An ionizable lipid compound, and methods of making and using the same

Abstract

The present application relates to the technical field of biological medicine, in particular to a kind of ionizable lipid compound and its preparation method and application.The present application provides a kind of ionizable lipid compound, the ionizable lipid compound has a hydrophilic core and three hydrophobic tail chains, chemical structure is as shown in formula (I):The ionizable lipid compound provided by the present application can form two-component lipid nanoparticles for safe, stable drug delivery with cholesterol or its derivative, and the formed lipid nanoparticles show high transfection efficiency in a variety of cell lines transfection, can transfect different site cells, and have high safety.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an ionizable lipid compound, its preparation method, and its application. Background Technology

[0002] Gene therapy offers novel approaches to treating major diseases, but the lack of safe and effective gene delivery systems remains a significant challenge. Research indicates that lipid nanoparticles can serve as non-viral gene carriers for the delivery of various genes, demonstrating promising applications. Typically, lipid nanoparticles contain components such as ionizable lipids, auxiliary lipids, cholesterol, and PEGylated lipids. Ionizable lipids, which are electrically neutral under physiological conditions (pH 7.4) but positively charged under acidic conditions, are a key component of the delivery system. Their molecular structure plays a decisive role in the safety, efficacy, and stability of the entire lipid nanoparticle. Furthermore, non-viral gene delivery systems like lipid nanoparticles often face challenges such as complex composition, poor stability, difficulty in quality control, and high cost, limiting their clinical application in gene therapy research and the treatment of major diseases. Therefore, the development of novel ionizable lipids is crucial for promoting the use of lipid nanoparticles for gene and other drug delivery and related disease treatment.

[0003] Currently, most commonly used ionizable lipids are compounds with a tertiary amine head and an alkyl hydrophobic tail, synthesized through multiple steps, and used for the construction of lipid nanoparticles and gene delivery. However, many ionizable lipids still face problems such as high toxicity, and they usually require multiple components, such as auxiliary lipids, cholesterol, and PEG lipids, to generate lipid nanoparticles capable of delivering nucleic acid drugs. The large number of components leads to safety risks and makes quality control difficult. In other words, existing ionizable lipids generally suffer from complex preparation processes, transfection / delivery efficiency that needs further improvement, high cytotoxicity, and low stability.

[0004] Therefore, there is an urgent need for an ionizable lipid compound that can form highly efficient, low-toxicity, stable, and easily prepared lipid nanoparticles. Summary of the Invention

[0005] In a first aspect, the present invention provides a novel ionizable lipid compound, characterized in that the ionizable lipid compound has a hydrophilic core and three hydrophobic tail chains, and is a peptide-like molecule with a branched structure, the chemical structure of which is shown in formula (I):

[0006]

[0007] R is independently selected from at least one of hydrogen atom, straight-chain alkyl, branched-chain alkyl, straight-chain alkenyl, branched-chain alkenyl, substituted alkynyl, cycloalkyl, phenyl, and heteroatom-containing aromatic group.

[0008] In some embodiments, all three hydrophobic tail chains are straight chains. In some embodiments, two of the three hydrophobic tail chains are symmetrical.

[0009] In some embodiments, R is independently selected from alkyl or heterocyclic compounds containing nitrogen, oxygen, or sulfur. In some embodiments, R is independently selected from piperazine, pyridinyl, pyrimidinyl, thiazolyl, imidazolyl, benzene ring, pyrrole, indole, acridine, ethylene oxide, furanyl, thiophene, or derivatives thereof.

[0010] In some embodiments, R is independently selected from any of the following structures:

[0011]

[0012] In some embodiments, R is independently selected from any of the following structures:

[0013]

[0014] In some embodiments, the ionizable lipid compound is selected from any one of the following formulas:

[0015]

[0016]

[0017]

[0018]

[0019] In some embodiments, the ionizable lipid compound is selected from any one of the following formulas:

[0020]

[0021]

[0022] As used herein, "alkyl" refers to straight-chain and branched alkyl and cycloalkyl groups, for example, having 1 to 40 carbon atoms, or 1 to 20 carbon atoms, or 1 to 12 carbon atoms, or 1 to 8 carbon atoms. Examples of straight-chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl. In this invention, "alkyl" includes n-alkyl, isoalkyl, and trans-isoalkyl, as well as other branched forms of alkyl groups. Representative substituted alkyl groups may be substituted once or multiple times by any group listed in this invention, such as amino, hydroxyl, cyano, carboxyl, nitro, thio, alkoxy, and halogen groups.

[0023] As used herein, "alkenyl" refers to straight-chain, branched, and cycloalkyl groups as defined in this invention, distinguished by the presence of at least one double bond between two carbon atoms. Thus, an alkenyl group can have 2 to 40 carbon atoms, or 2 to 20 carbon atoms, or 2 to 12 carbon atoms, or 2 to 8 carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl.

[0024] As used herein, "alkynyl" refers to straight-chain and branched alkyl groups as defined in this invention, distinguished by the presence of at least one triple bond between two carbon atoms. Therefore, an alkynyl group can have 2 to 40 carbon atoms, or 2 to 20 carbon atoms, or 2 to 12 carbon atoms, or 2 to 8 carbon atoms. Examples of alkynyl groups include, but are not limited to, -C≡CH, -C≡C(CH3), -C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and -CH2C≡C(CH2CH3).

[0025] As used herein, “cycloalkyl” refers to a cyclic alkyl group, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the cycloalkyl group may have 3 to 8-12 ring members. In some embodiments, the number of ring carbon atoms is in the range of 3 to 4, 5, 6, or 7. The cycloalkyl group may also include polycyclic cycloalkyl groups, such as norbornyl, adamantyl, borneol, camphenyl, isocamphenyl, and careyl, as well as fused rings, such as naphthyl. The cycloalkyl group may also include straight-chain or branched alkyl-substituted rings as defined herein. Representative substituted cycloalkyl groups may be monosubstituted or substituted more than once, including but not limited to 2,2-, 2,3-, 2,4-, 2,5-, or 2,6-disubstituted cyclohexyl or mono-, di-, or trisubstituted norbornyl or cycloheptyl, which may be substituted with, for example, amino, hydroxyl, cyano, carboxyl, nitro, thio, alkoxy, and halogen groups.

[0026] As used herein, “aryl” refers to a cyclic aromatic hydrocarbon that does not contain heteroatoms in its ring. Therefore, aryl includes, but is not limited to, phenyl, azulel, heptapenyl, biphenyl, indalpenyl, fluorenyl, phenanthrene, triphenylene, pyrene, tetraphenyl, biphenylene, anthracene, and naphthyl. In some embodiments, the aryl group contains 6 to 14 carbons in the cyclic portion of the group. The aryl group can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be monosubstituted or substituted more than once, such as 2-, 3-, 4-, 5-, or 6-substituted phenyl or 2-8-substituted naphthyl groups, which can be substituted by carbon or non-carbon groups as those listed herein.

[0027] As used herein, "aromatic heterocycle (containing heteroatomic aromatic group)" refers to an aromatic heterocycle having at least one heteroatomic ring member such as an O, S, or N atom. Aromatic heterocycle groups include monocyclic and polycyclic (e.g., having 2, 3, or 4 fused rings) systems. The cyclic N atom in an aromatic heterocycle group can also be oxidized to form an N-oxygen moiety. Examples of aromatic heterocycle groups include pyridyl, N-oxypyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furanyl, quinolinyl, isoquinolinyl, thiopheneyl, imidazolinyl, thiazolyl, indoleyl, pyrroleyl, oxazolyl, benzofuranyl, benzothiopheneyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indoleyl, 1,2,4-thiophenediazolyl, isothiazolyl, benzothiopheneyl, purine, carbazoleyl, benzimidazolinyl, and dihydroindoleyl. In some embodiments, the aromatic heteroyl group may have 1 to 20 carbon atoms, or 3 to 20 carbon atoms. In some embodiments, the aromatic heteroyl group contains 3-14, 3-7, or 5-6 cyclic atoms. In some embodiments, the aromatic heteroyl group has 1-4, 1-3, or 1-2 heteroatoms.

[0028] As used herein, a "heterocyclic group (heterocyclic compound)" refers to an aromatic or non-aromatic cyclic compound containing three or more ring members, one or more of which are heteroatoms, including but not limited to N, O, and S. Therefore, a heterocyclic group can be a cycloheteroalkyl group, a heteroaryl group, or, if polycyclic, any combination thereof. In some embodiments, a heterocyclic group may include 3 to 20 ring members, or 3 to 15 ring members. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. The heterocyclic ring may also include one or more double bonds. A "heterocyclic group" may include fused ring species, including fused rings comprising fused aromatic and non-aromatic groups.

[0029] As used herein, “halogenated,” “halogen,” or “halide” means, either by itself or as part of other substituents, a fluorine, chlorine, bromine, or iodine atom.

[0030] In some embodiments, n is selected from any integer between 5 and 30. In some embodiments, m is selected from any integer between 0 and 30. In some embodiments, n is selected from any integer between 5 and 20. In some embodiments, m is selected from any integer between 0 and 10.

[0031] In some embodiments, n is selected from any integer from 8 to 18. In some embodiments, m is selected from any integer from 6 to 8.

[0032] In some embodiments, the ratio of n to m is greater than 1. In some embodiments, the ratio of n to m includes (1.1-3):1. In some embodiments, the ratio of n to m is (9-11):7.

[0033] Secondly, the present invention provides a method for preparing the above-mentioned ionizable lipid compound, characterized by comprising the following steps:

[0034] Aldehydes, amines, and carboxylic acids are added to an organic solvent to carry out a first reaction, yielding the first reactant.

[0035] An isonitrile compound is added to the first reactant to carry out a second reaction, yielding a second reactant, namely the ionizable lipid compound.

[0036] The structural formula of the amine compound is shown in Formula A:

[0037]

[0038] The structural formula of the aldehyde compound is shown in Formula B:

[0039]

[0040] The structural formula of the isonitrile compound is shown in Formula C:

[0041]

[0042] The structural formula of the carboxylic acid compound is shown in Formula D:

[0043]

[0044] In some embodiments, n is selected from any integer between 5 and 30. In some embodiments, m is selected from any integer between 0 and 30. In some embodiments, n is selected from any integer between 5 and 20. In some embodiments, m is selected from any integer between 0 and 10.

[0045] In some embodiments, n is selected from any integer from 8 to 18. In some embodiments, m is selected from any integer from 6 to 8.

[0046] In some embodiments, the ratio of n to m is greater than 1. In some embodiments, the ratio of n to m includes (1.1-3):1. In some embodiments, the ratio of n to m is (9-11):7.

[0047] In some embodiments, the molar ratio of the amine compound, the aldehyde compound, the carboxylic acid compound, and the isonitrile compound is (0.5–2.0):(0.5–2.0):(0.5–2.0):(0.5–2.0).

[0048] In some embodiments, the molar ratio of the amine compound, the aldehyde compound, the carboxylic acid compound, and the isonitrile compound is 1:1.3:1:1.3.

[0049] In some embodiments, the reaction conditions for the first reaction include 10 to 60 minutes at room temperature.

[0050] In some embodiments, the reaction conditions for the second reaction include a reaction at 25–60°C for 6–24 hours.

[0051] In some embodiments, the amine compound is selected from any one of compounds A1-A11:

[0052]

[0053] In some embodiments, the aldehyde compound is selected from any one of compounds B1-B5:

[0054]

[0055] In some embodiments, the carboxylic acid compound is selected from any one of compounds D8-D18:

[0056]

[0057] In some embodiments, the isonitrile compound is selected from any one of compounds C8-C18:

[0058]

[0059] As used herein, "solvent" means a liquid that can dissolve solids, other liquids, or gases to form a solution. Non-limiting examples of solvents are organic compounds, water, alcohols, ionic liquids, and supercritical fluids.

[0060] Thirdly, the present invention provides a composition, characterized in that the composition comprises at least the above-mentioned ionizable lipid compound and sterol.

[0061] In some embodiments, sterols are used to provide membrane integrity and stability for lipid nanoparticles. In some embodiments, exemplary sterols are cholesterol or derivatives thereof. In some embodiments, the cholesterol or derivatives thereof include at least one selected from cholesterol, sitosterol, stigmasterol, cholic acid, deoxycholic acid, and cholesterol derivatives.

[0062] In some embodiments, non-limiting examples of the cholesterol derivative include: polar analogs, such as 5α-cholesterol, 5β-fecal alcohol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholesterol; nonpolar analogs, such as 5α-cholesterol, cholesterolenone, 5α-cholesterol, 5β-cholesterol, and cholesterol decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, such as cholesteryl-(4'-hydroxy)-butyl ether. In some embodiments, the cholesterol derivative is hemisuccinate cholesterol ester.

[0063] In some embodiments, the molar ratio of the ionizable lipid compound to cholesterol or its derivative in the composition includes (10-90):(10-50). In some embodiments, the molar ratio of the ionizable lipid compound to cholesterol or its derivative in the composition can be 80:20, 70:30, 60:40, or 50:50. It is important to emphasize that the ionizable lipid compound provided by this invention has high stability and easy self-assembly characteristics. The two-component lipid nanoparticles formed by combining it with sterols (e.g., cholesterol or its derivatives) already exhibit high transfection efficiency and excellent delivery efficiency, possessing low toxicity, specific targeting, and high stability, enabling efficient drug delivery and organ targeting.

[0064] In some embodiments, the composition may further comprise non-cationic lipids. The non-cationic lipids can be used to enhance fusion and also to enhance the stability of lipid nanoparticles during formation. In some embodiments, the non-cationic lipids may include amphiphilic lipids, neutral lipids, and anionic lipids. Therefore, non-cationic lipids can be neutral, uncharged, zwitterionic, or anionic lipids.

[0065] Exemplary noncationic lipids include, but are not limited to, distearyl-sn-glycerol-phosphoethanolamine, distearyl-phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylethanolamine (POP). E), Dioleoyl-phosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), Dipalmitoylphosphatidylethanolamine (DPPE), Dimyristoylphosphatidylethanolamine (DMPE), Distearate-phosphatidyl-ethanolamine (DSPE), Monomethyl-phosphatidylethanolamine (e.g., 16-O-monomethylPE), Dimethyl-phosphatidylethanolamine (e.g., 16-O-dimethylPE), 18-1-transPE, 1-stearoyl -2-Oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), lecithin choline (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearate phosphatidylglycerol (DSPG), disinylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), ditrans-oleoylphosphatidyl Ethanolamine (DEPE), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphatidylphosphate, lysophosphatidylcholine, dilinoleylphosphatidylcholine, or mixtures thereof. It should be understood that other diacylphosphatidylcholines and diacylphosphatidylethanolamine phospholipids may also be used. The acyl group in these lipids is preferably derived from a group having a C 10 -C 24The acyl group of the carbon chain fatty acid, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. In some embodiments, other examples of non-cationic lipids include non-phospholipids such as stearamine, dodecylamine, hexadecylamine, acetyl palmitate, glyceryl ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethoxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramides, sphingomyelin, etc.

[0066] In some embodiments, the non-cationic lipid is a phospholipid. In some embodiments, the non-cationic lipid is selected from the group consisting of: DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0067] In some embodiments, the composition may further include a targeting agent. In some embodiments, the targeting agent may be a ligand, oligosaccharide, cell surface receptor, peptide, lipoprotein, glycoprotein, hormone, vitamin, antibody or antibody fragment, prodrug, conjugate, or a combination thereof. In some embodiments, the targeting agent may bind directly to or only to the outer surface of the lipid nanoparticles via a crosslinking agent. In some embodiments, the targeting agent is used to deliver lipid nanoparticles to one or more specific tissues.

[0068] In some embodiments, the composition may further include a hydrogel material, such as gelatin.

[0069] Fourthly, the present invention provides a nanoparticle, characterized in that the nanoparticle comprises the above-described composition.

[0070] Fifthly, the present invention provides a method for preparing the above-mentioned nanoparticles, characterized by comprising the following steps:

[0071] The composition is dissolved in an organic solvent to obtain an organic phase;

[0072] The organic phase and the aqueous phase are mixed to obtain the nanoparticles.

[0073] In some embodiments, the aqueous phase is an acidic buffer solution.

[0074] In some embodiments, the volume ratio of the aqueous phase to the organic phase is (3-10):1.

[0075] In some embodiments, the mixing can be vortex mixing or microfluidic mixing.

[0076] In a sixth aspect, the present invention provides a drug-loaded nanoparticle, characterized in that the drug-loaded nanoparticle comprises the above-described composition and a drug.

[0077] In some embodiments, the drug comprises at least one of small molecule compounds, nucleic acid molecules, protein or polypeptide molecules, and gene editing complexes.

[0078] As used herein, “nucleic acid” or “nucleic acid molecule” includes polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or their analogues or modifications, such as single-stranded DNA or RNA, double-stranded DNA or RNA, multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derived nucleotide bases.

[0079] In some embodiments, the nucleic acid molecule includes DNA, RNA, or a combination thereof. In some embodiments, the nucleic acid molecule includes at least one of messenger RNA (mRNA), transfer RNA (tRNA), dsRNA, shRNA, DNA, plasmid DNA, siRNA, antisense oligonucleotide, aiRNA, and miRNA.

[0080] In some embodiments, the gene editing complex comprises mRNA / sgRNA or Cas9 / sgRNA.

[0081] In some embodiments, the mass ratio of the ionizable lipid compound to the drug is (1–50):1. In some embodiments, the mass ratio of the ionizable lipid compound to the drug may be 5:1, 10:1, 20:1, 30:1, or 40:1. In some embodiments, the drug is a nucleic acid molecule.

[0082] In a seventh aspect, the present invention provides a method for preparing the above-mentioned drug-loaded nanoparticles, characterized by comprising the following steps:

[0083] The composition is dissolved in an organic solvent to obtain an organic phase;

[0084] The drug was dissolved in an acidic buffer solution to obtain an aqueous phase;

[0085] The organic phase and the aqueous phase are mixed to obtain the drug-loaded nanoparticles.

[0086] In some embodiments, the volume ratio of the aqueous phase to the organic phase is (3-10):1.

[0087] In some embodiments, the mixing can be vortex mixing or microfluidic mixing.

[0088] In some embodiments, the drug is a nucleic acid molecule.

[0089] Eighthly, the present invention provides the use of the above-described ionizable lipid compounds, the above-described compositions, the above-described nanoparticles, or the above-described drug-loaded nanoparticles in the preparation of transfection formulations and / or drug delivery carriers.

[0090] In some embodiments, the transfection agent may be used in conjunction with an electrotransfection instrument.

[0091] In some embodiments, the drug comprises at least one of small molecule compounds, nucleic acid molecules, protein or polypeptide molecules, and gene editing complexes.

[0092] In some embodiments, the nucleic acid molecule includes DNA, RNA, or a combination thereof. In some embodiments, the nucleic acid molecule includes at least one of messenger RNA (mRNA), transfer RNA (tRNA), dsRNA, shRNA, DNA, plasmid DNA, siRNA, antisense oligonucleotide, aiRNA, and miRNA.

[0093] In some embodiments, the gene editing complex comprises mRNA / sgRNA or Cas9 / sgRNA.

[0094] In a ninth aspect, the present invention provides the use of the above-described ionizable lipid compounds, the above-described compositions, the above-described nanoparticles, or the above-described drug-loaded nanoparticles in the preparation of medicaments for the prevention and / or treatment of cancer, inflammation, autoimmune diseases, infections, mental illnesses, blood diseases, musculoskeletal diseases, or tissue repair.

[0095] In some embodiments, the above composition, the above nanoparticles, or the above drug-loaded nanoparticles may not contain one or more of non-cationic lipids, polyethylene glycol (PEG), and PEG-lipid conjugates.

[0096] Compared with the prior art, the beneficial technical effects of the present invention include at least the following aspects:

[0097] Although a series of ionizable lipid compounds have been reported in the prior art, they still require the cooperation of other components (such as non-cationic lipids, sterols, and polyethylene glycol (PEG) or PEG-lipid conjugates) to generate lipid nanoparticles that can safely and stably deliver drugs. In other words, the prior art has not reported any ionizable lipids that can form lipid nanoparticles that safely and stably deliver drugs by simply combining with cholesterol or its derivatives.

[0098] Compared to existing technologies (such as CN116554125A and CN116574070A) that construct ionizable lipid compounds with at least two hydrophilic cores, this invention constructs a branched peptide-like molecule. This ionizable lipid compound of formula I has a single hydrophilic core and three hydrophobic tail chains, wherein the two hydrophobic tail chains are symmetrical, and the R group can be a nitrogen-, oxygen-, or sulfur-containing alkyl or heterocyclic compound. Unexpectedly, the branched peptide-like molecule constructed in this invention can be used as an ionizable lipid for efficient gene delivery, exhibiting high stability, easy self-assembly, degradability, and low toxicity. It can be combined with cholesterol or its derivatives to form safe and stable two-component lipid nanoparticles for drug delivery. Furthermore, the formed lipid nanoparticles exhibit high transfection efficiency in various cell lines, can transfect cells at different sites, and have high safety, providing key technical support for the development of novel non-viral gene delivery systems.

[0099] Experiments of this invention demonstrate that the lipid nanoparticles of this invention require only two components to achieve drug delivery and organ targeting, exhibiting low toxicity and specific targeting. They can achieve mRNA expression in different organs in vivo through various administration methods. When used in conjunction with therapeutic genes, the drug-loaded nanoparticles of this invention can effectively kill tumor cells. Therefore, the ionizable lipid compounds of this invention can meet the needs of applications such as mRNA nucleic acid therapy, nucleic acid vaccines, and gene editing.

[0100] Furthermore, the ionizable lipid compounds of this invention feature mild reaction conditions, simple synthesis processes, good stability, and a wide range of raw material selection. This invention uses aldehydes, amines, carboxylic acids, and isonitriles as raw materials to synthesize the ionizable lipid compounds of Formula I in a one-step Ugi reaction. The lipid nanoparticles of this invention have a simple and low-cost preparation process and can be prepared on a large scale.

[0101] In summary, the branched peptide molecules constructed in this invention can be efficiently prepared using a one-pot method. When combined with substances such as cholesterol, they can form stable lipid nanoparticles for the efficient delivery of DNA, RNA, and small molecule drugs. They have advantages such as good stability, ease of preparation and quality control, and show great promise for translational applications in gene therapy and other fields. Attached Figure Description

[0102] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0103] Figure 1A The structural formula of the ionizable lipid compound synthesized in this invention is shown below;

[0104] Figure 1B The structural formula of the ionizable lipid compound synthesized in this invention is shown below;

[0105] Figure 1C The structural formula of the ionizable lipid compound synthesized in this invention is shown below;

[0106] Figure 1D The structural formula of the ionizable lipid compound synthesized in this invention is shown below;

[0107] Figure 1E The structural formula of the ionizable lipid compound synthesized in this invention is shown below;

[0108] Figure 1F The structural formula of the ionizable lipid compound synthesized in this invention is shown below;

[0109] Figure 2 The NMR spectrum is a representative of the ionizable lipid compound A1B2C14D14.

[0110] Figure 3 This is a representative mass spectrum of the ionizable lipid compound A1B2C14D14;

[0111] Figure 4 The NMR spectrum is a representative of the ionizable lipid compound A7B2C14D14.

[0112] Figure 5 This is a representative mass spectrum of the ionizable lipid compound A7B2C14D14;

[0113] Figure 6 This is a diagram showing the particle size distribution of the lipid nanoparticles synthesized in this invention.

[0114] Figure 7 This is a graph showing the DNA binding capacity of the lipid nanoparticles synthesized in this invention with pGFP at different mass ratios.

[0115] Figure 8This is a graph showing the effect of the lipid nanoparticles synthesized in this invention on cell viability.

[0116] Figure 9 This diagram shows the results of transfecting different cells with the lipid nanoparticles synthesized in this invention.

[0117] Figure 10 This diagram shows the results of transfecting different cells with the lipid nanoparticles synthesized in this invention.

[0118] Figure 11 The graph shows the transfection efficiency results for ionizable lipid compounds with different m values.

[0119] Figure 12 Transmission electron microscopy image of lipid nanoparticles formed from A7B2C14D14;

[0120] Figure 13 The NMR spectrum is a representative of the ionizable lipid compound A1B2C12D12.

[0121] Figure 14 The structural formula of the ionizable lipid compound synthesized in this invention is shown below;

[0122] Figure 15 The NMR spectrum is a representative of the ionizable lipid compound A11B1C14D14.

[0123] Figure 16 The transfection efficiency results are shown for ionizable lipid compounds with different m and n values.

[0124] Figure 17 The figure shows the in vitro mRNA delivery efficiency results for lipid nanoparticles with different m and n values. Detailed Implementation

[0125] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0126] In this document, the terms "upper," "lower," "inner," "outer," "front," "rear," "one end," and "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the present invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0127] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0128] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0129] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0130] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4%, more typically + / -3%, more typically + / -2%, even more typically + / -1%, and even more typically + / -0.5% of the value.

[0131] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0132] Example 1

[0133] This embodiment provides a method for preparing an ionizable lipid compound, wherein the synthetic route for the ionizable lipid compound is shown below:

[0134]

[0135] The specific preparation method includes: adding an amine compound (Formula A), an aldehyde compound (Formula B), and a carboxylic acid compound (Formula D) to an organic solvent (e.g., 0.5 mL methanol solution) at room temperature, reacting for 30 min at room temperature, then adding an isonitrile compound (Formula C), and reacting at 40 °C for 12 h. After the reaction, the product is separated and purified by chromatography, wherein the mobile phase is a mixture of methanol and dichloromethane. The molar ratio (feed ratio) of the aldehyde compound, the amine compound, the carboxylic acid compound, and the isonitrile compound is (0.5–2.0):(0.5–2.0):(0.5–2.0):(0.5–2.0) (e.g., 1 mmol: 1.3 mmol: 1 mmol: 1.3 mmol).

[0136] In some embodiments, the amine compound can be any one of compounds A1-A11:

[0137]

[0138] In some embodiments, the aldehyde compound can be any one of compounds B1-B5:

[0139]

[0140] In some embodiments, the carboxylic acid compound can be any one of compounds D8-D18:

[0141]

[0142] In some embodiments, the isonitrile compound can be any one of compounds C8-C18:

[0143]

[0144] Based on the above synthetic route, this invention synthesizes an ionizable lipid compound of formula I:

[0145]

[0146] Specifically, this invention synthesizes the following ionizable lipid compounds, including: A2B2C10D10, A3B2C10D10, A4B2C10D10, A1B2C10D10, A9B2C10D10, A2B2C12D12, A3B2C12D12, A1B2C14D14, A2B2C14D14, A3B2C14D14, A4B2C14D14, A5B2C14D14, A6B2C14D14, A7B2C14D14, A8B2C14D14, A9B2C14D14, A10B2C14D14, and A11B2. C14D14, A1B2C8D8, A7B2C8D8, A1B2C10D10, A7B2C10D10, A1B2C12D12, A4B2C12D12, A7B2C12D12, A1B2C16D16, A7B2C16D16, A1B2C18D18, A1B1C14D14, A9B1C14D14, A9B2C12D12, A11B1C14D14, A7B1C14D14, A11B3C14D14, A7B3C14D14, A11B4C14D14, A7B4C14D14, such as Figures 1A-1F and Figure 14 As shown.

[0147] It should be noted that the specific names of the ionizable lipid compounds are indicated by the specific raw materials used. For example, ionizable lipid compound A1B2C14D14 indicates that it is prepared using amine compound A1, aldehyde compound B2, carboxylic acid compound D14, and isonitrile compound C14 as raw materials. Therefore, those skilled in the art can determine the corresponding specific preparation methods based on the synthetic route of this invention and the naming of the ionizable lipid compounds, which will not be elaborated further in this invention.

[0148] The representative NMR and mass spectra of the ionizable lipid compound A1B2C14D14 synthesized in this invention are shown below. Figure 2 and Figure 3 As shown. The representative NMR and mass spectra of the ionizable lipid compound A7B2C14D14 synthesized in this invention are shown below. Figure 4 and Figure 5 As shown. The representative NMR spectra of the ionizable lipid compound A1B2C12D12 synthesized in this invention are as follows. Figure 13 As shown. The representative NMR spectra of the ionizable lipid compound A11B1C14D14 synthesized in this invention are as follows. Figure 15 As shown.

[0149] Example 2

[0150] Preparation and characterization of drug-loaded nanoparticles

[0151] The drug-loaded nanoparticles comprise an ionizable lipid compound and cholesterol or a derivative thereof. The specific preparation method includes: dissolving the ionizable lipid compound and cholesterol or a derivative thereof (e.g., cholesterol, or other cholesterol or derivative thereof) prepared in Example 1 in an organic solvent (e.g., ethanol) to obtain an organic phase. Dissolving nucleic acid molecules in an acidic buffer (e.g., sodium acetate buffer solution (25 mM, pH = 4.0)) to obtain an aqueous phase. The volume ratio of the aqueous phase to the organic phase can be (3–10):1 (e.g., 5:1). The molar ratio of the ionizable lipid compound to cholesterol or a derivative thereof can be (10–90):(10–50) (e.g., 60:40). The mass ratio of the ionizable lipid compound to the nucleic acid molecules can be (1–50):1 (e.g., 10:1).

[0152] After the organic phase and aqueous phase are vortexed using a pipette or mixed uniformly using microfluidics, drug-loaded nanoparticles are obtained. After standing for 15 minutes, they are dialyzed at 4°C for 30 minutes.

[0153] 100 μL of the prepared drug-loaded nanoparticles were diluted to 1 mL, and the particle size of the drug-loaded nanoparticles was measured using a nanoparticle size analyzer and a ZETA potentiometer. After the diluted drug-loaded nanoparticles were mixed evenly, they were slowly injected into the sample cell. The measurement temperature was set to 25 °C and the equilibration time was 10 s. The particle size was measured three times for each sample.

[0154] The particle size results of the drug-loaded nanoparticles are as follows: Figure 6 As shown in the figure. The results show that the different ionizable lipid compounds synthesized in this invention, when combined with cholesterol or its derivatives, can form drug-loaded nanoparticles of suitable size.

[0155] Agarose gel electrophoresis was used to investigate the DNA binding affinity of drug-loaded nanoparticles formed from the ionizable lipid compound A7B2C14D14 to pGFP under different mass ratios (0:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, w / w). Figure 7 As shown in the figure. The results show that although the drug-loaded nanoparticles formed in this invention contain only two components, an ionizable lipid compound and cholesterol or its derivatives, they still exhibit excellent ability to bind nucleic acid molecules.

[0156] 293T cells in logarithmic growth phase were collected and seeded at a density of 10,000 cells per well in 96-well plates. After 24 h of culture, lipid nanoparticles (formed from ionizable lipid compound A7B2C14D14) of different concentrations were added to the 96-well plates, with three parallel replicates. After 24 h of culture, the culture medium was aspirated, and DMEM high-glucose medium containing 10% CCK-8 was added to each well. The cells were incubated at 37°C for 30 min, and the absorbance of the samples was measured at 450 nm using a UV spectrophotometer to assess cell viability. Figure 8 As shown in the figure. The results show that the two-component lipid nanoparticles (empty carrier) have low toxicity and minimal impact on cell viability even at high concentrations, which is beneficial for further in vivo applications. The morphological characterization results of the above lipid nanoparticles by transmission electron microscopy are shown in the figure. Figure 12 As shown.

[0157] Example 3

[0158] Transfection of DNA-loaded drug-eluting nanoparticles into different cells

[0159] Take cells in the logarithmic growth phase, at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [insert density here] in 48-well plates in complete DMEM high-glucose medium. Green fluorescent protein particles (pGFP, 0.3 μg / well) were used as reporter genes. Drug-loaded nanoparticles (formed from the ionizable lipid compound A7B2C14D14) loaded with pGFP were added to the wells. After 6 h of incubation, the medium in the 48-well plates was replaced with complete DMEM medium, and the cells were incubated at 37°C in a 5% CO2 incubator for another 24 h. Subsequently, the expression of green fluorescent protein in the transfected cells was observed and photographed under a fluorescence microscope. Figure 9 As shown in the figure. The results show that although the drug-loaded nanoparticles synthesized in this invention have a simple composition, they can efficiently transfect different cells.

[0160] Transfection of drug-loaded nanoparticles containing mRNA into different cell types

[0161] Take cells in the logarithmic growth phase, at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [insert density here] in 48-well plates using DMEM high-glucose medium. The medium in the 48-well plates was then replaced with complete DMEM medium. Green fluorescent protein mRNA (GFP, 0.3 μg / well) was used as a reporter gene, and drug-loaded nanoparticles (formed from the ionizable lipid compound A7B2C14D14) loaded with mRNA were added to the wells. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. Subsequently, the expression of green fluorescent protein in the transfected cells was observed and photographed under a fluorescence microscope. Figure 10As shown in the figure. The results show that although the drug-loaded nanoparticles synthesized in this invention have a simple composition, they can efficiently transfect different cells.

[0162] Example 4

[0163] Animal experiments were conducted to detect the in vivo delivery and expression levels of intravenously injected mRNA.

[0164] Luciferase-mRNA was used as the model mRNA. Drug-loaded nanoparticles were prepared using ionizable lipid compounds A1B2C14D14 and A7B2C14D14 according to the method in Example 2. The specific method was as follows: 60 μL of drug-loaded nanoparticles containing 5 μg of Luciferase-mRNA were intravenously injected into 6-8 week old C57BL / 6 mice. Six hours later, 200 μL of 1×PBS solution containing 3 mg D-Luciferin potassiumsalt was injected intraperitoneally into the mice. After waiting 10 minutes, the in vivo mRNA expression was evaluated by bioluminescence imaging. The results are shown in Table 1. The drug-loaded nanoparticles formed by the ionizable lipid compounds (which can also be understood as cationic analogs) A1B2C14D14 and A7B2C14D14 were mainly expressed in the spleen after intravenous injection. The results show that although the drug-loaded nanoparticles synthesized in this invention have simple components, they can achieve efficient, safe, and stable delivery of nucleic acid molecules.

[0165] Table 1

[0166]

[0167] Example 5

[0168] Animal experiments to detect the in vivo delivery and expression levels of mRNA injected intramuscularly

[0169] Luciferase-mRNA was used as the model mRNA. Drug-loaded nanoparticles were prepared using ionizable lipid compounds A1B2C14D14 and A7B2C14D14 according to the method in Example 2. The specific method is as follows: 60 μL of drug-loaded nanoparticles containing 5 μg of Luciferase-mRNA were injected intramuscularly into 6-8 week old C57BL / 6 mice. Six hours later, 200 μL of 1×PBS solution containing 3 mg of D-Luciferin potassiumsalt was injected intraperitoneally into the mice. After waiting for 10 min, the in vivo mRNA expression was evaluated by bioluminescence imaging.

[0170] The results are shown in Table 2. The drug-loaded nanoparticles formed by ionizable lipid compounds A1B2C14D14 and A7B2C14D14 exhibited high fluorescence intensity after intramuscular injection. These results demonstrate that although the drug-loaded nanoparticles synthesized in this invention have simple components, they can achieve efficient, safe, and stable delivery of nucleic acid molecules.

[0171] Table 2

[0172]

[0173] Example 6

[0174] Animal experiments to detect the in vivo delivery and expression levels of intraperitoneally injected mRNA

[0175] Luciferase-mRNA was used as the model mRNA; drug-loaded nanoparticles were prepared using the ionizable lipid compound A7B2C14D14 according to the method in Example 2. The specific method is as follows: 60 μL of nanoparticles containing 5 μg Luciferase-mRNA or the drug-loaded nanoparticles prepared above were injected intraperitoneally into 6-8 week old C57BL / 6 mice. Six hours later, 200 μL of 1×PBS solution containing 3 mg D-Luciferin potassiumsalt was injected intraperitoneally into the mice. After waiting 10 minutes, the in vivo mRNA expression was evaluated by bioluminescence imaging. The results are shown in Table 3. The drug-loaded nanoparticles formed by the ionizable lipid compound were mainly expressed in the intestine after intraperitoneal injection. The results show that although the drug-loaded nanoparticles synthesized in this invention have simple components, they can achieve efficient, safe, and stable delivery of nucleic acid molecules.

[0176] Table 3

[0177]

[0178] Example 7

[0179] This embodiment tested the effect of the values ​​of m and / or n on the formation of drug-loaded nanoparticles from ionizable lipid compounds.

[0180] First, the following ionizable lipid compounds were tested:

[0181]

[0182] Take cells in the logarithmic growth phase, at a density of 1 × 10⁶ cells per well. 4Cells were seeded at a density suitable for DMEM high-glucose medium in 48-well plates. The medium in the 48-well plates was then replaced with DMEM. Green fluorescent protein mRNA (GFP, 0.3 μg / well) was added as a reporter gene to the wells, along with drug-loaded nanoparticles. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 hours. Subsequently, the expression of green fluorescent protein in the transfected cells was observed and photographed under a fluorescence microscope.

[0183] The results are as follows Figure 11 As shown, when m=0, drug-loaded nanoparticles formed by ionizable lipid compounds can transfect DC2.4 cells, but the efficiency is low; when m increases (for example, when increased to m=8), the transfection efficiency of drug-loaded nanoparticles formed by ionizable lipid compounds on DC2.4 cells is significantly improved.

[0184] The results are as follows Figure 16 As shown, compounds A1B2C14D14, A2B2C14D14, A3B2C14D14, A4B2C14D14, A5B2C14D14, A6B2C14D14, A7B2C14D14, and A8B2C14D14 all efficiently transfected B16 cells. These results indicate that ionizable lipid compounds with n set at 9-14 and m set at 6-8 exhibited higher transfection efficiency.

[0185] Log-phase 293T cells were seeded at a density of 10,000 cells per well in opaque white 96-well plates. Each experiment was performed in triplicate. After 24 hours of culture, the medium containing metabolic waste was discarded, and 100 μL of fresh medium containing 10% FBS was added. Drug-loaded nanoparticles containing Luciferase-mRNA were prepared according to the above method. The weight ratio of ionizable lipid compound to mRNA was 10 / 1, and 0.15 μg of mRNA was used per well. Drug-loaded nanoparticles formed from the ionizable lipid SM-102 were used as a control. The preparation method of SM102-LNP was as follows: SM-102, cholesterol, DSPC, and DMG-PEG2000 were mixed and dissolved in ethanol solution at a molar ratio of 50:10:38.5:1.5 to obtain the organic phase. The nucleic acid molecule (Luciferase-mRNA) was dissolved in an acidic buffer (e.g., sodium acetate buffer solution (25 mM, pH = 4.0)) to obtain the aqueous phase. The volume ratio of the aqueous phase to the organic phase was 5:1. The mass ratio of SM-102 to nucleic acid molecules was 10:1. After the organic phase and the aqueous phase were vortexed using a pipette or mixed uniformly using microfluidics, drug-loaded nanoparticles were obtained. After standing for 15 min, they were dialyzed at 4 °C for 30 min.

[0186] After adding the drug-loaded nanoparticles to each well, the cell culture plate was placed in an incubator and cultured for another 24 hours. Bio-Lumi was used.TM II. Firefly luciferase assay kit for detecting luciferase expression.

[0187] result( Figure 17 The results show that the two-component drug-loaded nanoparticles prepared from the ionizable compounds (e.g., A1B2C14D14, A7B2C14D14, A1B2C10D10, A7B2C12D12, A7B2C16D16) prepared in this invention exhibit high expression of fluorescent proteins and good delivery efficiency, even surpassing the performance of the well-known high-performance ionizable lipid SM-102.

[0188] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. An ionizable lipid compound, characterized in that, The ionizable lipid compound has a hydrophilic core and three hydrophobic tail chains, and its chemical structure is shown in formula (I): Wherein, R is independently selected from at least one of hydrogen atom, straight-chain alkyl, branched-chain alkyl, straight-chain alkenyl, branched-chain alkenyl, substituted alkynyl, cycloalkyl, phenyl, and heteroatom-containing aromatic group; n is any integer selected from 5 to 30; m is any integer from 0 to 30.

2. The ionizable lipid compound according to claim 1, characterized in that, R is independently selected from alkyl or heterocyclic compounds containing nitrogen, oxygen, or sulfur.

3. The ionizable lipid compound according to claim 2, characterized in that, R is independently selected from piperazinyl, pyridinyl, pyrimidinyl, thiazolyl, imidazolyl, benzene ring, pyrroleyl, indole, acridine, ethylene oxide, furanyl, thiopheneyl or derivatives thereof.

4. The ionizable lipid compound according to claim 1, characterized in that, R is independently selected from any of the following structures:

5. The ionizable lipid compound according to claim 1, characterized in that, The ionizable lipid compound is selected from any one of the following formulas:

6. A method for preparing the ionizable lipid compound according to any one of claims 1-5, characterized in that, Includes the following steps: Aldehydes, amines, and carboxylic acids are added to an organic solvent to carry out a first reaction, yielding the first reactant. An isonitrile compound is added to the first reactant to carry out a second reaction, thereby obtaining the second reactant, namely the ionizable lipid compound. The structural formula of the amine compound is shown in Formula A: The structural formula of the aldehyde compound is shown in Formula B: The structural formula of the isonitrile compound is shown in Formula C: The structural formula of the carboxylic acid compound is shown in Formula D: Where n is selected from any integer between 5 and 30; m is selected from any integer between 0 and 30.

7. The preparation method according to claim 6, characterized in that, The molar ratio of the amine compound, the aldehyde compound, the carboxylic acid compound, and the isonitrile compound is (0.5–2.0):(0.5–2.0):(0.5–2.0):(0.5–2.0).

8. A composition, characterized in that, The composition comprises at least the ionizable lipid compound and sterol as described in any one of claims 1-5; the sterol optionally includes cholesterol or a derivative thereof.

9. The composition according to claim 8, characterized in that, The cholesterol or its derivatives include at least one of cholesterol, sitosterol, stigmasterol, cholic acid, deoxycholic acid, and cholesterol derivatives.

10. A drug-loaded nanoparticle, characterized in that, The drug-loaded nanoparticles comprise the composition of claim 8 or 9, and a drug; the drug may optionally comprise at least one of a small molecule compound, a nucleic acid molecule, a protein or polypeptide molecule, or a gene editing complex.

11. The drug-loaded nanoparticles according to claim 10, characterized in that, The mass ratio of the ionizable lipid compound to the drug is (1-50):

1.

12. The method for preparing drug-loaded nanoparticles according to claim 10 or 11, characterized in that, Includes the following steps: The composition is dissolved in an organic solvent to obtain an organic phase; The drug was dissolved in an acidic buffer solution to obtain an aqueous phase; The organic phase and the aqueous phase are mixed to obtain the drug-loaded nanoparticles.

13. The use of the ionizable lipid compound of any one of claims 1-5, the composition of claim 8 or 9, or the drug-loaded nanoparticle of claim 10 or 11 in the preparation of transfection formulations and / or drug delivery carriers.

14. The use of the ionizable lipid compound of any one of claims 1-5, the composition of claim 8 or 9, or the drug-loaded nanoparticles of claim 10 or 11 in the preparation of a medicament for the treatment of cancer, inflammation, autoimmune diseases, infections, mental disorders, blood diseases, musculoskeletal diseases, or tissue repair.

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