CN116509816A - A kind of ionizable lipid nanoparticle and its preparation method and application - Google Patents

Abstract

The invention belongs to the field of biological medicine, and in particular relates to an ionizable lipid nanoparticle, a preparation method and application thereof. It includes ionizable lipids, helper lipids, cholesterol, and PEG lipids. The invention adjusts the structure of the ionizable lipid molecules and optimizes and screens the proportion of each component of LNP. The detection of the ionizable lipid nanoparticle of the invention finds that the particle size is mostly concentrated at 80-100nm and accords with the nano drug scale, so that the prepared LNP can smoothly pass through a cell gap, has excellent nucleic acid drug delivery performance, has an encapsulation rate of 60-95%, and has a transfection efficiency of 10-90%, and the LNP has good transfection performance.

Description

A kind of ionizable lipid nanoparticle and its preparation method and application

technical field

The invention belongs to the field of biomedicine, and specifically relates to an ionizable lipid nanoparticle and a preparation method and application thereof.

Background technique

Due to the dual characteristics of gene modification and traditional drugs, nucleic acid drugs have gradually become a hot spot in precision biomedicine and disease treatment. In recent years, with the continuous and in-depth research on disease gene-related mechanisms, nucleic acid drugs including siRNA, mRNA, plasmid DNA and other gene therapy have become a hot field for new drug development, and some of these nucleic acid drugs have been approved for marketing , for clinical treatment. Nucleic acid drugs are aimed at the post-transcriptional level of gene therapy, and there are abundant candidate targets, especially some genes whose protein targets are difficult to be drugged, with great potential for development and application.

However, nucleic acid drugs need to enter the interior of cells to better play their role in regulating gene expression. To achieve this goal, the existing technology uses lipid nanoparticles (LNP) to encapsulate and deliver RNA to specific parts of the body. LNP is mainly composed of the following four components: 1) "Ionizable (or cationic) lipid molecules" are the key to LNP technology, and its polarity will change with the change of pH value. Carrying a positive charge in the medium, it combines with electronegative nucleic acids to form nanoparticles through electrostatic interaction, which can protect nucleic acid drugs from degradation by nucleases and achieve stable delivery of nucleic acid drugs in vivo. It is an electrically neutral molecule at physiological pH, reducing toxic side effects. 2) Polyethylene glycol-modified lipid molecules. The modification of polyethylene glycol (PEG) has multiple functions, which can prevent the aggregation of LNP, make the particle size of LNP uniform, and reduce the clearance of the reticuloendothelial system. 3) Cholesterol, which increases the stability of LNP and promotes membrane fusion; 4) Phospholipid molecules, which provide structure for the LNP bilayer and assist in forming the complete structure of LNP.

In vivo, changes in the composition and ratio of LNP will have a profound impact on the delivery effect and the biological activity of mRNA in vivo. In addition, some mRNA needs to be delivered to specific tissues to effectively play its role. However, the existing LNP component schemes cannot give full play to the efficacy of mRNA-LNP preparations and are also difficult to achieve the purpose of targeted delivery. Therefore, it is necessary to continuously adjust the structure of ionizable lipid molecules and adjust the ratio of each component of LNP. Perform optimization screening.

Therefore, aiming at the above defects, the present invention proposes an ionizable lipid nanoparticle and its preparation method and application.

Contents of the invention

The purpose of the present invention is to provide an ionizable lipid nanoparticle and its preparation method and application, so as to solve the problem that the existing LNP component scheme cannot fully exert the effectiveness of the mRNA-LNP preparation and it is also difficult to achieve targeted delivery.

The technical scheme that the present invention solves the problems of the technologies described above is as follows:

A first aspect of the present invention provides an ionizable lipid nanoparticle, comprising a compound of the following formula (I):

or its isomers;

Wherein said R is -(CH ₂ ) _n CH ₃ .

As a preferred technical solution of the present invention, it also includes auxiliary lipids, cholesterol, and PEG lipids, wherein the auxiliary lipids include DMG-PEG.

As a preferred technical scheme of the present invention, the compound of the structure (I) is:

2,3-Dinonanolamidopropyl (2-(dimethylamino)ethyl) carbamate

2,3-Didecylamidopropyl (2-(dimethylamino)ethyl) carbamate

2,3-Eicosamidopropyl (2-(dimethylamino)ethyl) carbamate

2,3-Docosamidopropyl (2-(dimethylamino)ethyl) carbamate

2,3-Trisacamidopropyl (2-(dimethylamino)ethyl) carbamate

2,3-tetracosamidopropyl (2-(dimethylamino)ethyl) carbamate

2,3-Pentacosamidopropyl (2-(dimethylamino)ethyl) carbamate

2,3-Hexacosamidopropyl (2-(dimethylamino)ethyl) carbamate

Any one or any isomer of any of them.

A second aspect of the present invention provides a method for preparing the above-mentioned ionizable lipid nanoparticles, comprising the following steps:

Add 2,3-diaminopropionic acid methyl ester dihydrochloride and N,N-dimethylformamide into the flask, stir evenly, add triethylamine, add the initiator dropwise under ice bath conditions, and react at room temperature After 2 hours, TLC detected that the reaction of the raw materials was complete, and the reaction was stopped; water was added to the reaction system, followed by extraction with dichloromethane, the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to obtain the crude product , the obtained crude product is purified and separated by column chromatography to obtain product A;

Add product A and tetrahydrofuran into the reaction flask, and after cooling at -40°C for 10 minutes, add lithium borohydride solution dropwise. After 2 hours of reaction, TLC detects that the reaction of the raw materials is complete, and the reaction is stopped; water is added to the reaction system, followed by two Extract with methyl chloride, combine the organic phases, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, filter and concentrate in vacuo to obtain a crude product. The obtained crude product was purified and separated by column chromatography to obtain product B;

Add product B, pyridine and tetrahydrofuran to the reaction flask, cool in an ice bath for 10 minutes, add 4-nitrophenylcarbonyl chloride, react for 2 hours, TLC detects that the reaction of the raw materials is complete, stop the reaction; add to the reaction system water, followed by extraction with dichloromethane, combined organic phases, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to obtain product C;

Add product C, N,N-dimethylethane-1,2-diamine, triethylamine and dichloromethane into the reaction tube, stir at room temperature for 4 hours, TLC detects that the reaction of the raw materials is complete, stop the reaction, and pour into the reaction system Add water to the mixture, then extract with dichloromethane, combine the organic phases, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, filter and concentrate in vacuo to obtain a crude product, which is purified and separated by column chromatography to obtain the final product .

As a preferred technical solution of the present invention, the initiator is nonanoyl chloride, decanoyl chloride, undecanoyl chloride, dodecanoyl chloride, dodecanoyl chloride, tridecanoyl chloride, tetradecanoyl chloride, pentadecanoyl chloride , any one of hexadecanoyl chloride.

The third aspect of the present invention provides an application of ionizable lipid nanoparticles in tumor diagnosis and treatment

Beneficial effects of the present invention:

The invention adjusts the structure of the ionizable lipid molecule, and optimizes and screens the ratio of each component of the LNP. In order to realize the LNP component scheme can give full play to the effectiveness of the mRNA-LNP preparation and also better achieve the purpose of targeted delivery, by detecting the ionizable lipid nanoparticles of the present invention, it is found that the particle size is mostly concentrated in 80-100nm, It conforms to the scale of nanomedicine, indicating that the prepared LNP can pass through the intercellular space smoothly, has excellent nucleic acid drug delivery performance, and the encapsulation rate is between 60% and 95%, and the transfection rate is between 10% and 90%. Have good transfection performance.

Description of drawings

Fig. 1 is the particle size diagram of dynamic light scattering detection LNP in the embodiment 9 of the present invention;

Fig. 2 is the potential diagram of dynamic light scattering detection LNP in embodiment 9 of the present invention;

Fig. 3 is the measurement result figure of LNP encapsulation efficiency in the embodiment of the present invention 9;

FIG. 4 is a diagram showing the results of determination of LNP transfection rate in Example 9. FIG.

The technical solutions of the present invention will be described in further detail below through specific embodiments and examples.

Detailed ways

The technical solutions of the present invention are clearly and completely described below through specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

(1) Synthesis of methyl 2,3-dinonylamide propionate

Add 2,3-diaminopropionic acid methyl ester dihydrochloride (3.56mmol, 0.68g) and 50ml N,N-dimethylformamide into a 250ml round bottom flask, stir well and add triethylamine (35.56 mmol, 5 mL). Nonanoyl chloride (14.22 mmol, 3.28 mL) was added dropwise to the reaction solution in an ice bath, and reacted at room temperature for 2 hours. Thin layer chromatography (TLC) detected that the reaction of the starting material was complete, and the reaction was stopped. Add 100 mL of water to the reaction system, then extract with dichloromethane (50 mL*3), combine the organic phases, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, filter and concentrate in vacuo to obtain a crude product. The obtained crude product was purified and isolated by column chromatography (DCM/MeOH=30/1 to 10/1) to obtain methyl 2,3-dinonylamide propionate as a white solid (1.15 g, 81%). ¹ H NMR (600MHz, CDCl ₃ ) δ6.89(d, J=12Hz, 1H), 6.25(t, J=11.2Hz, 1H), 4.57(m, 1H), 3.72(s, 3H), 3.60( t, J＝9.0Hz, 2H), 2.11-2.21 (m, 4H), 1.58 (m, 4H), 1.21 (b, 20H), 0.82-0.85 (t, J＝11.2Hz, 6H) ppm; ¹³ C NMR(150MHz, CDCl ₃ )δ177.1, 145.0, 143.6, 143.5 141.5, 140.9, 136.4, 132.1, 130.2(2C), 128.5, 127.7(2C), 125.9, 123.6, 116.8, 112.4, 111.8, 71. 7, 41.8, 21.4ppm ; _HRMS (ESI): _m /z [M+H] ⁺ calcd for _{C22H43N2O4 399.5960} ; found 399.5962.

(2) Synthesis of N, N'-(3-hydroxypropane-1,2-diacyl) dinonanamide

Add 2,3-dinonylamide propionate methyl ester 1-1 (0.73mmol, 0.35g) and 10ml tetrahydrofuran into a 25ml reaction flask, and after cooling at -40°C for 10 minutes, dropwise add lithium borohydride solution (4N in THF, 0.22 mmol, 0.055 mL). After 2 hours of reaction, TLC detected that the reaction of the raw materials was complete, and the reaction was stopped. Add 15 mL of water to the reaction system, then extract with dichloromethane (20 mL*3), combine the organic phases, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, filter and concentrate in vacuo to obtain a crude product. The resulting crude product was purified and isolated by column chromatography (DCM/MeOH=30/1 to 10/1) to obtain N,N'-(3-hydroxypropane-1,2-diacyl)dinonamide as a white solid (0.21 g, 76%). ¹ H NMR (600MHz, CDCl ₃ )δ8.01(s,1H),5.52(s,1H),4.49(s,1H),3.50–3.33(m,5H),2.77(t,J＝6.6Hz, 2H),2.52(s,2H),2.23–2.17(m,2H),1.62–1.57(m,2H),1.39(m,2H),1.31–1.24(m,26H),0.89(t,J= 7.8Hz, 6H) ppm; ¹³ C NMR (125MHz, CDCl ₃ ) δ172.6, 63.7, 62.7, 47.4, 41.3, 36.5, 31.9 (2C), 30.8, 29.6, 29.3 (3C), 28.9, 28.6, 27.0, 25.6, 22.7 (2C), 14.1 (2C) ppm; HRMS _{( ESI} ): m/z [M+H]+calcd for _{C21H43N2O3 371.5860} ; found 371.5861.

(3) Synthesis of 2,3-dinonyl amidopropyl (4-nitrophenyl) carbonate

Add N, N'-(3-hydroxypropane-1,2-diacyl) dinonanamide 1-2 (0.57mmol, 0.21g), pyridine (0.74mmol, 60μL) and 5ml tetrahydrofuran into a 15ml reaction flask , after cooling in an ice bath for 10 minutes, 4-nitrophenylcarbonyl chloride (0.74 mmol, 0.15 g) was added. After 2 hours of reaction, TLC detected that the reaction of the raw materials was complete, and the reaction was stopped. Add 10 mL of water to the reaction system, then extract with dichloromethane (15 mL*3), combine the organic phases, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, filter and concentrate in vacuo to obtain the crude product 2,3-dinonamide Propyl (4-nitrophenyl) carbonate was a light yellow solid, and the crude product was directly used in the next reaction. HRMS ( _ESI ): m _/ z [M+H] ⁺ calcd for _{C21H43N2O3 536.3336} ; found 536.3337.

(4) Synthesis of 2,3-dinonyl amidopropyl (2-(dimethylamino) ethyl) carbamate

In a 10 ml reaction tube, add 2,3-disononamidopropyl (4-nitrophenyl) carbonate 1-3 (0.08 mmol, 42.8 mg), N, N-dimethyl ethane-1,2 - Diamine (0.12 mmol, 10.5 mg), triethylamine (0.24 mmol, 10 μL) and 1 mL of dichloromethane. After the reaction mixture was stirred at room temperature for 4 hours, TLC detected that the reaction of the raw materials was complete, and the reaction was stopped. Add 10 mL of water to the reaction system, then extract with dichloromethane (15 mL*3), combine the organic phases, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, filter and concentrate in vacuo to obtain a crude product. The resulting crude product was purified and separated by column chromatography (DCM/MeOH=30/1 to 10/1) to obtain the product 2,3-dinononamidopropyl (2-(dimethylamino)ethyl)carbamic acid The ester was a white solid (25 mg, 65%). ¹ H NMR (600MHz, CDCl ₃ ) δ7.17(d, J=7.8Hz, 1H), 6.89(t, J=6.6Hz, 1H), 6.03(t, J=6.6Hz, 1H), 4.21–4.11 (m,3H),3.50–3.33(m,4H),2.77(t,J＝6.6Hz,2H),2.52(s,6H),2.23–2.17(m,4H),1.62–1.57(m,4H ),1.31–1.24(m,20H),0.89(t,J＝7.8Hz,6H)ppm; ¹³ C NMR(125MHz,CDCl ₃ )δ173.7,172.6,157.6,62.3,60.3,50.7,46.7(2C), 41.7, 38.2, 36.8, 36.5, 31.9(2C), 29.3(2C), 28.9(2C), 28.6(2C), 25.6(2C), 22.7(2C), 14.1(2C)ppm; HRMS(ESI):m /z[M+H] ⁺ calcd for C ₂₈ H ₄₆ N ₃ O ₇ 536.6900; found 536.6904.

Example 2

Synthesis of 2,3-didecylamidopropyl (2-(dimethylamino)ethyl) carbamate

Using 2,3-diaminopropionic acid methyl ester dihydrochloride and decanoyl chloride as raw materials, a method similar to Example 1 can be used to prepare 2,3-didecyl amidopropyl (2-(dimethylamino) ethyl base) carbamate as a white solid (51%). ¹ H NMR (600MHz, CDCl ₃ ) δ7.15(d, J=7.8Hz, 1H), 6.90(t, J=6.6Hz, 1H), 6.03(t, J=6.6Hz, 1H), 4.21–4.11 (m,3H),3.61–3.29(m,4H),2.83(t,J＝6.6Hz,2H),2.48(s,6H),2.21–2.15(m,4H),1.68–1.43(m,4H ),1.28–1.18(m,24H),0.81(t,J＝7.8Hz,6H)ppm; ¹³ C NMR(125MHz,CDCl ₃ )δ173.8,172.5,157.8,62.4,60.1,50.9,46.5(2C), 41.5, 38.2, 36.8, 36.5, 31.8(2C), 29.1(2C), 28.5(2C), 28.3(2C), 25.5(2C), 22.8(2C), 14.2(2C) ppm; HRMS(ESI): m /z[M+H]+calcd for C ₂₈ H ₅₇ N ₄ O ₄ 513.7880; found 513.7881.

Example 3

Synthesis of 2,3-eicosamidopropyl (2-(dimethylamino)ethyl) carbamate

With 2,3-diaminopropionic acid methyl ester dihydrochloride and undecyl chloride as raw materials, the method similar to Example 1 can be used to prepare 2,3-eicosacamidopropyl (2-(di Methylamino)ethyl)carbamate as pale yellow solid (64%). ¹ HNMR (600MHz, CDCl ₃ ) δ7.23(d, J=7.8Hz, 1H), 6.79(t, J=6.6Hz, 1H), 6.10(t, J=6.6Hz, 1H), 4.31–4.14( m,3H),3.42–3.31(m,4H),2.61(t,J＝6.6Hz,2H),2.47(s,6H),2.27–2.12(m,4H),1.64–1.52(m,4H) ,1.31–1.24(m,28H),0.89(t,J=7.8Hz,6H)ppm; ¹³ C NMR(125MHz,CDCl ₃ )δ173.2,172.6,157.3,62.3,60.5,50.7,46.9(2C),41.7 ,38.3,36.7,36.5,31.9(2C),29.6(4C),29.3(2C),28.9(2C),28.6(2C),25.6(2C),22.7(2C),14.3(2C)ppm; HRMS( ESI): m/z [M+H]+calcd for C ₃₀ H ₆₁ N ₄ O ₄ 541.4963; found 541.4964.

Example 4

Synthesis of 2,3-docosamidopropyl (2-(dimethylamino)ethyl) carbamate

With 2,3-diaminopropionic acid methyl ester dihydrochloride and dodecanoyl chloride as raw materials, 2,3-docosacamidopropyl (2-(dicarbonyl) can be prepared by a method similar to Example 1 Methylamino)ethyl)carbamate as pale yellow solid (57%). ¹ HNMR (600MHz, CDCl ₃ ) δ7.16(d, J=7.8Hz, 1H), 6.84(t, J=6.6Hz, 1H), 6.08(t, J=6.6Hz, 1H), 4.35–4.09( m,3H),3.57–3.32(m,4H),2.74(t,J＝6.6Hz,2H),2.57(s,6H),2.33–2.20(m,4H),1.67–1.56(m,4H) ,1.29–1.23(m,32H),0.88(t,J=7.8Hz,6H)ppm; ¹³ C NMR(125MHz,CDCl ₃ )δ173.7,172.2,157.4,62.3,60.0,50.5,46.7(2C),41.7 ,38.3,36.8,36.5,31.9(2C),29.4(6C),29.3(2C),28.9(2C),28.6(2C),25.6(2C),22.7(2C),14.1(2C)ppm; HRMS( _ESI ): m/z [M+H]+calcd for _{C32H65N4O4 569.5006} ; found _569.5007 .

Example 5

Synthesis of 2,3-tricosamidopropyl (2-(dimethylamino)ethyl) carbamate

With 2,3-diaminopropionic acid methyl ester dihydrochloride and tridecyl chloride as raw materials, the method similar to Example 1 can be used to prepare 2,3-tricosacamidopropyl (2-(di Methylamino)ethyl)carbamate as a white solid (65%). ¹ HNMR (600MHz, CDCl ₃ ) δ7.17(d, J=7.8Hz, 1H), 6.89(t, J=6.6Hz, 1H), 6.03(t, J=6.6Hz, 1H), 4.21–4.11( m,3H),3.50–3.33(m,4H),2.77(t,J＝6.6Hz,2H),2.52(s,6H),2.23–2.17(m,4H),1.62–1.57(m,4H) ,1.31–1.24(m,36H),0.89(t,J=7.8Hz,6H)ppm; ¹³ C NMR(125MHz,CDCl ₃ )δ173.8,172.6,157.8,62.3,60.3,50.7,46.7(2C),41.7 ,38.3,36.8,36.5,31.9(2C),29.6(8C),29.3(2C),28.9(2C),28.6(2C),25.6(2C),22.7(2C),14.1(2C)ppm; HRMS( ESI): m/z [M+H]+calcd for C ₃₄ H ₆₉ N ₄ O ₄ 597.5319; found 597.5319.

Example 6

2,3-tetracosamidopropyl (2-(dimethylamino)ethyl) carbamate

With 2,3-diaminopropionic acid methyl ester dihydrochloride and tetradecanoyl chloride as raw materials, with a method similar to Example 1, 2,3-tetracosamidopropyl (2-( Methylamino)ethyl)carbamate as pale yellow solid (61%). ¹ HNMR (600MHz, CDCl ₃ ) δ7.17(d, J=7.8Hz, 1H), 6.89(t, J=6.6Hz, 1H), 6.03(t, J=6.6Hz, 1H), 4.21–4.11( m,3H),3.50–3.33(m,4H),2.77(t,J＝6.6Hz,2H),2.52(s,6H),2.23–2.17(m,4H),1.62–1.57(m,4H) ,1.31–1.24(m,40H),0.89(t,J=7.8Hz,6H)ppm; ¹³ C NMR(125MHz,CDCl ₃ )δ175.1,174.4,156.7,63.6,57.9,49.3,44.5(2C),41.0 ,37.5,36.7(2C),31.9(2C),29.8(4C),29.7(4C),29.6(2C),29.5(2C),29.4(2C),29.3(2C),25.8,25.7,22.7(2C ), 14.1 (2C) ppm; HRMS (ESI): m/z [M+H]+calcd for C ₃₆ H ₇₃ N ₄ O ₄ 625.5632; found 625.5635.

Example 7

2,3-Pentacosamidopropyl (2-(dimethylamino)ethyl) carbamate

With 2,3-diaminopropionic acid methyl ester dihydrochloride and pentadecanoyl chloride as raw materials, the method similar to Example 1 can be used to prepare 2,3-pentacosamidopropyl (2-(di Methylamino)ethyl)carbamate as a white solid (53%). ¹ HNMR (600MHz, CDCl ₃ ) δ7.14(d, J=7.8Hz, 1H), 6.79(t, J=6.6Hz, 1H), 6.10(t, J=6.6Hz, 1H), 4.27–4.10( m,3H),3.52–3.31(m,4H),2.79(t,J＝6.6Hz,2H),2.54(s,6H),2.25–2.15(m,4H),1.68–1.58(m,4H) ,1.31–1.25(m,44H),0.84(t,J=7.8Hz,6H)ppm; ¹³ C NMR(125MHz,CDCl ₃ )δ175.5,174.4,156.7,63.6,57.9,49.3,44.5(2C),41.0 ,37.5,36.7(2C),31.9(2C),29.8(4C),29.7(4C),29.6(4C),29.5(2C),29.4(2C),29.3(2C),25.8,25.7,22.7(2C ), 14.1 (2C) ppm; HRMS (ESI): m/z [M+H]+calcd for C ₃₈ H ₇₇ N ₄ O ₄ 653.5945; found 653.5947.

Example 8

2,3-Hexacetamidopropyl (2-(dimethylamino)ethyl) carbamate

Using 2,3-diaminopropionic acid methyl ester dihydrochloride and hexadecanoyl chloride as raw materials, a method similar to Example 1 can be used to prepare 2,3-hexacetamidopropyl (2-(dimethyl Amino)ethyl)carbamate as a white solid (64%). ¹ HNMR (600MHz, CDCl ₃ ) δ7.17(d, J=7.8Hz, 1H), 6.89(t, J=6.6Hz, 1H), 6.03(t, J=6.6Hz, 1H), 4.21–4.11( m,3H),3.50–3.33(m,4H),2.77(t,J＝6.6Hz,2H),2.52(s,6H),2.23–2.17(m,4H),1.62–1.57(m,4H) ,1.31–1.24(m,48H),0.89(t,J=7.8Hz,6H)ppm; ¹³ C NMR(125MHz,CDCl ₃ )δ175.0,174.3,156.8,63.5,58.0,50.1,44.9(2C),41.1 ,38.1,36.8,36.7(4C),31.9(4C),29.7(4C),29.7(2C),29.6(2C),29.4(2C),29.4,29.3(2C),29.3,25.8,25.7,22.7( 2C), 14.1 (2C) ppm; HRMS (ESI): m/z [M+H]+calcd for C ₄₀ H ₈₁ N ₄ O ₄ 681.6258; found 681.6259.

Example 9

The ionizable lipid molecules designed and synthesized in the embodiments of the present invention described above are mainly used to prepare LNP, and the specific operations are as follows:

Dissolve ionizable lipids, cholesterol, PEG, and helper lipids in ethanol, and dissolve mRNA encoding EGFP expression in citrate buffer at pH=4.0. The mixed lipid solution and mRNA solution were mixed according to the prescription in Table 1 by microfluidic method to obtain mRNA-LNP.

Synthesis of LNPs by Microfluidics

With the help of a microfluidic synthesizer (NanoAssemblr) and the accompanying microfluidic chip. After the channel of the microfluidic chip was pretreated, 900 μL of mRNA buffer solution was sucked into the syringe on one side, and 450 μL of ethanol solution containing each component was sucked into the syringe on the other side. The specific parameters are as follows: the total flow rate is 12ml/min, the aqueous phase/organic phase is 2:1, all the product LNP solutions are collected, and then diluted with 1×PBS buffer until the ethanol ratio in the solution is less than 0.5%.

In Table 1, the mRNA concentration is 1 mg/mL, the volume is 40 μL and the total volume of the aqueous phase is 900 μL, the ionizable lipid concentration is 10 mg/mL, the volume is 40 μL and the total volume of the organic phase is 450 μL, the cholesterol concentration is 5 mg/mL, The concentration of PEG was 5 mg/mL, the concentration of helper lipid was 5 mg/mL, the weight ratio of ionizable lipid to mRNA was about 10:1, and the mixing method was microfluidic. Through the 8 groups of samples formed, the following tests were carried out.

sample ionizable lipid cholesterol PEG Helper Lipid Sterol (μL) PEG (μL) DOPE (μL) Ethanol (μL) 1 1 cholesterol C14-PEG2K DOPE 50.5 8.2 48.5 152.8 2 2 cholesterol C14-PEG2K DOPE 50.5 8.2 48.5 152.8 3 3 cholesterol C14-PEG2K DOPE 50.5 8.2 48.5 152.8 4 4 cholesterol C14-PEG2K DOPE 50.5 8.2 48.5 152.8 5 5 cholesterol C14-PEG2K DOPE 50.5 8.2 48.5 152.8 6 6 cholesterol C14-PEG2K DOPE 50.5 8.2 48.5 152.8 7 7 cholesterol C14-PEG2K DOPE 50.5 8.2 48.5 152.8 8 8 cholesterol C14-PEG2K DOPE 50.5 8.2 48.5 152.8

Table 1

In order to measure the scale of LNP, the particle size and potential characterization of mRNA-LNP were carried out:

The particle size and potential of LNP were detected by dynamic light scattering using a Malvern Zetasizer Nano ZS in 90° backscatter detection mode. The results are shown in Figure 1 and Figure 2. The particle size range of LNP shown in Figure 1 is 70-110nm, most of which are concentrated in 80-100nm, which conforms to the scale of nano-drugs, indicating that the prepared LNP can pass through the intercellular space smoothly and has excellent nucleic acid drug delivery performance. The zeta potential shown in Figure 2 is between -6 and 6mV, which proves that the LNP preparation system is stable and not easy to aggregate or settle.

To measure the encapsulation efficiency of LNP, the encapsulation efficiency characterization of mRNA-LNP was performed:

Using Quant-iT ^™ RNA reagent and multimode microplate detection system Mutimode PlateReader (EnSight) were used to measure the encapsulation efficiency of LNP. Quant-iT ^TM /> RNA reagents are impermeable to LNP, so only free nucleic acids not entrapped by LNP can be bound. Triton-100 is often used as a surfactant as a demulsifier, and the LNP obtained by treating with 2% Triton-100 can release the entrapped nucleic acid and obtain the total nucleic acid amount. The drug loading amount was obtained by calculating the difference of the amount of nucleic acid before and after emulsion breaking, and then divided by the total amount of nucleic acid to obtain the encapsulation efficiency. It is measured that the encapsulation efficiency of this series of products is between 60% and 95%, and the results are shown in Figure 3.

To measure the transfection efficiency of LNP, perform the characterization of the transfection efficiency of mRNA-LNP:

Hep3B cells in the logarithmic growth phase were inoculated into 6-well cell plates (200,000 cells/well) and cultured overnight. When the cell density reached more than 80%, the medium was discarded and washed 3 times with 1X PBS, and cultured in 1 mL serum-free DMEM. The EGFPmRNA-LNP (1 μg/mL) solution prepared in the base was added to the cell wells and three replicate wells were set. After 6 hours, the medium was discarded and replaced with normal DMEM medium with serum to continue culturing for 24 hours, and the fluorescence ratio of the cells was detected by flow cytometry. The results are shown in Fig. 4, the transfection rate is between 10% and 90%, and the transfection performance is good.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (6)

1. An ionizable lipid nanoparticle comprising a compound of formula (I):

or an isomer thereof;

wherein R is- (CH) ₂ ) _n CH ₃ 。

2. The ionizable lipid nanoparticle of claim 1, further comprising a helper lipid, cholesterol, a PEG lipid, wherein the helper lipid comprises DMG-PEG.

3. The ionizable lipid nanoparticle of claim 1, wherein the compound of structure (I) is:

2, 3-dinonamide propyl (2- (dimethylamino) ethyl) carbamate,

2, 3-Didecanoamidopropyl (2- (dimethylamino) ethyl) carbamate,

2, 3-eicosamidopropyl (2- (dimethylamino) ethyl) carbamate,

2, 3-docosanamide propyl (2- (dimethylamino) ethyl) carbamate,

2, 3-Trieicosamidopropyl (2- (dimethylamino) ethyl) carbamate,

2, 3-tetracosamidopropyl (2- (dimethylamino) ethyl) carbamate,

2, 3-eicosamidopropyl (2- (dimethylamino) ethyl) carbamate,

An isomer of either or both of 2, 3-eicosamidopropyl (2- (dimethylamino) ethyl) carbamate.

4. A method for preparing the ionizable lipid nanoparticle of any one of claims 1-3, characterized in that the specific preparation steps comprise:

adding 2, 3-diamino methyl propionate dihydrochloride and N, N-dimethylformamide into a flask, uniformly stirring, adding triethylamine, dropwise adding an initiator under the ice bath condition, reacting for 2 hours at room temperature, detecting that the raw materials are completely reacted by TLC, and stopping the reaction; adding water into a reaction system, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, filtering, vacuum concentrating to obtain a crude product, and purifying and separating the obtained crude product by column chromatography to obtain a product A;

adding a product A and tetrahydrofuran into a reaction bottle, cooling at the temperature of minus 40 ℃ for 10 minutes, dropwise adding a lithium borohydride solution, reacting for 2 hours, detecting that the raw materials are completely reacted by TLC, and stopping the reaction; adding water into the reaction system, extracting with dichloromethane, combining organic phases, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, filtering, and concentrating in vacuum to obtain a crude product. Purifying and separating the crude product by a column chromatography to obtain a product B;

adding a product B, pyridine and tetrahydrofuran into a reaction bottle, cooling in an ice bath for 10 minutes, adding 4-nitrophenylcarbonyl chloride, reacting for 2 hours, detecting that the raw materials are completely reacted by TLC, and stopping the reaction; adding water into the reaction system, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, filtering, and vacuum concentrating to obtain a product C;

adding a product C, N, N-dimethylethyl-1, 2-diamine, triethylamine and dichloromethane into a reaction tube, stirring for 4 hours at room temperature, detecting that the raw materials are completely reacted by TLC, stopping the reaction, adding water into a reaction system, extracting with dichloromethane, combining organic phases, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, filtering, concentrating in vacuo to obtain a crude product, and purifying and separating the obtained crude product by a column chromatography to obtain a final product.

5. The method for preparing the ionizable lipid nanoparticle according to claim 4, wherein the initiator is any one of nonanoyl chloride, decanoyl chloride, undecanoyl chloride, dodecanoyl chloride, tridecanoyl chloride, tetradecanoyl chloride, pentadecanoyl chloride, hexadecanoyl chloride.

6. Use of the ionizable lipid nanoparticle according to any one of claims 1-3 for the diagnosis and treatment of tumors.