Hydrogen bond assisted two-component lipid nanoparticle as well as preparation method and application thereof

Abstract

The invention discloses hydrogen bond assisted two-component lipid nanoparticles as well as a preparation method and application thereof. The nanoparticle comprises a two-component lipid nanoparticle and a nucleic acid molecule, wherein the two-component lipid nanoparticle is prepared from the following raw materials: peptidyl ionizable lipid (PIL) and steroid. The invention innovatively introduces a hydrogen bond assisted assembly technology, and successfully develops a new method for preparing mRNA-LNP in a neutral environment. Experimental results show that compared with the traditional four-component LNP, the hydrogen bond assisted 2C-PIL LNP containing fewer amido bonds shows more excellent delivery efficiency, and efficient mRNA delivery in the spleen is realized. More importantly, the hydrogen bond assisted 2C-PIL LNP not only has good in-vivo biological safety, but also significantly simplifies the composition of the LNP, so that the preparation process is more convenient and efficient.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a hydrogen-bond-assisted two-component lipid nanoparticle as well as a preparation method and application thereof. BACKGROUND

[0002] With the success of the messenger ribonucleic acid (mRNA) vaccine for the novel coronavirus (COVID-19) and its application, mRNA therapy has rapidly developed in modern medical research and clinical applications. Compared with traditional small molecule drugs and antibody drugs, mRNA therapy has a series of significant advantages such as simple design, short research and development cycle, strong targeting, wide treatment field and long-acting, and has great therapeutic potential in the fields of mRNA vaccine, protein replacement therapy, cancer immunotherapy and gene editing. However, due to the large molecular weight and negative charge of naked mRNA, the intracellular delivery thereof faces the following challenges: 1) mRNA is easily degraded by ribonucleases (RNases) in the plasma and tissues, rapidly cleared by the liver and kidney, and recognized by the immune system; 2) mRNA is easily "stuck" in endosomes after entering cells and cannot function; 3) the large molecular weight and negative charge of mRNA make it unable to freely pass through biological membranes; 4) the off-target effect of mRNA can cause unexpected serious side effects. Therefore, the development of safe and efficient delivery systems is a hot research direction for current mRNA therapy.

[0003] At present, lipid nanoparticles (LNP) are one of the most commonly used carriers for delivering mRNA, opening up new avenues for many frontier application fields such as infectious disease treatment, tumor vaccine research and development, gene editing, and protein replacement therapy. However, due to the development stage of non-liver targeted delivery technology, when mRNA-LNP is applied to target tissues outside the liver, it faces many major challenges. Traditional LNP is mainly composed of four components, including cationic lipids, auxiliary phospholipids, cholesterol and polyethylene glycol lipids. Among them, ionizable cationic lipids play a crucial role, they have an amphiphilic structure, containing a tertiary amine head and a saturated or unsaturated alkyl tail, and their pKa value is usually between 6.2 and 6.5. This property allows ionizable cationic lipids to carry a positive charge in an acidic environment and restore electrical neutrality under physiological conditions. Based on this principle, mRNA-LNP is usually formulated in an acidic buffer, and the positively charged ionizable lipids encapsulate mRNA through electrostatic interactions. Subsequently, dialysis in a phosphate buffer makes mRNA-LNP electrically neutral, thereby reducing the safety risk after injection and ensuring its efficient escape from acidic endosomes. At present, this strategy has been applied to all FDA-approved LNP and mainstream LNP formulations. SUMMARY

[0004] In view of the defects of the prior art, the application provides a two-component lipid nanoparticle for encapsulating nucleic acid molecules based on hydrogen bonds, and the two-component lipid nanoparticle has high spleen targeting efficiency through optimization.

[0005] Firstly, the inventors used a peptide-based ionizable lipid (PIL) a12Orn5 synthesized by the research group in the early stage as a representative lipid to prepare a single-component mRNA-LNP to verify the formation of hydrogen bonds between the PIL lipid and mRNA. Secondly, in order to further improve the stability of the LNP and the encapsulation efficiency of the mRNA, the inventors added cholesterol to the prescription to prepare a two-component (2C) mRNA-LNP system. Thirdly, the two-component (2C) LNP prepared based on the PIL lipid library was systematically screened. Among the 25 2C-PIL LNP prescriptions with excellent properties tested in vivo, 20 2C-PIL LNP achieved significant mRNA expression in the spleen, among which a12K1 (containing 2 amide bonds) and a12Orn4 (containing 5 amide bonds) stood out and exhibited the highest expression level. In addition, when the biologically safe a12K1 was prepared into a hydrogen bond-driven 2C-LNP and a traditional four-component (4C)-LNP respectively, the former exhibited a significant advantage in the efficiency of spleen delivery. This key finding strongly indicates that the hydrogen bond-assisted LNP assembly strategy may be a highly potential and effective means to convert the originally "inactive" LNP into "active" LNP with high delivery capacity.

[0006] The hydrogen bond-driven 2C-LNP delivery platform based on the peptide-based ionizable lipid not only provides an innovative and highly potential solution for the spleen-targeted mRNA delivery, but also opens up a new research direction for the further optimization and improvement of the LNP.

[0007] The above-mentioned object of the application is realized by the following technical solutions.

[0008] In a first aspect, the application provides a two-component lipid nanoparticle for encapsulating nucleic acid molecules based on hydrogen bonds.

[0009] The two-component lipid nanoparticle for encapsulating nucleic acid molecules based on hydrogen bonds provided by the application comprises a two-component lipid nanoparticle and a nucleic acid molecule, and the two-component lipid nanoparticle is made of the following raw materials: a peptide-based ionizable lipid and a steroid.

[0010] The peptide-based ionizable lipid has the structural formula as shown in Formula I:

[0011]

[0012] In Formula I, i is an integer from 1 to 5, for example, i can be 1, 2, 3, 4, or 5;

[0013] m is an integer from 1 to 4, for example, m can be 1, 2, 3, or 4;

[0014] R1 is an alkyl chain with a carbon number of 6 to 18 or a hydroxyl-containing alkyl chain with a carbon number of 6 to 18.

[0015] Further, the molar ratio of the peptide-based ionizable lipid to the steroid can be 4:0-0:4, but not including 4:0 and 0:4, further 4:1-1:4, for example, 4:1-1:1, 6:4-1:1, 4:1, 6:4, 1:1, 4:6, or 2:8, preferably the molar ratio is 1:1.

[0016] Further, the steroid includes but is not limited to cholesterols and derivatives thereof, ergosterol, lanosterol, stigmasterol, sitosterol, avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydrolanosterol, dinosterol, epicholesterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, photosterol, phaeosterol, stigmastanol, stigmastanediol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid, etc. Among them, examples of cholesterol derivatives include but are not limited to 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ether, cholesteryl-(4'-hydroxy)-butyl ether, 6-ketocholstanol, 5α-cholestan, cholestenone, 5α-cholestanone, 5β-cholestanone, etc.

[0017] Further, the mass ratio of the peptide-based ionizable lipid to the nucleic acid molecule in the two-component lipid nanoparticle can be 60:1-10:1, preferably the mass ratio is 40:1.

[0018] Further, the nucleic acid molecule can be a chemically modified or unmodified nucleic acid molecule, for example, a DNA molecule or an RNA molecule.

[0019] In some embodiments of the present application, the chemically modified or unmodified DNA molecule can be any type of DNA molecule (but not limited to), including but not limited to linear or circular DNA, double-stranded or single-stranded or multi-stranded assembled DNA, coding or non-coding DNA, which is optionally selected from plasmid, oligodeoxynucleotide, genomic DNA, DNA probe, DNA aptamer, DNA nanoframe, DNA primer, homologous repair DNA template, immunostimulatory DNA, or a combination thereof.

[0020] In some embodiments of the present application, the chemically modified or unmodified RNA molecule can be any type of RNA molecule (but not limited to), including but not limited to messenger RNA (mRNA), small interfering RNA (siRNA), circular RNA (circRNA or oRNA), guide RNA (sgRNA), nicking guide RNA (nicking sgRNA), small hairpin RNA (shRNA), viral RNA, replicon RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), immune-stimulatory RNA (isRNA), microRNA (miRNA), small nuclear RNA (snRNA), RNA aptamer, antisense RNA, RNA nanoframe, RNA ribonucleoprotein complex, or a combination thereof. In some embodiments, the chemically modified messenger RNA (mRNA) is preferably any mRNA encoding a gene editing element. Such as luciferase mRNA (Luc-mRNA) or Cre recombinase mRNA (Cre mRNA).

[0021] In some embodiments of the present application, in R1, the hydroxyl group-containing alkyl chain having 6 to 18 carbon atoms can be selected from the following groups: -CH2CHOH(CH2) q CH2CH3, wherein q is an integer from 2 to 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, preferably q is an integer from 6 to 10.

[0022] In some embodiments of the present application, the peptide-based ionizable lipid can be a lipid of formula I, wherein m is an integer from 1 to 3, and R1is an alkyl chain having 12 carbon atoms, and i is 2, 4, or 5.

[0023] In some embodiments of the present application, the peptide-based ionizable lipid can be a lipid of formula I, wherein m is 4, and R1is an alkyl chain having 6 to 18 carbon atoms, and i is 1, 2, or 3.

[0024] In some embodiments of the present application, the peptide-based ionizable lipid can be a lipid of formula I, wherein m is 4, and R1is -CH2CHOH(CH2) q CH2CH3, wherein q is an integer from 6 to 10, and i is 2, 3, 4, or 5; further, q is 6, and i is 2 or 3; or q is 10, and i is 2, 3, 4, or 5.

[0025] In some embodiments of the present application, the LNP is a spleen-targeting LNP; further, the spleen is the only target organ for the LNP.

[0026] "Spleen targeting" in the present application refers to that when the LNP is delivered into a living body, the expression ratio in the spleen is more than 50%, for example, more than 70%, and more preferably more than 90%.

[0027] In the present application, the peptide-based ionizable lipid can be selected from any of the following structural formulae:

[0028]

[0029]

[0030]

[0031] In some embodiments of the present application, the peptide-based ionizable lipid is preferably any of the following lipids: a6K1, a8K1, a10K1, a12K1, a16K1, a18K1, a12K2, a16K3, a18K3, a12Dab2, a12Orn2, a12Dap4, a12Orn4, a12Orn5, e10K2, e10K3, e14K2, e14K3, e14K4, e14K5; and more preferably a12K1 or a12Orn4.

[0032] The peptide-based ionizable lipid described in the present application can be prepared according to the method described in the patent (application publication number: CN118852328A).

[0033] In a second aspect, the present application provides a preparation method of the two-component lipid nanoparticle encapsulating nucleic acid molecules based on hydrogen bonds according to the first aspect.

[0034] The preparation method of the two-component lipid nanoparticle encapsulating nucleic acid molecules based on hydrogen bonds provided by the present application comprises the following steps:

[0035] Step (A1): dissolving the peptide-based ionizable lipid and the steroid in a specific molar percentage in an organic solvent to obtain a lipid organic phase;

[0036] Step (A2): dissolving the nucleic acid molecules in appropriate RNase-free water to obtain a nucleic acid molecule solution;

[0037] Step (A3): mixing the lipid organic phase in step (A1) and the nucleic acid molecule solution in step (A2) and incubating to obtain a solution of the two-component lipid nanoparticle encapsulating nucleic acid molecules based on hydrogen bonds.

[0038] Preferably, the organic solvent includes any one or a combination of at least two of methanol, ethanol, propanol, tetrahydrofuran, and diethyl ether.

[0039] Preferably, the mass ratio of the peptide-based ionizable lipid to the nucleic acid molecule in the lipid organic phase to the nucleic acid molecule in the nucleic acid molecule solution is (60-10):1, more preferably 40:1.

[0040] Preferably, the volume ratio of the lipid organic phase to the nucleic acid molecule solution is 1:(1-6), more preferably 1:3.

[0041] Preferably, the incubation condition is standing incubation at room temperature for 10-30 min.

[0042] In a third aspect, the present application provides a pharmaceutical composition.

[0043] The pharmaceutical composition provided by the present application comprises the two-component lipid nanoparticle for encapsulating nucleic acid molecules based on hydrogen bonds according to the first aspect of the present application and a pharmaceutically acceptable carrier.

[0044] In a fourth aspect, the present application provides a method for delivering nucleic acid molecules to the spleen or preventing or treating diseases.

[0045] The method provided by the present application comprises administering an effective amount of the two-component lipid nanoparticle for encapsulating nucleic acid molecules based on hydrogen bonds according to the first aspect of the present application or the pharmaceutical composition according to the third aspect of the present application to a subject or tissue in need.

[0046] In a fifth aspect, the present application provides the use of the two-component lipid nanoparticle for encapsulating nucleic acid molecules based on hydrogen bonds according to the first aspect of the present application or the pharmaceutical composition according to the third aspect of the present application in the preparation of a medicament for treating and / or preventing diseases.

[0047] In a sixth aspect, the present application further provides a medicament for treating and / or preventing diseases, which comprises the two-component lipid nanoparticle for encapsulating nucleic acid molecules based on hydrogen bonds according to the first aspect of the present application or the pharmaceutical composition according to the third aspect of the present application.

[0048] In some embodiments of the present application, the disease is any disease for which the nucleic acid molecule of the present application can have a therapeutic or prophylactic effect. In certain embodiments, the disease is a genetic disease and the nucleic acid molecule is a gene therapy agent. In certain embodiments, the nucleic acid molecule is an mRNA capable of inducing an immune response. In certain embodiments, the disease is a disease requiring organ-targeted therapy. In certain embodiments, the disease is cancer.

[0049] The application deeply explores the application potential of mRNA therapy in the treatment of various diseases, and systematically studies and optimizes the key problems of LNP in mRNA delivery. mRNA therapy is highly concerned due to its ability to directly and quickly synthesize proteins in the cytoplasm, which effectively avoids the risk of insertional mutation, and shows great potential in the treatment of various diseases. However, the traditional mRNA-LNP preparation method has many limitations, such as the need for assembly in an acidic environment and neutralization by dialysis, which not only is cumbersome to operate, but also is high in cost, and seriously limits its wide application, especially in the targeted delivery of non-liver tissues.

[0050] To solve the above problems, the application innovatively introduces a hydrogen bond assisted assembly technology, and successfully develops a new method for preparing mRNA-LNP in a neutral environment. By synthesizing a series of PILs with diverse structures, the hydrogen bond interaction between the amide bond in PIL and the phosphate group of mRNA is utilized to realize efficient encapsulation of mRNA, and the needs of tissue targeted delivery are met by customized design. Combined with molecular dynamics simulation and in vivo screening, the research team successfully developed a 2C-PIL LNP targeting the spleen. Experimental results show that, compared with traditional four-component LNP, the PIL containing fewer amide bonds exhibits superior delivery efficiency, and realizes efficient mRNA delivery in the spleen. More importantly, the hydrogen bond assisted 2C-PIL LNP not only has good in vivo safety, but also significantly simplifies the composition of LNP, making the preparation process more convenient and efficient.

[0051] In summary, the 2C-PIL LNP developed based on hydrogen bond interaction effectively simplifies the preparation process of mRNA-LNP. The optimized 2C LNP platform provides an efficient and feasible method for spleen targeted mRNA delivery and LNP optimization, which has broad application prospects and is expected to play a role in the fields of cancer vaccine and autoimmune disease treatment. Future research will further explore the application potential of the platform in other tissue targeted delivery, in order to provide stronger support for the clinical application of mRNA therapy.

[0052] Compared with the prior art, the application has the following beneficial effects:

[0053] 1、The application utilizes the hydrogen bond interaction between the amide bond of peptide-based ionizable lipid (PIL) and the phosphate group of mRNA to realize efficient encapsulation and delivery of mRNA.

[0054] 2、Through in vivo screening of two-component PIL mRNA-LNP, it is found that 80% of the PIL mRNA-LNP has high spleen delivery efficiency.

[0055] 3. Two-component LNP of K1 series of hydrogen-bonding driven PILs (a6K1, a8K1, a10K1, a12K1, a16K1, a18K1) showed significant advantages in mRNA spleen delivery efficiency compared with traditional four-component LNP. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 Molecular dynamics simulation of hydrogen bonding interactions and their number distribution in 1C-a12Orn5 LNP (green: a12Orn5).

[0057] Figure 2 Characterization of particle size, polydispersity index and encapsulation efficiency of 1C-a12Orn5 LNP prepared with different mass ratios of a12Orn5 and mRNA.

[0058] Figure 3 Molecular dynamics simulation of hydrogen bonding interactions and their number distribution in 2C-a12Orn5 LNP (green: a12Orn5; magenta: cholesterol).

[0059] Figure 4 Molecular dynamics simulation of hydrogen bonding interactions and their number distribution in 2C-ALC-0315 LNP (yellow: ALC-0315; magenta: cholesterol).

[0060] Figure 5 Characterization of particle size, polydispersity index and encapsulation efficiency of 2C-a12Orn5 LNP and 2C-ALC-0315 LNP prepared with different molar ratios of lipid and cholesterol.

[0061] Figure 6 Bioluminescence imaging of 2C-a12Orn5 LNP and 2C-ALC-0315 LNP with different molar ratios in whole body and major organs of mice.

[0062] Figure 7 Synthetic route map of Nα-Fmoc protected alkylated amino acids (reductive amination).

[0063] Figure 8 Synthetic route map of Nα-Fmoc protected alkylated amino acids (epoxide ring opening).

[0064] Figure 9 Structural formulas of 43 kinds of PILs.

[0065] Figure 10 Characterization of particle size and encapsulation efficiency of 43 kinds of 2C-PIL LNP.

[0066] Figure 11 pKa values of 25 kinds of well-performing 2C-PIL LNP.

[0067] Figure 12 Bioluminescence imaging of mouse organs mediated by optimized 2C-PIL LNP.

[0068] Figure 13 Heatmap of in vivo mRNA expression level and organ specificity of optimized 2C-PIL LNP.

[0069] Figure 14 In vivo distribution fluorescence imaging and quantitative results of 2C-a12K1 LNP and 2C-a12Orn4 LNP.

[0070] Figure 15 Bioluminescence imaging of mouse organs mediated by 15% DOPS-ALC-0315 LNP and 2C-a12K1 LNP.

[0071] Figure 16 Bioluminescence quantification and organ-specific expression of mouse organs mediated by 15% DOPS-ALC-0315 LNP and 2C-a12K1 LNP.

[0072] Figure 17 Chemical structure of a12K1 and a12Orn4.

[0073] Figure 18 Bioluminescence imaging of mouse organs mediated by 2C-a12 LNP and 2C-a12Orn4 LNP, quantification of Luc-mRNA expression in spleen, and organ-specific expression.

[0074] Figure 19 Fold increase of tdTomato fluorescence intensity in organs of Ai9 mice after intravenous administration of 2C-a12K1 LNP and 2C-a12Orn4 LNP encapsulating Cre mRNA relative to PBS group.

[0075] Figure 20 Flow cytometry quantitative analysis of tdTomato expression in dendritic cells, macrophages, B cells, and T cells in spleen of mice treated with 2C-a12K1 LNP and 2C-a12Orn4 LNP.

[0076] Figure 21 Flow cytometry quantitative analysis of Cy5 expression in dendritic cells, macrophages, B cells, and T cells in spleen of mice treated with 2C-a12K1 LNP and 2C-a12Orn4 LNP.

[0077] Figure 22AST and ALT levels in mouse serum 3h and 24h after intravenous administration of different doses of 2C-a12K1 LNP and 2C-a12Orn4 LNP.

[0078] Figure 23 Bioluminescence imaging of mouse organs mediated by 4C-LNP and 2C-LNP encapsulating Luc-mRNA.

[0079] Figure 24 Quantification of mouse spleen mediated by 4C-LNP and 2C-LNP encapsulating Luc-mRNA.

[0080] Figure 25 Bioluminescence imaging of mouse muscle mediated by 4C-LNP and 2C-LNP encapsulating Luc-mRNA.

[0081] Figure 26 Quantification of mouse muscle mediated by 4C-LNP and 2C-LNP encapsulating Luc-mRNA.

[0082] Figure 27 NMR hydrogen spectrum of HB-free-a12K1.

[0083] Figure 28 Particle size, PDI and EE characterization of 2C-HB-free-a12K1 LNP and 2C-a12K1 LNP.

[0084] Figure 29 Bioluminescence imaging of mouse organs and quantification of Luc-mRNA expression in spleen mediated by 2C-HB-free-a12K1 LNP and 2C-a12K1 LNP. DETAILED DESCRIPTION

[0085] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0086] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0087] The peptide-based ionizable lipids (PILs) involved in the following examples have been patented (Application Publication No. CN118852328A), and can be prepared according to the methods described in the patent.

[0088] HB-free-a12K1 used in the following Example 5 was synthesized according to the following method:

[0089]

[0090] Fmoc-N-Me-Lys(Boc)-OH was first dissolved in dry DCM, and excess trifluoroacetic acid was added, and stirred at room temperature for 4 h to remove the Boc protecting group. After the reaction was completed, 3 equivalents of dodecanal and NaBH(OAc)3 were added and the reaction was continued at room temperature for 3 d to obtain the alkylated Fmoc-N-Me-lysine. Subsequently, acetylation was completed by solid-phase synthesis; finally, HB-free-a12K1 was obtained under EDC / DMAP ester condensation conditions.

[0091] Example 1, Preparation and characterization of One-component (1C)-a12Orn5 LNP

[0092] First, we successfully synthesized a PIL lipid, a12Orn5 lipid, using a solid-phase synthesis strategy, and its structure is as follows:

[0093]

[0094] The lipid structure contains six amide bonds, which can provide abundant hydrogen bond donors, so it is selected as a representative lipid for subsequent research.

[0095] Next, we used molecular dynamics simulation to explore the formation mechanism of hydrogen bonds between a12Orn5 and mRNA. The simulation results showed that the N-H bonds in a12Orn5 can form multiple hydrogen bonds with the oxygen atoms in the phosphate groups of mRNA. Figure 1 ).

[0096] Based on the above simulation results, we tried to assemble luciferase mRNA (Luc-mRNA) (SEQ ID NO: 1) using a12Orn5 lipid in RNase-free water and prepared One-component (1C)-a12Orn5 LNP with different mass ratios (a12Orn5:mRNA = 40:1, 20:1, 10:1).

[0097] The specific preparation method is as follows: prepare a lipid ethanol solution of a12Orn5 lipid, and mix the lipid ethanol solution with the RNase-free aqueous solution of mRNA at a mass ratio of a12Orn5 / mRNA = 40:1, 20:1 or 10:1 to prepare a series of a12Orn5 mRNA LNP, and its physicochemical properties were characterized.

[0098] Characterization results of the above LNP showed that when the mass ratio of lipid to mRNA was 40 / 1, 1C-a12Orn5 LNP exhibited smaller particle size (175.23 ± 2.8 nm), narrower polydispersity index (PDI = 0.114 ± 0.015), and higher encapsulation efficiency (58.96% ± 3.87%). In contrast, LNP with mass ratios of 20 / 1 and 10 / 1 performed poorly in terms of particle size distribution and encapsulation efficiency. Figure 2 This result indicates that in RNase-free water, PIL lipids successfully encapsulate mRNA with high efficiency through hydrogen bonding.

[0099] Example 2, Preparation and characterization of 2C-a12Orn5 LNP

[0100] To further improve the stability and mRNA encapsulation efficiency of LNP, we added cholesterol (Beijing Inokai Technology Co., Ltd.) to the prescription. We prepared lipid ethanol solutions with a12Orn5: cholesterol = 100:0 / 80:20 / 60:40 / 50:50 / 40:60 / 20:80 / 0:100 molar ratio, and rapidly prepared a series of 2C-a12Orn5 LNP in RNase-free water with a12Orn5 / Luc-mRNA = 40:1 mass ratio, and characterized the formation of hydrogen bonds and their physicochemical properties.

[0101] In addition, in order to more comprehensively evaluate the performance of 2C-a12Orn5 LNP, this study also selected FDA-approved ALC-0315 lipid as a control group, and prepared the corresponding 2C-ALC-0315 LNP using the same method.

[0102] Molecular dynamics simulation results showed that compared with 2C-ALC-0315 LNP, 2C-a12Orn5 LNP formed more hydrogen bonds with mRNA phosphate groups Figure 3 , 4). Physicochemical property characterization results showed that 2C-a12Orn5 LNP exhibited smaller and more uniform nanoparticle size, with more concentrated particle size distribution Figure 5 . In addition, 2C-a12Orn5 LNP also showed a significant advantage in mRNA encapsulation efficiency, with higher encapsulation efficiency than 2C-ALC-0315 LNP Figure 5 .

[0103] Example 3, In vivo evaluation of 2C-a12Orn5 LNP and 2C-ALC-0315 LNP

[0104] C57BL / 6 mice (n = 3) were administered 2C-a12Orn5 LNP and 2C-ALC-0315 LNP (both encapsulating Luc-mRNA) at a dose of 0.05 mg / kg (mRNA mass) via intramuscular injection (i.m.) and intravenous injection (i.v.), respectively, and the in vivo Luc mRNA expression was evaluated in detail 6 hours post-injection. The mice were imaged using an in vivo imaging system (IVIS), and the results showed that 2C-a12Orn5 LNP exhibited significant Luc-mRNA expression in both muscle and spleen at a molar ratio of a12Orn5 to cholesterol ranging from 50:50 to 20:80 Figure 6 ). In particular, the mRNA expression efficiency reached the highest at a molar ratio of a12Orn5 to cholesterol of 50:50, indicating that this formulation had the best delivery effect. In contrast, no significant mRNA expression was detected in all samples of 2C-ALC-0315 LNP Figure 6 ), indicating that ALC-0315 LNP could not effectively deliver and express mRNA under the conditions of this study.

[0105] In summary, these results strongly demonstrate that PIL-based 2C-LNP can effectively encapsulate mRNA through hydrogen bonding interactions and achieve efficient mRNA delivery in vivo.

[0106] Example 4, Construction of a PILs lipid library, 2C-PIL mRNA-LNP based on PILs and evaluation of their in vivo effects

[0107] We further synthesized a series of structurally diverse PILs lipid library and carried out high-throughput screening experiments. To achieve structural diversity of PILs, we first synthesized a variety of alkylated ionizable Fmoc-protected amino acids (AIFAs) building blocks through two main chemical reaction pathways. Specifically, through reductive amination reaction Figure 7 ) and epoxide ring-opening reaction Figure 8 ), we successfully synthesized a variety of AIFAs modules with different functional groups and chain lengths. Using the above-synthesized AIFAs building blocks, we further successfully synthesized 43 structurally diverse PILs through solid-phase synthesis strategy (SPSS) Figure 9PIL is named XmYi, where X represents the type of alkyl tail, a is the saturated alkyl chain obtained by aldehyde reduction and amination, e is the hydroxyl-containing alkyl chain obtained by epoxy ring opening, m represents the number of carbon atoms in the alkyl chain, Y represents lysine (K) or its structural analogues such as 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab) or ornithine (Orn), and i represents the number of AIFAs.

[0108] Next, we further constructed and characterized 43 2C-PIL mRNA-LNPs in detail, following the method in Example 2. Through systematic evaluation of the physicochemical properties of these LNPs, 25 high-performance LNPs were screened, exhibiting uniform particle size (<600 nm) and high encapsulation efficiency (>70%). Figure 10 The pKa of 25 high-performing LNPs was determined using the TNS fluorescent probe method. Figure 11 As shown, except for 2C-e14K4 LNP with a pKa of 5.89, the pKa of the other formulations were all in the range of 6.00-7.40, significantly higher than the reported pKa range of spleen-targeting SORT LNPs (2-6). This difference suggests that the mechanism of spleen-specific mRNA delivery mediated by 2C-PIL LNP may be fundamentally different from that of traditional 4C-LNP and SORT LNP. Subsequently, all 25 LNPs were used in vivo experiments in mice to systematically evaluate their efficiency and targeting of Luc-mRNA delivery. C57BL / 6 mice were administered 2C-PIL mRNA-LNP (n=3) via intravenous injection (iv), and the mice's bodies and major organs were imaged using IVIS 6 hours after injection. The results showed that 80% of 2C-PIL LNPs achieved significant mRNA expression in the spleen. Figure 12 This result indicates that the screened LNPs possess highly efficient spleen-targeted delivery capabilities in vivo. Quantitative analysis of mRNA expression levels in the liver, spleen, and lungs revealed that 2C-a12K1 LNP and 2C-a12Orn4 LNP exhibited high mRNA expression efficiency in the spleen, while their expression levels were relatively low in the liver and lungs, demonstrating good organ selectivity. Figure 13 The above results fully demonstrate that 2C-PIL LNP can achieve efficient and specific spleen-targeted mRNA delivery, representing a very promising spleen-targeted delivery strategy.

[0109] To investigate whether the mRNA delivery efficiency in the spleen depends only on the accumulation of mRNA-LNP in the spleen, we prepared 2C-a12K1 LNP and 2C-a12Orn4 LNP encapsulating Cy5-labeled mRNA as objects for in vivo distribution study. After 1 h of intravenous injection of mice, the main organs were removed and subjected to ex vivo fluorescence imaging. The results showed that both LNPs exhibited strong Cy5-mRNA signals in the spleen( Figure 14 ); however, significant fluorescence was also observed in non-splenic organs such as the liver and lungs, suggesting that functional delivery to the spleen is not linearly related to simple organ accumulation. Subsequently, we compared the spleen-targeted 2C-a12K1 LNPs with the classic spleen SORT LNPs (15% DOPS-ALC-0315). The data showed that the mRNA expression of 2C-a12K1 LNPs in the spleen was comparable to that of 15% DOPS-ALC-0315 LNPs, but the expression in off-target organs such as the liver and lungs was significantly reduced( Figure 15 , 16), highlighting its advantages of efficient delivery and specific targeting of the spleen. In summary, 2C-PIL LNPs can achieve efficient and precise mRNA delivery to the spleen, providing a promising new strategy for the development of spleen-targeted drug delivery systems.

[0110] Despite the significant difference in the number of hydrogen bond donors between a12K1 and a12Orn4 (2 amide bonds and 5 amide bonds, respectively), both LNPs exhibited excellent spleen-targeting delivery capabilities( Figure 17 , 18). To further investigate the differences between these two LNPs in spleen-targeted delivery, we used an Ai9 mouse model (the construction method of this model mouse is described in the following reference: Madisen L, Zeng H, et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci. 2010; 13(4): 451-456.) to further explore the cell types they transfect in the spleen. We administered 2C-a12K1 LNP (n = 3) or 2C-a12Orn4 LNP (n = 3) containing 0.3 mg / kg of Cre mRNA (SEQ ID NO: 2) to Ai9 mice via tail vein injection, and performed ex vivo imaging of the liver, spleen, lungs, heart, and kidneys 6 hours after injection. After delivery of Cre mRNA by both LNPs, we observed high and comparable fluorescence intensity in the spleen, confirming their specific targeting capability( Figure 19). In addition, strong tdTomato fluorescence signal was also detected in the spleen section, further confirming that the Cre mRNA gene was successfully edited Figure 19 Flow cytometry analysis showed that both 2C-a12K1 LNP and 2C-a12Orn4 LNP mainly delivered Cre mRNA to dendritic cells (11.93% and 12.03%, respectively) Figure 20 However, significant preference differences were observed in other cell populations: 2C-a12Orn4 LNP had higher transfection efficiency in macrophages (11.87% vs 5.25% for 2C-a12K1 LNP), while 2C-a12K1 LNP had higher transfection efficiency in T cells and B cells Figure 20 To further elucidate the differential mechanisms of the two formulations in cell type-specific transfection, we injected 2C-LNPs encapsulating Cy5-mRNA into mice via tail vein, and systematically compared their spleen uptake characteristics. One hour after administration, the spleen was taken to prepare a single cell suspension, and flow cytometry was used for quantitative analysis. The results were highly consistent with the gene editing performance: the uptake rate of 2C-a12K1 LNPs in B cells (20.60%) and T cells (2.70%) was significantly higher than that of 2C-a12Orn4 LNPs (B cells 5.42%, T cells 0.95%); the Cy5 + The proportion of 2C-a12Orn4 group increased slightly (71.00% vs 62.40%), but the difference was not statistically significant Figure 21 It is speculated that the functional mRNA delivery differences of the two 2C-LNPs in different cell types may be due to their significant differences in cell uptake efficiency. The above results show that although both LNP have high spleen targeting ability, their transfection efficiency in different cell types is significantly different, which may be related to the number and structural properties of their hydrogen bond donors.

[0111] We further systematically evaluated the in vivo biological safety of 2C-a12K1 LNP and 2C-a12Orn4 LNP at three different administration doses (0.5, 1 and 2 mg / kg, based on the mass of mRNA), and quantitatively detected the liver function of mice at 3h and 24h after administration, respectively. It should be noted that the blank LNP used in this safety evaluation does not encapsulate mRNA. However, the administration dose is calculated based on the mass of the encapsulated mRNA.

[0112] The detection results of liver function indicators (alanine aminotransferase ALT and aspartate aminotransferase AST) showed that 2C-a12K1 LNP had higher tolerance than 2C-a12Orn4 LNP Figure 22). There were no significant differences between the 2C-a12K1 LNP group and the physiological saline (PBS) control group in these indicators at all test doses Figure 22 ), indicating that 2C-a12K1 LNP had little effect on liver function after administration, and had good safety. These research results fully demonstrate that 2C-a12K1 LNP has high efficient spleen targeting ability and good in vivo biological safety, making it a promising carrier in mRNA delivery applications.

[0113] Example 5, Comparison of Delivery Efficiency and Specific Targeting of 2C-LNP and 4C-LNP

[0114] In view of the excellent biocompatibility and delivery efficiency of 2C-a12K1 LNP in vivo, this study further explores whether the delivery efficiency of a12K1 can be further improved in traditional four-component (4C)-LNP.

[0115] For this purpose, this study selected six PILs (a6K1, a8K1, a10K1, a16K1, a18K1) similar in structure to a12K1, prepared hydrogen-bonding-assisted 2C-LNP in aqueous solution, and prepared the corresponding 4C-LNP in acidic solution using the traditional method. The prescription composition and ingredient ratio of hydrogen-bonding-assisted 2C-LNP and traditional 4C-LNP are shown in Table 1.

[0116] Table 1

[0117]

[0118] In Table 1, Ionizablelipid represents PILs, DOPC is dioleoyl phosphatidylcholine, and DMG-PEG represents dimyristyl polyethylene glycol.

[0119] Subsequently, Luc-mRNA was loaded into the above carriers, respectively, wherein the total lipid mRNA mass in 4C-LNP = 40 / 1, and the 2PILs mass / mRNA mass in 2C-LNP = 40 / 1, and IVIS imaging analysis was performed at 6h after intravenous and intramuscular administration. Surprisingly, all 2C-LNPs achieved efficient mRNA delivery and specific targeting in the spleen ( Figure 23, 24, 25, 26), while all 4C-LNPs failed to achieve efficient transfection in any tissue in vivo. This result indicates that the hydrogen bond-assisted LNP assembly strategy has a significant advantage in improving mRNA delivery efficiency and targeting specificity, while the traditional 4C-LNP preparation method failed to achieve the expected delivery effect under the conditions of this study. To further verify the dominant role of hydrogen bonds in 2C-LNPs encapsulating and functionally delivering mRNA, we synthesized a control lipid HB-free a12K1 without any hydrogen bond donor group and performed a head-to-head comparison with a12K1 PIL Figure 27 ). The results showed that the mRNA encapsulation rate of 2C-HB-free a12K1 LNPs in water was only about 25%, much lower than that of 2C-a12K1 LNPs (about 100%, Figure 28 ). In addition, 2C-HB-free a12K1 LNPs failed to mediate mRNA expression in vivo, and no bioluminescence signal was detected in the spleen and other major organs Figure 29 ). These results further indicate that the amide bond in the PIL as a hydrogen bond donor is of critical importance for the efficient encapsulation of mRNA and its highly efficient delivery in vivo. In summary, this unexpected finding indicates that hydrogen bond-assisted LNP assembly can be an effective strategy for optimizing LNPs, which is expected to transform “inactive” LNPs into “active” delivery systems.

[0120] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, this application intends to include any changes, uses or improvements of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in this application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. A two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding, comprising a two-component lipid nanoparticle and a nucleic acid molecule, the two-component lipid nanoparticle is made from the following raw materials: a peptide-based ionizable lipid, a steroid; the peptide-based ionizable lipid has a structural formula as shown in Formula I: wherein i is an integer from 1 to 5; m is an integer from 1 to 4; R 1 is an alkyl chain with a carbon number of 6 to 18 or a hydroxyl-containing alkyl chain with a carbon number of 6 to 18; the molar ratio of the peptide-based ionizable lipid to the steroid is 4:0-0:4, but not including 4:0 and 0:4, further 4:1-1:4, preferably 1:1; and / or, the mass ratio of the peptide-based ionizable lipid in the two-component lipid nanoparticle to the nucleic acid molecule is 60:1-10:1; and / or, the two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding has a spleen targeting property; and / or, the nucleic acid molecule is mRNA. wherein 2.The two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding according to claim 1, wherein the steroid includes but is not limited to cholesterols and their derivatives, ergosterol, lanosterol, stigmasterol, sitosterol, avenasterol, beta-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydrolanosterol, dinosterol, epicholesterol, fucosterol, hexahydroergosterol, hydroxycholesterol, lumisterol, phaeosterol, poriferasterol, sitostanol, stigmastanol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid. 3.The two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding according to claim 1 or 2, wherein the peptide-based ionizable lipid is a lipid as follows: in Formula I, m is an integer from 1 to 3, R 1 is an alkyl chain with a carbon number of 12, and i is 2, 4 or 5. 4.The two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding according to claim 1 or 2, wherein the peptide-based ionizable lipid is a lipid as follows: in Formula I, m is 4, R 1 is an alkyl chain with a carbon number of 6 to 18, and i is 1, 2 or 3. 5.The two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding according to claim 1 or 2, wherein the peptide-based ionizable lipid is selected from any one of the following structural formulas:

2. The two-component lipid nanoparticle encapsulating a nucleic acid molecule based on hydrogen bonding according to claim 1, characterized in that: 6.The two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding according to any one of claims 1 to 5, wherein the nucleic acid molecule is mRNA. 7.A method for preparing the two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding according to any one of claims 1 to 6, comprising the following steps: step (A1): dissolving the peptide-based ionizable lipid and the steroid in an organic solvent at a specific molar percentage to obtain a lipid organic phase; step (A2): dissolving the nucleic acid molecule in appropriate RNase-free water to obtain a nucleic acid molecule solution; and step (A3): mixing the lipid organic phase in step (A1) and the nucleic acid molecule solution in step (A2) and incubating to obtain a solution of the two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding. 8.The method for preparing the two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding according to claim 7, wherein the organic solvent includes any one or a combination of at least two of the following: methanol, ethanol, propanol, tetrahydrofuran and diethyl ether. 9.The method for preparing the two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding according to claim 7 or 8, wherein the mass ratio of the peptide-based ionizable lipid in the lipid organic phase to the nucleic acid molecule in the nucleic acid molecule solution is (60-10):1, and the preferred mass ratio is 40:

1.

3. The two-component lipid nanoparticle encapsulating a nucleic acid molecule based on hydrogen bonding according to claim 1 or 2, characterized in that: 10.The method for preparing the two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding according to claim 7 or 8, wherein the volume ratio of the lipid organic phase to the nucleic acid molecule solution is 1:(1-6), and the more preferred volume ratio is 1:

3.

4. The two-component lipid nanoparticle encapsulating a nucleic acid molecule based on hydrogen bonding according to any one of claims 1-3, characterized in that: In the formula I, the alkyl chain having a hydroxyl group with a carbon number of 6 to 18 in R1is selected from the following groups: -CH2CHOH(CH2) q CH2CH3, wherein q is an integer from 2 to 14, preferably the q is an integer from 6 to 10.

5. The two-component lipid nanoparticle encapsulating a nucleic acid molecule based on hydrogen bonding according to any one of claims 1-4, characterized in that: 11.The method for preparing the two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding according to claim 7 or 8, wherein the incubation condition is to incubate at room temperature for 10-30 min. 12.The method for preparing the two-component lipid nanoparticle for encapsulating a nucleic acid molecule based on hydrogen bonding according to claim 7 or 8, wherein the nucleic acid molecule is mRNA. and / or the peptide-based ionizable lipid is a lipid of Formula I, wherein m is 4, and R1is -CH2CHOH(CH2) q CH2CH3, wherein q is an integer from 6 to 10, and i is 2, 3, 4, or 5; further, q is 6, and n is 2 or 3; or q is 10, and i is 2, 3, 4, or 5.

6. The two-component lipid nanoparticle encapsulating a nucleic acid molecule based on hydrogen bonding according to any one of claims 1-5, characterized in that: ​ ​ ​ ​ ​ 8. The method of claim 7, wherein the method is characterized by: ​ ​ ​ ​ ​ 9. A pharmaceutical composition comprising the two-component lipid nanoparticle encapsulating a nucleic acid molecule based on hydrogen bonds according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

10. Use of the two-component lipid nanoparticle encapsulating a nucleic acid molecule based on hydrogen bonds according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 9 for the manufacture of a medicament for the treatment and / or prevention of a disease.

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