gibco™ IMDM medium with GlutaMax™ (Gibco cat. No 31980097), supplemented with 7.5 mg / ml NaHCOs, 10 mM HEPES-Na pH 7, 50 ^M betamercaptoethanol, 50 U / ml penicillin, 50 ^g / mL streptomycin, and 10% fetal bovine serum, at 37 °C, 5% CO2. The cells are rinsed with phosphate-buffered saline (PBS, Cytiva, cat. No SH30526) harvested using 5 mM EDTA in PBS (PBS-EDTA), resuspended in the growth medium, inoculated in the white opaque 96-well cell culture plates (e.g., Costar cat. No 3917) in the wells in columns 3-10 of the plate at 15,000 cells / well in 0.1 ml of the medium and allowed to acclimate overnight. The media are aspirated, replaced with 0.1 ml of the media containing the desired concentration of mRNA LNP (0.1-1 ^g / mL mRNA, typically 0.3 ^g / mL mRNA - SEQ ID NO: 3 encoding SEQ ID NO: 2), and incubated at 37 °C, 5% CO2 for the desired transfection period, typically 24 hours. The mRNA can be an mRNA encoding a firefly luciferase protein sequence such as SEQ ID NO:2. Three to four hours prior to the end of the transfection period, the wells A2 and A11 of the plates are seeded with 0.1 ml of the cell suspension standard containing approximately 1,000,000 viable cells / mL (the viable cell concentration is accurately determined), and wells B2 to G2, and B11 to G11 are seeded with the serial dilutions of the standard at the dilution factor of 1.5 to generate eight cell quantity standards per plate in duplicate. In the wells A1-H1, A12-H12, 0.1 ml of the cell medium is added. At the end of the transfection period, 11 ^L of 20 mM aqueous solution of Hoechst 33342 DNA fluorescent dye (Ultrapure grade, cat. No 17533, AAT Bio, USA) is added to each well, carefully mixed by gentle tapping and swirling of the plate by hand, and the plate is 2026202200   20 Mar 2026 incubated at 37 °C, 5% CO2 for one hour to allow for the staining of the cellular DNA by added dye. Luciferase standards are prepared using recombinant firefly luciferase (cat. No. SRE0045, Sigma-Aldrich, USA) reconstituted to 2 mg / ml protein in 1 M Tris-HCl buffer pH 8.5 and stored at -80°C. An interim standard is prepared by diluting the reconstituted luciferase 20-fold (to 0.1 mg / ml) in PBS-EDTA containing 1 mg / ml nuclease- and protease-free bovine serum albumin (PBS-EDTA-BSA). Eight working standards (DA to DH) are prepared by further diluting the interim standard in PBS-EDTA-BSA, first to 1000 ng / mL (DA), and then serially diluting DA down to DH with the dilution factor of three per step. The interim and working standards are prepared fresh. At the end of one hour incubation, the media from the wells in columns 2-11 of the plate are aspirated, and 0.1 ml of PBS-EDTA is added. The plate is incubated at 37 °C, 5% CO2 for 10 min and on the microplate shaker at 700 rpm for 3 min to dislodge the cells. Then the media from the wells A1-H1, A12-H12 are aspirated and 0.1 ml aliquots of the luciferase working standards are added, from the highest (DA) in A1 and A12, to the lowest (DH) in H1 and H12, creating a series of eight luciferase standards per plate in duplicate (plate columns 1 and 12). The Hoechst 33342 fluorescence of the cell suspensions in the wells is determined using SpectraMax M2 multimode plate reader (Molecular Devices, USA) using excitation at 360 nm, emission at 461 nm, and nine-point well scan option. The plate is auto-mixed for 5 sec prior to measurements. The average fluorescence signal per each well is determined, and the fluorescence readings of the cell standard wells A2-H2, A11-H11 are used to generate the linear standard curve of the average fluorescence vs. cell concentration in the well (0.1 ml). The cell concentration in the 0.1 ml of the cell suspension at the end of transfection is determined from this standard curve for each well in a conventional manner. The luciferase concentration in the cell suspensions is determined using BrightGlo® Firefly Luciferase assay kit (Promega Corp., USA). To each of the luciferase standard wells, test sample wells, and blank (cells only) wells G2-H2, G11-H11 in the same plate, 0.1 mL of the reconstituted complete Luciferase substrate buffer (prepared according to the manufacturer’s recommendations) is added, the contents of the wells are thoroughly mixed, and the luminescence of the mixtures in the wells is determined at 10-15 min after the luciferase substrate addition using SpectraMax M2 multimode plate reader (Molecular Devices, USA) in the luminescence mode 2026202200   20 Mar 2026 under the instrument default settings. The readings of the duplicate standards in the wells A1-H1 and A12-H12, after subtraction of the blank readings of the wells G2-H2 and G11-H11, are used to generate the Luciferase standard curve linearly approximated in double-log coordinates using the minimization of the mean squared relative error. The amount of luciferase formed in each well is calculated from the luminescence readings (with blank readings subtracted) using the luciferase standard curve, normalized to the number of cells per well determined using the Hoechst 33342 method above, and presented in ng of luciferase per 1 million (106) cells. It was found that for the targeted LNP and the typical assay range of post-transfection cell concentrations of (99-1251)x103 cell / ml in 0.1 ml of the cell harvesting solution ( (9.9-125.1)x103 cells / well ), observed under the above assay conditions, the Luciferase expression normalized to the final number of the cells in the well was dependent on both initial (plating) cell density and final (post-transfection) cell density, and the dependence was in good agreement with the second-degree polynomial approximation against the logarithm of the post-transfection cell concentration (R2 =0.9925). Therefore, for targeted LNPs the assayed luciferase activities in each individual well were corrected to the standard post-transfection cell concentration of 400x103 cells / ml (cell density 40x103 cells / well) using the formula: Luc = Luc(D)*100 / (91.6xlog2(D)-659.2xlog(D)+1196.4), where D is the measured post-transfection cell concentration in 103 cells / ml, Luc(D) is the measured luciferase expression at the post-transfection cell concentration D. Example 6. In vitro transfection of Ffluc-mRNA LNPs composed of the ionizable lipids formulated in the presence and absence of 5 mol% DPPS-NH4. A mRNA encoding Ffluc-mRNA was formulated separately into LNPs comprising an ICL / DSPC / Chol / PEG-DMG (52 / 10 / 36.5 / 1.5 mol%) or ICL / DSPC / DPPS-NH4 / Chol / PEG-DMG (52 / 5 / 5 / 36.5 / 1.5 mol%) formulated at an N / P ratio of 5.25. The LNPs were prepared and characterized as described in Example 2 and 3 respectively. Particle size refers to the Z-Average particle size after freezing and thawing. Zeta refers to zeta potential measurements at either pH 5 or pH 7 in units of mV. mRNA encapsulation efficiency after freezing & thawing is referred to as (E.E,%). 2026202200   20 Mar 2026 Table 9A. Physicochemical properties and characterization of LNPs containing Ffluc mRNA comparing different ICLs in a PS-targeted and non-targeted form. ICL Targeted? Z-Ave PDI %E.E Zeta pH 5 Zeta pH 7 KC3-X1-PH12 No PS 84.54 0.006 95.71 ±0.27 17.41 3.66 KC3-X2-PH12 No PS 88.77 0.036 95.78 ±0.19 17.94 2.73 KC3-X1-PH5 No PS 85.84 0.06 96.64±0.28 20.69 4.61 KC3-OA No PS 81.68 0.077 97.20±0.39 16.76 5.09 KC3-X1-PH12 5mol% PS 87.15 0.065 96.78±0.08 19.84 -1.16 KC3-X2-PH12 5mol% PS 87.65 0.017 96.69±0.33 18.83 0.17 KC3-X1-AA 5mol% PS 86.16 0.052 96.47±0.20 19.62 -1.23 KC3-X1-PH5 5mol% PS 83.27 0.048 98.22±0.13 19.24 -2.66 KC3-OA 5mol% PS 82.93 0.028 97.90±0.09 15.77 -0.89 The formulations ranged in size from approximately 82 to 93 nm in diameter The % entrapped mRNA ranged from 94 - 98% and LNPs exhibited positive surface charge at pH 5 ~ +16-21 and a negative charge at pH 7 -3 to +5 mV. The activity of the LNPs was evaluated by transfection MutuDC1940 cells in vitro at 0.3 ugmL mRNA concentration for 24h as described in Example 5. Table 9B. In vitro activity of LNPs containing Ffluc mRNA comparing different ICLs in a PS-targeted or non-targeted format. ICL Targeted? Luc (ng / million cells) T / NT KC3-X1-PH12 No PS 5.03 ±0.16 KC3-X2-PH12 No PS 8.12 ±0.55 KC3-X1-PH5 No PS 12.40±1.15 KC3-OA No PS 26.89 ±4.67 KC3-X1-PH12 5mol% PS 334.27 ±17.96 66.5 KC3-X2-PH12 5mol% PS 447.74 ±43.72 55.2 2026202200   20 Mar 2026 ICL Targeted? Luc (ng / million cells) T / NT KC3-X1-PH5 5mol% PS 199.92 ±22.25 16.1 KC3-OA 5mol% PS 76.19 ± 6.52 2.8 It was found that the rank order of activity and the activity of the PS-targeted to nontargeted ratio (highest to lowest) was KC3-X2-PH12 > KC3-X1-PH12 > KC3-X1-PH5 > KC3-OA at 0.3 ugmL. Example 7. Preparation and analysis of PS-containing LNPs containing KC3-X1-PH12 at varying mol% encapsulating the mRNA FFluc which was used to evaluate the effect of ICL content on activity in MutuDC1940 murine dendritic cells in vitro. LNPs were prepared comprising 48, 52, 54, 56, 60 or 64 mol% KC3-X1-PH12 ionizable lipid with the composition KC3-X1-PH12 / DSPC / DPPS / Chol / PEG-DMG (x / 5 / 5 / (88.5-x) / 1.5 mol%) and an N / P ratio of 4.5 encapsulating firefly luciferase mRNA (FFluc) were prepared as described in Example 2 (using a ChipShop 187 chip to mix and prepare the LNPs at a flow rate of 18 ml / min.) where x represents the mol% ionizable lipid. The LNPs were used to evaluate the effect of altering the mol% of KC3-X1-PH12 on in vitro luciferase activity in vitro. The LNPs were characterized using the method of Example 3. Particle size refers to the Z-Average particle size after freezing and thawing. Zeta refers to zeta potential measurements at either pH 5 or pH 7 in units of mV. mRNA encapsulation efficiency is referred to as (E.E,%). Table 10A. Physicochemical properties and characterization of DPPS containing KC3-X1-PH12 LNPs containing FFluc mRNA Mol% Particle Size (nm) E.E,%, Stdev Zeta pH 5 Zeta pH 7 48 101.80 87.02 ± 0.76 18.68 -1.84 52 104.20 87.42 ± 0.54 16.04 -2.36 56 109.40 88.65 ± 0.45 15.34 -3.38 60 111.70 88.76 ± 0.52 18.70 -3.34 64 119.30 87.06 ± 0.67 13.73 -4.40 2026202200   20 Mar 2026 The formulations ranged in size from approximately 104 to 112 nm in diameter. The % entrapped mRNA ranged from % and LNPs exhibited a surface charge range at pH 5 from approximately +14 to + 19 mV and a range from -4 to -2 mV at pH 7. Table 10B. Luciferase expression in MutuDC 1940 cells after exposure to LNPs for 24h at 0.3 ugmL total (encapsulated + non-encapsulated) mRNA concentration. A frozen KC3-OA LNP formulation described in Example 11 was used in the luciferase assay as a control. Mol% Luciferase activity (ng / 10A6 cells) ± stdev Luciferase Activity KC3-X1-PH12 / KC3OA 48 421.7 ± 21.8 1.07 52 506.5 ± 24.8 1.29 56 662.4 ± 44.3 1.69 60 592.2 ± 27.8 1.51 64 417.8 ± 25.5 1.06 Example 8. Preparation and analysis of LNPs containing KC3X1-PH5,KC3-X1-AA and KC3-X1-PH12 at different N / P ratios encapsulating FFluc mRNA used to evaluate activity in vitro. LNPs were prepared as described in Example 2, comprising of ICL / DSPC / DPPS-NH4 / Chol / PEG-DMG (52 / 5 / 5 / 36.5 / 1.5 mol%) at varying N / P ratios containing the reporter mRNA FFluc and evaluated in vitro for luciferase activity. LNPs were characterized with the methods of Example 3. Particle size refers to the Z-Average particle size after freezing and thawing. Zeta refers to zeta potential measurements at either pH 5 or pH 7 in units of mV. mRNA encapsulation efficiency is referred to as (E.E,%). Table 11A. Physicochemical properties and characterization of KC3-X1-PH5 and KC3-X1-AA LNPs containing FFluc mRNA at different N / P ratios. ICL N / P Particle Size (nm) E.E,%, Stdev Zeta pH 5 Zeta pH 7 KC3-X1-PH5 4.0 114.60 95.63 ± 7.04 16.12 -7.35 KC3-X1-PH5 4.25 114.60 94.76 ± 7.04 18.28 -3.93 KC3-X1-PH5 5.0 116.10 95.76 ± 7.04 21.06 -3.50 KC3-X1-PH5 5.5 114.40 95.42 ± 7.04 22.90 -2.67 KC3-X1-PH5 6.0 111.90 95.00 ± 7.04 22.09 -2.91 KC3-X1-PH5 6.5 114.50 95.61 ± 7.04 26.79 -3.46 2026202200   20 Mar 2026 Table 11B. Luciferase expression (ng / million cells ± stdev) in MutuDC 1940 cells after exposure to LNPs for 24h at 0.1 pg / mL total (encapsulated + non-encapsulated) mRNA concentration. ICL N / P Luciferase Activity KC3-X1-PH5 4.0 103.7 ± 9.9 KC3-X1-PH5 4.25 118.9 ± 4.4 KC3-X1-PH5 5.0 138.9 ± 6.0 KC3-X1-PH5 5.5 142.8 ± 6.9 KC3-X1-PH5 6.0 146.6 ± 4.8 KC3-X1-PH5 6.5 157.8 ± 10.7 Table 11C Luciferase expression (ng / million cells ± stdev) in MutuDC 1940 cells after exposure to LNPs for 24h at 0.3 pg / mL total (encapsulated + non-encapsulated) mRNA concentration. ICL N / P Luciferase Activity KC3-X1-PH5 4.0 273.3 ± 19.8 KC3-X1-PH5 4.25 284.4 ± 13.4 KC3-X1-PH5 5.0 295.3 ± 9.3 KC3-X1-PH5 5.5 287. ± 12.4 KC3-X1-PH5 6.0 302.3 ± 13.1 KC3-X1-PH5 6.5 293.5 ± 11.8 LNP formulations prepared with KC3-X1-PH5 and KC3-X1-AA demonstrated peak luciferase activity at an N / P of 6 and 4.25, respectively. All formulations demonstrated greater than 90 % encapsulation efficiency and a particle size between 107 and 119 nm, independent of the N / P ratio. Table 12A. Physicochemical properties and characterization of KC3-X1-PH12 LNPs containing FFluc mRNA at different N / P ratios. Some formulations were prepared on more than one occasion. N / P Particle Size (nm) E.E,%, Stdev Zeta pH 5 Zeta pH 7 1.0 129.00 16.07 ± 0.48 -0.08 -27.50 2026202200   20 Mar 2026 N / P Particle Size (nm) E.E,%, Stdev Zeta pH 5 Zeta pH 7 2.0 151.20 56.87± 2.77 0.29 -6.67 3.0 109.50 76.03 ± 2.39 6.80 -1.54 3.25 110.20 82.23 ± 2.17 10.95 -1.50 3.5 109.70 89.15 ± 3.14 12.50 -2.95 3.75 111.30 93.00 ± 2.93 12.99 -3.60 4.0 107.90 92.08 ± 1.99 15.52 -4.01 4.25 107.40 92.27 ± 3.66 15.22 -5.62 4.5 107.30 92.25 ± 2.68 14.96 -5.37 4.75 110.40 92.06 ± 3.88 17.68 -8.92 5.0 108.80 92.53 ± 2.02 18.66 -6.93 4.0 108.30 89.58 ± 2.13 7.347 -5.356 4.25 104.10 88.59 ± 3.19 7.788 -6.262 4.5 105.30 89.21 ± 3.33 8.238 -6.534 4.75 110.20 89.29 ± 2.45 11.34 -4.663 5.0 105.50 88.37 ± 2.67 12.34 -6.036 5.0 114.60 89.71 ± 3.51 23.56 -0.36 5.25 113.40 91.80 ± 5.23 16.56 -2.33 5.50 108.60 89.57 ± 2.81 17.31 -2.29 5.75 109.30 90.33 ± 1.74 24.24 -1.67 6.00 110.40 91.54 ± 4.31 19.47 -3.63 Table 12B. Luciferase expression (ng / million cells ± stdev) of KC3-X1-PH12 LNPs in MutuDC 1940 cells after exposure to LNPs for 24h at 0.3 ugmL total (encapsulated + nonencapsulated) mRNA concentration. N / P Luc activity Rel. Luc. Activity 1.0 0.90 ± 0.13 1.00 2.0 1.89 ± 1.33 2.10 3.0 139.52 ± 5.09 155.02 3.25 252.30 ± 11.47 280.33 3.5 357.14 ± 11.79 396.82 3.75 439.23 ± 27.62 488.03 4.0 435.03 ± 38.70 483.37 4.25 441.10 ± 38.06 490.11 4.5 469.42 ± 37.90 521.58 4.75 400.85 ± 20.80 445.39 5.0 400.11 ± 36.32 444.57 4.0 536.19 ± 25.21 595.77 4.25 494.61 ± 42.91 549.57 2026202200   20 Mar 2026 N / P Luc activity Rel. Luc. Activity 4.5 471.19 ± 63.12 523.54 4.75 476.97 ± 60.50 529.97 5.0 498.15 ± 50.44 553.50 5.0 457.75 ± 28.64 508.61 5.25 429.82 ± 32.22 477.58 5.50 390.98 ± 19.98 434.42 5.75 379.55 ± 29.30 421.72 6.00 372.24 ± 26.92 413.60 LNP formulations prepared with KC3-X1-PH 12 demonstrated a peak luciferase activity at an N / P of 4.5 - 5.0 with activity dropping off rapidly below N / P 3.5, which also corresponded to increases in particle size and poly dispersity and a decrease in encapsulation efficiency. Example 10. Comparing the immunogenicity of a quadrivalent mRNA vaccine in mice using KC3-X1-PH12 LNPs and KC3-X2-PH12 LNPs composed with different ICL mol% and N / P ratios. Four mRNAs encoding influenza A subtypes H1 (SEQ ID NO: 13 encoding SEQ ID NO: 12), H3 (SEQ ID NO: 15 encoding SEQ ID NO: 14), influenza B / Victoria (SEQ ID NO: 17 encoding SEQ ID NO: 16) and RSVpreF (SEQ ID NOs: 10 encoding SEQ ID NO: 9) mRNAs were combined at equal weight ratios and encapsulated into LNPs comprising of KC3-X1-PH12 / DSPC / DPPS-NH4 / Chol / PEG-DMG where the ICL mol% was 52 or 56 mol%, DSPC and DPPS-NH4 were present at 5 mol% each, PEG-DMG was 1.5 mol% and the remainder lipid was cholesterol. Similar LNPs were prepared with 52 or 56 mol% KC3-X2-PH12 and N / P ratios of 4.5 or 5.25. KC3-X1-PH12 was also used at 52 or 56 mol% and an N / P ratio of 5.25 to encapsulate a single influenza subtype H5 mRNA (SEQ ID NO: 34). The LNPs were prepared as in Example 2 and analyzed as in Example 3. An outline of the composition for formulation and a panel of physicochemical characteristics are shown below in Table 14A. Additionally, a portion of the quadrivalent mRNA-LNPs made with 52 mol% KC3-X1-PH12 and 56 mol% KC3-X1-PH12 were lyophilized. The physicochemical characteristics of these mRNA-LNPs after reconstitution are shown below in Table 14B. LNPs were characterized as described in Example 3. Particle size refers to the Z-Average particle size after freezing and at an N / P ratio. Zeta refers to zeta potential measurements at either pH 5 or pH 7 in units of mV. 2026202200   20 Mar 2026 mRNA encapsulation efficiency after freezing & thawing is referred to as (E.E,%), PDI, polydispersity index. Table 14A. The physicochemical details of the KC3-X1-PH12 or KC3-X2-PH12 based LNP formulations. Group # ICL N / P mol % Z-Ave PDI %EE ± stdev Zeta pH 5 Zeta pH 7 1-4 KC3-X1-PH12 5.25 52 111.1 0.054 88.5 ± 0.25 16.76 0.47 5-8 KC3-X1-PH12 5.25 56 116.1 0.047 88.4 ± 0.29 19.70 0.12 9-10 KC3-X2-PH12 4.25 52 116.8 0.056 85.3 ± 1.01 9.13 0.30 11-12 KC3-X2-PH12 5.25 56 106.1 0.048 93.2 ± 0.75 15.49 0.62 13-14 KC3-X2-PH12 5.25 52 97.66 0.051 94.7 ± 0.29 15.20 -0.61 15-16 KC3-X1-PH12 5.25 52 114.1 0.044 88.2 ± 0.13 20.43 0.68 17-18 KC3-X1-PH12 5.25 56 108.5 0.057 89.1 ± 0.43 13.16 0.72 The formulations ranged in size from approximately 97 to 117 nm in diameter. The % entrapped mRNA ranged from 85 - 94% and LNPs exhibited positive surface charge at pH 5 of approximately ~ +9 to +20 mV and a charge at pH 7 -1 to +1 mV. Table 14B. Particle size (Z-ave), polydispersity (PDI) and mRNA encapsulation efficiency (%EE) of LNPs that were lyophilized and reconstituted (Post Lyo / R), made with either 52 or 56mol% KC3-X1-PH12. Compare post lyophilization and reconstitution characteristics shown for Groups 3-4 to pre lyophilization characteristics shown for Groups 1-4 in Table 14A. Likewise, compare post lyophilization and reconstitution characteristics shown for Groups 7-8 to pre lyophilization characteristics shown for Groups 5-8 in Table 14A. P ost Lyo / R Group # ICL N / P mol % Z-ave PDI %EE 3-4 KC3-X1-PH12 5.25 52 136.40 0.08 78.49 ± 1.07 7-8 KC3-X1-PH12 5.25 56 140.20 0.02 75.46 ± 1.60 After lyophilization, it was found that the particle size increased by about 25 nm for each, and the %EE was reduced by about 10-12% points. In vivo immunogenicity in mice. To measure the ability of these mRNA-LNPs to induce antibodies, BALB / c mice were immunized at a high and 3-fold lower dose in the caudal thigh in a volume of 50 uL. The 2026202200   20 Mar 2026 experimental design and dosing schema is shown in Figure 6A. Twenty-one days post vaccination, serum was collected and stored at -80°C until analysis. Mouse IgG endpoint antibody titers against the matching H1, H3, B-Victora, RSVpreF or H5 antigens were determined by standard ELISA. For H1-specific Ab titers, we found for KC3-X1-PH12 that immunogenicity of freeze-dried material was not deleterious for biological activity (Figure 6B and Figure 6C). This was true for 52 and 56 mol% ICL with N / P = 5.25, indicating that lyophilization may be an option for longer-term storage. Second, antibody titers were comparable between LNPs comprised of KC3-X1-PH12 and KC3-X2-PH12, showing that either orientation of the biodegradable ester bond can be used for PH12 with a KC3 headgroup. Third, we found for KC3-X1-PH12 that either 52 or 56 mol% with N / P = 5.25 elicited similar immune responses; the same was true for X2-PH12. For H3-specific Ab titers (Figure 6D and Figure 6E), results were similar to H1 titers: lyophilization of the mRNA-LPN was not deleterious to biological activity (H3 titers elicited by lyophilized material trended higher than for nonlyophilized, X1-PH12 and X2-PH12 were comparably immunogenic, and these ICLs can be used with either 52 or 56 mol% without impacting antibody titers. Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is therefore not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments. While specific embodiments of the subject disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this specification. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. All publications, patents and patent applications referenced in this specification are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application were specifically indicated to be so incorporated by reference.

Claims

1. A compound of Formula (I):(I)or a pharmaceutically acceptable salt thereof, whereinR1 and R2 are each independently C1-4 alkyl optionally substituted with hydroxyl;Z is *-(CH2)n-**, wherein * is a covalent bond to the 1,3-dioxolane ring and ** is a covalent bond to -NR1R2, and n is 2, 3 or 4;-Xi- and -X2- are the same of different and are each independently -(CH2)(X)-, wherein (x) is 7-10;-A1- and -A2- are the same or different and are each independently ester groups;-Y1 and -Y2 comprise one or more branched C5 hydrocarbon monomer units having a total of (y) carbon atoms each, wherein (y) is 10-17; andthe sum of (x)+(y) is 20-25.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein -Y1-and -Y2- each comprise a total of one, two or three isopentane (IPN) or 2-methylbut-2-ene (2MB) branched C5 hydrocarbon monomer units.

3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein -Y1-and -Y2- each identical.

4. The compound of claim 3, wherein -Xi- and -X2- are each independently -(CH2)io -.

5. The compound of claim 4, wherein n is 3.

6. The compound of claim 5, wherein R1 and R2 are each methyl.

7. The compound of claim 6, wherein A1 and A2 are both *-O-C(O)-**.2026202200   20 Mar 20268. The compound of claim 7, wherein the compound is KC3-X1-PH12:KC3-X1-PH12.

9. The compound of claim 6, wherein A1 and A2 are both *-C(O)-O-**.

10. The compound of claim 9, wherein the compound is KC3-X2-PH12:KC3-X2-PH12.

11. A lipid nanoparticle (LNP) composition comprising a mRNA nucleic acid encapsulated in lipidic nanoparticles formed from the compound of any one of claims 1-10, at a N / P ratio of 3.5 to 8 relative to the mRNA nucleic acid in a total amount of 40-65 mol% of the total lipid content of the LNP composition; a sterol in a total amount of 25-45 mol% of the total lipid content of the LNP composition; one or more phospholipids in a total amount of phospholipids of 5-25 mol% of the total lipid content of the LNP composition; and a PEG-containing conjugated lipid in a total amount of 0.5-5.0 mol% of the total lipid content of the LNP composition.2026202200   20 Mar 202612. The composition of claim 11, wherein the LNP composition is characterized by one or more of the following:a. two or more phospholipids comprise a total of 5 mol% of a phosphatidyl-L-serine (PS);b. the composition comprises the conjugated lipid in a total amount of 0.5-2.5 mol% of the total lipid content of the LNP composition;c. the two or more phospholipids consist of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dipalmitoylphosphatidyl-L-serine (DPPS), distearoylphosphatidyl-L-serine (DSPS), hydrogenated soy phosphatidylcholine (HSPC), or sphingomyelin (SM); andd. the PEG-containing conjugated lipid in the LNP is PEG(2000)-dimyristoylglycerol (PEG-DMG) or PEG(2000)-dilauroylglycerol (PEG-DLG).

13. The composition of claim 11, wherein the LNP composition is characterized by the following:a. two or more phospholipids comprise a total of 5 mol% of a phosphatidyl-L-serine (PS);b. the two or more phospholipids consist of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and dipalmitoylphosphatidyl-L-serine (DPPS) or distearoylphosphatidyl-L-serine (DSPS); andc. the composition comprises the conjugated lipid in a total amount of 0.5-2.5 mol% of the total lipid content of the LNP composition; andd. the PEG-containing conjugated lipid in the LNP is PEG(2000)-dimyristoylglycerol (PEG-DMG) or PEG(2000)-dilauroylglycerol (PEG-DLG).

14. The composition of claim 13, wherein the mRNA encodes: one or more antigens for RSV, one or more antigens for influenza IAV or IBV, an antigen present in HA, HA stabilized stem (Group 1 and 2), NA, M2, stabilized H5; one or more antigens for Mtb selected from the group consisting of: one or more individual Mtb proteins and / or a synthetic fusion protein, or one or more antigens for malaria selected from the group consisting of: circumsporozoite protein (CPS), RTS,S, R21, CSP-HBsAg fusion (hepatitis B surface Ag); Pfs25; PfGARP; CelTOS, RH5.1, and RH5.2.2026202200   20 Mar 202615. The lipid nanoparticle (LNP) composition of claim 11, comprising a mRNA nucleic acid encapsulated in lipidic nanoparticles consisting of:a. a KC3-X1-PH12 ionizable cationic lipidKC3-X1-PH12, at a N / P ratio of 5.25 relative to the mRNA nucleic acid in a total amount of 52-56 mol% of the total lipid content of the LNP composition;b.  1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a total amount ofphospholipids of 5mol% of the total lipid content of the LNP composition;c. dipalmitoylphosphatidyl-L-serine (DPPS) in a total amount of phospholipids of 5 mol% of the total lipid content of the LNP composition;d. PEG(2000)-dimyristoylglycerol (PEG-DMG) in a total amount of 1.5 mol% of the total lipid content of the LNP composition; ande. cholesterol in a total amount of 32.5 - 36.5 mol% of the total lipid content of the LNP composition.

16. The LNP composition of claim 15, wherein the composition is a vaccine and the mRNA encodes a vaccine antigen.

17. The LNP composition of claim 16, wherein the mRNA encodes an influenza antigen.

18. The LNP composition of claim 17, wherein the mRNA encodes multiple influenza antigens.

19. The LNP composition of claim 15, wherein the mRNA encodes a RSV antigen.

20. A lipid nanoparticle (LNP) vaccine composition, comprising a mRNA nucleic acid encoding one or more immunogenic proteins, encapsulated in lipidic nanoparticles consisting of:2026202200   20 Mar 2026a. an ionizable cationic lipid of Formula (I) at a N / P ratio of 3.25 - 8 relative to the mRNA nucleic acid,(I)or a pharmaceutically acceptable salt thereof, whereinR1 and R2 are each independently C1-4 alkyl optionally substituted with hydroxyl;Z is *-(CH2)n-**, wherein * is a covalent bond to the 1,3-dioxolane ring and ** is a covalent bond to -NR1R2, and n is 2, 3 or 4;-X1- and -X2- are the same of different and are each independently -(CH2)(x)-, wherein (x) is 7-10;-A1- and -A2- are the same or different and are each independently ester groups;-Y1 and -Y2 comprise one or more branched C5 hydrocarbon monomer units having a total of (y) carbon atoms each, wherein (y) is 10-17;wherein -Y1- and -Y2- each comprise a total of one, two or threeisopentane (IPN) or 2-methylbut-2-ene (2MB) branched C5 hydrocarbon monomer units; andthe sum of (x)+(y) is 20-25;b. a phosphocholine phospholipid in a total amount of phospholipids of (10-X) mol% of the total lipid content of the LNP composition;c. an anionic phospholipid in a total amount of phospholipids of X mol% of the total lipid content of the LNP composition, wherein X is 2.5-7.5;2026202200   20 Mar 2026d. a PEG-containing conjugated lipid in a total amount of 1.5-4.0 mol% of the total lipid content of the LNP composition; ande. cholesterol, in a molar ratio of the ionizable cationic lipid to the cholesterol of from 65:35 to 45:55.

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