CN117603298A - Multi-tail ionizable oligopeptide lipid and application thereof in RNA delivery - Google Patents
Multi-tail ionizable oligopeptide lipid and application thereof in RNA delivery Download PDFInfo
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- CN117603298A CN117603298A CN202311630927.9A CN202311630927A CN117603298A CN 117603298 A CN117603298 A CN 117603298A CN 202311630927 A CN202311630927 A CN 202311630927A CN 117603298 A CN117603298 A CN 117603298A
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- oligopeptide
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- XHFLOLLMZOTPSM-UHFFFAOYSA-M sodium;hydrogen carbonate;hydrate Chemical class [OH-].[Na+].OC(O)=O XHFLOLLMZOTPSM-UHFFFAOYSA-M 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 150000003512 tertiary amines Chemical group 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
Abstract
The invention discloses multi-tail ionizable oligopeptide lipids and their use in RNA delivery. The invention discloses multi-tail chain ionizable oligopeptide lipid, which takes tertiary amine groups as head groups, oligopeptide as connecting chains and three or four alkane chains as hydrophobic tail chains. The membrane destabilization of cell endosomes can be mediated by the protonated tertiary amine head group, and the endosome escape of the constructed lipid nanoparticle can be accelerated by means of the enhancement of membrane fusion of a plurality of alkane tail chains, so that the transfection efficiency is improved. The multi-tail chain ionizable oligopeptide lipid has the advantages of good biocompatibility, high safety, convenient synthesis method, low phase transition temperature, strong membrane fusion capability and the like, and the constructed lipid nanoparticle has a better RNA transfection effect and is superior to lipofectamine2000 which is a commercially available transfection reagent. The invention provides a novel oligopeptide lipid material, and provides a safe and effective non-viral vector platform for RNA delivery.
Description
Technical Field
The invention relates to the field of chemistry and preparations, in particular to multi-tail ionizable oligopeptide lipid and application thereof in RNA delivery.
Background
RNA drugs have wide application prospects in various gene-related diseases, and are currently in a high-speed development stage. However, RNA drugs have limitations such as easy clearance, easy degradation by nucleases, difficulty in acting across cell membranes and the like due to their hydrophilicity and electronegativity. Thus, there is a need to develop safe and efficient delivery vehicles to improve the patentability of RNA.
Common RNA gene delivery vectors mainly include viral vectors and non-viral vectors. Although the transfection efficiency of the viral vector is high, the defects of potential safety hazard, small loading capacity, high cost and the like limit the clinical production and application. The non-viral vector has the advantages of good safety, low cost, large loading capacity and the like, and is widely paid attention to. However, its low transfection efficiency is a major reason for limiting its development. The lipid nanoparticle (lipid nanoparticle, LNP) is a non-viral vector composed of ionizable lipid, neutral phospholipid, cholesterol and polyethylene glycol lipid, and the first siRNA systemic infusion drug (Onpattro) obtained in the first global model is to use LNP as a delivery vector, shows good safety and effectiveness, and is one of the most potential gene delivery vectors at present.
LNP, when taken up by cells, localizes to intracellular endosomes (pH 5.0-6.0) and the endosomes are further acidified to become lysosomes (pH 4.5-5.0). If LNP cannot escape from endosomes (endosomes or lysosomes) into the cytoplasm rapidly, the loaded RNA drug will be degraded by enzymes in the endosome and will fail, which is one of the key causes of low LNP transfection efficiency. Although LNP has made a breakthrough in siRNA and mRNA delivery, FDA has approved LNP with three ionizable lipid constructs MC3, SM-102 and ALC-0315 to be marketed, but studies have found that LNP transfection efficiency is still inadequate. The main reason is that after LNP is taken up by cells into endosomes (endosomes or lysosomes), only very little RNA can smoothly escape into cytoplasm to play a role, and the endosome escape efficiency is only 2% -15%. Therefore, increasing the endosomal escape efficiency of LNP is critical to increasing its transfection efficiency.
In addition, the degradability of ionizable lipids in vivo is also closely related to the in vivo safety of LNP, and increasing its biodegradability is of great importance for improving the safety of its in vivo application.
Disclosure of Invention
In view of the above-mentioned shortcomings in the art, the present invention aims to provide a multi-tail ionizable oligopeptide lipid, so as to enhance the escape capability of endosomes constructing LNP and improve the safety of LNP.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the first aim of the invention is to provide a multi-tail chain ionizable oligopeptide lipid, which has the structure shown in the general formula (I).
Wherein p=1 or 2;
q=an integer from 1 to 3;
r=an integer from 1 to 3;
wherein u represents an integer of 1 to 19;
preferably, in the multi-tail ionizable oligopeptide lipid, p=2; q=1; r=3; r1 and R2 are selected fromu=an integer of 7 to 10; r3 is selected from->u=7-10An integer; r4 is selected from->
The multi-tail chain ionizable oligopeptide lipid provided by the invention takes tertiary amine groups with pKa of 8-9 as head groups, so that the lipid can be rapidly protonated in early endosomes (pH 5.0-6.0), and RNA degradation is prevented from being accelerated due to long-term retention in the endosomes; the dipeptide formed by oligopeptide (glutamic acid (or aspartic acid) and lysine (or ornithine) is used as a connecting chain, and the ester bond is used as a connecting bond, so that the biocompatibility and biodegradability of the polypeptide are improved; three or four hydrophobic alkanes are used as tail chains, and the formation of inverted hexagonal phase is facilitated by increasing the volume of the tail of the ionizable lipid, so that the escape of endosomes is accelerated.
The second object of the present invention is to provide a method for synthesizing the above multi-tail ionizable oligopeptide lipid. The synthesis method is efficient and quick in synthesis, high in yield, environment-friendly in synthesis process and suitable for industrial scale-up production.
The synthesis method of the multi-tail chain ionizable oligopeptide lipid shown in the general formula I comprises the following synthesis steps:
a. mixing the dicarboxylic amino acid (I-1) with anhydrous toluene, adding p-toluenesulfonic acid monohydrate under stirring, heating in an oil bath to 100-160 ℃, and carrying out reflux reaction for 1-6 h. After cooling to room temperature, adding fatty alcohol, and continuing reflux reaction at 100-160 ℃ for overnight. Toluene was removed by rotary evaporation at 60℃to give the crude product. The crude product is redissolved in dichloromethane, washed by proper amount of water, saturated sodium bicarbonate water solution and saturated saline solution in sequence, and the organic phase is dried by anhydrous sodium sulfate, filtered and concentrated by suction, and purified by petroleum ether/ethyl acetate column chromatography to obtain the fatty alcohol-dicarboxylic amino acid (I-2).
Fatty alcohol-dicarboxylic amino acid (I-2) synthesis reaction formula:
b. dissolving N-boc-N' -fmoc-binary amino acid in dichloromethane, sequentially adding EDCI and HOBt at room temperature, and stirring for 0.5-3 h to obtain a reaction solution A; dissolving fatty alcohol-dicarboxylic amino acid (I-2) and methylene dichloride, and adding triethylamine at room temperature to obtain a reaction solution B. Reaction solution B was added dropwise to reaction solution a, and stirred at room temperature overnight. After the reaction, washing with a proper amount of water, 10% (w: w) citric acid aqueous solution and saturated saline water in turn, drying by anhydrous sodium sulfate, carrying out suction filtration and concentration, and purifying by petroleum ether/ethyl acetate column chromatography to obtain the N-boc-N' -fmoc-diamino amino acid-fatty alcohol-dicarboxylic amino acid (I-3).
N-boc-N' -fmoc-diamino amino acid-fatty alcohol-dicarboxylic amino acid (I-3) synthesis reaction formula:
c. dissolving N-boc-N' -fmoc-diamino amino acid-fatty alcohol-dicarboxylic amino acid (I-3) in methylene dichloride, dropwise adding trifluoroacetic acid at room temperature, and stirring for reaction for 0.5-3 h. After the reaction is finished, washing the mixture with a proper amount of water, saturated sodium bicarbonate solution and saturated saline water in sequence, drying the mixture with anhydrous sodium sulfate, concentrating and filtering the mixture to obtain Fmoc-protected binary amino acid-fatty alcohol-binary carboxyl amino acid (I-4).
Fmoc protected diamino amino acid-fatty alcohol-dicarboxylic amino acid (I-4) synthesis reaction formula:
d. dissolving fatty alcohol in dichloromethane, sequentially adding p-nitrophenyl chloroformate and DMAP at room temperature, and stirring at room temperature for reacting for 2-6 h to obtain a reaction solution A; fmoc-protected diamino amino acid-fatty alcohol-dicarboxylic amino acid (I-4) was dissolved in dichloromethane and triethylamine was added to obtain a reaction solution B. Reaction solution B was added dropwise to reaction solution a, and stirred at room temperature overnight. After the reaction is finished, washing the mixture with a proper amount of water, 10% (w: w) citric acid aqueous solution and saturated saline water in sequence, drying the mixture with anhydrous sodium sulfate, carrying out suction filtration and concentration, and purifying the mixture by petroleum ether/ethyl acetate column chromatography to obtain the Fmoc-protected fatty alcohol-dipeptide (I-5).
Fmoc protected fatty alcohol-dipeptide (I-5) Synthesis reaction formula:
e. fmoc-protected fatty alcohol-dipeptide (I-5) was dissolved in dichloromethane, 20% piperidine was added dropwise at room temperature, and the reaction was stirred at room temperature for 1-5 h. After the reaction, washing with a proper amount of water, 10% (w: w) citric acid aqueous solution and saturated saline water in sequence, drying with anhydrous sodium sulfate, carrying out suction filtration and concentration, and purifying by petroleum ether/ethyl acetate column chromatography to obtain the dipeptide-fatty alcohol (I-6).
Fatty alcohol-dipeptide (I-6) synthesis reaction formula:
f. the fatty alcohol-dipeptide (I-6) was dissolved in methylene chloride, and dibasic acid anhydride, triethylamine and DMAP were added in this order at room temperature to react overnight at room temperature. After the completion of the reaction, the mixture was washed with an appropriate amount of water, 10% (w: w) aqueous citric acid solution and saturated brine in this order, dried over anhydrous sodium sulfate, and concentrated by suction filtration to obtain carboxylated-fatty alcohol-dipeptide (I-7).
Carboxylated-fatty alcohol-dipeptide (I-7) synthesis reaction formula:
g. dissolving carboxylation-fatty alcohol-dipeptide (I-7) in dichloromethane, sequentially adding EDCI and HOBt at room temperature, and stirring for 1-3 h to obtain a reaction solution A; dissolving R4H in dichloromethane, and adding triethylamine at room temperature to obtain a reaction solution B; reaction solution B was slowly added dropwise to reaction solution a, and stirred at room temperature overnight. After the reaction, washing with a proper amount of water, 10% (w: w) citric acid aqueous solution and saturated saline water in sequence, drying with anhydrous sodium sulfate, filtering and concentrating, and purifying by dichloromethane/methanol column chromatography to obtain the multi-tail chain ionizable oligopeptide lipid (I).
Multi-tail ionizable oligopeptide lipid (I) synthesis reaction formula:
the third object of the invention is to provide an application of multi-tail chain ionizable oligopeptide lipid in preparing RNA-entrapped lipid nanoparticles. The RNA-entrapped lipid nanoparticle comprises multi-tail ionizable oligopeptide lipid, neutral phospholipid, cholesterol and polyethylene glycol lipid.
The molar ratio of the multi-tail chain ionizable oligopeptide lipid to the neutral phospholipid to the cholesterol to the polyethylene glycol lipid in the RNA-entrapped lipid nanoparticle is 10-40:10-60:0.5-10.
The neutral phospholipid is selected from one or more of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), dimyristoyl phosphatidylcholine (DMPC), soybean Phospholipid (SPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1-palmitoyl-2-oleoyl lecithin (POPC), dithianoyl lecithin (DEPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and dipalmitoyl phospholipid (DPPC).
The tail of the PEGylated lipid includes, but is not limited to, a length C 6 -C 20 Saturated or unsaturated alkane chains of (2) such as 1, 2-dimyristoyl-rac-glycerol-3-methoxy aggregation diol 2000 (DMG-PEG) 2000 ) Distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG) 2000 ) Cholesterol-polyethylene glycol 2000 (Chol-PEG) 2000 ) Bitetradecyl (4-methoxypolyethylene glycol-2000-4-oxobutanoyl) glutamic acid (PEG) 2000 -Suc-TA 2 ) Etc.
The RNA is selected from siRNA, shRNA, microRNA or mRNA, etc.
The ratio of nitrogen to phosphorus of the ionizable lipid to RNA is 1:1-30:1.
The lipid nanoparticle for encapsulating RNA has an average particle size of 100-1000 nm and a surface potential of-20 mV to +40 mV.
Preferably, the molar ratio of the multi-tail ionizable oligopeptide lipid to the neutral phospholipid to the cholesterol to the polyethylene glycol lipid is 20-35:20-40:0.5-2.5.
Preferably, the neutral phospholipid is selected from DOPE or DSPC.
Preferably, the pegylated lipid is selected from PEG 2000 -Suc-TA 2 。
Preferably, the RNA is selected from siRNA or mRNA.
Preferably, the ratio of nitrogen to phosphorus of the ionizable lipid to RNA is 3:1 to 11:1.
Preferably, the RNA-entrapped lipid nanoparticle has an average particle size of 100 to 300nm, a surface potential of-10 mV to +20 mV.
The fourth object of the present invention is to provide a method for preparing lipid nanoparticles by using the multi-tail ionizable oligopeptide lipid. The preparation method comprises the following steps: ethanol injection, microfluidic, T-tube mixing, or extrusion through a membrane.
Preferably, the preparation method of the lipid nanoparticle is selected from ethanol injection method or microfluidic method.
The process for preparing the lipid nanoparticle by the ethanol injection method comprises the following steps: weighing a proper amount of multi-tail chain ionizable oligopeptide lipid, neutral phospholipid, cholesterol and polyethylene glycol lipid, dissolving in absolute ethanol, and mixing according to a molar ratio to obtain an ethanol phase. At the same time, RNA was dissolved in 10mM citrate buffer (ph=4.0) as an aqueous phase. Under vigorous stirring, the ethanol phase is rapidly and uniformly injected into the water phase, and the volume ratio of the water phase to the ethanol phase is 1:1-4:1. After the injection is completed, the lipid nanoparticle solution can be obtained by dialysis with ultrapure water for 2 to 4 hours at room temperature, and the lipid nanoparticle solution is preserved at the temperature of 4 ℃ for standby.
The process for preparing the lipid nanoparticle by the microfluidic method comprises the following steps: weighing a proper amount of multi-tail chain ionizable oligopeptide lipid, neutral phospholipid, cholesterol and polyethylene glycol lipid, dissolving in absolute ethanol, and mixing according to a molar ratio to obtain an ethanol phase. RNA was dissolved in 10mM citrate buffer (ph=4.0) as an aqueous phase. Through the micro-fluidic device, the two phases are mixed in the micro-fluidic chip at the flow rate of 1-20 mL/min and the ratio of 1:1-4:1, the mixed solution is collected, the mixed solution is dialyzed for 2-4 h by ultrapure water at room temperature, and the lipid nanoparticle solution can be obtained and is preserved at the temperature of 4 ℃ for standby.
A fifth object of the present invention is to provide an application of delivering RNA-entrapped lipid nanoparticles to target cells for gene transfection.
The target cells are selected from primary immune cells or tumor cells, wherein the immune cells comprise T cells, neutrophils, macrophages, dendritic cells and the like, and the tumor cells comprise cervical cancer Hela cells, breast cancer MCF-7 cells, pancreatic cancer PANC-1 cells, liver cancer HepG2 cells, lung cancer A549 cells and the like.
The gene transfection effect comprises siRNA gene silencing effect, shRNA gene silencing effect, microRNA gene silencing effect, mRNA expression effect or CRISPR/Cas9 gene editing application.
Compared with the prior art, the invention has the following technical effects:
the multi-tail chain ionizable oligopeptide lipid provided by the invention has the advantages of good safety, strong membrane fusion capability, low phase transition temperature and the like, and is beneficial to improving the endosome escape efficiency of LNP.
The RNA-entrapped lipid nanoparticle constructed by the multi-tail-chain ionizable oligopeptide lipid has better gene transfection capability on different cells. Solves the problem of low cell transfection efficiency of the existing non-viral vector to a certain extent, and has important significance for developing gene delivery vectors with independent intellectual property rights in China.
Drawings
FIG. 1 is an agarose gel electrophoresis chart of lipid nanoparticle MFNP loaded with siRNA in example 11;
FIG. 2 is an agarose gel electrophoresis of lipid nanoparticle loaded mRNA (N/P=3, 5, 7) in example 12;
FIG. 3 is an agarose gel electrophoresis of lipid nanoparticle-loaded microRNAs (N/P=3, 5, 7) in example 13;
FIG. 4 is an in vitro safety evaluation of lipid nanoparticles in example 14;
FIG. 5 is the in vitro stability of lipid nanoparticles MF2NP, MF5NP in example 15;
FIG. 6 is the membrane fusion ability of the multi-tail ionizable oligopeptide lipid of example 16;
FIG. 7 is the phase inversion ability of the multi-tail ionizable oligopeptide lipid of example 17;
FIG. 8 shows the expression of green fluorescent protein (eGFP) after transfection of human cervical cancer Hela-eGFP cells with siRNA with lipid nanoparticles in example 18 (inverted fluorescence microscope, scale: 100 μm);
FIG. 9 shows the expression of green fluorescent protein (eGFP) after transfection of human cervical cancer Hela-eGFP cells with siRNA loaded with lipid nanoparticles in example 18 (flow cytometry);
FIG. 10 shows the expression of green fluorescent protein (eGFP) after transfection of the lipid nanoparticle-loaded mRNA into human primary T cells in example 19 (inverted fluorescence microscope, scale: 100 μm);
FIG. 11 shows green fluorescent protein (eGFP) expression (flow cytometry) following transfection of the lipid nanoparticle-loaded mRNA into human primary T cells in example 19;
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the invention in any way.
Example 1 preparation of Multi-tail ionizable oligopeptide lipid MF1
MF1 starting from compound I-7 (1 g,1.10 mmol) and 1- (3-aminopropyl) -4-methylpiperazine (337 mg,1.76 mmol). I-7 (1 g,1.10 mmol) was dissolved in methylene chloride, EDCI (337 mg,1.76 mmol) and HOBt (238 mg,1.76 mmol) were added in this order at room temperature and stirred for 1.5h to give a reaction solution A; 1- (3-Aminopropyl) -4-methylpiperazine (337 mg,1.76 mmol) was dissolved in dichloromethane, and triethylamine (222 mg, 2) was added at room temperature20 mmol) to obtain a reaction solution B; reaction solution B was slowly added dropwise to reaction solution a, and stirred at room temperature overnight. After the completion of the reaction, the reaction mixture was washed twice with an appropriate amount of water, twice with an aqueous solution of citric acid (10% (w: w)) and once with saturated brine, dried over anhydrous sodium sulfate, and concentrated by suction to give a crude product as a clear oil, which was purified by column chromatography (dichloromethane: methanol=5:1) to give 760mg of the product as a white powder. Yield: 65.9%. 1 H NMR(300MHz,CDCl 3 ):δ(ppm)8.0(m,2H,CONHCH),7.5(m,1H,CONHCH 2 ),4.51(m,1H,NHCHCO),4.47(m,1H,COOCH),4.44(m,1H,NHCHCO),4.13-4.06(t,4H,COOCH 2 ),3.42-3.18(m,2H,NHCH 2 CH 2 ),2.50(m,2H,COCH 2 CH 2 ),2.36-2.34(m,6H,NHCOCH 2 ),2.29(m,10H,NCH 2 ),2.14(m,3H,NCH 3 ),1.77(m,2H,NHCHCH 2 ),1.73(m,2H,NHCH 2 CH 2 ),1.60(dd,4H,COOCH 2 CH 2 ),1.49(dd,4H,COOCHCH 2 ),1.47-1.26(m,46H,CH 2 (myristoyl)),0.89(t,J＝6.9Hz,12H,CH 2 CH 3 ).HRMS,ESI + ,m/z:Calcd for C 59 H 112 N 6 O 9 [M+H] + ,1049.8561；found,1049.8545.
Example 2 preparation of Multi-tail ionizable oligopeptide lipid MF2
MF2 starting from I-7 (1 g,1.10 mmol) and 1- (3-aminopropyl) piperidine (156 mg,1.10 mmol), the process for preparing MF1 gives 678mg as a white powder. Yield: 66.7%.1HNMR (300 MHz, CDCl) 3 ):δ(ppm)8.0(m,2H,CONHCH),7.5(m,1H,CONHCH 2 ),4.51(m,1H,NHCHCO),4.47(m,1H,COOCH),4.44(m,1H,NHCHCO),4.13-4.06(t,4H,COOCH 2 ),3.42-3.18(m,4H,NHCH 2 CH 2 ),2.50(m,6H,NCH 2 ),2.36-2.34(m,6H,NHCOCH 2 ),2.29(m,2H,NHCHCH 2 ),1.77(m,2H,NHCHCH 2 ),1.73(m,2H,NHCH 2 CH 2 ),1.60(dd,4H,COOCH 2 CH 2 ),1.49(m,4H,NCH 2 CH 2 ),1.49(dd,4H,COOCHCH 2 ),1.47-1.26(m,48H,CH 2 (myristoyl)),0.89(t,J＝6.9Hz,12H,CH 2 CH 3 ).HRMS,ESI + ,m/z:Calcd for C 59 H 111 N 5 O 9 [M+H] + ,1034.8477；found,1049.8352.
Example 3 preparation of Multi-tail ionizable oligopeptide lipid MF3
MF3 starting from I-7 (1 g,1.10 mmol) and N-methyl-2- (2-aminoethyl) -pyrrolidine (170 mg,1.10 mmol), the process for preparing MF1 afforded 523mg as a clear oil. Yield: 62.3%. 1 H NMR(300MHz,CDCl 3 ):δ(ppm)8.0(m,2H,CONHCH),7.5(m,1H,CONHCH 2 ),5.49(m,1H,CONHCH),4.51(m,1H,NHCHCO),4.47(m,1H,COOCH),4.44(m,1H,NHCHCO),4.13-4.06(t,4H,COOCH 2 ),3.42-3.18(m,2H,NHCH 2 CH 2 ),2.40(m,4H,NCH 2 ),2.36-2.34(m,8H,NHCOCH 2 ),2.18(m,3H,NCH 3 ),1.77(m,2H,NHCHCH 2 ),1.69(m,4H,NHCHCH 2 ),1.60(dd,4H,COOCH 2 CH 2 ),1.53(dd,2H,NHCHCH 2 ),1.49(m,4H,NCH 2 CH 2 ),1.47-1.26(m,46H,CH 2 (myristoyl)),0.89(t,J＝6.9Hz,12H,CH 2 CH 3 ).HRMS,ESI + ,m/z:Calcd for C 56 H 105 N 5 O 9 [M+H] + ,992.7941；found,992.7926.
Example 4 preparation of Multi-tail ionizable oligopeptide lipid MF4
MF4 starting from I-7 (1 g,1.10 mmol) and 1-methylpyrrolidin-3-amine (180 mg,1.10 mmol), the process for preparing MF1 gives 660mg of the product as a white powder. Yield: 62.5%. 1 H NMR(300MHz,CDCl 3 ):δ(ppm)8.0(m,2H,CONHCH),7.5(m,1H,CONHCH 2 ),5.49(m,1H,CONHCH),4.51(m,1H,NHCHCO),4.47(m,1H,COOCH),4.44(m,1H,NHCHCO),4.13-4.06(t,4H,COOCH 2 ),3.69(m,1H,NHCHCH 2 ),3.42-3.18(m,2H,NHCH 2 CH 2 ),2.40(m,4H,NCH 2 CH 2 ),2.36-2.34(m,8H,NHCOCH 2 ),2.18(m,3H,NCH 3 ),1.77(m,2H,NHCHCH 2 ),1.69(m,2H,NHCH 2 CH 2 ),1.60(dd,4H,COOCH 2 CH 2 ),1.53(dd,4H,NHCHCH 2 ),1.49(m,4H,NCH 2 CH 2 ),1.47-1.26(m,46H,CH 2 (myristoyl)),0.89(t,J＝6.9Hz,12H,CH 2 CH 3 ).HRMS,ESI + ,m/z:Calcd for C 56 H 105 N 5 O 9 [M+H] + ,992.7941；found,992.7926.
Example 5 preparation of Multi-tail ionizable oligopeptide lipid MF5
MF5 starting from I-7 (1 g,1.10 mmol) and 3-diethylaminopropylamine (140 mg,1.10 mmol), the process for preparing MF1 afforded 678mg as a clear oil. Yield: 60.4%. 1 H NMR(300MHz,CDCl 3 ):δ(ppm)8.0(m,2H,CONHCH),7.5(m,1H,CONHCH 2 ),4.51(m,1H,NHCHCO),4.47(m,1H,COOCH),4.44(m,1H,NHCHCO),4.13-4.06(t,4H,COOCH 2 ),3.42-3.18(m,4H,NHCH2CH 2 ),3.01(m,2H,NCH 2 CH 3 ),2.50(m,2H,COCH 2 CH 2 ),2.36-2.34(m,8H,NHCOCH 2 ),1.77(m,2H,NHCHCH 2 ),1.73(m,2H,NHCH 2 CH 2 ),1.60(dd,4H,COOCH 2 CH 2 ),1.53(dd,2H,NHCHCH 2 ),1.49(m,4H,NCH 2 CH 2 ),1.47-1.26(m,48H,CH 2 (myristoyl)),0.89(t,J＝6.9Hz,12H,CH 2 CH 3 ).HRMS,ESI + ,m/z:Calcd for C 56 H 105 N 5 O 9 [M+H] + ,1022.8436；found,1022.8471.
Example 6 preparation of Multi-tail ionizable oligopeptide lipid MF6
MF6 starting from I-7 (1 g,1.10 mmol) and N- (3-aminopropyl) diethanolamine (180 mg,1.10 mmol), the process for preparing MF1 gave 553mg of the product as a white powder. Yield: 52.8%. 1 H NMR(300MHz,CDCl 3 ):δ(ppm)8.0(m,2H,CONHCH),7.5(m,1H,CONHCH 2 ),4.51(m,1H,NHCHCO),4.47(m,1H,COOCH),4.44(m,1H,NHCHCO),4.13-4.06(t,4H,COOCH 2 ),3.42(m,6H,CH 2 CH 2 OH),3.18(m,2H,NHCH 2 CH 2 ),2.40(m,2H,CH 2 CH 2 OH),2.37(m,6H,NCH 2 CH 2 ),2.36-2.34(m,8H,NHCOCH 2 ),1.77(m,2H,NHCHCH 2 ),1.73(m,2H,NHCH 2 CH 2 ),1.60(dd,4H,COOCH 2 CH 2 ),1.53(dd,2H,NHCHCH 2 ),1.49(m,4H,NCH 2 CH 2 ),1.47-1.26(m,46H,CH 2 (myristoyl)),0.89(t,J＝6.9Hz,12H,CH2CH3).HRMS,ESI + ,m/z:Calcd for C 58 H 111 N 5 O 11 [M+H] + ,1054.8376；found,1054.8348.
EXAMPLE 7 preparation of Multi-tail ionizable oligopeptide lipid MF7
MF7 starting from I-7 (1 g,1.10 mmol) and N, N-dimethyl-1, 3-diaminopropane (192 mg,1.10 mmol), the process for preparing MF1 afforded 682mg as a white powder. Yield: 66.7%. 1 H NMR(300MHz,CDCl 3 ):δ(ppm)8.0(m,2H,CONHCH),7.5(m,1H,CONHCH 2 ),4.51(m,1H,NHCHCO),4.47(m,1H,COOCH),4.44(m,1H,NHCHCO),4.13-4.06(t,4H,COOCH 2 ),3.42-3.18(m,4H,NHCH 2 CH 2 ),2.37(m,2H,NCH 2 CH 2 ),2.36-2.34(m,8H,NHCOCH 2 ),2.15(m,6H,NCH 3 ),1.77(m,2H,NHCHCH 2 ),1.73(m,2H,NHCH 2 CH 2 ),1.60(dd,4H,COOCH 2 CH 2 ),1.53(dd,2H,NHCHCH 2 ),1.49(m,4H,NCH 2 CH 2 ),1.47-1.26(m,46H,CH 2 (myristoyl)),0.89(t,J＝6.9Hz,12H,CH 2 CH 3 ).HRMS,ESI + ,m/z:Calcd for C 56 H 107 N 5 O 9 [M+H] + ,994.8241；found,994.8375.
Example 8 preparation of Multi-tail ionizable oligopeptide lipid MF8
MF8 starting from I-7 (1 g,1.10 mmol) and 4-dimethylaminopiperidine (194 mg,1.10 mmol), the process for preparing MF1 gave 714mg of the product as a pale yellow powder. Yield: 68.5%. 1 HNMR(300MHz,CDCl 3 ):δ(ppm)8.0(m,2H,CONHCH),7.5(m,1H,CONHCH 2 ),4.51(m,1H,NHCHCO),4.47(m,1H,COOCH),4.44(m,1H,NHCHCO),4.13-4.06(t,4H,COOCH 2 ),3.42-3.18(m,4H,NHCH 2 CH 2 ),2.37(m,2H,NCH 2 CH 2 ),2.36-2.34(m,8H,NHCOCH 2 ),2.15(m,6H,NCH 3 ),1.77(m,2H,NHCHCH 2 ),1.73(m,2H,NHCH 2 CH 2 ),1.60(dd,4H,COOCH2CH2),1.53(dd,2H,NHCHCH 2 ),1.49(m,4H,NCH 2 CH 2 ),1.47-1.26(m,46H,CH 2 (myristoyl)),0.89(t,J＝6.9Hz,12H,CH 2 CH 3 ).HRMS,ESI + ,m/z:Calcd for C 56 H 107 N 5 O 9 [M+H] + ,994.8241；found,994.8375.
Example 9 preparation of Multi-tail ionizable oligopeptide lipid MF9
MF9 starting from I-7 (1 g,1.10 mmol) and 4-methyl-1-piperazineethylamine (177 mg,1.10 mmol) the procedure for the preparation of MF1 gave 612mg as a clear oil. Yield: 64.7％。 1 H NMR(300MHz,CDCl 3 ):δ(ppm)8.0(m,2H,CONHCH),7.5(m,1H,CONHCH 2 ),4.51(m,1H,NHCHCO),4.47(m,1H,COOCH),4.44(m,1H,NHCHCO),4.13-4.06(t,4H,COOCH 2 ),3.18(m,2H,NHCH 2 CH 2 ),3.14(m,2H,NCH 2 CH 2 ),2.46(m,2H,NHCH 2 CH 2 ),2.36-2.34(m,8H,NHCOCH 2 ),2.29(m,8H,NCH 2 CH 2 ),2.14(t,3H,NCH 3 ),1.77(m,2H,NHCHCH 2 ),1.60(dd,4H,COOCH 2 CH 2 ),1.53(dd,2H,NHCHCH 2 ),1.49(m,4H,NCH 2 CH 2 ),1.47-1.26(m,46H,CH 2 (myristoyl)),0.89(t,J＝6.9Hz,12H,CH 2 CH 3 ).HRMS,ESI + ,m/z:Calcd for C 58 H 110 N 6 O 9 [M+H] + ,1035.8473；found,1035.8481.
example 10 preparation and characterization of lipid nanoparticles
The desired multi-tailed ionizable oligopeptide lipids MF1 to MF9, neutral phospholipids, cholesterol, and pegylated lipids were weighed according to the molar ratios in table 1 and dissolved in absolute ethanol as ethanol phase. eGFP-siRNA (ebo biotechnology ltd, guangzhou) was dissolved in 10mM citric acid buffer (ph=4.0) as an aqueous phase. The ethanol phase was rapidly and uniformly injected into the aqueous phase with vigorous stirring, the volume ratio of aqueous phase to ethanol phase being 3:1. After the injection is completed, the sample is dialyzed by ultrapure water for 2 to 4 hours at room temperature to obtain LNP solution, and the LNP solution is preserved at the temperature of 4 ℃ for standby. The particle size, polydispersity (PDI) and potential of LNP were measured using an Omni particle size potential analyzer and the results are shown in table 2.
TABLE 1 prescription of lipid nanoparticles of the invention
Table 2 properties of lipid nanoparticles of the invention (n=3)
The data show that the LNP particle size of the invention is between 100 and 200nm, and the polydispersity is less than 0.3, which indicates that the LNP particle size is uniform and regular; the potential is between +5 and +20mV, and weak positive electricity is shown.
Example 11 lipid nanoparticle Loading Capacity for siRNA
Lipid nanoparticles of different multi-tailed ionizable oligopeptide lipids loaded with siRNA were prepared using the ethanol injection method of example 10 with N/p=7. The ability of lipid nanoparticles to carry siRNA was examined by agarose gel electrophoresis, as shown in figure 1. The results show that most of the lipid nanoparticles of the invention can stably load siRNA at the time of N/P=7, and can be further used for cell transfection experiments.
Example 12 lipid nanoparticle Loading Capacity for mRNA
Lipid nanoparticles MF2NP and MF5NP loaded with eGFP-mRNA (sharp biotechnology ltd, guangzhou) were prepared at different nitrogen-to-phosphorus ratios (N/p=3, 5, 7) using the ethanol injection method of example 10. The ability of lipid nanoparticles to load mRNA was examined by agarose gel electrophoresis experiments, as shown in figure 2. The results show that the lipid nanoparticle can stably load mRNA under a certain N/P, and can be further used for cell transfection experiments.
Example 13 lipid nanoparticle Loading Capacity to microRNA
Lipid nanoparticles MF2NP and MF5NP loaded with microRNA-140 (sharp bio-technologies, inc., guangzhou) were prepared at different nitrogen-to-phosphorus ratios (N/p=3, 5, 7) using the ethanol injection method of example 10. The ability of lipid nanoparticles to load micrornas was examined by agarose gel electrophoresis experiments, as shown in figure 3. The result shows that the lipid nanoparticle can stably load microRNA under a certain N/P, and can be further used for cell transfection experiments.
Example 14 in vitro safety assessment of lipid nanoparticles
Lipid nanoparticles MF2 and MF5 were prepared using the method of example 10, and toxicity of the lipid nanoparticles to human embryonic kidney HEK293T cells was evaluated by the CCK8 method for comparison with the commercial transfection reagent Lipofectamine2000, as shown in fig. 4. The results show that the lipid nanoparticle provided by the invention has no toxic effect on human embryonic kidney HEK293T cells, and is equivalent to Lipofectamine2000.
Example 15 in vitro stability investigation of siRNA lipid nanoparticles
The siRNA loaded lipid nanoparticles MF2NP and MF5NP were prepared using the method of example 10, and after standing at 37 ℃ for 0, 1,2, 4, 8, 16, 24 hours, respectively, the particle diameter changes thereof were measured by Omni particle diameter meter, and the results are shown in fig. 5. The data show that the particle size of NF2NP and MF5NP is basically unchanged after 24h incubation in ultrapure water, and the lipid nanoparticle has good in-vitro stability.
Example 16 Membrane fusion Capacity of Multi-tail ionizable oligopeptides lipids
Anionic liposomes were prepared using thin film dispersion to mimic the endosomal membrane of cells. DOPC, DOPS, DOPE and cholesterol were weighed and mixed in the ratio DOPC: DOPS: DOPE: chol=40:20:20:15 (w: w: w, total lipid mass 10 mg), and 1% mol Rho-PE and 1% mol NBD-PE were blended, dissolved in a mixed solvent of 2mL of methanol and 3mL of chloroform, distilled under reduced pressure in a water bath at 40℃for 5 minutes, and the organic solvent was thoroughly removed by vacuum drying overnight. After drying, 3mL of purified water was added, hydrated at 37℃for 30min, and then sonicated in an ice bath for 15min. Then passing through a microporous filter membrane with the thickness of 0.45 mu m to obtain the anionic liposome solution. Pure water was added to the liposome solution for dilution to give a total lipid concentration of 792. Mu.M. The preparation of MF1NP to MF9NP was performed as in example 19, and the lipid nanoparticle LNP was diluted with PBS buffer (pH 7.4) and citrate buffer (pH 6.5 and pH 5.5, 20 mM), respectively, to give an ionizable lipid concentration of 95. Mu.M. 150. Mu.L of LNP solution and 7.5. Mu.L of anionic liposome solution were sequentially added to a 96-well plate, and after thorough mixing, incubated at 37℃for 5min, the fluorescence intensity was measured. Three replicates were set up for each sample with 150 μl of 0.5% triton X-100 solution treated anionic liposome solution as positive control and 150 μl of LPBS solution and 7.5 μl of anionic liposome solution as negative control. The membrane fusion ratio was calculated by measurement using an enzyme-labeled instrument (excitation wavelength: 480nm, emission wavelength: 538 nm). The results are shown in fig. 6, where 9 ionizable lipids have significant differences in membrane fusion capacities at pH 7.4 and pH 6.5-5.5, indicating that the lipid nanoparticles prepared from these ionizable lipids can maintain the relative integrity of the structure under physiological conditions, achieving rapid escape in early endosomes.
Example 17 phase inversion Capacity of Multi-tail ionizable oligopeptides lipids
The cationic liposomes of MF1, MF4, MF2 and MF5 were prepared by a thin film dispersion method, and were prescribed MT/MF: DOPE: DOPC: chol=20:20:20:40 (w: w: w), with a total lipid mass of 10mg. The lipids were precisely weighed, dissolved in chloroform, dried by spin-drying at 40℃for 10min and then dried under vacuum overnight. Followed by addition of 3mL D 2 O is hydrated for 30min, and ice bath ultrasonic treatment is carried out for 15min to obtain each cationic liposome. The method is used for preparing the anionic liposome simulating the endosome membrane, and the anionic liposome, the anionic liposome and the cationic liposome mixture are respectively recorded 31 PNMR spectra, results are shown in fig. 7. After the MF2 and MF5 liposome and the anionic liposome are mixed, 31 the asymmetry of the peak of the P NMR spectrum is reversed, which shows that the phase transition temperature of MF2 and MF5 is lower, the formation of inverted hexagonal phase can be better promoted, and the endosome escape of RNA is facilitated. The phase transition temperature Tc of each liposome was examined using Differential Scanning Calorimetry (DSC), and the results are shown in table 3, further demonstrating that MF2 and MF5 have lower phase transition temperatures.
TABLE 3 phase transition temperature Tc of ionizable lipids of the invention
EXAMPLE 18 transfection of siRNA lipid nanoparticles into cervical cancer Hela-eGFP cells
The siRNA loaded lipid nanoparticles MF1NP to MF9NP were prepared as in example 10. At transfection, 1X 10 5 Density of cells/mL human cervical cancer Hela cells stably expressing enhanced Green fluorescent protein (eGFP) were seeded in 24 well plates with 0.5mL of R containing 10% fetal bovine serum and 1% penicillin-streptomycin per wellPMI 1640 medium was incubated in a 37℃incubator containing 5% carbon dioxide for 24 hours, the medium was discarded, and 0.44 mM of PMI 1640 medium and 60. Mu.L of lipid nanoparticles were added to the medium, followed by further incubation for 6 hours. The culture medium was discarded, and 0.5mL of RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added thereto, followed by further culturing. After 48h, the expression of green fluorescent protein in Hela-eGFP cells was observed using an inverted fluorescent microscope and the Mean Fluorescence Intensity (MFI) in Hela-eGFP cells was quantitatively examined using a flow cytometer. In the experiment, a commercial transfection reagent Lipofectamine2000 is used as a positive control, and the gene silencing efficiency of the lipid nanoparticle provided by the invention is compared. The results are shown in fig. 8 and 9. The MF2NP, the MF3NP and the MF5NP can be used for efficiently transfecting Hela-eGFP cells, and the silencing efficiency of the MF5NP is obviously superior to that of positive control Lipofectamine2000.
EXAMPLE 19 transfection of mRNA lipid nanoparticles into human Primary CD3 + T cell
Lipid nanoparticles MF2NP and MF5NP bearing Green Fluorescent Protein (GFP) mRNA were prepared as in example 10. Extraction of human CD3 from human peripheral blood mononuclear cells using magnetic bead separation kit + T cells were resuspended in T cell medium containing human IL-2 (100 IU/mL) and human CD3/CD28 antibodies at 1X 10 in ultra low adsorption suspension cell six well plates 6 Plates were plated at 1X 10 per well after 4d incubation 6 Cell density of each mL was inoculated into 24-well plate, 0.5mL of T cell culture medium containing human IL-2 (100 IU/mL) and human CD3/CD28 antibody was added to each well, 100. Mu.L of LNP was added, and after thorough mixing, the mixture was placed in a 37℃and 5% CO2 cell incubator for cultivation, after 48 hours, GFP expression was observed by an inverted fluorescence microscope, and GFP was examined by flow cytometry + T cell positive rate and Mean Fluorescence Intensity (MFI) of GFP. In the experiment, a commercially available transfection reagent Lipofectamine2000 is used as a positive control, and the mRNA transfection efficiency of the lipid nanoparticle provided by the invention is compared. The results are shown in fig. 10 and 11. The MF2NP and MF5NP of the present invention can transfect human primary CD3 relatively high effectively + T cells and transfection efficiency was better than positive control Lipofectamine2000.
Claims (10)
1. A multi-tail ionizable oligopeptide lipid has a chemical structure represented by a general formula (I):
wherein p=1 or 2; q=an integer from 1 to 3; r=an integer from 1 to 3;
r1, R2, R3 are selected fromU1, u2 are independently integers selected from 1-19.
R4 is selected from Any one of the following.
2. The multi-tail ionizable oligopeptide lipid according to claim 1, characterized in that p = 2, q = 1, r = 2;
preferably, R1, R2 are selected fromu 1 An integer of =7 to 10;
preferably, R3 is selected fromu 2 An integer of =7 to 10;
preferably, R4 is selected from
3. The method for synthesizing the multi-tail ionizable oligopeptide lipid shown in the general formula I in claim 1, wherein the synthetic route is as follows:
4. use of a multi-tail ionizable oligopeptide lipid according to claims 1-2 for the preparation of RNA-entrapped lipid nanoparticles.
5. An RNA-entrapped lipid nanoparticle comprising the multi-tailed ionizable oligopeptide lipid of claims 1-2, neutral phospholipid, cholesterol, and pegylated lipid;
preferably, the molar ratio of the multi-tail ionizable oligopeptide lipid to the neutral phospholipid to the cholesterol to the polyethylene glycol lipid is 10-40:10-60:0.5-10;
further preferably, the molar ratio of the multi-tail ionizable oligopeptide lipid to the neutral phospholipid to the cholesterol to the pegylated lipid is 20-35:20-40:0.5-2.5.
6. The RNA-entrapped lipid nanoparticle of claim 5, wherein the neutral phospholipid is selected from one or more of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphocholine, dimyristoyl-phosphatidylcholine, soybean phospholipid, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-lecithin, sinapyl-lecithin, 1, 2-dioleoyl-sn-glycero-3-phosphocholine, dipalmitoyl-phospholipid, preferably DOPE, DSPC.
7. The RNA-entrapped lipid nanoparticle of claim 5, wherein the pegylated lipid has a tail comprising, but not limited to, a length C 6 -C 20 Preferably 1, 2-dimyristoyl-rac-glycerol-3-methoxy aggregation diol 2000, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol-polyethylene glycol 2000 orBitetradecyl (4-methoxypolyethylene glycol-2000-4-oxobutanoyl) glutamic acid.
8. The RNA-entrapped lipid nanoparticle of claim 5, wherein the RNA is selected from siRNA, shRNA, microRNA or mRNA, preferably siRNA or mRNA.
9. The RNA-entrapped lipid nanoparticle of claim 5, wherein the ratio of nitrogen to phosphorus of the ionizable lipid to RNA is 1:1 to 30:1, preferably 3:1 to 11:1.
10. Use of the lipid nanoparticle of any one of claims 5-9 in siRNA gene silencing, shRNA gene silencing, microRNA gene silencing, mRNA transfection, or CRISPR/Cas9 gene editing; preferably used for transfection of primary immune cells or tumor cells, wherein the immune cells comprise T cells, neutrophils, macrophages, dendritic cells and the like, and the tumor cells comprise any one of cervical cancer Hela cells, breast cancer MCF-7 cells, pancreatic cancer PANC-1 cells, liver cancer HepG2 cells and lung cancer A549 cells.
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