CN118178664A - A lipid material for nucleic acid delivery and its use - Google Patents

Abstract

The present invention provides a lipid material for nucleic acid delivery, wherein the lipid material comprises a compound having an I structure. The present invention also provides a use of a lipid material for nucleic acid delivery, for preparing a therapeutic drug selected from one or more of infectious diseases, tumor diseases, congenital genetic diseases and immune diseases. Through the lipid material provided by the present invention, a high-efficiency and low-toxic nucleic acid drug carrier strategy is adopted, and a novel ionizable lipid and an auxiliary lipid material are mixed to encapsulate nucleic acid drugs, thereby achieving efficient and safe delivery of nucleic acid drugs in vivo and improving the drugability of nucleic acid drugs.

Description

A lipid material for nucleic acid delivery and its use

Technical Field

The present invention relates to a pharmaceutical lipid material, in particular to a lipid material for nucleic acid delivery and application thereof.

Background Art

In recent years, nucleic acid drugs have received widespread attention due to their advantages such as small dosage, strong biological effects, and wide application range. At present, nucleic acid drugs are gradually being used in the treatment of polygenic diseases, such as genetic diseases, malignant tumors, metabolic diseases, and infectious diseases. For example, the mRNA vaccine that has been approved for marketing introduces mRNA encoding viral antigens into the antigen-presenting cells of the human body, thereby stimulating the body to produce neutralizing antibodies and exerting immunological effects; in addition, small nucleic acid drugs such as siRNA mainly participate in the formation of RNA-induced silencing complex (RISC) through the RNA interference process, thereby silencing the corresponding mRNA fragments and exerting their biological activity. Currently, there are also a number of siRNA drugs approved for marketing, mainly for the treatment of genetic diseases; in addition, ASO, pDNA and other active nucleic acid drugs are also used in the treatment of various diseases.

However, there are many problems in the in vivo application of nucleic acid drugs: for example, free nucleic acid molecules are easily degraded and destroyed by nucleases in the blood circulation system, thus losing their activity; at the same time, nucleic acid molecules are usually large in molecular weight and have strong negative charges, making them difficult to be phagocytosed and taken up by cells. Therefore, the clinical application of nucleic acid drugs is greatly limited.

In order to solve the above-mentioned problems of nucleic acid drugs, many nucleic acid drug delivery carriers have been developed one after another. Among them, cationic lipid-based nanoparticles are the most widely used. Their structure usually includes a hydrophilic head containing a cationic fragment, a hydrophobic tail chain, and a connecting part in the middle. Among them, the cationic fragment can bind to the nucleic acid drug through electrostatic interaction, thereby achieving the encapsulation of the nucleic acid drug. This type of delivery carrier usually has a strong nucleic acid drug encapsulation capacity and transfection ability, but the cationic fragments in the carrier material can also cause some serious in vivo safety issues, such as strong cytotoxicity and immunogenicity, easy adsorption by plasma proteins in the blood circulation, and easy accumulation in the liver.

Summary of the invention

In order to overcome the defects of the prior art, the present invention provides a lipid material for nucleic acid delivery and its use, adopts a high-efficiency and low-toxic nucleic acid drug carrier strategy, and utilizes a new type of ionizable lipid and auxiliary lipid material to mix and encapsulate nucleic acid drugs, thereby achieving efficient and safe delivery of nucleic acid drugs in the body and improving the drugability of nucleic acid drugs.

The present invention provides a lipid material for nucleic acid delivery, wherein the lipid material comprises a compound having structure I:

Wherein, C _n H _2n includes straight chain or branched alkyl carbon, and n is an integer between 0 and 10;

R _1a , R _1b , R _1c , R _1d are selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl; and/or R _1a , R _1b , R _1c , R _1d are selected from polycyclic carbon rings, nitrogen-containing polycyclic rings, oxygen-containing polycyclic rings; and/or R _1a , R _1b and R _1c , R _1d respectively form a closed ring with N;

L _1a , L _2a , L _1b , L _2b are one or more selected from —CC—, —(C═O)—O—, —O—(C═O)—, —(C═O)—NH—, —NH—(C═O)—, —OO—, and —SS—;

X is one or more of -CC-, -C=C-, -C=N-, -S-, -SS-, -SSS-, -Se-, -Se-Se-, -SC(CH3) ₂ -S-, -OO-;

R _2a and R _2b are saturated or unsaturated fatty chain structures containing 10 to 24 carbon atoms, including cholesterol derivatives and/or tocopherol derivatives.

In order to solve the above problems, the present invention adopts an ionizable tertiary amine structure, which can be positively charged under acidic conditions and uncharged under neutral conditions. That is, compared with other Gemini lipid materials (such as patents US20080112915A1 and WO2016197264A1), the lipid provided by the present invention has more advantages: the hydrophilic head of the material has an ionizable tertiary amine structure, which avoids the permanently charged quaternary amine structure, so that it is positively charged under acidic conditions, and can be used for the encapsulation of nucleic acid drugs and enhance the escape ability of drug delivery carriers in lysosomes; and under body fluid conditions (pH = 7.4), the potential of the nucleic acid drug delivery system prepared using the lipid material is nearly neutral, so that it has good safety and can effectively avoid the safety problems (such as cytotoxicity problems, etc.) caused by the cationic head of other Gemini lipid materials. This property enables the cationic material to reduce its toxicity to the human body while ensuring efficient transfection, thereby safely and efficiently delivering nucleic acid drugs to target organs or target cells.

At the same time, because the lipid material has a special "H-type" structure, compared with other lipid materials (including the cationic lipid material Dlin-MC3-DMA (MC3) that has been on the market), this type of material has a more significant cell transfection ability, so that nucleic acid drugs can play a better therapeutic effect. The inventors have proved through in vitro cell transfection experiments that the transfection ability of the tertiary amine head lipid material provided by the present invention is better than that of the cationic lipids on the market. At the same time, in vivo transfection experiments have found that H-type lipids have excellent spleen-specific transfection ability.

Preferably, the lipid material provided by the present invention has one or more of the following structures:

Preferably, the lipid material provided by the present invention has one or more of the following structures:

in

in

in

Preferably, the lipid material provided by the present invention has one or more of the following structures:

Preferably, the lipid material provided by the present invention can be prepared by methods including connecting R _2a , R _2b fatty chains, deprotection and/or condensation. Those skilled in the art can easily achieve the preparation and synthesis of H1 to H36 under the guidance of this application; at the same time, under the guidance of this application, the compounds in structures I to V can be easily obtained by connecting R _2a , R _2b fatty chains, deprotection, condensation, etc. through similar synthetic routes.

Preferably, the lipid material provided by the present invention further comprises one or more of neutral lipids, steroidal compounds, and/or polymer-conjugated lipids.

Preferably, the lipid material provided by the present invention, wherein the neutral lipid is selected from one or more of DSPC, DPPC, DMPC, DOPC, POPC, and DOPE, and the molar ratio of the compound to the neutral lipid is 1:1 to 10:1; the steroid compound is selected from sterol compounds, preferably cholesterol, and the molar ratio of the compound to the steroid compound is 1:1 to 10:1; the polymer-conjugated lipid is selected from PEGylated lipids, and the molar ratio of the compound to the polymer-conjugated lipid is 100:1 to 5:1. Most of the lipid nanoparticles prepared in this way are 100-250nm, have good assembly ability, PDI is 0.1-0.3, uniform particle size distribution, and good stability; the encapsulation rate is between 75%-95%, and has good nucleic acid encapsulation capacity; the Zeta potential is -15-+10mV, the pka is 5.5-7.0, and it is nearly neutral under the physiological environment of pH 7.4, and has good safety. These excellent nanoparticle properties make it applicable to the treatment of diseases in vivo and are conducive to subsequent large-scale production.

Preferably, the lipid material provided by the present invention, wherein the lipid material is used in combination with a nucleic acid drug to achieve nucleic acid drug delivery; the combined use includes being prepared into one or more pharmaceutical compositions with the nucleic acid drug; the nucleic acid drug is selected from one or more of ASO, siRNA, mRNA, miRNA and pDNA.

Preferably, the lipid material provided by the present invention, wherein the lipid material and the nucleic acid drug are prepared into lipid nanoparticles containing the nucleic acid drug, including mixing an aqueous solution of the nucleic acid drug with an ethanol solution of the lipid material; preferably, the mixing is carried out by a microfluidic device, a high-pressure microfluidizer, a high-pressure homogenizer and/or a T-tube mixer.

Preferably, the lipid material provided by the present invention, wherein the lipid material for nucleic acid delivery, is used to prepare a therapeutic drug for one or more selected from infectious diseases, tumor diseases, congenital genetic diseases and immune diseases.

After the inventors' creative work and multiple experiments, it was found that the lipid nanoparticles prepared by the lipid material have a better delivery effect in one or more intracellular nucleic acids (mRNA, siRNA and miRNA, etc.) than the lipid nanoparticles prepared by the commercially available cationic lipid materials (Dlin-MC3-DMA, ALC-0315, SM-102, etc.), and have obvious advantages. In addition, compared with the cationic lipid materials approved for marketing, which show a large amount of accumulation in the liver, the lipid material provided by the present invention shows the accumulation specificity in non-liver parts, such as the spleen targeted accumulation ability.

At the same time, by adopting the administration method of intravenous injection or intratumoral injection, the lipid nanoparticles containing nucleic acid drugs provided by the present invention, and the mRNA vaccines prepared therefrom, etc., exhibit excellent tumor inhibitory effects. Through their unique cell transfection ability and delivery effect, they can be used as effective therapeutic drugs for infectious diseases, tumor diseases, congenital hereditary diseases and immune diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

FIG1 is a synthetic route of H-type series lipid materials (taking lipid material H1 as an example);

FIG2 is a H1 nuclear magnetic resonance hydrogen spectrum of lipid materials;

FIG3 is a mass spectrum of lipid material H1;

FIG4 is a diagram showing the particle size and potential of representative lipid nanoparticles;

FIG5 shows the transfection of lipid nanoparticles encapsulating mRNA prepared by H-series lipid materials on 293T cells;

FIG6 shows the transfection of lipid nanoparticles encapsulating mRNA prepared by H-series lipid materials on U87 cells;

FIG7 shows the transfection of lipid nanoparticles encapsulating mRNA prepared by H-series lipid materials on Hela cells;

FIG8 shows the gene silencing of lipid nanoparticles encapsulating siRNA prepared by H-type series lipid materials on 293T cells;

FIG9 shows the transfection of lipid nanoparticles encapsulating mRNA prepared by H-type series lipid materials in mice;

FIG10 is a diagram showing the transfection of lipid nanoparticles encapsulating mRNA prepared by H-type lipid materials at the mouse spleen cell level;

FIG. 11 shows the tumor inhibition effect of mRNA tumor vaccine prepared with H-type lipid material in B16-OVA tumor-bearing mice by intravenous injection.

DETAILED DESCRIPTION

In order to further illustrate the present invention, examples are given below. It should be noted that these examples are purely illustrative. The purpose of providing these examples is to fully illustrate the meaning and content of the present invention, but the present invention is not limited to the scope of the examples.

Example 1

The present invention provides a lipid material for nucleic acid delivery, wherein the lipid material comprises a compound having structure I:

Wherein, C _n H _2n includes straight chain or branched alkyl carbon, and n is an integer between 0 and 10;

R _1a , R _1b , R _1c , R _1d are selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl; and/or R _1a , R _1b , R _1c , R _1d are selected from polycyclic carbon rings, nitrogen-containing polycyclic rings, oxygen-containing polycyclic rings; and/or R _1a , R _1b and R _1c , R _1d respectively form a closed ring with N;

L _1a , L _2a , L _1b , L _2b are one or more selected from —CC—, —(C═O)—O—, —O—(C═O)—, —(C═O)—NH—, —NH—(C═O)—, —OO—;

X is one or more of -CC-, -C=C-, -C=N-, -S-, -SS-, -SSS-, -Se-, -Se-Se-, -SC(CH3) ₂ -S-, -OO-;

R _2a and R _2b are saturated or unsaturated fatty chain structures containing 10 to 24 carbon atoms, including cholesterol derivatives and/or tocopherol derivatives.

In one embodiment, the lipid material provided by the invention, wherein the compound has one or more of the following structures:

In another embodiment, the lipid material provided by the present invention, wherein the compound has one or more of the following structures:

in

in

in

In another embodiment, the structure V lipid material can be the following structure:

Table 1 Structure V Specific structure of lipid material

The structural formulas of the above lipids can be obtained by combining the above lipids by those skilled in the art. The structural formulas H1 to H36 are listed as follows:

Example 2

This example is used to illustrate the synthesis routes of the lipids in Example 1. Other lipids can be obtained by similar routes. The specific synthesis routes are shown in Figure 1.

2.1 Synthesis of intermediate 1

Weigh 2.20gN, N'-bis(tert-butyloxycarbonyl)-L-cystine (5mmol), 2.68g (10mmol) oleyl alcohol, 1.22g (10mmol) 4-dimethylaminopyridine (DMAP) and 3.87g (30mmol) N, N-diisopropylethylamine (DIPEA) in a 100mL eggplant-shaped bottle, 15mL dichloromethane (DCM) as solvent, and stir at room temperature for 20min. Then add 15mL of 1.91g (10mmol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) in dichloromethane dropwise to the reaction bottle. React overnight at room temperature. After the reaction is complete, column chromatography purification is performed to obtain 2.35g white solid powder with a yield of 50.0%.

¹ H NMR (400MHz, DMSO) δ7.37 (d, J=7.9Hz, 2H), 5.33 (dd, J=12.5, 7.9Hz, 4H), 4.24 (d, J=4.6Hz, 2H), 4.05 ( d, J＝5.5Hz, 4H), 2.99 (ddd, J＝23.2, 13.7, 7.2Hz, 4H), 1.98 (d, J＝5.4Hz, 8H), 1.59-1.53 (m, 4H), 1.41-1.35 (m, 18H), 1.24 (s, 44H), 0.86 (t, J=6.6Hz, 6H).

2.2 Synthesis of intermediate 2

1.88 g (2.0 mmol) of intermediate 1 was weighed into a reaction bottle, and 20 mL of HCl/EA was added to the reaction bottle, and stirred at room temperature for 4 h. After the reaction was complete, the solvent was drained using an oil pump to obtain 1.13 g of white solid powder with a yield of 80.7%.

2.3 Synthesis of intermediate 3

Weigh 2.34g (5mmol) N, N'-bis (tert-butyloxycarbonyl) -L- homocystine, 2.68g (10mmol) oleyl alcohol, 1.22g (10, mmol) DMAP and 3.87g (30mmol) DIPEA in a 100mL eggplant-shaped bottle, 15mL DCM as solvent, and stir at room temperature for 20min. Then add 15mL of 1.91g (10mmol) EDCI DCM solution dropwise to the reaction bottle. React overnight at room temperature. After the reaction is complete, column chromatography purification is performed to obtain 1.98g white solid powder with a yield of 49.8%.

¹ H NMR (400MHz, DMSO) δ7.31 (d, J=7.8Hz, 2H), 5.32 (t, J=4.8Hz, 4H), 4.05 (d, J=5.8Hz, 4H), 4.02-3.97 ( m, 2H), 2.72 (s, 4H), 2.03-1.92 (m, 12H), 1.54 (d, J=6.0Hz, 4H), 1.38 (s, 18H), 1.24 (s, 44H), 0.86 (t , J=6.7Hz, 6H).

2.4 Synthesis of intermediate 4

1.94 g (2.0 mmol) of intermediate 3 was weighed into a reaction bottle, and 20 mL of HCl/EA was added to the reaction bottle, and stirred at room temperature for 4 h. After the reaction was complete, the solvent was drained using an oil pump to obtain 1.01 g of white solid powder with a yield of 70.0%.

2.5 Synthesis of compounds H1-H12

2.5.1 Synthesis of Compound H1

Weigh 206 mg (2.0 mmol) of dimethylglycine, 230 mg (2.0 mmol) of NHS and 282 mg (2.0 mmol) of EDCI in a 100 mL eggplant-shaped bottle, 15 mL of DCM as solvent, and stir at room temperature for 30 min. Then add 740 mg of intermediate 2 to the reaction bottle and react overnight at room temperature. After the reaction is complete, column chromatography purification is performed to obtain 457 mg of light yellow transparent oily liquid with a yield of 50.3%.

¹ H NMR (400 MHz, DMSO) δ 8.18 (d, J = 8.3 Hz, 2H), 5.40-5.26 (m, 4H), 4.63 (td, J = 8.3, 5.2 Hz, 2H), 4.04 (dt, J = 8.0, 5.4 Hz, 4H), 3.21-3.08 (m, 4H), 2.91 (q, J = 15.4 Hz, 4H), 2.24 (s, 12H), 2.11-1.80 (m, 8H), 1.55 (dd, J = 13.3, 6.4 Hz, 4H), 1.24 (s, 48H), 0.86 (t, J = 6.8 Hz, 6H). See Figures 2 and 3 for specific NMR and mass spectra.

2.5.2 Synthesis of Compound H2

306 mg (2.0 mmol) of 3-dimethylaminopropionic acid hydrochloride, 230 mg (2.0 mmol) of NHS and 282 mg (2.0 mmol) of EDCI were placed in a 100 mL eggplant-shaped bottle, 15 mL of DCM was used as solvent, and stirred at room temperature for 30 min. Subsequently, 740 mg of intermediate 2 was added to the reaction bottle, and the reaction was continued overnight at room temperature. After the reaction was complete, column chromatography was performed to purify the product, and 656 mg of a light yellow transparent oily liquid was obtained, with a yield of 70.1%.

¹ H NMR (400MHz, DMSO) δ8.18 (d, J=8.3Hz, 2H), 5.40-5.26 (m, 4H), 4.63 (td, J=8.3, 5.2Hz, 2H), 4.04 (dt, J =8.0, 5.4Hz, 4H), 3.21-3.08(m, 4H), 2.91(q, J＝15.4Hz, 4H), 2.24(s, 12H), 2.11-1.80(m, 8H), 1.55(dd, J=13.3, 6.4Hz, 4H), 1.24 (s, 48H), 0.86 (t, J=6.8Hz, 6H).

2.5.3 Synthesis of Compound H3

262 mg (2.0 mmol) of 4-(dimethylamino)butyric acid, 230 mg (2.0 mmol) of NHS and 282 mg (2.0 mmol) of EDCI were placed in a 100 mL eggplant-shaped bottle, 15 mL of DCM was used as solvent, and stirred at room temperature for 30 min. Subsequently, 740 mg of intermediate 2 was added to the reaction bottle, and the reaction was continued overnight at room temperature. After the reaction was complete, column chromatography was performed to purify the mixture, and 544 mg of a light yellow transparent oily liquid was obtained, with a yield of 56.3%.

¹ H NMR (400MHz, DMSO) δ8.43 (d, J=7.9Hz, 2H), 5.32 (s, 4H), 4.53 (s, 2H), 4.05 (s, 4H), 3.37 (d, J=6.9 Hz, 4H), 3.10 (d, J=12.8Hz, 2H), 2.98-2.90 (m, 2H), 2.28 (s, 12H), 2.16 (s, 4H), 1.99 (s, 8H), 1.69 (d , J=5.9Hz, 4H), 1.56 (s, 4H), 1.24 (s, 48H), 0.86 (s, 6H).

2.5.4 Synthesis of Compound H4

286 mg (2.0 mmol) of 3-pyrrolidin-1-yl propionic acid, 230 mg (2.0 mmol) of NHS and 282 mg (2.0 mmol) of EDCI were placed in a 100 mL eggplant-shaped bottle, 15 mL of DCM was used as solvent, and stirred at room temperature for 30 min. Subsequently, 740 mg of intermediate 2 was added to the reaction bottle, and the reaction was continued overnight at room temperature. After the reaction was complete, column chromatography was performed to purify the product, and 420 mg of a light yellow transparent oily liquid was obtained, with a yield of 42.4%.

¹ H NMR (400MHz, DMSO) δ8.18 (d, J=8.3Hz, 2H), 5.40-5.26 (m, 4H), 4.63 (td, J=8.3, 5.2Hz, 2H), 4.04 (dt, J =8.0, 5.4Hz, 4H), 3.21-3.08(m, 4H), 2.91(q, J＝15.4Hz, 4H), 2.24(s, 12H), 2.11-1.80(m, 8H), 1.55(dd, J=13.3, 6.4Hz, 4H), 1.24 (s, 48H), 0.86 (t, J=6.8Hz, 6H).

2.5.5 Synthesis of Compound H5

286 mg (2.0 mmol) of 1-methylpiperidine-4-carboxylic acid, 230 mg (2.0 mmol) of NHS and 282 mg (2.0 mmol) of EDCI were placed in a 100 mL eggplant-shaped bottle, 15 mL of DCM was used as solvent, and stirred at room temperature for 30 min. Subsequently, 740 mg of intermediate 2 was added to the reaction bottle, and the reaction was continued overnight at room temperature. After the reaction was complete, column chromatography was performed to purify the product, and 448 mg of a light yellow transparent oily liquid was obtained, with a yield of 45.3%.

¹ H NMR (400MHz, DMSO) δ8.38 (d, J=7.8Hz, 2H), 5.36-5.30 (m, 4H), 4.54-4.49 (m, 2H), 4.04 (dd, J=10.3, 6.3Hz , 4H), 3.10 (dd, J=13.8, 5.2Hz, 4H), 2.33 (s, 7H), 2.22 (s, 8H), 2.02-1.93 (m, 8H), 1.63 (dd, J=36.5, 24.3 Hz, 12H), 1.24 (s, 48H), 0.86 (t, J=4.8Hz, 6H).

2.5.6 Synthesis of Compound H6

344 mg (1.0 mmol) of 3-(4-methyl-1-piperazinyl) propionic acid, 230 mg (2.0 mmol) of NHS and 282 mg (2.0 mmol) of EDCI were placed in a 100 mL eggplant-shaped bottle, 15 mL of DCM was used as solvent, and stirred at room temperature for 30 min. Subsequently, 740 mg of intermediate 2 was added to the reaction bottle, and the reaction was continued overnight at room temperature. After the reaction was complete, column chromatography was performed to purify the product, and 650 mg of a light yellow transparent oily liquid was obtained, with a yield of 62.1%.

¹ H NMR (400MHz, DMSO) δ8.18 (d, J=8.3Hz, 2H), 5.40-5.26 (m, 4H), 4.63 (td, J=8.3, 5.2Hz, 2H), 4.04 (dt, J =8.0, 5.4Hz, 4H), 3.21-3.08(m, 4H), 2.91(q, J＝15.4Hz, 4H), 2.24(s, 12H), 2.11-1.80(m, 8H), 1.55(dd, J=13.3, 6.4Hz, 4H), 1.24 (s, 48H), 0.86 (t, J=6.8Hz, 6H).

2.5.7 Synthesis of Compound H7

206 mg (2.0 mmol) of dimethylglycine, 230 mg (2.0 mmol) of NHS and 282 mg (2.0 mmol) of EDCI were placed in a 100 mL eggplant-shaped bottle, 15 mL of DCM was used as solvent, and stirred at room temperature for 30 min. Subsequently, 768 mg of intermediate 4 was added to the reaction bottle and reacted overnight at room temperature. After the reaction was complete, column chromatography was performed to purify the product to obtain 284 mg of a light yellow transparent oily liquid with a yield of 30.3%.

¹ H NMR (400MHz, DMSO) δ8.11 (d, J=7.9Hz, 2H), 5.32 (s, 4H), 4.42 (s, 2H), 4.04 (d, J=12.0Hz, 5H), 2.91 ( dd, J=47.0, 14.9Hz, 5H), 2.78-2.63(m, 5H), 2.22(s, 12H), 2.15-1.92(m, 12H), 1.55(s, 4H), 1.24(s, 48H) ,0.86(s,6H).

2.5.8 Synthesis of Compound H8

306 mg (2.0 mmol) of 3-dimethylaminopropionic acid hydrochloride, 230 mg (2.0 mmol) of NHS and 282 mg (2.0 mmol) of EDCI were placed in a 100 mL eggplant-shaped bottle, 15 mL of DCM was used as solvent, and stirred at room temperature for 30 min. Subsequently, 768 mg of intermediate 4 was added to the reaction bottle, and the reaction was continued overnight at room temperature. After the reaction was complete, column chromatography was performed to purify the product, and 391 mg of a light yellow transparent oily liquid was obtained, with a yield of 40.5%.

¹ H NMR (400MHz, DMSO) δ8.58 (s, 2H), 5.32 (s, 4H), 4.36 (s, 2H), 4.03 (s, 5H), 3.02 (d, J=19.2Hz, 8H), 2.75(s, 5H), 2.53(d, J=9.6Hz, 12H), 2.03(d, J=42.1Hz, 13H), 1.56(s, 4H), 1.24(s, 48H), 0.86(s, 6H ).

2.5.9 Synthesis of Compound H9

262 mg (2.0 mmol) of 4-(dimethylamino)butyric acid, 230 mg (2.0 mmol) of NHS and 282 mg (2.0 mmol) of EDCI were placed in a 100 mL eggplant-shaped bottle, 15 mL of DCM was used as solvent, and stirred at room temperature for 30 min. Subsequently, 768 mg of intermediate 4 was added to the reaction bottle, and the reaction was continued overnight at room temperature. After the reaction was complete, column chromatography was performed to purify the mixture, and 498 mg of a light yellow transparent oily liquid was obtained, with a yield of 50.1%.

¹ H NMR (400MHz, DMSO) δ8.30 (d, J=7.3Hz, 2H), 5.32 (s, 5H), 4.35 (s, 2H), 4.09-3.97 (m, 4H), 2.29 (d, J =44.3Hz, 12H), 2.16(s, 4H), 1.98(s, 8H), 1.66(s, 4H), 1.55(s, 4H), 1.24(s, 48H), 0.86(s, 6H).

2.5.10 Synthesis of Compound H10

286 mg (2.0 mmol) of 3-pyrrolidin-1-yl propionic acid, 230 mg (2.0 mmol) of NHS and 282 mg (2.0 mmol) of EDCI were placed in a 100 mL eggplant-shaped bottle, 15 mL of DCM was used as solvent, and stirred at room temperature for 30 min. Subsequently, 768 mg of intermediate 4 was added to the reaction bottle, and the reaction was continued overnight at room temperature. After the reaction was complete, column chromatography was performed to purify the product, and 321 mg of a light yellow transparent oily liquid was obtained, with a yield of 31.6%.

¹ H NMR (400MHz, DMSO) δ8.18 (d, J=8.3Hz, 2H), 5.40-5.26 (m, 4H), 4.63 (td, J=8.3, 5.2Hz, 2H), 4.04 (dt, J =8.0, 5.4Hz, 4H), 3.21-3.08(m, 4H), 2.91(q, J＝15.4Hz, 4H), 2.24(s, 12H), 2.11-1.80(m, 8H), 1.55(dd, J=13.3, 6.4Hz, 4H), 1.24 (s, 48H), 0.86 (t, J=6.8Hz, 6H).

2.5.11 Synthesis of Compound H11

286 mg (2.0 mmol) of 1-methylpiperidine-4-carboxylic acid, 230 mg (2.0 mmol) of NHS and 282 mg (2.0 mmol) of EDCI were placed in a 100 mL eggplant-shaped bottle, 15 mL of DCM was used as solvent, and stirred at room temperature for 30 min. Subsequently, 768 mg of intermediate 4 was added to the reaction bottle, and the reaction was continued overnight at room temperature. After the reaction was complete, column chromatography was performed to purify the product, and 634 mg of a light yellow transparent oily liquid was obtained, with a yield of 62.3%.

¹ H NMR (400MHz, DMSO) δ8.24 (d, J=6.6Hz, 2H), 5.32 (s, 4H), 4.32 (s, 2H), 4.07-3.98 (m, 4H), 2.90 (d, J =9.6Hz, 4H), 2.71 (s, 4H), 2.28 (s, 8H), 2.20 (s, 2H), 2.10 (s, 8H), 1.98 (s, 6H), 1.68-1.53 (m, 12H) , 1.24(s, 48H), 0.85(s, 6H).

2.5.12 Synthesis of Compound H12

344 mg (1.0 mmol) of 3-(4-methyl-1-piperazinyl) propionic acid, 230 mg (2.0 mmol) of NHS and 282 mg (2.0 mmol) of EDCI were placed in a 100 mL eggplant-shaped bottle, 15 mL of DCM was used as solvent, and stirred at room temperature for 30 min. Subsequently, 768 mg of intermediate 4 was added to the reaction bottle, and the reaction was continued overnight at room temperature. After the reaction was complete, column chromatography was performed to purify the product, and 429 mg of a light yellow transparent oily liquid was obtained, with a yield of 39.9%.

¹ H NMR (400MHz, CDCl3) δ8.86 (s, 2H), 5.35 (d, J=16.7Hz, 4H), 4.68 (d, J=5.3Hz, 3H), 4.14 (d, J=3.8Hz, 4H), 3.65 (dd, J=14.5, 7.1Hz, 4H), 2.86-2.74 (m, 24H), 2.53 (s, 14H), 2.03 (d, J=5.4Hz, 8H), 1.66 (d, J =6.7Hz, 4H), 1.28 (s, 49H), 0.89 (d, J = 7.0Hz, 6H).

It should be noted that the embodiments of the present invention are not exhaustive, and those skilled in the art can realize the preparation and synthesis of H1 to H36 under the guidance of the present application; at the same time, under the guidance of the present application, the compounds in structures I to V can be easily obtained by similar synthetic routes through the methods of connecting _R2a , _R2b fatty chains, deprotection, condensation, etc.

Example 3

The lipid material provided by the present invention further comprises one or more of neutral lipids, steroidal compounds, and/or polymer-conjugated lipids. The neutral lipids are selected from one or more of DSPC, DPPC, DMPC, DOPC, POPC, and DOPE, and the molar ratio of the compound to the neutral lipid is 1:1 to 10:1; the steroidal compounds are selected from sterol compounds, preferably cholesterol, and the molar ratio of the compound to the steroidal compounds is 1:1 to 10:1; the polymer-conjugated lipids are selected from PEGylated lipids, and the molar ratio of the compound to the polymer-conjugated lipids is 100:1 to 5:1.

In one embodiment, the lipid material is used in combination with a nucleic acid drug to achieve nucleic acid drug delivery; the combined use includes preparing one or more pharmaceutical compositions; the nucleic acid drug is selected from one or more of ASO, siRNA, mRNA, miRNA and pDNA. The lipid material and the nucleic acid drug are prepared into lipid nanoparticles containing the nucleic acid drug, including mixing an aqueous solution of the nucleic acid drug with an ethanol solution of the lipid material; preferably, the mixing is performed by a microfluidic device, a high-pressure microfluidizer, a high-pressure homogenizer and/or a T-tube mixer.

In another embodiment, the lipid material for nucleic acid delivery is used to prepare a therapeutic drug for one or more selected from infectious diseases, tumor diseases, congenital genetic diseases and immune diseases.

This example mainly illustrates the use of the nucleic acid drug delivery vector for the delivery of nucleic acids such as ASO, siRNA, mRNA, miRNA and pDNA, and the preparation method of lipid nanoparticles containing the above-mentioned nucleic acid drugs. It also takes the particle size, polydispersity coefficient, Zeta potential, encapsulation efficiency and pKa determination method of the mRNA delivery vector as an example, which is not a complete enumeration.

3.1 Preparation of lipid nanoparticles

ASO, siRNA, mRNA, miRNA and/or pDNA are dissolved in 40mM citric acid buffer at pH=4, and the lipid material of Example 1 or Example 2 is mixed according to the prescription composition in Table 2 and dissolved in ethanol; according to the flow rate ratio of water phase: alcohol phase=1:3, lipid nanoparticles are prepared by rapid mixing through a microfluidic device, a high-pressure microfluidizer, a high-pressure homogenizer and/or a T-tube mixer. Subsequently, the prepared lipid nanoparticles are placed in a dialysis bag with a molecular weight cutoff of 3500, dialyzed overnight in a PBS buffer at pH=7.4 to remove free small molecules, ethanol and adjust the pH, and the obtained lipid nanoparticles are stored at 4°C.

3.2 Determination of particle size, polydispersity index (PDI) and zeta potential of lipid nanoparticles

The particle size, polydispersity index (PDI) and Zeta potential of the lipid nanoparticles were measured by dynamic light scattering using a Malvern Zetasizer Pro, wherein the particle size, PDI and Zeta potential of the lipid nanoparticles prepared from representative lipid materials included in the present invention are shown in Table 3 and FIG. 4 .

3.3 Determination of lipid nanoparticle encapsulation efficiency

The encapsulation efficiency of lipid nanoparticles was determined using the Quant-iT RiboGreen RNA Assay Kit RNA quantitative detection kit: the sample was diluted to a concentration of about 5 μg/mL in TE buffer solution, and the RiboGreen reagent was diluted in TE buffer at 1:200, 100 μL of the diluted sample was transferred to a 96-well plate and 100 μL of the diluted RiboGreen solution was added. Incubate at 37°C for 15 minutes. The fluorescence intensity was measured using a fluorescent plate reader (excitation wavelength 480 nm, emission wavelength 520 nm), and the free RNA concentration was calculated. The lipid nanoparticles prepared from the representative lipid materials included in the present invention are shown in Table 3.

3.4 Determination of pKa of lipid nanoparticles

The apparent pKa of lipid nanoparticles is determined by the fluorescence of 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS). A buffer solution (pH=2.5-11.0) containing 150mM sodium chloride, 10mM sodium phosphate, 10mM sodium citrate, and 10mM sodium borate is prepared. After the lipid nanoparticles are mixed with buffer solutions of different pH values, TNS is added. A fluorescence microplate reader is used to detect the fluorescence intensity at an excitation wavelength of 321nm and an emission wavelength of 445nm at room temperature. The fluorescence data is fitted and analyzed, and pKa is the pH value that produces half the maximum fluorescence intensity, wherein the pKa data of representative lipid nanoparticles included in the present invention are shown in Table 4.

Table 2 Lipid nanoparticle formulations containing lipid materials of the present invention

Table 3 Characterization of lipid nanoparticles comprising lipid materials of the present invention

Notes: Dipalmitoylcholine (DPPC), Distearoylcholine (DSPC), Dimyristoylphosphatidylcholine (DMPC), Dioleoylphosphatidylcholine (DOPC), Dioleoylphosphatidylethanolamine (DOPE), Phosphatidylcholine (POPC), Dimyristoylglycerol-Polyethylene Glycol 2000 (DMG-PEG2000).

Table 4 pKa values of lipid nanoparticles containing lipid materials of the present invention

Lipid materials pka L1 6.80 L2 8.97 L3 10.07 L4 6.60 L5 9.01 L6 8.30 L7 6.93 L8 9.65 L9 9.66 L10 7.00 L11 10.52 L12 8.55 L13 6.56 L14 6.18 L15 5.78 L16 5.99 L17 5.37 L18 5.81 L19 6.14 L20 6.42

Conclusion: Most of the lipid nanoparticles prepared in this way are 100-250m, have good assembly ability, PDI is 0.1-0.3, uniform particle size distribution, good stability; encapsulation efficiency is between 75%-95%, with good nucleic acid encapsulation capacity; Zeta potential is -15mV-+10mV, pka is 5.5-7.0, near neutral under pH7.4 physiological environment, and good safety. These excellent nanoparticle properties make it possible to be used in the treatment of diseases in vivo and are conducive to subsequent large-scale production.

Example 4

In this example, commercially available cationic lipids (MC3, ALC-0315 and SM-102) were used as positive controls to investigate the mRNA cell transfection efficiency of lipid nanoparticles formed by various H-type lipid materials.

HEK239T, U87MG and Hela cells were seeded in 96-well plates at a density of 10,000 cells per well, cultured overnight, and when the cell density reached more than 80%, lipid nanoparticles containing luciferase mRNA were added to each well. After 6 hours, the fluorescence intensity of the expressed luciferase protein was detected using a luciferase detection kit and a chemiluminescence instrument. The data are shown in Figures 5, 6 and 7.

Conclusion: Based on an incomplete enumeration, the lipid nanoparticles formed by compounds H1, H4, H7, H8, and H10 have a better mRNA delivery effect in one or more cells than lipid nanoparticles prepared by commercially available cationic lipids (MC3, ALC-0315, SM-102), and have obvious advantages.

Example 5

In this example, commercially available cationic lipid (MC3) was used as a positive control, and the gene silencing efficiency of lipid nanoparticles formed by various H-type lipid materials on 293T cells was investigated by qRT-PCR experiments.

293T cells were seeded in six-well plates at a density of 500,000 per well. After culturing for 24 hours, lipid particles containing EGFR siRNA were added to each well, where the final concentration of siRNA was set to 100 nM. Subsequently, the cells were incubated in the incubator for transfection for another 24 hours. After transfection, the old culture medium was discarded and RNA extraction was prepared. The six-well plate was removed from the incubator, 1 mL of TRIZOL reagent was added to each well, and then allowed to stand at 4 ° C for 30 minutes to promote cell lysis. Next, 200 μL of chloroform was added, vortexed vigorously for 30 seconds, and allowed to stand at room temperature for 15 minutes. Subsequently, RNA was extracted by centrifugation at 12000g for 15 minutes at 4 ° C, and dissolved with an appropriate amount of DEPC-treated water. An appropriate amount of RNA solution was taken, and its A280 and A260 values were determined using a NanoDrop ultra-micro spectrophotometer, and the RNA concentration was accurately read. The internal reference group and the target genome were set for reverse transcription and amplification. The parameters were set as follows: pre-denaturation at 95°C for 60 seconds; PCR cycle including denaturation at 95°C for 15 seconds and extension at 60°C for 60 seconds; and finally melting curve analysis. Data processing was performed using the MX3005P instrument.

Conclusion: Based on an incomplete enumeration, the gene silencing efficiency of lipid nanoparticles formed by lipid materials H1, H4, and H10 in 293T cells is better than that of lipid microparticles formed by commercially available cationic lipid material (MC3).

Example 6

Using fluorescence intensity to evaluate the mRNA delivery ability of lipid nanoparticles in vivo:

To evaluate the effective delivery of mRNA in vivo by lipid nanoparticles and the expression of corresponding encoded proteins, 6-8 weeks of female BALB/c tail vein injections were encapsulated with mRNA liposome nanoparticles expressing luciferase at a dosage of 0.5mg/kg. After 6 hours, luciferase substrate was injected into the peritoneal cavity of each mouse, and IVIS small animal optical in vivo imaging instrument (PerkinElme) was used to take fluorescent pictures of mice, and the fluorescence intensity of the whole body of mice was counted. The height of the fluorescence intensity represents the height of the expression of luciferase protein, i.e., reflects the efficiency of mRNA delivery in the body of lipid nanoparticles, and the data are shown in Figure 10.

Conclusion: Compared with the large accumulation of cationic lipids approved for marketing in the liver, lipid nanoparticles prepared from H-type lipid materials exhibit specific spleen targeted accumulation capabilities.

Example 7

To investigate the transfection of lipid nanoparticles prepared by H-type lipids in spleen tissue, 6-8 week old male C57BL/6J were injected with mRNA lipid nanoparticles encapsulating green fluorescent protein (EGFP) at a dose of 1.5 mg/kg via the tail vein. After 24 hours, the spleen was isolated, shredded with surgical shears, and digested in RPMI 1640 medium containing 1 mg/mL collagenase A and 10 mg/mL DNAse I to obtain a tissue suspension. Subsequently, the tissue suspension was passed through a 70 mm nylon cell strainer, centrifuged at 500 g for 7 min, lysed with red blood cell lysis buffer for 5 min, washed with PBS, and then counted, 6×10 ⁵ cells were taken, centrifuged at 500 g for 5 min, resuspended in PBS containing 2% BSA, stained with antibodies according to the designed flow color scheme, and analyzed by flow cytometry. The analysis results are shown in Figure 10.

Conclusion: Lipid nanoparticles prepared with H-type lipids were mainly transfected in DC cells in the spleen.

Example 8

4-6 week old C57BL/6 mice were injected with 2×10 ⁵ B16-OVA cells each and randomly divided into 4 groups, 8 mice in each group, half male and half female. On the sixth and tenth days after tumor inoculation, PBS solution, OVA protein solution (15 mg/kg), MC3-LNP (15 mg/kg) encoding OVA mRNA and lipid nanoparticles prepared by H-type lipids encoding OVA mRNA (15 mg/kg) were intravenously injected twice in total, and the tumor size was measured every day using a digital vernier caliper (tumor volume calculation formula = 0.5×long diameter×short diameter ² ), and the tumor inhibition curve was drawn, as shown in Figure 11.

Conclusion: The mRNA vaccine prepared with H-type lipid materials exhibited excellent tumor inhibitory effects.

The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (10)

1. A lipid material for nucleic acid delivery, characterized in that the lipid material comprises a compound having the structure I:

wherein C _nH_2n comprises a straight or branched alkyl carbon, n is an integer between 0 and 10;

R _1a、R_1b、R_1c、R_1d is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl; and/or R _1a、R_1b、R_1c、R_1d is selected from a multi-membered carbocyclic ring, a nitrogen containing multi-membered ring, an oxygen containing multi-membered ring; and/or R _1a、R_1b and R _1c、R_1d each form a closed loop with N;

L _1a,L_2a,L_1b,L_2b is selected from one or more of-C-, - (c=o) -O-, -O- (c=o) -, - (c=o) -NH-, -NH- (c=o) -, -O-, -S-;

-S-S-, -S-S-S-, -S-S-, -S-S-S-, -Se-, -one or more of Se-, -S-C (CH 3) ₂ -S-, -O-;

r _2a、R_2b is a saturated or unsaturated fatty chain structure containing 10-24 carbons, including cholesterol derivatives and/or tocopherol derivatives.

2. The lipid material of claim 1, wherein the compound has one or more of the following structures:

3. The lipid material of claim 1, wherein the compound has one or more of the following structures:

Wherein the method comprises the steps of

Wherein the method comprises the steps of

Wherein the method comprises the steps of

4. The lipid material of claim 1, wherein the compound has one or more of the following structures:

5. the lipid material of any one of claims 1 to 4, wherein the lipid material is prepared by a method comprising attaching an R _2a、R_2b fatty chain, deprotecting and/or condensing.

6. The lipid material of claim 1, further comprising one or more of a neutral lipid, a steroid, and/or a polymer conjugated lipid.

7. The lipid material of claim 6, wherein the neutral lipid is selected from one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and the molar ratio of the compound to the neutral lipid is 1:1 to 10:1; the steroid is selected from the group consisting of sterols, preferably from cholesterol, in a molar ratio of 1:1 to 10:1; the polymer conjugated lipid is selected from pegylated lipids, and the molar ratio of the compound to the polymer conjugated lipid is 100:1 to 5:1.

8. The lipid material of claim 1, wherein the lipid material achieves nucleic acid drug delivery by use in combination with a nucleic acid drug; the combination comprises the preparation of one or more pharmaceutical compositions; the nucleic acid drug is selected from one or more of ASO, siRNA, mRNA, miRNA and pDNA.

9. The lipid material of claim 8, wherein the lipid material is prepared with a nucleic acid drug to form lipid nanoparticles comprising the nucleic acid drug, comprising mixing an aqueous solution of the nucleic acid drug with an ethanol solution of the lipid material; preferably by microfluidic devices, high pressure microfluidic homogenizers, high pressure homogenizers and/or T-tube mixers.

10. The lipid material of claim 1, wherein the lipid material for nucleic acid delivery is used for the preparation of a therapeutic drug selected from one or more of infectious diseases, neoplastic diseases, congenital genetic diseases, and immune diseases.