Lipid, lipid nanoparticle including the same and use of lipid nanoparticle

Abstract

A lipid is represented by Formula (I), wherein each of the substituents in Formula (I) is given the definition as set forth in the Specification and Claims. A lipid nanoparticle including the lipid, a helper lipid, a steroid, a PEGylated lipid, a branched-chain ionizable cationic lipidoid with five hydroxyl groups, and a nucleic acid is also provided. A method for delivering a nucleic acid to a subject using a pharmaceutical composition including the lipid nanoparticle, and a pharmaceutical composition for delivering a nucleic acid to a subject are also provided.

Description

LIPID, LIPID NANOPARTICLE INCLUDING THE SAME AND USE OF LIPID NANOPARTICLECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 739,091, filed on December 26, 2024, the entire disclosure of which is incorporated by reference herein.FIELD

[0002] The disclosure relates to a lipid and a lipid nanoparticle including the lipid. The disclosure also relates to a method for delivering a nucleic acid to a subject using the lipid nanoparticle.BACKGROUND

[0003] Gene therapy and genetic vaccination are among the most promising and rapidly advancing techniques in the field of modern medicine. These approaches offer highly specific and individualized therapeutic options for a wide range of diseases. At the core of this concept, cellular machinery utilizes mRNA as a transient carrier of information to synthesize genetically encoded proteins. From a theoretical perspective, mRNA could potentially replace DNA or recombinantproteins for therapeutic applications. For instance, RNA interference (RNAi) agents, such as small interfering RNA (siRNA) and microRNA (miRNA), show great promise as therapeutic tools for treating various conditions, including cancers, infections, autoimmune disorders, and neurological diseases linked to undesirable gene expression.

[0004] To enable systemic delivery of the aforesaid RNA macromolecules, which are poorly permeable and easily degraded, a safe and effective delivery platform is essential. Nanocarriers, which are made from lipids and / or phospholipids (e.g., liposomes, lipid nanoparticles, and lipid nanoemulsions), are frequently used for RNA delivery due to their high biocompatibility, biodegradability, and established clinical history.

[0005] Genetic vaccination aims to elicit an immune response to selected antigens, such as bacterial surface components, viral particles, or tumor antigens. Genetic vaccines typically consist of genetically engineered nucleic acid molecules that encode peptide or protein fragments (antigens) representative of a pathogen or tumor antigen. These genetic vaccines, upon administration, are taken up by target cells, where the encoded proteins are produced. If these proteins are recognized as foreign substances by the patient's immune system, an immuneresponse is triggered.

[0006] Both DNA and RNA are nucleic acid molecules which can be used for genetic vaccination. DNA-based vaccines are relatively stable and easy to handle but carry the risk of undesired genomic integration, potentially causing mutagenic events, such as the loss of genetic function. Additionally, the generation of anti-DNA antibodies is a possible side effect. Another limitation of DNA-based vaccines is the restricted expression of the encoded peptide or protein, as the DNA must enter the nucleus for transcription before the resultant mRNA can be translated. The level of RNA transcription depends on the presence of specific transcription factors, which, if absent, will limit the amount of RNA produced.

[0007] Instead of DNA, use of RNA for gene therapy and genetic vaccination minimizes the risk of genomic integration and the formation of anti-DNA antibodies. However, RNA is inherently unstable and prone to degradation by ubiquitous ribonucleases. Therefore, there is a need for efficient mRNA delivery systems that can ensure the stability of the antigen, prevent early degradation, and facilitate efficient translation within cells. Additionally, reducing the required mRNA vaccine dose is important for addressing safety concerns and making vaccines more accessible, particularly in developing countries.

[0008] One of the key challenges in nucleic acid delivery is ensuring that the therapeutic molecules reach the correct cellular site and exert the desired biological effect. Despite the vast potential of nucleic acid-based therapeutics, there are significant obstacles to their effective delivery, including susceptibility to nuclease degradation in plasma, limited intracellular access, and poor release of internalized oligonucleotides into the cytoplasm.

[0009] In view of the aforesaid, there is still a need for developing improved ionizable lipids and lipid nanoparticles which allow effective and safe delivery of nucleic acids.SUMMARY

[0010] Therefore, in a first aspect, the present disclosure provides a lipid which can alleviate at least one of the drawbacks of the prior art. The lipid is represented by Formula (I),R^* ^^ -O~~L3”~Y”~L4 — Z ^N— L-|— X— L2-N^TR20) |nFormula (I), pyrrolidine has two oxygen substitutions respectively at positions 3 and 4 in a cis or trans configuration, each of R1and R2is independently a Ci-Ce alkyl, or R1and R2collectively form a cycloalkyl ring or a heterocycloalkyl ring, Liis a Ci-Ce linear alkyl, L2is a C2-C6linear alkyl, L3is a C2-C6linear alkyl, L4is a C0-C6linear alkyl, X is an ester (-C(=O)-O- or O-C(=O)-), Y is an ester (-C(=O)-O-or O-C(=O)-), and Z is selected from the group consisting of -CHR3R4, -(alkyne)-RS, -C=C-R6, -C=C-CH2-C=C-R7, and -C=C-CH2-C=C-CH2-C=C-R8, where each of R3, R4, R5, R6, R7, and R8is independently a C5-C10 linear alkyl.

[0011] In a second aspect, the present disclosure provides a lipid nanoparticle, which can alleviate at least one of the drawbacks of the prior art. The lipid nanoparticle includes the aforesaid lipid, a helper lipid, a steroid, a polyethylene glycol-conjugated (PEGylated) lipid, and a branched-chain ionizable cationic lipidoid with five hydroxyl groups.

[0012] In a third aspect, the present disclosure provides a pharmaceutical composition for delivering a nucleic acid to a subject, which can alleviate at least one of the drawbacks of the prior art. The pharmaceutical composition includes the aforesaid lipid nanoparticle which further includes the nucleic acid that is encapsulated within the lipid nanoparticle.

[0013] In a fourth aspect, the present disclosure provides a method for delivering a nucleic acid to a subject, which can alleviate at least one of the drawbacks of the prior art, and which includes administering to the subject in needthereof the aforesaid pharmaceutical composition.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Other features and advantages of the disclosure will become apparent in the following detailed description of the embodiment(s) with reference to the accompanying drawings. It is noted that various features may not be drawn to scale.

[0015] FIG. 1 shows the hydrodynamic size, the polydispersity index (PDI), and the encapsulation efficiency determined in each of the messenger ribonucleic acid-containing lipid nanoparticles (mRNA-LNPs) of Examples 1 to 21 and Comparative Example 1 (EX1 to EX21 and CE1) as described in sections A and C of “Characterization of mRNA-LNP or lipid in mRNA-LNP,” infra.

[0016] FIG. 2 shows the acid dissociation constant (pKa) determined in each of the lipids of EX1 to EX21 and D-Lin-MC3-DMA (an ionizable cationic lipid for CE1) and the zeta potential determined in each of the mRNA-LNPs of EX1 to EX21 and CE1 as described in sections B and D of “Characterization of mRNA-LNP or lipid in mRNA-LNP,” infra.

[0017] FIG. 3 shows the corrected mean EGFP fluorescence intensity determined in each group as described in section A of " Property evaluation,” infra.DETAILED DESCRIPTION

[0018] It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art.

[0019] For the purpose of this specification, it will be clearly understood that the word “comprising" means “including but not limited to", and that the word “comprises” has a corresponding meaning.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the present disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. Indeed, the present disclosure is in no way limited to the methods and materials described.

[0021] As used herein, the term “3,4-oxygen substituted pyrrolidine isomer” refers to an isomer having a pyrrolidine ring which is a saturated five-membered ring containing one nitrogen atom. The 3,4-oxygen substituted pyrrolidine isomer has two oxygen substitutions respectively at positions 3 and 4 that can be either on the same side (i.e., in a cis configuration) or on opposite sides (i.e., in a transconfiguration) of the ring plane.

[0022] As used herein, the term “c / s-3,4-substituted pyrrolidine" refers to both oxygen substituents (e.g., -OH) point in the same direction (both up or both down) relative to the mean plane of the ring in the c / s-isomer, and can be represented as (3R, 4S)-pyrrolidine derivative or (3S, 4R)-pyrrolidine derivative (i.e., the enantiomer of the 3R and 4S isomer).

[0023] As used herein, the term “trans-3, 4-substituted pyrrolidine” refers to the oxygen substituents point in opposite directions (one up and one down) relative to the mean plane of the ring in the ans-isomer, and can be represented as (3R, 4R)-pyrrolidine derivative or (3S, 4S)-pyrrolidine derivative (i.e., the enantiomer of the 3R, 4R isomer).

[0024] The aforesaid two pairs of enantiomers (i.e., 3R, 4S / 3S, 4R and 3R, 4R / 3S, 4S) represent the maximum number of stereoisomers for a 3,4-disubstituted pyrrolidine with two different chiral centers.

[0025] As used herein, the term “alkyl" refers to a saturated, straight- or branched-chain hydrocarbon radical composed only of carbon and hydrogen.

[0026] As used herein, the term “cycloalkyl” refers to a saturated monocyclic or polycyclic hydrocarbon ring system which typically contains 3 to 10carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0027] As used herein, the term “heterocycloalkyl” refers to a saturated monocyclic, bicyclic, or spiro ring system containing 3 to 10 ring atoms, where at least one ring atom is a heteroatom such as nitrogen, oxygen, or sulfur, with the remainder being carbon.

[0028] The present disclosure provides a lipid which is represented byR^ O~~L3’~~Y’~~L4“~~ZZN— L-(— X — L2-NQTFormula (I),R2L3”Y~~L4~“Z (|).

[0029] In Formula (I), pyrrolidine has two oxygen substitutions respectively at positions 3 and 4 in a cis or trans configuration. Each of R1and R2is independently a Ci-Ce alkyl, or R1and R2collectively form a cycloalkyl ring or a heterocycloalkyl ring. Li is a Ci-Cs linear alkyl. L2 is a C2-C6 linear alkyl. L3 is a C2-Cs linear alkyl. La is a Co-Ce linear alkyl. X is an ester (-C(=O)-O- or O-C(=O)-). Y is an ester (-C(=O)-O- or O-C(=O)-). Z is selected from the group consisting of -CHR3R4, -(alkyne)-R5, -C=C-R6, -C=C-CH2-C=C-R7, and -C=C-CH2-C=C-CH2-C=C-R8, where each of R3, R4, R5, R6, R7, and R8is independently a C5-C10 linear alkyl.R^ZN-|- p2

[0030] According to the present disclosure,in Formula (I) isselected from the group consisting of?

[0031] According to the present disclosure, Z in Formula (I) is selected

[0032] According to the present disclosure, the lipid may be selected from the group consisting of: (1) (((3R,4S)-1-(2-((4-(dimethylamino)butanoyl)oxy) ethyl)pyrrolidine-3.4-diyl)bis(oxy))bis(hexane-6.1 -diyl)bis(2-hexyldecanoate); (2) (((3R,4S)-1-(3-(3-(dimethylamino)propoxy)-3-oxopropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(2-hexyldecanoate); (3) (((3R,4S)-1 -(4-(3- (dimethylamino)propoxy)-4-oxobutyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl) bis(2-hexyldecanoate); (4) di(heptadecan-9-yl) 6,6'-(((3S,4R)-1 -(3-((4-(dimethylamino)butanoyl)oxy)propyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate; (5) (((3R,4S)-1-(3-((4-(diethylamino)biitanoyl)oxy)propyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1 -diyl)bis(2-hexyldecanoate); (6) (((3R,4S)-1-(3-((4-(pyrrolidin-1 -yl)butanoyl)oxy)propyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6, 1 -diyl) bis(2-hexyldecanoate); (7) di(heptadecan-9-yl) 6,6'-(((3S,4R)-1 -(3-(2-(dimethylamino)acetoxy)propyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate; (8) 5-(((3S,4S)-1-(3-((4-(dimethylamino)butanoyl)oxy)propyl)-4-((5-(((8Z,11Z)-octadeca-8,11-dienoyl)oxy)pentyl)oxy)pyrrolidin-3-yl)oxy)pentyl (7Z,10Z)-octadeca-7,10-dienoate; (9) di(dec-3-yn-1 -yl) 6,6'-(((3S,4R)-1-(3-((4-(dimethylamino)butanoyl)oxy)propyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate; (10) di(heptadecan-9-yl) 5,5'-(((3S,4S)-1-(2-((4-(dimethylamino)butanoyl)oxy) ethyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate; (11) di((9Z, 12Z)-octadeca-9, 12-dien-1-yl) 6,6'-(((3S,4R)-1-(2-((4-(dimethylamino)butanoyl)oxy)ethyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate; (12) (((3R,4S)-1-(2-((4-(dimethylamino)butanoyl) oxy)ethyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5, 1-diyl)(9Z,9'Z, 12Z,12'Z)-bis(octadeca-9,12-dienoate); (13) di(dec-3-yn-1 -yl) 6,6'-(((3S,4R)-1-(2-((3- (dimethylamino)propanoyl)oxy)ethyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate;(14) di(dec-3-yn-1 -yl) 6,6’-(((3S,4R)-1-(3-((3-(dimethylamino)propanoyl)oxy) propyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate; (15) di(dec-3-yn-1 -yl) 6,6'-(((3S,4R)-1-(2-((4-(dimethylamino)butanoyl)oxy)ethyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate; (16) di(dec-3-yn-1 -yl) 5,5’-(((3S,4S)-1 -(3-((3-(dimethyla ino)propanoyl)oxy)propyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate; (17) di(non-2-yn-1 -yl) 6,6'-(((3R,4S)-1-(3-((3-(dimethylamino)propanoyl)oxy) propyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate; (18) (((3R,4R)-1-(3-((3-(dimethylamino)propanoyl)oxy)propyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl) (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate); (19) di(non-3-yn-1 -yl) 6,6'-(((3S,4R)-1-(3-((3-(dimethylamino)propanoyl)oxy)propyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate; (20) (((3R,4S)-1-(2-((3-(dimethylamino)propanoyl) oxy)ethyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5, 1 -diyl) (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate); and (21) di(dec-3-yn-1-yl) 6,6'-(((3S,4R)-1-(3-((3-(diethylamino)propanoyl)oxy)propyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate.

[0033] According to the present disclosure, a protonated form of the lipid has an acid dissociation constant (pKa) ranging from 5.0 to 7.0.

[0034] The present disclosure also provides a lipid nanoparticle (LNP), which includes the aforesaid lipid, a helper lipid, a steroid, a polyethylene glycol-conjugated (PEGylated) lipid, and a branched-chain ionizable cationic lipidoid with five hydroxyl groups.

[0035] In certain embodiments, the helper lipid may be selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and combinations thereof. The steroid may be selected from the group consisting of campesterol, cholesterol, beta-sitosterol (^-sitosterol), stigmasterol, ergosterol, lanosterol, and combinations thereof. The PEGylated lipid may be selected from the group consisting of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol-2000)] (DSPE-PEG2000), GalNAc PEG lipid (e.g, DSPE-PEG2000-triGalNAc), and combinations thereof. The branched-chain ionizable cationic lipidoid with five hydroxyl groups may be C12-200.

[0036] According to the present disclosure, the LNP has a hydrodynamic size ranging from 83 nm to 140 nm.

[0037] According to the present disclosure, the LNP has a polydispersity index (PDI) ranging from 0.02 to 0.22.

[0038] According to the present disclosure, the LNP has a zeta potential ranging from -6.5 mV to 9 mV.

[0039] According to the present disclosure, the LNP has an encapsulation efficiency ranging from 93.5 % to 99.2%.

[0040] According to the present disclosure, the LNP further includes a nucleic acid that is encapsulated within the lipid nanoparticle.

[0041] According to the present disclosure, the nucleic acid may be selected from the group consisting of non-replicating mRNA, self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), microRNA (miRNA), and combinations thereof.

[0042] According to the present disclosure, the lipid nanoparticle can protect the nucleic acid from degradation, facilitate cellular uptake, and release therapeutic levels of the nucleic acid into the cytoplasm.

[0043] The present disclosure also provides a pharmaceutical composition for delivering a nucleic acid to a subject, which includes the aforesaid LNP that includes the nucleic acid encapsulated within the LNP.

[0044] According to the present disclosure, the pharmaceutical composition may be formulated into a parenteral dosage form using technologywell-known to those skilled in the art. To be specific, the pharmaceutical composition may be formulated into an injection (e.g., a sterile aqueous solution or a dispersion) and may be administered via one of the following parenteral routes: intravenous injection, intramuscular injection, and subcutaneous injection.

[0045] According to the present disclosure, the dose and frequency of administration of the pharmaceutical composition may vary depending on the following factors: the severity of the illness or disorder to be treated, routes of administration, and age, physical condition and response of the subject to be treated. The selection for the dose and frequency of administration are within the expertise and routine skills of those skilled in the art.

[0046] The present disclosure also provides a method for delivering a nucleic acid to a subject, which includes administering to the subject in need thereof the aforesaid pharmaceutical composition.

[0047] According to the present disclosure, the lipid, when incorporated into the LNP, can exhibit the efficacy of optimizing the drug-to-lipid ratio, and can protect the nucleic acid from degradation and clearance in serum, and thus achieves the goal of systemic and local delivery of the nucleic acid. In addition, the LNP facilitates intracellular delivery of the nucleic acid and is well-tolerated, andhence can provide an adequate therapeutic index. As a result, when a patient is treated with the LNP containing an effective dose of the nucleic acid, the risk of toxicity or undesirable side effects can be reduced.

[0048] The disclosure will be further described by way of the following examples. However, it should be understood that the following examples are solely intended for the purpose of illustration and should not be construed as limiting the disclosure in practice.EXAMPLESPreparation of lipid:Examples 1 to 21 (EX1 to EX21)

[0049] The lipids of EX1 to EX21 were synthesized by synthetic route A or synthetic route B as depicted in Schemes 1 and 2, respectively.

[0050] Scheme 1(Step 1} {Step 2} (Step 3) Br— Lj-CN / ^.0-1,-CN BocN KOH - *>• BocN TBAB, NaOH(act)V'A,O-L3-CN EtOH, H2O U / T) (Step 4) (Step 5} R? 1. Z-LrOH, EDCI. DMAP, CH2CI2, N — L-4 — X — Lg— 8f (5) 2. TFA, CH2CI2K2CO3, OMFR^ N-M-X-UrN J R* ’O-L3J3-U-Z (6)

[0051] Scheme 2 (Step 1) (Step 2) (Step 3)Br — Lj — OBn ’Li— O Bn ^^. O-Lj-OH - >. Pd / C, H2BocN Y BocN / J TBAB. NaOH(aq)O~La — OBn EtOH, EA (1 / r; (7) (a) O-Lj-OH (Step 4) R1(Step s) o 4,cn^, Y— Lf-~~X 1. HO L4-Z, EDCI,nDMMAA DP, CHJCIJ EJXR(5) - 2. TFA, CH2CI2K2COS, DMFJI.a-Lj-o^u-z N-LrX-L24l QT ^O-Ls-O U-Z(“) Y ynthetic route A

[0052] The synthetic route A includes the following steps 1 to 5.

[0053] < Step 1: Preparation of 3,4-dihydroxy-pyrrolidine-1 -carboxylic acidtert-butyl ester in cis (1) or trans (1’) configuration(1) Preparation of c / s-3,4-dihydroxy-pyrrolidine-1 -carboxylic acid tert-butyl ester (1)

[0054] First, osmium tetroxide (4.46 mL of a 4 % aqueous solution) was added to a solution of N-methylmorpholine-N-oxide (NMO) in water (50 mL) and acetone (24 mL). Nest, the resultant solution was allowed to stir for 30 minutes after which a solution of 2,5-dihydro-pyrrole-1 -carboxylic acid tert-butyl ester (19.8 g, 117.0 mmol) in acetone (24 mL) was added over 2 hours. Afterwards, the resultant reaction mixture was allowed to stir for 20 hours after which sodium bisulfite (5 g) was added in one portion. The resultant suspension was then stirred for 15 minutes, and subsequently filtered and concentrated under vacuum to remove the acetone. Thereafter, the resultant aqueous solution was adjusted to pH 2 by the addition of sulfuric acid and was subsequently extracted using ethyl acetate (EtOAc, 50 mL each time) three times, followed by collecting the resultant organic phases (i.e., a combined organic phase) from the three extractions. Subsequently, the combined organic phase was washed with brine, dried over MgSC, filtered, and concentrated under vacuum in sequence, so as to obtain 24.8 g (quantitative) of c / 's-3,4-di hyd roxy-py rrol id in e- 1 -carboxylic acid tert-butyl ester (1 ) which was present in a light-yellow oil form.(2) Preparation of trans-3,4-dihydroxy-pyrrolidine-1 -carboxylic acid ferf-butyl ester (1!)

[0055] First, a solution of 6-oxa-3-aza-bicyclo[3.1.0]hexane-3-carboxylic acid tert-butyl ester (obtained commercially, 1.63 g, 8.80 mmol) in dioxane (8 ml) was added 2 M NaOH (30 mL), followed by stirring at 95°C for 24 hours. Nest, the resultant mixture was evaporated and extracted with EtOAc (10 mL each time) three times, followed by collecting the resultant organic phases (i.e., a combined organic phase) from the three extractions. Subsequently, the combined organic phase was washed with brine, dried over MgSO4, filtered, and concentrated under vacuum in sequence. Afterward, the resultant crude product was triturated with EtOAc, filtered, and washed with EtOAc in sequence, so as to obtain frans-3,4-dihydroxy-pyrrolidine-1 -carboxylic acid ferf-butyl ester (1’) which was present in a light-yellow solid form.

[0056] < Step 2: Alkylation of 3,4-dihydroxy-pyrrolidine-1 -carboxylic acid ferf-butyl ester in cis (1) or trans (1’) configuration >

[0057] First, the 3, 4-dihydroxy-pyrrolidine-1 -carboxylic acid ferf-butyl ester in cis (1) or trans (1’) configuration (1 equiv.) obtained in step 1, bromoalkyl nitrile (2.5-3.0 equiv.), and tetrabutylammonium bromide (TBAB, 0.05-0.1 equiv.) wereadded to a solution containing toluene or dichloromethane (CH2CI2) and 50% aqueous sodium hydroxide (NaOH). Next, the resultant mixture was stirred vigorously at a temperature ranging from a room temperature to 50°C for 4-24 hours until completion by thin layer chromatography (TLC). Afterwards, the resultant mixture was quenched with water, followed by separating the organic layer, washing with brine, drying over magnesium sulfate (MgSO4), and concentrating in sequence. Subsequently, the result crude ether was purified by column chromatography, so as to obtain a first intermediate (2) which was present in a light-yellow oil form.

[0058] < Step 3: Nitrile hydrolysis>

[0059] First, a solution of the nitrile (2) (i.e., the first intermediate obtained in step 2, 1 equiv.) in ethanol (10 volumes) was added to 10% aqueous potassium hydroxide (KOH, 2-5 volumes, 2-3 equiv.), followed by refluxing or heating to 80°C-100°C. Next, the resultant solvent was monitored by TLC until completion (4-16 hours), followed by cooling, and evaporating in sequence. Subsequently, the resultant residue was diluted with water, and then washed with an organic solvent (e.g., ether) to remove impurities, followed by acidifying the aqueous layer therein with concentrated hydrochloric acid (HCI) to reach a pH of 1-2, extracting with asolvent (e.g., dichloromethane), drying over anhydrous sodium sulfate (Na2SO4), and concentrating under reduced pressure in sequence, so as to provide a second intermediate (3) which was present in a light-yellow oil form.

[0060] < Step 4: Esterification and ferf-butoxycarbonyl (Boc) deprotection>

[0061] First, a solution of alcohol (3.0 equiv.) in dichloromethane (CH2Cl2) was added, under argon (Ar) gas, to a solution of the dicarboxylic acid derivative (3) (i.e., the second intermediate obtained in step 3, 1.0 equiv.), 4-dimethylaminopyridine (DMAP, 0.5 equiv.), N, N-diisopropylethylamine (2.0 equiv.), and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDCI HCI, 3.0 equiv.) in dichloromethane (CH2Cl2). Next, the resultant mixture was stirred for 24 hours at room temperature, followed by diluting with dichloromethane (CH2Cl2), washed sequentially with saturated aqueous sodium bicarbonate (NaHCO3) solution twice, water twice, saturated aqueous sodium chloride (NaCI) solution twice, drying over anhydrous sodium sulfate (Na2SC>4) and concentrating under reduced pressure in sequence, so as to obtain a solution of crude tert-butoxycarbonyl (Boc) pyrrolidine in dichloromethane. Afterwards, the solution of crude Boc pyrrolidine in dichloromethane was added to trifluoroacetic acid (TFA, 2-3 equiv.). Subsequently, the resultant mixture was stirred at room temperaturefor 2~4 hours until completion by TLC. Thereafter, the resultant mixture was quenched with water, followed by neutralizing with aqueous saturated sodium bicarbonate (NaHCO3) solution to pH 8, separating the organic layer, washing with brine, drying over magnesium sulfate (MgSO4), and concentrating in sequence. Subsequently, the resultant crude ether was purified by column chromatography, so as to obtain a third intermediate (4) which was present in a light-yellow oil form.

[0062] < Step 5: Alkylation of pyrrolidine>

[0063] First, the pyrrolidine derivative (4) (i.e., the third intermediate obtained in step 4, 1 equiv.) and alkyl halide (5) (1.1-1.5 equiv) were dissolved in dimethylformamide (DMF), optionally with addition of potassium carbonate (K2CO3, 1.5 equiv.). Next, the resultant mixture was stirred at a temperature ranging room temperature to 60°C for 2-12 hours until completion by TLC. Afterwards, the resultant mixture was quenched with water or saturated sodium bicarbonate (NaHCOs), followed by extracting with EtOAc, washing the organic layer with brine, drying over anhydrous Na2SO4, and concentrating in sequence. Subsequently, the resultant residue was purified by column chromatography, so as to obtain the lipid (6) (i.e., the lipid of a respective one of EX1 to EX21) which was present in a light-yellow oil form.2. Synthetic route B

[0064] The synthetic route B includes the following steps 1 to 5.

[0065] < Step 1: Preparing 3,4-dihydroxy-pyrrolidine-1 -carboxylic acid tert-butyl ester in cis (1) or trans (1’) configuration>

[0066] Step 1 of the synthetic route B was equivalent to step 1 of the synthetic route A, and was performed to obtain the 3,4-dihydroxy-pyrrolidine-1 -carboxylic acid tert-butyl ester in cis (1) or trans (V) configuration.

[0067] < Step 2: Alkylation of 3,4-dihydroxy-pyrrolidine-1 -carboxylic acid tert-butyl ester in cis (1) or trans (1’) configuration>

[0068] Step 2 of the synthetic route B was similar to step 2 of the synthetic route A, except that in step 2 of the synthetic route B, the bromoalkyl nitrile (2.5-3.0 equiv.) used in the synthetic route A was replaced by benzyl bromoalkyl ether (2.5-3.0 equiv.), so as to obtain a first intermediate (7) which was present in a lightyellow oil form.

[0069] < Step 3: Hydrogenation>

[0070] First, the N-benzyl substrate (7) (i.e., the first intermediate obtained in step 2, 1 equiv.) was dissolved in a solvent (e.g., ethanol and ethyl acetate) in a flask. Next, palladium on carbon (Pd / C) catalyst (5-10 mol%, 10% Pd loading) wasadded into the resultant solvent in the flask, followed by evacuating the flask, and then the flask was backfilled 2-3 times with hydrogen gas (H2) from a balloon to establish a hydrogen atmosphere. Afterwards, the resultant solvent was stirred vigorously at room temperature until reaction completion typically for 2-12 hours, as monitored by TLC or liquid chromatography-mass spectrometry (LC-MS). Subsequently, the resultant solvent was filtered through Celite® diatomaceous earth to remove catalyst therein, so as to obtain a filtrate. Thereafter, the Celite® diatomaceous earth was washed with one or more solvents selected from ethanol and ethyl acetate to collect the resultant residue liquid from the Celite® diatomaceous earth, followed by combining the filtrate and the residue liquid, so as to obtain a combined liquid. Afterwards, the combined liquid was concentrated, followed by purifying by chromatography in sequence, so as to obtain a second intermediate (8) which was present in a light-yellow oil form.

[0071] < Step 4: Esterification and Boc deprotection>

[0072] Step 4 of the synthetic route B was similar to step 4 of the synthetic route A, except that in the initial procedure of step 4 of the synthetic route B, the solution of alcohol (3.0 equiv.) in CH2CI2 used in the synthetic route A was preplaced by a solution of carboxylic acid (3.0 equiv.) in CH2CI2, and thedicarboxylic acid derivative (3) (i.e., the second intermediate of the synthetic route A, 1.0 equiv.) used in the synthetic route A was replaced by a diol derivative (8) (i.e. the second intermediate obtained in step 3 of the synthetic route B, 1.0 equiv.), so as to obtain a third intermediate (9) which was present in a light-yellow oil form.

[0073] < Step 5: Alkylation of pyrrolidine>

[0074] Step 5 of the synthetic route B was similar to step 5 of the synthetic route A, except that in step 5 of the synthetic route B, the third intermediate (1 equiv.) (4) of the synthetic route A was replaced by the third intermediate (9) (1 equiv.) of the synthetic route B, so as to obtain the lipid (10) (i.e., the lipid of a respective one of EX1 to EX21) which was present in a light-yellow oil form.

[0075] The structure and chemical name of the lipid of the respective one of EX1 to EX21 synthesized by the synthetic route A or the synthetic route B are shown in Table 1 below. In addition, the lipid of the respective one of EX1 to EX21 was characterized by proton nuclear magnetic resonance (1H-NMR) spectroscopy and liquid chromatography-electrospray ionization-mass (LC-ESI-MS) spectroscopy respectively using an NMR spectrometer (Manufacturer: Bruker, Model no.: 600MHz, AVANCE III) and a LC-ESI-MS spectrometer (Manufacturer: SCIEX, Model no.: API 3200LC / MS), so as to obtain a1H-NMR spectrum data andLC-ESI-MS spectrum data, respectively, as shown in Table 2 below.

[0076] Table 1Lipid Structure Chemical nameo(((3R,4S)-1-(2-((4- O ~s (dimethylamino)butanoyl)oxy) EX1 \ O ethyl)pyrrolidine-3,4-diyl)bis (oxy))bis(hexane-6, 1 -diyl)bis(2- o hexyldecanoate)0 ((((3R,4S)-1-(3-(3- (dimethylamino)propoxy)-3- EX2 x^x-x oxopropyl)pyrrolidine-3,4- LXXA / XXXX diyl)bis(oxy))bis(hexane-6,1-diyl) bis(2-hexyldecanoate) y'xX^OyKx^-X'xX ((((3R,4S)-1-(4-(3- (dimethylamino)propoxy)-4- EX3 N-T Y_ozX_N / "'['' 6 '--^o o oxobutyl)pyrrolidine-3,4-diyl)bis kxx^V^ (oxy))bis(pentane-5, 1 -diyl) bis(2- hexyldecanoate) di(heptadecan-9-yl) 6,6'- (((3S,4R)-1-(3-((4- EX4 V-^p-OX“-N^X (dimethylamino)butanoyl)oxy)pro pyl)pyrrolidine-3,4- °^VC0CC' diyl)bis(oxy))dihexanoate0j / SZ'XoApz'v'x / X (((3R,4S)-1-(3-((4-(diethylamino) — \ r~\ / --r>® butanoyl)oxy)propyl)pyrrolidine- EX5 N-yV-0 -N To ’--^O 3,4-diyl)bis(oxy))bis(hexane-6,1- diyl)bis(2-hexyldecanoate) 0q(((3R,4S)-1-(3-((4-(pyrrolidin-1- yl)butanoyl)oxy)propyl)pyrrolidine EX6 ■ '^"^Yx-Cg -3,4-diyl)bis(oxy))bis(hexane-6,1- diyl) bis(2-hexyldecanoate)d i(heptadecan-S-yl) 6,6’- (((3S,4R)-1-(3-(2-(dimethylamino) EX7 / >-X y<> r-\ V-N z-> r >O' acetoxy)propyi)pyrrolidine-3,4- '°x,z',Xz-z'--vzX-vzXx diyl)bis(oxy))dihexanoate5-(((3S,4S)-1-(3-((4- (dimethylamino)butanoyl)oxy)pro pyl)-4-((5-(((8Z,11Z)-octadeca- EX88, 11 -dienoyl)oxy)pentyi)oxy) pyrrolidin-3-yl)oxy)pentyl (7Z,10Z)-octadeca-7,10-dienoate I 9fZSZVV^ di(dec-3-yn-1-yl) 6,6’-(((3S,4R)-1 - (3-((4-(dimethylamino)butanoyl) EX9 ) (10 0 oxy)propyl)pyrrolidine-3,4- ^^o o=-C ° diyl)bis(oxy))dihexanoate i Jd i(heptadecan-9-yf) 5,5’- po % \~-^I < (((3S,4S)-1-(2-((4- EX10 Vf<~ °>(dimethylamino)butanoyl)oxy)ethy l)pyrrolidine-3,4- ^Xo < diyl)bis(oxy))dipentanoate <di((9Z,12Z)-octadeca-9,12-dien- c°1-yf) 6,6’-(((3S,4R)-1-(2-((4- f ZMEX11 (dimethylamino)butanoyl)oxy)ethy ' o '-%l)pyrrolidine-3,4-diyl)bis(oxy)) dihexanoate(((3R,4S)-1-(2-((4- (dimethylamino)butanoyl)oxy)ethy EX12 l)pyrrolidine-3,4-diyl)bis(oxy))bis '■ * 'A> o (pentane-5,1-diyl) (9Z,9'Z,12Z,12'Z)-bis(octadeca-9, 12-dienoate) di(dec-3-yn-1-yl) 6,6’-(((3S,4R)-1 - (2-((3-(dimethylamino)propanoyl) EX13oxy)ethyl)pyrrolidine-3,4-diyl)bis <>v"\}(oxy))dihexanoatedi(dec-3-yn-1-yl) 6,6’-(((3S,4R)-1 - (3-((3-(dimethylamino)propanoyl) EX14 " KVNA oxy)propyl)pyrrolidine-3,4- diyl)bis(oxy))dihexanoate g Xdi(dec-3-yn-1-yl) 6,6'-(((3S,4R)-1 - (2-((4-(dimethylamino)butanoyl) EX15oxy)ethyl)pyrrolidine-3,4-diy!)bis (oxy))dihexanoateXdi(dec-3-yn-1-yl) 5,5'-(((3S,4S)-1 - (3-((3-(dimethylamino)propanoyl) EX16p o X z oxy)propyl)pyrrolidine-3,4-d iyl) O O >~~^ bis(oxy))dipentanoate? \o o=~ odi(non-2-yn-1-yl) 6,6'-(((3R,4S)-1- (3-((3-(dimethylamino)propanoyl) EX17 oxy)propyl)pyrrolidine-3,4-diyl)bis (oxy))dihexanoate(((3R,4R)-1-(3-((3- > < (dimethylamino)propanoyl)oxy)pr EX18 opyl)pyrrolidine-3,4-diyl)bis(oxy))y bis(hexane-6, 1 -diyl) (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate) di(non-3-yn-1-yl) 6,6’-(((3S,4R)-1 - o (3-((3-(dimethylamino)propanoyl) EX19 -N K--0 Vtf Jo A) oxy)propyl)pyrrolidine-3,4-diyl)bis (oxy))dihexanoate(((3R,4S)-1-(2-((3- (dimethylamino)propanoyl)oxy)et N-, 0-. / -v0 0hyl)pyrrolidine-3,4-diyl)bis(oxy)) EX20 ' H 'X T0 " A? 0 bis(pentane-5, 1 -diyl) (9Z,9'Z, 12Z,12'Z) -bis(octadeca-9,12- dienoate)di(dec-3-yn-1-yl) 6,6'-(((3S,4R)-1 - (3-((3-(diethylamino)propanoyl) EX21 r~n X-0 '''—N'y / } O' oxy)propyl)pyrrolidine-3,4-d iyl) bis(oxy))dihexanoate

[0077] Table 2Lipid1H-NMR spectrum data LC-ESI-MS spectrum data1H NMR (600 MHz, CDCl3) δ 4.18 (t, J = 5.9 LCMS (ESI) m / z:Hz, 2H), 4.08 (t, J = 6.7 Hz, 4H), 3.89 (td, J = [M+H]+Calcd for C57H103N2O8+C56H109N2O8+4.2, 2.1 Hz, 2H), 3.53 (dt, J = 9.0, 6.7 Hz, 2H), 937.81;3.45 (dt, J = 9.1, 6.7 Hz, 2H), 3.13 (ddt, J = Found 938.110.3, 5.8, 4.1 Hz, 2H), 2.77 (t, J = 5.9 Hz, 2H),EX1 2.60 - 2.50 (m, 2H), 2.38 (t, J = 7.4 Hz, 2H),2.36 - 2.28 (m, 4H), 2.26 (s, 6H), 1.82 (p, J =7.4 Hz, 2H), 1.61 (tdd, J = 20.6, 10.1, 5.7 Hz,12H), 1.48 - 1.41 (m, 4H), 1.39 (h, J = 3.0 Hz,8H), 1.33 - 1.24 (m, 40H), 0.89 (td, J = 7.1,1.8 Hz, 12H)1H NMR (600 MHz, CDCl3) δ 4.14 (t, J = 6.5 LCMS (ESI) m / z:Hz, 2H), 4.08 (t, J = 6.7 Hz, 4H), 3.88 (td, J = [M+H]+Calcd for C57H103N2O8+C56H109N2O8+4.2, 2.1 Hz, 2H), 3.52 (dt, J = 9.1, 6.7 Hz, 2H), 937.81;3.45 (dt, J = 9.1, 6.7 Hz, 2H), 3.07 (ddt, J = Found 938.810.2, 5.8, 4.1 Hz, 2H), 2.81 (t, J = 7.5 Hz, 2H),EX22.56 - 2.43 (m, 4H), 2.38 - 2.29 (m, 4H), 2.25(s, 6H), 1.82 (dd, J = 8.0, 6.6 Hz, 2H), 1.67 - 1.57 (m, 12H), 1.47 - 1.42 (m, 4H), 1.39 (h, J= 2.9 Hz, 8H), 1.33 - 1.24 (m, 40H), 0.89 (td,J = 7.0, 1.8 Hz, 12H)1H NMR (600 MHz, CDCl3) δ 4.12 (td, J = 6.5, LCMS (ESI) m / z:2.0 Hz, 2H), 4.06 (td, J = 6.7, 1.9 Hz, 4H), [M+H]+Calcd for C57H103N2O8+C55H107N2O8+3.88 (q, J = 5.4 Hz, 2H), 3.52 (qd, J = 7.7, 6.7, 923.79;1.9 Hz, 2H), 3.44 (td, J = 8.4, 7.9, 5.8 Hz, 2H), Found 924.03.15 - 3.08 (m, 2H), 2.57 - 2.46 (m, 4H), 2.42EX3- 2.36 (m, 2H), 2.37 - 2.28 (m, 4H), 2.28 (s.6H), 1.82 (dq, J = 14.1, 6.9 Hz, 4H), 1.61(ddp, J = 28.0, 15.2, 7.2 Hz, 12H), 1.42 (tt, J= 8.8, 4.7 Hz, 8H), 1.25 (s, 40H), 0.88 (dd, J= 8.0, 5.9 Hz, 12H)1H NMR (500 MHz, CDCh) 5 4.85 (p, J = 6.2 LCMS (ESI) m / z:Hz, 2H). 4.12 (q, J = 7.2, 6.6 Hz, 2H), 3.88 (t. [M+H]+Calcd for C57H103N2O8+C59H115N2O8+J = 4.3 Hz, 2H), 3.47 (ddt, J = 43.4, 9.1, 6.7 979.86;Hz, 4H), 3.14 (s, 2H), 2.60 (s, 2H). 2.50 (s,EX4 Found 980.32H), 2.40 - 2.34 (m, 12H), 2.28 (t, J = 7.6 Hz,4H), 1.62 (dp, J = 14.4, 7.2, 6.8 Hz, 8H), 1.50(q, J = 6.7, 6.3 Hz, 8H), 1.42 - 1.32 (m, 4H),1.31 - 1.21 (m, 50H), 0.87 (t, J = 6.9 Hz, 12H)1H NMR (600 MHz, CDCl3) δ 4.09 (dt, J = LCMS (ESI) m / z:22.1, 6.6 Hz, 6H), 3.88 (t, J = 4.4 Hz, 2H), [M+H]+Calcd for C57H103N2O8+C59H115N2O8+3.48 (ddt, J = 38.9, 9.2, 6.7 Hz, 4H), 3.07 (dd, 979.86;J = 9.5, 5.5 Hz, 2H), 2.65 - 2.42 (m, 10H), Found 980.3EX52.39 - 2.23 (m, 4H), 1.81 (qt, J = 8.0, 4.6 Hz,4H), 1.70 - 1.52 (m, 12H), 1.47 - 1.41 (m,4H), 1.38 (p, J = 3.3 Hz, 8H), 1.27 (s, 40H),1.07 (t, J = 7.2 Hz, 6H), 0.94 - 0.82 (m, 12H)1H NMR (600 MHz, CDCl3) δ 4.12 (t, J = 6.5 LCMS (ESI) m / z:Hz, 2H), 4.08 (t, J = 6.7 Hz, 4H), 3.89 (t, J = [M+H]+Calcd for C57H103N2O8+C59H113N2O8+4.4 Hz, 2H), 3.53 (dt, J = 9.3, 6.7 Hz, 2H), 977.84;3.45 (dt, J = 9.3, 6.7 Hz, 2H), 3.08 (dd, J = Found 978.19.6. 5.2 Hz, 2H), 2.67 - 2.44 (m, 10H), 2.39EX6 (t, J = 7.4 Hz, 2H), 2.33 (tt, J = 9.3, 5.3 Hz,2H), 1.90 (q, J = 7.5 Hz, 4H), 1.82 (q, J = 6.9Hz, 4H), 1.62 (tt, J = 13.9, 6.7 Hz, 12H), 1.45(ddd, J = 14.3, 8.0, 4.8 Hz, 4H), 1.39 (p, J =3.2 Hz, 8H), 1.27 (s, 40H), 0.90 (td, J = 7.0,1.7 Hz, 12H)1H NMR (500 MHz, CDCl3) δ 4.86 (p, J = 6.2 LCMS (ESI) m / z:Hz, 2H), 4.17 (t, J = 6.6 Hz, 2H), 3.86 (s, 2H), [M+H]+Calcd for C57H103N2O8+C52H101N2O8+3.47 (ddt, J = 41.7, 9.1, 6.7 Hz, 4H), 3.15 (s, 951.83;2H), 3.05 (s, 2H), 2.49 (d, J = 40.9 Hz, 4H). Found 952.1EX7 2.35 (s, 6H), 2.28 (t, J = 7.6 Hz, 4H), 1.84 - 1.77 (m, 2H), 1.62 (dp, J = 14.4, 7.2, 6.8 Hz,8H), 1.50 (q, J = 6.1 Hz, 8H), 1.42 - 1.33 (m,4H), 1.32 - 1.18 (m, 48H), 0.87 (t, J = 6.9 Hz,12H)1H NMR (600 MHz, CDCl3) δ 5.44 - 5.31 (m, LCMS (ESI) m / z:8H), 4.07 (t, J = 6.7 Hz, 4H), 3.92 (qd, J = 5.6, [M+H]+Calcd for C57H103N2O8+C59H107N2O8+3.9 Hz, 2H), 3.55 (dt, J = 9.1, 6.5 Hz, 2H), 971.79;3.47 (dt, J = 9.0, 6.5 Hz, 2H), 2.79 (t, J = 6.9 Found 972.0Hz, 4H), 2.65 (t, J = 7.6 Hz, 2H), 2.60 - 2.52EX8 (m, 4H), 2.42 (d, J = 4.9 Hz, 4H), 2.30 (t, J =7.6 Hz, 4H), 2.19 (s, 6H), 2.07 (qd, J = 7.3,1.5 Hz. 8H), 1.90 (dp, J = 30.0, 7.1, 6.7 Hz,4H), 1.70 - 1.59 (m, 12H), 1.46 - 1.40 (m,4H), 1.39 - 1.27 (m, 30H), 0.91 (t, J = 7.0 Hz,6H)1H NMR (600 MHz, CDCl3) δ 4.18 (s, 2H), LCMS (ESI) m / z:4.12 (t, J = 7.0 Hz, 4H), 3.99 (s, 2H), 3.56 (q, [M+H]+Calcd for C57H103N2O8+C45H79N2O8+J = 7.3, 6.8 Hz, 2H), 3.47 (q, J = 7.3 Hz, 2H), 775.58;2.68 (s, 6H), 2.48 (tt, J = 7.1, 2.4 Hz, 6H),EX9 Found 776.22.32 (t, J = 7.5 Hz, 4H), 2.13 (tt, J = 7.1, 2.4Hz, 10H), 1.62 (dp, J = 27.3, 7.1, 6.7 Hz, 8H),1.50 - 1.41 (m, 4H), 1.41 - 1.32 (m, 8H), 1.31- 1.20 (m, 8H), 0.89 (t, J - 7.1 Hz, 6H)1H NMR (600 MHz, CDCI3) δ 4.96 – 4.80 (m, LCMS (ESI) m / z:2H), 4.21 (t, J = 6.0 Hz, 2H), 3.88 - 3.75 (m, [M+H]+Calcd for C57H103N2O8+C58H113N2O8+2H), 3.47 (ddt, J = 27.2, 9.2, 6.3 Hz, 4H), 2.98 937.81;- 2.84 (m, 2H), 2.79 - 2.63 (m, 2H), 2.58 (dd, Found 938.2EX10J = 10.0, 4.0 Hz, 2H), 2.41 - 2.29 (m, 8H),2.26 (s, 6H), 1.85 - 1.78 (m, 2H), 1.76 - 1.58(m, 12H), 1.57 - 1.44 (m, 8H), 1.39 - 1.11 (m,48H), 0.98 - 0.75 (m, 12H)1H NMR (600 MHz, CDCI3) 5 5.46 - 5.30 (m, LCMS (ESI) m / z:8H), 4.18 (t, J = 5.9 Hz. 2H). 4.07 (t, J = 6.8 [M+H]+Calcd for C57H103N2O8+C60H109N2O8+Hz, 4H), 3.88 (ddt, J = 8.3, 5.8, 2.9 Hz, 2H), 985.81;3.52 (dt, J = 9.0, 6.7 Hz, 2H), 3.45 (dt, J = Found 986.1EX11 9.2, 6.7 Hz, 2H), 3.12 (ddt, J = 10.2, 5.8, 4.1Hz, 2H), 2.86 - 2.70 (m. 6H), 2.61 - 2.51 (m,2H), 2.38 (t, J = 7.5 Hz, 2H), 2.31 (t, J = 7.5Hz, 6H), 2.24 (s, 6H), 2.07 (qd, J = 7.1, 1.3Hz, 8H), 1.68 - 1.60 (m, 12H), 1.43 - 1.27 (m,38H), 0.91 (t, J = 7.0 Hz, 6H)1H NMR (600 MHz, CDCI3) 5 5.46 - 5.25 (m, LCMS (ESI) m / z:8H), 4.18 (t, J = 5.9 Hz. 2H). 4.07 (t, J = 6.8 [M+H]+Calcd for C57H103N2O8+C58H105N2O8+Hz, 4H), 3.89 (pd, J = 5.8, 2.9 Hz, 2H), 3.53 957.78;(dt, J = 9.1, 6.6 Hz, 2H), 3.46 (dt, J = 9.1, 6.6 Found 958.2.Hz, 2H), 3.12 (ddt, J = 10.2, 5.7, 4.1 Hz, 2H),EX12 2.85 - 2.72 (m, 6H), 2.61 - 2.50 (m, 2H). 2.38(t, J = 7.5 Hz, 2H), 2.31 (dd, J = 7 A, 2.5 Hz,4H), 2.24 (s, 6H), 2.07 (qd, J = 7.1, 1.5 Hz.8H), 1.81 (p, J = 7.5 Hz, 2H), 1.73 (s, 2H),1.70 - 1.59 (m. 14H), 1.46 - 1.40 (m. 4H),1.40 - 1.25 (m, 30H), 0.91 (t, J = 7.0 Hz. 6H)1H NMR (600 MHz, CDCI3) 6 4.17 (t. J = 5.9 LCMS (ESI) m / z:Hz, 1 H), 4.12 (td, J = 7.1, 0.6 Hz, 4H), 3.90 - [M+H]+Calcd for C57H103N2O8+C43H75N2O8+3.84 (m. 2H), 3.63 - 3.57 (m, 1H), 3.55 - 3.39 747.54;(m, 4H), 3.13 - 3.03 (m, 2H), 2.75 (t, J = 5.9 Found 748.1EX13 Hz, 1H), 2.69 (t, J = 5.3 Hz, 1H), 2.65 - 2.58(m, 2H), 2.56 - 2.44 (m. 6H), 2.31 (td, J = 7.6,2.2 Hz, 4H), 2.24 (d, J = 4.4 Hz, 4H), 2.13 (tt,J = 7.2, 2.4 Hz, 4H), 1.68 - 1.56 (m, 11H),1.53 - 1.41 (m, 4H), 1.42 - 1.23 (m, 17H),0.89 (t, J = 7.1 Hz, 6H)1H NMR (600 MHz, CDCI3) 54.12 (td, J = 6.8, LCMS (ESI) m / z: 3.9 Hz, 6H), 3.86 (ddt, J = 8.3, 5.7, 2.8 Hz, [M+H]’ Calcd for C44H77N2CV 2H), 3.50 (dt, J = 9.1, 6.7 Hz, 2H), 3.43 (dt, J 761.56;= 9.1, 6.6 Hz, 2H), 3.07 (s, 2H), 2.61 (t, J = Found 761.97.3 Hz, 2H). 2.57 - 2.49 (m, 2H). 2.51 - 2.43EX14 (m, 8H), 2.31 (t, J = 7.6 Hz, 4H), 2.24 (s, 6H),2.13 (tt, J = 7.2, 2.4 Hz, 4H), 1.84 - 1.76 (m,2H), 1.68 - 1.56 (m, 8H), 1.46 (tt, J = 7.5, 6.6Hz, 4H), 1.41 - 1.22 (m, 16H), 0.89 (t, J = 7.1Hz, 6H)1H NMR (600 MHz, CDCI3) 54.18 - 4.10 (m, LCMS (ESI) m / z: 6H), 3.86 (td, J = 4.1, 2.1 Hz, 2H), 3.50 (dt, J [M+H]+Calcd for C57H103N2O8+C44H77N2O8+= 9.1, 6.7 Hz, 2H), 3.43 (dt, J = 9.2, 6.7 Hz, 761.56;2H), 3.09 (ddt, J = 10.2, 5.8, 4.1 Hz, 2H), 2.74 Found 761.9(t, J = 5.9 Hz, 2H), 2.56 - 2.50 (m, 2H), 2.48EX15(tt, J = 7.1, 2.4 Hz, 4H). 2.34 (dt, J = 29.4, 7.5Hz, 8H), 2.25 (s, 6H), 2.13 (tt, J = 7.1, 2.4 Hz.4H), 1.81 (p, J = 7.5 Hz, 2H), 1.68 - 1.56 (m,8H), 1.50 - 1.42 (m, 4H), 1.42 - 1.22 (m,16H), 0.88 (t, J = 7.1 Hz, 6H)1H NMR (600 MHz, CDCI3) 54.15 - 4.09 (m, LCMS (ESI) m / z: 6H), 3.81 - 3.75 (m, 2H), 3.49 - 3.38 (m, 4H). [M+H]+Calcd for C57H103N2O8+C42H73N2O8+2.84 (dd, J = 9.7, 6.1 Hz, 2H), 2.61 (t, J = 7.3 733.53;Hz. 2H), 2.54 - 2.43 (m, 8H), 2.34 (dd. J = Found 733.9EX16 8.5, 6.4 Hz, 4H), 2.24 (s, 6H), 2.13 (tt, J = 7.2,2.4 Hz. 4H), 1.82 (dqd, J = 8.7, 6.6. 2.0 Hz,2H), 1.73 - 1.64 (m, 6H), 1.64 - 1.58 (m, 4H),1.50 - 1.42 (m. 4H). 1.40 - 1.22 (m, 12H).0.89 (t. J = 7.1 Hz, 6H)1H NMR (600 MHz, CDCI3) 64.68 (q, J = 2.2 LCMS (ESI) m / z:Hz, 4H), 4.14 (td, J = 6.5, 1.9 Hz, 2H), 3.88 [M+H]+Calcd for C57H103N2O8+C42H73N2O8+(tt, J = 4.3, 1.9 Hz, 2H), 3.52 (dtd, J = 8.8, 733.53;6.7, 2.0 Hz, 2H), 3.48 - 3.43 (m, 2H), 3.07 Found 733.9(ddd, J = 9.7, 4.1, 1.9 Hz, 2H), 2.62 (td, J =7.3, 2.0 Hz, 2H). 2.55 (ddd, J = 8.2, 6.8, 1.8EX17 Hz, 2H), 2.51 - 2.44 (m, 4H), 2.36 (td, J = 7.6,1.9 Hz, 4H), 2.26 (d. J = 2.0 Hz, 6H), 2.23(ddq, J = 6.9, 4.3, 2.2 Hz, 4H), 1.81 (pd, J =6.7, 3.3 Hz, 2H), 1.65 (dddd, J = 28.9, 12.8,8.2, 6.2 Hz, 10H). 1.53 (pd, J = 7.2. 2.0 Hz.4H), 1.43 - 1.37 (m, 8H), 1.35 - 1.27 (m, 8H),0.91 (td, J = 7.1, 2.0 Hz, 6H)1H NMR (600 MHz, CDCI3) 5 5.45 - 5.28 (m, LCMS (ESI) m / z:8H), 4.14 (td, J = 6.8, 4.2 Hz. 2H). 4.07 (t, J [M+H]+Calcd for C57H103N2O8+C60H109N2O8+= 6.7 Hz, 4H), 3.86 - 3.79 (m, 2H), 3.45 (qt, 985.81;J = 9.3, 6.7 Hz, 4H), 2.89 (ddd, J = 23.7, 9.9, Found 986.16.0 Hz, 2H), 2.79 (t, J = 6.7 Hz, 4H), 2.63 (q,EX18J = 9.1, 8.2 Hz, 2H), 2.54 - 2.48 (m, 4H). 2.31(t, J = 7.6 Hz, 4H), 2.27 (s, 6H), 2.07 (q. J =7.0 Hz, 8H), 1.89 - 1.80 (m, 2H), 1.69 - 1.56(m, 14H), 1.35 (dq, J = 31.0, 7.2, 5.3 Hz,36H), 0.91 (t, J = 6.8 Hz, 6H)1H NMR (500 MHz, CDCI3) 64.12 (td, J = 6.8, LCMS (ESI) m / z:4.8 Hz, 6H), 3.90 - 3.82 (m, 2H), 3.50 (dt, J = [M+H]+Calcd for C57H103N2O8+C42H73N2O8+9.0, 6.7 Hz, 2H), 3.42 (dt, J = 9.1, 6.6 Hz, 2H). 733.53;3.06 (dd, J = 9.9, 5.1 Hz, 2H), 2.60 (t, J = 7.2 Found 733.9.EX19 Hz, 2H), 2.56 - 2.41 (m, 10H), 2.31 (t, J = 7.5Hz, 4H), 2.24 (s, 6H), 2.12 (tq, J = 7.7, 2.6Hz, 4H). 1.79 (p, J = 6.8 Hz, 2H), 1.67 - 1.56(m, 8H), 1.52 - 1.23 (m, 16H), 0.89 (t, J = 6.9Hz, 6H)1H NMR (600 MHz, CDCI3) 5 5.51 - 5.20 (m, LCMS (ESI) m / z:8H), 4.07 (td, J = 6.8. 2.5 Hz, 4H), 3.95 - 3.83 [M+H]+Calcd for C57H103N2O8+(m, 2H), 3.66 - 3.60 (m, 1H), 3.57 - 3.43 (m, 943.76;4H), 3.10 (dddd, J = 34.6, 9.9, 4.3, 1.7 Hz, Found 943.72H), 2.81 - 2.76 (m, 5H), 2.74 - 2.68 (m, 1 H),EX20 2.67 - 2.60 (m, 2H), 2.57 - 2.50 (m, 2H), 2.30(t, J = 7.6 Hz. 4H), 2.26 (s, 6H), 2.07 (qd, J =7.3, 1.5 Hz, 8H), 1.64 (dddd, J = 18.4, 11.0,7.7, 3.3 Hz, 12H), 1.43 (dddd, J = 9.6, 7.1,4.7. 2.8 Hz, 4H), 1.40 - 1.26 (m, 30H). 0.91(t, J = 7.0 Hz. 6H)1H NMR (600 MHz, CDCI3) 54.18 - 4.08 (m, LCMS (ESI) m / z:6H), 3.88 (td, J = 4.3, 2.1 Hz, 2H), 3.52 (dt, J [M+H]+Calcd for C57H103N2O8+C46H81N2O8+= 8.9, 6.7 Hz, 2H), 3.45 (dt, J = 9.1, 6.6 Hz, 789.59;2H), 3.07 (ddt, J = 8.7, 5.9, 4.3 Hz, 2H), 2.81 Found 790.1(dd, J = 8.1, 6.9 Hz, 2H), 2.57 - 2.53 (m, 4H),EX21 2.53 - 2.44 (m, 8H), 2.34 (t. J = 7.6 Hz, 4H),2.15 (tt, J = 7.2, 2.4 Hz, 4H), 1.89 - 1.78 (m,4H), 1.69 - 1.60 (m, 8H), 1.49 (p, 7 = 7.1 Hz,4H), 1.43 - 1.36 (m, 8H), 1.34 - 1.27 (m, 8H),1.05 (t, J = 7.1 Hz, 6H), 0.91 (t, J = 7.1 Hz,6H)Preparation of messenger ribonucleic acid-containing lipid nanoparticle(mRNA-LNP):EX 1 to 21 and Comparative Example 1 (EX1 to EX21 and CE1 )

[0078] The lipid of the respective one of EX1 to EX21 as described aboveor D-Lin-MC3-DMA (an ionizable cationic lipid for CE1) was used to prepare anmRNA-LNP of a respective one of EX1 to EX21 and CE1. In brief, first, the lipid ofthe respective one of EX1 to EX21 or D-Lin-MC3-DMA (for CE1 ), a branched-chainionizable cationic lipidoid with five hydroxyl groups, a helper lipid, a steroid, and apolyethylene glycol-conjugated (PEGylated) lipid were dissolved in ethanol, andmolar ratios thereof for preparing the mRNA-LNP of the respective one of EX1 toEX21 and CE1 are shown in Table 3 below. Next, the resultant lipid-ethanol solutionwas rapidly mixed with an aqueous buffer (e.g., citrate buffer solution) containingthe nucleic acid drug, as shown in Table 4 below, using a microfluidic device undercontrolled conditions to allow the lipid of the respective one of EX1 to EX21 or D-Lin-MC3-DMA (for CE1), the branched-chain ionizable cationic lipidoid with fivehydroxyl groups, the helper lipid, a steroid, and the PEGylated lipid to self¬assemble into the mRNA-LNP of the respective one of EX1 to EX21 and CE1encapsulating the nucleic acid drug. Afterwards, the ethanol in the resultantmixture containing the mRNA-LNP was removed, followed by conducting solventexchange and subsequent concentration using a tangential flow filtration device(e.g., an ultrafiltration system) with a buffer solution (e.g., phosphate-bufferedsaline (PBS)).

[0079] Table 3Molar ratio Materials for preparing mRNA-LNP of the respective one of(based on a total amount of EX1 to EX21 and CE1 mRNA-LNP as 100%) Lipid of the respective one of EX1 toEX21 (for mRNA-LNP of the respectiveIonizable cationic one of EX1 to EX21) 10%lipidD-Lin-MC3-DMA (for mRNA-LNP of CE1)Branched-chainionizable cationicC12-200 35% lipidoid with fivehydroxyl groupsDMG-PEG2000 1.5% PEGylated lipidDSPE-PEG2000-triGalNAc 0.25%Helper lipid DOPE 16% Steroid Campesterol 37.25%

[0080] Table 4For use in the mRNA Catalog No. Manufacturer following experiments CleanCap® EGFP mRNATriLink In vitro cell-based (5-methoxyuridine (5moU)- L-7201modified) BioTechnologies assay In vitro cell-based CleanCap® FLuc mRNATriLink assay and in vivo (5-methoxyuridine (5moU)- L-7202 BioTechnologies transfection modified)efficiency assayCharacterization of mRNA-LNP or lipid in mRNA-LNPA. Determination of hydrodynamic size and polydispersity index (PDI)

[0081] The mRNA-LNP of the respective one of EX1 to E21 and CE1 wassubjected to determination of particle size (presented as hydrodynamic size (z-average diameter)) and distribution (presented as PDI) by dynamic light scattering(DLS) using Zetasizer Pro Blue (Manufacturer: Malvern Panalytical). The resultsare shown in FIG. 1.B. Determination of zeta potential

[0082] The mRNA-LNP of the respective one of EX1 to E21 and CE1 wassubjected to determination of surface potential (presented as zeta potential) byelectrophoretic light scattering (ELS) using the Zetasizer Pro Blue. The results are shown in FIG. 2.C. Determination of encapsulation efficiency

[0083] The mRNA-LNP of the respective one of EX1 to E21 and CE1 was subjected to determination of encapsulation efficiency by a fluorescence dye method (e.g., a RiboGreen RNA assay) using A Quant-it™ RiboGreen assay kit (Manufacturer: ThermoFisher). In brief, first, the mRNA-LNP of the respective one of EX1 to E21 and CE1 was diluted with equal volume of Tris / EDTA (TE) buffer or TE buffer containing 2% Triton X-100, so as to obtain a sample. Next, a RiboGreen reagent was added to the sample, and then incubation was performed at 37°C in absence of light for 15 minutes. Thereafter, fluorescence intensity (Ex / Em 480 / 520 nm) was measured using a microplate reader (Manufacturer: Tecan Group Ltd., with excitation wavelength of 480 nm and emission wavelength of 520 nm) to determine total mRNA concentration and non-encapsulated mRNA concentration of the mRNA-LNP of the respective one of EX1 to E21 and CE1. Afterwards, the encapsulation efficiency (E. E) for the mRNA-LNP of the respective one of EX1 to E21 and CE1 was calculated by substituting the total mRNA concentration and non-encapsulated mRNA concentration thus determined into the following Equation (1):E. E (%) = (Total mRNA concentration - Non-encapsulated mRNA concentration) / (Total mRNA concentration)x100% (1)

[0084] The results are shown in FIG. 1.D. Determination of acid dissociation constant (pKa)

[0085] The mRNA-LNP of the respective one of EX1 to E21 and CE1 was subjected to determination of pKa value of the lipid of the respective one of EX1 to E21 or D-Lin-MC3-DMA (for CE1) therein by 6-(p-toluidino)-2-naphthalenesulfonyl chloride (TNS) binding assay to evaluate physicochemical properties thereof. To be specific, first, buffers with several pH range (i.e., 20 mM citrate buffers ranging from pH 3.0 to 5.5, 20 mM sodium phosphate buffers ranging from pH 6.0 to 8.0, and 20 M Tris buffers ranging from pH 8.5 to 10.0) were prepared. Next, the mRNA-LNP of the respective one of EX1 to E21 and CE1 (having 0.5 mM total lipid concentration) was mixed with the aforesaid buffers, followed by addition of a TNS reagent to a final concentration of 6 pM in a total volume of 0.2 mL, and the resultant mixture was then kept for a reaction to proceed for ten minutes. Afterwards, fluorescence intensity was measured using an Infinite M200 PRO multimode plate reader (with excitation wavelength of 321 nm and emission wavelength of 447 nm) to determine the pKa value of the lipid of the respective oneof EX1 to E21 or D-Lin-MC3-DMA (for CE1). The results are shown in FIG. 2. Results

[0086] As shown in FIG. 1, the hydrodynamic size, the PDI, and the encapsulation efficiency of the mRNA-LNP of the respective one of EX1 to E21 respectively ranged from 83 nm to 140 nm, 0.02 to 0.22, and 93.5 % to 99.2%. The hydrodynamic size, the PDI, and the encapsulation efficiency of the mRNA-LNP of CE1 is 94.1 nm, 0.11, and 88.13%, respectively.

[0087] As shown in FIG. 2, the pKa value of the lipid of the respective one of EX1 to E21 in the mRNA-LNP of the respective one of EX1 to E21 and the zeta potential of the mRNA-LNP of the respective one of EX1 to E21 respectively ranged from 5.0 to 7.0 and -6.5 mV to 9 mV. The pKa value of the D-Lin-MC3-DMA in the mRNA-LNP of CE1 and the zeta potential of the mRNA-LNP of CE1 is 5.91 and -2.24 mV, respectively.Property evaluationA. In vitro cell-based assay for EGFP mRNA transfection in HepG2 ceils Experimental Materials:

[0088] 1. Cell line used in this experiment was human hepatocellular carcinoma cell line 2 (HepG2) which is obtained from the American Type CultureCollection (ATCC, Manassas, Va., USA). The HepG2 were grown in a 10-cm Petri dish containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, and were cultivated in an incubator at 37°C with 5% CO2. Subsequently, medium change was performed every two to three days. Cell passage was performed when the cultured cells reached 80% to 90% confluence.

[0089] 2. mRNA used in this experiment was the CleanCap® EGFP mRNA (stock concentration: 1000 ug / mL) described as in Table 4 above.

[0090] 3. Delivery vehicles used in this experiment were the mRNA-LNP of EX7 (hereafter abbreviated as “inventive mRNA-LNP’’) containing the lipid of EX7 of present disclosure as described in “Preparation of mRNA-LNP” above, and an mRNA-LNP containing a lipid of Example 11 of US 20250034088 A1 (hereafter abbreviated as “comparative mRNA-LNP”). The structures of the lipid of EX7 of present disclosure in the inventive mRNA-LNP and the lipid of Example 11 of US 20250034088 A1 in the comparative mRNA-LNP are shown in Table 5 below. In this experiment, the comparative mRNA-LNP was prepared in accordance with the procedures and materials as described for the inventive mRNA-LNP above, except that the lipid of EX7 of present disclosure used to prepare the inventive mRNA-LNP was replaced by the lipid of Example 11 of US 20250034088 A1.

[0091] Table 5Lipid Structuref'- —Lipid of EX7 of present disclosure ininventive mRNA-LNPOLipid of Example 11 of US20250034088 A1 in comparative HO N TmRNA-LNP L Z-x.

[0092] 4. An inventive mRNA-LNP solution and a comparative mRNA-LNPsolution used in this experiment were prepared by respectively dissolving anappropriate amount of the inventive mRNA-LNP and the comparative mRNA-LNPin an appropriate amount of PBS at a pH value of 7.2.Experimental Procedures:

[0093] First, the HepG2 cells prepared in subsection 1 of “ExperimentalMaterials" of section A of “Property evaluation” were divided into 2 groups,including an experimental group and a comparative group. The HepG2 cells of eachgroup were seeded at a concentration of 5×104cells / mL into each well of a 12-wellplate containing DMEM supplemented with 10% FBS and 1%penicillin / streptomycin, followed by cultivation in an incubator (37°C, 5% CO2) for overnight, so as to ensure that the HepG2 cells of each group reach approximately 70% to 80% confluence prior to cell transfection.

[0094] Next, the used culture medium in each well of the 12-well plate was replaced by 900 uL of fresh complete DMEM supplemented with 10% FBS and 1% penicillin / streptomycin to stabilize the culture environment. When the HepG2 cells of each group reached approximately 80% confluence, the HepG2 cells of the experimental group and the comparative group were respectively treated with 100 uL of the inventive mRNA-LNP solution and 100 uL of the comparative mRNA-LNP solution as described in subsection 4 of “Experimental Materials” of section A of “Property evaluation.” so that each group has a final CieanCap® EGFP mRNA concentration of 1.0 pg / mL. Subsequently, the 12-well plate was gently rocked back and forth to ensure uniform distribution of the inventive mRNA-LNP solution or the comparative mRNA-LNP solution in the culture medium, followed by cultivation in an incubator (37°C, 5% CO2) for 24 hours.

[0095] Afterwards, the resultant cell culture of each group was subjected to determination of EGFP fluorescence expression using an EVOS™ M5000 imaging system under unified image acquisition condition, so as to obtain capturedimages. Thereafter, the captured images were quantitatively analyzed usingImageJ software by selecting region of interests (ROI), calculating meanfluorescence intensity (MFI), and performing background subtraction to obtain thecorrected mean EGFP fluorescence intensity for each of the experimental groupand the comparative group, as shown in FIG. 3 and Table 6 below, thereby enablingcomparison of transfection efficiency between the two groups.Results:

[0096] Table 6Comparative group Experimental group Mean 42.09 98.83 Standard deviation (SD) 2.54 16.36 Fold change compared with1 2.3comparative group

[0097] As shown in FIG. 3, in combination with Table 6, the corrected meanEGFP fluorescence intensity determined in the experimental group showed anapproximately 2.3-fold increase compared with the comparative group. Theseresults demonstrate that the lipid of EX7 of present disclosure, which has twotertiary amines, enables the inventive mRNA-LNP including the lipid of EX7 ofpresent disclosure to exhibit a transfection efficiency more than two-fold higherthan that of the comparative mRNA-LNP including the lipid of Example 11 of US 20250034088 A1. Accordingly, the inventive mRNA-LNP can demonstrate superior liver-targeted transfection efficacy compared with the comparative mRNA-LNP.B. In vitro cell-based assay for evaluation of cell viability Experimental Materials:

[0098] 1. Cell line used in this experiment was HepG2 as described in subsection 1 of " Experimental Materials” of section A of " Property evaluation.”

[0099] 2. mRNA used in this experiment was the CleanCap® FLuc mRNA as listed in Table 4 above.

[0100] 3. Delivery vehicles used in this experiment were the mRNA-LNPs of EX1 to E21 and CE1 as described in “Preparation of mRNA-LNP” above.

[0101] 4. An mRNA-LNP solution of a respective one of EX1 to E21 and CE1 used in this experiment was prepared by dissolving an appropriate amount of the mRNA-LNP of a respective one of EX1 to E21 and CE1, as described in subsection 3 of “Experimental Materials” of section B of “Property evaluation,” in an appropriate amount of PBS at a pH value of 7.2.Experimental Procedures:

[0102] The cell viability was evaluated using a CCK-8 assay. In brief, theHepG2 cells were divided into 22 groups, including a comparative group and experimental groups 1 to 21. The HepG2 cells of each group were seeded into each well of a 96-well plate containing DMEM supplemented with 10% FBS and 1% penicillin / streptomycin, followed by cultivation in an incubator (37°C, 5% CO2) for overnight. Afterwards, the used culture medium in each well of the 96-well plate was replaced by fresh complete DMEM supplemented with 10% FBS and 1% penicillin / streptomycin and the mRNA-LNP solution of the respective one of EX1 to E21 and CE1 as described in subsection 4 of “Experimental Materials" of section B of “Property evaluation” with a final CleanCap® FLuc mRNA concentration of 0.1 pg / well, followed by cultivation in an incubator (37°C, 5% CO2) for 18 hours. Thereafter, the resultant cell culture of each group was added with a cell counting kit-8 (CCK-8) reagent, followed by cultivation in an incubator (37°C, 5% CO2) for 30 minutes. Subsequently, the resultant cell culture of each group was subjected to absorbance measurement at a wavelength of 450 nm (OD450) using a microplate reader so as to determine cell viability of each group using techniques well-known to those skilled in the art. The results are shown in Table 7 below.Results:

[0103] Table 7Group mRNA-LNP solution used for Cell viability (%) treatmentmRNA-LNP solution of CE1Comparative group 91.4 ± 3.3 including D-Lin-MC3-DMAmRNA-LNP solution of EX1Experimental group 1 89.4 ± 4.2 including lipid of EX1mRNA-LNP solution of EX2Experimental group 2 including lipid of EX2 95.3 ± 6.0 mRNA-LNP solution of EX3Experimental group 3 including lipid of EX3 95.1 ± 2.2 mRNA-LNP solution of EX4Experimental group 4 91.4 ± 2.2 including lipid of EX4mRNA-LNP solution of EX5Experimental group 5 85.1 ± 2.0 including lipid of EX5Experimental group 6 mRNA-LNP solution of EX6 88.8 ± 0.9 including lipid of EX6mRNA-LNP solution of EX7Experimental group 7 including lipid of EX7 92.0 ± 6.9 mRNA-LNP solution of EX8Experimental group 8 including lipid of EX8 107.0 ± 9.7 mRNA-LNP solution of EX9Experimental group 9 114.0 ± 17.7 including lipid of EX9mRNA-LNP solution of EX10Experimental group 10 99.0 ± 1.7 including lipid of EX10mRNA-LNP solution of EX11Experimental group 11 100.0 ± 1.3 including lipid of EX11mRNA-LNP solution of EX12Experimental group 12 99.4 ± 1.1 including lipid of EX12mRNA-LNP solution of EX13Experimental group 13 98.7 ± 1.8 including lipid of EX13mRNA-LNP solution of EX14Experimental group 14 96.1 ± 1.4 including lipid of EX14mRNA-LNP solution of EX15Experimental group 15 96.0 ± 4.1 including lipid of EX15mRNA-LNP solution of EX16Experimental group 16 95.0 ± 3.3 including lipid of EX16mRNA-LNP solution of EX17Experimental group 17 111.0 ± 6.2 including lipid of EX17mRNA-LNP solution of EX18Experimental group 18 110.0 ± 12.6including lipid of EX18Experimental group 19 mRNA-LNP solution of EX19 102.2 ± 4.2 including lipid of EX19Experimental group 20 mRNA-LNP solution of EX20 93.8 ± 5.3including lipid of EX20mRNA-LNP solution of EX21Experimental group 21 including lipid of EX21 92.7 ± 12.7

[0104] Referring to Table 7, the experimental groups 1 to 21 and the comparative group, which were respectively treated with the mRNA-LNP solutions of EX1 to EX21 and CE1, exhibit relative high cell viability, with determined values thereof ranging from 83.1% to 131.7%. These results indicate that the lipids of EX1 to EX21, when respectively assembled into the mRNA-LNPs of EX1 to EX21, are capable of exhibiting cell viability comparable to that of the conventional lipid (i.e., D-Lin-MC3-DMA) which was assembled into the mRNA-LNP of CE1.C. In vivo transfection efficiency assayExperimental Materials:

[0105] 1. Mice used in this experiment were male BALB / c mice (7-8 weeks old, with a body weight approximately ranging from 17.8 g to 19.7 g) purchased from BioLasco Co., Ltd. All the mice were housed in an animal room with an independent air conditioning system under the following laboratory conditions: an alternating 12-hour light and 12-hour dark cycle, a temperature maintained at 20° C to 26 °C, and a relative humidity maintained at 30% to 70%. The mice wereprovided with reverse osmosis water and fed ad libitum. All experimental procedures involving the mice were in compliance with the legal provision of the Institutional Animal Care and Use Committee (IACUC), and were carried out according to the guidelines of IACUC Animal Experiment no. 2024-R403-030.

[0106] 2. mRNA used in this experiment was the CleanCap® FLuc mRNA as listed in Table 4 above.

[0107] 3. Delivery vehicles used in this experiment were the mRNA-LNPs of EX1 to E21 as described in “Preparation of mRNA-LNP” above and ONPATTRO® patisiran (a well-known commercial mRNA-LNP, in which the LNP includes the D-Lin-MC3-DMA, and which was used in a positive control group in this experiment).

[0108] 4. An mRNA-LNP solution of a respective one of EX1 to EX21 and positive control example (PC) used in this experiment was prepared by dissolving an appropriate amount of the mRNA-LNP of a respective one of EX1 to EX21 and the ONPATTRO® patisiran as described in subsection 3 of “Experimental Materials” of section C of “Property evaluation” in an appropriate amount of PBS at a pH value of 7.2.Experimental Procedures:

[0109] First, the mice as described in subsection 1 of “ExperimentalMaterials” of section C of “Property evaluation” were randomly divided into 22 groups (n= 3 mice in each group), including a positive control group and experimental groups 1 to 21. Next, the mice in the positive control group and experimental groups 1 to 21 were respectively administered the mRNA-LNP solution of PC and EX1 to EX21 prepared in subsection 4 of “Experimental Materials” of section C of “Property evaluation” via tail vein injection with a final CleanCap® FLuc mRNA concentration of 2.5 pg in each mouse.

[0110] After 6 hours of administration, each mouse in the positive control group and experimental groups 1 to 21 was subjected to determination of bioluminescence (deriving from the CleanCap® FLuc mRNA) distribution using an in vivo imaging system (IVIS), and the resultant luminescence intensity detected from different organs of each mouse were recorded. Afterwards, the relative luminescence intensity in the liver of the mice of each group was calculated by substituting the thus detected luminescence intensity from the liver into the following Equation (2):Relative luminescence Intensity = Luminescence intensity from liver of each group / Luminescence intensity from liver of positive control group (2)

[0111] The results are shown in Table 8 below, and illustrates efficiency oftransfection targeting the liver among the groups.Results:

[0112] Table 8mRNA-LNP solution used for Relative luminescence Grouptreatment intensity mRNA-LNP solution of PCPositive control group 1.0including D-Lin-MC3-DMAmRNA-LNP solution of EX1Experimental group 1 **including lipid of EX1mRNA-LNP solution of EX2Experimental group 2 ***including lipid of EX2mRNA-LNP solution of EX3Experimental group 3 *including lipid of EX3mRNA-LNP solution of EX4Experimental group 4 *including lipid of EX4mRNA-LNP solution of EX5Experimental group 5 **including lipid of EX5mRNA-LNP solution of EX6Experimental group 6 **including lipid of EX6mRNA-LNP solution of EX7Experimental group 7 ***including lipid of EX7mRNA-LNP solution of EX8Experimental group 8 *including lipid of EX8mRNA-LNP solution of EX9Experimental group 9 ***including lipid of EX9mRNA-LNP solution of EX10Experimental group 10 ***including lipid of EX10mRNA-LNP solution of EX11Experimental group 11 ***including lipid of EX11Experimental group 12 mRNA-LNP solution of EX12 ***including lipid of EX12Experimental group 13 mRNA-LNP solution of EX13including lipid of EX13mRNA-LNP solution of EX14Experimental group 14 ***including lipid of EX14mRNA-LNP solution of EX15Experimental group 15 **including lipid of EX15Experimental group 16 mRNA-LNP solution of EX16 * including lipid of EX16Experimental group 17 mRNA-LNP solution of EX17 ***including lipid of EX17mRNA-LNP solution of EX18Experimental group 18 including lipid of EX18mRNA-LNP solution of EX19Experimental group 19 ***including lipid of EX19mRNA-LNP solution of EX20Experimental group 20 *including lipid of EX20mRNA-LNP solution of EX21Experimental group 21 *including lipid of EX21Note:(1) The symbol "*" indicates a 2.5- to 3.5-fold increase compared with the positive control group.(2) The symbol “**” indicates a 3.5- to 5.0-fold increase compared with the positive control group.(3) The symbol “***” indicates more than 5.0-fold increase compared with the positive control group.

[0113] Referring to Table 8, compared with the positive control group, therelative luminescence intensity in the liver of the mice in the experimental groups1 to 21 exhibited 2.5-fold increase or greater. These results demonstrate that thelipids of EX1 to EX21, when respectively assembled into the mRNA-LNPs of EX1to EX21, are capable of exhibiting superior efficiency of transfection targeting theliver compared with the conventional lipid (i.e., D-Lin-MC3-DMA) assembled intothe ONPATTRO® patisiran (i.e., the well-known commercial mRNA-LNP).

[0114] Summarizing the above test results, it is clear that by virtue of thelipid of the present disclosure (i.e., EX1 to EX21) having the specific structure, themRNA-LNP, in which the LNP includes the lipid of the present disclosure, are capable of exhibiting excellent efficiency of transfection targeting the liver and excellent cell viability, and hence can deliver nucleic acids effectively and safely to a subject.

[0115] In the description above, for the purposes of explanation, numerous specific details have been set forth in order to provide a thorough understanding of the embodiment(s). It will be apparent, however, to one skilled in the art, that one or more other embodiments may be practiced without some of these specific details. It should also be appreciated that reference throughout this specification to “one embodiment,’’ “an embodiment,” an embodiment with an indication of an ordinal number and so forth means that a particular feature, structure, or characteristic may be included in the practice of the disclosure. It should be further appreciated that in the description, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of various inventive aspects; such does not mean that every one of these features needs to be practiced with the presence of all the other features. In other words, in any described embodiment, when implementation of one or more features or specific details doesnot affect implementation of another one or more features or specific details, said one or more features may be singled out and practiced alone without said another one or more features or specific details. It should be further noted that one or more features or specific details from one embodiment may be practiced together with one or more features or specific details from another embodiment, where appropriate, in the practice of the disclosure.

[0116] While the disclosure has been described in connection with what is(are) considered the exemplary embodiment(s), it is understood that this disclosure is not limited to the disclosed embodiment(s) but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.

Claims

WHAT IS CLAIMED IS:

1. A lipid, which is represented by Formula (I):R1 / ,0— L3~Y— L4~Z L2-NQT R2 1 x / 1whereinpyrrolidine has two oxygen substitutions respectively at positions 3 and 4 in a cis or trans configuration,each of R1and R2is independently a Ci-Ce alkyl, or R1and R2collectively form a cycloalkyl ring or a heterocycloalkyl ring,L1is a C1-C6linear alkyl,L2is a C2-C6linear alkyl,L3is a C2-C6linear alkyl,L4is a C0-C6linear alkyl,X is an ester (-C(=O)-O- or O-C(=O)-),Y is an ester (-C(=O)-O- or O-C(=O)-), andZ is selected from the group consisting of -CHR3R4, -(alkyne)-R5, -C=C-R6, -C=C-CH2-C=C-R7, and -C=C-CH2-C=C-CH2-C=C-R8, where each of R3, R4, R5, R6, R7, and R8is independently a C5-C10 linear alkyl.

2. The lipid as claimed in claim 1, whereinis selected from the group consisting of3. The lipid as claimed in claim 1, wherein Z is selected from the group4. The lipid as claimed in claim 1, which is selected from the group consisting of:(1) (((3R,4S)-1-(2-((4-(dimethylamino)butanoyl)oxy) ethyl)pyrrolidine-3,4- diyl)bis(oxy))bis(hexane-6,1-diyl)bis(2-hexyldecanoate);(2) (((3R,4S)-1-(3-(3-(dimethylamino)propoxy)-3-oxopropyl)pyrrolidine-3,4-diyl)bis(oxy)) bis(hexane-6,1-diyl)bis(2-hexyldecanoate);(3) (((3R,4S)-1-(4-(3-(dimethylamino)propoxy)-4-oxobutyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl) bis(2-hexyldecanoate);(4) di(heptadecan-9-yl) 6,6'-(((3S,4R)-1-(3-((4-(dimethylamino)butanoyl)oxy) propyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate;(5) (((3R,4S)-1-(3-((4-(diethylamino)butanoyl)oxy)propyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(2-hexyldecanoate);(6) (((3R,4S)-1-(3-((4-(pyrrolidin-1-yl)butanoyl)oxy)propyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl) bis(2-hexyldecanoate);(7) di(heptadecan-9-yl) 6,6'-(((3S,4R)-1-(3-(2-(dimethylamino)acetoxy)propyl) pyrrolidine-3,4-diyl)bis(oxy))dihexanoate;(8) 5-(((3S,4S)-1-(3-((4-(dimethylamino)butanoyl)oxy)propyl)-4-((5-(((8Z,11Z)-octadeca-8,11-dienoyl)oxy)pentyl)oxy)pyrrolidin-3-yl)oxy)pentyl (7Z,10Z)-octadeca-7,10-dienoate;(9) di(dec-3-yn-1-yl) 6,6'-(((3S,4R)-1-(3-((4-(dimethylamino)butanoyl)oxy) propyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate;(10) di(heptadecan-9-yl) 5,5'-(((3S,4S)-1-(2-((4-(dimethylamino)butanoyl)oxy)ethyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate;(11) di((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl) 6,6’-(((3S,4R)-1-(2-((4- (dimethylamino)butanoyl)oxy)ethyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate; (12) (((3R,4S)-1-(2-((4-(dimethylamino)butanoyl)oxy)ethyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5, 1 -diyl)(9Z,9'Z, 12Z,12'Z)-bis(octadeca-9,12-dienoate); (13) di(dec-3-yn-1-yl) 6,6'-(((3S,4R)-1-(2-((3-(dimethylamino)propanoyl)oxy) ethyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate;(14) di(dec-3-yn-1-yl) 6,6'-(((3S,4R)-1-(3-((3-(dimethylamino)propanoyl)oxy) propyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate;(15) di(dec-3-yn-1 -yl) 6,6'-(((3S,4R)-1-(2-((4-(dimethylamino)butanoyl)oxy) ethyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate;(16) di(dec-3-yn-1 -yl) 5,5'-(((3S,4S)-1-(3-((3-(dimethylamino)propanoyl)oxy) propyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate;(17) di(non-2-yn-1-yl) 6,6'-(((3R,4S)-1-(3-((3-(dimethylamino)propanoyl)oxy) propyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate;(18) (((3R,4R)-1-(3-((3-(dimethylamino)propanoyl)oxy)propyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6, 1 -diyl) (9Z,9’Z,12Z,12'Z)-bis(octadeca-9,12-dienoate); (19) di(non-3-yn-1-yl) 6,6'-(((3S,4R)-1-(3-((3-(dimethylamino)propanoyl)oxy)propyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate;(20) (((3R,4S)-1-(2-((3-(dimethylamino)propanoyl)oxy)ethyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5, 1 -diyl) (9Z,9'Z,12Z, 12'Z)-bis(octadeca-9, 12-dienoate); and(21) di(dec-3-yn-1-yl) 6,6'-(((3S,4R)-1-(3-((3-(diethylamino)propanoyl)oxy) propyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate.

5. The lipid as claimed in claim 1, wherein a protonated form of the lipid has an acid dissociation constant (pKa) ranging from 5.0 to 7.0.

6. A lipid nanoparticle, comprising:the lipid as claimed in claim 1;a helper lipid;a steroid;a polyethylene glycol-conjugated (PEGylated) lipid; anda branched-chain ionizable cationic lipidoid with five hydroxyl groups.

7. The lipid nanoparticle as claimed in claim 6, which has a hydrodynamic size ranging from 83 nm to 140 nm.

8. The lipid nanoparticle as claimed in claim 6, which has a polydispersity index (PDI) ranging from 0.02 to 0.22.

9. The lipid nanoparticle as claimed in claim 6, which has a zeta potential ranging from -6.5 mV to 9 mV.

10. The lipid nanoparticle as claimed in claim 6, which has an encapsulation efficiency ranging from 93.5 % to 99.2%.

11. The lipid nanoparticle as claimed in claim 6, which further includes a nucleic acid that is encapsulated within the lipid nanoparticle.

12. The lipid nanoparticle as claimed in claim 11, wherein the nucleic acid is selected from the group consisting of non-replicating mRNA, self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), microRNA (miRNA), and combinations thereof.

13. A method for delivering a nucleic acid to a subject, comprising administering to the subject in need thereof a pharmaceutical composition including the lipid nanoparticle as claimed in claim 11 or 12.

14. The method as claimed in claim 13, wherein the pharmaceutical composition is in a parenteral dosage form.

15. A pharmaceutical composition for delivering a nucleic acid to a subject, comprising the lipid nanoparticle as claimed in claim 11 or 12.

16. The pharmaceutical composition as claimed in claim 15, which is in aparenteral dosage form.

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