Lipid compounds, nanoparticles thereof, and applications

Abstract

The present application relates to an ionizable lipid compound, a corresponding lipid nanoparticle and application thereof. The ionizable lipid compound of the present application contains a piperazine structure and multiple alkyl chains, which reduces toxic side effects while taking into account delivery efficiency and targeting, and facilitates large-dose and repeated administration. The ionizable lipid compound and the lipid nanoparticle thereof can be applied to delivery of small molecule drugs and various nucleic acid drugs (siRNA, DNA, mRNA, miRNA, ASO, etc.).

Description

Technical Field

[0001] This invention relates to a lipid compound, its nanoparticles, and their applications, belonging to the field of drug delivery or formulation technology. Background Technology

[0002] After decades of research, nucleic acid drugs, including DNA, mRNA, and small nucleic acid molecules (siRNA, miRNA, antisense molecules, ribozymes, and aptamers), are gradually being developed for use in preventive vaccines, therapeutic drugs, and other fields.

[0003] These nucleic acid drugs typically need to be delivered into cells to exert their effects. However, due to their susceptibility to enzymatic degradation and in vivo clearance, as well as their high negative charge making them difficult for cells to take up, delivery vectors are required for encapsulation and delivery. Commonly used delivery vectors fall into two categories: viral vectors and non-viral vectors. Viral vectors have relatively high transfection efficiency but suffer from drawbacks such as high toxicity, strong immune responses, small gene capacity, poor targeting, and complex preparation processes. Non-viral nanoparticle delivery vectors are widely used due to their low immunogenicity, good biocompatibility, and high transfection efficiency. These delivery vectors include polymers, liposomes and lipid nanoparticles, and inorganic nanocarriers. In particular, lipid nanoparticles (LNPs) based on cationic or ionizable lipids have become more mature in application due to the widespread use and safety verification of mRNA COVID-19 vaccines. Traditional LNPs consist of four components: cationic or ionizable lipids, cholesterol, cofactor phospholipids, and polyethylene glycol lipids. Among these, cationic or ionizable lipids are the core of the LNP delivery system, playing a crucial role in encapsulating nucleic acids, cellular uptake, and intracellular release, and even in targeting.

[0004] Different nucleic acid drugs have different delivery requirements, and existing delivery systems are not perfect. For example, the commercially available MC3 is specifically designed for liver-specific diseases using small nucleic acid drugs, while SM-102 and ALC-0315 are specifically designed for mRNA COVID-19 vaccines. In these scenarios, the required doses are often not very high, and the toxic side effects of lipids are usually low. However, in other applications, such as mRNA-based gene editing therapies, the required doses are much higher, leading to a significant increase in the amount of lipid injected, often resulting in potential cumulative toxicity and metabolic burden. To meet the delivery needs of different nucleic acid drugs and improve delivery efficiency while reducing toxicity, it is necessary to develop new ionizable lipid compounds. Summary of the Invention

[0005] The main objective of this invention is to overcome the problems existing in the prior art and propose an ionizable lipid compound containing a piperazine structure and multiple alkyl chains, which reduces toxic side effects while balancing delivery efficiency and targeting. Corresponding lipid nanoparticles and their applications are also proposed.

[0006] The technical solution of this invention to solve its technical problem is as follows:

[0007] A compound of formula I, which is a cationic lipid compound or an ionizable lipid compound:

[0008]

[0009] in:

[0010] n1, n2, n3, n4, and n5 are all integers; where n1 = 0 to 6, n2 = 1 to 10, n3 = 1 to 10, n4 = 1 to 5, and n5 = 1 to 5.

[0011] X1 and X2 are independently selected from -C-, -C(O)O-, -O-, -SC(O)O-, -OC(O)S-, -OC(O)O-, -OC(O)-, -SC(O-), -C(O)-S, -C(O-), -OC(S-), -C(S)O-, -SS-, or -S(O). 0-2 -;

[0012] R1 and R2 are independently selected from C. 2-16 Alkyl, C 2-16 alkenyl, or C 2-16 alkynyl group;

[0013] R1 and R2 are optionally substituted by one or more R', and / or one or more methylene units in R1 and R2 are optionally and independently substituted by -NR''-; wherein,

[0014] R' is independently selected from H, C 1-14 Alkyl, -L a -OR a -L a -SR a , or -L a -NR a ';

[0015] R'' is independently selected from H or C 1-14 alkyl;

[0016] L a and L b Each is independently selected from chemical bonds or C 1-14 Alkylene;

[0017] L aand L b Each is independently selected from chemical bonds or C 1-14 Alkylene;

[0018] R a and R a 'Each is independently selected from H or C 1-14 alkyl.

[0019] The compound of Formula I of this invention is an aminolipid compound based on piperazine. The presence of the piperazine structure is beneficial for the preparation of stable lipid nanoparticles (LNPs), which have high encapsulation efficiency for active molecules, high biosafety for in vivo delivery, and can improve targeted delivery to the liver.

[0020] Preferably, R1 and R2 are each independently selected from one of the following structures:

[0021] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,

[0022] , ,

[0023] , ,

[0024] ;

[0025] t is an integer, and t = 0 to 4.

[0026] More preferably, t=0 or 1.

[0027] Preferably, the compound of formula I is one of the following representative compounds:

[0028] M10: ;

[0029] M2: ;

[0030] M3: ;

[0031] M4: ;

[0032] M5: ;

[0033] M6: ;

[0034] M7: ;

[0035] M8: .

[0036] Stereoisomers, tautomers, isotopic variants, solvates, or pharmaceutically acceptable salts of the compounds of Formula I described above.

[0037] The present invention also proposes:

[0038] Lipid nanoparticles containing a compound of Formula I described above, or a stereoisomer, tautomer, isotope variant, solvate, or pharmaceutically acceptable salt thereof.

[0039] Preferably, the lipid nanoparticles are solid lipid nanoparticles containing one or more lipids, structured lipid nanoparticles, liposomes, multilayer lipid vesicles, or micelles. Preferably, the composition of the lipid nanoparticles is as follows:

[0040] (a) A compound of formula I or its stereoisomers, tautomers, isotopic variants, solvates, or pharmaceutically acceptable salts, in a molar percentage of 20-70%.

[0041] (b) Phospholipids with a molar percentage of 5-30%;

[0042] (c) Steroids with a molar percentage of 20-60%;

[0043] (d) Hidden lipids with a molar percentage of 0-5%;

[0044] Furthermore, the total molar percentage of the above components is 100%.

[0045] More preferably, the phospholipid is selected from DSPC, DOPC, DOPE, DMPE, DMPC, DPPC, 4ME-PC, Egg-PC, or DOPG; the steroid is cholesterol; and the occult lipid is DMG-PEG2000 or ALC-0159. More preferably, the molar percentage of the compound of formula I or its stereoisomers, tautomers, isotopic variants, solvates, or pharmaceutically acceptable salts is 30-60%; or, the molar percentage of the phospholipid is 5-17%; or, the molar percentage of the steroid is 30-50%; or, the molar percentage of the occult lipid is 2-3%.

[0046] More preferably, the molar percentage of the compound of formula I or its stereoisomers, tautomers, isotopic variants, solvates, or pharmaceutically acceptable salts is 35-45%.

[0047] Preferably, the lipid nanoparticles include an internal aqueous phase in which hydrophilic substances are dissolved.

[0048] More preferably, the hydrophilic cargo is an oligonucleotide; or, the hydrophilic cargo is a therapeutic agent composed of polynucleotides, hydrophobic and / or hydrophilic small molecule compounds or bioactive lipids; or, the hydrophilic cargo is a hydrophilic small molecule drug (such as doxorubicin).

[0049] More preferably, the therapeutic agent comprises a polynucleotide, which is DNA or RNA, selected from siRNA, miRNA, pri-miRNA, messenger RNA, CRISPR nucleic acid, single-stranded RNA, CRISPR-RNA, trans-activating crRNA, plasmid DNA, transfer RNA, antisense oligonucleotide, guide RNA, double-stranded DNA, single-stranded DNA, single-stranded RNA, or double-stranded RNA.

[0050] More preferably, the RNA is a self-replicating mRNA, a non-self-replicating mRNA, or a circular RNA.

[0051] Preferably, the lipid nanoparticles further include an internal oil phase containing lipid-soluble cargoes.

[0052] More preferably, the lipid-soluble cargo is a bioactive lipid or bioactive lipid; the bioactive lipid is a fatty acid, monoglyceride, lauric acid, linoleic acid, oleic acid, C6 ceramide, or sphingolipid.

[0053] Preferably, the N / P molar ratio of the lipid nanoparticles is 4, 5, 6, 7, 8, 9 or 10.

[0054] Preferably, when the lipid nanoparticles are administered to mammals, at least 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the therapeutic agent contained in the lipid nanoparticles is delivered to the liver; or, when the lipid nanoparticles are administered to mammals, up to 30%, 25%, 20%, 15%, 10%, or 5% of the therapeutic agent contained in the lipid nanoparticles is delivered to the spleen or other non-hepatic organ combinations.

[0055] Preferably, the average size of the lipid nanoparticles is less than 200 nm, less than 150 nm, less than 100 nm, less than 90 nm, or less than 80 nm; or, the lipid nanoparticles have a polydispersity index (PDI) between 0.10 and 0.25, or between 0.15 and 0.20.

[0056] The present invention also proposes:

[0057] The use of the compound of Formula I or the lipid nanoparticles described above in the preparation of drugs.

[0058] Preferably, the drug is a pharmaceutical composition or pharmaceutical preparation, and the administration method is systemic administration; the systemic administration includes pulmonary administration, intravenous injection, subcutaneous injection, catheter injection, nasopharyngeal administration, or oral / gastrointestinal administration.

[0059] The ionizable lipid compound of this invention contains a piperazine structure and multiple alkyl chains. In in vivo delivery studies, it has shown superior performance compared to existing technologies MC3 and SM-102, achieving both delivery efficiency and targeting while reducing toxicity and facilitating high-dose and repeated administration. This ionizable lipid compound and its lipid nanoparticles can be used to deliver small molecule drugs and various nucleic acid drugs (siRNA, DNA, mRNA, miRNA, ASO, etc.). Attached Figure Description

[0060] Figure 1 The compound M10 in Example 1 of this invention 1 H-NMR spectrum.

[0061] Figure 2 The compound M10 in Example 1 of this invention 13 C-NMR spectrum.

[0062] Figure 3 This is the mass spectrum of compound M10 in Example 1 of the present invention.

[0063] Figure 4 The images shown in Table 1 of Example 2 of this invention are in vivo fluorescence imaging images of each formulation.

[0064] Figure 5The images show the fluorescence imaging of ex vivo organs for each formulation in Example 2 of this invention. The organs in the images are the liver, lung, kidney, spleen, and heart.

[0065] Figure 6 for Figure 5 Statistical graph of fluorescence intensity in various organs.

[0066] Figure 7 for Figure 5 Heatmap of expression intensity in various organs.

[0067] Figure 8 The above are in vivo fluorescence imaging images of each formulation in Example 2 of the present invention.

[0068] Figure 9 The images show fluorescence imaging of ex vivo organs for each formulation in Example 2 of this invention. The organs in the images are the liver, lung, kidney, spleen, and heart.

[0069] Figure 10 for Figure 9 Statistical graph of fluorescence intensity in various organs.

[0070] Figure 11 for Figure 9 Heatmap of expression intensity in various organs.

[0071] Figure 12 This is a schematic diagram of the editing efficiency test results in Embodiment 3 of the present invention.

[0072] Figure 13 The images show the H&E staining results of the heart, liver, spleen, lungs, and kidneys in each group of Example 4 of this invention. Detailed Implementation

[0073] In specific implementation, one embodiment of the present invention involves a synthetic compound of formula I or a stereoisomer, tautomer, isotopic variant, solvate, or pharmaceutically acceptable salt of formula I:

[0074]

[0075] in:

[0076] (1) n1, n2, n3, n4 and n5 are all integers; among them, n1 = 0 to 6, n2 = 1 to 10, n3 = 1 to 10, n4 = 1 to 5, and n5 = 1 to 5.

[0077] (2) X1 and X2 are independently selected from -C-, -C(O)O-, -O-, -SC(O)O-, -OC(O)S-, -OC(O)O-, -OC(O)-, -SC(O-), -C(O)-S, -C(O-), -OC(S)-, -C(S)O-, -SS-, or -S(O). 0-2-

[0078] (3) R1 and R2 are each independently selected from C 2-16 Alkyl, C 2-16 alkenyl, or C 2-16 alkynyl group;

[0079] R1 and R2 are optionally substituted by one or more R', and / or one or more methylene units in R1 and R2 are optionally and independently substituted by -NR''-; wherein,

[0080] R' is independently selected from H, C 1-14 Alkyl, -L a -OR a -L a -SR a , or -L a -NR a ';

[0081] R'' is independently selected from H or C 1-14 alkyl;

[0082] L a and L b Each is independently selected from chemical bonds or C 1-14 Alkylene;

[0083] L a and L b Each is independently selected from chemical bonds or C 1-14 Alkylene;

[0084] R a and R a 'Each is independently selected from H or C 1-14 alkyl.

[0085] Compound I is a cationic lipid compound or an ionizable lipid compound. This compound is a piperazine-derived aminolipid compound. The presence of the piperazine structure is beneficial for the preparation of stable lipid nanoparticles (LNPs), which have high encapsulation efficiency for active molecules, high biosafety for in vivo delivery, and can improve targeted delivery to the liver.

[0086] In some embodiments, R1 and R2 of the compound of formula I are each independently selected from one of the following structures:

[0087] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,

[0088] , ,

[0089] , ,

[0090] ;

[0091] t is an integer, and t = 0 to 4 (t is preferably 0 or 1).

[0092] In some embodiments, the compound of formula I is one of the following representative compounds:

[0093] M10: ;

[0094] M2: ;

[0095] M3: ;

[0096] M4: ;

[0097] M5: ;

[0098] M6: ;

[0099] M7: ;

[0100] M8: .

[0101] In another embodiment of the invention, a method for preparing a piperazine-derived lipid compound is disclosed, comprising the following steps:

[0102] Step 1: React R1-COOH with Br-L1-OH to obtain R1-COO-L1-Br, and / or react R2-COOH with Br-L2-OH to obtain R2-COO-L2-Br; during the reaction, dichloromethane (DCM) is used as the reaction solvent, and 2 eq of 1,3-dicyclohexylcarbodiimide (DCC) and 0.5 eq of 4-dimethylaminopyridine (DMAP) are added, and the esterification reaction is carried out overnight at room temperature (i.e., ambient temperature). The reaction formula is as follows:

[0103] ;

[0104] .

[0105] Step 2: React the compound of formula II or tert-butyl 1-carboxylic acid of formula II with R1-COO-L1-Br and / or R2-COO-L2-Br to obtain the target compound of formula III (Note: Compound III is a substructure of compound I); during the reaction, ethanol (EtOH) is used as the reaction solvent, and 0.2 eq of potassium iodide (KI) and 4 eq of N,N-diisopropylethylamine (DIPEA) are added, and the amination reaction is carried out under reflux for 72 h. The reaction formula is as follows:

[0106] ,

[0107] or ,

[0108] in,

[0109] L1 and L2 are each independently selected from C 1-10 Alkyl groups, L3, are independently selected from chemical bonds or C. 1-6 alkyl;

[0110] R1 and R2 are independently selected from C. 2-16 Alkyl, C 2-16 alkenyl, or C 2-16 alkynyl group;

[0111] R1 and R2 are optionally substituted by one or more R', and / or one or more methylene units in R1 and R2 are optionally and independently substituted by -NR''-; wherein,

[0112] R' is independently selected from H, C 1-14 Alkyl, -L a -OR a -L a -SR a , or -L a -NR a ';

[0113] R'' is independently selected from H or C 1-14alkyl;

[0114] L a and L b Each is independently selected from chemical bonds or C 1-14 Alkylene;

[0115] L a and L b Each is independently selected from chemical bonds or C 1-14 Alkylene;

[0116] R a and R a 'Each is independently selected from H or C 1-14 alkyl.

[0117] In another embodiment of the invention, lipid nanoparticles (LNPs) containing the compound of Formula I above are involved.

[0118] Within the scope of this invention, the term "lipid nanoparticles" includes solid lipid nanoparticles composed of one or more lipids, structured lipid nanoparticles, liposomes or multilayer lipid vesicles, and micelles.

[0119] In some embodiments, the LNP described above comprises: (a) a molar percentage of about 20-70%, about 30-60%, or about 35-45% of a cationic compound or ionizable lipid compound synthesized in this invention, i.e., a compound of formula I; (b) a molar percentage of about 5-30% (e.g., about 5-17%, or about 16%) of phospholipids, optionally such as DSPC, DOPC, DOPE, DMPE, DMPC, DPPC, 4ME-PC, Egg-PC, DOPG; (c) a molar percentage of about 20-60% (e.g., about 30-50%, or about 47.5%) of steroids (e.g., cholesterol); and (d) a molar percentage of about 0-5% (e.g., about 2-3%, or about 1.5%) of occult lipids (e.g., DMG-PEG2000, ALC-0159); the total molar percentage is 100%.

[0120] In some implementations, the LNP includes an internal aqueous phase that can dissolve hydrophilic cargo molecules, such as oligonucleotides, in the tLCNP.

[0121] In some embodiments, the hydrophilic cargo is a therapeutic agent composed of polynucleotides (including DNA and RNA, such as mRNA, siRNA, miRNA, ASO, and pDNA), hydrophobic and / or hydrophilic small molecule compounds (such as those with a molecular weight of less than 1000 Da, 750 Da, 500 Da, 250 Da, or 100 Da), or bioactive lipids (such as fatty acids or monoglycerides).

[0122] In some implementations, the therapeutic agent includes polynucleotides such as siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), a type of regularly spaced short palindromic repeat (CRISPR) associated nucleic acid, single-stranded RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA).

[0123] In some implementations, the RNA is self-replicating mRNA, non-self-replicating mRNA, or circular RNA.

[0124] In some embodiments, the cargo is a small molecule. In some embodiments, the small molecule is a hydrophilic drug dissolved in the aqueous phase of the LNP.

[0125] In some implementations, the small molecule includes doxorubicin (a small molecule antitumor chemical drug and also a hydrophilic drug).

[0126] In some implementations, the LNP also includes an internal oil phase that can hold fat-soluble cargo.

[0127] In some embodiments, the cargo is a bioactive lipid. Fatty acids, monoglycerides, and other bioactive lipids may also be incorporated into the LNP system of this invention.

[0128] In some implementations, the bioactive lipids include lauric acid, linoleic acid, oleic acid, C6 ceramide, or sphingolipids.

[0129] In some implementations, the N / P molar ratio of the LNP is approximately 4, 5, 6, 7, 8, 9, or 10, for example, 7.

[0130] In some implementations, when LNP is administered to mammals, at least about 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the therapeutic agent is delivered to the liver.

[0131] In some implementations, when LNP is administered to mammals, no more than about 30%, 25%, 20%, 15%, 10%, or 5% of the therapeutic agent is delivered to the spleen (or other non-hepatic organ combination).

[0132] In some implementations, when LNP is administered to mammals, approximately 1-15%, approximately 2-10%, or approximately 3-5% of the therapeutic agent is delivered to the lungs.

[0133] In some implementations, the average size of the LNP described above is less than about 200 nanometers, less than about 150 nanometers, less than about 100 nanometers, less than about 90 nanometers, or less than about 80 nanometers.

[0134] In some embodiments, the LNP described above has a polydispersity index (PDI) between about 0.10 and 0.25 or between about 0.15 and 0.20.

[0135] In another embodiment of the invention, the use of the above-described LNP for delivering a bioactive agent into a living organism for therapeutic purposes is described.

[0136] Another embodiment of the invention relates to a pharmaceutical composition / formulation comprising the LNP described above, and a pharmaceutically acceptable carrier or excipient.

[0137] In some embodiments, the pharmaceutical composition / formulation is formulated for systemic administration, such as intravenous injection / administration.

[0138] The phrase "systemic drug delivery" as used above refers to its generally accepted meaning in the art. This term generally refers to a method or technique for delivering molecules, drugs, formulations, or compounds in a manner that results in the drug being absorbed systemically or accumulated in the bloodstream and then distributed throughout the body. Systemic drug delivery includes intracorporeal drug delivery.

[0139] In some embodiments, systemic administration includes pulmonary administration (inhalation, nebulization, etc.), intravenous injection, subcutaneous injection, catheter injection, nasopharyngeal administration, or oral / gastrointestinal administration, all of which are commonly known in the art.

[0140] Other non-limiting examples of systemic drug delivery methods of the present invention include oral administration, sublingual administration, parenteral administration (i.e., intravenous injection, intraperitoneal injection, subcutaneous injection, or intramuscular injection), local rectal administration, or other local administration methods that enable the drug delivery composition to be absorbed or accumulated in the bloodstream and then distributed throughout the body.

[0141] In another embodiment of the invention, a method for preparing the LNP of the present invention is disclosed, the method comprising mixing an organic liquid phase and an aqueous phase in an organic solvent (such as ethanol) miscible with water, the organic liquid phase comprising a compound of formula I of the present invention, phospholipids, steroids and occult lipids, and the aqueous phase comprising a therapeutic agent disclosed in the present invention to form the LNP.

[0142] In some embodiments, the preparation method is performed in a microfluidic mixing device or chip. Automated microfluidic devices or microfluidic chips are rapid and efficient methods for preparing LNPs. Using these devices allows for rapid mixing in a highly controllable and reproducible manner, resulting in uniform LNPs and high encapsulation efficiency. In these devices, single streams of an ethanol lipid mixture and an aqueous oligonucleotide solution are rapidly mixed. The resulting mixture forms the LNP being tested and is collected in a collection tube. The LNP can be fine-tuned by changing parameters such as the flow rate ratio and the total flow rate.

[0143] In some embodiments, the preparation method is carried out in a T-type or Y-type mixer. These mixers can be assembled using inexpensive materials commonly used in the laboratory. The T-type or Y-type connected mixer can be fitted with two inlets, each connected to a syringe containing a lipid mixture or oligonucleotide solution, and one outlet leading the LNPs to a collection tube. An inlet flow rate can be optionally controlled using a syringe pump.

[0144] In some embodiments, the preparation method employs an ethanol injection method. An ethanol-lipid mixture and an aqueous oligonucleotide solution are mixed using a magnetic stirring plate. While continuously stirring, the ethanol-lipid mixture is injected into an acidic oligonucleotide aqueous solution, and stirring continues for 30 minutes. Alternatively, a simpler method is manual stirring. The ethanol-lipid mixture is transferred into the acidic oligonucleotide aqueous solution and rapidly stirred for 15 seconds using a pipette. The mixture is then allowed to stand for 10 minutes.

[0145] In some embodiments, the preparation method further includes conditions or equipment such as extrusion or high-pressure microfluidics to reduce particle size and produce a uniform particle size distribution.

[0146] In some embodiments, the preparation method further includes dialyzing the LNP in a storage buffer using an appropriate molecular weight cutoff (MWCO) tube. This step removes unencapsulated cargo, excess lipid components, and ethanol from the final preparation. Dialysis also adjusts the pH of the LNP from the acidic preparation buffer to the neutral storage solution.

[0147] In some embodiments, the preparation method further includes filtration to eliminate bacteria in the LNP solution. In some embodiments, the filtration step removes bacteria or other contaminants by using a 0.22 µm filter.

[0148] In some embodiments, the preparation method further includes stepwise purification of LNP using a tangential flow filtration (TFF) system.

[0149] In some embodiments, the organic solvent in the organic liquid phase is composed of ethanol, or is substantially composed of ethanol.

[0150] In some embodiments, the organic solvent in the organic liquid phase is composed of methanol, or is substantially composed of methanol.

[0151] In some embodiments, the organic solvent in the organic liquid phase is composed of other alcohols or other organic phases, such as tetrahydrofuran, dimethylformamide, chloroform, etc.

[0152] In some embodiments, the organic liquid phase and the aqueous phase are mixed in the microfluidic device, with selectable volume ratios of 1:2, 1:3, 1:4, and 1:5; and / or an optional total flow rate of approximately 10-25 mL / min (e.g., approximately 15 mL / min).

[0153] In some embodiments, the aqueous phase includes 10 mM, 25 mM, or 50 mM citrate buffer (pH 3.0, 4.0, 5.0, or 6.0).

[0154] In some implementations, stepwise purification is performed in a TFF system containing 3-10% sucrose citrate buffer (pH 5.0) and Tris-HCl buffer (pH 7.2).

[0155] In some embodiments, the preparation method further includes measuring the particle size of the LNP (e.g., average particle size and / or particle size distribution). In some embodiments, the particle size is determined by dynamic light scattering (DLS) measurements, for example using a Zetasizer NanoZS from Malvern Instruments Ltd.

[0156] In some embodiments, the preparation method also includes measuring the encapsulation efficiency (EE%) of the cargo (such as mRNA).

[0157] In some implementations, encapsulation efficiency (EE%) is determined by, for example, the RIBOGREEN® assay. RIBOGREEN® is a dye that fluoresces when it binds to single-stranded mRNA but cannot enter LNPs.

[0158] In some embodiments, the preparation method further includes imaging the LNP sample using cryo-electron microscopy (Cryo-EM) to study its nanoprecipitate core structure. Cryo-electron microscopy images can reveal that mRNA is encapsulated within a substance resembling LNPs, exhibiting a multi-layered core structure.

[0159] The above-mentioned LNP preparation method is simple, the raw materials are readily available, the reaction conditions are mild, the yield is high, and the cost is low. When used as an excipient in pharmaceutical formulations, it has the advantages of low toxicity.

[0160] The present invention will be further described in detail below with reference to the embodiments. However, the present invention is not limited to the examples given.

[0161] Example 1

[0162] This example describes the synthesis of an ionizable lipid compound, M10.

[0163] In the synthetic route:

[0164] Step 1: Compound L1 is obtained by reacting compound A with compound B. Dichloromethane (DCM) is used as the reaction solvent, and 2 eq of 1,3-dicyclohexylcarbodiimide (DCC) and 0.5 eq of 4-dimethylaminopyridine (DMAP) are added. The reaction is carried out overnight at room temperature. The reaction formula is as follows:

[0165] .

[0166] The second step involves reacting compound C with 3 eq of compound L1 to obtain the ionizable lipid compound M10. Ethanol (EtOH) was used as the reaction solvent, and 0.2 eq of potassium iodide (KI) and 4 eq of N,N-diisopropylethylamine (DIPEA) were added. The reaction was refluxed for 72 h. The reaction equation is as follows:

[0167] .

[0168] The resulting ionizable lipid compound M10 1 H-NMR spectrum 13 C-NMR spectrum and mass spectrum, such as Figures 1 to 3 As shown.

[0169] Example 2

[0170] The specific details of this embodiment are as follows:

[0171] (1) In vitro transcription of mRNA

[0172] Traditional luciferase mRNA manufacturing involves three steps: First, amplification, extraction, and purification of plasmid DNA encoding luciferase; then, linearization of the plasmid followed by purification by chromatography and / or lithium chloride / ethanol precipitation; finally, using the linearized plasmid DNA as a template, in vitro enzyme digestion to synthesize mRNA, followed by purification and storage at -80°C for later use.

[0173] (2) Preparation of mRNA@LNP

[0174] Using a typical microfluidic device, ethanol and aqueous phases in a 1:3 volume ratio were rapidly mixed at a total flow rate of 12 mL / min to generate mRNA@LNP. The aqueous phase was a 50 mM citrate buffer (pH 5.0) containing mRNA or other nucleic acid molecules; the ethanol phase consisted of ionizable lipid compounds, cholesterol (Chol), DSPC, and DMG-PEG2000 in a predetermined molar ratio. The resulting mRNA@LNP was purified by dialysis or a TFF system in citrate buffer (pH 5.0) and Tris-HCl buffer (pH 7.2) containing 7.5% sucrose. The mRNA@LNP was then diluted to the target concentration and stored at 2–8°C for short-term storage (less than 3 months) and at -80°C for long-term storage (more than 3 months).

[0175] (3) Characterization of mRNA@LNP

[0176] The nanoparticle size and polydispersity index (PDI) of the above mRNA@LNP were determined by dynamic light scattering (DLS), and the mRNA concentration was determined by the Ribogreen® assay. Ethanol was then removed using a TFF purification process, and the mRNA@LNP was converted to a sucrose-containing Tris-HCl buffer suitable for intravenous injection. Finally, the prepared product was characterized by DLS and the Ribogreen® assay to determine its size and encapsulation efficiency (EE) / concentration.

[0177] (4) In vivo assessment of mRNA@LNP

[0178] Animal experiments were conducted to determine the in vivo expression efficiency and organ distribution of the test substance LNP using the delivery system. All animal experiments were approved by the Animal Care and Use Committee of Hangzhou Normal University and complied with relevant Chinese government regulations on animal care and use. BALB / c mice or C57BL / 6 (female, 6-8 weeks old) were randomly divided into n=3 groups. Mice were intravenously injected with luciferase mRNA@LNP (mRNA dose: 5 μg), dispersed in Tris-HCl buffer (pH 7.2) containing 7.5% sucrose. Six hours after injection, mice were intraperitoneally injected with D-luciferin potassium (150 mg / kg, intraperitoneal injection). Imaging was performed using the IVIS Lumina system 10 minutes later, followed by organ isolation and imaging.

[0179] First, LNP formulations were screened and optimized based on the ionizable lipid compound M10. The formulations shown in Table 1 were prepared according to (1) and (2) above, then characterized according to (3), and finally evaluated for in vivo delivery in animals according to (4). Among them, MC3 and SM-102 (note: dilinoleylmethyl-4-dimethylaminobutyrate, DLin-MC3-DMA, abbreviated as MC3; 1-octylnonyl-8-[(2-hydroxyethyl)[6-O-6-(undecapoxy)hexyl]amino]octanoate, code name SM-102) were used as controls in the prior art. Formulations with different molar ratios were screened using the ionizable lipid compound M10. The characterization results of each formulation are shown in Table 1. The imaging and data analysis of Luciferase mRNA expression in animals for each formulation are shown in Table 1. Figures 4 to 7 As shown.

[0180] Table 1. Formulations and characterization results of each LNP

[0181] MC3-F0 MC3:DSPC: Chol: DMG-PEG =50: 10: 38.5: 1.5 73.23 / 0.153 89.11 SM-102-F0 SM-102:DSPC: Chol: DMG-PEG =50: 10: 38.5: 1.5 65.51 / 0.148 97.91 M10-F1 M10:DSPC: Chol: DMG-PEG =45: 13: 40.5: 1.5 71.26 / 0.136 86.93 M10-F2 M10:DSPC: Chol: DMG-PEG =40: 13: 45.5: 1.5 68.48 / 0.173 88.32 M10-F3 M10:DSPC: Chol: DMG-PEG =40: 13: 44.5: 2.5 73.46 / 0.185 86.67 M10-F4 M10:DSPC: Chol: DMG-PEG =38: 16: 44.5: 1.5 83.56 / 0.149 88.81 M10-F5 M10: DOPE: Chol: DMG-PEG =40: 13: 44.5: 2.5 64.29 / 0.134 90.70 M10-F6 M10: DOPC: Chol: DMG-PEG =40: 13: 44.5: 2.5 63.52 / 0.161 97.26 M10-F7 M10: DOPE: Chol: DMG-PEG =35: 16: 46.5: 2.5 89.45 / 0.173 91.82 M10-F8 M10: DOPC: Chol: DMG-PEG =35: 16: 46.5: 2.5 84.26 / 0.127 92.93

[0182] Next, one formulation was selected from Table 1 as a base, and different ionizable lipid compound molecules were replaced and tested. The formulations shown in Table 2 were prepared according to (1) and (2) above, then characterized according to (3), and finally evaluated for in vivo delivery in animals according to (4). Ionizable lipid compounds M10, M2, M4, and M8 were used for testing. The characterization results of each formulation are shown in Table 2. Imaging and data analysis of Luciferase mRNA expression in animals for each formulation are as follows. Figures 8 to 11 As shown. Note: Since M10-F2 in Table 2 was prepared anew and new mice were used in the experiment, it is normal for the results to differ from the corresponding results above.

[0183] Table 2. Formulations and characterization results for replacing different ionizable lipid compound molecules.

[0184] M10-F2 M10:DSPC: Chol: DMG-PEG =40: 13: 45.5: 1.5 73.23 / 0.153 89.11 M2-F2 M2:DSPC: Chol: DMG-PEG =40: 13: 45.5: 1.5 65.51 / 0.148 97.91 M4-F2 M4:DSPC: Chol: DMG-PEG =40: 13: 45.5: 1.5 71.26 / 0.136 86.93 M8-F2 M8:DSPC: Chol: DMG-PEG =40: 13: 45.5: 1.5 68.48 / 0.173 81.32

[0185] Based on the above results, it can be seen that M2, M4, M8, and M10 all have relatively reliable drug physicochemical characterization data and in vivo delivery efficiency, among which M10 performs better.

[0186] Example 3

[0187] This embodiment is an example of the application of LNP based on the cationic liposomes of the present invention in gene editing.

[0188] The specific details of this embodiment are as follows:

[0189] 1. Experiments on the delivery of mRNA and sgRNA of Cas9, a gene editor targeting PCSK9.

[0190] By intravenously injecting different lipid nanoparticles, the mRNA and sgRNA encoding Cas9 were delivered to mouse hepatocytes. Mutations were introduced by cleaving at the PCSK9 gene location, which prevented the normal transcription of PCSK9 protein and reduced protein expression. The editing efficiency was calculated by sequencing and RT-qPCR.

[0191] By analyzing the PCSK9 gene sequence, two sgRNAs targeting this region were designed (synthesized by Nanjing GenScript Biotech Co., Ltd.), and their sequences are as follows:

[0192] PCSK9-sgRNA1: 5'-UGGAGCUGACGGUGCCCAUG-3' (SEQ ID NO.1)

[0193] PCSK9-sgRNA2: 5'-UGGGCACCGUCAGCUCCAGG-3' (SEQ ID NO.2)

[0194] (1) Lipid nanoparticles containing the cationic liposomes of the present invention, which encapsulate the mRNA and sgRNA of Cas9 (using the same formulation as M10-F2 in Example 1, and M2, M3, M4, M5, M6, M7, M8, and M10 respectively), were injected via the tail vein into 6-7 week old female C57BL / 6 mice (purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.).

[0195] (2) Lipid nanoparticles containing MC3 (using the same formulation as MC3-F0 in Example 1) and lipid nanoparticles containing SM-102 (using the same formulation as SM-102-F0 in Example 1) were administered to mice of similar age and sex as positive controls in a similar manner.

[0196] (3) PBS buffer was injected via the tail vein into mice of similar age and sex as a negative control.

[0197] 2. Editing efficiency test

[0198] One week after administration to mice, the editing efficiency was tested. The mice were sacrificed, liver tissue was collected, lysed, and the genome and RNA were extracted. The efficiency was analyzed by sequencing and RT-qPCR.

[0199] The results are as follows Figure 12As shown in the figure, the expression level of group NC is used as the standard, and the relative expression levels of PCSK9 in other groups are calculated with reference to this standard. Based on these results, it can be concluded that lipid nanoparticles containing the cationic liposomes of this invention can deliver mRNA in vivo and achieve editing efficiencies no less than those of the positive controls MC3 and SM-102.

[0200] Example 4

[0201] This embodiment is for testing the biosafety of the lipid nanoparticle formulation of the present invention.

[0202] ICR mice were randomly divided into 5 groups of 10 mice each. Lipid nanoparticles (LNPs) prepared according to the M10-F2 formulation in Example 1 were administered via tail vein at doses of 1 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / kg, and 20 mg / kg (qd / 3d / iv), respectively. After 3 consecutive days of administration, 3 mice from each group were randomly selected, and plasma was collected from each group. The mice were then dissected, and tissue samples from the heart, liver, spleen, lungs, and kidneys were collected. These samples were subjected to blood biochemistry and routine blood tests, as well as hematoxylin-eosin (H&E) staining of the organs for observation. The remaining 7 mice from each group were observed for 21 days.

[0203] The results are shown in Tables 3 to 7, and Figure 13 As shown in the figure. After intravenous administration of different concentrations of LNP, the blood routine test results of each group were all within the reference range; the H&E staining results showed no obvious abnormalities; no mice died within 21 days; and there were no abnormalities in the mice's drinking water and diet. The above results indicate that the above-mentioned LNP has good safety at concentrations up to 20 times the effective concentration.

[0204] Table 3. Blood routine test results of the 1 mg / kg group

[0205] White blood cell count WBC 2.9 10^9 / L 0.8-10.6 Lymphocyte count Lymph# 2.3 10^9 / L 0.6-8.9 Monocyte count Mon# 0.1 10^9 / L 0.04-1.4 neutrophil count Gran# 0.5 10^9 / L 0.23-3.6 lymphocyte percentage Lymph% 78.3 % 40-92 percentage of monocytes Mon% 3.4 % 0.9-18 neutrophil percentage Gran% 18.3 % 6.5-50 Red blood cell count RBC 6.38 10^12 / L 6.5-11.5 hemoglobin HGB 118 g / L 110-165 Hematocrit HCT 31.4 % 35-55 Mean corpuscular volume MCV 49.3 fL 41-55 Mean corpuscular hemoglobin content MCH 18.4 pg 13-18 Mean corpuscular hemoglobin concentration MCHC 375 g / L 300-360 Red blood cell distribution width RDW 14.9 % 12-19 Platelet count PLT 900 10^9 / L 400-1600 Mean platelet volume MPV 5.4 fL 4.0-6.2 Platelet distribution width PDW 16.2 12.0-17.5 Platelet-weighted hematocrit PCT 0.486 % 0.100-0.780

[0206] Table 4. Blood routine test results of the 2 mg / kg group

[0207] White blood cell count WBC 3.4 10^9 / L 0.8-10.6 Lymphocyte count Lymph# 2.6 10^9 / L 0.6-8.9 Monocyte count Mon# 0.1 10^9 / L 0.04-1.4 neutrophil count Gran# 0.7 10^9 / L 0.23-3.6 lymphocyte percentage Lymph% 74.9 % 40-92 percentage of monocytes Mon% 3.7 % 0.9-18 neutrophil percentage Gran% 21.4 % 6.5-50 Red blood cell count RBC 6.91 10^12 / L 6.5-11.5 hemoglobin HGB 124 g / L 110-165 Hematocrit HCT 33.5 % 35-55 Mean corpuscular volume MCV 48.5 fL 41-55 Mean corpuscular hemoglobin content MCH 17.9 pg 13-18 Mean corpuscular hemoglobin concentration MCHC 370 g / L 300-360 Red blood cell distribution width RDW 15.3 % 12-19 Platelet count PLT 940 10^9 / L 400-1600 Mean platelet volume MPV 6.8 fL 4.0-6.2 Platelet distribution width PDW 16.6 12.0-17.5 Platelet-weighted hematocrit PCT 0.639 % 0.100-0.780

[0208] Table 5. Blood routine test results of the 5 mg / kg group

[0209] White blood cell count WBC 2.2 10^9 / L 0.8-10.6 Lymphocyte count Lymph# 1.7 10^9 / L 0.6-8.9 Monocyte count Mon# 0.1 10^9 / L 0.04-1.4 neutrophil count Gran# 0.4 10^9 / L 0.23-3.6 lymphocyte percentage Lymph% 78.8 % 40-92 percentage of monocytes Mon% 3.7 % 0.9-18 neutrophil percentage Gran% 17.5 % 6.5-50 Red blood cell count RBC 7.4 10^12 / L 6.5-11.5 hemoglobin HGB 141 g / L 110-165 Hematocrit HCT 36.8 % 35-55 Mean corpuscular volume MCV 49.8 fL 41-55 Mean corpuscular hemoglobin content MCH 19 pg 13-18 Mean corpuscular hemoglobin concentration MCHC 383 g / L 300-360 Red blood cell distribution width RDW 14.3 % 12-19 Platelet count PLT 1128 10^9 / L 400-1600 Mean platelet volume MPV 7.2 fL 4.0-6.2 Platelet distribution width PDW 16.9 12.0-17.5 Platelet-weighted hematocrit PCT *** % 0.100-0.780

[0210] Note: *** indicates that the detection range is below the minimum and no detection was detected.

[0211] Table 6. Blood routine test results of the 10mg / kg group

[0212] White blood cell count WBC 3.4 10^9 / L 0.8-10.6 Lymphocyte count Lymph# 2.7 10^9 / L 0.6-8.9 Monocyte count Mon# 0.1 10^9 / L 0.04-1.4 neutrophil count Gran# 0.6 10^9 / L 0.23-3.6 lymphocyte percentage Lymph% 78.7 % 40-92 percentage of monocytes Mon% 3.3 % 0.9-18 neutrophil percentage Gran% 18 % 6.5-50 Red blood cell count RBC 7.5 10^12 / L 6.5-11.5 hemoglobin HGB 137 g / L 110-165 Hematocrit HCT 35.7 % 35-55 Mean corpuscular volume MCV 47.6 fL 41-55 Mean corpuscular hemoglobin content MCH 18.2 pg 13-18 Mean corpuscular hemoglobin concentration MCHC 383 g / L 300-360 Red blood cell distribution width RDW 14.9 % 12-19 Platelet count PLT 776 10^9 / L 400-1600 Mean platelet volume MPV 5.9 fL 4.0-6.2 Platelet distribution width PDW 16.5 12.0-17.5 Platelet-weighted hematocrit PCT 0.457 % 0.100-0.780

[0213] Table 7. Blood routine test results of the 20mg / kg group

[0214] White blood cell count WBC 2.5 10^9 / L 0.8-10.6 Lymphocyte count Lymph# 2.1 10^9 / L 0.6-8.9 Monocyte count Mon# 0.1 10^9 / L 0.04-1.4 neutrophil count Gran# 0.3 10^9 / L 0.23-3.6 lymphocyte percentage Lymph% 83.9 % 40-92 percentage of monocytes Mon% 3.1 % 0.9-18 neutrophil percentage Gran% 13 % 6.5-50 Red blood cell count RBC 8.34 10^12 / L 6.5-11.5 hemoglobin HGB 152 g / L 110-165 Hematocrit HCT 40.6 % 35-55 Mean corpuscular volume MCV 48.8 fL 41-55 Mean corpuscular hemoglobin content MCH 18.2 pg 13-18 Mean corpuscular hemoglobin concentration MCHC 374 g / L 300-360 Red blood cell distribution width RDW 15.6 % 12-19 Platelet count PLT 967 10^9 / L 400-1600 Mean platelet volume MPV 5.4 fL 4.0-6.2 Platelet distribution width PDW 16.5 12.0-17.5 Platelet-weighted hematocrit PCT 0.522 % 0.100-0.780

[0215] In addition to the embodiments described above, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.

Claims

1. A compound of formula I, which is a cationic lipid compound or an ionizable lipid compound: The compound of formula I is one of the following representative compounds: M10： ； M2： ； M3： ； M4： ； M5： ； M6： ； M7： ； M8： 。 2. A tautomer of the compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof.

3. Lipid nanoparticles containing a compound of formula I according to claim 1 or a tautomer of a compound of formula I according to claim 2, or a pharmaceutically acceptable salt thereof.

4. The lipid nanoparticles according to claim 3, characterized in that, The lipid nanoparticles are solid lipid nanoparticles containing one or more lipids, structured lipid nanoparticles, liposomes, multilayer lipid vesicles, or micelles.

5. The lipid nanoparticles according to claim 3, characterized in that, The lipid nanoparticles have the following composition: (a) A compound of formula I or its tautomers, or a pharmaceutically acceptable salt, in a molar percentage of 20-70%; (b) Phospholipids with a molar percentage of 5-30%; (c) Steroids with a molar percentage of 20-60%; (d) Hidden lipids with a molar percentage of 0-5%; Furthermore, the total molar percentage of the above components is 100%.

6. The lipid nanoparticles according to claim 5, characterized in that, The phospholipids are selected from DSPC, DOPC, DOPE, DMPE, DMPC, DPPC, 4ME-PC, Egg-PC, or DOPG; the steroids are cholesterol; and the occult lipids are DMG-PEG2000 or ALC-0159.

7. The lipid nanoparticles according to claim 5, characterized in that, The molar percentage of the compound of Formula I or its tautomers or pharmaceutically acceptable salts is 30-60%; or the molar percentage of the phospholipid is 5-17%; or the molar percentage of the steroid is 30-50%; or the molar percentage of the occult lipid is 2-3%.

8. The lipid nanoparticles according to claim 5, characterized in that, The molar percentage of the compound of Formula I or its tautomers, or pharmaceutically acceptable salts, is 35-45%.

9. The lipid nanoparticles according to claim 3, characterized in that, The lipid nanoparticles include an internal aqueous phase in which hydrophilic cargoes are dissolved.

10. The lipid nanoparticles according to claim 9, characterized in that, The hydrophilic cargo is an oligonucleotide; or, the hydrophilic cargo is a therapeutic agent composed of polynucleotides, hydrophobic and / or hydrophilic small molecule compounds or bioactive lipids; or, the hydrophilic cargo is a hydrophilic small molecule drug.

11. The lipid nanoparticles according to claim 10, characterized in that, The therapeutic agent comprises a polynucleotide, which is DNA or RNA, selected from siRNA, miRNA, pri-miRNA, messenger RNA, CRISPR nucleic acid, single-stranded RNA, CRISPR-RNA, trans-activating crRNA, plasmid DNA, transfer RNA, antisense oligonucleotide, guide RNA, double-stranded DNA, single-stranded DNA, single-stranded RNA, or double-stranded RNA.

12. The lipid nanoparticles according to claim 11, characterized in that, The RNA is a self-replicating mRNA, a non-self-replicating mRNA, or a circular RNA.

13. The lipid nanoparticles according to claim 9, characterized in that, The lipid nanoparticles also include an internal oil phase containing lipid-soluble cargoes.

14. The lipid nanoparticles according to claim 13, characterized in that, The lipid-soluble cargo is a bioactive lipid or bioactive lipid; the bioactive lipid is a fatty acid, monoglyceride, lauric acid, linoleic acid, oleic acid, C6 ceramide, or sphingolipid.

15. The lipid nanoparticles according to claim 3, characterized in that, The N / P molar ratio of the lipid nanoparticles is 4, 5, 6, 7, 8, 9 or 10.

16. The lipid nanoparticles according to any one of claims 3 to 15, characterized in that, When the lipid nanoparticles are administered to mammals, at least 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the therapeutic agent contained in the lipid nanoparticles is delivered to the liver; or, when the lipid nanoparticles are administered to mammals, up to 30%, 25%, 20%, 15%, 10%, or 5% of the therapeutic agent contained in the lipid nanoparticles is delivered to the spleen or other non-hepatic organ combinations.

17. The lipid nanoparticles according to any one of claims 3 to 15, characterized in that, The lipid nanoparticles have an average size of less than 200 nm, less than 150 nm, less than 100 nm, less than 90 nm, or less than 80 nm; or, the lipid nanoparticles have a polydispersity index (PDI) between 0.10 and 0.25, or between 0.15 and 0.

20.

18. Use of the compound of Formula I according to claim 1 or the lipid nanoparticles according to any one of claims 3 to 17 in the preparation of a pharmaceutical product.

19. The use according to claim 18, characterized in that, The drug is a pharmaceutical composition or pharmaceutical preparation, and the administration method is systemic administration; the systemic administration includes pulmonary administration, intravenous injection, subcutaneous injection, catheter injection, nasopharyngeal administration, or oral / gastrointestinal administration.

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