Lipid nanoparticles, formulations, methods of making and uses thereof

Abstract

A kind of lipid nanoparticle, preparation, its preparation method and purposes.The present application is by the ursolic acid identified from natural product library incorporation biodegradable cationic phosphamide derivative LNP preparation, develop a kind of non-inflammatory LNP (NIF-LNP).Compared with the LNP containing ALC-0315, the lung protein expression of NIF-LNP is significantly enhanced by 40 times, and does not cause significant reactivity.In addition, we found that the five-component LNP formed by loading natural products such as UA into LNP can deliver mRNA and siRNA, and the introduction of UA can significantly improve the gene knockdown efficiency of siRNA.In mouse asthma model, LNP4 and LNP6 loaded with Muc5ac-siRNA show good therapeutic effect.

Description

Technical Field

[0001] This article generally relates to ionizable lipid nanoparticle technology, with a particular focus on lipid nanoparticles, formulations, their preparation methods, and applications. Background Technology

[0002] mRNA-based therapies have revolutionized treatment for a range of diseases, from genetic disorders to infectious diseases and malignancies. Lipid nanoparticles (LNPs), proven effective in developing mRNA vaccines against COVID-19, are the only clinically approved mRNA vectors and show great potential in protein replacement therapies and gene editing. However, clinical application of lipid-60-formulated RNA vaccines has been associated with dose-limiting systemic inflammatory responses in humans, causing numerous side effects including pain, swelling, and fever. Compared to vaccines that elicit a strong immune response with minimal antigen, therapeutic mRNAs require up to 1,000 times more protein to meet the therapeutic window. This increased demand can lead to higher reactivity—an immune response characterized by inflammation and side effects—and may conversely reduce mRNA transfection efficiency by activating endogenous RNA sensors. The inherent reactivity of LNPs exacerbates the progression of many chronic diseases that are typically associated with inflammation, posing an additional challenge to treating chronic diseases with mRNA therapies. Studies have found that reactivity is significantly enhanced in the context of chronic diseases in animal models, sometimes even leading to death. Therefore, a key step in unlocking the full potential of mRNA as a therapeutic agent is to improve mRNA delivery efficiency while minimizing reactivity. Summary of the Invention

[0003] Based on this, this application provides a lipid nanoparticle (LNP), comprising: At least one ionizable lipid, optionally comprising about 20 mol% to about 70 mol% of LNP; and At least one compound capable of activating V-ATPase, optionally, the compound capable of activating V-ATPase accounts for about 2.5 mol% to about 50 mol% of LNP.

[0004] In some embodiments, the lipid nanoparticles have ionizable lipids in a molar ratio of about 20%-70%, 30%-50%, 33%-47%, 35%-45%, 38%-43%, or 40%-41%.

[0005] In some embodiments, the lipid nanoparticles have a molar ratio of about 2.5%-50%, 10%-30%, 13%-27%, 15%-25%, 18%-22%, or 20%-21% of a compound capable of activating V-ATPase.

[0006] In some implementations, the compounds that can activate V-ATPase are selected from ursolic acid, oleanolic acid, cholic acid, crataegolic acid, and crosolic acid and their derivatives.

[0007] In some implementations, the compound capable of activating V-ATPase is selected from compounds of the following formula: In formula (I-1), Y is -O- or -NH-, Z is hydrogen or C1-C6 alkylene or a single bond, and R1 is hydrogen, carboxyl or sulfonic acid or absent (when Z is hydrogen). R2 and R3 are each independently hydrogen or methyl; R4 and R5 are each independently hydrogen or methyl; R6 is hydroxyl or hydrogen, R7 is hydroxymethyl or methyl, and R8 is hydroxyl or hydrogen.

[0008] In some embodiments, in formula (I-1), Y is -O- or -NH-, Z is hydrogen, -CH2-, -CH2-CH2-, -CH2-CH2-CH2- or -CH2-CH2-CH2-CH2-, and R1 is hydrogen, carboxyl or sulfonic acid group or absent (when Z is hydrogen); in some instances, Y is -O- or -NH-, Z is hydrogen, and R1 is absent; in some instances, Y is -O- or -NH-, Z is hydrogen, -CH2-, -CH2-CH2-, -CH2-CH2-CH2- or -CH2-CH2-CH2-CH2-, and R1 is hydrogen, carboxyl or sulfonic acid group or absent (when Z is hydrogen).

[0009] In some embodiments, in formula (I-1), R2 and R3 are both methyl groups; R4 and R5 are both hydrogen groups; R6 and R8 are both hydrogen groups; and R7 is a methyl group.

[0010] In some embodiments, in formula (I-1), R2 is methyl and R3 is hydrogen; R4 is methyl and R5 is hydrogen; R6 and R8 are both hydrogen; and R7 is methyl.

[0011] In some embodiments, in formula (I-1), R2 and R3 are both methyl groups; R4 and R5 are both hydrogen groups; R6 is a hydroxyl group; R8 is hydrogen; and R7 is a methyl group.

[0012] In some embodiments, in formula (I-1), R2 is methyl and R3 is hydrogen; R4 is methyl and R5 is hydrogen; R6 is hydroxyl, R8 is hydrogen; and R7 is methyl.

[0013] In some embodiments, in formula (I-1), R2 is methyl and R3 is hydrogen; R4 is methyl and R5 is hydrogen; R6 is hydroxyl, R8 is hydroxyl; and R7 is hydroxymethyl.

[0014] In some embodiments, formula (I-1) is one of the following compounds: .

[0015] In some implementations, the compound capable of activating V-ATPase is selected from compounds of the following formula: In formula (I-2), Y is -O- or -NH-, Z is hydrogen or C1-C6 alkylene, and R1 is hydrogen, carboxyl or sulfonic acid group or absent (Z is hydrogen). One of R3 and R4 is hydrogen and the other is either hydrogen or hydroxyl; R2 and R5 are each independently hydrogen or hydroxyl.

[0016] In some embodiments, in formula (I-2), Y is -O- or -NH-, Z is hydrogen, -CH2-, -CH2-CH2-, -CH2-CH2-CH2- or -CH2-CH2-CH2-CH2-, and R1 is hydrogen, carboxyl or sulfonic acid group or absent (Z is hydrogen); in some instances, Y is -O- or -NH-, Z is hydrogen, and R1 is hydrogen; in some instances, Y is -O- or -NH-, Z is -CH2-, -CH2-CH2-, -CH2-CH2-CH2- or -CH2-CH2-CH2-CH2-, and R1 is carboxyl or sulfonic acid group.

[0017] In some implementations, in formula (I-2), R3 is a hydroxyl group, R4 is hydrogen, R2 is a hydroxyl group, and R5 is hydrogen.

[0018] In some implementations, in formula (I-2), R3 is hydrogen, R4 is hydroxyl, and R2 and R5 are both hydrogen.

[0019] In some implementations, in formula (I-2), R3 is a hydroxyl group, R4 is hydrogen, and R2 and R5 are both hydrogen.

[0020] In some implementations, R3, R4, R2, and R5 in formula (I-2) are all hydrogen.

[0021] In some implementations, the compound capable of activating V-ATPase is selected from compounds of the following formula: .

[0022] In some embodiments, ionizable lipids refer to lipids that carry a positive charge under acidic conditions and can be protonated, including ionizable lipids with unsaturated tails (e.g., MC3, OF-02, A6, or A18-Iso5-2DC18) and ionizable lipids with multiple tails (e.g., 98N). 12-5, C12-200 or cKK-E12), ionizable polymeric lipids (e.g., 7C1, G0-C14), biodegradable ionizable lipids (e.g., L319, 304O) 13 OF-Deg-Lin or 306-O12B), ionizable lipids in branched tails (e.g., 306O). 110 Or FTT5).

[0023] In some implementations, the ionizable lipids are selected from the group consisting of: 3-(docomonasylamino)-N1,N1,4-tridodecyl-1-piperazine ethylamine (KL10), N1-[2-(docomonasylamino)ethyl]-N1,N4,N4-docomonasyl-1,4-piperazine diethylamine (KL22), 14,25-docomonasyl-15,18,21,24-tetraaza-octacosane (KL25), 1,2-dilinolenicyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinolenicyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinolenicyl-4-(2-dimethylaminoethyl)- [1,3]-Dioxolane (DLin-KC2-DMA), 1,2-diolenyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA) (2R)) and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)).

[0024] In some embodiments, the ionizable lipid is a phosphoramide compound comprising one or more tertiary amino groups, said phosphoramide compound being as described in formula (I-3), or a stereoisomer thereof, a prodrug, or a pharmaceutically acceptable salt thereof: In equation (I-3), n is an integer between 0 and 5; L1 and L2 are each independently C5-C30 Alkyl, C5-C 40 alkenyl, or -R'-M-R''; R1 and R2 are each independently C4-C 30 Alkyl, C2-C 40 Alkenyl, or -R'-M-R''; or, R1 and R2 together with their respective attached N and L4 form 5- to 8-membered diazacycloalkyl groups; R3 and R4 are each independently C1-C 30 Alkyl, C2-C 40 Alkenyl, or -R'-M-R''; or, R3 is H, C1-C 30 Alkyl, C2-C 40 Alkenyl, or -R'-M-R'', while R4 is C1-C 30 Alkyl, C2-C 40 Alkenyl or -R'-M-R''; R' is C1-C 24 Alkylene or C2-C 24 alkenyl; M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -SS-, -C(O)N(R a )-、-N(R a )C(O)-、-OC(O)N(R a )-、-N(R a )C(O)O-、-N(R a )C(O)N(R a )-、-C(OC(O)-R a (H)-C 1-6 Alkylene -OC(O)-, -C(O)OC 1-6 Alkylenes -C(O)-O-, -CH(OH)-, -P(O)(OR b O-, C6-C 10 arylene and 5- to 10-membered heteroarylene; R''、R a and R b Each is independently selected from hydrogen, C1-C 30 Alkyl and C2-C 40 alkenyl; L3, L4, and L5 are each independently C1-C 12 Alkylene, C2-C 12alkenyl, C3-C8 cycloalkylene, -AYB-, -AY-, or -YB-, where A and B are each independently C1-C 12 Alkylene or C2-C 12 The alkylene group, Y, is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene, or heteroarylene. As disclosed by the inventors in WO2024120455A1, the entire contents are incorporated herein by reference.

[0025] In some embodiments, the ionizable lipid is selected from one of the following compounds: PL10; PL11; PL12; PL13; PL14; PL17; PL18; PL19; PL20; PL21; PL22; PL23; PL24 PL26 PL27 PL28; PL29; PL30; PL31; PL32; PL33; PL34; PL35; PL36; PL38; PL41; PL44; PL45; PL53; PL54; PL55; PL56; PL57; PL58; PL59; PL60; PL61; PL78; PL79; PL93; PL94; PL95; PL96; PL97; PL103 Or its stereoisomers, prodrugs, or pharmaceutically acceptable salts.

[0026] In some embodiments, the ionizable lipid is PL32, ALC-0315, SM-102, ALC366, DODMA, C12-200, cKK-E12, YSK05, LP01, TT3, CL4H6, Lipid 5, ECO, OF-02, 306Oi10, 4A3-SC8 or HY-501, preferably, PL32.

[0027] As used in this article, the structural formula of the ionizable lipid PL32 is shown below: .

[0028] The synthesis method of PL32 is as follows: First, 1,8-octanediol (730 mg, 5 mmol) was dissolved in 5 mL of THF. Then, NaH (240 mg, 10 mmol) was added under ice bath conditions and the reaction was carried out for 10 minutes. Next, 4-methoxybenzyl chloride (783 mg, 5 mmol) was added dropwise to the system and the reaction was carried out overnight at room temperature. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with DCM, concentrated under reduced pressure, and purified by column chromatography. The product PL30-1 was obtained by elution with petroleum ether:ethyl acetate = 5:1. PL30-1 was dissolved in acetone, and Jones' reagent was added dropwise under ice bath conditions until the system turned brownish-yellow. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with DCM, concentrated under reduced pressure, and purified by column chromatography. The product PL30-2 was obtained by elution with petroleum ether:ethyl acetate = 5:1.

[0029] PL30-2 (560 mg, 2 mmol) and 3-undecyl alcohol (344 mg, 2 mmol) were dissolved in 10 mL of DCM, followed by the addition of DCC (412 mg, 2 mmol) and DMAP (24 mg, 0.2 mmol), and the reaction was carried out overnight at room temperature. After the reaction was completed, the mixture was filtered, washed with DCM, concentrated under reduced pressure, and purified by column chromatography. The product PL32-1 was obtained by elution with petroleum ether:ethyl acetate at a ratio of 25:1. PL32-1 (434 mg, 1 mmol) was then added to a mixture of DCM / H2O (10 mL / 1 mL), followed by the addition of DDQ (227 mg, 1 mmol), and the reaction was carried out at room temperature for 4 h. After the reaction was completed, water was added, and the mixture was extracted with ethyl acetate. The mixture was washed several times with water, the organic phase was concentrated, and purified by column chromatography. The product PL32-2 was obtained by elution with petroleum ether:ethyl acetate at a ratio of 25:1. 1 H NMR (400 MHz, CDCl3): 4.86-4.80 (m, 1H), 3.98 (t, J =8Hz, 2H), 2.31 (t, J =8Hz, 2H), 1.69-1.51(m, 8H), 1.36-1.28 (m, 18H), 0.91-0.87(m,6H).

[0030] First, PL32-2 (628 mg, 2 mmol) was dissolved in 5 mL of DCM, and 2 mmol of triethylamine was added to prepare a solution. Then, the above solution was slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) under ice bath (0 °C) with nitrogen protection. The reaction was carried out at -20 °C for 2 h, followed by dropwise addition of the above system to a dichloromethane mixture of 1,4-bis(3-aminopropyl)piperazine (400 mg, 2 mmol) and triethylamine (2 eq), and the reaction was carried out at room temperature for 2 h. After the reaction was complete, the mixture was washed with saturated sodium bicarbonate and concentrated under reduced pressure to obtain the crude product. Purification was achieved by column chromatography, eluting with dichloromethane:methanol:ammonia = 45:3:1 to obtain the liquid compound PL32-3. PL32-3 (873 mg, 1 mmol) was dissolved in DMF, and 1-bromododecane (747 mg, 3 mmol) and K2CO3 (414 mg, 3 mmol) were added, respectively, and the mixture was reacted overnight at room temperature. After the reaction was complete, the reaction was quenched with water, extracted with dichloromethane, washed three times with water, and concentrated under reduced pressure to obtain the crude product. Purified by column chromatography, eluted with dichloromethane:methanol = 20:1, to give liquid compound PL32 (665.43 mg, 55%). 1H NMR (400 MHz, CDCl3): 4.85-4.81(m, 2H), 4.00-3.94 (m, 4H), 3.03-2.96 (m, 4H), 2.47-2.29(m, 20H), 1.69-1.51(m,20H), 1.36-1.28 (m, 76H), 0.92-0.87 (m, 18H).

[0031] In some implementations, the LNP also includes: At least one auxiliary lipid, optionally, said auxiliary lipid comprising about 0 mol% to about 60 mol% of said LNP. At least one sterol, optionally, said sterol comprising about 0 mol% to about 60 mol% of said LNP. At least one PEG-modified lipid, optionally, the PEG-modified lipid comprising about 0.2 mol% to about 20 mol% of the LNP. In some embodiments, the lipid nanoparticles have an accessory lipid in a molar ratio of about 0%-60%, 5%-55%, 10%-50%, 15%-45%, 20%-40%, 25%-35%, or 27%-30%. In some embodiments, the lipid nanoparticles have a sterol in a molar ratio of about 0%-60%, 5%-55%, 10%-50%, 15%-45%, 20%-40%, 25%-35%, or 27%-30%. In some embodiments, the lipid nanoparticles have a PEG-modified lipid in a molar ratio of about 0.2%-20%, 1%-17%, 2%-15%, 3%-12%, 5%-10%, or 7%-8%.

[0032] In some embodiments, the sterol is selected from one or more of the following: cholesterol, β-sitosterol, stigmasterol, lanosterol, ergosterol, campesterol, cholesterol hemisuccinate, PEGylated cholesterol, oxosterol, oxidized cholesterol derivatives (e.g., cholesterol hemisuccinate), and phytosterol esters (e.g., β-sitosterol oleate); preferably, the sterol is cholesterol.

[0033] In some embodiments, the adjuvant lipid is selected from the group consisting of: distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), bis(pentadecanoyl)phosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), arachidoylphosphatidylcholine (DAPC), dibenzylphosphatidylcholine (DBPC), bis(triacyl)phosphatidylcholine (DTPC), bis(tetracosyl)phosphatidylcholine (DLPC), palmitoyloleyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauryl-phosphatidylethanolamine (DLPE), diphytyl-phosphatidylethanolamine (DPyPE), and combinations thereof; preferably, the auxiliary lipid is distearylphosphatidylcholine (DSPC) or dioleoylphosphatidylethanolamine (DOPE).

[0034] In some embodiments, the PEG-modified lipid is selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and any mixture thereof; preferably, the PEG-modified lipid is polyoxazoline, polyglycerol, poly(N-vinylpyrrolidone), polydopamine, polycarboxybetaine, polysulfobetaine, or polycaprolactone-polyethylene glycol; preferably, the PEG-modified lipid is DMG-PEG. 2000 .

[0035] In some embodiments, the ionizable lipid is PL32, the cofactor lipid is distearylphosphatidylcholine (DSPC) or dioleoylphosphatidylethanolamine (DOPE), and the PEG-modified lipid is DMG-PEG. 2000 Compounds that can activate V-ATPase account for approximately 20 mol of LNP.

[0036] In some implementations, the molar ratio of ionizable lipids: cofactor lipids: cholesterol: PEG-modified lipids: compounds capable of activating V-ATPase is 42.5:8.5:32.725:1.275:15.

[0037] In some implementations, LNPs also contain mRNA or siRNA.

[0038] In some embodiments, the siRNA is a siRNA targeting the Muc5ac gene; preferably, the sequence of the siRNA is derived from 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO: 65-68; preferably, the sequence of the siRNA is the sequence shown in SEQ ID NO: 65-68.

[0039] Wherein, SEQ ID NO:65 is the sense strand sequence of a human-derived sequence, which can also be represented as: (5'-3')(invAb)sgcuguucuGfCfGfacuacuacaas(invAb); SEQ ID NO:66 is the antisense strand sequence of a human-derived sequence, which can also be represented as: (5'-3') cPrpusUfsgsUfaGfuAfgUfcGfcAfgAfaCfaGfsc; SEQ ID NO:67 is the sense strand sequence of the mouse-derived sequence, which can also be represented as: (invAb) sccauacagCfAfGfuacaguuacas (invAb); SEQ ID NO:68 is the antisense strand sequence of the mouse-derived sequence, which can also be represented as: (5'-3') cPrpusGfsusAfaCfuGfuAfcUfgCfuGfuAfuGfsg; In this representation, a, c, g, and u in the sequence represent 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methylguanosine, and 2'-O-methyluridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoroadenosine, 2'-fluorocytidine, 2'-fluoroguanosine, and 2'-fluorouridine, respectively; s represents a thiophosphate bond; and cPrpu represents 5'-cyclopropylphosphonate-2'-O-methyluridine.

[0040] LNPs can be prepared using microfluidic mixers or micromixers. Exemplary microfluidic mixers may include, but are not limited to, slit interdigitated micromixers, including, but not limited to, those manufactured by Microinnova and / or staggered herringbone micromixers (SHM).

[0041] Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometric titration (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size.

[0042] In some embodiments, the lipid nanoparticles may have a diameter of about 1 nm to about 100 nm, such as, but not limited to, about 1 nm to about 20 nm, about 1 nm to about 30 nm, about 1 nm to about 40 nm, about 1 nm to about 50 nm, about 1 nm to about 60 nm, about 1 nm to about 70 nm, about 1 nm to about 80 nm, about 1 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 10 nm, about 5 nm to about 20 nm, and about 5 nm to about 30 nm. Approximately 5nm to approximately 40nm, approximately 5nm to approximately 50nm, approximately 5nm to approximately 60nm, approximately 5nm to approximately 70nm, approximately 5nm to approximately 80nm, approximately 5nm to approximately 90nm, approximately 10 to approximately 20nm, approximately 10 to approximately 30nm, approximately 10 to approximately 40nm, approximately 10 to approximately 50nm, approximately 10 to approximately 60nm, approximately 10 to approximately 70nm, approximately 10 to approximately 80nm, approximately 10 to approximately 90nm, approximately 20 to approximately 30nm, approximately 20 to approximately 40nm, approximately 20 to approximately 50nm, approximately 20 to approximately 60nm, approximately 20 to approximately 70nm, approximately 20 to approximately 80nm, approximately 20 to approximately 90nm, approximately 20 to approximately 100nm, approximately 30 to approximately 40nm, approximately 30 to approximately 50nm, approximately 30 to approximately 60nm, approximately 30 to approximately 70nm, approximately 30 to approximately 80nm, approximately 30 to approximately 90nm, approximately 30 to approximately 100nm, approximately 40 to approximately 50nm, approximately 40 to approximately 60nm, approximately 40 to approximately 70nm, approximately 40 to approximately 80nm, approximately 40 to approximately 90nm, about 40 to about 100nm, about 50 to about 60nm, about 50 to about 70nm, about 50 to about 80nm, about 50 to about 90nm, about 50 to about 100nm, about 60 to about 70nm, about 60 to about 80nm, about 60 to about 90nm, about 60 to about 100nm, about 70 to about 80nm, about 70 to about 90nm, about 70 to about 100nm, about 80 to about 90nm, about 80 to about 100nm and / or about 90 to about 100nm.

[0043] In some embodiments, the lipid nanoparticles may have a diameter of about 10 to 500 nm. In one embodiment, the lipid nanoparticles may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm, or greater than 1000 nm.

[0044] The lipid nanoparticles described herein can be prepared by any method known or subsequently developed in the field of pharmacology. Typically, such preparation methods involve the steps of associating the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if desired and / or wishfully, dispensing, shaping, and / or packaging the product into desired single- or multi-dose units.

[0045] The lipid nanoparticles described herein can be prepared, packaged, and / or marketed in large quantities as a single unit dose and / or as multiple single unit doses. The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any other components in the formulations described herein may vary depending on the identity, size, and / or condition of the subject being treated, and further depend on the route of administration of the composition.

[0046] As used herein, pharmaceutically acceptable excipients include, but are not limited to, any and all solvents, dispersion media or other liquid carriers, dispersing or suspending agents, diluents, granulators and / or dispersants, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, binders, lubricants or oils, colorants, sweeteners or flavorings, stabilizers, antioxidants, antimicrobial or antifungal agents, osmolarity regulators, pH regulators, buffers, chelating agents, cryoprotectants and / or fillers, suitable for the desired specific dosage form.

[0047] On the other hand, this application also provides a method for preparing an LNP formulation, comprising: dialyzing the LNP described herein in a phosphate buffer-sucrose solution, and then nebulizing or lyophilizing the dialyzed LNP.

[0048] In some implementations, the sucrose concentration in the phosphate buffer-sucrose solution is about 9% (w / w), and the dialysis time is about 16 hours.

[0049] In some embodiments, the LNP formulation further comprises siRNA targeting the Muc5ac gene; preferably, the siRNA sequence is shown in SEQ ID NO: 65-68.

[0050] On the other hand, this application also provides an LNP formulation prepared by the preparation method described herein.

[0051] On the other hand, this application also provides the use of the LNP or LNP formulation described herein in the preparation of a medicament for treating asthma.

[0052] In some implementations, the drug is administered via nebulization or inhalation.

[0053] On the other hand, this application also provides a method for treating asthma, comprising administering, by nebulization or inhalation, to a subject an LNP or LNP formulation described herein containing siRNA targeting the Muc5ac gene, preferably having the sequence of siRNA as shown in SEQ ID NO: 65-68.

[0054] On the other hand, this application also provides a pentacyclic triterpenoid compound, said compound being selected from the following formula: In formula (I-1), Y is -O- or -NH-, Z is a C3-C6 alkylene group, and R1 is a carboxyl group or a sulfonic acid group; R2 and R3 are each independently hydrogen or methyl; R4 and R5 are each independently hydrogen or methyl.

[0055] In some embodiments, in formula (I-1), Y is -O- or -NH-, Z is -CH2-CH2-CH2- or -CH2-CH2-CH2-CH2-, and R1 is a carboxyl or sulfonic acid group; in some instances, Y is -O- or -NH-, Z is -CH2-CH2-CH2- or -CH2-CH2-CH2-CH2-, and R1 is a carboxyl or sulfonic acid group.

[0056] In some implementations, in formula (I-1), R2 and R3 are both methyl groups; R4 and R5 are both hydrogen groups.

[0057] In some implementations, in formula (I-1), R2 is methyl and R3 is hydrogen; R4 is hydrogen and R5 is methyl.

[0058] In some embodiments, formula (I-1) is one of the following compounds: .

[0059] On the other hand, this application provides the use of the above-mentioned pentacyclic triterpenoid compounds as activators of V-ATPase.

[0060] This application identifies a series of compounds from a natural product library that can activate the V-ATPase complex. When incorporated into LNP, these compounds can significantly increase the release of encapsulated mRNA or siRNA and significantly reduce the inflammatory response caused by LNP delivery.

[0061] This application describes the development of a non-inflammatory lung protein (LNP) (NIF-LNP) by incorporating ursolic acid, identified from a natural product library, into a biodegradable cationic phosphoramide-derived LNP formulation. Compared to LNPs containing ALC-0315, NIF-LNP significantly enhanced lung protein expression by 40-fold without inducing significant reactivity. Our Crispr-KO mechanism study revealed that ursolic acid promotes endosomal acidification by activating the V-ATPase complex, a pivotal element in LNP transport and inflammation control. Furthermore, we identified NR1D1, an intracellular circadian rhythm regulatory gene encapsulated within NIF-LNP, which has significant therapeutic effects on bronchopulmonary dysplasia and pulmonary fibrosis. To improve clinical feasibility, we developed a lyophilized formulation that remains stable for over 90 days and ensures efficient nebulization in preclinical mouse, rat, and canine models. In conclusion, this V-ATPase-activated nebulized NIF-LNP provides a viable strategy for treating various chronic inflammatory lung diseases.

[0062] Furthermore, we found that the five-component LNP formed by encapsulating natural products such as UA into LNPs can deliver both mRNA and siRNA. The introduction of UA can significantly improve the gene knockdown efficiency of siRNA. In a mouse asthma model, LNP4 loaded with Muc5ac-siRNA showed good therapeutic effects.

[0063] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the embodiments described in the description and the accompanying drawings. Attached Figure Description

[0064] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0065] Figure 1The development of a PL lipid library for lung-targeted RNA delivery and its inflammatory assessment are illustrated in Example 1 of this application. (a) Schematic diagram of the phosphorus oxychloride-mediated multicomponent reaction (PMR) in phosphoramide lipid synthesis. (b) and (c) Structures of the three components of the PL lipids used in the synthesis and screening process. (d) Preparation of PL LNPs via a microfluidic device. (e) Expression of mLuc 6 hours after intratracheal administration of PL LNPs, pristine MC3, ALC-0315, or cKK-E12 LNPs (0.2 mg / kg, n=3 per group, one representative mouse shown per group). (f) Quantitative analysis of (e). (g) Schematic diagram of LNP-induced lung inflammation and bronchoalveolar lavage fluid (BAL) collection. (h) Correlation analysis between cytokine secretion and mLuc expression after intratracheal administration of PL LNPs. The concentrations of IL-1β, IL-6, and TNF-α in BAL collected 24 hours after intratracheal injection of PL LNPs (n=3 per group, representing the mean) were measured using ELISA. (i) Schematic diagram showing a positive correlation between cytokine concentrations and luciferase expression. (j) Chemical structure of PL32. (k) Gross anatomical images and H&E staining of the lungs of healthy mice and ALI mice after intratracheal instillation of PBS or a single dose of 0.6 mg / kg mLuc (MC3 LNPs, ALC-0315 LNPs, and PL32 LNPs). Scale bar, 100 μm. Arrows indicate areas of hepatization and inflammation. (l) Survival curves of bleomycin-induced pulmonary fibrosis in mice after two doses of mLuc LNPs treatment (0.6 mg / kg, n=8 per group). Data are expressed as mean ± standard error (SEM).

[0066] Figure 2The construction of NIF LNPs with improved mRNA transfection efficiency in Example 2 of this application is shown. (a) Preparation of non-inflammatory NAIP LNPs by adding NAIP to PL32 LNPs. (b) In vitro expression of mLuc delivered in A549 cells by the NAIP-LNP formulation. Expression was measured using a BioTek microplate reader after incubation of 100 ng mLuc LNPs for 24 hours (n=3). (c) Chemical structure of the primary candidate NAIP. (d) Expression of mLuc 6 hours after intratracheal administration of representative NAIP-LNPs and original PL32 LNPs (0.2 mg / kg, n=4 per group). Lung tissue was lysed and luminescence was detected using a BioTek microplate reader. Quantification of mLuc expression is shown on the right side of (e). (f)(d) Relative mRNA expression of IL-1β and CXCL1 in mouse lung tissue (n=3). (g) Quantitative analysis of neutrophils, macrophages, and M1- / M2-macrophages in (d) mouse lung tissue by flow cytometry (n=3 mice per group). (h) and (i) Levels of inflammatory cytokines in BAL of IPF and ALI mice 24 hours after intratracheal instillation of PL32 LNPs and P-UA LNPs (0.6 mg / kg, n=3 mice per group). (j) Anatomical examination and H&E staining of lung tissue. Scale bar, 100 μm. (k) Detection of inflammatory cytokines in human PBMCs treated with PL32 LNPs and P-UA LNPs for 24 hours using a flow cytometry array (CBA)-I kit (n=3 independent wells). (l) Schematic diagram of lung mRNA expression and inflammation after administration of non-inflammatory P-UA LNPs and conventional PL32 LNPs. Data are expressed as mean ± standard error (SEM).

[0067] Figure 3This paper illustrates how NIF LNPs in Example 3 of this application improve mRNA expression and alleviate inflammatory damage by activating V-ATPase. (a) Uptake of PL32 and P-UA LNPs encapsulating Cy3-labeled mLuc in vitro. LNPs were incubated with A549 cells for 2 hours, after which cells were collected and analyzed by flow cytometry (n=3). (b) Confocal image of intracellular distribution of LNPs in A549 cells. mLuc was labeled with Cy3, lipids with Bodipy, and lysosomes with lysotracker. The experiment was repeated three times with similar results. Scale bar, 20 μm. (c) Schematic diagram of a genome-wide CRISPR knockout (KO) screening method based on flow cytometry. (d) Hit rate of the top 10 gRNAs enriched in the low-GFP population of PL32 and P-UA LNPs. (e) String-based protein interactions based on the top 50 gRNAs enriched in the low-GFP P-UA LNP population. (f) GO analysis of gRNA in a low-GFP population of P-UA LNPs. (g) Decreased mLuc expression of P32LNP and P-UA LNP in major gene knockdown cell lines. Genes associated with glycosaminoglycan pathway, Golgi in vivo transport, and vascular acidification were knocked down using siRNA (n=3). (h) Endosomal acidity was measured in different groups using lysotracker. A549 cells were treated with 200 ng mLucLNPs for 2 h. The relative fluorescence intensity of lysotracker was quantified in the images. Scale bar, 20 μm. (i) V-ATPase activity (left) and concentration (right) in purified endosomals were assessed by ultracentrifugation after 24 hours of treatment with PL32 and P-UA LNPs in A549 cells (n=3). (j) IF staining of Alix spots was performed after treating A549 cells with different treatment methods. The figure below shows the number of ALIX spots per cell per image (n=3 per group). Scale bar, 50 μm. (k) Schematic diagram of UA liposome administration with PL32 LNPs and P-UA LNPs, respectively. (l) Activation of the ROS system in RAW264.7 after treatment with P32 LNPs, P-UA LNPs, and UA liposomes alone with PL32 LNPs. Scale bar, 10 μm. Quantitative results are shown in the images. (m) Downstream activation of the NF-κB pathway after treatment with PL32, P-UA LNPs, and UA liposomes alone with PL32 LNPs. Top: Quantitative results; Bottom: Bands (n=3). (n) Schematic diagram of the mechanism by which UA promotes mRNA expression and reduces inflammation. Data are expressed as mean ± standard deviation (SEM).

[0068] Figure 4This paper illustrates the use of NIF LNPs to alleviate lung injury in a BPD rat model as described in Example 4 of this application. (a) Schematic diagram of BPD model construction. (b) Differential proteomic analysis of lung tissues from BPD rats and healthy rats (n=6). (c) Relative mRNA expression of NR1D1 in healthy rats and BPD rats at 7, 14, 21, and 28 days under 80% oxygen exposure (n=5 at days 7 and 14, n=3 at days 21 and 28). (d) Immunofluorescence staining of NR1D1 in lung tissues of healthy rats and BPD rats at 21 days under 80% oxygen exposure. One representative image is shown for each group (n=5 mice per group). (e) Treatment regimens including intratracheal administration of PBS, mFGF10 prepared with PL32 LNPs, mFGF10 and mNR1D1 prepared with P-UA LNPs (0.6 mg / kg), blank PL32 LNPs, and P-UA LNPs at the same lipid dose. (f) Growth rate of BPD rats after different treatment regimens (n=6 per group). (g) Levels of inflammatory cytokines in BAL of BPD rats (n=6 per group) in all treatment groups and (h) Levels of ROS and SOD in lung tissue. (i) H&E and CD31 staining of lung tissue of BPD rats in all groups after treatment. Scale bar, 100 μm. (n=6 per group) (j) Mean linear intercept (MLI), radial alveolar count (RAC), and CD31 coverage % area in the lungs of BPD rats in all groups after treatment. MLI and RAC were quantified in four randomly selected fields of view for each rat, and CD31 was quantified in three randomly selected fields of view (n=6 rats per group). (k) IF staining of NR1D1 in lung tissue of BPD rats in the P-UA mNR1D1 treatment group. The same experiment was repeated three times, and one representative image is shown each time. (l) Heatmap of bulk transcriptome in the lungs of BPD rats treated with PBS, blank P-UA LNPs, and mNR1D1 LNPs (n=4 per group). (m) Bubble plots from KEGG analysis show key altered pathways in lung differentiation of BPD rats treated with PBS, blank P-UA LNPs, and mNR1D1 LNPs. The size and color of the dots reflect the gene ratio and p-value of altered genes in each pathway. Data are expressed as mean ± standard error (SEM).

[0069] Figure 5This paper illustrates the use of NIF LNPs to alleviate lung injury in an IPF mouse model according to Example 5 of this application. (a) Treatment regimens involving intratracheal administration of the same lipid doses of PBS, mNR1D1 prepared with PL32 LNPs, mNR1D1 prepared with P-UA LNPs (0.6 mg / kg), and empty P-UA LNPs. (b) Survival curves of mice in each treatment group and those treated with mNR1D1 and PL32 LNPs after four doses (0.6 mg / kg, n=8 per group). (c) Levels of inflammatory cytokines in the BAL of IPF mice in each treatment group (n=5). (d) Flow cytometry analysis of the proportion of infiltrating immune cells in the lung tissue of each treatment group (PBS group n=4 mice, P-UA group n=3 mice, P-UA mNR1D1 and WT groups n=5 mice). T cells were labeled CD45+CD11b-CD3+; CD4 T cells were labeled CD45+CD11b-CD3+CD4+; macrophages were labeled CD45+CD11b+F4 / 80high; neutrophils were labeled CD45+CD11b+F4 / 80medLy6G+; PMN MDSCs were labeled CD45+CD11b+F4 / 80lowLy6clowLy6G+; representative images of (e) Masson's trichrome staining, (f) Sirius red staining, (g) H&E staining, and (h) LOX and (i) IF staining of α-SMA in IPF mice (n=5) after different treatments. Scale bar, 100 μm. (j) Quantitative analysis of (e), (f), (h), and (i) in three randomly selected fields of view for each mouse (n=5 biologically independent mice per group, mean ± SEM). (k) Flow cytometry analysis of IL-4 (top) and IL-17 (bottom) positive cells in the CD45+ group (n=5). (l) Quantitative analysis of (k). Data are expressed as mean ± SEM.

[0070] Figure 6The preparation of nebulizable and lyophilized stable NIF LNPs in Example 6 of this application is shown. (a) Schematic diagram of the integrated process of LNP preparation, lyophilized storage, and nebulized inhalation remodeling. (b) Quantitative analysis of protein expression in lungs treated with P-UA LNPs of all formulations 6 hours after nebulized inhalation administration (n=3, 0.2 mLuc / kg). (c) Photographs of lyophilized and remodeled optimized P-UA LNPs. (d) Lung IVIS images of healthy mice treated with mLuc containing P-UA LNPs before and after lyophilization (n=3, 0.2 mLuc / kg). (e) Size changes of P-UA LNPs over 3 months after lyophilization. (f) Schematic diagram and treatment regimen of treating BPD rats with mNR1D1 P-UA LNPs via nebulized inhalation. (g) H&E staining of the lungs of BPD rats (n=3) treated with mNR1D1 P-UA LNPs via nebulized inhalation. Scale bar, 100 μm. (h) Quantitative analysis of radial alveolar count (RAC) and mean linear intercept (MLI) is shown on the right. (n=3, 5 fields of view randomly selected per mouse). (i) Treatment regimen of IPF mice treated with mNR1D1 P-UA LNPs via nebulization. (j) Masson tricolor and Sirius red staining of the lungs of IPF mice (n=3) treated with mNR1D1 P-UA LNPs via nebulization. Scale bar, 100 μm. (k) Quantitative analysis of collagen coverage area is shown on the right. (n=3, 5 fields of view randomly selected per mouse). (l) Schematic diagram assessing mLuc expression in nebulized LNPs in beagle dogs. (m) IVIS images of the lungs of beagle dogs treated with PL32 LNPs and P-UA LNPs containing mLuc via nebulization (0.3 mg / kg). Quantitative analysis results of the lungs 12 hours after administration are shown on the right. Twenty-one lung tissue samples were collected from the lungs of seven lobes of a dog, lysed, and analyzed for luminescence using a BioTek microplate reader. (n) H&E staining of lungs in beagle dogs treated with PL32 LNPs and P-UA LNPs containing mLuc via nebulization. Scale bar, 100 μm. (o) Levels of inflammatory cytokines and CRP protein in BAL after beagle dogs treated with PL32 LNPs and P-UA LNPs containing mLuc via nebulization. (p) Percentages of neutrophils and leukocytes in the blood of beagle dogs treated with PL32 LNPs and P-UA LNPs containing mLuc via nebulization. Wild-type beagle dogs were used as controls. Data are presented as mean ± standard deviation (SEM).

[0071] Figure 7The structures of phosphoramide lipids (PL) and the protein expression and inflammation of PL LNPs in embodiments of this application are shown. (a) Structures of PL and commercially available ionizable lipids. (b) mLuc expression 6 hours after intratracheal injection of PL LNP and raw MC3, ALC-0315 and cKK-E12 LNPs (0.2 mg / kg, n=3 per group). (c) Levels of inflammatory cytokines in BAL collected 24 hours after intratracheal injection of PL LNP and raw MC3, ALC-0315 and cKK-E12 LNPs (0.2 mg / kg, n=3 per group). (d) Relative mLuc expression of inflammatory cytokines in IFN-γ and LPS-induced M1 macrophages after 24 hours of treatment with PL LNP and commercially available LNPs (n=3 per group). (e) and (f) Levels of inflammatory cytokines in BAL collected 24 hours after intratracheal injection of PL LNPs and commercially available LNPs in LPS-induced acute lung injury (ALI) mice and mice treated with bleomycin for 21 days (0.6–1.2 mg / kg, n=3 per group). Inflammatory cytokine concentrations in control ALI and IPF mice, as well as wild-type (WT) mice, are indicated by dashed lines (n=3 per group). Data are presented as mean ± SEM.

[0072] Figure 8This paper illustrates the addition of natural anti-inflammatory products (NAIPs) to PL32 LNPs to alleviate inflammation in embodiments of this application. (a) Structures of four types of NAIPs. (b) Photograph of empty PL-32 LNPs (without mRNA) bound to NAIPs. (c) In vitro expression of mLuc in A549 cells with a NAIP-LNPs formulation containing 30 mol% NAIPs. Expression was measured using a BioTek microplate reader after incubation with 100 ng mLuc LNPs for 24 hours (n=3). (d) Schematic diagram of the measurement of inflammatory cytokines and their relative mRNA expression after treatment of IFN-γ and LPS-induced M1 macrophages with NAPI LNPs and PL-32 LNPs for 24 hours (n=3). (e) In vitro expression of mLuc in HULEC-5a (pulmonary microvascular endothelial cells), BEAS-2B (bronchial epithelial cells), AT-II (pulmonary epithelial cells), HUVEC (umbilical vein endothelial cells), and RAW264.7 (immune cells) using a leading NAIP-LNP formulation. Expression was measured using a BioTek plate reader after 24 hours of incubation with 100 ng mLuc LNPs (n=3). (f) In vitro expression of mGFP in A549 cells using PL32 LNPs and P-UA LNPs. Expression was measured by flow cytometry after 24 hours of incubation with 100–400 ng mGFP LNPs (n=3). (g) In vivo expression of mLuc in C57BL / 6 mice via intratracheal injection of a leading NAIP-LNP formulation. Protein expression in lung tissue was measured at 2 and 6 hours post-administration (0.2 mg / kg, n=4 per group) using in vivo imaging. (h) Figure 2 The level of IL-6 in the serum of mice in group d. (i) Analysis of the effect of flow cytometry on the IL-6 level. Figure 2 (d) Quantitative analysis of T cells in lung tissue was performed. (j) Inflammatory cytokines in human PBMCs treated with PL32 LNPs and P-UA LNPs for 24 hours were detected using a flow cytometry microsphere array (CBA)-I kit (n=3). Data are presented as mean ± SEM.

[0073] Figure 9This paper illustrates the mechanism by which P-UA LNPs improve mRNA expression and alleviate inflammation in the embodiments of this application. (a) Representative FACS analysis of GFP+ cells in different cell populations in the lungs of healthy C57BL / 6 mice. Mice were given mCre LNPs (n=3) 48 hours before sacrifice. (b) Quantitative analysis of (a). (c) Percentage of positive cells for each cell in (a) (n=3). (d) H3 lentivirus-transduced A549 cells were given PL32 LNPs and P-UA LNPs (3 μg / culture dish) containing mEGFP for 24 hours. GFP-positive cells were divided into four groups according to GFP expression. Genomic DNA was extracted from cells in each group, amplicon libraries were prepared, and sequencing was performed to determine the enrichment of sgRNAs. (e) Hit counts of the top 20 gRNAs enriched in the GFP-low and GFP-high populations of PL32 and P-UA LNPs, respectively. (f) Interaction network of leading candidates in P32 LNPs generated by STRINGdb (top 50 hits, p < 0.05). Line thickness indicates confidence for each interaction. The minimum interaction requirement score for the network was set to 0.4. (g) ClueGo analysis of leading candidates in P32 LNPs and P-UA LNPs (top 50 hits, p < 0.05). (h) KEGG and Reactome analysis of the top 50 gRNAs enriched in the GFP-low population in the PL32 and P-UA LNP groups. (i) Validation of siRNA knockdown efficiency in A549 cells (n=3, MC3LNPs containing siRNA incubated with A549 cells for 48 hours). (j) Cellular uptake of DiD-labeled P32 LNPs and P-UALNPs for mLuc delivery in major gene knockdown cell lines. Genes associated with glycosaminoglycan pathways, angiogenesis, and Golgi in vivo transport were knocked down with siRNA (n=3). (k) Levels of inflammatory cytokines in BAL collected from healthy mice following intratracheal injection of PL32 LNP, P-UA LNP, and PL32 LNP+UA liposomes (0.8 mg / kg, n=3). In vitro (l) and in vivo (m) P-UA LNP transport efficiency and mLuc transport efficiency of UA liposomes delivered with PL32 LNP and UA liposomes, respectively. Quantitative analysis is shown on the right (n=3, A549 cells, 1 μg / mL mLuc in vitro and 0.2 mg / kg in vivo). Data are presented as mean ± SEM.

[0074] Figure 10This paper illustrates the treatment of BPD with NIF LNPs in embodiments of this application. (a) and (b) In vivo mLuc expression in the lungs of BPD rats 14 days after intratracheal injection of PL32 and P-UA LNP (0.2 mg / kg, n=4 rats per group, 3 rats shown in in vivo imaging). (c) Changes in body weight of BPD rats after treatment (n=6). (d) White blood cell (WBC) counts in the blood of BPD rats in all treatment groups (n=4 rats). (e) Levels of IL-6 and CXCL1 in BAL of BPD rats in all treatment groups (n=6 rats). (f) Representative H&E staining of lung tissue from BPD rats in the P-UA mNR1D1 treatment group and other control groups, at high magnification (scale bar = 500 μm). (g) Western blotting and gray-scale quantitative analysis of α-SMA in lung tissue of BPD rats in all treatment groups (n=3). Data are presented as mean ± SEM.

[0075] Figure 11 This illustration shows the treatment of IPF with NIF LNPs in embodiments of this application. (a) and (b) In vivo expression of mLuc in lung tissue of IPF mice treated with bleomycin for 21 days, 6 hours after intratracheal injection of PL32 and P-UA LNP (0.2 mg / kg, 3 mice per group). (c) Weight changes in all treatment groups of IPF mice (n=6 mice). (d) Representative H&E, Sirius red, and Masson staining of lung tissue from PL32 mNR1D1-treated IPF mice. Scale bar, 100 μm. (e) IF staining of collagen I in lung tissue from all treatment groups of IPF mice. Scale bar, 100 μm. Quantitative analysis was performed on three randomly selected fields of view for each mouse (n=5 mice per group). Data are expressed as mean ± SEM.

[0076] Figure 12The following images illustrate the preparation of stable P-UA LNPs by nebulization lyophilization in embodiments of this application. (a) Changes in particle size and encapsulation efficiency of P-UA LNPs in each formulation before and after nebulization (n=3). (b) IVIS images of the lungs of healthy mice treated with mLuc-containing P-UA LNPs under different protectants and dialysis conditions (n=3). (c) Lyophilization process of optimized P-UA LNP formulations. (d) Particle size, encapsulation efficiency, and zeta potential of P-UA LNPs before and after lyophilization (n=3). (e) and (g) Photographs of simultaneous inhalation administration of P-UA LNPs to three BPD rats and IPF mice using a custom device. (f) and (h) Changes in body weight of BPD rats and IPF mice treated with nebulized P-UA mNR1D1 (n=3). (i) Blood levels of IL-6, IL-1β, TNF-α, and CRP in beagle dogs treated with nebulized PL32 and P-UA LNPs (0.3 mg / mLuc / kg). Data are presented as mean ± standard deviation (SEM).

[0077] Figure 13 The changes in protein expression of PL32 LNP after the addition of 20%mol ursolic acid and oleanolic acid derivative in Example 7 of this application are shown.

[0078] Figure 14 The changes in protein expression of PL32 LNP after the addition of 20%mol cholic acid and its derivatives in Example 7 of this application are shown.

[0079] Figure 15 The results of examining inflammation activation were shown in Example 7 of this application, in which NK92mi cell supernatant treated with PL32 LNP and PL32-UAS LNP was added to RAW-ISG cells.

[0080] Figure 16 The example in Example 7 of this application shows the detection of INFγ and TNFα gene levels by qPCR 24 hours after administration of PL32 LNP and PL32-UAS LNP (1 μg / mL mFluc) to NK92mi cells.

[0081] Figure 17 The knockdown efficiency of different LNP formulations in A549 cells (siRNA concentration of 100 nM) in Example 8 of this application is shown. 1-16 represent 16 LNP formulations, and SM102 (17) is the positive control. "-" represents the untreated group; "+" represents free siRNA containing the avβ6 target.

[0082] Figure 18The siRNA encapsulation efficiency of the three LNPs and SM102 LNPs prepared by the Harvard Bioscience microfluidic device in Example 8 of this application is shown.

[0083] Figure 19 The siRNA particle size characteristics of three types of LNPs and SM102 LNPs prepared by the Harvard Bioscience microfluidic device in Example 8 of this application are shown.

[0084] Figure 20 The cytotoxicity of the three LNPs and SM102 LNPs in A549 cells (siRNA concentrations of 50, 100, and 200 nM) in Example 8 of this application is shown.

[0085] Figure 21 The encapsulation efficiency of siRNA for two types of LNPs and SM102 in Example 8 of this application is shown.

[0086] Figure 22 The particle size characteristics of two types of LNP and SM102 in Embodiment 8 of this application are shown.

[0087] Figure 23 The encapsulation efficiency of siRNA for two types of LNP and SM102 under different preparation conditions is shown in Example 8 of this application.

[0088] Figure 24 The siRNA administration regimen in Example 8 of this application is shown (TA is the administration siRNA, and HDM is the administration dust mite protein).

[0089] Figure 25 The survival rate of mice during drug administration is shown in Example 8 of this application.

[0090] Figure 26 The changes in body weight of mice during administration are shown in Example 8 of this application.

[0091] Figure 27 The expression of the Muc5ac gene in the lungs of mice after drug administration is shown in Example 8 of this application.

[0092] Figure 28 The protein expression level of Muc5ac in the lung lavage fluid of mice after administration in Example 8 of this application is shown (ELISA detection).

[0093] Figure 29 The protein expression level of Muc5ac in mouse lung tissue after administration in Example 8 of this application is shown (WB detection).

[0094] Figure 30The illustration shows the different LNPs loaded with Cy5-siRNA in Example 8 of this application and the fluorescence distribution of Cy5-siRNA solution (Sol) in mouse lungs.

[0095] Figure 31 This illustrates the distribution of different LNPs carrying Cy5-siRNA and Cy5-siRNA solution (Sol) in different cells of the mouse lung in Example 8 of this application.

[0096] Figure 32 The following example illustrates the detection of Muc5ac, IFN-γ, TNF-α, IL-13, and IL-17 levels in mouse lung lavage fluid and IgE levels in mouse serum using ELISA after treatment in Example 8 of this application.

[0097] Figure 33 The H&E staining of lung tissues in each group after treatment is shown in Example 8 of this application.

[0098] Figure 34 PAS staining of lung tissues in each group after treatment is shown in Example 8 of this application.

[0099] Figure 35 The image shows Muc5ac immunofluorescence staining of lung tissues from each group after treatment in Example 8 of this application (red: Muc5ac, blue: DAPI). Detailed Implementation

[0100] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application pertains. When a quantity, concentration, or other value or parameter is expressed as a range, preferred range, or preferred upper and lower numerical limits, it should be understood that this is equivalent to specifically disclosing any range by combining any pair of upper or preferred values ​​with any lower or preferred value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range.

[0101] When used with a numerical variable, the terms "about" or "approximately" usually mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within the 95% confidence interval of the mean) or within ±10% of the specified value, or a wider range.

[0102] The expression "comprising," or similar expressions such as "including," "containing," and "having," is open-ended and does not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0103] The expression "at least one" or "one or more" indicates 1, 2, 3, 4, 5, 6, 7, 8, 9 or more kinds.

[0104] This application describes several embodiments, but these descriptions are exemplary and not limiting, and it will be apparent to those skilled in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature or element of any embodiment may be used in combination with, or may replace, any feature or element of any other embodiment.

[0105] This application includes and contemplates combinations of features and elements known to those skilled in the art. The embodiments, features, and elements disclosed in this application can also be combined with any conventional features or elements to form unique inventive solutions. Any feature or element of any embodiment can also be combined with features or elements from other inventive solutions to form another unique inventive solution. Therefore, it should be understood that any feature shown and / or discussed in this application can be implemented individually or in any suitable combination. Therefore, the embodiments are not limited except by the limitations imposed by the appended claims and their equivalents. Furthermore, various modifications and changes can be made within the scope of the appended claims.

[0106] Furthermore, in describing representative embodiments, the specification may have presented methods and / or processes as a specific sequence of steps. However, the method or process should not be limited to the specific order of steps described herein, to the extent that it does not depend on such a specific order. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation of the claims. Moreover, the claims concerning the method and / or process should not be limited to the steps performed in the written order, and those skilled in the art will readily understand that these orders can be varied and still remain within the spirit and scope of the embodiments of this application.

[0107] Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental materials in the following embodiments without specified sources are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, then generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer are followed. Unless otherwise defined or stated, all technical and scientific terms used in this application have the same meaning as those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this application.

[0108] Example The materials used in the embodiments are shown below: Reagents, materials and instruments Commercially available ionizable lipids containing DLin-MC3-DMA (MC3) and 6-((2-hexyldecanoyl)oxy)-N-(6-(((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexane-1-ammonium (ALC-0315) were purchased from Avanti PolarLipids, Inc. 2,5-piperazinedione and 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl] (cKK-E12) were purchased from MedChemExpress (MCE) Life Science Reagents. Cholesterol-containing co-lipids, 1,2-bisoctadecanoyl-sn-glycerol-3-phosphocholine (DSPC), and 1,2-dimyristicoyl-racemic-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG) were also available. 2000All reagents were purchased from AVT (Shanghai) Pharmaceutical Technology Co., Ltd. 1,2-Dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) was purchased from Avanti Polar Lipids, Inc. Anti-inflammatory natural products were a gift from Professor Xue Qiao. Firefly luciferase mRNA (mLuc) was provided by Proxybio. All plasmid sequences were provided by Genscript Biotech Co., Ltd. Primers and siRNA were provided by Tsingke Biotechnology Co., Ltd. All cell lines and fetal bovine serum (FBS) were purchased from Procell Life Sciences & Technology Co., Ltd. Other reagents required for basic culture were purchased from Meilunbio Co., Ltd. ELISA kits for mouse IL-6, TNF-α, IL-1β, and IFN-γ, and ELISA kits for rat IL-1β, IL-6, TNF-α, and CXCL1 were purchased from Solarbio Science & Technology Co., Ltd. Microfluidic chips for preparing LNPs were purchased from Micro&Nano Biologics Co., Ltd. The freeze dryer used in this study was a LyoBeta-15 II 050-359 freeze dryer manufactured by Telstar Co., Ltd.

[0109] Experimental methods mNR1D1, mFGF10, and mCre were synthesized using a T7 polymerase-mediated in vitro transcription (IVT) system with a linearized pUC57 plasmid vector containing a T7 promoter, 5' and 3' untranslated regions (UTRs), and a 100 nt Poly A tail. CleanCap AG 5' capping (Cap1) and 1-methylpseudouridine UTP were added to the transcription reaction. Urate-5' triphosphate (UTP) was completely replaced by 1-methylpseudouridine UTP. The IVT reaction was performed according to the manufacturer's (Hongene Biotech Inc., China) instructions.

[0110] 1. Preparation and characterization of lipid nanoparticles An aqueous solution containing mRNA was mixed with ionizable lipids, DSPC, cholesterol, and DMG-PEG. 2000LNPs were prepared by microfluidic mixing of an ethanol solution containing natural products at a 3:1 ratio. Formulations are shown in Table 1. Organic solvents were removed by dialyzing overnight with 1×PBS phosphate buffer at 4°C. For nebulized administration, LNPs were dialyzed in 1×PBS, 0.1×PBS, sucrose phosphate buffer, sucrose Tris buffer, and sodium acetate buffer. 2% w / v excipients were added to each LNP formulation. Then, 500 μl of dialyzed LNPs were loaded into an Aeroneb vibrating mesh nebulizer placed on ice, and the condensed LNPs were collected in 1.5 mL microcentrifuge tubes. The mRNA concentration and encapsulation efficiency of the LNPs were determined using the Quant-it RiboGreen RNA Detection Kit (Invitrogen). The hydrodynamic diameter and zeta potential of the LNPs were determined using a dynamic light scattering instrument (Zetasizer Nano ZSP, Malvern) in 1×PBS and 4 mM KCl.

[0111] Table 1: Formulations of PL32 LNPs and PL32 LNPs with added NAIP. 2. Preparation of UA liposomes Liposomes were prepared using the ethanol infusion method. DSPC, cholesterol, and UA were mixed at a mass ratio of 15:5:1.5 to form the organic phase. Then, 200 μL of the organic phase was mixed with 600 μL of pure water. The mixture was dialyzed overnight in PBS to remove the ethanol.

[0112] 3. Cell culture and assay A549 lung epithelial cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Procell, China) and penicillin and streptomycin (Meilunbio, China). RAW 264.7 was cultured in heat-inactivated DMEM medium. HULEC5A was cultured in DMEM medium supplemented with 10% FBS and 1% Anti-Anti. BEAS-2B was cultured in BEGM (bronchial epithelial cell growth medium). AT2 was cultured in DMEM medium supplemented with 10 ng / ml hEGF, 5 μg / ml insulin, 10% FBS, and 1% Anti-Anti. HUVECs were cultured in complete HUVEC medium produced by Wuhan Presella Biotechnology Co., Ltd.

[0113] 4. In vitro inflammation detection using empty LNPs Empty nanoparticles (LNPs) were prepared using the method described above, without the need to add mRNA to the aqueous phase. RAW 264.7 macrophages were seeded in 24-well plates overnight, and IFN-γ (2.5 ng / mL) and LPS (200 ng / mL) were added to induce macrophage differentiation into the M1 type. Then, 10 μL of empty nanoparticles were added to the cells, and the cells were cultured for 24 hours. Cells were lysed with Trizol, and RNA was extracted for qPCR detection.

[0114] 5. Quantitative real-time PCR (qPCR) assay RNA was extracted and purified from lung tissue and cultured cells using the TransZol Up Plus RNA Kit (TransGen Biotech). cDNA was reverse transcribed using the HiScript III All-in-one RT SuperMix Perfect (Vazyme Biotech Co., Ltd). qPCR was performed on a QuantStudio™ 6Flex using the Sybr green method with QuantStudio Real-Time PCR software v1.7.1 on qPCR SuperMix (TransGen Biotech). All primers are listed in Table 2 below. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Quantification was performed using 2^-ΔΔct values.

[0115] Table 2: qPCR primers and siRNA sequences (SEQ ID NO: 1-52) 6. In vitro transfection For Luc protein transfection, cells were seeded in 96-well plates overnight, and LNPs containing mLuc were added to each well at a concentration of 0.1 µg mLuc. After 24 hours, transfection efficiency was measured using a Firefly-Glo luciferase reporter gene assay kit (YeasenBiotechnology Co., Ltd.) on a BioTek synergy H1 microplate reader according to the kit instructions.

[0116] For GFP protein transfection, cells were seeded in 96-well plates overnight, and LNPs containing mGFP were added to each well at concentrations of 0.1 µg, 0.2 µg, and 0.4 µg mRNA. After 4 hours, the LNPs were removed, and fresh culture medium was added. After 24 hours, the fluorescence signal of the GFP protein was measured using flow cytometry.

[0117] 7. Cytokine Measurement in PBMCs Human peripheral blood mononuclear cells (PBMCs) were purchased from Milestone Biotech (PBLP-08B). 1 × 10⁶ cells were used. 5 PBMCs were seeded at 1 per well in 96-well plates, followed by the addition of LNP at a concentration of 1 μg RNA per well. After 24 hours, the plates were centrifuged at 4°C for 3 minutes (500g), and the supernatant was collected. Cytokines such as IL-1β, IL-2, IL-4, IL-6, IL-8, TNF-α, and IFN-γ were measured using the Human Inflammatory Cytokine Flow Cytometry Bead Array (CBA)-I kit according to the manufacturer's instructions.

[0118] 8. In vitro cellular uptake Twenty-four hours before the experiment, A549 cells were seeded in 96-well plates at a density of approximately 10 cells per well. 4 Cells were used to knock down LNP-related proteins using an MC3 formulation containing siRNA. 48 hours before LNP incubation, 200 nM MC3 LNP (siRNA) was added to the cells and incubated at 37°C. After LNP pre-incubation, the supernatant was aspirated and the cells were washed twice with PBS. Then, PL32LNP and P-UA LNP encapsulating Cy3-mLuc (equivalent to 0.1 μg RNA per well) were added to each well. After incubation at 37°C for 2 hours, the wells were washed three times with cold PBS and the culture medium was replaced with fresh medium. Cell uptake was assessed by flow cytometry.

[0119] 9. Live-cell imaging of mRNA release Intracellular mRNA release of LNPs was observed using an inverted Zeiss LSM880 confocal microscope. An Airyscan array detector (Carl Zeiss AG) was used to enhance and visualize cytoplasmic Cy3-mLuc signaling in live cells. Intracellular behavior of LNPs and mRNA was tracked using 5% BODIPY-liposomes and Cy3-mLuc. Cells were treated with LNP-labeled mRNA at a concentration of 1 μg / mL and incubated at 37°C for 2 h. Subsequently, the medium was replaced with fresh medium containing 1 μg / mL Hoechst 33342 and lysotracker, and endosome release was observed using a 100×Plan-Neofluar 1.3 numerical aperture (NA) oil immersion objective. The field of view (FOV) for all experiments was set to 354, 25 μm × 354, 25 μm (full field of view). All channels were pinholes set to 1 Airy Unit (AU). Acquired image data were processed and analyzed using Zen 2.3 software (blue version). Colocalization of Cy3-mLuc and lysotracker fluorescence signals was quantified using the Pearson correlation coefficient (PCC) implemented in ImageJ 1.53u.

[0120] 10. Genome-wide CRISPR screening of P32 and P-UA LNPs A whole-genome H3 human CRISPR KO library targeting >18,000 genes (117,587 sgRNAs) was obtained from Addgene (#133914) and constructed in the pLentiCRISPRv2 vector. Plasmid amplification and viral packaging were performed according to published protocols for maintaining library representativeness. Approximately 200 million A549 cells were seeded in 15 cm culture dishes (approximately 14 million cells per dish) with 25 mL of complete RPMI 1640 medium (RPMI 1640 medium containing 10 v / v% fetal bovine serum and 1 v / v% penicillin / streptomycin) and incubated overnight at 37°C and 5% CO2 to allow cell adhesion. The next morning, polybrene and the H3 lentiviral library were added to the culture dishes. The final concentration of polybrene was 8 μg / mL. The volume of lentivirus added was determined by titrating the virus under the same conditions to infect approximately 24% of the cells. This was the standard for lentivirus whole-genome knockout screening to ensure that the majority of cells were infected with a single virus. Twenty-four hours after infection, the viral medium was removed from the culture dish and replaced with fresh complete medium. The cells were incubated for another 24 hours to induce puromycin resistance gene expression, and then 2 μg / mL puromycin was added to select for infection. Three days after puromycin selection, all remaining cells were collected and pooled to obtain the A549-H3 library cell line. A549-H3 cells were cultured in 25 mL of complete RPMI 1640 medium in each culture dish, with 1 μg / mL puromycin added. The cells were passaged every 2–3 days, with each passage retaining at least 30 million cells to maintain the representativeness of the library.

[0121] A549-H3 cells were passaged approximately two times to achieve a stable CRISPR KO phenotype. Cells were then collected and divided into three replicates (approximately 30 million cells per replicate, 14 million cells per 15 cm dish). One replicate served as an unclassified control for quality control, while the other two replicates were transfected with P32 at a dose of 3 μg / dish or with P-UA LNP loaded with mGFP, respectively. Twenty-four hours after transfection, cells were collected and sorted using flow cytometry (FACS) based on EGFP expression. The lowest and highest 10%–15% of cells in each replicate were isolated, and genomic DNA was extracted using the TIANamp Genomic DNA Kit (Tiangen #DP304), along with the unclassified control.

[0122] sgRNA barcodes were amplified from genomic DNA using a 2×EasyTaq® PCR SuperMix (TransGen#AS111-11). The amplification process followed a published protocol and consisted of three rounds of PCR. The first round of PCR amplified the sequence from the U6 promoter to the EF1a promoter, spanning the gRNA sequence on the modified pLentiCRISPRv2 backbone. The second round of PCR used F+R primer pairs to add an 8nt index to each sample for multiplex analysis and to increase base diversity in each Illumina sequencing cycle. The third round of PCR primers were equivalent to Illumina P5 and P7 adapters. All primer sequences used are listed in Table 3. The third PCR product was purified using a TIANgel purification kit (Tiangen#DP219) and submitted to Sequanta Technologies Corporation for sequencing. The amplicon library was sequenced using an Illumina NovaS4. Data analysis was performed using MAGeCK version 0.5.9.3, as reported in our previous publications, for quality control and to detect differentially enriched genes and pathways between the low-expression and high-expression groups.

[0123] Table 3: PCR Oligo sequences used for CRISPR screening (SEQ ID NO:53-64) 11. Detect the acidity of the internal compartments. 24 hours before the experiment, A549 cells were inoculated at ~2×10⁻⁶. 4 Cells were seeded at a density of 1:1 in 8-well plates. V-ATPase-associated proteins were knocked down using LNPs with MC3 formulations containing siRNA (ATP6V0B and ATP6V1B2). 200 nM MC3 LNPs (containing siRNA) were added to the cells and incubated at 37°C for 48 h, followed by LNP incubation. Subsequently, cells were treated with 2 μg / mL mLuc LNPs and incubated at 37°C for 30 min. The medium was then replaced with fresh medium containing 1 μg / mL Hoechst 33342 and lysotracker. Lysotracker signaling in the cells was detected using confocal microscopy.

[0124] 12. In vitro measurement of V-ATPase concentration and activity Cells in 15 cm culture dishes were treated with 1 μg / mL LNP containing mLuc and incubated at 37°C for 24 h. Endosomes were purified using an endosome extraction kit according to the manufacturer's instructions. Briefly, cells were harvested by centrifugation (850 × g, 2 min, 4°C) and homogenized on ice using a Dounce homogenizer. Cell debris and organelles were then removed using a commercially available endosome extraction kit. The isolated endosomes were washed three times with PBS to remove residual contaminants. The coarsely extracted endosomes were further separated by centrifugation at a density gradient of 145,000 × g for 2 h at 4°C. The harvested endosomes were then washed three times. The concentration of V-ATPase was assessed by ELISA according to the manufacturer's instructions. ATP hydrolytic activity was determined using the previously described coupled spectrophotometric method. In short, the endosomes were suspended in ATPase assay buffer (50 mM NaCl, 30 mM KCl, 20 mM HEPES-NaOH, pH 7.0, 10% (v / v) glycerol, 1 mM MgCl2, 1.5 mM phosphoenolpyruvate, 0.35 mM NADH, 20 U / ml). -1 Pyruvate kinase and 10 U / ml -1 Add 5 μM concanavalin A (used to calculate V-ATPase-specific ATP hydrolysis activity) or DMSO to lactate dehydrogenase and preheat at 37°C for 10 minutes. Add 5 mM ATP to start the assay and continuously record OD341 using a BioTeksynergy H1 microplate reader.

[0125] 13. Alix puncta imaging Cells were treated with 1 μg / mL mLuc of LNP and LK (60 μM) at 37°C for 30 min, followed by fixation with 4% fresh paraformaldehyde for 15 min. For the siRNA knockdown group, cells were treated with siRNA containing 200 nM MC3 LNP at 37°C for 48 h, followed by LNP incubation. The samples were then incubated with 5% BSA for 1 h. After washing with PBS, cells were co-cultured overnight at 4°C with a 1:250 primary antibody. Unbound antibodies were washed three times with PBS. Next, a 1:1000 secondary antibody was added to the cells, and the cells were incubated at 37°C for 1.5 h. Unbound antibodies were washed three times with PBS. Cell nuclei were labeled with DAPI. Images were acquired using Vectra Polaris and Phenochart 1.0.8 software (Akoya, USA). Quantitative results are expressed as the number of ALIX dots per cell per image. (n=3 per group) 14. ROS Detection Cells were treated with liposomes containing 2 μg / mL LNPs or mLuc-containing LNPs + UA and incubated at 37°C for 4 hours. Dihydroethidium (DHE) probes were loaded into the cells and incubated at 37°C for 30 minutes. ROS signals were detected by confocal microscopy.

[0126] 15. Western Imprint Cells and lung tissue were mixed with cold PBS and Ripa lysis buffer containing protease inhibitors, respectively. Protein loading buffer was added to the protein solution, and the mixture was heated at 95°C for 5 minutes. After electrophoresis and membrane transfer, the protein-containing PVDF membrane was incubated with 5% BSA for 1 hour. After washing with TBST, the PVDF membrane was incubated overnight at 4°C with a 1:1000 primary antibody. Unbound antibodies were washed three times with TBST. Next, a 1:10000 secondary antibody was added to the cells, and the mixture was incubated at 37°C for 1 hour. Unbound antibodies were washed three times with TBST. The protein bands were visualized using chemiluminescence imaging.

[0127] 16. Histological and immunofluorescence staining.

[0128] Hematoxylin-eosin (H&E) staining, Sirius red staining, and Masson's trichrome staining were performed by Wuhan Saiwei Biotechnology Co., Ltd. Images were captured using a WS-10 tissue paraffin scanner equipped with NDP View 2 (Zhiyue Medical Technology, Hefei), and quantitative analysis was performed using Image J 1.53u. All statistical analyses were performed according to the manufacturer's (Abcam) protocol, in 3-4 different regions of 5-6 biologically independent specimens. Frozen or paraffin sections of lung tissue and cells grown on slides were used for immunofluorescence staining. Primary and secondary antibodies and their corresponding dilutions are listed in Table 4. Immunofluorescence images were scanned using Vectra Polaris and Phenochart 1.0.8 software (Akoya, USA). Quantitative analysis was performed using Image J 1.53u, using the same methods described above.

[0129] Table 4: Antibody Information 17. Animal models All animal studies complied with the ethical guidelines approved by the Peking University Animal Protection and Use Committee. Bronchopulmonary dysplasia model: 20-day-old gestation rats were intraperitoneally injected with LPS (0.25 mg / kg). Postpartum rats and newborn mice were cultured in an incubator with 80% oxygen for 21 days. Acute lung injury model: C57BL / 6 mice were anesthetized and intratracheally injected with 50 μL LPS (2.5 mg / kg). Pulmonary fibrosis model: Mice were anesthetized and intratracheally injected with 50 μL bleomycin sulfate (2 U / kg).

[0130] 18. In vivo expression and inflammation detection of PL LNPs mLuc PL LNPs were administered intratracheally to healthy mice. Six hours post-administration, bioluminescence imaging was performed using an IVIS imaging system (Perkin Elmer) to assess lung Luc protein expression. Twenty-four hours later, mice were anesthetized via intraperitoneal injection of chloral hydrate. A longitudinal incision was made in the neck with surgical scissors, and the muscles were dissected to expose the trachea. The trachea was opened through a small incision, and an indwelling needle was inserted approximately 1 cm into the trachea. Then, 800 μL of PBS was injected into the lungs twice, and bronchoalveolar lavage fluid (BAL) was collected. The obtained BAL solution was centrifuged at 500 g for 3 minutes, and the supernatant was collected for ELISA analysis according to the manufacturer's instructions.

[0131] 19. In vivo expression and inflammation detection of NAIP LNPs mLuc NAIP LNPs were administered intratracheally to healthy mice (0.2 mg / kg). Bioluminescence imaging was performed using an IVIS imaging system (Perkin Elmer) at 2 h and 6 h post-administration. Mice were then sacrificed, and blood and lung tissue were collected. 50 mg of lung tissue was lysed using a 1x Promega Luciferase Assay System cell culture lysis reagent. To detect luciferase expression, 5 µL of lysis supernatant and 50 µL of luciferin solution were added to white 96-well plates, and bioluminescence was measured using a BioTek Synergy H1 microplate reader. 50 mg of lung tissue was lysed with Trizol, and mRNA was extracted for qPCR detection. The remaining lung tissue was digested and broken down into single-cell suspensions, and the infiltration of immune cells in the lung tissue was detected by flow cytometry. Blood was centrifuged at 8000 rpm for 3 minutes, and serum was collected for IL-6 level determination by ELISA.

[0132] 20. In vivo expression and inflammatory response of UA-containing ALC-0315 and cKK-E12 LNPs Healthy mice were intratracheally injected with mLuc LNPs containing ALC-0315 and cKK-E12 (with or without added UA). Bioluminescence imaging was performed using an IVIS imaging system (Perkin Elmer) at 6 and 24 hours post-administration. Mice were then sacrificed, and bronchoalveolar lavage fluid (BAL), blood, and lung tissue were collected. 50 mg of lung tissue was lysed with Trizol, and mRNA was extracted for qPCR detection. Blood was centrifuged at 8000 rpm for 3 minutes, and serum was collected for IL-6 level determination by ELISA. The obtained BAL was centrifuged at 500 g for 3 minutes, and the supernatant was collected for ELISA detection according to the manufacturer's instructions.

[0133] 21. In vivo expression in BPD rats and IPF mice A BPD rat model was established using the previous method, followed by 14 days of hyperoxia treatment. mLuc LNPs (0.2 mg / kg) were administered intratracheally to the BPD rats. Six hours post-administration, bioluminescence imaging was performed using an IVIS imaging system (Perkin Elmer) to assess lung Luc protein expression. An IPF mouse model was also established using the previous method. mLuc LNPs (0.2 mg / kg) were administered intratracheally to the IPF mice. Six hours post-administration, bioluminescence imaging was performed using an IVIS imaging system (Perkin Elmer) to assess lung Luc protein expression.

[0134] 22. mRNA delivery and expression in LoxP-EGFP mice To assess mRNA expression in different lung cells, mCre LNPs (0.8 mg / kg) were administered intratracheally to LoxP-EGFP reporter mice. After 48 hours of treatment, cells were isolated and stained, followed by flow cytometry analysis. Lung perfusion: Left atrial perforation was performed, and 5 ml of dPBS was infused through the right ventricle until the lung tissue was completely white. Lung tissue was digested using 2500 U collagenase I, 30 U neutral protease, and 500 U deoxyribonuclease (DNAse) I. The isolated lung tissue was digested in 5 ml of enzyme solution using a gentleMACS™ Octo dissociator, following the following digestion steps: Lung_01_02 digested 3 times, m_LIDK_1 digested once at 37℃, and Lung_01_02 digested 3 times. The cell suspension was filtered through a 40 μm filter and resuspended in pre-chilled 1×RBC lysis buffer and dPBS. Cell types were distinguished by specific markers using flow cytometry (epithelial cells: CD326+, immune cells: CD326- and CD45+, endothelial cells: CD326-, CD45- and CD31+, stromal cells: CD326-, CD45- and CD31-). The percentage of EGFP+ cells in each cell population was measured and quantified.

[0135] 23. Treatment efficacy research A BPD rat model was established using the previously described method (21 days). On days 3, 8, 13, and 18, mice were administered PBS, PL32 LNP containing mFGF10, P-UA LNP containing mFGF10 and mNR1D1 (at a concentration of 0.6 mg / kg), and empty PL32 LNP and P-UA LNP at the same lipid concentrations via intratracheal administration, respectively. Rats were weighed every two days. On day 24, rats were sacrificed, and blood, bronchoalveolar lavage fluid (BAL), and lung tissue were collected for leukocyte detection, ELISA analysis, tissue sectioning, and ROS and SOD measurements.

[0136] A C57BL / 6J mouse model of IPF was established using previous methods. On days 3, 8, 13, and 18 after bleomycin administration, mice were administered PBS, PL32 and P-UA LNPs containing mNR1D1 (0.6 mg / kg), and empty PL32 LNPs and P-UA LNPs with the same lipid concentration via tracheal infusion. Mice were sacrificed on day 3 after the last administration, and bronchoalveolar lavage fluid (BAL) and lung tissue were collected. The concentrations of IL-1β, IL-6, and TNF-α in BAL were determined using ELISA. Lung tissue was used for immunocytology detection, and flow cytometry and section staining were performed.

[0137] 24. Lyophilized LNP P-UA LNPs containing mLuc were prepared using microfluidic technology and dialyzed overnight in sucrose phosphate buffer. The dialyzed LNPs were diluted with sucrose phosphate buffer to a concentration of 0.04 mg / mL mLuc. For the freeze-drying method, 1 mL of the diluted mLuc LNP (containing 40 μg mLuc) was placed into a 7 mL Type I glass vial. The freeze-drying procedure is as follows: Figure 12 As described in c in the text.

[0138] 25. Inhalation administration of LNP mLuc-containing LNPs for nebulized inhalation were prepared according to the previously described method. The LNPs were nebulized using an Aeroneb vibrating mesh nebulizer, and the nebulized LNP droplets were collected. The concentration of mLuc was determined using the Quant-it RiboGreen RNA assay. The mLuc-containing LNPs were administered intratracheally to healthy mice. Six hours after administration, the mice were euthanized, and their lungs were removed. Bioluminescence imaging was performed using an IVIS imaging system (Perkin Elmer) to assess the expression of Luc protein in the lungs.

[0139] To investigate the inhalation efficacy of PL32 and P-UA LNPs in large animals, male beagle dogs (approximately 8 kg, Marshall BioResources) were acclimatized for at least 14 days prior to the experiment. The beagle dogs were anesthetized with isoflurane in oxygen using a nasal-only mask. An AerogenSolo nebulizer was connected to the nasal mask, and PL32 and P-UA LNPs containing mLuc were nebulized at a dose of 0.3 mg / kg and inhaled by the dogs. Dogs were sacrificed 12 h after administration, and bioluminescence images and luciferase expression were detected using the same method described above. Additionally, blood, BAL, and lung tissue were collected for complete blood count, ELISA, and HE staining. Lyophilized mNR1D1 P-UA LNPs were reconstituted in nuclease-free water. Three BPD rats or IPF mice were placed in the nebulizer. Add 0.04 mg / mL mNR1D1 P-UA LNPs to the AerogenSolo nebulizer, and administer the nebulized dose at a dose of 8 mg / kg mNR1D1 per mouse / rat. The treatment regimen is the same as described above.

[0140] Example 1. Construction of a phosphoramide lipid (PL) library for RNA delivery to the lungs and assessment of inflammation. The low degradation rate and prolonged tissue accumulation of ionizable cationic lipids are key issues leading to the overactivation of innate immunity by LNPs. In our previous study, we identified PL14, an ionizable cationic lipid with a piperazine-centered phosphoramide core (PL) structure, which showed more than 60-fold higher intra-articular transfection efficiency after local administration compared to commercially available MC3 LNP formulations. To enhance the in vivo degradation and clearance of PL14 lipids, we constructed and purified a panel of biodegradable PLs by introducing various ester and amide bonds into their head and tail for in situ lung mRNA delivery. Figure 1 ac and Figure 7 (a) In order to identify the leading candidate, we followed the classic formulation ratio (PLs:DSPC:cholesterol:DMG-PEG). 2000 =50:10:38.5:1.5) to prepare a mixture containing ionizable PL and other auxiliary lipids [including 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG)]. 2000 LNPs were loaded with firefly luciferase mRNA (mLuc) ( ) Figure 1 (d) The encapsulation efficiency of LNPs containing ionizable PL (PL LNPs) was between 65% and 80%. Six hours after intratracheal administration, transfection efficacy was measured by luminescent signals in the lungs using IVIS imaging technology. Figure 1 e and f in Figure 7 (b) In the first round of screening, compared to PL14, we found that introducing degradable linkers (PL24 and PL26) near the piperazine core reduced mLuc delivery efficiency. With the core structure fixed, we found that adding ester bonds at the distal end of the phosphoramide linker reduced mLuc delivery efficiency, while adding branched ester bond tails near the phosphoramide bonds significantly increased mLuc expression in the lungs. Finally, we identified PL32 (R2a-Head1-R1b) as an optimal candidate lipid. Figure 1 Among them, b and c), it showed the highest protein expression efficiency, with protein expression efficiency increased by nearly 14-fold, 6-fold, and 2-fold respectively compared with LNPs formulated with MC3, ALC-0315, and cKK-E12. Figure 1 e and f in Figure 7 (b) in the middle.

[0141] To investigate the immunogenicity of PL lipids, we collected bronchoalveolar lavage (BAL) fluid 24 hours after mLuc LNP treatment and analyzed cytokine levels. Figure 1 To mitigate the inherent immunogenicity of mRNA, we used 100% N1-methylpseudouridine-modified mRNA in this study. Compared to control mice, we observed significantly elevated levels of IL-1β, IL-6, and TNF-α after administration of mLuc LNP (g). Figure 7 (c) Furthermore, a strong positive logarithmic linear correlation was found between cytokine secretion and the efficiency of LNP delivery of mLuc (c). Figure 1 (h and i in the text). We also evaluated the inflammatory response of empty PL LNPs and other reported LNP formulations without mRNA (e.g., formulations containing MC3, ALC-0315, and cKK-E12) by incubation with pro-inflammatory macrophages. The previously mentioned LNP-induced pro-inflammatory cytokines were measured by qPCR. These LNPs consistently caused significant upregulation of inflammatory genes, indicating the generation of inflammation (h and i in the text). Figure 7 (d in the text). This finding suggests that ionizable cationic lipid carriers are the main driver of the observed reactivity. Notably, among the LNPs tested, the leading LNP PL32 ( Figure 1 (j) showed the highest mRNA in vivo delivery efficiency and a good inflammatory expression ratio ( Figure 1 (eh in the middle).

[0142] Gene replacement therapy typically requires repeated administration of high doses to achieve satisfactory therapeutic protein levels. However, such treatments may exacerbate lung injury in diseases characterized by inflammation. To investigate this, we administered a single high-dose dose of mLuc (0.6 and 1.2 mg / kg) via intratracheal infusion directly into the lungs of LPS-induced acute lung injury (ALI) mice or bleomycin-induced idiopathic pulmonary fibrosis (IPF) mice using MC3, ALC-0315, or PL32 formulations. BAL samples collected 24 hours post-administration showed a 2-6 fold increase in cytokine levels in ALI and IPF mice compared to healthy controls. Figure 7 The results (e and f in the original text) confirmed the establishment of the model. High-dose LNP treatment further amplified the increase in cytokine secretion, increasing it 4-28 times, and in a dose-dependent manner. Figure 7 (e and f in the text). Gross anatomical examination of the lungs of ALI mice treated with 0.6 mg / kg mLuc of LNP revealed severe "hepatization," a clear sign of lung inflammation. Figure 1 (k in the text). Further lung histological analysis confirmed that after LNP injection, there was increased immune cell infiltration, alveolar rupture, and thickening of the alveolar septa. Figure 1 More importantly, repeated injections of LNPs encapsulating GMP-grade mLuc at a dose of 0.6 mg / kg into IPF mice resulted in a 100% mortality rate. Figure 1 (l in the text). This suggests that the severe reactivity caused by ionizable cationic LNPs may be harmful to patients with chronic lung inflammation, posing a significant obstacle to RNA nanotherapy for lung-related damage.

[0143] Example 2. Integrating natural products into lipid nanoparticles (LNPs) can promote mRNA expression and alleviate inflammation. In order to reduce reactivity, promote mRNA expression, and break the positive correlation between the two ( Figure 1 In section i), we explored the integration of hydrophobic natural anti-inflammatory products (NAIPs) with relatively low reported toxicity into LNP formulations. We introduced 22 natural anti-inflammatory products (NAIPs) as additional ingredients in LNP formulations, comprising four main subclasses: (I) flavonoids, (II) tetracyclic triterpenoids, (III) pentacyclic triterpenoids, and (IV) other phenolic compounds. Figure 2 a and Figure 8 (a) These NAIPs, comprising 20% ​​of the total lipid content, are complementary to our proprietary ionizable cationic lipid PL32, eliminating the need for RNA loading. Of these, 17 NAIPs were successfully encapsulated in LNPs, forming stable NAIP LNPs, which were used in subsequent experiments. Figure 8 (b) in the middle.

[0144] We evaluated the ability of NAIPs to reduce the inflammatory response induced by LNPs (without mRNA) in pro-inflammatory macrophages. Both PL32 LNPs and NAIP LNPs were applied to pro-inflammatory macrophages and activated with LPS and IFN-γ for 24 hours. Figure 8 c). and Figure 7 Consistent with the results in d, PL32 LNPs induced significant upregulation of inflammatory cytokines such as IL-1β, IL-6, TNF-α, and CCL5, with increases ranging from 10-fold to 57-fold. Figure 8 (c) Conversely, incorporating NAIPs into LNPs significantly reduced these inflammatory responses. Notably, compared to PL32 LNPs, the addition of isoflavones, ursolic acid, protopanaxadiol, and forsythoside A to LNPs resulted in a 3-10 fold reduction in IL-1β and IL-6 levels, approaching the levels of wild-type (WT) M1 macrophages. Figure 8 (c) These effects are similar to, and in some cases even exceed, the anti-inflammatory effects of dexamethasone (Dex) added to LNP at an equivalent dose. Figure 8 (c in the text)

[0145] Next, we evaluated the transfection efficiency of NAPI LNPs encapsulating mLuc in A549 lung epithelial cells. The concentrations of NAIPs ranged from 10 mol% to 30 mol% of the total lipid content. Except for LNPs containing rhein, most NAIP LNPs had similar particle sizes, ranging from 100 to 200 nm, with encapsulation efficiency (EE) between 65% and 80%. Furthermore, the addition of acid-derived NAIPs reduced surface charge, likely due to rearrangement of the polar head groups of the surface carboxyl groups caused by electrostatic repulsion during dialysis. For protein expression, we found that the addition of most flavonoids and pentacyclic triterpenoids to PL32 LNPs significantly promoted mLuc expression. Figure 2 b and Figure 8 Specifically, the addition of 10% citronellol isoflavone (CAL), 20% asiatic acid (AA), 20% ursolic acid (UA), and 20% oleanolic acid (OA) increased the expression levels of PL32 LNP by 6.2-fold, 8.7-fold, 14.3-fold, and 12.8-fold, respectively, compared to the original PL32 LNP. Figure 2 b in Figure 8 (d) This trend weakens after adding 30% of AA, UA, and OA. Figure 8 In contrast, LNPs containing Dex showed reduced mLuc expression across all test ranges (d). Figure 2 b in Figure 8Interestingly, among the NAIPs (AA, UA, OA) with the lowest inflammation levels and the highest mLuc expression, they share a common structural feature: the presence of a C-3 hydroxyl group and a C-28 carboxyl group ( ). Figure 2 c). 3-hydroxy esterification in OA and its 3-acetate derivatives reduces mLuc expression and increases inflammation ( Figure 2 b, c and Figure 8 (c, d). Furthermore, compared to α-amyrin, the removal of the C-28 carboxyl group in UA is detrimental to mLuc expression and inflammation relief. Additionally, the introduction of a hydroxyl group at the C-16 position is detrimental to LNP encapsulation, as demonstrated by a comparison of UA with styracidium acid. Figure 2 c and Figure 8 (b) in the middle.

[0146] We further verified whether adding NAPI could improve mRNA expression or encapsulate mRNA encoding other reporter genes in other cell models. Figure 8 (e and f in the original text). Lead candidate NAIP LNPs (P-CAL, P-AA, P-UA, and P-OA LNPs) containing 10 mol% CAL, 20 mol% AA, 20 mol% UA, and 20 mol% OA were formulated and compared with the control PL32 LNP. All five formulations had a particle size of approximately 120 nm, a polydispersity index (PDI) below 0.2, and encapsulation efficiency exceeding 65%. P-AA, P-UA, and P-OA LNPs exhibited a higher negative charge due to the presence of carboxyl groups. Similar trends were observed in pulmonary microvessels and umbilical endothelial cells, lung and bronchial epithelial cells, and immune cells. Figure 8 (e). P-UA LNP can also enhance the delivery of GFP mRNA (mGFP). In A549 cells, a dose of 0.1-0.4 μg mGFP can increase mGFP expression by 3-6 times, and mGFP expression is dose-dependent. Figure 8 (f in the text). We then intratracheally instilled LNPs containing mLuc into healthy mice (0.2 mg / kg) and assessed protein expression in the lungs at 2 and 6 hours. P-CAL, P-AA, P-OA, and P-UALNPs significantly enhanced luciferase expression in the lungs, increasing it by 2 to 11 times compared to the original PL32 formulation. Figure 2 d, e and Figure 8Notably, P-UA LNP significantly increased lung protein expression by 40-fold compared to conventional ALC-0315 LNP. This observation is consistent with in vitro transfection studies in A549 cells, highlighting the great potential of NAIP LNP in enhancing local mRNA delivery efficiency. Furthermore, we collected blood and lung tissue to assess the inflammatory response. Compared to PL32 LNP, P-UA LNP exhibited the strongest anti-inflammatory activity, significantly reducing gene expression of IL-6 and CXCL1 in the lungs, decreasing neutrophil and macrophage infiltration, reducing the M1 / M2 macrophage ratio in the lungs, and downregulating serum IL-6 levels (g). Figure 2 f, g and Figure 8 (h and i in the text). Based on these findings, P-UA LNP was identified as the optimal formulation.

[0147] Next, we compared the effects of UA-coated and conventional Dex on LNP-mediated mRNA expression and in vivo inflammation. We found that incorporating Dex into PL32 LNPs negatively impacted lung tissue mRNA transfection, resulting in a slight, but not significant, decrease in Luc protein expression. Conversely, coating PL32 LNPs with UA enhanced protein expression by more than 10-fold. Figure 2 (d and e in the original text). To assess the anti-inflammatory effect, we examined the levels of pro-inflammatory cytokines in bronchoalveolar lavage fluid (BAL) 24 hours after LNP treatment. Both P-UA LNP and P-Dex LNP exhibited potent anti-inflammatory activity, significantly reducing pro-inflammatory cytokine levels compared to PL32 LNP. Furthermore, we found that the addition of UA to ALC-0315 and cKK-E12 also significantly enhanced protein expression, increasing it 3-5 times after 24 hours compared to the original formulation. The addition of UA persistently and effectively alleviated the pulmonary inflammatory response, as evidenced by the reduction in BAL, serum, and pulmonary levels of pro-inflammatory cytokines (e.g., IL-1β, IL-6, and CXCL1). In conclusion, these results indicate that the addition of UA is a general strategy to enhance the safety and efficacy of LNP.

[0148] We further investigated the effects of a high dose of 0.6 mg / kg P-UA LNP on mice with acute lung injury (ALI) and idiopathic pulmonary fibrosis (IPF). The results contradicted our previous observations on PL32 LNP. Figure 7 (e and f in the text), P-UA LNP treatment did not lead to significant changes in cytokine (i.e., IL-1β, IL-6, and TNF-α) levels in BAL. Figure 2 In addition, gross anatomical examination of the lung tissue and H&E staining did not show any signs of hepaticization, increased immune cell infiltration, or alveolar damage in patients treated with P-UA LNP. Figure 2 (j) These findings indicate that even at high doses, P-UA LNP does not exacerbate the inflammatory condition in mice with pre-existing lung inflammation. We further evaluated the inflammatory response induced by PL32 LNP and P-UA LNP in human peripheral blood mononuclear cells (PBMCs). Figure 2 k in (the value of k). (The value of k in 1×10) 5 PBMCs were treated with 1 μg mLuc (encapsulated in PL32 LNPs or P-UA LNPs) for 24 hours. Consistent with mouse studies, the concentrations of IL-1β, IL-6, IL-8, and TNF-α were significantly increased in the PL32 LNPs-treated group. Conversely, the levels of these cytokines in the P-UA LNP-treated group were significantly lower than those in the PL32 LNPs-treated group, indicating that the low-inflammatory properties of P-UA LNPs can be readily applied to humans. Figure 2 k and Figure 8 (j in the text). These results confirm that UA successfully reduced reactivity and increased mRNA expression. P-UA LNPs, as NIF LNPs (non-inflammatory LNPs), show great potential in the treatment of inflammatory lung diseases. Figure 2 (l in the middle).

[0149] Example 3. NIF P-UA LNPs improve mRNA expression and alleviate inflammatory damage by activating V-ATPase. Typically, LNP-induced inflammatory responses depend on ionizable lipids and are positively correlated with mRNA expression levels. However, P-UA LNPs defy this pattern, achieving high protein expression efficiency with minimal inflammatory response. To explore the underlying mechanisms behind this difference, we first used Cy3-labeled mLuc (Cy3-mLuc) to assess the cellular uptake and endosome escape capabilities of PL32 LNPs and P-UA LNPs. Flow cytometry analysis showed that P-UA LNPs significantly enhanced endocytosis efficiency, increasing the percentage of Cy3-positive cells from approximately 43% to approximately 70%. Figure 3 (a) Furthermore, we assessed the endosome release kinetics of mRNA LNPs using a triple labeling method involving BODIPY-lipids, Cy3-mLuc, and Lysotracker staining of endosomes. After 2 hours of incubation with A549 cells, confocal microscopy revealed that the mRNA in the P-UA LNP-treated group was clearly separated from the Lysotracker, indicating rapid degradation and release of mRNA from endosomes (a). Figure 3 (b) In contrast, approximately 70% of the mRNA in the PL32 LNP-treated group remained colocalized with endosomes.

[0150] To identify specific cell types leading to high expression of P-UA LNP mRNA, we employed a genetically engineered mouse model with a loxP stop codon upstream of the GFP-2A luciferase sequence. Targeted cellular expression of luciferase and GFP was achieved via cleavage by the cre recombinase (delivered by mCre-encoding Cre mRNA). Flow cytometry results showed that P-UA LNP exhibited higher in vivo transfection efficiency across various cell types compared to PL32 LNP. Figure 9 In the study of UA (ac), epithelial cells and immune cells were identified as the main targets of these two LNPs. This indicates that the enhanced expression induced by UA is a universal effect, rather than cell type specific, which is consistent with... Figure 8 The results were consistent with those in the previous section. Next, we developed a flow cytometry-based whole-genome CRISPR knockout (KO) screening method to identify key genes leading to differences in mRNA transfection efficiency between PL32 and P-UA LNP. To this end, we transduced A549 cells into an H3 lentiviral library containing a wide range of guide RNAs (gRNAs) targeting over 18,000 different genes and containing a total of 108,000 unique gRNAs. After two transduction passages, cells were exposed to mGFP encapsulated within PL32 and P-UA LNP for 24 hours. Figure 3 (c) Subsequently, cells were divided into four groups according to GFP expression levels, from low to high ( Figure 9 (d) Then, the MAGeCK-RRA method was used to assess the difference in sgRNA enrichment between the high (group IV) and low (group I) GFP expression levels. Subsequently, sgRNAs were sorted according to their enrichment in the low GFP expression group to identify genes that play a positive regulatory role in both LNP types. Figure 3 (c in the text)

[0151] Comprehensive utilization of gene ontology (GO) Figure 3 f), Kyoto Encyclopedia of Genes and Genomes (KEGG) Figure 9 h) and Reactome ( Figure 9 h), and STRING-based protein interaction assessment ( Figure 3 e and Figure 9 In f), it was found that genes involved in the biosynthesis of glycosaminoglycans and the biosynthesis of heparan sulfate proteoglycans had a significant impact on the transfection efficiency of PL32 LNPs and P-UA LNPs. Figure 3 df and Figure 9These results are consistent with previous studies that suggest that ionizable cationic liposomes interact with negatively charged proteoglycans on the cell surface, thereby promoting the internalization of nanoparticles by lung epithelial and endothelial cells. We further validated this hypothesis by knocking down three key genes involved in heparin sulfate biosynthesis—EXT1, EXT2, and SLC35B2. The results showed that knockdown of these genes led to reduced cellular uptake of PL32 and P-UA LNP, thereby affecting the expression of the encapsulated mRNA ( eh). Figure 9 i, j and Figure 3 In addition, genes associated with endoplasmic reticulum-Golgi transport function (i.e., TMEM165) and those involved in tRNA biosynthesis have been identified as having an impact on RNA expression promoted by PL32 and P-UA LNP. Figure 9 i, j and Figure 3 (g in the original text). This result was expected because these processes are crucial for mRNA translation and overall mRNA expression efficiency.

[0152] Notably, we found that genes associated with vesicle acidification (such as ATP6V0B and ATP6V1B2) and genes regulating vesicle transport (including TRAPCC4) specifically affected P-UA LNP-mediated mRNA expression (compared to PL32 LNP). Figure 3 df and Figure 9 (eh). STRING-based protein-protein interaction analysis showed that among the top 50 differentially expressed genes, 11 genes interacted closely and belonged to the ATPase-coupled transmembrane transporter family ( ). Figure 3 (e). These genes use the energy generated by ATP hydrolysis to transport protons from the cytoplasm to organelles (such as endosomes), thereby affecting the organization of lysosomes (e). Figure 9 g), macroautophagy ( Figure 3 f, GO analysis) and vesicle recycling process ( Figure 9 (h, KEGG, and Reactome analyses were performed). To further investigate this, we performed siRNA-mediated knockdown of the V-ATPase subunit encoding genes ATP6V0B and ATP6V1B2 (KO efficiency is shown in [reference needed]). Figure 9 Importantly, we found that knockdown of these vesicle acidification-related genes led to a significant downregulation (more than 10-fold change) of mLuc expression in P-UA LNPs compared to PL32 LNPs. Figure 3Notably, the gRNA of ATP6AP1 (a component of vacuolar ATPase and a surface receptor for prorenin) was enriched in the low-expression population along with another surface protein, TRAPCC4. Knockdown of these membrane proteins led to reduced uptake of P-UA LNPs and subsequent decreased mLuc expression. Figure 9 j and Figure 3 The presence of g in the results indicates that these proteins are involved in the cellular uptake of P-UA LNP. In summary, the siRNA knockdown study is consistent with the CRISPR screening results, highlighting the close relationship between V-ATPase activity and the enhanced transfection efficiency induced by P-UA LNP.

[0153] Previously, it was known that V-ATPase could regulate endosome pH, so we sought to investigate how PL32 and P-UA LNPs affected endosome acidity. By using the pH-sensitive dye lysotracker in A549 cells, we detected a decrease in fluorescence intensity after treatment with PL32 LNPs (…). Figure 3 The decrease in fluorescence intensity indicates a decrease in endosome acidity, which may be due to endosome rupture induced by the cationic charge present in PL32. On the other hand, we found that P-UA LNPs can increase endosome acidity. This increase in acidity may enhance the fusion process between ionizable cationic lipids and the endosome membrane, thereby promoting the release of mRNA into the cytoplasm and maintaining endosome homeostasis, such as... Figure 3 As shown in b. To further investigate the mechanism by which P-UA LNPs regulate endosome pH and balance mRNA expression with immunogenicity, we first evaluated their effects on V-ATPase activity and expression levels. Our results indicate that P-UA LNPs did not significantly affect endosome V-ATPase levels, but may act as V-ATPase agonists, significantly enhancing enzyme activity (…). Figure 3(i) This enhancement is consistent with the results of CRISPR knockout studies and supports our conclusion that P-UA LNPs activate V-ATPase and the resulting endosome acidification, which is crucial for enhancing protein expression. Furthermore, V-ATPase-driven endosome acidification is essential for maintaining endosome homeostasis. Previous studies have shown that when endosomes undergo minor rupture with a slight dissipation of the pH gradient, ESCRT proteins (such as Alix) rapidly accumulate at the rupture site to promote repair. Conversely, severe endosome damage (characterized by large-area rupture and a significant increase in pH) fails to recruit ESCRT proteins. Based on the intrinsic role of V-ATPase in maintaining endosome acidity, we hypothesize that acid-driven endosome stability can mitigate membrane damage caused by LNPs. To test our hypothesis, we investigated the recruitment of Alix to endosomes after LNP treatment. Using chloroquine as a potent endosome disruptor, we observed minimal or no ESCRT recruitment (expressed as the smallest Alix point). Conversely, treatment with P-UA LNP significantly enhanced ESCRT recruitment compared to PL32 LNP, indicating the presence of small perforations. Figure 3 The results (j) support our hypothesis. Furthermore, we knocked down the genes ATP6V0B and ATP6V1B2, which encode the V-ATPase subunit, and observed a significant decrease in endosome acidity and ESCRT recruitment after gene knockdown. Figure 3 j in the middle.

[0154] To confirm that the reduced ESCRT recruitment was due to severe endosomal damage, we measured the production of reactive oxygen species (ROS) after LNP treatment. Endosomal damage is typically associated with increased ROS production. Indeed, we observed elevated ROS levels in the PL32 LNP group, while ROS levels were significantly reduced when UA was encapsulated in PL32 LNP or co-delivered with PL32 LNP in liposome form. Figure 3 In addition, the activation of NF-κB (a key mediator of cationic lipid-related inflammatory responses) (k and l). Figure 3 The effect of UA on the m) was weakened by the addition of UA. In summary, our results suggest that P-UA LNPs may act as potential V-ATPase agonists to maintain endosomal homeostasis and alleviate inflammation caused by membrane perturbations, while simultaneously promoting LNP uptake, membrane fusion, and release via small ruptures. However, the potential mechanisms underlying the coordination of UA-V-ATPase interactions and V-ATPase-ESCRT require further investigation.

[0155] In addition, we evaluated the effects of delivery of UA liposomes and PL32 LNPs, respectively, on protein expression and inflammation. Figure 9(km in the text). Although co-administration of PL32 LNP with UA liposomes reduced immunogenicity compared to PL32 LNP alone (likely due to the direct anti-inflammatory effect of UA), the immune response was still higher than that of co-encapsulated P-UA LNP. Furthermore, UA liposomes alone failed to enhance protein expression relative to P32 LNP, either in vitro or in vivo. Figure 9 The presence of "l" and "m" in the text collectively demonstrates the superior efficacy of a single-carrier P-UA formulation. There are two possible reasons why UA liposomes fail to improve PL32 LNP-mediated protein expression. First, liposomes may compete for cellular uptake receptors required for LNP internalization. Second, delivery alone may lead to UA liposomes and PL32 LNPs being sorted into different endosomes, thereby disrupting the synergistic effect of V-ATPase-mediated mRNA release and inflammation relief. To verify the first hypothesis, we compared P32 LNPs alone with a combination of P32 LNPs and blank liposomes. The latter combination resulted in decreased mRNA expression, while delivery of UA liposomes alone only partially salvaged this decrease (…). Figure 9 In stark contrast, P-UA LNPs exhibited the best performance, with significantly enhanced mRNA expression and reduced immunogenicity (l). Figure 9 (k and l in the text). These findings highlight the criticality of co-delivery within a single nanoparticle system for achieving maximum benefits.

[0156] Example 4. NIF P-UA LNP showed significant efficacy in treating bronchopulmonary dysplasia. Bronchopulmonary dysplasia (BPD) is a serious lung disease affecting preterm infants, leading to persistent impairment of lung function and neurodevelopment. Despite improved neonatal care increasing survival rates, the prevalence of BPD continues to rise. A key driver of BPD is the inflammatory response, which exacerbates lung damage, thus highlighting the importance of innovative treatments for managing this disease. In this study, we used a well-established hyperoxia / inflammation-induced bronchopulmonary dysplasia (BPD) rat model, which exhibits key pathological features similar to those of clinical BPD in infants, particularly the characteristic simplification of alveolar structure. Figure 4 As shown in a and b in the figure, compared with the normoxic control group, we observed a significant downregulation of the circadian clock protein NR1D1 ( Figure 4 (a and b in the original text). NR1D1 is crucial for regulating circadian rhythms, inflammation, oxidative stress, pulmonary fibrosis, and mitigating lung injury; its mRNA level is continuously downregulated with prolonged hyperoxia exposure. Figure 4 In the c), protein levels also decrease accordingly ( Figure 4(d in the original text). To directly enhance intracellular NR1D1 expression, we proposed an mRNA therapy. In vitro validation on A549 cells showed that NR1D1 was successfully upregulated and activated downstream pathways after mNR1D1 delivery. Given the widespread reduction of NR1D1 in various lung cell types in damaged tissues ( Figure 4 (d) We explored the potential of P-UA LNP as an mRNA vector for BPD treatment. Compared with the original PL32 LNP, P-UA LNP increased luciferase expression in rat BPD lungs by 9.3-fold ( Figure 10 (a and b in the text) suggests that they may be effective delivery vehicles for mNR1D1 therapy in BPD.

[0157] We further investigated the therapeutic potential of using P-UA LNPs encapsulating mNR1D1 for NR1D1 protein replacement therapy in rats with BPD. FGF10, a growth factor effective in BPD treatment, was used as a positive control. Figure 4 (e). Five therapeutic agents, including empty LNPs and RNA-loaded LNPs, were administered intratracheally to BPD rats, with normoxic rats serving as a control. BPD rats exhibited stunted growth compared to healthy rats. While empty LNPs and PL32 LNPs loaded with mFGF10 showed minimal effects, P-UA LNPs containing mFGF10 and mNR1D1 significantly promoted growth, with weight gain similar to that of healthy rats. Figure 4 f and Figure 10 c). Empty PL32 LNPs induced a 1.4 to 4-fold upregulation of inflammatory cytokines, while empty P-UALNPs alleviated inflammation and oxidative stress by reducing inflammatory cytokines (IL-1β, IL-6, CXCL1, and TNF-α), white blood cell (WBC) counts, ROS levels, and enhancing SOD activity. Figure 4 g and h in Figure 10 (d and e in the text). P-UA LNPs containing mFGF10 and mNR1D1 further enhanced the therapeutic effect on inflammation. BPD rats exhibited characteristic alveolar structural simplification and reduced angiogenesis, while P-UA LNPs loaded with mFGF10 and mNR1D1 effectively alleviated these symptoms, restoring lung structure to near-normal levels, and showed better efficacy than PL32 LNPs containing mNR1D1. Figure 4 i and j in Figure 10 (f in the text). P-UA LNPs containing mNR1D1 also exhibited strong anti-fibrotic effects, reducing α-SMA expression (f in the text). Figure 10 (g in the text). The upregulation of NR1D1 levels after P-UAmNR1D1 treatment was also verified by immunofluorescence (IF). Figure 4k in the middle.

[0158] Transcriptomic analysis of the lungs of BPD rats showed that mNR1D1 P-UA LNP treatment modulated inflammatory pathways, including those involving IL-17 and TNF-α, as well as ROS-related pathways. Figure 4 In addition, mNR1D1 LNP promoted angiogenesis, reduced the expression of myofibroblast-related genes, and increased lipid droplet formation, suggesting that the transition from myofibroblasts to adipocytes is beneficial to the stability of the lung microenvironment. As expected, mNR1D1 LNP treatment reduced the expression of BPD-related genes (m). Figure 4 (l and m in the text). Therefore, P-UA LNP encapsulating mNR1D1 provides a promising treatment option for BPD.

[0159] Example 5. NIF P-UA LNP showed improvement in the treatment of pulmonary fibrosis. Previous studies have shown that circadian rhythm disruption can affect the progression of idiopathic pulmonary fibrosis (IPF), and NR1D1 plays a key role in regulating fibroblast differentiation triggered by TGF-β54. Based on the established antifibrotic efficacy of NR1D1 in treating BPD (…), Figure 10 Based on the potential of NIF LNP delivery of mNR1D1 for treating IPF, and considering its potential in treating lung inflammation and fibrosis caused by cigarette smoke, we investigated the potential of NIF LNP delivery of mNR1D1 for treating IPF.

[0160] Consistent with our findings in healthy animals, we observed a 13.4-fold increase in luciferase expression in IPF lungs after administration of P-UA LNP compared to the original PL32 LNP. Figure 11 (a and b in the text). During the establishment of the IPF model, mice were administered PBS, empty P-UA LNP, mNR1D1 PL32 LNP, and mNR1D1 P-UA LNP intratracheally, respectively. The body weight and survival rate of the mice were then closely monitored. Figure 5 a, b and Figure 11 c). The results showed that mNR1D1 delivered via inflammatory PL32 LNP did not improve IPF status, but instead led to significant weight loss and death in mice ( Figure 5 b in Figure 11 (c and d in the original text). Conversely, compared to the PBS-treated IPF group, empty P-UA LNP significantly reduced pro-inflammatory cytokines by nearly 3-fold without causing any death ( Figure 5(c) The addition of mNR1D1 to P-UA LNP further enhanced the anti-inflammatory effect, resulting in a 7- to 21-fold reduction in cytokine levels. Flow cytometry analysis showed that, compared with empty P-UA LNP, mNR1D1 LNP was more effective in inhibiting T cell infiltration and reducing macrophage and neutrophil levels. Figure 5 (d in the text). Furthermore, the increased accumulation of monocyte-derived suppressor cells (MDSCs) in IPF lungs is associated with activation of lung fibroblasts. We observed that after treatment with mNR1D1 P-UA LNP, MDSC levels decreased to levels similar to the untreated group ( Figure 5 (d in the text)

[0161] To further understand the function of NR1D1, we examined two proteins regulated by NR1D1, α-SMA and lysyl oxidase (LOX). We found that these proteins were significantly inhibited by mNR1D1 P-UA LNPs, and their downstream type I collagen was significantly reduced, while the P-UA vector group did not show this effect. Figure 5 hj and Figure 11 (e). Therefore, this produces a significant anti-fibrotic effect, which was confirmed by histological evaluation using H&E, Masson's trichrome, and Sirius red staining. Figure 5 (eg and j in the text). Given that NR1D1 is known to affect TGF-β40 function, we observed a significant reduction in CD4+IL-17 and CD4+IL-4 secreting cells, which have been shown to be regulated by the TGF-β pathway (eg and j in the text). Figure 5 (k and l in the text). In general, mNR1D1 encapsulated in non-inflammatory LNPs reduces inflammation and fibrosis, effectively reversing IPF to a near-healthy state.

[0162] Example 6. Optimization of NIF LNPs nebulization and lyophilization processes to improve clinical feasibility. Nebulization is a primary treatment route for neonates with bronchopulmonary dysplasia (BPD) and one of the most promising clinical drug delivery routes for patients with idiopathic pulmonary fibrosis (IPF). Therefore, we explored efficient drug delivery of P-UA LNP via nebulization technology. Figure 6 (a) However, we found that protein expression of P-UA LNP was significantly reduced after nebulization, which was due to aggregation, disintegration and premature mRNA leakage caused by high shear force (a). Figure 6 (b) To enhance the stability of LNPs during nebulization, we used sodium acetate (NaAc) as the LNP buffer and hydrophilic polyethylene glycols (PEG6K and PEG20K) as stabilizing excipients (see Table 5). Although these modifications maintained the particle size and mLuc encapsulation efficiency, they did not significantly improve protein expression after nebulization. Figure 6 b and Figure 12 (ab in the text). We hypothesize that the inherent surface charge of P-UA LNPs helps them stabilize through electrostatic repulsion. Using a low-salt dialysis buffer, we enhanced the electrostatic repulsion and improved nebulization stability by eliminating the charge shielding effect of high ion concentrations in PBS. Figure 12 (a) Notably, we found that all three low-salt buffers improved the encapsulation efficiency of mLuc in LNPs, increasing the encapsulation efficiency to approximately 85% before and after nebulization. Figure 12 (a) Specifically, compared with PBS-buffered P-UA LNPs, phosphate-buffered saline-sucrose (PB-sucrose) low-salt dialysis buffer increased protein expression by 7.7-fold, with no significant effect on particle size after nebulization. Figure 6 b in Figure 12 (a and b in the text).

[0163] Table 5: Dialysis conditions and protectants for P-UA LNPs Since sucrose has already been used as a freeze-drying preservative, we will next freeze-dry the optimized LNP formulation. Figure 12 In the lyophilization cycle shown in c, all P-UA LNPs exhibited a dense, white, and uniform lyophilized cake appearance. After reconstitution with nuclease-free water, no significant loss of protein expression was observed in the lyophilized product. Figure 6 c, d and Figure 12 (d) Furthermore, after storage at 4°C, the lyophilized vials retained their elegant cake-like structure without showing signs of collapse, shrinkage, or cracking. Upon reconstitution, particle size and polydispersity remained stable for at least 3 months. Figure 6 Next, we evaluated the therapeutic effects of lyophilized and nebulizable mNR1D1 P-UA LNPs in BPD and IPF models via nebulization. We used a nebulizer to administer mNR1D1-encapsulated P-UA LNPs at an adjusted dose of 80 mg / kg to BPD rats and IPF mice. Figure 12 The results showed that mNR1D1 P-UA LNPs effectively alleviated BPD in rats and IPF in mice, and significantly reduced alveolar structural simplification and collagen deposition. Figure 6 fk in Figure 12 (f and h in the text). Notably, in beagle dogs with respiratory systems similar to humans, lung protein expression was increased more than 5-fold with mLuc-loaded P-UA LNPs compared to PL32 LNPs. Figure 6The results, consistent with those in mice and rats, showed that P-UA LNPs were significantly associated with reduced inflammation, manifested as decreased white blood cell count and neutrophil percentage in the blood, decreased levels of inflammatory cytokines and C-reactive protein (CRP), and reduced immune cell infiltration in the blood, bronchoalveolar lavage fluid (BAL), and lungs. Figure 6 np and Figure 12 (i) In summary, we optimized the preparation process to enable P-UA LNPs for nebulized drug delivery and long-term lyophilized storage, thereby greatly enhancing their clinical translation potential.

[0164] Example 7. Oleanolic acid, ursolic acid derivatives, and bile salts enhance LNP expression and inhibit inflammation. Based on the ability of carboxyl-containing natural products such as ursolic acid and oleanolic acid to alleviate LNP inflammatory response and enhance LNP protein expression efficiency, we further optimized the structures of oleanolic acid and ursolic acid, synthesizing a series of derivatives. In addition, we also investigated some bile acid derivatives with similar results to ursolic acid. The effects of oleanolic acid, ursolic acid derivatives, and bile acid derivatives on LNP protein expression efficiency and inflammation were explored. The structures of these derivatives are shown below: The synthetic methods for these compounds are as follows: Synthesis of OAC1C: Accurately weigh 0.50 g of oleanolic acid (1.09 mmol, 1 equivalent) and dissolve it in tetrahydrofuran. Then, add 0.30 g of K₂CO₃ (2.18 mmol, 2 equivalent) and 0.22 g of ethyl bromoacetate (1.31 mmol, 1.2 equivalent) sequentially. The mixture is stirred overnight at 40 °C, and the reaction is monitored using a thin-layer chromatography plate. After the reaction is complete, the reaction system is cooled to room temperature and slowly added dropwise to ice water, resulting in the formation of a white precipitate. Filter the precipitate and collect the filter cake (0.44 g, white solid). Dissolve the filter cake in a 1:1 methanol / tetrahydrofuran mixture, add 0.10 g of lithium hydroxide (5 equivalent), and stir the mixture at room temperature for 2 h. After the reaction is complete, adjust the pH to 3-4 using 1M hydrochloric acid aqueous solution. A white solid precipitates out. Filter the precipitate, collect the filter cake, and dry it to obtain a pure white solid powder, OAC₁C (0.38 g, yield 64.30%). 1H NMR (400 MHz, Methanol-d4) δ 5.20 (t, J = 3.6 Hz, 1H), 4.34 (q, J= 15.1 Hz, 2H), 3.10 (dd, J = 11.3, 4.8 Hz, 1H), 2.85 (dd, J = 14.0, 4.5 Hz,1H), 1.99 (td, J = 13.7, 12.8, 4.0 Hz, 1H), 1.90 - 1.76 (m, 3H), 1.75 - 1.22(m, 13H), 1.20 - 1.05 (m, 5H), 1.05 - 0.99 (m, 1H), 0.92 (s, 3H), 0.91 - 0.84 (m, 9H), 0.72 (s, 3H), 0.70 (s, 4H). 13 C NMR (101 MHz, MeOD) δ 178.97, 174.35,145.11, 123.64, 79.67, 63.48, 56.76, 49.64, 49.43, 49.21, 49.06, 49.00,48.79, 48.57, 48.36, 47.89, 47.29, 42.83, 42.67, 40.61, 39.87, 39.83, 38.16,34.95, 33.95, 33.67, 33.22, 31.60, 28.82, 28.80, 27.86, 26.54, 24.53, 24.12,24.05, 19.51, 17.62, 16.35, 15.94. ESI-MS: m / z: 514.3582 [MH]-, C 32 H 50 O5(514.3658).

[0165] Synthesis of OAC3C: The synthesis of OAC3C is the same as that of OAC1C, using oleanolic acid and ethyl 4-bromobutyrate as raw materials. The product is a pure white solid with a yield of 67.52%. 1¹H NMR (400 MHz, Chloroform-d) δ 5.31 (d, J = 3.7 Hz, 1H), 4.09 (hept, J = 5.7, 5.2 Hz, 2H), 3.24 (dd, J = 11.1, 4.7 Hz, 1H), 2.87 (dd, J = 13.9, 4.5 Hz, 1H), 2.46 (t, J = 7.4 Hz, 2H), 1.97 (h, J = 6.1, 5.1 Hz, 3H), 1.89 (dq, J = 6.8, 3.3 Hz, 2H), 1.76 - 1.26 (m, 16H), 1.24 - 1.20 (m, 1H), 1.17 (d, J = 4.8 Hz, 1H), 1.14 (s, 3H), 1.12 - 1.04 (m, 1H), 1.00 (s, 3H), 0.94 (s, 3H), 0.91 (s, 6H), 0.79 (s, 3H), 0.73 (s, 3H). 13 ¹³C NMR (101 MHz, CDCl₃) δ 178.36, 177.81, 143.91, 122.64, 79.26, 77.48, 77.16, 76.84, 63.20, 55.35, 47.75, 46.92, 45.98, 41.86, 41.49, 39.48, 38.87, 38.57, 37.16, 33.99, 33.24, 32.88, 32.62, 30.83, 30.75, 28.25, 27.78, 27.23, 26.04, 23.99, 23.77, 23.54, 23.15, 18.47, 17.18, 15.74, 15.42. ESI-MS: m / z: 541.3898 [M-H]⁻, C 34 H 54 O₅(542.3971).

[0166] Synthesis of OAS: Accurately weigh 0.45 g of oleanolic acid (1.0 mmol, 1 equivalent) and dissolve it in an appropriate amount of DMF. Add 0.28 g of potassium carbonate (2.0 mmol, 2 equivalent), and slowly add 0.25 g of sodium 3-bromopropylsulfonate (1.1 mmol, 1.1 equivalent) dissolved in DMF dropwise while stirring at room temperature. Then, heat the reaction system to 40 °C and stir overnight. Monitor the reaction with a thin-layer chromatography plate. After the reaction is complete, remove DMF by rotary evaporation. Add water to the residue, sonicate to prepare a suspension, and adjust the pH to 3-4 with 1M HCl. A white solid precipitates out. Filter and collect the filter cake, then dry to obtain a pure white solid product, OAS (0.33 g, yield 57.06%). 1 H NMR (400 MHz, Methanol-) d 4) δ 5.32 - 5.24 (m, 1H), 0.78 - 0.74 (m, 6H), 4.13 (t, J = 6.3 Hz, 2H), 3.14 (dd, J = 11.3, 4.8 Hz, 1H), 2.98 - 2.79 (m, 1H), 2.10 (ddd, J =14.1, 7.9, 6.3 Hz, 2H), 2.06 - 1.82 (m, 4H), 1.78 - 1.26 (m, 15H), 1.25 -1.04 (m, 6H), 0.97 (s, 3H), 0.94 (d, J = 2.4 Hz, 6H), 0.91 (s, 3H), 0.76 (d, J = 2.9 Hz, 6H). 13 C NMR (101 MHz, MeOD) δ 179.34, 144.94, 124.02, 79.70,64.43, 56.74, 49.64, 49.54, 49.43, 49.21, 49.00, 48.79, 48.57, 48.36, 48.13,47.08, 42.86, 42.79, 40.65, 39.82, 38.16, 34.80, 33.98, 33.72, 33.52, 31.58,28.77, 28.75, 27.86, 26.42, 25.74, 24.52, 24.03, 23.99, 19.48, 17.88, 16.31,15.90. ESI-MS: m / z: 577.3567 [MH]-, C 33 H 54O6S (578.3641). Synthesis of UAC1C: The synthesis of UAC1C is the same as that of OAC1C, using ursolic acid and ethyl bromoacetate as raw materials. The product is a pure white solid with a yield of 58.75%. 1 H NMR (400 MHz, Chloroform- d ) δ 5.26 (t, J = 3.7 Hz, 1H), 4.59 (d, J = 16.3 Hz, 1H), 4.54 (d, J = 16.3 Hz, 1H), 3.23 (dd, J = 10.8, 4.8 Hz, 1H), 2.25 (d, J = 11.3 Hz, 1H), 2.04 (td, J = 13.3, 12.8, 4.4 Hz, 1H), 1.90 (dd, J = 8.9, 3.7 Hz, 2H), 1.85 - 1.41 (m, 12H), 1.33 (tdd, J = 18.9, 14.0, 8.8 Hz,8H), 1.08 (s, 3H), 0.98 (s, 3H), 0.94 (d, J = 6.1 Hz, 3H), 0.91 (s, 3H), 0.86(d, J = 6.4 Hz, 3H), 0.78 (s, 3H), 0.73 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ177.03, 172.95, 137.98, 125.92, 79.41, 77.48, 77.16, 76.84, 60.36, 55.32,52.86, 48.29, 47.67, 42.18, 39.65, 39.20, 38.91, 38.82, 38.75, 37.06, 36.49,33.14, 30.78, 28.24, 28.11, 27.11, 24.33, 23.63, 23.43, 21.32, 18.44, 17.15,17.09, 15.79, 15.60. ESI-MS: m / z: 514.3582 [MH]-, C32 H 50 O5 (514.3658).

[0167] Synthesis of UAC3C: The synthesis of UAC3C is the same as that of OAC1C, using ursolic acid and ethyl 4-bromobutyrate as raw materials. The product is a pure white solid with a yield of 71.36%. 1 H NMR (400 MHz, Chloroform- d ) δ 5.27 - 5.21 (m, 1H), 4.10 - 3.95 (m,2H), 3.22 (dd, J = 10.9, 4.8 Hz, 1H), 2.35 (t, J = 7.4 Hz, 2H), 2.21 (d, J =11.1 Hz, 1H), 1.99 (td, J = 13.2, 4.4 Hz, 1H), 1.90 (t, J = 6.6 Hz, 4H), 1.75(td, J = 13.5, 4.4 Hz, 1H), 1.71 - 1.41 (m, 11H), 1.31 (td, J = 18.9, 18.0,7.0 Hz, 8H), 1.07 (s, 3H), 0.99 (s, 3H), 0.94 (d, J = 6.0 Hz, 3H), 0.91 (s,3H), 0.85 (d, J = 6.3 Hz, 3H), 0.78 (s, 3H), 0.73 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 179.44, 177.93, 138.25, 125.74, 79.17, 77.48, 77.16, 76.84, 63.61,55.29, 52.93, 48.25, 47.63, 42.15, 39.63, 39.17, 38.99, 38.84, 38.73, 37.05,36.86, 33.15, 31.91, 30.80, 28.27, 28.08, 27.21, 24.41, 24.30, 23.68, 23.41,21.33, 18.44, 17.20, 17.16, 15.81, 15.59. ESI-MS: m / z: 541.3893 [MH]-,C 34 H 54 O5 (542.3971).

[0168] Synthesis of UAS: The synthesis of UAS is the same as that of OAS, using ursolic acid and sodium 3-bromopropylsulfonate as raw materials. The product is a pure white solid with a yield of 62.40%. 1 H NMR (400 MHz, Methanol- d 4) δ 5.27 (t, J = 3.5 Hz, 1H), 4.10 (td, J =6.3, 2.8 Hz, 2H), 3.15 (dd, J = 11.0, 4.8 Hz, 1H), 2.97 - 2.78 (m, 2H), 2.24(dd, J = 11.2, 1.7 Hz, 1H), 2.14 - 2.01 (m, 3H), 2.00 - 1.91 (m, 2H), 1.83(td, J = 13.7, 4.7 Hz, 1H), 1.72 - 1.46 (m, 10H), 1.47 - 1.25 (m, 6H), 1.12(s, 3H), 0.97 (d, J = 4.5 Hz, 9H), 0.88 (d, J = 6.4 Hz, 3H), 0.78 (d, J = 4.1Hz, 6H). 13C NMR (101 MHz, MeOD) δ 179.12, 139.52, 127.24, 79.65, 64.42,56.70, 54.31, 49.64, 49.58, 49.47, 49.43, 49.21, 49.00, 48.79, 48.57, 48.36,43.20, 40.84, 40.37, 40.32, 39.96, 39.82, 38.09, 37.98, 34.26, 31.67, 29.08,28.79, 27.87, 25.68, 25.29, 24.35, 24.17, 21.54, 19.45, 17.95, 17.62, 16.39,16.03. ESI-MS: m / z: 577.3559 [MH]-, C 33 H 54 O6S (578.3641). Among them, OAC1C, UAC1C, and OAC3C are reported compounds, while the other derivatives are novel compounds synthesized independently.

[0169] The protein expression changes of PL32 LNP after adding 20% ​​mol ursolic acid and oleanolic acid derivatives are as follows: Figure 13 As shown in the figure (A549 cells, 1 μg / mL mFluc, 24 h after drug administration), the protein expression changes of PL32 LNP after the addition of 20% mol cholic acid and its derivatives are as follows. Figure 14 As shown (A549 cells, 1 μg / mL mFluc, administered for 24 h). This demonstrates that the introduction of bile acids, ursolic acid, oleanolic acid, and bile acid derivatives can enhance the protein expression efficiency of LNP-delivered mRNA.

[0170] We investigated the anti-inflammatory effects of UAS derivatives as an example. We found that introducing UAS into PL32 LNP cells in the NK92mi cell line effectively inhibited the activation of the INF-α and β pathways. Figure 15As shown, we treated NK92mi cells with PL32 LNP (PL32) and PL32 LNP containing UAS (PL32-UAS), respectively. After 24 h, the cell supernatant was added to RAW-ISG cells and incubated for 24 h. The bioluminescence intensity of the RAW-ISG cell supernatant was then measured. Stronger bioluminescence indicates a stronger ability to activate the INF-α and β pathways. Compared to PL32 LNP, the bioluminescence of PL32-UAS LNP was significantly reduced, demonstrating that the introduction of UAS can inhibit the LNP inflammatory response. Furthermore, we used qPCR to detect changes in the expression of inflammatory factors in NK92mi cells after administration of PL32 LNP and PL32-UAS LNP. Figure 16 As shown, we found that PL32-UAS LNPs had lower expression levels of INFγ and TNFα compared to PL32 LNPs. This also indicates that the introduction of UAS can inhibit LNP inflammation.

[0171] In summary, we found that adding ursolic acid, oleanolic acid derivatives, cholic acid and its derivatives to LNPs can also enhance expression and inhibit inflammation.

[0172] Example 8. LNP-delivered siRNA containing natural products for the treatment of asthma In previous studies, we found that introducing anti-inflammatory natural products such as ursolic acid could effectively inhibit LNP-induced inflammation and improve the protein transfection efficiency of the encapsulated mRNA. Based on this, we further explored whether LNPs containing such natural products are suitable for siRNA delivery. We investigated the application of LNPs containing natural products in the treatment of asthma, using Muc5ac as the therapeutic target.

[0173] 1. Preliminary prescription screening for LNP Experimental Content and Methods (1) Prescription construction With cationic lipids, co-lipids, cholesterol, DMG-PEG 2000 Formulas 1-12 were prepared using natural anti-inflammatory acid derivatives (including ursolic acid, crosolic acid, and hawthorn acid) in molar proportions of 42.5%, 8.5%, 32.725%, 1.275%, and 15%, respectively. Cationic lipids, co-lipids, cholesterol, and DMG-PEG were also included. 2000 Formulas 13-17 (without acid) were prepared in molar ratios of 50%, 10%, 38.5%, 1.5%, and 0.

[0174] The volume ratio of the aqueous phase to the ethanol organic phase was 3:1. The final concentration of siRNA was 0.072 mg / mL, and the volume of 50 mM citrate (pH 4) was 1 / 5 of the volume of the aqueous phase.

[0175] A total of 17 formulations were prepared, with ionizable cationic lipids, auxiliary lipids, and the fifth component - acid as variables. The specific formulation details are shown in Table 6 below (SM102 is the control): Table 6: Lipid formulations used in 17 LNPs (2) Evaluation of siRNA delivery and knockdown activity 1) Cell treatment: Inoculation density: 2×10 6 Cells / well (plate type: 24-well plate); Incubation conditions: 100 nM siRNA, 48 h; Groups: (a) -: blank control group, (b) +: siRNA4 with target added (not loaded with LNP), (17): commercially available SM102 formulation, others: LNP formulations 1-16.

[0176] 2) qPCR validation: The detection method is the Sybr Green assay. Prepare the reaction system; set up the qPCR program and run it on the machine: denaturation (95℃) → annealing (55-60℃) → extension (72℃).

[0177] Experimental results The expression status of Muc5ac mRNA in A549 cells (reflecting siRNA knockdown activity) is shown in the following results. Figure 17 The siRNAs with added targets showed low in vitro activity (A549 cells lack the avβ6 receptor); the green column group showed better knockdown efficiency than the SM102 formulation, and three of them will be selected for further screening.

[0178] 2. Screening of physicochemical properties of three LNP formulations Experimental Content and Methods (1) Preparation of LNP formulation (a) Organic phase preparation: cationic lipids, cholesterol, DSPC / DOPE, DMG-PEG 2000 Lipids and (acids) are dissolved in ethanol at a predetermined molar ratio. The mixture is then vortexed.

[0179] (b) Aqueous phase preparation: The final concentration of siRNA is 0.072 mg / mL, and the volume of 50 mM citrate (pH 4) is 1 / 5 of the volume of the aqueous phase. Prepare the aqueous phase.

[0180] (c) Microfluidic mixing: The organic phase and aqueous phase were injected into the microfluidic chip at a flow rate ratio of 3:1. The mixture was collected and dialyzed with PBS to remove ethanol.

[0181] (2) Determination of physicochemical properties (a) Encapsulation efficiency: direct determination method Dilute LNP 100-fold and add it by volume to a black 96-well plate, dividing it into two groups to measure total RNA and free RNA, respectively. Add equal volumes of TE and TX to each group and shake. Then add RiboGreen fluorescent dye diluted 200-400-fold to each well, incubate for 5 minutes, and measure the fluorescence intensity using a microplate reader (excitation wavelength 485nm, emission wavelength 515nm). Calculate the encapsulation efficiency based on the RNA standard curve using the following formula: (b) Particle size and PDI: The particle size and zeta potential of each group of LNPs were determined using a Zetasizer Pro nanoparticle size potential analyzer. When measuring particle size, 1 mL of the formulation was added to a particle size cup for testing (n=3).

[0182] Experimental results Encapsulation efficiency results are as follows Figure 18 As shown, the particle size results are as follows Figure 19 As shown.

[0183] 3. Cytotoxicity assessment Experimental Content and Methods MTT assay for cytotoxicity (1) Cell seeding: A549 cells were seeded at a rate of 5 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of 100 μL per well in 96-well plates. The plates were incubated at 37°C for 24 h.

[0184] (2) Cell drug delivery - LNP treatment Experimental groups: Culture medium containing LNP was added (siRNA final concentration: 50, 100, 200 nM). Three replicates were set for each concentration (n=3). Blank control group: Culture medium only (no cells). Negative control group: Cells + culture medium (no LNP). Incubation conditions: 37℃, 5% CO2, treatment time 24 h.

[0185] (3) MTT staining: Add 10 μL of MTT solution (5 mg / mL, prepared in PBS) to each well and incubate in the dark for 4 h. Carefully aspirate the culture medium, add 100 μL of DMSO to each well, and shake for 10 min to dissolve the formazan crystals.

[0186] (4) Absorbance detection: The absorbance at 570 nm was detected by an enzyme-linked immunosorbent assay (ELISA) reader.

[0187] (5) Calculate cell viability: Experimental results Cell survival rate (reflecting cytotoxicity) 24 hours after drug administration, such as Figure 20As shown. Based on cytotoxicity experiments, two LNPs with lower toxicity (1, 2) were finally screened. LNP 9 was discarded due to its higher cytotoxicity.

[0188] 4. Optimize LNP preparation conditions to increase siRNA drug loading concentration. Based on the two LNP formulations (1 and 2) screened earlier, we changed the microfluidic equipment and used the Myanna microfluidic preparation system to prepare LNPs to optimize their formulation properties (particle size, encapsulation efficiency, etc.) and increase the ratio of siRNA to lipids, thereby further reducing the inflammatory response of LNPs.

[0189] Experimental methods and procedures (1) Adjust the N / P ratio of LNP to 3:1 (originally 6:1), and the final concentration of siRNA to 0.144 mg / mL. Use Myanna microfluidic control to prepare LNP: total volume 0.6 mL, total flow rate: 12 mL / min, aqueous phase: organic phase = 3:1.

[0190] (2) Collect the mixture and remove ethanol by dialysis with PBS. Determine the particle size and encapsulation efficiency of LNPs in accordance with the method described above.

[0191] Experimental results Encapsulation efficiency results are as follows Figure 21 As shown, the particle size results are as follows Figure 22 As shown, after preparation using Myanna microfluidics, the particle size of LNPs can reach below 200 nm; the encapsulation efficiency is above 60% (and can still be further optimized).

[0192] 5. Encapsulation efficiency further optimized. We aim to further improve the encapsulation efficiency of LNPs by changing the preparation conditions. Experimental methods and procedures (1) Process optimization Table 7: Optimization of Microfluidic LNP Fabrication Process (2) Optimization of organic solvent removal methods The sodium chloride in the dialysis medium PBS was replaced with an equimolar amount of sucrose (phosphate concentration remained unchanged), and the effects of ultrafiltration and dialysis on removing the organic phase were compared. Four groups were set up for the experiment: PBS ultrafiltration, PBS dialysis, sucrose ultrafiltration, and sucrose dialysis. The encapsulation efficiency of each of the four groups was measured.

[0193] Experimental results Encapsulation efficiency results are as follows Figure 23 As shown, after optimization, the encapsulation efficiency of the experimental group in which sodium chloride in PBS was replaced with sucrose was significantly improved, and the encapsulation efficiency of the candidate formulation could be increased from 60% to over 80%, meeting the drug administration requirements for animal experiments.

[0194] 6. In vivo siRNA gene knockdown efficiency Based on two previously screened LNP formulations (1, 2), the in vivo knockout efficiency of Muc5ac was evaluated by administering siRNA LNPs via the trachea to induce high expression of Muc5ac in the lungs of mice through house dust mite protein. The final formulation was screened using commercially available formulation SM102 and target-tipped siRNA TD-PC as controls.

[0195] Experimental methods and procedures Administration method such as Figure 24 As shown in Table 8, the grouping and treatment (TD-PC is the targeted modified siRNA, i.e., T-siRNA) are as follows.

[0196] Table 8: Grouping and Processing LNP4 (Prescription 1) is prescription number 1 in Table 6; LNP5 (Prescription 2) is prescription number 2 in Table 6.

[0197] Experimental results Survival rate and weight change, such as Figure 25 and Figure 26 As shown. The inhibitory effect of Muc5ac expression is as follows. Figure 27 As shown, changes in Muc5ac protein levels are as follows: Figure 28 and Figure 29 As shown.

[0198] Overall, both LNPs were superior to SM102 in terms of safety, with LNP4 showing the best performance at the protein level.

[0199] 7. Lung distribution of Cy5-siRNA in vivo Experimental methods and procedures Establishment of a mouse asthma model Female balb / c mice weighing 18-22g were administered 50 μL of a dust mite protein solution via trachea on days 1, 8, 9, 10, 11, 12, and 13.

[0200] Cy5-siRNA lung distribution The house dust mite-infected mice were divided into four groups and administered intratracheal solutions containing Cy5-siRNA (LNP4, LNP5, SM102, and Cy5-siRNA, respectively, 5 μg siRNA / mouse). Six hours after administration, the mice were sacrificed, and in vivo imaging analysis of the Cy5 fluorescence signal in the lungs was performed. Lung tissue was digested, separated into single-cell suspensions, and flow cytometry was used to analyze the signal intensity of Cy5 in different cell types (including epithelial cells, endothelial cells, immune cells, and stromal cells).

[0201] Experimental results Depend on Figure 30 and Figure 31 It was found that the fluorescence intensity of all three LNPs (including SM102) was higher than that of Cy5-siRNA Sol. Flow cytometry results showed that siRNA was distributed in epithelial cells, endothelial cells, immune cells, and stromal cells. Among them, LNP4 showed the strongest total Cy5 fluorescence signal in the lungs.

[0202] 8. In vivo asthma treatment effect Experimental methods and procedures Establishment and administration of mouse asthma model Female Balb / c mice weighing 18-22g were administered 50 μL of a dust mite protein solution containing 50 μg via trachea on days 1, 8, 9, 10, 11, 12, and 13. On days 13, 16, and 19, PBS, three types of LNPs, and a targeted modified siRNA (T-siRNA) were administered via trachea. The mice were sacrificed on day 21, and blood, bronchoalveolar lavage fluid, and lung tissue were collected for ELISA testing and section staining.

[0203] Experimental results like Figures 32-35 As shown, siRNA treatment significantly reduced the expression level of Muc5ac in mouse bronchoalveolar lavage fluid and lung tissue. The knockdown efficiency was LNP4 > T-siRNA > SM-102 LNP. Based on its good gene knockdown efficiency, LNP4 demonstrated a good therapeutic effect on asthma, significantly downregulating asthma-related indicators such as IFN-γ, IgE, TNF-α, IL-13, and IL-17. Its therapeutic efficiency was significantly higher than that of the SM-102 group and the T-siRNA group. H&E and PAS staining also confirmed its therapeutic effect.

[0204] Conclusion: We found that the five-component LNP formed by encapsulating natural products such as UA into LNPs can deliver both mRNA and siRNA. The introduction of UA can significantly improve the gene knockdown efficiency of siRNA. In a mouse asthma model, LNP4 loaded with Muc5ac-siRNA showed good therapeutic effects.

[0205] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A lipid nanoparticle (LNP), comprising: At least one ionizable lipid, optionally, said ionizable lipid constitutes about 20 mol% to about 70 mol% of said LNP; and At least one compound capable of activating V-ATPase, optionally, the compound capable of activating V-ATPase accounts for about 2.5 mol% to about 50 mol% of the LNP.

2. The LNP according to claim 1, wherein, The compounds that can activate V-ATPase are selected from ursolic acid, oleanolic acid, cholic acid, crataegolic acid, and crosolic acid and their derivatives.

3. The LNP according to claim 1 or 2, wherein, The compound capable of activating V-ATPase is selected from compounds of the following formula: In formula (I-1), Y is -O- or -NH-, Z is hydrogen or C3-C6 alkylene, and R1 is hydrogen, carboxyl or sulfonic acid group or absent (when Z is hydrogen). R2 and R3 are each independently hydrogen or methyl; R4 and R5 are each independently hydrogen or methyl; R6 is hydroxyl or hydrogen, R7 is hydroxymethyl or methyl, and R8 is hydroxyl or hydrogen. Alternatively, in formula (I-1), Y is -O- or -NH-, Z is hydrogen, -CH2-, -CH2-CH2-CH2- or -CH2-CH2-CH2-CH2-, and R1 is hydrogen, carboxyl or sulfonic acid group or absent (when Z is hydrogen); or, Y is -O- or -NH-, Z is hydrogen, -CH2- or -CH2-CH2-CH2-, and R1 is hydrogen, carboxyl or sulfonic acid group or absent (when Z is hydrogen). Alternatively, in formula (I-1), R2 and R3 are both methyl groups; R4 and R5 are both hydrogens; R6 and R8 are both hydrogens; and R7 is a methyl group. Alternatively, in formula (I-1), R2 is methyl and R3 is hydrogen; R4 is methyl and R5 is hydrogen; R6 and R8 are both hydrogen; R7 is methyl; Alternatively, in formula (I-1), R2 and R3 are both methyl groups; R4 and R5 are both hydrogens; R6 is a hydroxyl group; R8 is hydrogen; and R7 is a methyl group. Alternatively, in formula (I-1), R2 is methyl and R3 is hydrogen; R4 is methyl and R5 is hydrogen; R6 is hydroxyl, R8 is hydrogen; and R7 is methyl. Alternatively, in formula (I-1), R2 is methyl and R3 is hydrogen; R4 is methyl and R5 is hydrogen; R6 is hydroxyl, R8 is hydroxyl; and R7 is hydroxymethyl. In formula (I-2), Y is -O- or -NH-, Z is hydrogen or C1-C6 alkylene, and R1 is hydrogen, carboxyl, sulfonic acid, or absent (Z is hydrogen). One of R3 and R4 is hydrogen and the other is either hydrogen or hydroxyl; R2 and R5 are each independently hydrogen or hydroxyl.

4. The LNP according to any one of claims 1-3, wherein, The compound that can activate V-ATPase is selected from compounds of the following formula: 。 5. The LNP according to any one of claims 1-4, wherein, The ionizable lipid is a phosphoramide compound containing one or more tertiary amino groups, said phosphoramide compound being as described in formula (I-3), or a stereoisomer thereof, a prodrug, or a pharmaceutically acceptable salt thereof: In equation (I-3), n is an integer between 0 and 5; L1 and L2 are each independently C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-M-R''; R1 and R2 are each independently C4-C 30 Alkyl, C2-C 40 Alkenyl, or -R'-M-R''; or, R1 and R2 together with their respective attached N and L4 form 5- to 8-membered diazacycloalkyl groups; R3 and R4 are each independently C1-C 30 Alkyl, C2-C 40 Alkenyl, or -R'-M-R''; or, R3 is H, C1-C 30 Alkyl, C2-C 40 Alkenyl, or -R'-M-R'', while R4 is C1-C 30 Alkyl, C2-C 40 Alkenyl or -R'-M-R''; R' is C1-C 24 Alkylene or C2-C 24 alkenyl; M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -SS-, -C(O)N(R a )-、-N(R a )C(O)-、-OC(O)N(R a )-、-N(R a )C(O)O-、-N(R a )C(O)N(R a )-、-C(OC(O)-R a (H)-C 1-6 Alkylene -OC(O)-, -C(O)OC 1-6 Alkylenes -C(O)-O-, -CH(OH)-, -P(O)(OR b O-, C6-C 10 arylene and 5- to 10-membered heteroarylene; R''、R a and R b Each is independently selected from hydrogen, C1-C 30 Alkyl and C2-C 40 alkenyl; L3, L4, and L5 are each independently C1-C 12 Alkylene, C2-C 12 alkenyl, C3-C8 cycloalkylene, -AYB-, -AY- or -YB-, where A and B are each independently C1-C 12 Alkylene or C2-C 12 The alkylene group, where Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene; Preferably, the ionizable lipid is selected from one of the following compounds: PL10, PL11, PL12, PL13, PL14, PL17, PL18, PL19, PL20, PL21, PL22, PL23, PL24, PL26, PL27, PL28, PL29, PL30, PL31, PL32, PL33, PL34, PL35, PL36, PL38, PL41, PL44, PL45, PL53, PL54, PL55, PL56, PL57, PL58, PL59, PL60, PL61, PL78, PL79, PL93, PL94, PL95, PL96, PL97 and PL103; Preferably, the ionizable lipid is PL32.

6. The LNP according to any one of claims 1-4, wherein, The ionizable lipids are unsaturated tail ionizable lipids, multi-tailed ionizable lipids, ionizable polymeric lipids, biodegradable ionizable lipids, or branched tail ionizable lipids. Preferably, the ionizable lipid is selected from the group consisting of: 3-(eicosylamino)-N1,N1,4-tridodecyl-1-piperazine ethylamine (KL10), N1-[2-(eicosylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazine diethylamine (KL22), 14,25-tetraaza-octacosane (KL25), 1,2-dilinolenicyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinolenicyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinolenicyl-4-(2-dimethylaminoethyl)- [1,3]-Dioxolane (DLin-KC2-DMA), 1,2-diolenyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA) (2R)) and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)).

7. The LNP according to claim 6, wherein, The ionizable lipids are ALC-0315, SM-102, ALC366, DODMA, C12-200, cKK-E12, YSK05, LP01, TT3, CL4H6, Lipid 5, ECO, OF-02, 306Oi10, 4A3-SC8 or HY-501.

8. The LNP according to any one of claims 1-7, wherein, The LNP also includes: At least one auxiliary lipid, optionally, said auxiliary lipid comprising about 0 mol% to about 60 mol% of said LNP. At least one sterol, optionally, said sterol comprising about 0 mol% to about 60 mol% of said LNP. At least one PEG-modified lipid, optionally, the PEG-modified lipid comprising about 0.2 mol% to about 20 mol% of the LNP.

9. The LNP according to any one of claims 1-8, wherein, The sterol is selected from one or more of the following: cholesterol, β-sitosterol, stigmasterol, lycopene, ergosterol, campesterol, cholesterol hemisuccinate, PEGylated cholesterol, oxosterol, oxidized cholesterol derivatives, and phytosterol esters; preferably, the sterol is cholesterol.

10. The LNP according to any one of claims 1-9, wherein, The auxiliary lipids are selected from the group consisting of: distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), bis(pentadecanoyl)phosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), arachidoylphosphatidylcholine (DAPC), dibenzylphosphatidylcholine (DBPC), bis(triacyl)phosphatidylcholine (DTPC), bis(tetracosyl)phosphatidylcholine (DLPC), palmitoyloleyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauryl-phosphatidylethanolamine (DLPE), diphytyl-phosphatidylethanolamine (DPyPE), and combinations thereof; preferably, the auxiliary lipid is distearylphosphatidylcholine (DSPC) or dioleoylphosphatidylethanolamine (DOPE).

11. The LNP according to any one of claims 1-10, wherein, The PEG-modified lipid is selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and any mixture thereof; preferably, the PEG-modified lipid is polyoxazoline, polyglycerol, poly(N-vinylpyrrolidone), polydopamine, polycarboxybetaine, polysulfobetaine, or polycaprolactone-polyethylene glycol; preferably, the PEG-modified lipid is DMG-PEG. 2000 .

12. The LNP according to any one of claims 1-11, wherein, The ionizable lipid is PL32, the auxiliary lipid is distearylphosphatidylcholine (DSPC) or dioleoylphosphatidylethanolamine (DOPE), and the PEG-modified lipid is DMG-PEG. 2000 The compound that can activate V-ATPase accounts for approximately 20 mol of the LNP.

13. The LNP according to any one of claims 1-12, wherein, The molar ratio of the ionizable lipid, the auxiliary lipid, the cholesterol, the PEG-modified lipid, and the compound capable of activating V-ATPase is 42.5:8.5:32.725:1.275:

15.

14. The LNP according to any one of claims 1-13, wherein, The LNP also contains mRNA or siRNA.

15. The LNP according to claim 14, wherein, The siRNA is a siRNA targeting the Muc5ac gene; preferably, the sequence of the siRNA is derived from 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 65-68; preferably, the sequence of the siRNA is as shown in SEQ ID NO: 65-68.

16. A method for preparing an LNP formulation, comprising: The LNP according to any one of claims 1-15 was dialyzed in a phosphate buffer-sucrose solution, and then the dialyzed LNP was nebulized or lyophilized.

17. The preparation method according to claim 16, wherein, The sucrose concentration in the phosphate buffer-sucrose solution was approximately 9% (w / w), and the dialysis time was approximately 16 hours.

18. An LNP formulation prepared by the preparation method according to claim 16 or 17.

19. The LNP formulation according to claim 18, wherein, The LNP formulation further comprises siRNA targeting the Muc5ac gene; preferably, the sequence of the siRNA is shown in SEQ ID NO: 65-68.

20. Use of the LNP of claim 15 or the LNP formulation of claim 19 in the preparation of a medicament for treating asthma.

21. The use according to claim 20, wherein the drug is administered by atomization or inhalation.

22. A pentacyclic triterpenoid compound, said compound being selected from the following formula: In formula (I-1), Y is -O- or -NH-, Z is a C3-C6 alkylene group, and R1 is a carboxyl group or a sulfonic acid group; R2 and R3 are each independently hydrogen or methyl; R4 and R5 are each independently hydrogen or methyl; R6 is hydroxyl or hydrogen, R7 is hydroxymethyl or methyl, and R8 is hydroxyl or hydrogen. Alternatively, in formula (I-1), Y is -O- or -NH-, Z is -CH2-CH2-CH2- or -CH2-CH2-CH2-CH2-, and R1 is a carboxyl group or a sulfonic acid group; or, Y is -O- or -NH-, Z is -CH2-CH2-CH2- or -CH2-CH2-CH2-CH2-, and R1 is a carboxyl group or a sulfonic acid group; Alternatively, in formula (I-1), R2 and R3 are both methyl groups; R4 and R5 are both hydrogens; R6 and R8 are both hydrogens; and R7 is a methyl group. Alternatively, in formula (I-1), R2 is methyl and R3 is hydrogen; R4 is methyl and R5 is hydrogen; R6 and R8 are both hydrogen; and R7 is methyl.

23. The compound according to claim 22, or one of the following compounds: 。 24. Use of the compound according to claim 22 or 23 as an activator of V-ATPase.

Images