Compound containing chloroquine structure, composition containing compound and application of compound

Abstract

The invention discloses a chloroquine structure-containing compound, a composition containing the chloroquine structure-containing compound and application of the chloroquine structure-containing compound and the composition. The invention provides a compound as shown in a formula I or pharmaceutically acceptable salt thereof. The compound containing the chloroquine structure provided by the invention is a nano material synthesized from chloroquine and a derivative thereof and a lipid tail, and the nano material can be applied to gene therapy, drug delivery and the like. The LNP formed by the nano material and mRNA has a novel nano space structure different from that of the traditional LNP, and has better pharmaceutical properties compared with the traditional LNP. The LNP has an immunosuppression function, can avoid causing redundant inflammatory reactions, and has higher safety.

Description

[0001] This application claims priority to Chinese Patent Application No. 2024106568712, filed on May 24, 2024. This application incorporates the entirety of the aforementioned Chinese Patent Application. TECHNICAL FIELD

[0002] The present application relates to compounds, compositions and methods for delivering therapeutic, diagnostic or prophylactic drugs, such as nucleic acids. In particular, it relates to a compound containing a chloroquine structure, a composition containing the same and its use. BACKGROUND

[0003] mRNA-based therapy shows broad application prospects in the fields of prophylactic vaccines against infectious diseases, tumor therapeutic vaccines, protein supplementation, protein replacement, etc. Although mRNA vaccines based on lipid nanoparticle delivery systems (LNPs) have been successful in the prevention of infectious diseases, the inflammatory side effects induced by LNP-mRNA vaccines in clinical applications are worrisome. However, in the application scenarios of mRNA, such as protein supplementation, protein replacement, and combination with other therapies, the inflammatory reactions induced by mRNA and LNP themselves are redundant, and such inflammatory side effects are particularly a limiting factor for the application of mRNA therapy in other fields. Therefore, the development of a delivery system without inflammatory side effects is particularly important for the improvement of the safety of mRNA drugs and the expansion of application scenarios. SUMMARY

[0004] The technical problem to be solved by the present application is that the existing technology based on lipid nanoparticle delivery systems is prone to induce inflammation. To this end, the present application provides a compound containing a chloroquine structure, a composition containing the same and its use. The compound containing a chloroquine structure provided by the present application is a nanomaterial synthesized from chloroquine and its derivatives and a lipid tail, which can be used in gene therapy, drug delivery, etc. The lipid nanoparticle (LNP) formed by the nanomaterial and mRNA has a novel nano-space structure different from traditional LNP, and has better formulation properties than traditional LNP. The LNP has an immunosuppressive function, which can avoid inducing redundant inflammatory reactions and has higher safety.

[0005] The present application provides a compound as shown in formula I or a pharmaceutically acceptable salt thereof,

[0006]

[0007] wherein:

[0008] T is C 1-30 alkyl, C 2-30 alkenyl, C 2-30 alkynyl or

[0009] X is a single bond, O, S or N(Rx ); R x For H or C 1-10 alkyl;

[0010] L 1 It is a single bond, -(CH2) n1 -、 X and L 1 Not both are single bonds; A 1 Terminal A is connected to X. 2 and A 3 The end is connected to the Y-axis;

[0011] A 1 -(CH2) n2 -;

[0012] A 2 and A 3 Independently a single bond or -(CH2) n2 -;

[0013] M 1 and M 2 Independently O or NH;

[0014] M 3 for O, S or N(R) x );

[0015] Y is N or CR Y ;R Y For H, C 1-30 Alkyl, C 2-30 alkenyl, C 2-30 acetylinyl

[0016] R 1 and R 2 Independently for C 1-30 Alkyl, C 2-30 alkenyl, C 2-30 acetylinyl

[0017] D 1 D 2 and D 3 Independently O or NH;

[0018] B 1 and B 3 Independently -(CH2) n3 -;

[0019] B 2 Independently for C 1-30 Alkyl, C 2-30alkenyl or C 2-30 alkynyl;

[0020] L 2 is K 1 is attached to Y, K 2 is attached to Z 1 ;

[0021] K 1 and K 3 are independently -(CH2) n4 -;

[0022] K 2 are independently a single bond or -(CH2) n4 -;

[0023] H 1 , H 2 , H 3 , H 4 and H 5 are independently O or NH;

[0024] H 6 are independently O, S or NH;

[0025] Z 1 is independently N or CH;

[0026] R 3 and R 4 are independently C 1-30 alkyl, C 2-30 alkenyl or C 2-30 alkynyl;

[0027] n1, n2, n3 and n4 are independently integers from 1 to 10.

[0028] In a certain preferred embodiment, in the compound of formula I, or a pharmaceutically acceptable salt thereof, certain groups are defined as follows, and the definition of groups not mentioned is as described in any of the embodiments of the present application (hereinafter this paragraph is referred to as "in a certain embodiment").

[0029] In a certain embodiment, T, R Y , B 2 , R 1 , R 2 , R 3 and R 4 , the C 1-30 alkyl is independently a linear alkyl or a branched alkyl, preferably a linear alkyl.

[0030] In a certain embodiment, T, R Y , B2 , R 1 , R 2 , R 3 , and R 4 , the C 1-30 alkyl is independently C 4-20 alkyl, for example C 4-18 alkyl, and further for example C 4-10 alkyl.

[0031] In a certain embodiment, T, R Y , B 2 , R 1 , R 2 , R 3 , and R 4 , the C 1-30 alkyl is independently C

[0032]

[0033] In a certain embodiment, T, R Y , B 2 , R 1 , R 2 , R 3 , and R 4 , the number of alkenyl groups in the C 2-30 alkenyl group is independently 1, 2, 3, 4, or 5, for example 1, 2, or 3.

[0034] In a certain embodiment, T, R Y , B 2 , R 1 , R 2 , R 3 , and R 4 , the C 2-30 alkenyl is independently a straight chain alkenyl or a branched chain alkenyl, preferably a straight chain alkenyl.

[0035] In a certain embodiment, T, R Y , B 2 , R 1 , R 2 , R 3 , and R 4 , the C 2-30 alkenyl is independently C 4-20 alkenyl, for example C 4-20 alkenyl, and further for example C 8-20 alkenyl, and more for example

[0036]

[0037] In a certain embodiment, T, R YB 2 R 1 R 2 R 3 and R 4 In, the C 2-30 The alkynyl group can be either a straight-chain alkynyl group or a branched-chain alkynyl group, wherein the C 2-30 The number of alkyne bonds in the alkenyl group is independently 1, 2, 3, 4, or 5, preferably C. 4-20 Straight-chain acetylene group.

[0038] In one implementation scheme, R x In, the C 1-10 Alkyl group is C 1-6 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.

[0039] In one implementation, in T, the C 2-30 The alkenyl group is C 4-20 Straight-chain alkenyl, the C 4-20 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3.

[0040] In one implementation scheme, R 1 and R 2 In, the C 1-30 Alkyl groups are independently C 4-20 Straight-chain alkyl groups.

[0041] In one implementation scheme, R 1 and R 2 In, the C 2-30 The alkenyl group is independently C 4-20 Straight-chain alkenyl, the C 4-20 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3.

[0042] In one implementation scheme, R 3 and R 4 In, the C 1-30 Alkyl groups are independently C 4-20 Straight-chain alkyl groups, such as C 4-10 Straight-chain alkyl groups.

[0043] In one implementation scheme, R Y B in 2 In, the C 2-30 The alkenyl group is C 4-20 Straight-chain alkenyl, the C 4-20 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3.

[0044] In one implementation scheme, R 1 and R 2 B in2 In certain embodiments, the C 1-30 alkyl is independently C 4-20 linear alkyl.

[0045] In certain embodiments, R 1 and R 2 B 2 In certain embodiments, the C 2-30 alkenyl is independently C 4-20 alkenyl, e.g., C 4-20 linear alkenyl, the number of alkenyl groups in said C 4-20 linear alkenyl is 1, 2, or 3.

[0046] In certain embodiments, T is C 2-30 alkenyl, or Preferably, T is C 4-20 linear alkenyl, or the number of alkenyl groups in said C 4-20 linear alkenyl is 1, 2, or 3; more preferably, T is

[0047] In certain embodiments, T is C 1-30 alkyl, C 2-30 alkenyl, or C 2-30 alkynyl.

[0048] In certain embodiments, X is a single bond or O; preferably, X is a single bond.

[0049] In certain embodiments, L 1 is a single bond, -(CH2) n1 -, Preferably, L 1 is

[0050] In certain embodiments, n1 and n2 are independently 1, 2, 3, 4, 5, 6, 7, or 8; preferably, n1 and n2 are independently 1, 2, 3, or 4.

[0051] In certain embodiments, M 1 is O.

[0052] In certain embodiments, M 3 is independently or N(R x ).

[0053] In certain embodiments, R x is H.

[0054] In certain embodiments, R Y is H, preferably, H or Ideally, D 2 For O; better yet, R Y For H.

[0055] In one implementation scheme, R Y In the middle, B 2 Independently for C 2-30 Alkenyl; preferably, B 2 Independently for C 4-20 Straight-chain alkenyl, the C 4-20 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3.

[0056] In one implementation scheme, R 1 and R 2 Independently for C 1-30 Alkyl, C 2-30 alkenyl,

[0057] Or, R 1 and R 2 Independently for C 1-30 Alkyl, C 2-30 alkenyl, C 2-30 acetylinyl

[0058] Ideally, R 1 and R 2 Independently for C 4-20 straight-chain alkyl, C 4-20 straight-chain alkenyl, Better yet, R 1 and R 2 Independently

[0059] In one implementation scheme, D 1 D 2 and D 3 It is O.

[0060] In one implementation, n3 is 1, 2, 3, 4, 5, 6, 7 or 8.

[0061] In one implementation scheme, R 1 and R 2 In the middle, B 2 Independently for C 1-30 Alkyl or C 2-30 Alkenyl; preferably, B 2 Independently for C 4-20 Straight-chain alkyl or C 4-20 Straight-chain alkenyl, the C 4-20 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3.

[0062] In one implementation scheme, L 2 Independently

[0063] In one implementation scheme, H 1 H 2 H 3 and H 5 It is O.

[0064] In one implementation scheme, H 6 Independently Or NH.

[0065] In one implementation scheme, K 2 It is a single key.

[0066] In one implementation, n4 is independently 1, 2, 3, 4, 5, 6, 7 or 8; preferably, n4 is independently 1, 2, 3 or 4.

[0067] In one implementation scheme, Z 1 For CH.

[0068] In one implementation scheme, R 3 and R 4 Independently for C 1-30 Alkyl; preferably, R 3 and R 4 Independently for C 4-20 Straight-chain alkyl groups, for example, C 4-10 Straight-chain alkyl groups.

[0069] In one implementation scheme, L 1 For single bond,

[0070] Preferably, bit "1" is connected to X, and bit "2" is connected to A. 3 The terminal is connected to Y; more preferably, L 1 for The "1" bit is connected to X, and the "2" bit is connected to Y.

[0071] In one implementation, -XL 1 - for single bond, O,

[0072] Preferably, the "2" bit and the Y end are connected to Y; more preferably, -XL 1 -for The "2" bit is connected to Y.

[0073] In one implementation scheme, R YFor H or

[0074] In one implementation scheme, B 2 Independently

[0075] In one implementation scheme, K 1 Independently

[0076] In one implementation scheme, K 2 Independently for single bonds,

[0077] In one implementation scheme, K 3 Independently

[0078] In one implementation scheme, L 2 Independently

[0079] Preferably, bit "1" is connected to Y, and bit "2" is connected to Z. 1 Connection; better yet, L 2 1 Bit "1" is connected to Y, and bit "2" is connected to Z. 1 Connection. In one implementation, R 3 and R 4 Independently Better yet, R 3 and R 4 for In one implementation scheme, R 1 and R 2 Independently

[0080] Ideally, R 1 and R 2 Independently

[0081] In one embodiment, the compound as shown in Formula I is selected from any of the following:

[0082] Option 1: The compound shown in Formula I is the compound shown in Formula I-1:

[0083]

[0084] R 1 and R2 Independently for C 1-30 Alkyl, C 2-30 alkenyl, C 2-30 acetylinyl X, L 1 Y, D 1 D 2 D 3 B 1 B 2 and B 3 The definition is as described in any embodiment of the present invention;

[0085] Option 2: The compound shown in Formula I is a compound shown in Formula I-2:

[0086]

[0087] X, L 1 Y, L 2 Z 1 R 3 and R 4 The definition is as described in any embodiment of the present invention;

[0088] Option 3: The compound shown in Formula I is a compound shown in Formulas I-3:

[0089]

[0090] R Y R 1 and R 2 Independently for C 1-30 Alkyl, C 2-30 alkenyl, C 2-30 acetylinyl X, L 1 D 1 D 2 D 3 B 1 B 2 and B 3 The definition is as described in any embodiment of the present invention.

[0091] In one embodiment, the compound shown in Formula I is a compound shown in Formula I-2:

[0092]

[0093] X is a single bond;

[0094] L 1 for

[0095] A 1 -(CH2) n2 -;A 2 -(CH2) n2 -;

[0096] n2 can be 1, 2, 3 or 4 independently;

[0097] M 1 It is O;

[0098] Y is either N or CH;

[0099] L 2 Independently K 1 and K 3 Independently -(CH2) n4 -;

[0100] K 2 Independently a single bond or -(CH2) n4 -;

[0101] n4 can be 1, 2, 3 or 4 independently;

[0102] H 1 H 3 and H 4 It is O;

[0103] Z 1 For CH;

[0104] R 3 and R 4 Independently for C 4-10 Straight-chain alkyl groups.

[0105] In one embodiment, the compound shown in Formula I is any of the following compounds:

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] The present invention also provides a lipid carrier comprising substance Z, said substance Z being a compound of formula I as described in any one of the present invention or a pharmaceutically acceptable salt thereof.

[0116] In a preferred embodiment, the lipid carrier further includes a diluent. The diluent is a conventional diluent in the art, such as citrate buffer or ethanol.

[0117] In a preferred embodiment, the lipid carrier further includes phospholipids. The phospholipids are conventional phospholipids in the art, being amphoteric accessory molecules that facilitate the fusion of lipid particles and cell membranes. The phospholipids may be phospholipid molecules having an electrically polar end and a nonpolar end of a fatty acid chain, such as distearate phosphatidylcholine (DSPC).

[0118] In a preferred embodiment, the lipid carrier further includes a PEG lipid (polyethylene glycol-modified lipid). The PEG lipid is a lipid molecule modified with a hydrophilic end of polyethylene glycol. The PEG lipid is preferably selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, such as PEG-modified dimyristoylglycerol (DMG PEG2000).

[0119] In a preferred embodiment, the lipid carrier further includes sterols. The sterols are conventional sterols in the art, including animal, plant, or fungal sterols. Preferably, the sterols are selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, and α-tocopherol, such as cholesterol.

[0120] In a preferred embodiment, the lipid carrier comprises the diluent, substance Z, the phospholipid, the PEG lipid, and the sterol.

[0121] In a preferred embodiment, the molar ratio of substance Z to the volume ratio of the diluent is (5-25) mmol:1L; for example, (10-15) mmol:1L; or, for another example, 12 mmol:1L.

[0122] In a preferred embodiment, the total molar mass of substance Z, the phospholipid, the PEG lipid, and the sterol is 100%, and the molar content of substance Z is 30 mol% to 60 mol%; for example, 40 mol% to 55 mol%; or, for example, 50 mol%.

[0123] In a preferred embodiment, the molar ratio of substance Z to phospholipid in the lipid carrier is 1-25:1, preferably 2-10:1, for example 5:1.

[0124] In a preferred embodiment, the molar ratio of substance Z to sterol in the lipid carrier is (0.5-3):1, preferably (0.5-3):1, for example 1.3:1.

[0125] In a preferred embodiment, the molar ratio of substance Z to PEG lipid in the lipid carrier is 20-50:1, preferably 20-40:1, for example 33:1.

[0126] In this invention, molar content refers to the percentage of a substance in the total amount of the lipid carrier, and the sum of the molar contents of all components in the lipid carrier does not exceed 100 mol.

[0127] In a preferred embodiment, the lipid carrier is composed of the substance Z, the diluent, the phospholipid, the PEG lipid, and the sterol.

[0128] In a preferred embodiment, the lipid carrier is composed of the substance Z, the phospholipid, the PEG lipid, and the sterol.

[0129] The present invention also provides a lipid nanoparticle comprising a therapeutic agent and / or a preventive agent as well as the aforementioned lipid carrier.

[0130] In a preferred embodiment, the therapeutic agent and / or preventive agent is one or more nucleic acids. The nucleic acid is a conventional nucleic acid in the art. The therapeutic agent and / or preventive agent may be single-stranded deoxyribonucleic acid (DNA), double-stranded DNA, small interfering RNA (siRNA), asymmetric double-stranded small interfering RNA (aiRNA), microRNA (miRNA), small hairpin RNA (shRNA), circular RNA (circRNA), transfer RNA (tRNA), messenger RNA (mRNA), and other forms of nucleic acid molecules known in the art, preferably mRNA, such as firefly luciferase (Fluc) mRNA, Luc mRNA synthesized from unmodified uracil (U), or poly(I:C) mRNA.

[0131] In a preferred embodiment, the therapeutic and / or preventive agent is VZV gE mRNA (varicella-zoster virus vaccine RNA), preferably having a nucleic acid sequence as shown in SEQ ID NO:1.

[0132] The present invention also provides a composition comprising substance Z, said substance Z being a compound as described above as shown in Formula I or a pharmaceutically acceptable salt thereof.

[0133] In a preferred embodiment, the composition further includes one or more of a diluent, phospholipids, PEG lipids, sterols, and therapeutic and / or preventative agents.

[0134] In a preferred embodiment, the diluent, phospholipid, PEG lipid, sterol, and therapeutic and / or preventative agent in the composition are as described above.

[0135] In a preferred embodiment, in the composition, substance Z forms a lipid carrier as described above with one or more of the diluent, phospholipid, PEG lipid, and sterol.

[0136] In a preferred embodiment, the lipid carrier in the composition forms lipid nanoparticles with the therapeutic agent and / or preventive agent as described above.

[0137] In a preferred embodiment, the encapsulation rate of the therapeutic agent and / or preventive agent in the composition is at least 94.5%, preferably at least 95%, for example 95.6%.

[0138] In a preferred embodiment, the composition contains a therapeutic and / or preventive agent with a particle size of 50-70 nm, for example 50-60 nm, or even 55 nm or 59 nm.

[0139] The present invention provides the use of the compound of Formula I or a pharmaceutically acceptable salt thereof or the lipid carrier thereof in the preparation of a medicament for the prevention and / or treatment of inflammation, for example, infectious inflammation.

[0140] This invention provides the use of the compound of Formula I or a pharmaceutically acceptable salt thereof or the lipid carrier thereof in the preparation of a medicament for the prevention and / or treatment of inflammation caused by the nucleic acid.

[0141] This invention provides the use of the compound of Formula I or a pharmaceutically acceptable salt thereof or the lipid carrier thereof in the preparation of a medicament for the prevention and / or treatment of inflammation caused by elevated levels of MCP-1, IL-6, ISG54 or NF-κB expression.

[0142] In a preferred embodiment, the elevated expression levels of MCP-1, IL-6, ISG54, or NF-κB are defined as an elevation of MCP-1, IL-6, ISG54, or NF-κB expression levels induced by the nucleic acid.

[0143] The present invention provides a method for preventing and / or treating said inflammation, comprising administering to a subject (a therapeutically effective amount) of the compound of Formula I or a pharmaceutically acceptable salt thereof or the lipid carrier thereof.

[0144] Unless otherwise specified, the terms used in this invention have the following meanings:

[0145] The term "pharmaceutical acceptable" means that something is relatively non-toxic, safe, and suitable for patient use.

[0146] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base.

[0147] In structural fragments This refers to the structural segment being connected to the rest of the molecule through this site. For example, To correct one's own shortcomings.

[0148] The term "alkyl" refers to an alkyl group having a specified number of carbon atoms (e.g., C40, C50, C6 ... 1-30 Alkyl groups are saturated, linear or branched, and include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0149] The term "alkylene" refers to a divalent group that is attached to the rest of the molecule by two single bonds, and the rest of the definition is the same as that of the term "alkyl".

[0150] The term "alkenyl" refers to a group having at least two carbon atoms (e.g., C4, C5, C6, C7, C8, C9 ... 2-30 An unsaturated monovalent hydrocarbon group, which is straight-chain or branched, contains one or more carbon-carbon double bonds and no carbon-carbon triple bonds, wherein the one or more carbon-carbon double bonds may be located at the end of the group or inside the group.

[0151] The term "alkynyl" refers to a group having at least two carbon atoms (e.g., C32-C4 ... 2-30 An unsaturated monovalent hydrocarbon group, which is straight-chain or branched, contains one or more carbon-carbon triple bonds and has no carbon-carbon double bonds, wherein the one or more carbon-carbon triple bonds may be located at the end of the group or inside the group.

[0152] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention. The reagents and raw materials used in the present invention are all commercially available.

[0153] The positive and progressive effects of this invention are as follows: the lipid nanoparticles (LNPs) formed by the nanomaterials and mRNA provided in this application are a novel nanostructure that differs from traditional LNPs, and possess one or more superior formulation properties compared to traditional LNPs:

[0154] (1) High drug loading capacity;

[0155] (2) The formulation has strong stability;

[0156] (3) High expression level in vivo;

[0157] (4) Effectively inhibits the decrease in mRNA expression caused by repeated administration;

[0158] (5) It has immunosuppressive function, significantly inhibiting the release of inflammatory factors induced by mRNA LNPs, thus avoiding redundant inflammatory responses and exhibiting higher safety. For example, it can inhibit...

[0159] It can inhibit the release of MCP-1 and IL-6; it can also inhibit the expression of ISG54 and NF-κB. Attached Figure Description

[0160] Figure 1 Gel electrophoresis image of the freeze-thaw process of HL-3LNP for Luc mRNA.

[0161] Figure 2 In vivo fluorescence imaging of HL-3LNP and HL-6LNP of Luc mRNA.

[0162] Figure 3 In vivo fluorescence imaging of HL-3LNP of Luc mRNA administered 4 times at 3-day intervals.

[0163] Figure 4 The relative percentage of in vivo fluorescence intensity of HL-3LNP of Luc mRNA relative to the fluorescence intensity of SM-102LNP of Luc mRNA in an experiment administered 4 times at 3-day intervals. Detailed Implementation

[0164] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0165] Example 1: Synthesis of Lipid 1

[0166] The synthetic route for Lipid 1 is as follows:

[0167]

[0168] 335.9 mg (1 mmol) of hydroxychloroquine (HCQ) and 284.5 mg (1 mmol) of isostearic acid (h1) were weighed and dissolved in 20 mL of anhydrous dichloromethane (DCM). Separately, 309.5 mg (1.5 mmol) of dicyclohexylcarbodiimide (DCC) and 24.4 mg (0.2 mmol) of 4-dimethylaminopyridine (DMAP) were added to the solution. The reaction was carried out at room temperature for 6 h. The reaction was monitored by TLC, with the developing solvent being DCM:MeOH (V / V) = 20:1.

[0169] After the reaction was complete, insoluble matter in the reaction solution was filtered off, and the reaction solvent was removed by vacuum distillation. 15 mL of water and 15 mL of ethyl acetate (EA) were added, and the mixture was extracted. After standing and separation, the organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by column chromatography using DCM:MeOH (V / V) = 50:1 as the eluent. The solvent was removed by vacuum distillation, yielding 447.8 mg of a pale yellow oily liquid, which was Lipid 1, with a yield of 74.3%.

[0170] H-NMR characterization is as follows: δ H (600MHz, CDCl3)8.52–8.50(1H,m,J 5.4),7.94(1H,d,J 2.2),7.70(1H,d,J 9.0),7.34(1H,dd,J 9.0,2.2),6.42(1H,d,J 5.5),4.14(2H,t,J 6.2),3.75–3.65(1H,m),2.73–2.46(7H,m),2.26(1H,t,J 7.6),1.62–1.54(4H,m,J 13.7,7.1),1.31(3H,t,J 6.2),1.29–1.14(32H,m),1.00(3H,t,J 7.1),0.91–0.79(6H,m,J 12.4,10.7,9.5,6.2).

[0171] Example 2: Synthesis of Lipid 6

[0172] The synthetic route for Lipid 6 is as follows:

[0173]

[0174] (1) Composition of Compound 1:

[0175] Weigh out 1.2823 g (5 mmol) of heptadecanol (h2), 1.0007 g (10 mmol) of succinic anhydride (h3) and 0.1222 g (1 mmol) of DMAP, dissolve them in 20 mL of DCM, and react at room temperature for 12 h.

[0176] After the reaction was complete, the reaction solvent was removed under reduced pressure, and the product was separated by column chromatography with PE (petroleum ether):EA (V / V) = 1:1 as the eluent. The solvent was removed under reduced pressure to obtain 1.6846 g of product Compound 1, with a yield of 94.5%.

[0177] (2) Synthesis of Lipid 6:

[0178] HCQ 335.9 mg (1.5 mmol) and Compound 1 356.5 mg (1 mmol) were dissolved in 20 mL of anhydrous DCM. DCC 309.5 mg (1.5 mmol) and DMAP 24.4 mg (0.2 mmol) were also added to the solution. The reaction was carried out at room temperature for 6 hours. The reaction was monitored by TLC, with the developing solvent being DCM:MeOH (V / V) = 20:1.

[0179] After the reaction was complete, insoluble matter in the reaction solution was filtered off, and the reaction solvent was removed by vacuum distillation. 15 mL of water and 15 mL of EA were added, followed by extraction. The mixture was allowed to stand and separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated. The product was separated by column chromatography using DCM:MeOH (V / V) = 45:1 as the eluent. The solvent was removed by vacuum distillation, yielding 485.6 mg of a pale yellow oily liquid, which was Lipid 6, with a yield of 72.0%.

[0180] H-NMR characterization is as follows: δ H (600MHz, CDCl3)8.51(1H,d,J 5.4),7.95(1H,d,J 2.1),7.71(1H,d,J9.0),7.35(1H,dd,J 8.9,2.2),6.42(1H,d,J 5.5),4.90–4.82(1H,m),4.16(2H,t),3.75–3.65(1H,m),2.71–2.46(11H,m),1.54–1.45(4H,m),1.32(3H,d,J 6.3),1.30–1.20(28H,m),1.00(3H,t,J 7.1),0.87(6H,td,J 7.0,4.0).

[0181] Example 3: Synthesis of Lipid 10

[0182] The synthetic route for Lipid 10 is as follows:

[0183]

[0184] (1) Composition of Compound 2:

[0185] Weigh 1.0639 g (3 mmol) of 2-decyl-1-tetradecyl alcohol (h4), 0.6004 g (6 mmol) of h3 and 0.1222 g (1 mmol) of DMAP, dissolve them in 20 mL of DCM, and react at room temperature for 12 h.

[0186] After the reaction was complete, the reaction solvent was removed under reduced pressure, and the product was separated by column chromatography with PE:EA (V / V) = 1:1 as the eluent. The solvent was removed under reduced pressure to obtain 1.2659 g of product Compound 2, with a yield of 92.8%.

[0187] (2) Synthesis of Lipid 10:

[0188] HCQ 335.9 mg (1.5 mmol) and Compound 2 454.7 mg (1 mmol) were dissolved in 20 mL of anhydrous DCM. DCC 309.5 mg (1.5 mmol) and DMAP 24.4 mg (0.2 mmol) were also added to the solution. The reaction was carried out at room temperature for 6 hours. The reaction was monitored by TLC, with the developing solvent being DCM:MeOH (V / V) = 20:1.

[0189] After the reaction was complete, insoluble matter in the reaction solution was filtered off, and the reaction solvent was removed by vacuum distillation. 15 mL of water and 15 mL of EA were added, followed by extraction. The mixture was allowed to stand and separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated. The product was separated by column chromatography using DCM:MeOH (V / V) = 45:1 as the eluent. The solvent was removed by vacuum distillation, yielding 590.3 mg of a pale yellow oily liquid, which was Lipid 10, with a yield of 76.4%.

[0190] H-NMR characterization is as follows: δ H (600MHz, CDCl3)8.51(1H,d,J 5.4),7.94(1H,d,J 2.2),7.70(1H,d,J9.0),7.34(1H,dd,J 8.9,2.2),6.42(1H,d,J 5.5),4.16(2H,t,J 6.2),3.97(2H,d,J 5.8),3.74–3.64(1H,m),2.72–2.47(13H,m),1.32(3H,d,J 6.4),1.30–1.20(45H,m),1.00(3H,t,J 7.1),0.88(6H,t,J 7.0).

[0191] Example 4: Synthesis of Lipid 21

[0192] The synthetic route for Lipid 21 is as follows:

[0193]

[0194] (1) Composition of Compound 3:

[0195] Weigh 0.5306 g (5 mmol) of 2-hydroxymethyl-1,3-propanediol (h5), 3.0949 g (15 mmol) of DCC, and 0.1222 g (1 mmol) of DMAP and dissolve them in 15 mL of a mixed solvent of anhydrous DCM and N,N-dimethylformamide (DMF) (volume ratio 2:1). Separately weigh 3.5654 g (10 mmol) of Compound 1 and dissolve it in 25 mL of anhydrous DCM. Slowly add the solution to the mixed solution of h5 using a dropping funnel and react at room temperature for 12 h.

[0196] After the reaction was complete, insoluble matter in the reaction solution was filtered off, and the reaction solvent was removed by vacuum distillation. PE was redissolved to precipitate insoluble impurities. The product was separated by column chromatography with PE:EA (V / V) = 7:1 as the eluent. The solvent was removed by vacuum distillation to obtain 2.0786 g of colorless liquid, which is Compound 3, with a yield of 53.1%.

[0197] (2) Composition of Compound 4:

[0198] Weigh 1.5664 g (2 mmol) of Compound 3, 0.6109 g (4 mmol) of h3, and 0.0611 g (0.5 mmol) of DMAP, dissolve them in 20 mL of DCM, and react at room temperature for 12 h. After the reaction is complete, remove the reaction solvent under reduced pressure, separate the product by column chromatography with PE:EA (V / V) = 5:1 as the eluent, and remove the solvent under reduced pressure to obtain 1.5033 g of colorless semi-solid, which is Compound 4, with a yield of 85.1%.

[0199] (3) Synthesis of Lipid 21:

[0200] HCQ 335.9 mg (1.5 mmol) and Compound 4 883.3 mg (1 mmol) were dissolved in 20 mL of anhydrous DCM. DCC 309.5 mg (1.5 mmol) and DMAP 24.4 mg (0.2 mmol) were also added to the solution. The reaction was carried out at room temperature for 12 h. The reaction was monitored by TLC, with the developing solvent being DCM:MeOH (V / V) = 20:1.

[0201] After the reaction was complete, insoluble matter in the reaction solution was filtered off, and the reaction solvent was removed by vacuum distillation. 15 mL of water and 15 mL of EA were added, followed by extraction. The mixture was allowed to stand and separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated. The product was separated by column chromatography using DCM:MeOH (V / V) = 60:1 as the eluent. The solvent was removed by vacuum distillation, yielding 679.9 mg of a pale yellow oily liquid, which was Lipid 21, with a yield of 56.6%.

[0202] H-NMR characterization is as follows: δH (600MHz, CDCl3) 8.50 (1H, d, J 5.5), 7.97 (1H, d, J 1.9), 7.73 (1H, d, J 9.0), 7.36 (1H, dd, J 8.9, 2.1), 6.43 (1H, d, J 5.6), 4.86 (2H, 28, 37, J 6.2), 4.19–4.12 (8H, m), 3.70 (1H, d, J 3.2), 2.73–2.49 (19H, m), 2.42–2.37 (1H, m, J 6.0), 1.77–1.58 (4H, m), 1.50 (8H, d, J 6.0), 1.33 (3H, d, J 6.4),1.31–1.21(48H,m),1.01(3H,t,J 7.1),0.87(12H,t,J 7.0).

[0203] Example 5: Synthesis of Lipid 29

[0204] The synthetic route for Lipid 29 is as follows:

[0205] (1) Composition of Compound 5

[0206] Weigh 2.5647 g (10 mmol) of h2 and 2.0238 g (20 mmol) of triethylamine (TEA) and dissolve them in 20 mL of anhydrous DCM. Stir and pre-cool in an ice bath. Separately weigh 1.3576 g of acryloyl chloride (h6) and dissolve it in 25 mL of anhydrous DCM. Slowly add it dropwise to the mixed solution of h2 using a dropping funnel. React in an ice bath for 4 h.

[0207] After the reaction was complete, insoluble matter in the reaction solution was filtered off, and the reaction solvent was removed by vacuum distillation. PE was redissolved to precipitate insoluble impurities. The product was separated by column chromatography, with PE:DCM (V / V) = 4:1 as the eluent. The solvent was removed by vacuum distillation to obtain 2.7465 g of colorless liquid, which is Compound 5, with a yield of 88.4%.

[0208] (2) Composition of Compound 6

[0209] Weigh out 1.8361 g (6 mmol) of Compound 5, 0.1832 g (3 mmol) of ethanolamine (h7) and 0.3036 g (3 mmol) of TEA, dissolve them in 30 mL of isopropanol, and reflux at 80 °C for 18 h.

[0210] After the reaction was complete, the reaction solvent was removed under reduced pressure, and the product was separated by column chromatography with PE:EA (V / V) = 6:1 as the eluent. The solvent was removed under reduced pressure to obtain 1.6842 g of light yellow liquid, which is Compound 6, with a yield of 82.3%.

[0211] (3) Composition of Compound 7

[0212] Weigh 1.3643 g (2 mmol) of Compound 6, 0.6109 g (4 mmol) of h3, and 0.0611 g (0.5 mmol) of DMAP, dissolve them in 20 mL of DCM, and react at room temperature for 12 h. After the reaction is complete, remove the reaction solvent under reduced pressure, separate the product by column chromatography with PE:EA (V / V) = 4:1 as the eluent, and remove the solvent under reduced pressure to obtain 1.4460 g of white solid, which is Compound 7, with a yield of 92.4%.

[0213] Synthesis of Lipid 29:

[0214] HCQ 335.9 mg (1.5 mmol) and Compound 7 782.2 mg (1 mmol) were dissolved in 20 mL of anhydrous DCM. DCC 309.5 mg (1.5 mmol) and DMAP 24.4 mg (0.2 mmol) were also added to the solution. The reaction was carried out at room temperature for 12 h. The reaction was monitored by TLC, with the developing solvent being DCM:MeOH (V / V) = 20:1.

[0215] After the reaction was complete, insoluble matter in the reaction solution was filtered off, and the reaction solvent was removed by vacuum distillation. 15 mL of water and 15 mL of EA were added, and the mixture was extracted. After standing and separation, the organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by column chromatography using DCM:MeOH (V / V) = 60:1 as the eluent. The solvent was removed by vacuum distillation, yielding 540.1 mg of a pale yellow oily liquid, which was Lipid 29, with a yield of 49.1%.

[0216] H-NMR characterization is as follows: δH (600MHz, CDCl3) 8.51 (1H,d,J 5.4), 7.95 (1H,d,J 2.1), 7.71 (1H,d,J 9.0), 7.35 (1H,dd), 6.42 (1H,d,J 5.5), 4.89–4.82 (2H,m), 4.19–4.10 (4H,m), 3.70 (1H,d,J 3.0), 2.86–2.39 (21H,m), 1.50 (8H,d,J 6.1), 1.32 (3H,d,J 6.3), 1.30–1.20 (52H,m), 1.00 (3H,t,J 9.1,5.1), 0.88 (12H,td,J 6.9,2.2).

[0217] Example 6: Synthesis of Lipid 47

[0218] The synthetic route for Lipid 47 is as follows:

[0219]

[0220] (1) Composition of Compound 8

[0221] Weigh 1.4204 g (2 mmol) of SM-102, 0.6109 g (4 mmol) of h3, and 0.0611 g (0.5 mmol) of DMAP, dissolve them in 20 mL of DCM, and react at room temperature for 12 h. After the reaction is complete, remove the reaction solvent under reduced pressure, separate the product by column chromatography, using DCM:MeOH (V / V) = 20:1 as the eluent, and remove the solvent under reduced pressure to obtain 1.5087 g of a pale yellow oily liquid, which is Compound 8, with a yield of 93.1%.

[0222] (2) Synthesis of Lipid 47

[0223] HCQ 335.9 mg (1.5 mmol) and Compound 8 810.25 mg (1 mmol) were dissolved in 20 mL of anhydrous DCM. DCC 309.5 mg (1.5 mmol) and DMAP 24.4 mg (0.2 mmol) were also added to the solution. The reaction was carried out at room temperature for 12 h. The reaction was monitored by TLC, with the developing solvent being DCM:MeOH (V / V) = 20:1.

[0224] After the reaction was complete, insoluble matter in the reaction solution was filtered off, and the reaction solvent was removed by vacuum distillation. 15 mL of water and 15 mL of EA were added, and the mixture was extracted. After standing and separation, the organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by column chromatography using DCM:MeOH (V / V) = 35:1 as the eluent. The solvent was removed by vacuum distillation, yielding 515.6 mg of a pale yellow oily liquid, which was Lipid 47, with a yield of 45.7%.

[0225] H-NMR characterization is as follows: δ H(600MHz, CDCl3)8.51(1H,d,J 5.4),7.94(1H,d,J 2.1),7.71(1H,dd,J9.0,4.6),7.34(1H,dd,J 8.9,2.1),6.42(1H,d,J 5.5),4.89–4.83(1H,m),4.19–4.09(4H,m),4.08–4.03(2H,m),3.70(1H,d,J 2.8),2.72–2.38(17H,m),2.32–2.25(4H,m),1.77–1.55(14H,m),1.50(4H,d,J 5.9),1.33–1.23(51H,m),1.00(3H,t,J 9.1,5.1),0.89–0.86(9H,m,J 7.2,4.5,1.9).

[0226] Example 7: Preparation of HL-3LNP and HL-6LNP of Luc mRNA

[0227] Lipid 29 (HL-3), DSPC (purchased from Iveto (Shanghai) Pharmaceutical Technology Co., Ltd.), Chol (purchased from Iveto (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000 (purchased from Iveto (Shanghai) Pharmaceutical Technology Co., Ltd.) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to prepare a mixed lipid solution with a HL-3 molar concentration of 12 mM. Luciferase (Luc) mRNA (Shanghai Weihuan Biotechnology Co., Ltd.) was dispersed in citrate buffer to prepare an acidic mRNA solution with a mass concentration of 0.22 mg / mL. Using a microfluidic device, the mRNA solution and the mixed lipid-ethanol solution (HL-3 molecule to mRNA nucleotide molar ratio 6:1) were mixed at a flow rate of 12 mL / min and a volume ratio of 3:1 (i.e., mRNA solution: mixed lipid-ethanol solution 3:1). The mixture was dialyzed on 0.01M PBS for 12-24 h to remove ethanol. After dialyzing, the LNP solution was concentrated by ultrafiltration (MWCO 10 kDa) and filtered through a 0.22 μm sterile filter to obtain HL-3LNP of Luc mRNA.

[0228] The preparation methods for HL-6LNP, DLin-MC3-DMA(MC3)LNP (abbreviated as MC3 LNP) and SM-102LNP formulations of Luc mRNA are the same as above. The hydroxychloroquine-derived lipid used in HL-6LNP is Lipid 21.

[0229] Example 8: Formulation characterization of HL-3LNP and HL-6LNP of Luc mRNA

[0230] The LNP formulation was diluted to an mRNA concentration of 0.01 mg / mL. The average particle size and polydispersity index (PDI) of LNP were determined using a Malvern particle size analyzer. The encapsulation efficiency (EE) of LNP was determined using a quantitative ribonucleic acid assay kit (Quant-it RibogreenRNA). The results are shown in Table 1.

[0231] Table 1. Characterization parameters of MC3 LNP, SM-102 LNP, HL-3 LNP and HL-6 LNP formulations

[0232] LNP (n=3) MC3 SM-102 HL-3 HL-6 Particle size (nm) 68.77±0.85 66.59±0.18 54.16±0.27 58.77±1.14 PDI 0.16±0.03 0.09±0.01 0.09±0.01 0.23±0.01 Encapsulation efficiency (%) 93.21 94.53 95.64 94.67

[0233] Example 9: Detection of the epigenetic pKa of HL-3 LNP and HL-6 LNP of Luc mRNA

[0234] To determine the apparent pKa of LNPs, LNPs were incubated with TNS (2-p-toluamino-6-naphthalenesulfonic acid) under different pH conditions, and the fluorescence signal of TNS was detected. For example, in a low pH environment, conventionally ionizable lipids dissociate, and the LNP surface becomes positively charged, interacting with the negatively charged TNS, causing it to enter a hydrophobic environment and produce fluorescence. In a high pH environment, the LNP becomes negatively charged, and the fluorescence signal weakens. Based on this, the pH value at which 50% of the ionizable lipids are charged is defined as the apparent pKa. A series of buffer solutions with a pH range of 2.0-12.0 were prepared by adding 2M sodium hydroxide and 2M hydrochloric acid to a basic buffer solution (10mM sodium phosphate, 10mM sodium borate, 10mM sodium citrate, 150mM sodium chloride). Add 94 μL of the aforementioned buffer solutions at different pH values ​​to each well of a 96-well black-background plate, followed by 4 μL of LNP solution (diluted with PBS buffer (pH 7.4) to a mRNA concentration of 0.05 mg / mL) and 2 μL of TNS solution (300 μM TNS molar concentration, 10% DMSO / water (V / V) solvent). After adding the LNP solution, gently shake to mix thoroughly, incubate at room temperature in the dark for 5 min, and measure the fluorescence intensity at an excitation wavelength of 325 nm and an emission wavelength of 435 nm using enzyme labeling. Fluorescence intensity is plotted on the Y-axis, buffer pH on the X-axis, and the LogEC50 value represents the apparent pKa of the LNP to be tested.

[0235] The apparent pKa of SM-102LNP, HL-3LNP and HL-6LNP prepared in Example 7 was tested using the method described above, and the results are shown in Table 2.

[0236] Table 2. Apparent pKa test data for SM-102LNP, HL-3LNP, and HL-6LNP (n=3)

[0237]

[0238]

[0239] The results showed that the hydroxychloroquine-derived lipid LNP had a completely opposite pKa curve to the traditional ionizable lipid SM-102-LNP. Based on the analysis of the LNP structure and the TNS detection principle, it can be seen that the hydroxychloroquine-derived lipid LNP is a nanoparticle with a novel spatial structure that is different from the traditional LNP.

[0240] Example 10: Freeze-thaw stability of HL-3LNP in Luc mRNA

[0241] The newly prepared HL-3LNP and SM-102LNP of Luc mRNA, prepared according to Example 7, were subjected to repeated freeze-thaw cycles (FT) at -20°C and -80°C five times. Particle size, PDI, and encapsulation efficiency were measured, and the results are shown in Tables 3, 4, and 5, respectively. The results indicate that HL-3LNP of Luc mRNA exhibits more stable formulation properties than SM-102LNP of Luc mRNA during freeze-thaw cycles, suggesting that the nanostructure of hydroxychloroquine-derived lipid particles is more stable than that of the traditional ionizable lipid SM-102-LNP.

[0242] Table 3. Freeze-thaw stability test of HL-3LNP of Luc mRNA at -20℃

[0243]

[0244]

[0245] Table 4. Freeze-thaw stability test of SM-102LNP of Luc mRNA at -20℃

[0246]

[0247] Table 5. Freeze-thaw stability test of HL-3LNP of Luc mRNA at -80℃

[0248]

[0249] In the above freeze-thaw experiments, mRNA integrity was simultaneously detected. LNP freeze-thawed samples were treated with a Triton X-100 gel at 70°C for 3 min, and mRNA integrity was detected by Bio-Rad gel electrophoresis. The results are as follows: Figure 1 As shown in the figure. The results indicate that the HL-3 LNP of Luc mRNA was not destroyed after Triton treatment, demonstrating the strong stability of hydroxychloroquine-derived lipid LNPs.

[0250] The freeze-thaw stability and apparent pKa experiments described above both indicate that the hydroxychloroquine-derived lipid LNP has a novel nanostructure that is distinct from traditional LNP.

[0251] Example 11: In vivo expression of HL-3LNP and HL-6LNP of Luc mRNA in Luc mRNA

[0252] HL-3LNP, HL-6LNP, DLin-MC3-DMA(MC3)LNP, and SM-102LNP formulations of Luc mRNA were prepared according to the method in Example 7. Three male BALB / c mice (purchased from Beijing Huafukang Biotechnology Co., Ltd., weighing 20-25g) were used in each group. Each BALB / c mouse was injected intravenously with 5μg of Luc mRNA (100μL of LNP solution with a mRNA concentration of 0.05mg / mL). Four hours later, 3mg of D-fluorescein potassium salt (200μL of solution with a concentration of 15mg / mL in PBS buffer) was injected intraperitoneally. Bioluminescence signals were detected using an IVIS small animal in vivo imaging system. Control was physiological saline. Results are as follows: Figure 2 As shown in Table 6.

[0253] Table 6. In vivo expression detection results of Luc mRNA formulations containing HL-3LNP, HL-6LNP, MC3LNP, and SM-102LNP.

[0254]

[0255]

[0256] The results showed that the in vivo expression of hydroxychloroquine-derived lipid LNP mRNA was significantly better than that of MC3 and comparable to that of SM-102.

[0257] Example 12: Repeated-dose trial of HL-3LNP

[0258] The HL-3LNP, DLin-MC3-DMA(MC3)LNP, and SM-102LNP formulations of Luc mRNA synthesized from unmodified uracil (U) were prepared according to the method in Example 7. Administration and detection methods were as described in Example 11. Three mice were administered the drug four times per group, with each dose administered three days apart (3-day gap). Four hours after administration, bioluminescent signals were detected, and mouse body weight changes were monitored. The results are shown in Tables 7 and 8. Figure 3 , Figure 4 As shown.

[0259] Table 7 Results of in vivo bioluminescence signal detection after repeated administration of the formulation.

[0260]

[0261] Table 8 Results of monitoring body weight changes in mice after repeated administration of the formulation.

[0262]

[0263] Hydroxychloroquine-derived lipids effectively inhibited the decline in mRNA expression levels after repeated administration and improved safety, which is related to hydroxychloroquine's inhibitory effect on immune activation and inflammatory responses. These results suggest that HL-LNP has promising applications in the field of protein supplementation requiring repeated administration.

[0264] Example 13: Cytokine detection after a single dose of HL-3LNP

[0265] The HL-3LNP, MC3LNP, and SM-102LNP formulations of unmodified uracil (U) synthesized Luc mRNA (Shanghai Weihuan Biotechnology Co., Ltd.) were prepared according to the method in Example 7. Six male BALB / c mice (Beijing Huafukang Biotechnology Co., Ltd., weighing 20-25g) were injected intramuscularly into each mouse with 25μg of Luc mRNA (250μL of LNP solution with an mRNA concentration of 0.1mg / mL). Serum was collected from the orbital blood of the mice at 6h and 48h after administration. The concentrations of MCP-1 and IL-6 in the mouse serum were detected using the Luminex Assay R&D SYSTEMS kit, and the results are shown in Table 9.

[0266] Table 9. Concentrations of MCP-1 and IL-6 in mouse serum after a single HL-3LNP administration.

[0267]

[0268] The results showed that HL-3 lipids significantly inhibited the release of inflammatory factors induced by LNP, and the serum levels of MCP-1 and IL-6 were significantly lower than those in the SM-102 group.

[0269] Example 14: Inhibition of innate immune response induced by HL lipids at the cell line level in poly(I:C)

[0270] The poly(I:C) (Sigma-Aldrich) formulations of HL-3LNP, HL-6LNP and SM-102LNP were prepared according to the method of Example 7, with the concentration of poly(I:C) controlled at 250 ng / μL.

[0271] The RPMI 1640 medium (Invitrogen) suspension of THP1-Dual Cells (InvivoGen) was seeded into 96-well plates at a rate of 50,000 cells / 180 μL / well for transfection with poly(I:C).

[0272] The LNP formulation of poly(I:C) was diluted tenfold with an 8% sucrose Tris-acetic acid solution to a poly(I:C) concentration of 25 ng / μL, and then set aside for use.

[0273] Take 1 μg of poly(I:C) and add it to 20 μL of Opti-MEM (Invitrogen) and mix well. Separately, take 4 μL of Lipofectamine 2000 (Invitrogen) and add it to 20 μL of Opti-MEM and mix well. Combine the above Opti-MEM solutions, shake gently to mix well, and let stand at room temperature for 10 min before use.

[0274] The above-mentioned poly(I:C) samples were added to THP1-Dual 96-well plates, 20 μL per well (i.e., 500 ng of poly(I:C) per well), with three replicates per group, and incubated in a 37℃ CO2 incubator for 24 h.

[0275] Add 200 ng of hairpinRNA (Shanghai Weihuan Biotechnology Co., Ltd.) to 20 μL of Opti-MEM and mix well. Separately, add 1 μL of Lipofectamine 2000 to 20 μL of Opti-MEM and mix well. Combine the Opti-MEM solutions, gently shake to mix, and let stand at room temperature for 10 min. Add the prepared hairpinRNA sample to a THP1-Dual 96-well plate, 20 μL per well (500 ng poly(I:C) per well), with three replicates as the hairpinRNA control group. Incubate at 37℃ in a CO2 incubator for 24 h.

[0276] Another 8% sucrose Tris-acetic acid solution was added to THP1-Dual 96-well plates, 20 μL per well, with three replicates, serving as the 8% Tris AC control group. The plates were incubated in a 37℃ CO2 incubator for 24 h.

[0277] RPMI 1640 medium was added to THP1-Dual 96-well plates, 20 μL per well, with three replicates, serving as the untreated control group. The plates were incubated in a 37℃ CO2 incubator for 24 h.

[0278] After 24 hours, cell viability was detected using the CellTiter-Glo 3D Cell Viability Assay Kit (Promega (Beijing) Biotechnology Co., Ltd.), and the results are shown in Table 10.

[0279] 1.5 mL of QUANTI-Luc 4Reagent (20X) (InvivoGen) was diluted 20-fold with 2.35 mL of sterile water to obtain the QUANTI-Luc solution. 20 μL of cell supernatant from a 96-well plate was added to a 96-well white plate, followed by 20 μL of QUANTI-Luc solution. After gentle mixing, bioluminescence was immediately detected. This indicated the activation of the Lucia luciferase reporter gene controlled by ISG54, thus characterizing ISG54 activation. The results are shown in Table 10.

[0280] Add 1 mL of QUANTI-Blue Reagent (InvivoGen) and 1 mL of QUANTI-Blue Buffer (InvivoGen) to 98 mL of sterile water and mix well to obtain the QUANTI-Blue solution. Add 20 μL of cell supernatant from a 96-well plate to a 96-well clear plate, and then add 180 μL of QUANTI-Blue solution. After gently mixing, immediately measure the absorbance at 620 nm. This absorbance represents the NF-κB-activated secretory embryonic alkaline phosphatase (SEAP) reporter gene, thus characterizing NF-κB activation, as shown in Table 10.

[0281] Table 10

[0282]

[0283] The results showed that SM-102LNP loaded with poly(I:C) induced high levels of ISG54 and NF-κB, while HL-3LNP and HL-6LNP were at levels comparable to the blank control, indicating that HL lipids have a significant inhibitory effect on the innate immune response induced by poly(I:C) at the cell line level.

[0284] Example 15. Toxicity of HL lipid inhibitor poly(I:C) in mice

[0285] The mixed nucleic acid formulations of HL-6LNP and SM-102LNP co-loaded with poly(I:C) (Sigma-Aldrich) and Luc mRNA (Shanghai Weihuan Biotechnology Co., Ltd.) were prepared according to the method in Example 7, wherein the mass ratio of poly(I:C) to Luc mRNA was 1:1, and the concentrations of both poly(I:C) and mRNA were controlled at 0.05 mg / mL.

[0286] Male BALB / c mice (Beijing Huafukang Biotechnology Co., Ltd., weighing 20-25g) were injected intravenously via the tail vein with the above-mentioned mixed nucleic acid preparation. The dosage was 5 μg poly(I:C) and 5 μg Luc mRNA per mouse (i.e., 100 μL of the above-mentioned mixed nucleic acid preparation), with 5 mice in each group. Luc expression was detected at 4, 24, and 48 hours after administration, as described in Example 11, and the results are shown in Table 11. At 4 hours after administration, orbital blood was collected from the mice to separate serum, and the concentrations of serum cytokines were detected using the Luminex Assay Multifactor Detection Kit (R&DSYSTEMS), and the results are shown in Table 12. The survival status of the mice was observed and recorded daily. All mice in the SM-102 group died after 48 hours, while all mice in the HL-6 group survived well after 216 hours.

[0287] Table 11

[0288] Time point SM-102 (mean ± SD, p / s) HL-6 (mean ± SD, p / s) 4h 8.314 x 10 10 ± 4.088 x 10 10 ]]> 3.242 x 10 10 ± 9.859 x 10 9 ]]> 24h 5.102 x 10 9 ± 2.621 x 10 9 ]]> 1.361 x 10 9 ± 9.318 x 10 8 ]]> 48h All mice died 1.144 x 10 8 ±4.810 x 10 7 ]]>

[0289] Table 12

[0290] Cytokines SM-102 (mean ± SD, pg / mL) HL-6 (mean ± SD, pg / mL) IL-6 23882.18±11228.18 8934.14±2625.82 TNF-a 1747.17±407.32 1146.2±561.69 IFN-g 3491.91±1386.57 1316.3±233.92 MCP-1 18694.64±21860.04 9359.72±5459.27 IL-2 28.57±1.71 16.33±6.86

[0291] The above results indicate that intravenous injection of conventional lipid LNPs encapsulating poly(I:C) was lethal to mice, while all mice injected intravenously with HL-LNP@poly(I:C) survived, demonstrating that HL lipids significantly inhibited the lethal toxicity of poly(I:C) in mice. Cytokine monitoring showed that poly(I:C) encapsulated in conventional lipid LNPs induced a severe cytokine storm in mice, while the introduction of HL lipids significantly reduced cytokine levels and protected the mice's survival.

[0292] Example 16. Application of HL lipids in varicella-zoster virus (VZV) vaccine

[0293] VZV gE mRNA (Shanghai Bluebird Biopharmaceutical Co., Ltd.) HL-3LNP, HL-6LNP, and SM-102LNP were prepared according to the method in Example 7, and the poly(I:C) and VZV gE mRNA concentrations were controlled at 0.02 mg / mL. Four groups were set up using a placebo of 8% sucrose Tris-acetic acid solution: placebo, SM-102-gE, HL-3-gE, and HL-6-gE. Each group consisted of 5 male BALB / c mice (Beijing Huafukang Biotechnology Co., Ltd., weighing 20-25 g). Each immunization dose was 2 μg mRNA (100 μL of the above gE mRNA preparation) injected intramuscularly. The placebo group was injected with the same volume of 8% sucrose Tris-acetic acid solution.

[0294] The administration and sampling procedures were as follows: On Day 0, Prime immunization was performed, and blood was collected 6 hours after administration. Cytokines were detected using the Luminex Assay Multifactor Detection Kit (R&D SYSTEMS) (Prime-6h), and the results are shown in Table 13. On Day 20, blood was collected, and the VZV gE binding antibody titer was detected by ELISA (Prime-Week 3), and the results are shown in Table 14. On Day 21, Boost immunization was performed, and blood was collected 6 hours after administration to detect cytokines (Boost-6h). On Day 35, blood was collected, and the VZV gE binding antibody titer was detected by ELISA (Boost-Week 2). Mice were sacrificed, and spleens were collected. VZV gE-specific cellular responses were detected by ELISA, and the results are shown in Table 15.

[0295] The ELISA method for detecting VZV gE binding antibody titers is as follows: Recombinant VZV gE antigen (ACRO Biosystems) was dissolved in 50 μL / well coating buffer and coated into 96-well plates at a concentration of 100 ng / well, then incubated overnight at 4°C. Subsequently, 2% bovine serum albumin blocking buffer was prepared using PBS solution containing 0.1% Tween 20 (PBST), and the antigen-coated plates were blocked at room temperature for 1 hour. After an initial 250-fold dilution of serum samples, 11 serial dilutions were performed using PBS. After washing the plates with PBST, serially diluted serum was added, and the plates were incubated at room temperature for 2 hours. To detect gE-specific IgG antibody levels, HRP-labeled goat anti-mouse IgG secondary antibody (Proteintech) was added and incubated at 37°C for 1 hour, followed by color development with tetramethylbenzidine (TMB) substrate solution (Invitrogen). The reaction was terminated by adding 1N sulfuric acid after approximately 10 minutes, and the absorbance was measured at 450 nm using a microplate reader.

[0296] The ELISpot assay for VZV gE-specific cell responses was performed as follows: Spleen cells were stimulated for 1 hour with a gE peptide library (GenScript, 2 μg / mL) and anti-mouse CD28 antibody (BD Bioscience), followed by 5 hours of culture with a monensin-containing protein transport inhibitor (BD Bioscience). One million cells were seeded per well into ELISpot plates (MABTECH) pre-coated with IFN-γ or IL-2 specific antibodies and cultured at 37°C for 48 hours. The wells were then washed with PBS, and biotinylated anti-IFN-γ or anti-IL-2 antibodies were added, followed by incubation at room temperature for 2 hours, and then reaction with streptavidin-alkaline phosphatase (ALP) for 1 hour at room temperature. After adding substrate solution to initiate the spot colorimetric reaction, the wells were rinsed with deionized water. Finally, the plates were allowed to air dry in the dark, and spot counting was performed using an ELISpot analyzer (AT-SPOT, SINSAGE).

[0297] Table 13

[0298]

[0299] Table 14

[0300] Antibody titer (mean ± SD) Placebo SM-102-gE HL-3-gE HL-6-gE Prime-Week 3 250±0 22400±8763.5 8800±4381.7 8400±4979.9 Boost-Week 2 300±111.8 179200±70108.4 179200±70108.4 128000±0

[0301] Table 15

[0302] SFU / 10 6 cells (mean ± SD) Placebo SM-102-gE HL-3-gE HL-6-gE IFN-g 0±0 3121.2±704.9 2952.2±841.3 2447.8±1171.4 IL-2 0±0 1695.2±303.4 1683.2±288.1 1489.6±634.1

[0303] As shown in Table 14, the HL-gE vaccine and the SM-102-gE vaccine induced comparable levels of humoral immune response; as shown in Table 15, the HL-gE vaccine and the SM-102-gE vaccine induced comparable levels of cellular immune response. Regarding inflammatory cytokines, HL lipids significantly inhibited LNP-mRNA-induced cytokines at levels comparable to placebo, while these inflammatory cytokines can induce systemic inflammatory side effects in the application of vaccines for the prevention of infectious diseases. These results indicate that HL lipid-based mRNA vaccines can induce specific immune responses comparable to those in positive controls, while simultaneously inhibiting the activation of innate immunity by LNP-mRNA and reducing the release of pro-inflammatory cytokines, demonstrating the promising application prospects of HL lipids in mRNA vaccines.

[0304] VZV gE mRNA sequence (SEQ ID NO.:01):

[0305]

Claims

1. A compound as shown in Formula I or a pharmaceutically acceptable salt thereof, in: T is C 1-30 Alkyl, C 2-30 alkenyl, C 2-30 acetylinyl or X is a single bond, O, S, or N(R) x ); R x For H or C 1-10 alkyl; L 1 It is a single bond, -(CH2) n1 -、 X and L 1 Not both are single bonds; A 1 Terminal A is connected to X. 2 and A 3 The end is connected to the Y-axis; A 1 -(CH2) n2 -; A 2 and A 3 Independently a single bond or -(CH2) n2 -; M 1 and M 2 Independently O or NH; M 3 for O, S or N(R) x ); Y is N or CR Y ;R Y For H, C 1-30 Alkyl, C 2-30 alkenyl, C 2-30 acetylinyl R 1 and R 2 Independently for C 1-30 Alkyl, C 2-30 alkenyl, C 2-30 acetylinyl D 1 D 2 and D 3 Independently O or NH; B 1 and B 3 Independently -(CH2) n3 -; B 2 Independently for C 1-30 Alkyl, C 2-30 alkenyl or C 2-30 alkynyl group; L 2 for K 1 Terminal is connected to Y, K 2 End and Z 1 connect; K 1 and K 3 Independently -(CH2) n4 -; K 2 Independently a single bond or -(CH2) n4 -; H 1 H 2 H 3 H 4 and H 5 Independently O or NH; H 6 Independently O, S, or NH; Z 1 Independently N or CH; R 3 and R 4 Independently for C 1-30 Alkyl, C 2-30 alkenyl or C 2-30 alkynyl group; n1, n2, n3, and n4 are independent integers from 1 to 10.

2. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1) T, R Y B 2 R 1 R 2 R 3 and R 4 In, the C 1-30 The alkyl group is independently a straight-chain alkyl group or a branched alkyl group, preferably a straight-chain alkyl group; (2) T, R Y B 2 R 1 R 2 R 3 and R 4 In, the C 1-30 Alkyl groups are independently C 4-20 Alkyl, such as C 4-18 Alkyl groups, for example, C 4-10 alkyl; (3) T, R Y B 2 R 1 R 2 R 3 and R 4 In, the C 2-30 The number of alkenyl bonds in the alkenyl group can be 1, 2, 3, 4 or 5 independently, for example 1, 2 or 3; (4) T, R Y B 2 R 1 R 2 R 3 and R 4 In, the C 2-30 The alkenyl group can be a straight-chain alkenyl or a branched alkenyl, preferably a straight-chain alkenyl; (5) T, R Y B 2 R 1 R 2 R 3 and R 4 In, the C 2-30 The alkenyl group is independently C 4-20 alkenyl groups, such as C 4-20 Alkenyl, for example C 8-20 alkenyl; (6) T, R Y B 2 R 1 R 2 R 3 and R 4 In, the C 2-30 The alkynyl group can be either a straight-chain alkynyl group or a branched-chain alkynyl group, wherein the C 2-30 The number of alkyne bonds in the alkenyl group is independently 1, 2, 3, 4, or 5, preferably C. 4-20 Straight-chain alkynyl groups; and (7)R x In, the C 1-10 Alkyl group is C 1-6 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl; Preferably, the compound shown in Formula I satisfies one or more of the following conditions: (1) In T, the C 2-30 The alkenyl group is C 4-20 Straight-chain alkenyl, the C 4-20 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3; (2)R 1 and R 2 In, the C 1-30 Alkyl groups are independently C 4-20 Straight-chain alkyl; (3)R 1 and R 2 In, the C 2-30 The alkenyl group is independently C 4-20 Straight-chain alkenyl, the C 4-20 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3; (4)R 3 and R 4 In, the C 1-30 Alkyl groups are independently C 4-20 Straight-chain alkyl groups, such as C 4-10 Straight-chain alkyl; (5)R Y B in 2 In, the C 2-30 The alkenyl group is C 4-20 Straight-chain alkenyl, the C 4-20 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3; (6)R 1 and R 2 B in 2 In, the C 1-30 Alkyl groups are independently C 4-20 Straight-chain alkyl groups; and (7)R 1 and R 2 B in 2 In, the C 2-30 The alkenyl group is independently C 4-20 Straight-chain alkenyl, the C 4-20 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3; Preferably, the compound shown in Formula I satisfies one or two of the following conditions: (1) T, R Y B 2 R 1 R 2 R 3 and R 4 In, the C 1-30 Alkyl groups are independently (2) T, R Y B 2 R 1 R 2 R 3 and R 4 In, the C 2-30 Alkene independently 3. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1) T represents scheme 1 or scheme 2. Option 1: T is C 1-30 Alkyl, C 2-30 alkenyl or C 2-30 alkynyl group; Option 2: T is C 2-30 alkenyl or Preferably, T is C 4-20 straight-chain alkenyl or The C 4-20 The number of alkene bonds in the straight-chain alkenyl group is 1, 2, or 3; more preferably, T is... (2) X is a single bond or O; preferably, X is a single bond; (3)R x For H; (4)L 1 It is a single bond, -(CH2) n1 -、 Ideally, L 1 for (5) n1 and n2 are independently 1, 2, 3, 4, 5, 6, 7 or 8; preferably, n1 and n2 are independently 1, 2, 3 or 4; (6)M 1 It is O; (7)M 3 Independently or N(R) x ); (8)R Y For H, Ideally, R Y For H or Better yet, R Y For H; (9)R Y In the middle, B 2 Independently for C 2-30 Alkenyl; preferably, B 2 Independently for C 4-20 Straight-chain alkenyl, the C 4-20 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3; (10)R 1 and R 2 Independently for C 1-30 Alkyl, C 2-30 alkenyl, Or, R 1 and R 2 Independently for C 1-30 Alkyl, C 2-30 alkenyl, C 2-30 acetylinyl Ideally, R 1 and R 2 Independently for C 4-20 straight-chain alkyl, C 4-20 straight-chain alkenyl, Better yet, R 1 and R 2 Independently (11)D 1 D 2 and D 3 It is O; (12) n3 is 1, 2, 3, 4, 5, 6, 7 or 8; (13)R 1 and R 2 In the middle, B 2 Independently C 1-30 Alkyl or C 2-30 Alkenyl; preferably, B 2 Independently for C 4-20 Straight-chain alkyl or C 4-20 Straight-chain alkenyl, the C 4-20 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3; (14)L 2 Independently (15)H 1 H 2 H 3 and H 5 It is O; (16)H 6 Independently Or NH; (17)K 2 It is a single bond; (18) n4 is independently 1, 2, 3, 4, 5, 6, 7 or 8; preferably, n4 is independently 1, 2, 3 or 4; (19)Z 1 For CH; (20)R 3 and R 4 Independently for C 1-30 Alkyl; preferably, R 3 and R 4 Independently for C 4-20 Straight-chain alkyl groups, for example, C 4-10 Straight-chain alkyl groups.

4. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1)L 1 For single bond, Preferably, bit "1" is connected to X, and bit "2" is connected to A. 3 The terminal is connected to Y; more preferably, L 1 for The "1" bit is connected to X, and the "2" bit is connected to Y; (2)B 2 Independently (3)K 1 Independently (4)K 2 Independently for single bonds, (5)K 3 Independently (6)R 3 and R 4 Independently Better yet, R 3 and R 4 for Preferably, the compound shown in Formula I satisfies one or more of the following conditions: (1)-XL 1 - for single bond, O, Preferably, bit "2" and the Y terminal are connected to Y; more preferably, -XL 1 -for The "2" bit is connected to Y; (2)L 2 Independently Preferably, bit "1" is connected to Y, and bit "2" is connected to Z. 1 Connection; better yet, L 2 for Bit "1" is connected to Y, and bit "2" is connected to Z. 1 connect; (3)R Y For H or More preferably, in the compound shown in Formula I, R 1 and R 2 Independently For example, R 1 and R 2 Independently 5. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound shown in Formula I is selected from any of the following schemes: Option 1: The compound shown in Formula I is the compound shown in Formula I-1: R 1 and R 2 Independently for C 1-30 Alkyl, C 2-30 alkenyl, C 2-30 acetylinyl X, L 1 Y, D 1 D 2 D 3 , B 1 B 2 and B 3 The definition is as described in any one of claims 1-4; Option 2: The compound shown in Formula I is a compound shown in Formula I-2: X, L 1 Y, L 2 Z 1 R 3 and R 4 The definition is as described in any one of claims 1-4; Option 3: The compound shown in Formula I is a compound shown in Formulas I-3: R Y R 1 and R 2 Independently for C 1-30 Alkyl, C 2-30 alkenyl, C 2-30 acetylinyl X, L 1 D 1 D 2 D 3 B 1 B 2 and B 3 The definition is as described in any one of claims 1-4; Preferably, the compound shown in Formula I is a compound shown in Formula I-2: X is a single bond; L 1 for A 1 -(CH2) n2 -;A 2 -(CH2) n2 -; n2 can be 1, 2, 3 or 4 independently; M 1 It is O; Y is either N or CH; L 2 Independently K 1 and K 3 Independently -(CH2) n4 -; K 2 Independently a single bond or -(CH2) n4 -; n4 can be 1, 2, 3 or 4 independently; H 1 H 3 and H 4 It is O; Z 1 For CH; R 3 and R 4 Independently for C 4-10 Straight-chain alkyl groups.

6. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound shown in Formula I is any of the following compounds:

7. A lipid carrier comprising substance Z, said substance Z being a compound of Formula I as described in any one of claims 1-6 or a pharmaceutically acceptable salt thereof; Preferably, the lipid carrier satisfies one or more of the following conditions: (1) The lipid carrier further includes a diluent; for example, the diluent is citrate buffer or ethanol; (2) The lipid carrier also includes phospholipids; for example, distearate phosphatidylcholine; (3) The lipid carrier further includes PEG lipids; for example, the PEG lipids are selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol; another example is PEG-modified dimyristoylglycerol; (4) The lipid carrier also includes sterols, for example, the sterols are selected from animal, plant or fungal sterols; for example, the sterols are selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassinosteroidol, tomatine, ursolic acid and α-tocopherol, and even more so, cholesterol; Preferably, the lipid carrier comprises the diluent, substance Z, the phospholipid, the PEG lipid, and the sterol; More preferably, the lipid carrier satisfies one or more of the following conditions: (1) The total molar mass of the substance Z, the phospholipid, the PEG lipid and the sterol is 100%, and the molar content of the substance Z is 30 mol% to 60 mol%; for example, 40 mol% to 55 mol%; or even 50 mol%; (2) In the lipid carrier, the molar ratio of substance Z to phospholipid is 1-25:1, preferably 2-10:1, for example 5:1; (3) In the lipid carrier, the molar ratio of substance Z to sterol is (0.5-3):1, preferably (0.5-3):1, for example 1.3:1; (4) In the lipid carrier, the molar ratio of substance Z to PEG lipid is 20-50:1, preferably 20-40:1, for example 33:1; (5) The molar ratio of substance Z to the volume of the diluent is (5-25) mmol:1L; for example, (10-15) mmol:1L; or for example, 12 mmol:1L. (6) The lipid carrier is composed of the substance Z, the diluent, the phospholipid, the PEG lipid and the sterol; or the lipid carrier is composed of the substance Z, the phospholipid, the PEG lipid and the sterol.

8. A lipid nanoparticle comprising a therapeutic agent and / or a preventive agent and a lipid carrier as described in claim 7; preferably, the therapeutic agent and / or preventive agent is one or more nucleic acids; more preferably, the therapeutic agent and / or preventive agent is single-stranded deoxyribonucleic acid, double-stranded DNA, small interfering RNA, asymmetric double-stranded small interfering RNA, microRNA, small hairpin RNA, circular RNA, transfer RNA, or messenger RNA, most preferably mRNA, such as firefly luciferase mRNA, unmodified uracil-synthesized Luc mRNA, polyinosinic-polycytidylic acid, or VZV gE mRNA.

9. A composition comprising substance Z, said substance Z being a compound of Formula I as described in any one of claims 1-6 or a pharmaceutically acceptable salt thereof; Preferably, the composition further comprises one or more of a diluent, phospholipids, PEG lipids, sterols, and therapeutic and / or preventive agents; said diluent, phospholipids, PEG lipids, sterols, and therapeutic and / or preventive agents as described in claim 8; More preferably, the lipid carrier satisfies one or more of the following conditions: (1) In the composition, the substance Z forms a lipid carrier as described in claim 7 with one or more of the diluent, phospholipid, PEG lipid and sterol; preferably, the lipid carrier forms lipid nanoparticles as described in claim 8 with the therapeutic agent and / or preventive agent; (2) In the composition, the encapsulation rate of the therapeutic agent and / or preventive agent is at least 94.5%, preferably at least 95%, for example 95.6%; (3) In the composition, the particle size of the therapeutic agent and / or preventive agent is 50-70 nm, for example 50-60 nm, or even 55 nm or 59 nm.

10. The use of a compound of Formula I as described in any one of claims 1-6, or a pharmaceutically acceptable salt thereof, or a lipid carrier as described in claim 7, in the preparation of a medicament for the prevention and / or treatment of inflammation, for example, said inflammation being infectious inflammation.

11. The use of a compound of Formula I as described in any one of claims 1-6, or a pharmaceutically acceptable salt thereof, or a lipid carrier as described in claim 7, in the preparation of a medicament for the prevention and / or treatment of inflammation caused by nucleic acids, wherein the nucleic acid is defined as described in claim 8.

12. The use of a compound of Formula I as described in any one of claims 1-6, or a pharmaceutically acceptable salt thereof, or a lipid carrier as described in claim 7, in the preparation of a medicament for the prevention and / or treatment of inflammation caused by elevated levels of MCP-1, IL-6, ISG54, or NF-κB, for example, the elevated levels of MCP-1, IL-6, ISG54, or NF-κB being induced by the nucleic acid as described in claim 8.

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