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CN116554046B - Ionizable lipid compound and lipid nanoparticle thereof - Google Patents

Ionizable lipid compound and lipid nanoparticle thereof Download PDF

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CN116554046B
CN116554046B CN202310406097.5A CN202310406097A CN116554046B CN 116554046 B CN116554046 B CN 116554046B CN 202310406097 A CN202310406097 A CN 202310406097A CN 116554046 B CN116554046 B CN 116554046B
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lipid
mrna
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nucleic acid
ionizable
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CN116554046A (en
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刘滨磊
郑小迪
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Wuhan Binhui Biotech Co ltd
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Wuhan Binhui Biotech Co ltd
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Abstract

The invention discloses a novel ionizable lipid compound, enriches the types of the ionizable lipid compound, provides more choices for providing novel vaccines or medicines, such as oncolytic virus medicines, novel coronavirus vaccines or medicines and cell transfection reagents, and has the advantages of uniform size, good stability, high encapsulation efficiency, high delivery efficiency and low toxicity of the formed lipid nanoparticles, thereby having important significance for the development and application of the fields of oncolytic virus medicines, novel coronavirus vaccines or medicines, cell transfection reagents, antitumor medicines and the like.

Description

Ionizable lipid compound and lipid nanoparticle thereof
Technical Field
The invention belongs to the field of biological medicine and biotechnology, and in particular relates to an ionizable lipid compound and lipid nanoparticles thereof.
Background
Exogenous biomolecules and some drug molecules are difficult to penetrate the cell membrane to reach the cytoplasm to achieve a therapeutic effect, for example, mRNA is a high-negative-charge biomolecule, and the barrier of the cell membrane must be overcome to translate into protein to perform biological functions, so that the therapeutic application of the biomolecule is an important challenge in vivo for efficient delivery.
Lipid Nanoparticles (LNP) are a novel nucleic acid biomolecule delivery technology, which provides a protective shell for nucleic acid biomolecules, for example mRNA, which can protect mRNA from rnase decomposition and mRNA molecules from TLRs recognition, avoiding the over-activation of the innate immune system; LNP has received attention for great success as a COVID-19mRNA vaccine delivery platform.
The ionizable lipid compound can affect the overall formulation and biological properties of the LNP, is a key component in the LNP, can self-assemble with mRNA into virus-sized particles, and can release mRNA from endosomes into the cytoplasm to achieve therapeutic or immune effects.
Over the last 50 years, significant systematic research has been devoted to the design of ideal ionizable lipid compounds, some of which have been FDA approved for the delivery of nucleic acid molecules, such as the ionizable lipid compounds DLin-MC3-DMA, SM-102 and ALC-0315.
However, there is also a need for extensive research to design low-toxicity ionizable lipid compounds that are capable of efficiently delivering different types of nucleic acid molecules.
Disclosure of Invention
In order to solve the above technical problems, a novel, high delivery efficiency and low toxicity ionizable lipid compound is found, and in a first aspect, the present invention provides an ionizable lipid compound comprising an ethanolamine head group, one bromate branching tail and another bromate straight tail or branching tail, wherein the ionizable lipid compound is one or more of the compounds represented by the following structural formulas:
in a second aspect, the present invention provides a process for the preparation of an ionizable lipid compound of the first aspect, comprising the steps of:
S1, preparing a first bromate forking tail chain: the bromoacid and alcohol are subjected to condensation reaction under the action of a catalyst to form an ester bond so as to obtain a first bromoacid ester forked tail chain;
preparation of S2, substituted ((2-hydroxyethyl) amino) acid ester: nucleophilic substitution of SN2 is carried out on the ethanolamine head group and the first bromate forked tail chain obtained in the S1 under high temperature and normal pressure, and the ethanolamine head group and the first bromate forked tail chain are connected to obtain substituted ((2-hydroxyethyl) amino) acid ester;
S3, preparing a second bromate straight tail chain or a second bromate branched tail chain: the bromoacid and alcohol are subjected to condensation reaction under the action of a catalyst to form an ester bond so as to obtain a second bromoacid ester straight tail chain or branched tail chain;
S4, carrying out SN2 nucleophilic substitution reaction on the substituted ((2-hydroxyethyl) amino) acid ester prepared in S2 and the second bromo acid ester straight tail chain or the branched tail chain prepared in S3 at high temperature and normal pressure to obtain the ionizable lipid compound.
In a third aspect, the present invention provides, on the basis of the ionizable lipid compound of the first aspect, a lipid nanoparticle comprising the ionizable lipid compound of the first aspect.
The lipid nanoparticle also comprises nucleic acid molecules and auxiliary molecules; the nucleic acid molecules include one or more of RNA, mRNA, siRNA, DNA; the auxiliary molecule is at least one or more of distearoyl phosphatidylcholine, cholesterol and 1, 2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000;
In some embodiments, the nucleic acid molecule is one of an RNA virus whole genome RNA positive strand or an RNA virus whole genome RNA negative strand or Fluc-mRNA or novel coronavirus spike protein mRNA or DNA virus whole genome nucleic acid DNA or type II herpes simplex virus gD envelope glycoprotein mRNA;
In some embodiments, the molar ratio of the ionizable lipid compound to the auxiliary molecule is that of an ionizable lipid compound: distearoyl phosphatidylcholine: cholesterol: 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 was 50:10:38.5:1.5;
preferably, the particle size of the lipid nanoparticle is 60 to 100nm.
The lipid nanoparticle is capable of delivering a nucleic acid molecule to the liver or spleen.
In a fourth aspect, the present invention provides a lipid nanoparticle having oncolytic activity, comprising the ionizable lipid compound of the first aspect and the RNA viral whole genome RNA positive strand or RNA viral whole genome RNA negative strand or DNA viral whole genome nucleic acid DNA or herpes simplex virus type II gD envelope glycoprotein mRNA of the third aspect, to form a lipid nanoparticle having oncolytic activity.
In a fifth aspect, the present invention provides a method for preparing a lipid nanoparticle according to the third aspect, comprising the steps of:
S5, preparing liposome raw material solution: mixing an ionizable lipid compound with a helper molecule according to the ionizable lipid compound: distearoyl phosphatidylcholine: cholesterol: 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 was 50:10:38.5:1.5, dissolving and mixing;
S6, preparing a nucleic acid preparation: dissolving the nucleic acid molecule of the third aspect in 20-30 mM sodium acetate buffer solution with pH of 4.0-6.0, wherein the concentration of the nucleic acid molecule is about 0.06 mg/mL-0.15 mg/mL;
S7, preparation of lipid nano particles: rapidly and uniformly mixing the liposome raw material solution prepared in the step S5 and the nucleic acid preparation prepared in the step S6 to form uniform and stable lipid nano particles; the volume ratio of the liposome raw material solution to the nucleic acid preparation is 2:1 to 6:1, a step of; the total mixing speed of the liposome raw material solution and the nucleic acid preparation is 8-20 mL/min;
s8, transformation of pH environment of the lipid nanoparticle: rapidly converting the solution environment of the lipid nano-particles from pH 4.0-6.0 in S6 to pH 7.0-7.4; the lipid nanoparticle converted to pH 7.0-7.4 is concentrated to 200-400 mM.
Preferably, in some embodiments, the pH in the preparation of the S6 nucleic acid formulation is 5.0; the concentration of sodium acetate buffer was 25mM; the concentration of the nucleic acid molecules was 0.1mg/ml.
Preferably, in some embodiments, the volume ratio of liposome stock solution to nucleic acid formulation in S7 is 4:1, a step of; the total mixing rate of the liposome raw material solution and the nucleic acid preparation is 12mL/min.
In a sixth aspect, the invention provides the use of an ionizable lipid compound according to the first aspect for the preparation of a cell transfection reagent, a novel coronavirus vaccine or drug, an oncolytic virus drug or an antitumor drug.
In a seventh aspect, the present invention provides an application of the lipid nanoparticle according to the third aspect in preparing a cell transfection reagent, a novel coronavirus vaccine or drug, an oncolytic virus drug or an antitumor drug.
In an eighth aspect, the present invention provides a vaccine or medicament comprising an ionizable lipid compound according to the first aspect.
The vaccine or the medicine is injection; administration is by local intramuscular, subcutaneous, endothelial, intratumoral injection, infusion or intravenous injection.
In some embodiments, it is preferred that the mode of administration is by topical intramuscular or intravenous administration.
In a ninth aspect, the present invention provides an oncolytic virus drug comprising the lipid nanoparticle having oncolytic activity of the fourth aspect.
In a tenth aspect, the present invention provides a novel coronavirus vaccine or medicament comprising the ionizable lipid compound of the first aspect and a novel coronavirus spike protein mRNA; the ionizable lipid compound and helper molecule encapsulate novel coronavirus spike protein mRNAs.
In an eleventh aspect, the present invention provides a cell transfection reagent comprising an ionizable lipid compound of the first aspect and Fluc-mRNA; the ionizable lipid compound is packaged with a helper molecule that is Fluc-mRNA.
The invention provides a new ionizable lipid compound, enriches the types of the ionizable lipid compound, provides more choices for providing new vaccines or medicines, such as oncolytic virus medicines, novel coronavirus vaccines or medicines and cell transfection reagents, and has the advantages of uniform size, good stability, high encapsulation efficiency, high delivery efficiency and low toxicity of the formed lipid nanoparticles, thereby having important significance for the development and application of the fields of oncolytic virus medicines, novel coronavirus vaccines or medicines, cell transfection reagents, antitumor medicines and the like.
The lipid nanoparticle comprising the ionizable lipid compound provided by the invention can be used for delivering nucleic acid molecules such as Fluc-mRNA or novel coronavirus spike protein mRNA or II type herpes simplex virus gD envelope glycoprotein mRNA into a body through various administration modes such as local muscle, subcutaneous, endothelial, intratumoral, perfusion and the like, and also can be used for delivering nucleic acid molecules such as Fluc-mRNA or novel coronavirus spike protein mRNA or II type herpes simplex virus gD envelope glycoprotein mRNA into the body through systemic administration modes such as intravenous injection and the like, can even target liver or spleen, can effectively express therapeutic protein drugs or antigens in cells in the body, and plays a role in preventing or treating diseases. Furthermore, the lipid nanoparticle of the present invention is suitable for delivering nucleic acid molecules such as novel coronavirus spike protein mRNA into the body to function as a prophylactic or therapeutic vaccine.
Drawings
The present disclosure will be described in further detail below in conjunction with the drawings and preferred embodiments, but those skilled in the art will appreciate that these drawings are drawn for the purpose of illustrating the preferred embodiments only and thus should not be taken as limiting the scope of the present disclosure. Moreover, unless specifically indicated otherwise, the drawings are merely schematic representations, not necessarily to scale, of the compositions or constructions of the described objects and may include exaggerated representations.
The hydrogen profile of the compound heptadec-9-yl 8- ((2-hydroxyethyl) 6- (((9 cis, 12 cis) -octadecadien-1-yl) oxy) 6-oxyethyl) amino) octanoate of fig. 1;
the carbon spectrum of the compound heptadec-9-yl 8- ((2-hydroxyethyl) 6- (((9 cis, 12 cis) -octadecadien-1-yl) oxy) 6-oxyethyl) amino) octanoate of figure 2;
The mass spectrum of the compound heptadec-9-yl 8- ((2-hydroxyethyl) 6- (((9 cis, 12 cis) -octadecadien-1-yl) oxy) 6-oxyethyl) amino) octanoate of fig. 3;
the hydrogen profile of the compound heptadec-9-yl 8- ((3- ((6- (heptanoyloxy) hexyl) oxy) -3-oxopropyl) (2-hydroxyethyl) amino) octanoate of fig. 4;
the carbon spectrum of the compound heptadec-9-yl 8- ((3- ((6- (heptanoyloxy) hexyl) oxy) -3-oxopropyl) (2-hydroxyethyl) amino) octanoate of FIG. 5;
The mass spectrum of the compound heptadec-9-yl 8- ((3- ((6- (heptanoyloxy) hexyl) oxy) -3-oxopropyl) (2-hydroxyethyl) amino) octanoate;
the hydrogen spectrum of the compound heptadec-9-yl 8- ((2-hydroxyethyl) (8- ((2-octyldodecyl) oxy) -8-oxooctyl) amino) octanoate of fig. 7;
the carbon spectrum of the compound heptadec-9-yl 8- ((2-hydroxyethyl) (8- ((2-octyldodecyl) oxy) -8-oxooctyl) amino) octanoate of fig. 8;
FIG. 9 in vivo transfection assay live imaging;
FIG. 10 is an in vivo distribution experimental fluorescence expression diagram;
FIG. 11 is a graph showing the persistence of fluorescence expression;
FIG. 12 ELISA detection chart of lipid nanoparticle immune C57BL/6 mouse ELISA experiments;
FIG. 13 is a graph showing the results of lipid nanoparticle immune C57BL/6 mice neutralizing antibody titer detection;
FIG. 14 ELIspot spot plot of lipid nanoparticle immunization C57BL/6 mice ELISA spot assay;
FIG. 15Lipid-VI-S δT -mRNA immune C57BL/6 mouse ELISA detection IgG antibody pattern;
FIG. 16Lipid-VI-S δT -mRNA immunoSyrian hamster ELISA assay IgG antibody profile;
FIG. 17Lipid-VI-S δT -mRNA immunization of Syrian hamster ELISPot spot assay;
FIG. 18Lipid-12-S δT -mRNA immunoSyrian hamster ELISA assay IgG antibody profile;
FIG. 19Lipid-12-S δT -mRNA immunization of Syrian hamster ELISPot spot assay;
FIG. 20 shows the results of toxicity experiments on lipid nanoparticle LNP-S δT -mRNA.
Detailed Description
The present disclosure is described in detail below with reference to fig. 1 to 20.
In order to make the objects, technical solutions and advantages of the present disclosure more apparent, the present disclosure will be further described in detail with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present disclosure.
EXAMPLE 1 preparation of ionizable Lipid Compound Lipid-VI
Firstly, 8-bromooctanoic acid and heptadecane-9-alcohol undergo a condensation reaction under the action of a catalyst to form an ester bond so as to obtain the first branched tail chain heptadecane-9-yl 8-bromooctanoate. And secondly, carrying out SN2 nucleophilic substitution on the head group ethanolamine and the heptadecane-9-yl 8-bromocaprylate under high temperature and normal pressure, connecting the head group with a forked tail chain to obtain the heptadecane-9-yl 8- ((2-hydroxyethyl) amino) caprylate, and simultaneously, carrying out condensation reaction on the 6-bromohexanoic acid and the linolenol to form an ester bond to obtain a second straight tail chain [ (9 cis, 12 cis) -octadecadien-1-yl ] 6-bromohexanoic acid ester. And finally, carrying out SN2 nucleophilic substitution reaction on the forked tail chain connected with the head group and the straight tail chain at high temperature and normal pressure to obtain the Lipid-VI.
Synthetic route to ionizable Lipid compound Lipid-VI:
step 1: synthesis of heptadec-9-yl 8-bromooctanoate
8-Bromooctanoic acid (1 eq.) and heptadecan-9-ol (1.3 eq.) were added to a 25ml round bottom flask, 5ml Dichloromethane (DCM) was added, and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (1.3 eq.) 4-Dimethylaminopyridine (DMAP) (0.2 eq.), N-Diisopropylethylamine (DIPEA) (4 eq.) were added sequentially and reacted at room temperature for 42h. After the reaction is finished, the solvent is evaporated under reduced pressure in vacuum, the organic phase is extracted for 2 to 3 times by saturated sodium bicarbonate after being diluted by ethyl acetate, then the organic phase is washed by saturated sodium chloride solution, the organic phase is collected and dried by anhydrous Na2SO4, the crude product is obtained by evaporation under reduced pressure in vacuum, and petroleum ether: ethyl acetate=10:1 tlc plates, rf=0.7. Petroleum ether: ethyl acetate=150:1 was purified by column chromatography on silica gel to give a colorless oil in 50-65% yield. The hydrogen, carbon and mass spectra data for the heptadec-9-yl 8-bromooctanoate compound are shown below:
1H NMR(400MHz,CDCl3)δ4.87(dd,J＝6.3Hz,1H),3.40(t,J＝6.8Hz,2H),2.28(t,J＝7.4Hz,2H),1.90–1.80(m,2H),1.62(dd,J＝9.7,4.7Hz,2H),1.51(d,J＝5.6Hz,4H),1.46–1.39(m,2H),1.38–1.18(m,30H),0.88(t,J＝6.8Hz,6H).
13C NMR(101MHz,CDCl3)δ173.50,74.17,34.63,34.16,33.73,32.73,31.85,29.53,29.49,29.22,28.95,28.42,28.00,25.32,25.01,22.64,14.06.
MS(ESI):483.3(C25H49BrO2,M+Na+)
step 2: synthesis of heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate
Heptadec-9-yl 8-bromooctanoate (1 eq.) and ethanolamine (20 eq.) were added to a 10ml round bottom flask, 2ml ethanol was added and reacted at 62 ℃ for 48h. After the reaction is finished, cooling to room temperature, evaporating the solvent under reduced pressure in vacuum, diluting with ethyl acetate, extracting the organic phase with water for 2-3 times, washing with saturated sodium chloride solution, collecting the organic phase, drying with anhydrous Na 2SO4, evaporating under reduced pressure in vacuum to obtain a crude product, namely dichloromethane: methanol=10:1 tlc plates, R f =0.5. Silica gel column separation and purification, methanol: dichloromethane=1 to 10% gradient elution to obtain yellowish oily matter with yield of 30 to 40%. The hydrogen, carbon and mass spectra data for the compound heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate are shown below:
1H NMR(400MHz,CDCl3)δ0.82–0.93(t,J＝6.7Hz,6H),1.21–1.29(m,30H),1.33–1.36(d,J＝3.7Hz,4H),1.45–1.56(d,J＝6.1Hz,4H),2.22–2.34(t,J＝7.5Hz,2H),2.78–2.89(m,2H),2.93–3.03(t,J＝5.0Hz,2H),3.78–3.89(t,J＝5.0Hz,2H),4.81–4.91(m,1H).
13C NMR(101MHz,CDCl3)δ173.55,74.20,58.75,58.20,50.57,48.74,34.58,34.10,31.82,29.65,29.49,29.45,29.19,28.98,28.87,27.52,26.74,25.28,24.95,22.61,18.33,14.03.
MS(ESI):442.4(C27H55NO3,M+H+)。
step 3: synthesis of [ (9 cis, 12 cis) -octadecadien-1-yl ] 6-bromohexanoate
6-Bromohexanoic acid (1 eq.) and linolenic alcohol (1.3 eq.) were added to a 25ml round bottom flask, 5ml Dichloromethane (DCM) was added, and EDCI (1.3 eq.), DMAP (0.2 eq.), DIPEA (4 eq.) were added in sequence and reacted at room temperature for 42h. After the reaction is finished, the solvent is evaporated under reduced pressure in vacuum, the organic phase is extracted for 2 to 3 times by saturated sodium bicarbonate and then washed by saturated sodium chloride solution, the organic phase is collected and dried by anhydrous Na 2SO4, and the crude product is obtained by evaporation under reduced pressure in vacuum, petroleum ether: ethyl acetate=10:1 tlc plates, rf=0.7. Petroleum ether: ethyl acetate=150:1 was purified by column chromatography on silica gel to give a colorless oil in 50-60% yield.
The synthetic hydrogen spectrum, carbon spectrum and mass spectrum data of the compound [ (9 cis, 12 cis) -octadecadien-1-yl ] 6-bromohexanoate are shown as follows:
1H NMR(400MHz,CDCl3)δ0.85–0.93(t,J＝6.7Hz,3H),1.26–1.39(m,16H),1.43–1.53(m,2H),1.59–1.69(dd,J＝7.9,15.5Hz,4H),1.73–1.94(dt,J＝7.4,34.3Hz,2H),1.95–2.13(d,J＝6.9Hz,4H),2.24–2.38(t,J＝7.4Hz,2H),2.72–2.83(t,J＝6.4Hz,2H),3.35–3.45(t,J＝6.7Hz,1H),3.49–3.59(t,J＝6.7Hz,1H),4.01–4.12(t,J＝6.7Hz,2H),5.31–5.43(m,4H).
13C NMR(101MHz,CDCl3)δ173.49,130.06,127.93,64.50,44.70,34.10,33.36,32.41,31.52,29.63,29.40,29.34,29.21,28.65,27.67,27.21,26.40,25.93,25.64,24.13,22.55,14.03.
MS(ESI):465.2(C24H43BrO2,M+Na+)。
Step 4: synthesis of ionizable Lipid Compound Lipid-VI-heptadec-9-yl 8- ((2-hydroxyethyl) 6- (((9 cis, 12 cis) -octadecadien-1-yl) oxy) 6-oxyethyl) amino) octanoate
Heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate (1 eq.) was reacted with [ (9 cis, 12 cis) -octadecadien-1-yl ] 6-bromohexanoate (1.2 eq.) in a 10ml round bottom flask, DIPEA (1.2 eq.) and 2ml ethanol at 65 ℃ for 48h. After the reaction is finished, cooling to room temperature, evaporating the solvent under reduced pressure in vacuum, diluting with ethyl acetate, extracting the organic phase with water for 2-3 times, washing with saturated sodium chloride solution, collecting the organic phase, drying with anhydrous Na 2SO4, evaporating under reduced pressure in vacuum to obtain a crude product, namely dichloromethane: methanol=10:1 tlc plate detection, rf=0.6. Silica gel column separation and purification, methanol: dichloromethane=1 to 10% gradient elution to obtain yellowish oily matter with yield of 25 to 30%. The synthetic hydrogen spectrum of the compound heptadec-9-yl 8- ((2-hydroxyethyl) 6- (((9 cis, 12 cis) -octadecadien-1-yl) oxy) 6-oxyethyl) amino) caprylate is shown in figure 1, the carbon spectrum is shown in figure 2, and the mass spectrum is shown in figure 3.
1H NMR(400MHz,CDCl3)δ0.82–0.94(t,J＝6.2Hz,9H),1.16–1.40(m,50H),1.44–1.53(m,7H),1.56–1.69(dd,J＝4.8,11.1Hz,6H),2.00–2.10(q,J＝6.8Hz,4H),2.24–2.35(dt,J＝7.5,9.3Hz,4H),2.43–2.52(dt,J＝5.1,8.1Hz,4H),2.57–2.66(t,J＝5.3Hz,2H),2.72–2.83(t,J＝6.4Hz,2H),3.49–3.59(t,J＝5.3Hz,2H),4.01–4.09(t,J＝6.8Hz,2H),4.82–4.91(m,1H),5.31–5.42(tt,J＝6.2,11.3Hz,4H).
13C NMR(101MHz,CDCl3)δ173.56,130.19,128.03,74.12,64.44,58.31,55.70,53.93,53.75,34.67,34.25,34.14,31.84,31.52,29.63,29.52,29.48,29.40,29.33,29.21,28.66,27.20,26.92,25.92,25.63,25.30,25.09,24.84,22.64,22.55,14.06.
MS(ESI):804.7(C51H97NO5,M+H+)。
EXAMPLE 2 preparation of the ionizable Lipid compound Lipid-12
Firstly, 8-bromooctanoic acid and heptadecane-9-alcohol undergo a condensation reaction under the action of a catalyst to form an ester bond so as to obtain the first branched tail chain heptadecane-9-yl 8-bromooctanoate. And secondly, carrying out SN2 nucleophilic substitution on the head group ethanolamine and heptadecane-9-yl 8-bromocaprylate under high temperature and normal pressure, connecting the head group and a forked tail chain to obtain heptadecane-9-yl 8- ((2-hydroxyethyl) amino) caprylate, and simultaneously, carrying out condensation reaction on 6-bromohexanoic acid and oleyl alcohol to form an ester bond to obtain a second straight tail chain [ (9 cis) -nonadecen-1-yl ] 6-bromohexanoic acid ester. And finally, carrying out SN2 nucleophilic substitution reaction on the forked tail chain connected with the head group and the straight tail chain at high temperature and normal pressure to obtain the Lipid-12.
Step 1: synthesis of heptadec-9-yl 8-bromooctanoate
Synthesis of heptadec-9-yl 8-bromooctanoate is described in example 1, step 1 of Lipid-VI.
Step 2: synthesis of heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate
Synthesis of heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate see step 2 of example 1 Lipid-VI.
Step 3: synthesis of [ (9 cis) -nonadecen-1-yl ] 6-bromohexanoate
6-Bromohexanoic acid (1 eq.) and oleyl alcohol (1.3 eq.) were added to a 25ml round bottom flask, 5ml Dichloromethane (DCM) was added, and EDCI (1.3 eq.), DMAP (0.2 eq.), DIPEA (4 eq.) were added in sequence and reacted at room temperature for 42h. After the reaction is finished, the solvent is evaporated under reduced pressure in vacuum, the organic phase is extracted for 2 to 3 times by saturated sodium bicarbonate and then washed by saturated sodium chloride solution, the organic phase is collected and dried by anhydrous Na 2SO4, and the crude product is obtained by evaporation under reduced pressure in vacuum, petroleum ether: ethyl acetate=10:1 tlc plates, rf=0.7. Petroleum ether: ethyl acetate=150:1 was purified by column chromatography on silica gel to give a colorless oil in 50-65% yield.
1H NMR(400MHz,CDCl3)δ0.89～0.96(t,3H),1.26～1.38(m,26H),1.541.57(d,2H),1.66～1.69(d,2H),1.78～1.80(d,2H),1.94～1.97(dd,4H),2.23～2.25(d,2H),3.30～3.31(d,2H),4.06～4.08(d,2H),5.42(s,1H),5.44(s,1H).
13C NMR(101MHz,CDCl3)δ172.11,131.65,131.57,66.81,33.82,33.71,33.02,32.54,30.48,30.46,30.42,30.41,30.33,30.32,30.31,30.05,30.03,29.89,28.13,27.33,27.29,26.52,24.41,23.17,14.02.
MS(ESI):482.5(C25H47BrO2，M+Na+)。
Step 4: synthesis of Lipid-12- (heptadec-9-yl) cis-8- ((2-hydroxyethyl) (6- (nonadecen-1-yl) oxy) -6-oxyethyl) amino) octanoate
Synthesis of heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate (1 eq.) with [ (9 cis) -nonadecen-1-yl ] 6-bromohexanoate (1.2 eq.) was added to a 10ml round bottom flask, DIPEA (1.2 eq.) and 2ml ethanol were added and reacted at 65℃for 48h. After the reaction is finished, cooling to room temperature, evaporating the solvent under reduced pressure in vacuum, diluting with ethyl acetate, extracting the organic phase with water for 2-3 times, washing with saturated sodium chloride solution, collecting the organic phase, drying with anhydrous Na2SO4, and evaporating under reduced pressure in vacuum to obtain a crude product, namely dichloromethane: methanol=10:1 tlc plates, rf=0.5. Silica gel column separation and purification, methanol: dichloromethane=1 to 10% gradient elution to obtain yellowish oily matter with yield of 25 to 30%. Hydrogen, carbon, mass spectral data of Lipid-12- (heptadec-9-yl) cis-8- ((2-hydroxyethyl) (6- (nonadecen-1-yl) oxy) -6-oxyethyl) amino) octanoate are as follows:
1H NMR(400MHz,CDCl3)δ0.90～0.98(ddd,9H),1.26～1.40(m,60H),1.53～1.58(m,6H),1.66～1.70(dd,4H),1.96～1.98(dd,4H),2.14(s,1H),2.24～2.25(dd,4H),2.35～2.38(dt,4H),2.55～2.56(d,2H),3.62～3.64(d,2H),3.94(s,1H),4.07～4.10(d,2H),5.40(s,1H),5.42(s,1H).
13C NMR(101MHz,CDCl3)δ172.06,172.04,131.73,131.71,75.58,66.70,62.71,57.12,54.21,54.20,34.61,34.60,33.91,33.61,32.52,32.50,32.49,30.42,30.41,30.40,30.38,30.34,30.33,30.32,30.31,30.30,30.28,30.27,30.05,30.04,30.02,30.01,29.99,29.93,29.72,29.71,29.70,28.02,27.41,27.31,27.29,26.51,25.40,25.11,24.31,24.29,23.10,23.09,23.11,14.02,14.01,14.00.
MS(ESI)：843.4(C52H101NO5，M+Na+)。
EXAMPLE 3 preparation of the ionizable Lipid compound Lipid-13
Firstly, performing condensation reaction on 8-bromooctanoic acid and heptadecane-9-alcohol under the action of a catalyst to form an ester bond to obtain first branched tail chain heptadecane-9-yl 8-bromooctanoate;
Secondly, performing SN2 nucleophilic substitution on the head group ethanolamine and heptadec-9-yl 8-bromooctanoate at high temperature and normal pressure, and connecting the head group with a forked tail chain to obtain heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate;
then, carrying out condensation reaction on 1, 6-hexanediol and heptanoic acid to form ester bonds so as to obtain 6-hydroxyheptanoic acid hexyl ester; the 6-hydroxyheptanoic acid hexyl ester and 3-bromopropionic acid undergo a condensation reaction to form an ester bond to obtain a second straight tail 6- ((3-bromopropionyl) oxy) -heptanoic acid hexyl ester;
And finally, carrying out SN2 nucleophilic substitution reaction on the forked tail chain connected with the head group and the straight tail chain at high temperature and normal pressure to obtain the Lipid-13.
Step 1: synthesis of heptadec-9-yl 8-bromooctanoate
Synthesis of heptadec-9-yl 8-bromooctanoate is described in example 1, step 1 of Lipid-VI.
Step 2: synthesis of heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate
Synthesis of heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate see step 2 of example 1 Lipid-VI.
Step 3: synthesis of 6-hydroxyhexyl heptanoate
6-Hydroxyhexyl heptanoate was synthesized using 1, 6-hexanediol and heptanoic acid, see step 1 in the preparation of the ionizable Lipid compound Lipid-VI, example 1. The hydrogen and carbon spectrum data for the compound hexyl 6-hydroxyheptanoate are shown below:
1H NMR(400MHz,CDCl3)δ0.83–0.94(m,3H),1.23–1.44(m,10H),1.53–1.69(m,6H),1.69–1.78(s,1H),2.24–2.35(t,J＝7.5Hz,2H),3.59–3.68(t,J＝6.6Hz,2H),4.02–4.11(t,J＝6.7Hz,2H).
13C NMR(101MHz,CDCl3)δ13.85–14.08,22.34–22.55,24.85–25.04,25.27–25.47,25.61–25.83,28.51–28.71,28.71–28.89,31.32–31.52,32.47–32.68,34.27–34.47,62.58–62.84,64.09–64.30,173.93–174.13.
Step 4: synthesis of 6- ((3-bromopropionyl) oxy) -heptanoic acid hexyl ester
6- ((3-Bromopropionyl) oxy) -heptanoic acid hexyl ester was synthesized using 6-hydroxyheptanoic acid hexyl ester and 3-bromopropionic acid, synthetic method see step 3 in the preparation of ionizable Lipid compound Lipid-12 of example 2. The hydrogen and carbon spectrum data for compound 6- ((3-bromopropionyl) oxy) -heptanoic acid hexyl ester are shown below:
1H NMR(400MHz,CDCl3)δ0.84–0.94(m,3H),1.23–1.36(m,7H),1.36–1.45(p,J＝3.8Hz,4H),1.57–1.74(dq,J＝7.4,15.6Hz,6H),2.25–2.33(t,J＝7.5Hz,2H),2.88–2.95(t,J＝6.8Hz,1H),3.55–3.62(t,J＝6.8Hz,1H),4.02–4.18(dt,J＝6.6,27.5Hz,4H).
13C NMR(101MHz,CDCl3)δ13.85–14.05,22.35–22.52,24.86–25.03,25.41–25.64,25.47–25.65,25.77–25.96,28.36–28.55,28.43–28.64,28.69–28.88,31.32–31.52,34.23–34.48,37.68–37.87,63.94–64.15,64.79–64.97,76.55–76.87,76.87–77.19,77.19–77.50,173.76–173.95.
step 5: synthesis and Synthesis of heptadec-9-yl 8- ((3- ((6- (heptanoyloxy) hexyl) oxy) -3-oxopropyl) (2-hydroxyethyl) amino) octanoate
Heptadec-9-yl 8- ((3- ((6- (heptanoyloxy) hexyl) oxy) -3-oxopropyl) (2-hydroxyethyl) amino) octanoate was synthesized using heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate and 6- ((3-bromopropionyl) oxy) -heptanoate, see step 4 in the preparation of the ionizable Lipid compound Lipid-12, for a synthetic procedure. The hydrogen spectrum of the compound heptadec-9-yl 8- ((3- ((6- (heptanoyloxy) hexyl) oxy) -3-oxopropyl) (2-hydroxyethyl) amino) octanoate is shown in figure 4, the carbon spectrum is shown in figure 5, and the mass spectrum is shown in figure 6.
1H NMR(400MHz,CDCl3)δ0.86–0.90(m,9H),1.21–1.34(d,J＝14.8Hz,40H),1.37–1.41(dt,J＝3.5,7.6Hz,6H),1.49–1.53(d,J＝6.0Hz,3H),1.59–1.65(m,8H),2.25–2.32(q,J＝7.3Hz,5H),2.42–2.48(m,4H),2.55–2.61(t,J＝5.2Hz,2H),2.77–2.83(t,J＝6.8Hz,2H),3.52–3.59(t,J＝5.2Hz,2H),3.61–3.69(t,J＝6.5Hz,1H),4.03–4.11(m,5H),4.82–4.91(m,1H).
13C NMR(101MHz,CDCl3)δ13.87–14.09,13.96–14.18,22.36–22.55,22.55–22.74,24.85–25.03,25.03–25.20,25.20–25.41,25.28–25.48,25.48–25.70,25.51–25.69,25.70–25.83,26.93–27.13,27.13–27.32,28.39–28.58,28.45–28.65,28.58–28.72,28.72–28.91,29.07–29.28,29.11–29.32,29.39–29.58,29.40–29.64,31.34–31.53,31.73–31.96,32.53–32.69,32.69–32.95,34.01–34.25,34.25–34.50,34.55–34.78,49.16–49.44,53.74–54.00,55.58–55.86,58.64–58.92,62.65–62.89,63.99–64.23,64.08–64.27,64.38–64.66,74.00–74.25,172.69–172.92,173.47–173.68,173.82–174.04.
MS(ESI):726.6230(C43H83NO7,M+H+)。
EXAMPLE 4 preparation of the ionizable Lipid compound Lipid-14
Firstly, performing condensation reaction on 8-bromooctanoic acid and heptadecane-9-alcohol under the action of a catalyst to form an ester bond to obtain first branched tail chain heptadecane-9-yl 8-bromooctanoate;
Secondly, performing SN2 nucleophilic substitution on the head group ethanolamine and heptadec-9-yl 8-bromooctanoate at high temperature and normal pressure, and connecting the head group with a forked tail chain to obtain heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate;
Then, performing condensation reaction on 8-bromooctanoic acid and 2-octyldodecanol to form an ester bond to obtain a second branch tail chain 2-octyldodecyl 8-bromooctanoate;
and finally, carrying out SN2 nucleophilic substitution reaction on the forked tail chain connected with the head group and the branched tail chain at high temperature and normal pressure to obtain the Lipid-14.
Step 1: synthesis of heptadec-9-yl 8-bromooctanoate
Synthesis of heptadec-9-yl 8-bromooctanoate is described in example 1, step 1 of Lipid-VI.
Step 2: synthesis of heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate
Synthesis of heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate see step 2 of example 1 Lipid-VI.
Step 3: synthesis of 2-octyl dodecyl 8-bromooctanoate
Synthesis of 2-octyldodecyl 8-bromooctanoate Using 8-bromooctanoate with 2-octyldodecanol see step 3 of example 2 Lipid-12. The hydrogen and carbon spectrum data of the compound 2-octyl dodecyl 8-bromooctanoate are shown below ：1H NMR(400MHz,CDCl3)δ0.82–0.94(t,J＝6.7Hz,6H),1.19–1.38(m,36H),1.39–1.49(dd,J＝5.8,9.6Hz,2H),1.58–1.67(td,J＝5.6,8.6,10.0Hz,3H),1.71–1.91(dt,J＝7.3,34.5Hz,2H),2.25–2.35(t,J＝7.5Hz,2H),3.34–3.43(t,J＝6.8Hz,1H),3.49–3.56(t,J＝6.7Hz,1H),3.93–4.01(d,J＝5.8Hz,2H).
13C NMR(101MHz,CDCl3)δ13.96–14.16,22.54–22.78,24.82–25.00,26.61–26.81,27.89–28.08,28.32–28.48,28.48–28.63,28.85–29.04,28.90–29.04,29.20–29.39,29.24–29.42,29.44–29.64,29.48–29.69,29.47–29.80,29.84–30.06,31.20–31.44,31.73–32.06,32.48–32.63,32.66–32.80,33.59–33.78,34.23–34.48,37.23–37.45,44.82–45.08,67.01–67.24,76.53–76.83,76.85–77.10,77.10–77.33,77.17–77.46,173.73–174.01.
Step 4: synthesis of heptadec-9-yl 8- ((2-hydroxyethyl) (8- ((2-octyldodecyl) oxy) -8-oxooctyl) amino) octanoate
Heptadec-9-yl 8- ((2-hydroxyethyl) (8- ((2-octyldodecyl) oxy) -8-oxooctyl) amino) octanoate was synthesized using heptadec-9-yl 8- ((2-hydroxyethyl) amino) octanoate and 2-octyldodecyl 8-bromooctanoate, see step 4 of example 2 Lipid-12. The hydrogen spectrum of the compound heptadec-9-yl 8- ((2-hydroxyethyl) (8- ((2-octyldodecyl) oxy) -8-oxooctyl) amino) octanoate is shown in figure 7, and the carbon spectrum is shown in figure 8.
1H NMR(400MHz,CDCl3)δ0.83–0.93(t,J＝6.6Hz,12H),1.22–1.35(d,J＝8.8Hz,68H),1.40–1.54(dt,J＝6.7,23.0Hz,8H),1.56–1.69(t,J＝7.2Hz,5H),2.23–2.34(q,J＝7.6Hz,4H),2.39–2.50(t,J＝7.5Hz,4H),2.54–2.63(t,J＝5.4Hz,2H),3.49–3.58(t,J＝5.4Hz,2H),3.92–4.00(d,J＝5.7Hz,2H),4.81–4.92(m,1H).
13C NMR(101MHz,CDCl3)δ13.94–14.23,22.50–22.82,24.88–25.05,25.05–25.21,25.21–25.42,26.58–26.82,26.83–27.16,27.16–27.41,29.02–29.31,29.12–29.29,29.20–29.36,29.31–29.43,29.43–29.53,29.36–29.75,29.53–29.67,29.53–29.79,29.86–30.05,31.15–31.42,31.77–31.94,31.74–32.07,34.04–34.25,34.30–34.50,34.56–34.80,37.18–37.44,53.74–54.04,55.45–55.73,58.16–58.44,66.98–67.21,74.01–74.24,173.46–173.73,173.90–174.11.
Example 5 preparation of lipid nanoparticles
S5, preparing liposome raw material solution: dissolving and mixing any one or more ionizable lipid compounds of examples 1-4 with distearoyl phosphatidylcholine (DSPC), 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) and cholesterol according to a molar ratio of 50:10:1.5:38.5 respectively to obtain a liposome raw material solution, controlling the sum of the concentration of each component to be 12.5mM, completely dissolving and mixing, and then placing the liposome raw material solution in a dark place at the temperature of minus 4 ℃;
S6, preparing a nucleic acid preparation: mRNA was dissolved in 25mM sodium acetate buffer pH 5.0 to prepare a nucleic acid preparation having a final concentration of about 0.1 mg/mL;
The mRNA is one of Fluc-mRNA (firefly luciferase mRNA) or gD ED -mRNA (mRNA of II type herpes simplex virus envelope glycoprotein gD) or S δT -mRNA (mRNA of novel coronavirus spike protein).
S7, preparation (encapsulation) of lipid nanoparticles: the liposome raw material solution and the nucleic acid preparation prepared by the method are mixed uniformly rapidly by a split type direct injection pump or a vortex method under the conditions that the volume ratio of two phases is about 4:1 and the total speed of the two phases is 12mL/min, so that uniform and stable lipid nano particles are formed;
The lipid nanoparticle is a Fluc-mRNA lipid nanoparticle (LNP-Fluc-mRNA) or a gD ED -mRNA lipid nanoparticle (LNP-gD ED -mRNA) or an S δT -mRNA lipid nanoparticle (LNP-S δT -mRNA);
S8, transformation of pH environment of the lipid nanoparticle: rapidly changing the lipid nanoparticle environment from pH 5.0 to pH 7.0-7.4; specifically, the lipid nanoparticle solution prepared in S3 is diluted by a PBS buffer solution with pH of 7.2 or a Tris-HCl buffer solution with pH of about 7.4-7.6 for 10 times of volume, then concentrated by an ultrafiltration tube with 10KD, the rotation speed of a centrifuge is 4000rpm, after 2-3 liquid changes, the pH of the solution environment of the lipid nanoparticle is about 7.2-7.4, and the lipid nanoparticle solution is placed in the environment of-20 ℃ for standby.
Particle size, polydispersity index (PDI) of the Fluc-mRNA lipid nanoparticles (LNP-Fluc-mRNA) were measured using a Zetasizer Pro (Malvern, worcestershire, UK). Particle size was measured after 50-fold dilution of lipid nanoparticle solution with 0.9% sodium chloride solution; the determination of the encapsulation efficiency is carried out on a Qubit 4 fluorometer by using Qubit RNABR quantitative detection kit; the particle size, PDI, encapsulation efficiency and potential were measured as shown in table 1.
TABLE 1 physical parameters of lipid nanoparticles
As is clear from Table 1, the Lipid-VI-Fluc-mRNA Lipid nanoparticles, the Lipid-12-Fluc-mRNA Lipid nanoparticles, the Lipid-13-Fluc-mRNA Lipid nanoparticles, the Lipid-14-Fluc-mRNA Lipid nanoparticles had particle diameters in the range of 70 to 150nm, the particle diameters were uniform, the encapsulation efficiency was 90% or more, and the electric potential was-26.48 to-2.094.
Example 6 in vivo transfection experiments
LNP-Fluc-mRNA solution with pH of 7.2-7.4 after liquid change is inoculated into leg muscles of female Babl/c mice for 6-8 weeks in an intramuscular injection mode, LNP-Fluc-mRNA solution with the injection amount of 30 mu g mRNA is respectively injected into left and right hind limbs, 200 mu L (15 mg/mL) of D-fluorescein potassium salt substrate is injected into the abdominal cavity of the mice after 4 hours, and the in-vivo fluorescence expression condition of the mice is observed through a living animal imaging system (Perkinelmer) after 6-10 minutes, and the result is shown in figure 9.
As is clear from FIG. 9, in addition to liposome Lipid-13, lipid-VI, lipid-12 and Lipid-14 all achieved 1X 10 8 or more in vivo in mice, demonstrating good transfection in mice.
Example 7 in vivo distribution experiment
Intramuscular injection of LNP-Fluc-mRNA solution into each hind leg of female Balb/c mice for 6-8 weeks, with an injection amount calculated to reach 30 μg mRNA; or by intravenous injection of LNP-Fluc-mRNA solution into the tail of the mouse, the injection amount being calculated to reach 30. Mu.g mRNA. After 4h of injection, the mice were dissected and examined for fluorescent expression of organs of heart, liver, spleen, lung, kidney, and the results are shown in fig. 10, wherein fluorescent expression of lipid nanoparticles is mainly concentrated in liver and spleen, whether intramuscular Injection (IM) or intravenous Injection (IV); the fluorescence expression level of intravenous Injection (IV) is higher than that of intramuscular Injection (IM) in terms of expression intensity.
Example 8 fluorescent expression persistence experiments
Female Balb/c mice were given intramuscular LNP-Fluc-mRNA solutions to each hind leg for 6-8 weeks, calculated as 30. Mu.g mRNA. Detecting the fluorescent expression condition of mRNA in the mouse body at different time points of 4h, 8h, 24h, 48h, 72h and 96h, integrating the fluorescent intensity inside the circle and obtaining a fluorescent intensity value, and quantitatively representing the expression quantity of luciferase and drawing a bar graph. Fluorescence intensity is closely related to the delivery efficiency of liposomes, with stronger fluorescence representing higher delivery efficiency of ionizable lipid compounds.
After intramuscular injection of LNP-Fluc-mRNA solution in mice, the fluorescent expression of mRNA at different time points is shown in FIG. 11, and compared with SM-102 encapsulated Fluc-mRNA Lipid nanoparticles of Moderna company, the fluorescent expression of Lipid-VI and Lipid-14 encapsulated Fluc-mRNA Lipid nanoparticles after 24 hours is stronger than that of SM102 and can last for 96 hours, which shows that Lipid-VI liposome and Lipid-14 liposome have good mRNA protection effect.
EXAMPLE 9 lipid nanoparticle LNP-gD ED -mRNA immunized C57BL/6 mice
Referring to characterization data of Fluc-mRNA Lipid nanoparticles, lipid-VI, lipid-12, lipid-14, and SM102 were selected as positive controls for liposome encapsulation delivery of mRNA of herpes simplex virus type 2 (HSV-2) envelope glycoprotein gD (LNP-gD ED -mRNA) immunization C57BL/6 mice experiments.
LNP-gD ED -mRNA is Lipid-VI-gD ED -mRNA or Lipid-12-gD ED -mRNA or Lipid-14-gD ED -mRNA or SM102-gD ED -mRNA.
ELISA (enzyme-Linked immunosorbent assay)
Three mice were immunized on day 0, day 14 and day 28 with C57BL/6, the orbitals on day 0, 21, 35, 63 were bled, serum isolated, and IgG antibody levels in the mouse serum were detected by ELISA. mu.L of gD protein (1 ng/. Mu.L) was coated on 96-well plates and kept at 4℃overnight. Plates were washed 1 time with wash solution PBST (PBS+0.05% Tween-20) and blocked with 1% BSA for 2 hours at 37 ℃. After washing the plates 3 times, the plates were incubated with 1000, 10000, 100000, 1000000 fold diluted mouse serum for 1 hour at 37 ℃. Plates were washed 3 times, HRP-labeled goat anti-mouse IgG was added, and incubated at 37 ℃ for 1 hour. The plate was washed 5 times, developed by adding TMB in the dark, followed by the addition of 2M sulfuric acid to terminate the development, and the plate was read at an absorbance of 450 nm.
As shown in FIG. 12, the highest level of IgG antibodies in the Lipid-VI-gD ED -mRNA compared to SM102-gD ED -mRNA, indicated that Lipid-VI stimulated faster production of IgG antibodies and continued high expression in the body. Over time, the IgG antibody levels of Lipid-12-gD ED -mRNA and Lipid-14-gD ED -mRNA were comparable to SM102-gD ED -mRNA at 63 days.
Neutralizing antibody titre assay
The neutralizing antibody titer is detected by taking mouse serum from 21 st day and 35 th day in ELISA. Vero cells were seeded into 96-well plates at 2 x 10 4 cells per well in advance. The serum ratio of the rats was diluted in multiple gradients, and 4 duplicate wells were set for each dilution. Herpes simplex virus type 2 (HSV-2) virus was diluted to 100 viruses per well. Diluted serum and virus were mixed in a volume ratio of 1:1 were added to a new 96-well plate, and incubated in an incubator at 37℃with 5% CO 2 for 1 hour. The Vero cell supernatant was removed, the incubated neutralization liquid was added, and the mixture was placed in an incubator at 37℃with 5% CO 2 for 48 hours. After the culture is finished, the cells are placed in a high content living cell monitoring station to observe green fluorescence, and the titer of the neutralizing antibody is calculated according to a formula LgND 50 =L+d (s-0.5).
As shown in FIG. 13, the results of the neutralizing antibody titer experiments show that Lipid-VI can stimulate the organism to produce neutralizing antibodies earlier, and the neutralizing antibody titer is not reduced significantly with the increase of time, and after three immunization needles, the neutralizing antibody titer of Lipid-12 is equivalent to SM102, and the neutralizing antibody titer of Lipid-14 is slightly higher than SM102.
ELISA spot test (ELIspot)
Mice were sacrificed by cervical method at day 68, spleens were ground and sieved, lysis was terminated with PBS after addition of erythrocyte lysate, complete medium was added for resuspension after centrifugation of the supernatant, cell counts were performed, and single cell suspensions were seeded at 2 x 10 5 cells per well onto PVDF 96-well plates pre-coated with IFN- γ. The experimental group was stimulated with herpes simplex virus type 2 (HSV-2) (10 6 CCID 50/well) inactivated by UV light for 30min, PMA and ionomycin as positive control/complete medium (no antigen) as negative control, cells were incubated with antigen for 48h, after which the plates were washed 5 times with PBS and incubated with detection antibody (MTH 29-biotin) at 37℃for 1.5 h. Plates were washed 5 times and incubated with the addition of Streptavidin-ALP for 1 hour at 37 ℃. The plate was washed 5 times, developed by adding BCIP/NBT-plus under dark conditions, followed by washing with water injection to terminate the development. The plates were left in the shade at room temperature and naturally dried, and the number of spots was counted using an automatic ELISPot counter, as shown in FIG. 14, and the Lipid-VI-gD ED -mRNA Lipid nanoparticles, lipid-12-gD ED -mRNA Lipid nanoparticles, lipid-14-gD ED -mRNA Lipid nanoparticles, SM102-gD ED -mRNA Lipid nanoparticles were each capable of stimulating the body to produce cellular immunity, with the exception that Lipid-12-gD ED -mRNA Lipid nanoparticles produced relatively few specific spots, and the number of specific spots of other Lipid nanoparticles was comparable to that of SM102 groups, indicating comparable ability to stimulate cellular immunity.
EXAMPLE 10 lipid nanoparticle LNP-S δT -mRNA immunized C57BL/6 mice
Lipid-VI-S δT -mRNA immunized C57BL/6 mice
Lipid-VI encapsulates the mRNA of New coronal spike protein (Lipid-VI-S δT -mRNA) after immunization of C57BL/6 mice on day 0, 14 two needles, the orbitals on days 14, 21, 28 were bled, serum isolated and the IgG antibody levels in the mouse serum were detected by ELISA. The spike protein was coated on 96-well plates and kept at4℃overnight for further use. Plates were washed 1 time with PBST (PBS+0.05% Tween-20) and blocked with 1% BSA for 2 hours at 37 ℃. After washing the plates 3 times, the plates were incubated with 1000-fold diluted mouse serum for 1 hour at 37 ℃. Plates were washed 3 times, HRP-labeled goat anti-mouse IgG was added, and incubated at 37 ℃ for 1 hour. Washing the plate for 5 times, adding TMB to develop color under the dark condition, then adding 2M sulfuric acid to stop developing color, and reading the plate under the absorbance of 450 nm; ELISA detection results As shown in FIG. 15, the level of IgG antibodies in the serum of C57BL/6 mice was detected by ELISA after two needles of mice were immunized with mRNA of the Lipid-VI encapsulated novel crown spike protein, and it was found that the level of IgG expression in the mice was gradually increased as the time after the second needle was inoculated was prolonged.
EXAMPLE 11 lipid nanoparticle LNP-S δT -mRNA immunization of Syrian hamsters
Lipid-VI-S δT -mRNA immunization of syrian hamsters
Lipid-VI encapsulates the mRNA of the novel crown spike protein after immunization of syrian hamsters on day 0, day 21, hearts were bled on days 28, 35, serum was isolated and IgG antibody titers in hamster serum were detected by ELISA. The spike protein was coated on 96-well plates and kept at 4℃overnight for further use. Plates were washed 1 time with PBST and blocked with 1% bsa for 2 hours at 37 ℃. After washing the plates 3 times, the plates were incubated with hamster serum diluted at different fold for 1 hour at 37 ℃. Plates were washed 3 times, HRP-labeled rabbit anti-syrian hamster IgG added and incubated for 1 hour at 37 ℃. The plate was washed 5 times, developed by adding TMB in the dark, followed by the addition of 2M sulfuric acid to terminate the development, and the plate was read at an absorbance of 450 nm. When the OD value was greater than 2.1 times the average OD value of the negative control, the antibody titer was calculated as the highest serum dilution. As shown in FIG. 16, lipid-VI encapsulated new crown spike mRNA can generate strong humoral immune response after immunization of two needles of syrian hamster; at the same dose, igG titers were higher than or comparable to SM 102.
Hamsters were sacrificed on day 51, spleens were ground and sieved, lysis was terminated with PBS after addition of erythrocyte lysate, complete medium was added for resuspension after centrifugation of the supernatant, cell counts were performed, and single cell suspensions were seeded at 40 ten thousand cells per well onto PVDF 96-well plates pre-coated with IFN- γ. After 48h incubation of cells with antigen, the plates were washed 5 times with PBS and incubated for 1.5 hours at 37℃with the addition of detection antibody (MTH 29-biotin) using the spike protein stimulus as the experimental group and PMA and ionomycin as positive control/complete medium (no antigen) as negative control. Plates were washed 5 times and incubated with the addition of Streptavidin-ALP for 1 hour at 37 ℃. The plate was washed 5 times, developed by adding BCIP/NBT-plus under dark conditions, followed by washing with water injection to terminate the development. Placing the plate in a shade place at room temperature, naturally airing, and counting the number of spots by using an automatic ELISPot counter; the ELISPot spot detection results are shown in FIG. 17, and the Lipid-VI-S δT -mRNA can induce strong antigen-specific T cell immune response, and is obviously superior to SM102.
Lipid-12-S δT -mRNA immunization of syrian hamsters
Lipid-12 encapsulates the mRNA of the novel crown spike protein, after two needles of the syrian hamster are immunized on day 0 and day 21, hearts are taken on day 28 and day 35, serum is separated, igG antibody titer in the hamster serum is detected by ELISA, the detection method and Lipid-VI-S δT -mRNA are immunized on the syrian hamster, and the detection result is shown in FIG. 18, and a strong humoral immune response can be generated after the Lipid-12 encapsulates the mRNA of the novel crown spike protein and the two needles of the syrian hamster are immunized; at high doses, igG titers were significantly higher than SM102.
Hamsters were sacrificed on day 42 for ELISpot detection as compared to the ELISpot detection of the Lipid-VI-S delta T-mRNA immunized syrian hamsters, as shown in figure 19, and at the same dose, lipid-12 induced a stronger antigen-specific T cell immune response than SM 102.
Example 12 toxicity experiment of lipid nanoparticle LNP-S δT -mRNA
Encapsulating mRNA of new coronal spike protein (LNP-S δT -mRNA) with Lipid-VI, SM102 and ALC-0315, setting a low dose group and a high dose group, administering for 48 hours by intramuscular injection, killing animals, collecting skin and muscle of spleen, liver, heart and injection sites, embedding paraffin, slicing, staining with H & E, and detecting the Lipid nanoparticle, wherein liver of Lipid-VI-S δT -mRNA group except for the confluent area is hyperemia, the morphology of liver cells is complete, nuclei are not enlarged, and the morphology of liver lobules is complete as shown in FIG. 20; ALC-0315-S δT -mRNA myocardial interval rupture is obvious, and other organ sections have no obvious lesions, which indicates that Lipid-VI has no obvious toxicity.
The foregoing has outlined rather broadly the more detailed description of the present disclosure in order that the detailed description of the principles and embodiments of the disclosure may be implemented in order that the detailed description may be better understood, or in order that the present disclosure may be better understood. It should be noted that it would be apparent to those skilled in the art that various improvements and modifications could be made to the present disclosure without departing from the principles of the present disclosure, and such improvements and modifications would be within the scope of the claims of the present disclosure.

Claims (1)

1. The application of lipid nanoparticles in preparing a novel coronavirus vaccine or an oncolytic virus vaccine or an anti-tumor vaccine is characterized in that the lipid nanoparticles comprise an ionizable lipid compound, an auxiliary molecule and a nucleic acid molecule; wherein,
The ionizable lipid compound is one or more of compounds represented by the following structural formula:
（Lipid-VI）、（Lipid-12）、
（Lipid-13）、（Lipid-14）；
The auxiliary molecule is distearoyl phosphatidylcholine, cholesterol and 1, 2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000; the ionizable lipid compound and the helper molecule are in the form of an ionizable lipid compound: distearoyl phosphatidylcholine: cholesterol: 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000=50: 10:38.5:1.5, wherein the sum of the concentrations of the components in the liposome forming solution is 12.5 mM;
The nucleic acid molecule is mRNA of novel coronavirus spike protein or mRNA of II type herpes simplex virus gD envelope glycoprotein; the nucleic acid molecules and the liposome form lipid nano particles, wherein the volume ratio of the nucleic acid molecule solution to the liposome solution when the nucleic acid molecule solution and the liposome solution are uniformly mixed is 1:4, the total speed of the uniform mixing is 12 mL/min;
the particle size of the lipid nanoparticle is 60-100 nm.
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