WO2025200517A1 - Lipid material for nucleic acid delivery and use thereof - Google Patents

Abstract

The present invention provides a lipid material for nucleic acid delivery, wherein the lipid material comprises a compound having structure I. The present invention also provides use of the lipid material for nucleic acid delivery in the preparation of a therapeutic drug for one or more selected from an infectious disease, a tumor disease, a congenital hereditary disease, and an immune disease. By means of the lipid material provided in the present invention and adopting a nucleic acid drug carrier strategy with high efficiency and low toxicity, a novel ionizable lipid and an auxiliary lipid material are mixed to encapsulate nucleic acid drugs, so that efficient and safe delivery of the nucleic acid drugs in vivo is achieved, and the druggability of the nucleic acid drugs is improved.

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 attracted widespread attention due to their advantages such as small dosage, strong biological effects, and wide application range. Currently, nucleic acid drugs are gradually being used to treat multi-gene 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 body's antigen-presenting cells, 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, active nucleic acid drugs such as ASO and pDNA are also used in the treatment of various diseases.

However, the in vivo application of nucleic acid drugs faces many challenges: free nucleic acid molecules are easily degraded and destroyed by nucleases in the bloodstream, thus losing their activity; and nucleic acid molecules are generally large in molecular weight and have a strong negative charge, making them difficult to be phagocytosed and taken up by cells. Therefore, the clinical application of nucleic acid drugs is greatly limited.

To address the aforementioned issues with nucleic acid drugs, numerous nucleic acid drug delivery vectors have been developed. Cationic liposomes are the most widely used type of nucleic acid drug delivery vector. Their structure typically includes a hydrophilic head containing a cationic segment, a hydrophobic tail, and a connecting portion therebetween. The cationic segment can bind to the nucleic acid drug through electrostatic interactions, thereby achieving encapsulation of the nucleic acid drug. This type of delivery vector typically possesses strong nucleic acid drug encapsulation and transfection capabilities, but the cationic segments in the carrier material can also cause serious in vivo safety issues, such as high 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 shortcomings of the existing technology, the present invention provides a lipid material for nucleic acid delivery and its use, adopting a high-efficiency and low-toxic nucleic acid drug carrier strategy, using 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 , and R _1d are selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and n-octyl; and/or R _1a , R _1b , R _1c , and R _1d are selected from a polycyclic carbon ring, a nitrogen-containing polycyclic ring, and an oxygen-containing polycyclic ring; and/or R _1a , R _1b , R _1c , and R _1d each form a closed ring with N;

L _1a , L _2a , L _1b , and 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 10-24 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, thereby being positively charged under acidic conditions and can be used for the encapsulation of nucleic acid drugs and enhancing the escape ability of drug delivery carriers in lysosomes; and under body fluid conditions (pH = 7.4), the delivery system prepared using the lipid material has a near-neutral potential, which makes the delivery system of the nucleic acid drug have good safety and can avoid the safety issues (such as cytotoxicity issues, etc.) caused by the cationic head of other Gemini lipid materials to a limited extent. 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 advantage in cell transfection ability, thereby enabling nucleic acid drugs to exert the greatest drug therapeutic effect. It is particularly noteworthy that this structure does not affect its transfection ability for nucleic acid drugs, but is more conducive to the transfection of nucleic acid drugs. The inventors have demonstrated 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:

Where R1＝

Where Linker =

Where R2＝

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

Preferably, the lipid material provided herein can be prepared by methods including attaching _R2a and _R2b fatty chains, deprotection, and/or condensation. Based on the teachings of this application, those skilled in the art can easily synthesize H1 to H36. Furthermore, based on the teachings of this application, compounds in Structures I to V can all be readily obtained using similar synthetic routes by attaching _R2a and _R2b fatty chains, deprotection, and condensation.

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

Preferably, the lipid material provided by the present invention comprises one or more of the neutral lipid selected from 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 100-250 nm, have good assembly ability, a PDI of 0.1-0.3, a uniform particle size distribution, and good stability; an encapsulation efficiency between 75% and 95%, and good nucleic acid encapsulation capacity; a Zeta potential of -15-+10 mV, a pka of 5.5-7.0, which is near neutral under a physiological pH of 7.4 and has good safety. These excellent nanoparticle properties make them suitable for the treatment of in vivo diseases 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, comprising 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.

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

Through the inventors' creative work and numerous experiments, they discovered that lipid nanoparticles prepared from this lipid material are significantly superior to lipid nanoparticles prepared from commercially available cationic lipid materials (Dlin-MC3-DMA, ALC-0315, SM-102, etc.) in delivering one or more intracellular nucleic acids (mRNA, siRNA, and miRNA, etc.). Furthermore, compared to approved cationic lipid materials that exhibit significant accumulation in the liver, the lipid material provided by this invention exhibits specific accumulation in non-liver sites, such as the spleen.

At the same time, the lipid nanoparticles containing nucleic acid drugs provided by the present invention, which are injected intravenously or intratumorally, and the mRNA vaccines prepared therefrom, have shown 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 following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

Figure 1 shows the synthesis route of H-type series lipid materials (taking lipid material H1 as an example);

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

FIG3 is a mass spectrum of lipid material H1;

Figure 4 shows the particle size and potential of representative lipid nanoparticles;

FIG5 shows the transfection of mRNA-encapsulated lipid nanoparticles prepared from H-series lipid materials into 293T cells;

FIG6 shows the transfection of mRNA-encapsulated lipid nanoparticles prepared from H-series lipid materials into U87 cells;

FIG7 shows the transfection of mRNA-encapsulated lipid nanoparticles prepared from H-series lipid materials into Hela cells;

FIG8 shows the gene silencing effect of siRNA-encapsulated lipid nanoparticles prepared from H-series lipid materials on 293T cells;

FIG9 shows the transfection of mRNA-encapsulated lipid nanoparticles prepared from H-series lipid materials in mice;

FIG10 shows the transfection of mRNA-encapsulated lipid nanoparticles prepared with H-type lipid materials at the mouse spleen cell level;

Figure 11 shows the tumor inhibition effect of mRNA tumor vaccine prepared with H-type lipid material in B16-0VA tumor-bearing mice after intravenous injection.

DETAILED DESCRIPTION

To further illustrate the present invention, the following examples are provided. It should be noted that these examples are purely illustrative. The purpose of providing these examples is to fully illustrate the significance and content of the present invention, but they are not intended to limit the present invention 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 , and R _1d are selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and n-octyl; and/or R _1a , R _1b , R _1c , and R _1d are selected from a polycyclic carbon ring, a nitrogen-containing polycyclic ring, and an oxygen-containing polycyclic ring; and/or R _1a , R _1b , R _1c , and R _1d each form a closed ring with N;

L _1a , L _2a , L _1b , and L _2b are one or more selected from —CC—, —(C═O)—O—, —O—(C═O)—, —(C═O)—NH—, —NH—(C═O)—, and —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 10-24 carbon atoms, including cholesterol derivatives and/or tocopherol derivatives.

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

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

Where R1＝

Where Linker =

Where R2＝

In another embodiment, the lipid material of structure V may be as follows:

Table 1 Specific structure of lipid material V

The structural formulas of the above lipids can be obtained by combining them 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 using similar routes. The specific synthesis routes are shown in Figure 1.

2.1 Synthesis of intermediate 1

2.20 g N,N'-bis(tert-butyloxycarbonyl)-L-cystine (5 mmol), 2.68 g (10 mmol) oleyl alcohol, 1.22 g (10 mmol) 4-dimethylaminopyridine (DMAP), and 3.87 g (30 mmol) N,N-diisopropylethylamine (DIPEA) were weighed into a 100 mL eggplant flask with 15 mL of dichloromethane (DCM) as solvent. The mixture was stirred at room temperature for 20 min. 15 mL of a dichloromethane solution of 1.91 g (10 mmol) 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) was then added dropwise to the reaction flask. The reaction was allowed to proceed overnight at room temperature. After completion of the reaction, the product was purified by column chromatography to obtain 2.35 g of a 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(dd d,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

Weigh 1.88 g (2.0 mmol) of intermediate 1 into a reaction flask, add 20 mL of HCl/EA, and stir at room temperature for 4 h. After the reaction is complete, the solvent is pumped dry using an oil pump to yield 1.13 g of a white solid powder (80.7% yield).

2.3 Synthesis of intermediate 3

Weigh 2.34 g (5 mmol) of N,N'-bis(tert-butyloxycarbonyl)-L-homocystine, 2.68 g (10 mmol) of oleyl alcohol, 1.22 g (10 mmol) of DMAP, and 3.87 g (30 mmol) of DIPEA into a 100 mL flask with 15 mL of DCM as solvent. Stir at room temperature for 20 min. Then, add 15 mL of a DCM solution of 1.91 g (10 mmol) of EDCI dropwise to the reaction flask. Allow the reaction to proceed overnight at room temperature. After the reaction is complete, purify the product by column chromatography to obtain 1.98 g of a 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

Weigh 1.94 g (2.0 mmol) of intermediate 3 into a reaction flask, add 20 mL of HCl/EA, and stir at room temperature for 4 h. After the reaction is complete, the solvent is pumped dry using an oil pump to yield 1.01 g of a white solid powder (70.0% yield).

2.5 Synthesis of Compounds 1-12

2.5.1 Synthesis of Compound H1

206 mg (2.0 mmol) of dimethylglycine, 230 mg (2.0 mmol) of NHS, and 282 mg (2.0 mmol) of EDCI were weighed into a 100 mL flask with 15 mL of DCM as solvent and stirred at room temperature for 30 min. Subsequently, 740 mg of intermediate 2 was added to the reaction flask and allowed to react overnight at room temperature. After the reaction was complete, column chromatography purification was performed to obtain 457 mg of a light yellow, transparent oily liquid in a yield of 50.3%.

^1H 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). Specific NMR and mass spectra are shown in Figures 2 and 3.

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 vial with 15 mL of DCM as solvent and stirred at room temperature for 30 min. 740 mg of intermediate 2 was then added to the reaction flask and allowed to react overnight at room temperature. After completion of the reaction, the product was purified by column chromatography to yield 656 mg of a light yellow, transparent oily liquid in a 70.1% yield.

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 flask with 15 mL of DCM as solvent and stirred at room temperature for 30 min. 740 mg of intermediate 2 was then added to the reaction flask and allowed to react overnight at room temperature. After completion of the reaction, the product was purified by column chromatography to yield 544 mg of a light yellow, transparent oily liquid in 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＝6.9Hz,4H),3.10(d,J＝12.8Hz,2 H),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-ylpropionic acid, 230 mg (2.0 mmol) of NHS, and 282 mg (2.0 mmol) of EDCI were placed in a 100 mL vial with 15 mL of DCM as solvent and stirred at room temperature for 30 min. 740 mg of intermediate 2 was then added to the reaction flask and allowed to react overnight at room temperature. After completion of the reaction, the product was purified by column chromatography to yield 420 mg of a light yellow, transparent oily liquid in a 42.4% yield.

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 flask with 15 mL of DCM as solvent and stirred at room temperature for 30 min. Subsequently, 740 mg of intermediate 2 was added to the reaction flask and allowed to react overnight at room temperature. After completion of the reaction, the product was purified by column chromatography to yield 448 mg of a light yellow, transparent oily liquid in a 45.3% yield.

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＝36.5,24.3Hz,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 flask with 15 mL of DCM as solvent and stirred at room temperature for 30 min. 740 mg of intermediate 2 was then added to the flask and allowed to react overnight at room temperature. After completion of the reaction, the product was purified by column chromatography to yield 650 mg of a light yellow, transparent oily liquid in 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 vial with 15 mL of DCM as solvent and stirred at room temperature for 30 min. Subsequently, 768 mg of intermediate 4 was added to the reaction flask and allowed to react overnight at room temperature. After completion of the reaction, column chromatography purification afforded 284 mg of a light yellow, transparent oily liquid in a 30.3% yield.

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 vial with 15 mL of DCM as solvent and stirred at room temperature for 30 min. Subsequently, 768 mg of intermediate 4 was added to the reaction flask and allowed to react overnight at room temperature. After completion of the reaction, the product was purified by column chromatography to afford 391 mg of a light yellow, transparent oily liquid in a 40.5% yield.

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 flask with 15 mL of DCM as solvent and stirred at room temperature for 30 min. 768 mg of intermediate 4 was then added to the reaction flask and allowed to react overnight at room temperature. After completion of the reaction, column chromatography purification afforded 498 mg of a light yellow, transparent oily liquid in a 50.1% yield.

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-ylpropionic acid, 230 mg (2.0 mmol) of NHS, and 282 mg (2.0 mmol) of EDCI were placed in a 100 mL vial with 15 mL of DCM as solvent and stirred at room temperature for 30 min. Subsequently, 768 mg of intermediate 4 was added to the reaction flask and allowed to react overnight at room temperature. After completion of the reaction, the product was purified by column chromatography to yield 321 mg of a light yellow, transparent oily liquid in 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 flask with 15 mL of DCM as solvent and stirred at room temperature for 30 min. Subsequently, 768 mg of intermediate 4 was added to the reaction flask and allowed to react overnight at room temperature. After the reaction was complete, column chromatography purification was performed to obtain 634 mg of a light yellow, transparent oily liquid in 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 flask with 15 mL of DCM as solvent and stirred at room temperature for 30 min. 768 mg of intermediate 4 was then added to the flask and allowed to react overnight at room temperature. After completion of the reaction, the product was purified by column chromatography to yield 429 mg of a light yellow, transparent oily liquid in a 39.9% yield.

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＝14.5,7.1 Hz,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, under the guidance of this application, realize the preparation and synthesis of H1 to H36. At the same time, under the guidance of this application, the compounds in structures I to V can be easily obtained by similar synthetic routes through the methods of attaching _R2a and _R2b fatty chains, deprotection, condensation, etc.

Example 3

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

In one embodiment, a lipid material is used in combination with a nucleic acid drug to achieve nucleic acid drug delivery; the combined use includes preparation into 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, comprising mixing an aqueous solution of the nucleic acid drug with an ethanol solution of the lipid material; the mixing is preferably performed using 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 uses 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 list.

3.1 Preparation of lipid nanoparticles

ASO, siRNA, mRNA, miRNA, and/or pDNA were dissolved in 40 mM citric acid buffer at pH 4. The lipid material from Example 1 or Example 2 was mixed according to the formulation in Table 2 and dissolved in ethanol. The mixture was rapidly mixed using a microfluidic device, a high-pressure microfluidizer, a high-pressure homogenizer, and/or a T-tube mixer at a flow rate ratio of 1:3 for the aqueous phase to the alcohol phase to prepare lipid nanoparticles. The prepared lipid nanoparticles were then placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed overnight against PBS buffer at pH 7.4 to remove free small molecules and ethanol, and the pH was adjusted. The resulting lipid nanoparticles were 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 lipid nanoparticles were determined by dynamic light scattering using a Malvern Zetasizer Pro. The particle size, PDI and zeta potential of lipid nanoparticles prepared from representative lipid materials included in the present invention are shown in Table 3 and Figure 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: the sample was diluted to a concentration of about 5 μg/mL in TE buffer solution, and the RiboGreen reagent was diluted 1:200 in TE buffer. 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 fluorescence plate reader (excitation wavelength 480 nm, emission wavelength 520 nm), and the free RNA concentration was calculated. The lipid nanoparticles prepared from 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 the lipid nanoparticles was determined by fluorescence analysis of 2-(p-toluidinyl)-6-naphthalenesulfonic acid (TNS). A buffer solution (pH = 2.5-11.0) containing 150 mM sodium chloride, 10 mM sodium phosphate, 10 mM sodium citrate, and 10 mM sodium borate was prepared. The lipid nanoparticles were mixed with the buffer solution at different pH values, and then TNS was added. Fluorescence intensity was measured at room temperature using a fluorescence microplate reader at an excitation wavelength of 321 nm and an emission wavelength of 445 nm. Fluorescence data were fitted and analyzed, and the pKa was determined as the pH value that produced half-maximal fluorescence intensity. The pKa data for representative lipid nanoparticles of 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

Conclusion: The majority of lipid nanoparticles prepared in this manner were 100-250 nm in size, exhibited good assembly ability, a PDI of 0.1-0.3, a uniform particle size distribution, and excellent stability. Encapsulation efficiencies ranged from 75% to 95%, demonstrating excellent nucleic acid encapsulation capacity. Zeta potentials ranged from -15mV to +10mV, and pKa values of 5.5-7.0, near-neutral at a physiological pH of 7.4, indicating good safety. These excellent nanoparticle properties make them suitable for in vivo disease treatment and facilitate subsequent large-scale production.

Example 4

This example mainly uses commercially available cationic lipids (MC3, ALC-0315, and SM-102) 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 and cultured overnight. When the cell density reached over 80%, lipid nanoparticles containing luciferase mRNA were added to each well. After 6 hours, the fluorescence intensity of the expressed luciferase protein was measured using a luciferase assay kit and a chemiluminescence analyzer. 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 better mRNA delivery effects 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 a six-well plate at a density of 500,000 per well. After culturing for 24 hours, lipid particles containing EGFR siRNA were added to each well, with the final concentration of siRNA set to 100 nM. Subsequently, the cells were incubated in the incubator for another 24 hours for transfection. 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 12,000 g for 15 minutes at 4°C and dissolved in an appropriate amount of DEPC-treated water. An appropriate amount of RNA solution was taken, and its A280 and A260 values were measured using a NanoDrop ultra-micro spectrophotometer, and the RNA concentration was accurately read. The internal reference group and target genome were set for reverse transcription and amplification. The following parameters were set: initial denaturation at 95°C for 60 seconds; PCR cycles consisted of denaturation at 95°C for 15 seconds and extension at 60°C for 60 seconds; and finally, melting curve analysis was performed. Data processing was performed using an 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 effectiveness of lipid nanoparticles in delivering mRNA in vivo and expressing the corresponding encoded protein, 6-8 week old female BALB/c mice were injected with mRNA liposome nanoparticles expressing luciferase at a dose of 0.5 mg/kg. After 6 hours, each mouse was intraperitoneally injected with luciferase substrate, and an IVIS small animal optical in vivo imaging instrument (PerkinElme) was used to take fluorescent images of the mice and calculate the fluorescence intensity of the whole body of the mice. The level of fluorescence intensity represents the level of expression of luciferase protein, which reflects the efficiency of mRNA delivery by lipid nanoparticles in vivo. The data are shown in Figure 10.

Conclusion: Compared with the approved cationic lipid materials that show a large accumulation in the liver, lipid nanoparticles prepared from H-type lipid materials show specific spleen-targeted accumulation ability.

Example 7

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

Conclusion: Lipid nanoparticles prepared with H-type lipids are mainly transfected into 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 four groups of eight mice, half male and half female. On the sixth and tenth days after tumor inoculation, mice were intravenously injected with PBS solution, OVA protein solution (15 mg/kg), MC3-LNPs (15 mg/kg) encoding OVA mRNA, and lipid nanoparticles prepared with H-type lipids encoding OVA mRNA (15 mg/kg), respectively. Tumor size was measured daily using a digital vernier caliper (tumor volume calculation formula = 0.5 × long diameter × short diameter ² ), and tumor inhibition curves were plotted, as shown in Figure 11.

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

The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims (10)

A lipid material for nucleic acid delivery, characterized in that 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 , and R _1d are selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and n-octyl; and/or R _1a , R _1b , R _1c , and R _1d are selected from a polycyclic carbon ring, a nitrogen-containing polycyclic ring, and an oxygen-containing polycyclic ring; and/or R _1a , R _1b , R _1c , and R _1d each form a closed ring with N; L _1a , L _2a , L _1b , and 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 10-24 carbon atoms, including cholesterol derivatives and/or tocopherol derivatives. The lipid material according to claim 1, wherein the compound has one or more of the following structures:

The lipid material according to claim 1, wherein the compound has one or more of the following structures:
in
in
in
The lipid material according to claim 1, wherein the compound has one or more of the following structures:

The lipid material according to any one of claims 1 to 4, characterized in that the lipid material is prepared by a process comprising attaching R _2a and R _2b fatty chains, deprotection and/or condensation. The lipid material of claim 1, wherein the lipid material further comprises one or more of a neutral lipid, a steroid, and/or a polymer-conjugated lipid. The lipid material according to claim 6, characterized in that 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. The lipid material according to claim 1, wherein 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; and the nucleic acid drug is selected from one or more of ASO, siRNA, mRNA, miRNA, and pDNA. The lipid material according to claim 8, characterized in that the lipid material and the nucleic acid drug are prepared into lipid nanoparticles containing the nucleic acid drug, comprising mixing an aqueous solution of the nucleic acid drug with an ethanol solution of the lipid material; preferably, mixing is performed by a microfluidic device, a high-pressure microfluidizer, a high-pressure homogenizer and/or a T-tube mixer. The lipid material according to claim 1, wherein the lipid material for nucleic acid delivery is used to prepare a therapeutic drug selected from one or more of infectious diseases, tumor diseases, congenital genetic diseases and immune diseases.