CN116589435A - Ionizable lipids and compositions thereof for nucleic acid delivery - Google Patents

Abstract

The present invention relates to ionizable lipids and compositions thereof for nucleic acid delivery, belonging to the field of pharmaceutical chemistry. The invention provides a compound shown as a formula I. The invention also provides the use of the compounds as nucleic acid delivery vehicles. The compounds of the present invention, when used as delivery vehicles for nucleic acid drugs, exhibit greater delivery efficiency and greater safety than conventional materials.

Description

Ionizable lipids and compositions thereof for nucleic acid delivery

Technical Field

The invention relates to ionizable lipids and compositions thereof for nucleic acid delivery, and belongs to the field of medicinal chemistry.

Background Art

Nucleic acid drugs include DNA, antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), miRNA mimics, antimiRs, ribozymes, mRNA, aptamers, plasmids, CRISPR RNA, etc. The application of nucleic acid drugs is limited by the instability of their chemical properties. They are easily degraded into single nucleotides by nucleases in vitro and in vivo, and lose their efficacy.

The application of nucleic acid drugs often requires special delivery vectors, including viral vectors and non-viral vectors. Commonly used viral vectors include retroviruses, lentiviruses, adeno-associated viruses, etc. Viral vectors can successfully invade cells with their natural cell infection activity and have high transport efficiency. However, factors such as immunogenicity, limited loading capacity, and complex production processes limit their clinical application. Non-viral vectors are a type of gene delivery vector that has been widely studied and has good application prospects. They mainly load mRNA through the adsorption of cations formed by delivery materials and mRNA phosphate ions to form structures such as liposomes or nanoparticles, protect it from degradation by nucleases and change its entry pathway into cells. The advantages are that the vectors are relatively easy to obtain, have low immunogenicity, and are relatively safe.

Traditional non-viral nucleic acid delivery materials are mostly cationic lipids or cationic polymers. Due to their strong positive charge, they are easily adsorbed by plasma proteins in the body and then taken up by the reticuloendothelial system, which destroys the nucleic acid drugs they carry. Among non-viral vectors, lipid nanoparticles based on ionizable lipids are the most studied. Nanoparticles prepared by ionizable lipid materials are positively charged in an acidic environment in vitro, and load nucleic acid drugs through electrostatic adsorption of nucleic acids. After entering the neutral environment in the body, they are electrically neutral, avoiding adsorption of plasma proteins and capture by the reticuloendothelial system. Based on this, ionizable lipid nanoparticles have a very broad prospect in the field of nucleic acid delivery.

However, the clinical application of ionizable lipid nanoparticles is relatively rare, and the core difficulty lies in the development of safe and effective ionizable lipids. Therefore, the development of ionizable lipid nucleic acid delivery materials with higher delivery efficiency and safety is of great significance for the widespread application of nucleic acid drug gene therapy.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the object of the present invention is to provide an ionizable lipid for nucleic acid delivery.

The present invention provides a compound represented by formula I, or a pharmaceutically acceptable salt, isomer, deuterated substance or prodrug thereof:

Where a is 1, 2 or 3;

X ₁ and X ₂ are independently selected from N or C;

_L1 , _L2 , _L3 , and _L4 are independently selected from _-ReCH (OH)-, _-ReC (=O)-, _-ReC (=O)O-, _-ReOC (=O)-, _-ReC (=O)S-, _-ReSC ( ₌ O ₎ -, -ReC(=O)NRa-, _-ReNRaC (=O)-, -ReNRaC(=O)O-, -ReOC( ₌ O) _NRa- , _{-ReO- ,} -Re _{- OO-} , _-ReS- , -Re _{- SS-} , -Re _{- SSS-} , -ReCH(OH) _CH2O- , _-ReCH (OH) _CH2S- , or are absent, _{and Re} is or does not exist, k is an integer greater than 1, and _Ra is -H, substituted or unsubstituted alkyl;

R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from C ₁ -C ₃₀ straight-chain alkyl, C ₁ -C ₃₀ branched alkyl, C ₂ -C ₃₀ straight-chain alkenyl, C ₂ -C ₃₀ branched alkenyl, C ₂ -C ₃₀ straight-chain alkynyl, or C ₂ -C ₃₀ branched alkynyl;

_G1 , _G2 , _G3 and _G4 are independently selected from _-Rc- , _-RcCH (OH) _Rd- , _-RcC (＝O) _Rd- , -RcC( _＝ O)ORd- _{, -RcOC} (＝ _O )Rd-, -RcC(＝O)SRd- _{, -RcSC} (＝O)Rd-, -RcC(＝O)N( _{Rb )} Rd-, -RcN( _Rb )C(＝ _O )Rd-, _-RcN ( _Rb )C(＝O) _{ORd-, -RcOC (} ＝ _O )N( _Rb ) _{Rd- , -RcORd-} , _{-Rc - OORd-} , _-RcSRd- , -Rc _{- SSRd-} , -Rc _{- SSSRd-} or are absent _{; Rb} is -H, _substituted or unsubstituted alkyl _; , R _d is independently selected from or does not exist, n is an integer greater than 1;

R ₅ and R ₆ are independently selected from -H, substituted or unsubstituted alkyl;

Q ₁ and Q ₂ are independently selected from O or S.

The lipid nanoparticles formed by the above-mentioned ionizable lipids are positively charged in an acidic environment in vitro, and the electrostatic adsorption of nucleic acids realizes the loading of nucleic acid drugs. After entering the neutral environment in the body, they are electrically neutral, avoiding the adsorption of plasma proteins and the capture of the reticuloendothelial system. The lipid delivery carrier formed by the above-mentioned ionizable lipids has stronger targeting to the liver and lungs. The nucleic acid drugs carried by it, such as mRNA, are more highly expressed in receptor cells, and the antigens expressed by the antigen mRNA are more strongly presented in the body. It can be used for the in vivo delivery of nucleic acid drugs such as mRNA to achieve the up-regulation or down-regulation of the corresponding genes, or to deliver antigen mRNA to express antigens in vivo to achieve immunotherapy, or to deliver mRNA encoding antibodies to express antibodies in vivo. At the same time, the inventors of the present application also unexpectedly found that after the lipid carrier formed by the above-mentioned ionizable lipids is loaded with drugs, when administered to mice by intramuscular injection, its local irritation is significantly lower than that of the positive control carrier, and has no significant effect on the weight growth of mice, while the positive control carrier has an inhibitory effect on the weight growth of mice.

Among them, the writing order of the L1, L2, L3, and L4 connecting bonds defined above corresponds from left to right to the nitrogen-proximal end to the nitrogen-distal end.

The writing order of the G1, G2, G3, and G4 connecting bonds defined above, from left to right, corresponds to the direction of the main chain of Formula I from left to right.

According to an embodiment of the present invention, _X1 and _X2 are both N. The inventors found that when _X1 and _X2 are both N, the lipid nanoparticles formed by the above compounds have better pharmaceutical properties, have better encapsulation effect on mRNA, and further, can express mRNA more efficiently in experimental animals.

According to an embodiment of the present invention, the compound has a structure shown in Formula II, or is a pharmaceutically acceptable salt, stereoisomer, deuterated substance or prodrug of the structure shown in Formula II.

According to an embodiment of the present invention, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

According to an embodiment of the present invention, k is 1.

According to an embodiment of the present invention, L1, L2, L3, and L4 are independently selected from -CH(OH)-, -C(＝O)-, -CH2C(＝O)O-, -C(＝O)O-, -OC(＝O)-, -C(＝O)S-, -SC(＝O)-, -CH2C(＝O)NRa-, -C(＝O)NRa-, -NRaC(＝O)-, -NRaC(＝O)O-, -OC(＝O)NRa-, -CH2O-, -O-, -CH2-O-O-, -CH2S-, -S-, -CH2-S-S-, -CH2-S-S-S-, -CH(OH)CH2O-, -CH(OH)CH2S- or do not exist, and Ra is -H, substituted or unsubstituted alkyl.

According to an embodiment of the present invention, L ₁ , L ₂ , L ₃ , and L ₄ are independently selected from -C(＝O)-, -C(＝O)NR _a -, -CH ₂ C(＝O)NR _a -, -NR _a C(＝O)-, -C(＝O)O-, -CH ₂ C(＝O)O-, -OC(＝O)-, -CH ₂ O-, -O-, -CH ₂ S-, -S-, -CH(OH)-, -CH(OH)CH ₂ O-, -CH(OH)CH ₂ S- or are absent, and _Ra is -H or unsubstituted alkyl.

According to an embodiment of the present invention, the _Ra is -H or an unsubstituted _C1 - _C6 alkyl group.

According to an embodiment of the present invention, the _Ra is -H.

According to an embodiment of the present invention, L ₁ , L ₂ , L ₃ , and L ₄ are independently selected from -C(＝O)-, -C(＝O)NH-, -CH ₂ C(＝O)NH-, -C(＝O)O-, -CH ₂ C(＝O)O-, -CH ₂ O-, -CH ₂ S-, -CH(OH)-, -CH(OH)CH ₂ O-, or are absent; preferably, L ₁ , L ₂ , L ₃ , and L ₄ are independently selected from -C(＝O)NH-, -C(＝O)O-, -CH(OH)-, -CH(OH)CH ₂ O-, or are absent.

According to an embodiment of the present invention, _L1 and _L2 are selected from the same group and or _L3 and _L4 are selected from the same group.

According to an embodiment of the present invention, _L1 and _L3 are selected from the same group and or _L2 and _L4 are selected from the same group.

According to an embodiment of the present invention, L ₁ , L ₂ , L ₃ and L ₄ are selected from the same group.

According to an embodiment of the present invention, R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from C ₁ -C ₃₀ straight-chain alkyl, C ₂ -C ₃₀ straight-chain alkenyl, and C ₂ -C ₃₀ straight-chain alkynyl.

According to an embodiment of the present invention, R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from unsubstituted C ₁ ˜C ₃₀ straight-chain alkyl groups.

According to an embodiment of the present invention, R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from unsubstituted C ₈ ˜C ₁₈ straight-chain alkyl groups.

According to an embodiment of the present invention, R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from unsubstituted C ₁₀ ˜C ₁₄ straight-chain alkyl groups.

According to an embodiment of the present invention, R ₁ and R ₂ are selected from the same group and or R ₃ and R ₄ are selected from the same group.

According to an embodiment of the present invention, R ₁ and R ₃ are selected from the same group and or R ₂ and R ₄ are selected from the same group.

According to an embodiment of the present invention, R ₁ , R ₂ , R ₃ and R ₄ are selected from the same group.

According to an embodiment of the present invention, G ₁ , G ₂ , G ₃ , and G ₄ are independently selected from or absent, n3 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

According to an embodiment of the present invention, G ₃ and G ₄ do not exist.

According to an embodiment of the present invention, G ₁ and G ₂ are independently selected from -CH ₂ - or -CH ₂ CH ₂ -.

According to an embodiment of the present invention, R ₅ and R ₆ are independently selected from -H, unsubstituted C ₁ ～C ₆ alkyl or C ₁ ～C ₆ alkyl substituted with -OH.

According to an embodiment of the present invention, R ₅ and R ₆ are independently selected from -H, methyl, ethyl, propyl, hydroxymethyl, hydroxyethyl or hydroxypropyl.

According to an embodiment of the present invention, R ₅ and R ₆ are selected from the same group.

According to a specific embodiment of the present invention, the compound has one of the following structures:

Pharmaceutically acceptable salts, stereoisomers, deuterated substances or prodrugs thereof.

In another aspect, the present invention provides use of the aforementioned compound, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance or prodrug thereof as a drug delivery carrier.

Furthermore, the active ingredient of the drug is selected from at least one of nucleic acids, small molecule drugs, protein drugs, and polypeptides.

Specifically, the active ingredient of the drug is selected from nucleic acids.

Specifically, the drug has heart, liver, spleen, lung or kidney targeting.

Preferably, the drug targets the lungs and spleen.

In another aspect, the present invention provides a drug delivery carrier. According to an embodiment of the present invention, the drug delivery carrier comprises the aforementioned compound, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance or prodrug thereof.

In another aspect, the present invention provides a drug complex. According to an embodiment of the present invention, the drug complex comprises a carrier and an active ingredient, wherein the carrier is connected to the active ingredient, the carrier comprises a cationic lipid, and the cationic lipid comprises the aforementioned compound, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance or prodrug thereof.

According to a specific embodiment of the present invention, the active ingredient is selected from at least one of nucleic acids, small molecule drugs, protein drugs, polypeptides, and antibodies.

According to a specific embodiment of the present invention, the active ingredient of the drug is selected from nucleic acids;

According to a specific embodiment of the present invention, the nucleic acid is selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, and aptamer.

According to a specific embodiment of the present invention, the nucleic acid is mRNA;

According to a specific embodiment of the present invention, the delivery vector is connected to the mRNA via an ionic bond.

According to a specific embodiment of the present invention, the drug complex targets the lung and spleen.

According to a specific embodiment of the present invention, the drug complex exists in a form selected from lipid nanoparticles LNP, PLGA nanoparticles, micelles, liposomes, core-shell nanoparticles, and polymer nanoparticles.

According to a specific embodiment of the present invention, the drug complex is in the form of lipid nanoparticles LNP.

According to a specific embodiment of the present invention, the pharmaceutical composition further comprises an excipient, and optionally, the excipient comprises at least one selected from neutral phospholipids, steroids, and pegylated lipids.

According to a specific embodiment of the present invention, the neutral phospholipid is selected from at least one of DOPE, DSPC, DOPC, DSPE, DMPC, DMPE, DPPC, DPPE, DEPC, HSPC, and POPC.

According to a specific embodiment of the present invention, the neutral phospholipid is DOPE.

According to a specific embodiment of the present invention, the molar ratio of the compound, or its pharmaceutically acceptable salt, isomer, deuterated substance or prodrug to the neutral phospholipid is 1:10 to 10:1.

According to a specific embodiment of the present invention, the steroid is selected from at least one of cholesterol, sitosterol, stigmasterol, lanosterol, ergosterol and fucoxanthol.

According to a specific embodiment of the present invention, the steroid is cholesterol.

According to a specific embodiment of the present invention, the molar ratio of the compound, or its pharmaceutically acceptable salt, isomer, deuterated substance or prodrug to the steroid is 1:10 to 10:1.

According to a specific embodiment of the present invention, the PEGylated lipid is selected from at least one of DMG-PEG and DSPE-PEG;

Preferably, the PEGylated lipid is DMG-PEG2000.

According to a specific embodiment of the present invention, the molar ratio of the compound, or its pharmaceutically acceptable salt, isomer, deuterated substance or prodrug to the PEGylated lipid is 5:1 to 1000:1.

According to a specific embodiment of the present invention, the molar ratio of the compound, or a pharmaceutically acceptable salt, isomer, deuterated substance or prodrug thereof to the PEGylated lipid is 10:1 to 20:1.

According to a specific embodiment of the present invention, the nucleic acid is selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, and aptamer.

According to a specific embodiment of the present invention, the nucleic acid is mRNA.

On the other hand, the present invention provides a lipid nanoparticle. According to an example of the present invention, the lipid nanoparticle comprises a lipid nanocapsid and a nucleic acid, wherein the nucleic acid is encapsulated in the lipid nanocapsid, and the lipid nanocapsid comprises a cationic lipid, wherein the cationic lipid comprises the aforementioned compound, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance or prodrug thereof.

According to a specific embodiment of the present invention, the average particle size of the lipid nanoparticles is 40nm to 500nm, Di(90)

≤500nm, polydispersity coefficient ≤30%.

In another aspect, the present invention provides a pharmaceutical composition, which, according to a specific embodiment of the present invention, comprises the aforementioned drug complex or the aforementioned lipid nanoparticle.

According to a specific embodiment of the present invention, it further includes a pharmaceutically acceptable excipient.

According to a specific embodiment of the present invention, the composition is an injection.

According to a specific embodiment of the present invention, the composition is suitable for intravenous or intramuscular injection.

On the other hand, the present invention provides use of the aforementioned drug complex or the aforementioned lipid nanoparticle or the aforementioned drug composition in preparing a drug for treating or preventing a disease.

According to a specific embodiment of the present invention, the disease is a heart, liver, spleen, lung or kidney related disease, preferably, the disease is a spleen or lung related disease.

According to a specific embodiment of the present invention, the disease is an infectious disease, cancer and proliferative disease, genetic disease, autoimmune disease, diabetes, neurodegenerative disease, cardiovascular and renal vascular disease and metabolic disease.

According to a specific embodiment of the present invention, the infectious disease is selected from: diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, or various herpes.

According to a specific embodiment of the present invention, the cancer is a solid tumor, preferably, the cancer is liver cancer or lung cancer.

According to a specific embodiment of the present invention, the drug treats or prevents a disease by presenting an antigen and/or activating an immune response.

The present invention provides a novel ionizable lipid, whose hydrophilic center is composed of 4 tertiary amine effective nitrogens and whose hydrophobic tail is composed of 4 saturated or unsaturated fatty chains. The novel ionizable lipid provided by the present invention is positively charged in an acidic environment and almost uncharged in a neutral environment. By utilizing this property, nucleic acid drugs can be loaded in an acidic buffer system. After the nucleic acid drugs are loaded, they enter the neutral environment in the body, and the lipid nanoparticles are electrically neutral, which can effectively avoid the adsorption of nucleic acid drug complexes by plasma proteins, thereby achieving a higher delivery efficiency of nucleic acid drugs and significantly improving safety.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Particle size, PDI and potential of LNPs@mRNA prepared in Example 4;

FIG. 2 shows the encapsulation efficiency of LNPs@mRNA prepared in Example 4;

Figure 3 LNPs@mRNA cell-level expression of reporter genes: (A) LNPs@EGFP mRNA transfected HEK293T cells; (B) LNPs@EGFP mRNA transfected DC2.4 cells; (C) LNPs@FLuc mRNA transfected DC2.4 cells;

Figure 4 MTT assay for 4N4T-LNPs@FLuc mRNA cytotoxicity;

Fig. 5 Bioluminescence images of LNPs@FLuc mRNA expression and distribution;

Fig. 6 Statistics of LNPs@FLuc mRNA expression and distribution after intravenous administration;

Figure 7 Activation effect of LNPs@OVA mRNA vaccine on BMDC:

Fig. 8 TNF-α cytokine secretion of BMDCs treated with LNPs@OVA mRNA vaccine.

DETAILED DESCRIPTION

The present invention may be implemented in other specific forms without departing from the basic attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features in any other embodiment or multiple other embodiments to obtain other embodiments. The present invention includes other embodiments obtained by such combination.

All publications and patents mentioned in this disclosure are hereby incorporated by reference into this disclosure in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this disclosure, then the purposes and terms of this disclosure shall prevail.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Unless otherwise specified, all technical and scientific terms used herein have the common meaning in the field to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

Except in the working examples or otherwise indicated, all numbers of quantitative properties such as dosage stated in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range recited in the application is intended to include all subranges within the range and any combination of the respective endpoints of the range or subrange.

The words "include", "contain" or "comprises" and the like used in the present disclosure mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unrecorded elements. The terms "contain" or "includes" used herein may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

The compounds and derivatives provided in the present invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

The term "alkyl" is a group of a straight or branched saturated hydrocarbon group. Examples of C1-C6 alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5) and n-hexyl (C6).

The term "alkenyl" refers to a straight or branched hydrocarbon group containing at least one double bond. Examples of alkenyl groups include, but are not limited to, vinyl, allyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-2-enyl, pent-3-enyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl.

The term "alkynyl" refers to a straight or branched hydrocarbon group containing at least one triple bond. Examples of alkynyl include, but are not limited to, ethynyl, propargyl, but-1-ynyl, but-2-ynyl, pent-1-ynyl, pent-2-ynyl, pent-3-ynyl, hex-1-ynyl, hex-2-ynyl, hex-3-ynyl, hex-4-ynyl.

The term "pharmaceutically acceptable" means that a certain carrier, excipient, salt, etc. is generally chemically or physically compatible with other ingredients constituting a certain pharmaceutical dosage form and physiologically compatible with receptors. Specifically, the compound or complex is chemically and/or toxicologically compatible with other ingredients constituting the formulation and/or with humans or mammals who use it to prevent or treat diseases or conditions.

The term "subject" or "patient" in this application includes humans and mammals.

The term "treatment" as used herein refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of the disease to cure, alleviate, mitigate, improve or affect the disease or the symptoms of the disease. In the context of this application, unless otherwise specifically stated, the term "treatment" may also include prevention.

The term "pharmaceutically acceptable salt" refers to acidic and/or basic salts formed by the compounds of the present invention with inorganic and/or organic acids and bases, and also includes zwitterionic salts (inner salts), and also includes quaternary ammonium salts, such as alkylammonium salts. These salts can be directly obtained in the final separation and purification of the compound. It can also be obtained by mixing the above-mentioned compound with a certain amount of acid or base appropriately (e.g., equivalent). These salts may form a precipitate in the solution and be collected by filtering, or be recovered after solvent evaporation, or be obtained by freeze drying after reaction in an aqueous medium. The salt described in the present invention can be a hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

It should be further understood that the compound of formula I or its pharmaceutically acceptable salt can be isolated in the form of a solvate, and therefore any such solvate is included within the scope of the present invention. For example, the compound of formula I or its pharmaceutically acceptable salt can exist in an unsolvated form and in a solvated form formed with a pharmaceutically acceptable solvent (such as water, ethanol, etc.).

Certain compounds disclosed herein may exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers and enantiomers. Therefore, compounds claimed in the present disclosure also include racemic mixtures, single stereoisomers and optically active mixtures. It should be understood by those skilled in the art that one stereoisomer may have better efficacy and/or lower side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by chiral source synthesis, chiral catalysis, chiral resolution and other methods. Racemates can be chirally resolved by chromatographic resolution or chemical resolution. For example, chiral acid resolution reagents such as chiral tartaric acid and chiral malic acid can be added to form salts with the compounds disclosed herein, and the physical and chemical properties of the products, such as different solubility, can be used for separation.

The present invention also includes all suitable isotopic variants of the disclosed compounds. An isotopic variant is defined as a compound in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass common or predominantly present in nature. Examples of isotopes that can be introduced into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, and oxygen, such as 2H (deuterium), 3H (tritium), 11C, 13C, 14C, 15N, 17O, and 18O, respectively.

A "therapeutically effective amount" is an amount of a therapeutic agent that ameliorates a disease or symptom when administered to a patient. A "prophylactically effective amount" is an amount of a prophylactic agent that prevents a disease or symptom when administered to a subject. The amount of a therapeutic agent that constitutes a "therapeutically effective amount" or the amount of a prophylactic agent that constitutes a "prophylactically effective amount" varies with the therapeutic agent/prophylactic agent, the disease state and its severity, the age, weight, etc. of the patient/subject to be treated/prevented. A person of ordinary skill in the art can routinely determine a therapeutically effective amount and a prophylactically effective amount based on his or her knowledge and the present disclosure.

In this application, when the name of a compound is inconsistent with the structural formula, the structural formula shall prevail.

It should be understood that the term "compounds of the present disclosure" used in the present application may include, depending on the context, compounds of Formula I, solvates thereof, pharmaceutically acceptable salts thereof, stereoisomers thereof, and mixtures thereof.

As used herein, the term cationic lipid refers to a lipid that has a positive charge at a selected pH value.

Cationic liposomes easily bind to negatively charged nucleic acids, that is, they interact with negatively charged phosphate groups in nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs). LNPs are currently one of the mainstream delivery vectors.

The present invention provides a novel ionizable lipid having a structure shown in formula (I) or formula (II), wherein the hydrophilic center is composed of four tertiary amine effective nitrogens, and the hydrophobic tail is composed of four saturated or unsaturated fatty chains. The novel ionizable lipid provided by the present invention is positively charged in an acidic environment and almost uncharged in a neutral environment. By utilizing this property, nucleic acid drugs can be loaded in an acidic buffer system. After the nucleic acid drugs are loaded, they enter the neutral environment in the body, and the lipid nanoparticles are positively charged, which can effectively avoid the adsorption of nucleic acid drug complexes by plasma proteins, thereby achieving a higher delivery efficiency of nucleic acid drugs and significantly improving safety.

Another aspect of the present disclosure provides a drug complex comprising a carrier, wherein the carrier comprises a cationic lipid, and the cationic lipid comprises the above-mentioned compound of formula (I) or formula (II) or a pharmaceutically acceptable salt or stereoisomer thereof.

In one embodiment, the complex is a nanoparticle preparation, the average size of the nanoparticle preparation is 40nm to 500nm, preferably 100nm to 205nm; the polydispersity coefficient of the nanoparticle preparation is ≤50%, preferably ≤30%, more preferably ≤25%.

In one embodiment of the complex/carrier disclosed herein, the cationic lipid is selected from one or more of the compounds of formula (I) or formula (II) above or their pharmaceutically acceptable salts or stereoisomers, deuterated derivatives. In one embodiment, the cationic lipid is selected from the compounds of formula (I) above.

In another embodiment of the complex/carrier disclosed herein, the cationic lipid comprises: (a) one or more selected from the above-mentioned compound of formula (I) or its deuterated substance, pharmaceutically acceptable salt or stereoisomer; (b) one or more other ionizable lipid compounds different from (a). (b) The cationic lipid compound can be a commercially available cationic lipid or a cationic lipid compound reported in the literature.

In one embodiment, the molar ratio of the cationic lipid to the carrier is 30% to 70%, such as 35%, 45%, 50%, 55%, 60%, or 65%.

The carrier can be used to deliver active ingredients such as therapeutic or prophylactic agents. The active ingredient can be encapsulated in the carrier or combined with the carrier.

For example, the therapeutic agent or preventive agent includes one or more of a nucleic acid molecule, a small molecule compound, a macromolecular compound, a polypeptide, an antibody or a protein. The nucleic acid includes, but is not limited to, single-stranded DNA, double-stranded DNA and RNA. Suitable RNA includes, but is not limited to, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.

The carrier may include a neutral lipid, such as a neutral phospholipid. Neutral lipids in the present disclosure refer to lipids that are uncharged or exist in the form of zwitterions at a selected pH value and play an auxiliary role. The neutral lipids may adjust the fluidity of the nanoparticles to a lipid bilayer structure and improve efficiency by promoting lipid phase transition, and may also affect the specificity of the target organ.

In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1:1 to 10:1, such as about 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1. In a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 5:1.

For example, the neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols, and derivatives thereof.

The carrier component comprising the cationic lipid complex may include one or more neutral lipid phospholipids, such as one or more (poly) unsaturated lipids. The phospholipids may be assembled into one or more lipid bilayers. In general, the phospholipids may include a phospholipid portion and one or more fatty acid portions.

The neutral lipid part can be selected from the non-limiting group of the following composition: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. The fatty acid part can be selected from the non-limiting group of the following composition: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid. Also encompassed are non-natural species including natural species with modification and replacement, the modification and replacement include branching, oxidation, cyclization and alkynes. For example, phospholipids can be functionalized with one or more alkynes (e.g., one or more double bonds are replaced by triple bond alkenyl) or cross-linked with the one or more alkynes. Under appropriate reaction conditions, alkynyl may undergo copper-catalyzed cycloaddition reactions when exposed to azide. These reactions can be used to functionalize the lipid bilayer of the complex to facilitate membrane permeation or cellular recognition, or to couple the complex to useful components such as targeting or imaging moieties (eg, dyes).

The neutral lipids that can be used in these complexes can be selected from the non-limiting group consisting of: 1,2 dilinoleoyl sn glycerophosphocholine (DLPC), 1,2 dimyristoyl sn glycerophosphocholine (DMPC), 1,2 dioleoyl sn glycerophosphocholine (DOPC), 1,2 dipalmitoyl sn glycerophosphocholine (DPPC), 1,2 distearoyl sn glycerophosphocholine (DSPC), 1,2 diundecanoyl sn glycerophosphocholine (DUPC), 1 palmitoyl 2 oleoyl sn glycerophosphocholine (POPC), 1,2 dioctadecenyl sn glycerophosphocholine (18:0 Diether PC), 1 oleoyl 2 cholesteryl hemisuccinoyl sn glycerophosphocholine (OChemsPC), 1 hexadecyl sn glycerophosphocholine (C16 Lyso PC), 1,2-linolenoyl sn glycerol 3 phosphocholine, 1,2-arachidonoyl sn glycerol 3 phosphocholine, 1,2-docosahexaenoyl sn glycerol 3 phosphocholine, 1,2-dioleoyl sn glycerol 3 phosphoethanolamine (DOPE), 1,2-diphytanoyl sn glycerol 3 phosphoethanolamine (ME 16.0PE), 1,2-distearoyl sn glycerol 3 phosphoethanolamine, 1,2-dilinoleoyl sn glycerol 3 phosphoethanolamine, 1 ,2-Dilinolenoyl sn-glycerol-3-phosphoethanolamine, 1,2-Diarachidonoyl sn-glycerol-3-phosphoethanolamine, 1,2-Didocosahexaenoyl sn-glycerol-3-phosphoethanolamine, 1,2-Dioleoyl sn-glycerol-3-phosphoethanolamine (1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine

(DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-oleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.

In some embodiments, the neutral lipid comprises DSPC.In certain embodiments, the neutral lipid comprises DOPE.

In some embodiments, the neutral lipids include both DSPC and DOPE.

The carrier of the complex comprising cationic lipids may also include one or more structural lipids, such as steroids. Structural lipids in the present disclosure refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids.

In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 1:1 to 5:1, for example, about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1.

The structured lipid can be selected from, but is not limited to, the group consisting of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, alpha tocopherol, corticosteroids, and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or a combination thereof.

The carrier of the complex containing cationic lipids can also include one or more polymer conjugated lipids, such as pegylated lipids. Polymer conjugated lipids mainly refer to lipids modified by polyethylene glycol (PEG). Hydrophilic PEG stabilizes LNPs, regulates nanoparticle size by limiting lipid fusion, and increases the half-life of nanoparticles by reducing nonspecific interactions with macrophages.

In one embodiment, the polymer conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. The molecular weight of PEG-modified PEG is usually 350-5000Da.

For example, the polymer conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE PEG2000), dimyristoylglycerol 3 methoxy polyethylene glycol 2000 (DMG PEG2000) and methoxy polyethylene glycol ditetradecanoyl acetamide (ALC 0159).

In one embodiment of the complex/carrier of the present disclosure, the polymer-conjugated lipid is DM GPEG2000.

In one embodiment of the complex/carrier of the present disclosure, the carrier comprises a neutral lipid, a structural lipid and a polymer-conjugated lipid, and the molar ratio of the ionizable lipid, the neutral lipid, the structural lipid and the polymer-conjugated lipid is 50:10:38.5:1.

Active pharmaceutical ingredients

Pharmaceutically active ingredients and alternatively referred to as "active agents" may include one or more therapeutic and/or prophylactic agents. In one embodiment, the mass ratio of the ionizable lipid to the therapeutic or prophylactic agent is 0.1:1-1000:1.

The therapeutic agent or preventive agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein.

For example, the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

The vectors disclosed herein can deliver therapeutic agents and/or prophylactic agents to mammalian cells or organs, and thus the disclosure also provides methods for treating diseases or disorders in mammals in need thereof, which methods comprise administering a complex or pharmaceutical composition comprising a therapeutic agent and/or prophylactic agent to the mammal and/or contacting mammalian cells with the complex or pharmaceutical composition.

The therapeutic and/or preventive agent can be a substance that causes a desired change in a cell or organ or in other body tissues or systems after delivery to the cell or organ. Such species can be used to treat one or more diseases, disorders or conditions. In some embodiments, the therapeutic and/or preventive agent is a small molecule drug that can be used to treat a specific disease, disorder or condition. Examples of drugs that can be used for the complex include, but are not limited to, anti-neoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate and streptozotocin), anti-tumor agents (e.g., actinomycin D), D), vincristine, vinblastine, cytosine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blocking agents (e.g., propranolol, timolol, and labetalol), antihypertensive agents (e.g., clonidine and hydralazine), ne), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics/antibacterials (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), B)), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma drugs, vitamins, sedatives and imaging agents.

In some embodiments, the therapeutic and/or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and/or another therapeutic and/or prophylactic agent.Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC 1065), and analogs or homologues thereof. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. Vaccines include compounds and preparations that can provide immunity to one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis, and may include mRNA encoding infectious disease-derived antigens and/or epitopes. Vaccines may also include compounds and preparations that guide an immune response against cancer cells and may include mRNA encoding tumor cell-derived antigens, epitopes, and/or new epitopes. Compounds that cause an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, vaccines and/or compounds that can cause an immune response are administered intramuscularly by including a complex according to formula (I). Other therapeutic and/or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,

and 5-fluorouracil (dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, razepamycin (CC 1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin), n)) and doxorubicin), antibiotics such as dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and antimitotic agents such as vincristine, vinblastine, taxol, and maytansines.

In other embodiments, the therapeutic and/or prophylactic agent is a protein. Therapeutic proteins that can be used in the nanoparticles of the present disclosure include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony stimulating factor (G CSF), granulocyte macrophage colony stimulating factor (GM CSF), factor VIR, luteinizing hormone releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (eg, ribonucleic acid or deoxyribonucleic acid).

The broadest meaning of the term "polynucleotide" includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used according to the present disclosure include, but are not limited to, one or more of: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger RNA (mRNA), its hybrid; RNAi inducing factor; RNAi factor; siRNA; shRNA; miRNA; antisense RNA; ribozyme; catalytic DNA; RNA that induces triple helix formation; aptamer, etc. In some embodiments, the therapeutic agent and/or preventive agent is RNA. The RNA that can be used in the complex and method described herein can be selected from the group consisting of, but not limited to, shortmer, antagomir, antisense RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA) and mixtures thereof. In certain embodiments, RNA is mRNA.

In certain embodiments, the therapeutic and/or preventive agent is mRNA. mRNA can encode any polypeptide of interest, including any natural or non-natural polypeptide or otherwise modified polypeptide. The polypeptide encoded by mRNA can have any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by mRNA can have a therapeutic effect when expressed in a cell.

In other embodiments, therapeutic and/or preventive agents are siRNA. siRNA can selectively reduce the expression of a gene of interest or down-regulate the expression of the gene. For example, the selection of siRNA can make the gene silencing relevant to a specific disease, disease or condition after the compound comprising the siRNA is administered to a subject in need. siRNA can include a sequence complementary to the mRNA sequence of a gene or protein of interest to encode. In some embodiments, siRNA can be an immunomodulatory siRNA.

In certain embodiments, the therapeutic and/or preventive agent is sgRNA and/or cas9 mRNA. sgRNA and/or cas9 mRNA can be used as a gene editing tool. For example, the sgRNAcas9 complex can affect the mRNA translation of a cell gene.

In some embodiments, the therapeutic and/or preventive agent is shRNA or its encoding vector or plasmid. shRNA can be produced inside the target cell after the appropriate construct is delivered to the nucleus. The construct and mechanism associated with shRNA are well-known in the relevant field.

The complex/carrier of the present disclosure can deliver active ingredients, including therapeutic agents or prophylactic agents, to a subject or patient. The therapeutic agent or prophylactic agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein. Therefore, the complex of the present disclosure can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs. Due to the wide variety of the above-mentioned therapeutic agents or prophylactic agents, the complex of the present disclosure can be used to treat or prevent a variety of diseases or conditions.

In one embodiment, the disease or disorder is characterized by a malfunction or aberrant protein or polypeptide activity.

For example, the disease or disorder is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

In one embodiment, the infectious disease is selected from the group consisting of diseases caused by coronavirus, influenza virus, or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, and various herpes.

The complex may include one or more components other than those described in the preceding section. For example, the complex may include one or more hydrophobic small molecules, such as vitamins (eg, vitamin A or vitamin E) or sterols.

The complex may also include one or more permeability enhancing molecules, carbohydrates, polymers, surface modifiers or other components. The permeability enhancing molecules may be, for example, molecules described in U.S. Patent Application Publication No. 2005/0222064. The carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

Surface-altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiol

The surface-altering agent can be placed within and/or on the surface of the nanoparticles of the complex (e.g., by coating, adsorption, covalent attachment or other methods).

The complex can also include one or more functionalized lipids. For example, lipids can be functionalized with alkynyl groups, which may undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized with one or more groups that can effectively promote membrane penetration, cell recognition or imaging in this way. The surface of the complex can also be coupled to one or more useful antibodies. Functional groups and conjugates that can be used for targeted cell delivery, imaging and membrane penetration are well known in the art.

In addition to these components, the complex can further form a pharmaceutical composition. For example, the pharmaceutical composition can include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as but not limited to one or more solvents, dispersion media, diluents, dispersing aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, adhesives, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc. Excipients such as starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and/or combinations thereof.

In some embodiments, a pharmaceutical composition comprising one or more lipids described herein may also include one or more adjuvants, such as glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxyribonucleotides (e.g., class A or class B), poly (I:C), aluminum hydroxide, and Pam3CSK4.

Pharmaceutical compositions of the present disclosure can be made into preparations in the form of solid, semisolid, liquid or gas, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. Pharmaceutical compositions of the present disclosure can be prepared by methods well known in the pharmaceutical field. For example, sterile injection solutions can be prepared by mixing the required amount of therapeutic agents or preventive agents with the required various other ingredients mentioned above into suitable solvents such as sterile distilled water, and then filtering and sterilizing. Surfactants can also be added to promote the formation of uniform solutions or suspensions.

For example, the complexes, pharmaceutical compositions of the present disclosure may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

In one embodiment, the pharmaceutical composition is administered intravenously.

The complexes and pharmaceutical compositions of the present disclosure are administered in a therapeutically effective amount, which may vary not only with the specific agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be determined at the discretion of the attending physician or clinician.

The present invention is further described below in conjunction with the examples, but the present invention is not limited to the following examples. If no specific conditions are specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

Example 1 Synthesis of ionizable lipid compounds

1. Synthesis of Compound I-1

The synthetic route is as follows:

(1) Synthesis of Compound 1:

Add anhydrous piperazine 2a (1.0 equiv.) and anhydrous potassium carbonate (5.0 equiv.) to an eggplant-shaped flask and dissolve in ultra-dry dichloromethane. Separately, take acryloyl chloride 1b (3.0 equiv.) and dissolve in ultra-dry dichloromethane and add to a dropping funnel. In an ice-water bath, add the acryloyl chloride solution in the dropping funnel dropwise to the eggplant-shaped flask. After reacting in an ice-water bath for 6 hours, stop the reaction and remove the reaction solvent by rotary evaporation. Separate the crude product by silica gel column chromatography with an eluent of DCM/MeOH=40:1 (V/V) to purify a white solid product, namely compound 4, with a yield of 87%. Take an appropriate amount of the product and dissolve it in deuterated chloroform for nuclear magnetic resonance analysis.

(2) Synthesis of Compound 2:

Compound 4 (1.0 equiv.) and tert-butyl 2-(methylamino)ethylcarbamate 2c (2.0 equiv.) were dissolved in anhydrous ethanol and refluxed in an oil bath at 80°C for 36 h. After the reaction, the reaction solvent was removed by rotary evaporation and silica gel column chromatography was performed with an eluent of DCM/MeOH=20:1-15:1 (V/V) (1% NH3·H2O) to purify a white solid product, namely compound 5, with a yield of 71%. An appropriate amount of the product was dissolved in deuterated chloroform for nuclear magnetic resonance analysis.

(3) Synthesis of Compound I-1:

Compound 2 (2 mmol, 1.0 equiv.), 4 mL TFA and 4 mL DCM were mixed and stirred at room temperature for 2 h to remove the Boc protecting group. TFA/DCM was removed by rotary evaporation to obtain a light yellow oil, which was dissolved in isopropanol, and anhydrous potassium carbonate was added. The remaining TFA was neutralized by stirring at room temperature until the isopropanol solution was alkaline. 1,2-epoxytetradecane 3a (12 mmol, 6.0 equiv.) was added to the solution and refluxed at 90 ° C in an oil bath for 36 h. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and the product was separated and purified by silica gel column chromatography. The eluent was DCM/MeOH = 25:1-15:1 (V/V) (1% NH3·H2O). The eluent was dried to obtain a light yellow semi-solid, i.e., the final product I-1, with a yield of 35%.

Take an appropriate amount of the product and dissolve it in deuterated chloroform for NMR analysis. Take an appropriate amount of the product and dissolve it in deuterated chloroform for NMR analysis. The results are as follows: 1H NMR (400MHz, CDCl3) δ (ppm) = 5.19 (s, 2H), 3.75–3.42 (m, 8H), 3.23 (d, J = 5.0, 4H), 2.88–2.16 (m, 30H), 1.47–1.16 (m, 92H), 0.89 (t, J = 6.8, 12H). The results show that I-1 was successfully synthesized.

2. Synthesis of Compound II-1

The synthetic route is as follows:

(1) The syntheses of Compound 1 and Compound 2 are similar to those described in the synthesis of Compound I-1.

(2) Synthesis of compound II-1: Compound 2 (2 mmol, 1.0 equiv.), 4 mL TFA and 4 mL DCM were mixed and stirred at room temperature for 2 h to remove the Boc protecting group. TFA/DCM was removed by rotary evaporation to obtain a light yellow oil, which was dissolved in acetonitrile, and anhydrous potassium carbonate was added. The remaining TFA was neutralized by stirring at room temperature until the acetonitrile solution became alkaline. Tetradecane bromide 3b (12 mmol, 6.0 equiv.) was added to the solution and refluxed at 90°C in an oil bath for 48 h. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and the product was separated and purified by silica gel column chromatography. The eluent was DCM/MeOH = 25:1-20:1 (V/V) (1% NH3·H2O). The eluent was dried by rotary evaporation to obtain a light yellow semi-solid, i.e., the final product II-1, with a yield of 34%.

An appropriate amount of the product was dissolved in deuterated chloroform for NMR analysis, and the results were as follows: 1H NMR (400MHz, CDCl3) δ (ppm) = 3.57 (dd, J = 48.8, 18.3, 8H), 2.86–2.21 (m, 30H), 1.55–1.18 (m, 96H), 0.89 (t, J = 6.8, 12H). The results showed that II-2 was successfully synthesized.

Example 2 Synthesis of positive control compound

The positive control of the present invention is selected from one or more of C12-200 (MW = 1136.96), SM-102 (MW = 710.18), ALC-0315 (MW = 766.29), and DLin-MC3-DMA (MC3, MW = 642.09). Among them, C12-200 and MC3 are classic mRNA delivery lipids, SM-102 is an ionizable lipid used in Moderna's marketed mRNA vaccine products, and ALC-0315 is an ionizable lipid used in CureVac and BioNTeach's marketed mRNA vaccine products.

Its structure is:

The synthetic route was reported in the literature and was commissioned to a third party company for synthesis or obtained commercially.

Example 3 Synthesis of other exemplary compounds of the present invention

The compound of the present invention, in terms of its overall structure, can be divided into a hydrophilic center composed of four tertiary amines (N) and a hydrophobic tail composed of four saturated fatty chains (T). The positive charge and hydrophilic and hydrophobic properties of each ionizable lipid molecule are comparable, ensuring that their mRNA loading capacities are comparable. The differences in fine structures constitute the differences in specific compounds.

The synthesis of the compounds of the present invention is mainly divided into two steps: first, constructing the 4N positive center through amide formation and Michael addition; second, connecting the 4T hydrophobic tail through epoxy ring opening reaction, alkylation reaction or Michael addition reaction. Other exemplary compounds disclosed in the present invention are prepared by similar routes described in Example 1 using different starting materials.

The structural formulas of other compounds of the present invention are as follows:

Compared with other ionizable lipid molecules disclosed in the art, such as the synthetic route of C12-200 reported in the literature, which requires the use of a relatively dangerous Raney Nickel hydrogenation reduction reaction, the synthetic route of the compound of the present invention is simple and easy, the structural characterization is clear, and the compound yield is sufficient.

Example 4 Preparation of LNPs@mRNA and investigation of its pharmaceutical properties

Furthermore, the inventors constructed an mRNA delivery system LNPs@mRNA based on the ionizable lipids prepared in Example 1, and investigated its formulation properties such as particle size, potential, encapsulation efficiency, TEM, etc. to evaluate its formulation properties.

1. Preparation of LNPs@mRNA

(1) Solution preparation: Dissolve ionizable lipids, DOPE (or DSPC), Chol, and DMG-PEG2000 in anhydrous ethanol to make the concentration of ionizable lipids 10 mg/mL. The ionizable lipids prepared in Example 1 of the present invention and the prescriptions of C12-200, MC3, SM-102, and ALC-0315 have a molar ratio of ionizable lipids, DSPC, cholesterol (Chol), and DMG-PEG2000 of 50:10:38.5:1.5. mRNA is diluted to an appropriate concentration with PBS buffer (prepared with RNase-free water) for use. In order to ensure that the performance of the positive control is maximized, the C12-200, MC3, SM-102, and ALC-0315 formulas are selected from the preferred ratios disclosed in known literature with reference to the prior art.

(2) LNP preparation: The ionizable lipid solution obtained in step (1) was mixed with the mRNA solution. Microfluidic process parameters: the volume ratio of the ethanol phase to the water phase was 1:3, and the flow rate was 9 mL/min.

(3) Ultrafiltration: Dilute the initial LNP preparation 25 times with PBS buffer and ultrafilter to the initial volume through an ultrafiltration cup to obtain the final LNP preparation. During the ultrafiltration process, the ethanol in the initial preparation is removed. Ultrafiltration process parameters: filter membrane 100 kDa, air pressure 0.2 MPa, rotation speed 100-200 rpm.

2. Investigation of the pharmaceutical properties of LNPs@mRNA

Particle size potential determination: Take an appropriate amount of LNPs@mRNA preparation and dilute it 10 times with purified water, and use Malvern nanoparticle size potential analyzer to detect its particle size, particle size distribution, ζ potential and other preparation properties.

Transmission electron microscopy characterization: Dilute LNPs@mRNA with purified water to make the mass concentration of nanoparticles 2 mg/mL, carefully drop it onto the TEM-specific copper grid, let it stand for 2 minutes, then use filter paper to absorb the excess liquid, add 2% phosphotungstic acid negative stain for 3 minutes, then absorb the excess dye, blow dry with an ear bulb, load the sample for detection and take pictures.

Encapsulation efficiency detection: Solution preparation: Dilute LNPs@mRNA with RNase-free water to an mRNA concentration of 0.1μg/μL. Under light-proof conditions, take an appropriate amount of RiboGreen dye and dilute it 400 times with 1×TE buffer, and store it in the dark for use. Membrane permeation: Add 25μL of diluted mRNA preparation and 75μL of 1×TE buffer to the sample well, and add 25μL of diluted mRNA preparation, 25μL of 1×TE buffer and 50μL of 1% Triton to the total mRNA well. Incubate the above 96-well plate at 37℃ in the dark for 10min. Color development: After 10min, add 100μL of diluted RiboGreen dye to each well in the dark and shake it to make it uniform. Microplate reader detection conditions: excitation wavelength 485nm, emission wavelength 528nm. Calculation formula: Encapsulation efficiency (%) = (1-sample well fluorescence value/total mRNA fluorescence value) × 100%.

The experimental results are shown in Figure 1: the LNPs prepared from different ionizable lipids prepared in Example 1 of the present invention have similar particle sizes, with average particle sizes of about 100 nm, Di(90) within 500 nm, and PDI below 0.3, indicating that the LNP particle size distribution is uniform. The formulation potential is about 5 mV.

The LNPs@mRNA prepared from different ionizable lipids prepared in Example 1 of the present invention are solid spheres with a particle size of about 100 nm under TEM. A fingerprint-like structure is found, which is a representative feature of the LNP nanostructure.

The encapsulation efficiency test results are shown in FIG2 . Compared with the positive controls MC3, ALC-0315, and SM-102, the encapsulation efficiency of LNPs@mRNA prepared by the ionizable lipid of Example 1 of the present invention is significantly higher than that of the positive controls.

The above test results show that the LNPs@mRNA prepared from the ionizable lipids of the present invention has good nanoformulation properties, especially the encapsulation efficiency is better than that of the positive controls MC3, ALC-0315, and SM-102.

Example 5 Cell transfection and cytotoxicity evaluation of LNPs@FLuc mRNA

The above examples verify the pharmaceutical properties of the ionizable lipid nanoparticles provided by the present invention. Furthermore, the inventors used EGFP mRNA and FLuc mRNA as reporter genes to evaluate the in vitro effect and safety of the LNPs@mRNA provided by the present invention through transfection experiments and toxicity experiments on HEK293T and DC2.4.

LNPs@EGFP mRNA transfection experiment: HEK293T and DC2.4 cells were plated. Then, LNPs@EGFP mRNA, positive control preparations MC3-LNPs@EGFP mRNA, ALC-0315-LNPs@EGFP mRNA, and SM-102-LNPs@EGFP mRNA were prepared according to the method described in Example 4; the mRNA concentration of the controlled administration preparation was 0.02 μg/μL, and 50 μL was administered to each well of the 24-well cell plate, i.e., 1 μg/well, and continued to be cultured in an incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, the GFP positivity rate and mean fluorescence intensity (MFI) were detected by flow cytometry.

LNPs@FLuc mRNA transfection experiment: HEK293T and DC2.4 cells were plated. LNPs@FLuc mRNA, positive control preparations MC3-LNPs@FLuc mRNA, ALC-0315-LNPs@FLuc mRNA, and SM-102-LNPs@FLuc mRNA were prepared according to the method described in Example 4; the mRNA concentration of the controlled administration preparation was 0.02 μg/μL, and 50 μL was administered to each well of the 24-well cell plate, i.e., 1 μg/well, and continued to be cultured in an incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, 10 μL of substrate luciferin (luciferin potassium salt, 15 mg/mL) was added to each well, and the bioluminescence was detected by an ELISA instrument after standing for 10 minutes away from light.

Cytotoxicity experiment: LNPs@FLuc mRNA and positive control preparation C12-200-LNPs@FLuc mRNA were selected to investigate the MTT toxicity of DC2.4 cells. The key experimental parameters are as follows: 10 μL was administered to each well of a 96-well plate; incubated in an incubator at 37°C and 5% CO2 for 24 hours; 5 mg/mL MTT solution, 40 μL of the above MTT solution was added to each well, and incubated in an incubator at 37°C and 5% CO2 for 4 hours.

The results of the cell transfection experiment are shown in FIG3 : on HEK-293T and DC2.4, the II-1@EGFP mRNA of the present invention has a stronger mRNA expression ability at the cellular level than the positive controls MC3 and ALC-0315.

The results of the MTT cytotoxicity experiment are shown in Figure 4: LNPs@FLuc mRNA had no obvious toxicity to DC2.4 cells after incubation for 24 h.

Example 6 In vivo expression and distribution of LNPs@mRNA

Furthermore, the ability of the LNPs@mRNA of the present invention to deliver mRNA in vivo was investigated.

In vivo expression investigation by intravenous injection: LNPs@Fluc mRNA preparation and positive control preparation C12-200@Fluc mRNA were prepared according to the method described in Example 4, and the mRNA concentration of the preparation was adjusted to 0.05 mg/mL with PBS solution (prepared with RNase-free water). At the same time, the osmotic pressure of the preparation was adjusted to isotonicity, and 200 μL, i.e. 10 μg FLuc mRNA/mouse, was injected into the tail vein of each C57BL/6 mouse, 3 mice per group, and PBS was used as a negative control. After administration, the mice were kept on a normal diet. 6 hours after administration, 200 μL of substrate solution (15 mg/mL, luciferin potassium salt) was injected intraperitoneally. The timing started after the injection of the substrate, and euthanasia was performed 10 minutes later. The heart, liver, spleen, lungs, and kidneys were quickly dissected, and the bioluminescence intensity of each organ in vitro was detected in the IVIS instrument, with an exposure time of 60 s. After imaging, the Total flux of each organ was counted.

In vivo expression investigation by intramuscular injection: LNP@Fluc mRNA, positive control preparations MC3-LNPs@Fluc mRNA, ALC-0315-LNPs@Fluc mRNA, and SM-102-LNPs@Fluc mRNA were prepared according to the method described in Example 4, and the mRNA concentration of the preparation was adjusted to 0.1 mg/mL with a PBS solution (prepared with RNase-free water). At the same time, the osmotic pressure of the preparation was adjusted to isotonicity, and 200 μL, i.e., 20 μg FLuc mRNA/mouse, was injected into the hind leg muscle of each BALB/c mouse, 3 mice per group, and PBS was used as a negative control. After administration, the mice were kept on a normal diet. 8 hours after administration, 200 μL of substrate solution (15 mg/mL, potassium salt of fluorescein) was injected intraperitoneally, i.e., 3 mg/mouse. The timing started after the injection of the substrate, and the mice were placed in the gas anesthesia device 10 minutes later. After complete anesthesia, the bioluminescence intensity of the whole body of the living body was detected in the IVIS instrument, and the exposure time was 60 s. After recovery, the normal diet was maintained. The above anesthesia and testing were repeated 24 hours and 48 hours after administration. The total flux of the whole body was calculated and the luminescence intensity-time curve was drawn.

The in vivo expression results of the intravenous injection route show (as shown in Figures 5 and 6): Unexpectedly, compared with the control C12-200, the LNPs@mRNA of the present invention is unexpectedly highly expressed in the lungs via the intravenous injection route, which will be beneficial for nucleic acid drugs targeting the lungs; at the same time, there is also a certain expression level in the spleen, suggesting that the LNPs@mRNA of the present invention can also be used for nucleic acid drugs via intravenous administration.

The above test results suggest that the LNPs of the present invention can be further used for the preparation of nucleic acid drugs targeting lung organs.

Example 7 Immune anti-tumor effect of LNPs@OVA mRNA

An E.G7-OVA (Ovalbumin) tumor model was further constructed, and OVA-mRNA was selected as a model antigen to evaluate the efficacy of LNPs@mRNA as an mRNA tumor vaccine delivery system, thereby screening out the mRNA tumor vaccine delivery system with high tumor inhibition effect in the present invention, and investigating the potential influence of the ionizable lipid chemical structure of the present invention on the effect of LNPs@mRNA tumor vaccine.

Among them, the method for establishing the E.G7-OVA tumor model is as follows: When E.G7-OVA cells are cultured to the logarithmic growth phase, the cells are collected, washed with sterile PBS, centrifuged and the supernatant is discarded, and the cells are resuspended with sterile PBS to adjust the cell concentration to 10 ⁷ /mL. Each male C57BL/6 mouse is subcutaneously inoculated with 100 μL of E.G7-OVA cells in the right ribs, that is, 10 ⁶ /mouse. The growth status of the mice and the size of the subcutaneous tumor are observed. After 5 days of inoculation, a small tumor visible to the naked eye is formed, and the immunotherapy experiment can be carried out. Compared with normal mice, there is no significant difference in the activity, appetite, defecation and other reactions of tumor-bearing mice. References: Luo, X.; Li, B.; Zhang, .; Siegwart, DJ, Theranostic dendrimer-based lipid nanoparticles containing PEGylated BODIPY dyes for tumor imaging and systemic mRNA delivery in vivo. JControl Release 2020, 325, 198-205.

E. G7-OVA cells (mouse T lymphoma cells) were purchased from ATCC and cultured in 1640 complete medium (containing 10% FBS and 1% P/S) at 37°C in a 5% CO2 incubator. Cells were passaged when the cell density reached 80-90%.

The experimental scheme is as follows: LNPs@OVAmRNA was prepared by microfluidic control agent technology, and its activation effect on BMDC in vitro and its effect on cytokine secretion were investigated.

BMDC maturation and activation investigation method: cell plating. LNPs@OVAmRNA and positive control preparation C12-200-LNPs@OVAmRNA were prepared according to the method described in Example 4, and the mRNA concentration of the administration preparation was controlled to be 0.05μg/μL. 20μL was administered to each well, i.e. 1μg/well, and incubated in an incubator at 37°C and 5% CO2 for 24h. Staining and detection: After the incubation, the cells in the 24-well plate were collected into a flow tube, and the cells were washed twice with pre-cooled 1mL PBS at 1500rpm for 5min. 100μL of the above cell suspension and 1μL Anti-mouse CD16/32 were added to each flow tube, mixed and incubated at 4°C for 15min to block nonspecific binding. Add 1 μL of PE-anti-mouse CD11c, FITC-anti-mouse CD80, APC/Cy7-anti-mouse CD86 and APC-anti-mouse H-2Kb bound to SIINFEKL flow cytometry antibodies to the corresponding flow tubes and incubate at 4°C for 40 min. After incubation, wash the cells twice with 1 mL of pre-cooled PBS at 1500 rpm for 5 min, then add 300 μL of PBS and mix well, and detect the maturation and activation state of BMDCs by flow cytometry.

Cytokine detection: Detection of LNPs@OVA mRNA vaccine stimulating BMDC maturation and activation. At the same time, after the incubation, collect the cell supernatant and dilute it 5 times with the sample diluent in the ELISA kit for testing. Dilute the standard sample and enzyme-labeled antibody according to the kit instructions, establish a standard working curve of TNF-α, and detect the level of BMDC secretion of TNF-α in the cell supernatant by the double antibody sandwich method.

The results showed that the mRNA vaccine based on piperazine-containing ionizable lipids and its derivatives or analogs of the present invention was superior to the positive control C12-200 in terms of BMDC maturation and activation (as shown in FIG. 7 ) and the secretion of cytokine TNF-α (as shown in FIG. 8 ).

It should be noted that, in the drawings of this specification, unless otherwise specified, ns or no mark means no significant difference; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

It should be noted that the specific features, structures, materials or characteristics described in this specification may be combined in any one or more embodiments in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments and features of the different embodiments described in this specification without contradiction.

Claims (21)

1. The compound shown in formula I, or its pharmaceutically acceptable salt, isomer, deuterated substance or prodrug: Wherein, a is 1, 2 or 3; X ₁ and X ₂ are independently selected from N or C; L ₁ , L ₂ , L ₃ , L ₄ are independently selected from -R _e CH(OH)-, -R _e C(=O)-, -R _e C(=O)O-, - R _e OC(=O)-, -R _e C(=O)S-, -R _e SC(=O)-, -R _e C(=O)NR _a -, -R _e NR _a C(= O)-,- R _e NR _a C(=O)O-, -R _e OC(=O)NR _a -, -R _e O-, -R _e -OO-, -R _e S-, -R _e -SS-, -R _e -SS- S-, -R _e CH(OH)CH ₂ O-, -R _e CH(OH)CH ₂ S- or absent, R _e is Or does not exist, k is an integer greater than 1, R _a is -H, substituted or unsubstituted alkyl; R ₁ , R ₂ , R ₃ , R ₄ are independently selected from C ₁ -C ₃₀ straight chain alkyl, C ₁ -C ₃₀ branched alkyl, C ₂ -C ₃₀ straight chain alkenyl, C ₂ -C ₃₀ Branched alkenyl, C ₂ -C ₃₀ straight chain alkynyl or C ₂ -C ₃₀ branched alkynyl; G ₁ , G ₂ , G ₃ , G ₄ are independently selected from -R _c -, -R _c CH(OH)R _d -, -R _c C(=O)R _d -, - R _c C(=O)OR _d -, -R _c OC(=O)R _d -, -R _c C(=O)SR _d -, -R _c SC(=O)R _d -, - R _c C(=O)N(R _b )R _d -, -R _c N(R _b )C(=O)R _d -, -R _c N(R _b )C(=O)OR _d -, - R _c OC(=O)N(R _b )R _d -, -R _c OR _d -, -R _c -OOR _d -, -R _c SR _d -, -R _c -SSR _d -, -R _c - SSS- R _d - or absent, R _b is -H, substituted or unsubstituted alkyl, R _c , R _d are independently selected from Or does not exist, n is an integer greater than 1; R ₅ and R ₆ are independently selected from -H, substituted or unsubstituted alkyl; Q ₁ and Q ₂ are independently selected from O or S. 2. The compound according to claim 1, characterized in that, it has the structure shown in formula II, or is a pharmaceutically acceptable salt, stereoisomer, deuterated substance or prodrug of the structure shown in formula II, 3. The compound according to claim 1 or 2, wherein said k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, the k is 1. 4. The compound according to claim 1 or 2, wherein said L1, L2, L3, L4 are independently selected from -CH(OH)-, -C(=O)-, -CH2C(=O )O-, -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -CH2C(=O)NRa-, -C( =O)NRa-, -NRaC(=O)-, -NRaC(=O)O-, -OC(=O)NRa-, -CH2O-, -O-, -CH2-O-O-, -CH2S-, -S-, -CH2-S-S-, -CH2-S-S-S-, -CH(OH)CH2O-, -CH(OH)CH2S- or absent, Ra is -H, substituted or unsubstituted alkyl; Preferably, said L ₁ , L ₂ , L ₃ , and L ₄ are independently selected from -C(=O)-, -C(=O)NR _a -, -CH ₂ C(=O)NR _a -, -NR _a C(=O)-, -C(=O)O-, -CH ₂ C(=O)O-, -OC(=O)-, -CH ₂ O-, -O-, -CH ₂ S-, -S-, -CH(OH)-, -CH(OH)CH ₂ O-, -CH(OH)CH ₂ S- or absent, R _a is -H or unsubstituted alkyl. 5. The compound according to any one of claims 1-4, wherein said R _a is -H or unsubstituted C ₁ -C ₆ alkyl; Preferably, said R _a is -H. 6. The compound according to claim 1 or 2, wherein said L ₁ , L ₂ , L ₃ , and L ₄ are independently selected from -C(=O)-, -C(=O)NH- , -CH ₂ C(=O)NH-, -C(=O)O-, -CH ₂ C(=O)O-, -CH ₂ O-, -CH ₂ S-, -CH(OH)- , -CH(OH)CH ₂ O- or absent; Preferably, L ₁ , L ₂ , L ₃ , L ₄ are independently selected from -C(=O)NH-, -C(=O)O-, -CH(OH)-, -CH(OH)CH ₂ O- or not present. 7. The compound according to any one of claims 1 to 6, wherein said L _{and L} are selected from the same group and/or L _{and L} are selected from the same group; Optionally, said L ₁ and L ₃ are selected from the same group and/or L ₂ and L ₄ are selected from the same group; Preferably, said L ₁ , L ₂ , L ₃ and L ₄ are selected from the same group. 8. The compound according to claim 1 or 2, wherein said R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from C ₁ -C ₃₀ straight chain alkyl, C ₂ -C ₃₀ straight chain Alkenyl, C ₂ -C ₃₀ straight chain alkynyl; Preferably, the R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from unsubstituted C ₁ -C ₃₀ linear alkyl groups; Preferably, the R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from unsubstituted C ₈ -C ₁₈ linear alkyl groups; Preferably, the R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from unsubstituted C ₁₀ -C ₁₄ linear alkyl groups. 9. The compound according to any one of claims ₁ to 8, wherein R and _R are selected from the same group and/or _R and _R are selected from the same group; Optionally, said R ₁ and R ₃ are selected from the same group and or R ₂ and R ₄ are selected from the same group; Preferably, the R ₁ , R ₂ , R ₃ and R ₄ are selected from the same group. 10. The compound according to claim 1 or 2, wherein said G ₁ , G ₂ , G ₃ , G ₄ are independently selected from or absent, n3 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, said G ₁ and G ₂ are independently selected from -CH ₂ - or -CH ₂ CH ₂ -; Preferably, the G ₃ and G ₄ do not exist. 11. The compound according to claim 1 or 2, wherein said R ₅ and R ₆ are independently selected from -H, unsubstituted C ₁ -C ₆ alkyl or -OH substituted C ₁ -C ₆ alkyl; Preferably, the R ₅ and R ₆ are independently selected from -H, methyl, ethyl, propyl, hydroxymethyl, hydroxyethyl or hydroxypropyl; Preferably, the R ₅ and R ₆ are selected from the same group. 12. A compound, characterized in that it has one of the following structures; , A pharmaceutically acceptable salt, stereoisomer, deuterated substance or prodrug thereof. 13. The compound according to any one of claims 1 to 12, or the use of its pharmaceutically acceptable salt, isomer, deuterated substance or prodrug in the preparation of a drug delivery carrier; further, the active ingredient of the drug Optionally selected from at least one of nucleic acid, small molecule drug, protein drug, polypeptide; preferably, the active ingredient of the drug is selected from nucleic acid; preferably, the drug has heart, liver, spleen, lung or kidney targeting Sex; preferably, the drug targets the lung, spleen. 14. A drug delivery carrier, characterized by comprising the compound according to any one of claims 1-12, or a pharmaceutically acceptable salt, stereoisomer, deuterated compound or prodrug thereof. 15. A drug complex, characterized in that it comprises a carrier and an active ingredient, the carrier is connected to the active ingredient, the carrier includes a cationic lipid, and the cationic lipid includes any one of claims 1-12. The compound, or its pharmaceutically acceptable salt, stereoisomer, deuterated substance or prodrug; Optionally, the active ingredient of the drug is selected from nucleic acid, small molecule drug, protein drug, polypeptide, At least one of the antibodies; preferably, the active ingredient of the drug is selected from nucleic acids; optionally, the nucleic acids are selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, and aptamers; optionally , the nucleic acid is mRNA; optionally, the delivery carrier is linked to the mRNA through an ionic bond; further, the drug targets the lung and spleen; Optionally, the preparation form of the drug complex is selected from one of lipid nanoparticles LNP, PLGA nanoparticles, micelles, liposomes, core-shell nanoparticles, and polymer nanoparticles; Preferably, the preparation form of the drug complex is lipid nanoparticle LNP. 16. The drug complex according to claim 15, wherein the pharmaceutical composition also contains at least one excipient in neutral phospholipids, steroids, and pegylated lipids; Optionally, the neutral phospholipid is selected from DOPE, DSPC, DOPC, DSPE, DMPC, At least one of DMPE, DPPC, DPPE, DEPC, HSPC, POPC; Preferably, the neutral phospholipid is DOPE; Optionally, the steroid is selected from cholesterol, sitosterol, stigmasterol, lanosterol, ergosterol At least one of alcohol and fucosterol; Preferably, the steroid is cholesterol; Optionally, the pegylated lipid is selected from at least one of DMG-PEG and DSPE-PEG; Preferably, the PEGylated lipid is DMG-PEG2000. 17. The drug complex as claimed in claim 15, characterized in that, the compound, or its pharmaceutically acceptable salt, isomer, deuterium or prodrug, has a molar ratio of 1:10～10:1. 18. The drug complex as claimed in claim 15, characterized in that, the compound, or its pharmaceutically acceptable salt, isomer, deuterium or prodrug and the pegylated lipid The molar ratio is 5:1～1000:1; Preferably, the molar ratio of the compound, or its pharmaceutically acceptable salt, isomer, deuterated substance or prodrug to the pegylated lipid is 10:1-20:1. 19. The drug complex according to claim 15, wherein the nucleic acid is selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, and aptamers; Preferably, the nucleic acid is mRNA. 20. The use of the drug complex according to any one of claims 15 to 19 in the preparation of medicines, which are used to treat or prevent diseases; Optionally, the disease is heart, liver, spleen, lung or kidney-related diseases; Preferably, the disease is spleen or lung related disease; Optionally, the disease is an infectious disease, cancer and proliferative disease, genetic disease, autoimmune disease, diabetes, neurodegenerative disease, cardiovascular and renovascular disease and metabolic disease; Preferably, the infectious disease is selected from: diseases caused by coronavirus, influenza virus or HIV virus, infantile pneumonia, Rift Valley fever, yellow fever, rabies, or multiple herpes; Preferably, the cancer is a solid tumor; Preferably, the cancer is liver cancer or lung cancer. 21. The use according to claim 20, characterized in that the drug treats or prevents diseases by presenting antigens and/or activating immune responses.