CN118178664A - Lipid material for nucleic acid delivery and application thereof - Google Patents
Lipid material for nucleic acid delivery and application thereof Download PDFInfo
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- CN118178664A CN118178664A CN202410347218.8A CN202410347218A CN118178664A CN 118178664 A CN118178664 A CN 118178664A CN 202410347218 A CN202410347218 A CN 202410347218A CN 118178664 A CN118178664 A CN 118178664A
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- 150000002632 lipids Chemical class 0.000 title claims abstract description 170
- 239000000463 material Substances 0.000 title claims abstract description 97
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 65
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 65
- 239000003814 drug Substances 0.000 claims abstract description 51
- 150000001875 compounds Chemical class 0.000 claims abstract description 35
- 238000002360 preparation method Methods 0.000 claims abstract description 9
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- 201000010099 disease Diseases 0.000 claims abstract description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 6
- 208000016361 genetic disease Diseases 0.000 claims abstract description 6
- 206010010356 Congenital anomaly Diseases 0.000 claims abstract description 5
- 208000026278 immune system disease Diseases 0.000 claims abstract description 5
- 230000001613 neoplastic effect Effects 0.000 claims abstract description 3
- 239000002105 nanoparticle Substances 0.000 claims description 53
- 108020004999 messenger RNA Proteins 0.000 claims description 21
- -1 alkyl carbon Chemical compound 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 14
- 230000007935 neutral effect Effects 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
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- 101710158773 L-ascorbate oxidase Proteins 0.000 claims description 5
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims description 5
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 claims description 4
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 claims description 4
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
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- JTNKXYWGZCNBCH-UHFFFAOYSA-N 3-(dimethylamino)propanoic acid;hydron;chloride Chemical compound Cl.CN(C)CCC(O)=O JTNKXYWGZCNBCH-UHFFFAOYSA-N 0.000 description 2
- BRSKDXVJFXXUKX-UHFFFAOYSA-N 3-pyrrolidin-1-ylpropanoic acid Chemical compound OC(=O)CCN1CCCC1 BRSKDXVJFXXUKX-UHFFFAOYSA-N 0.000 description 2
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- FFDGPVCHZBVARC-UHFFFAOYSA-N N,N-dimethylglycine Chemical compound CN(C)CC(O)=O FFDGPVCHZBVARC-UHFFFAOYSA-N 0.000 description 2
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention provides a lipid material for nucleic acid delivery, wherein the lipid material comprises a compound having the structure of I. The invention also provides the use of a lipid material for nucleic acid delivery for the preparation of a therapeutic agent selected from one or more of infectious diseases, neoplastic diseases, congenital genetic diseases and immune diseases. The lipid material provided by the invention adopts a high-efficiency low-toxicity nucleic acid drug carrier strategy, and utilizes a novel ionizable lipid and auxiliary lipid material to mix and encapsulate the nucleic acid drug, so that the high-efficiency and safe delivery of the nucleic acid drug in vivo is realized, and the drug property of the nucleic acid drug is improved.
Description
Technical Field
The present invention relates to a pharmaceutical lipid material, in particular to a lipid material for nucleic acid delivery and the use thereof.
Background
In recent years, nucleic acid drugs have received extensive attention due to advantages of small dosage, strong biological effect, wide application range, and the like. Currently, nucleic acid drugs are gradually applied to the treatment of polygenic diseases such as genetic diseases, malignant tumors, metabolic diseases and infectious diseases. mRNA vaccines, which are currently approved for sale, stimulate the body to produce neutralizing antibodies by introducing mRNA encoding viral antigens into antigen presenting cells of the body, thereby exerting an immunological effect; in addition, small nucleic acid drugs such as siRNA participate in the formation of RNA-induced silencing complex (RNA-induced silencing complex, RISC) mainly through the RNA interference process, thereby silencing the corresponding mRNA fragment and exerting the biological activity thereof, and a plurality of siRNA drugs are marketed in batches at present and mainly used for the treatment of hereditary diseases; in addition, nucleic acid drugs having activity such as ASO and pDNA are also used for the treatment of various diseases.
However, there are a number of problems with the in vivo use of nucleic acid pharmaceuticals: such as free nucleic acid molecules, are easily degraded and destroyed by nucleases in the blood circulatory system, thereby losing activity; meanwhile, the nucleic acid molecules have large molecular weight and strong negative charge, so that the nucleic acid molecules are difficult to phagocytose and ingest by cells. Thus, the clinical use of nucleic acid pharmaceuticals is greatly limited.
In order to solve the above-mentioned problems of nucleic acid drugs, a number of nucleic acid drug delivery vehicles have been developed successively, wherein cationic lipid-based nanoparticles are most widely used, and the structures thereof generally include a hydrophilic head containing a cationic fragment, a hydrophobic tail, and a linking moiety therebetween. Wherein, the cation fragment can be combined with the nucleic acid medicine through electrostatic action, thereby realizing the entrapment of the nucleic acid medicine. Such delivery vehicles generally have strong nucleic acid drug entrapment and transfection capabilities, but cationic fragments in the carrier material also cause serious in vivo safety problems, such as strong cytotoxicity and immunogenicity, easy adsorption by plasma proteins in the blood circulation, easy liver accumulation, etc.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a lipid material for nucleic acid delivery and application thereof, adopts a high-efficiency low-toxicity nucleic acid drug carrier strategy, and utilizes a novel ionizable lipid and auxiliary lipid material to mix and encapsulate nucleic acid drugs, thereby realizing high-efficiency and safe delivery of the nucleic acid drugs in vivo and improving the drug properties of the nucleic acid drugs.
The present invention provides a lipid material for nucleic acid delivery, wherein the lipid material comprises a compound having the structure I:
wherein C nH2n comprises a straight or branched alkyl carbon, n is an integer between 0 and 10;
R 1a、R1b、R1c、R1d is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl; and/or R 1a、R1b、Rlc、R1d is selected from a multi-membered carbocyclic ring, a nitrogen containing multi-membered ring, an oxygen containing multi-membered ring; and/or R 1a、R1b and R 1c、R1d each form a closed loop with N;
L 1a,L2a,L1b,L2b is selected from one or more of-C-, - (c=o) -O-, -O- (c=o) -, - (c=o) -NH-, -NH- (c=o) -, -O-, -S-;
-S-S-, -S-S-S-, -S-S-, -S-S-S-, -Se-, -one or more of Se-, -S-C (CH 3) 2 -S-, -O-;
r 2a、R2b is a saturated or unsaturated fatty chain structure containing 10-24 carbons, including cholesterol derivatives and/or tocopherol derivatives.
In order to solve the above problems, the present invention employs an ionizable tertiary amine structure that can be positively charged under acidic conditions and uncharged under neutral conditions. That is, compared to other gemini lipid materials (e.g., patent US20080112915A1, WO2016197264 A1), the lipid provided by the present invention has the advantage of: the hydrophilic head of the material has an ionizable tertiary amine structure, and avoids a permanently charged quaternary amine structure, so that the material is positively charged under an acidic condition, and can be used for entrapment of nucleic acid medicines and enhancing escape capacity of a medicine delivery carrier in a lysosome; under the condition of body fluid (pH=7.4), the potential of a nucleic acid drug delivery system prepared by using the lipid material is near neutral, so that the nucleic acid drug delivery system has good safety, and the safety problem (such as cytotoxicity problem and the like) caused by the cationic head of other gemini lipid materials can be effectively avoided. This property allows the cationic material to reduce its toxicity to humans while ensuring efficient transfection, thereby safely and efficiently delivering nucleic acid drugs to target organs or cells.
Meanwhile, as the lipid material has a special structure of H shape, compared with other lipid materials (including cationic lipid materials Dlin-MC3-DMA (MC 3) and the like which are already marketed), the lipid material has more remarkable cell transfection capability, so that the nucleic acid medicine can exert better therapeutic effect. The inventor proves that the transfection ability of the tertiary amine head lipid material provided by the invention is superior to that of cationic lipid on the market through in vitro cell transfection experiments, and meanwhile, the in vitro transfection experiments show that the H-type lipid has excellent spleen specific transfection ability.
Preferably, the present invention provides a lipid material wherein the compound has one or more of the following structures:
preferably, the present invention provides a lipid material wherein the compound has one or more of the following structures:
Wherein the method comprises the steps of
Wherein the method comprises the steps of
Wherein the method comprises the steps of
Preferably, the present invention provides a lipid material wherein the compound has one or more of the following structures:
Preferably, the lipid material provided by the application, wherein the lipid material can be prepared by means of a method comprising connecting R 2a、R2b fatty chains, deprotection and/or condensation and the like. The preparation and synthesis of H1-H36 can be easily realized by a person skilled in the art under the teaching of the application; meanwhile, under the teaching of the application, the compounds in the structures I to V can be easily obtained by means of R 2a、R2b fatty chains, deprotection, condensation and the like in a similar synthetic route.
Preferably, the present invention provides a lipid material, wherein the lipid material further comprises one or more of a neutral lipid, a steroid, and/or a polymer conjugated lipid.
Preferably, the lipid material provided by the invention, wherein the neutral lipid is selected from one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and the molar ratio of the compound to the neutral lipid is 1:1 to 10:1; the steroid is selected from the group consisting of sterols, preferably from cholesterol, the molar ratio of compound to steroid being 1:1 to 10:1, a step of; the polymer conjugated lipid is selected from pegylated lipids, and the molar ratio of the compound to the polymer conjugated lipid is 100:1 to 5:1. Most of lipid nano particles prepared by the method are 100-250nm, have good assembly capacity, PDI is 0.1-0.3, and the particle size distribution is uniform and the stability is good; the encapsulation rate is 75-95%, and the nucleic acid encapsulation capacity is good; the Zeta potential is-15- +10mV, the pka is 5.5-7.0, and the pH value is near neutral under the physiological environment of 7.4, and the safety is good. These excellent nanoparticle properties make it applicable in the treatment of diseases in vivo and facilitate subsequent mass production.
Preferably, the lipid material provided by the invention, wherein the lipid material is used in combination with a nucleic acid drug to achieve nucleic acid drug delivery; the combination includes the preparation of one or more pharmaceutical compositions with nucleic acid agents; the nucleic acid drug is selected from one or more of ASO, siRNA, mRNA, miRNA and pDNA.
Preferably, the lipid material provided by the invention is prepared into lipid nano particles containing nucleic acid medicines by mixing lipid materials with nucleic acid medicines, wherein the lipid nano particles are prepared by mixing aqueous solutions of the nucleic acid medicines with ethanol solutions of the lipid materials; preferably by microfluidic devices, high pressure microfluidic homogenizers, high pressure homogenizers and/or T-tube mixers.
Preferably, the lipid material provided by the invention, wherein the lipid material for nucleic acid delivery is used for preparing one or more therapeutic drugs selected from infectious diseases, tumor diseases, congenital genetic diseases and immune diseases.
Through creative labor and multiple experiments of the inventor, the lipid nanoparticle prepared by the lipid material is found to have better delivery effect in one or more intracellular nucleic acids (mRNA, siRNA, miRNA and the like) than the lipid nanoparticle prepared by the commercial cationic lipid material (Dlin-MC 3-DMA, ALC-0315, SM-102 and the like), and has obvious advantages. Moreover, compared to the large amounts of accumulation at liver sites exhibited by cationic lipid materials marketed in bulk, the lipid materials provided by the present invention exhibit accumulation specificity at non-liver sites, such as spleen targeting accumulation capacity.
Meanwhile, by adopting an intravenous injection or intratumoral injection administration mode, the lipid nanoparticle containing the nucleic acid drug provided by the invention has excellent tumor inhibition effect on mRNA vaccine and the like, and can be used as an effective therapeutic drug for infectious diseases, tumor diseases, congenital genetic diseases and immune diseases through the unique cell transfection capability and delivery effect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the synthesis route of H-type series lipid materials (taking lipid material H1 as an example);
FIG. 2 is a chart of H1 NMR hydrogen spectra of lipid materials;
FIG. 3 is a mass spectrum of lipid material H1;
FIG. 4 is a graph of particle size and potential of representative lipid nanoparticles;
FIG. 5 shows transfection of mRNA-entrapped lipid nanoparticles prepared from an H-series lipid material onto 293T cells;
FIG. 6 shows transfection of mRNA-encapsulated lipid nanoparticles prepared from an H-type series of lipid materials onto U87 cells;
FIG. 7 shows transfection of mRNA-entrapped lipid nanoparticles prepared from the H-series lipid material onto HeLa cells;
FIG. 8 shows gene silencing of siRNA-encapsulated lipid nanoparticles on 293T cells prepared from H-type series lipid materials;
FIG. 9 shows transfection of mRNA-entrapped lipid nanoparticles prepared from the H-series lipid material in mice;
FIG. 10 shows transfection of mRNA-encapsulated lipid nanoparticles prepared from H-type lipid material at the spleen cell level in mice;
FIG. 11 shows tumor inhibition of mRNA tumor vaccine prepared by intravenous injection of H-type lipid material in B16-OVA tumor-bearing mice.
Detailed Description
In order to further clarify the present invention, examples are given below. It should be noted that these examples are purely illustrative. These examples are given for the purpose of fully illustrating the meaning and content of the patent of the invention and are not to be construed as limiting the invention to the described examples.
Example 1
The present invention is a lipid material for nucleic acid delivery, wherein the lipid material comprises a compound having the structure I:
wherein C nH2n comprises a straight or branched alkyl carbon, n is an integer between 0 and 10;
R 1a、R1b、R1c、R1d is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl; and/or R 1a、R1b、R1c、R1d is selected from a multi-membered carbocyclic ring, a nitrogen containing multi-membered ring, an oxygen containing multi-membered ring; and/or R 1a、R1b and R 1c、R1d each form a closed loop with N;
L 1a,L2a,L1b,L2b is selected from one or more of-C-, - (c=o) -O-, -O- (c=o) -, - (c=o) -NH-, -NH- (c=o) -, -O-;
-S-S-, -S-S-S-, -S-S-, -S-S-S-, -Se-, -one or more of Se-, -S-C (CH 3) 2 -S-, -O-;
r 2a、R2b is a saturated or unsaturated fatty chain structure containing 10-24 carbons, including cholesterol derivatives and/or tocopherol derivatives.
In one embodiment, the invention provides a lipid material wherein the compound has one or more of the following structures:
In another embodiment, the present invention provides a lipid material, wherein the compound has one or more of the following structures:
Wherein the method comprises the steps of
Wherein the method comprises the steps of
Wherein the method comprises the steps of
In yet another embodiment, the structure V lipid material may be of the structure:
TABLE 1 Structure V specific Structure of lipid Material
The structural formulae of the above lipids can be combined by those skilled in the art, and the structural formulae of H1 to H36 are not fully enumerated below:
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example 2
This example is intended to illustrate the synthetic route for each lipid in example 1, and other lipids can be obtained using similar routes, with specific synthetic routes being shown in FIG. 1.
2.1 Synthesis of intermediate 1
2.20G of N, N' -bis (t-butoxycarbonyl) -L-cystine (5 mmol), 2.68g (10 mmol) of oleyl alcohol, 1.22g (10 mmol) of 4-Dimethylaminopyridine (DMAP) and 3.87g (30 mmol) of N, N-Diisopropylethylamine (DIPEA) are weighed into a 100mL eggplant-shaped bottle, 15mL of Dichloromethane (DCM) are taken as solvent and stirred at room temperature for 20min. Subsequently, 15mL of a 1.91g (10 mmol) methylene chloride solution of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) was added dropwise to the reaction flask. The reaction was carried out overnight at room temperature. After completion of the reaction, column chromatography purification was performed to obtain 2.35g of a white solid powder in a yield of 50.0%.
1H NMR(400MHz,DMSO)δ7.37(d,J＝7.9Hz,2H),5.33(dd,J＝12.5,7.9Hz,4H),4.24(d,J＝4.6Hz,2H),4.05(d,J＝5.5Hz,4H),2.99(ddd,J＝23.2,13.7,7.2Hz,4H),1.98(d,J＝5.4Hz,8H),1.59-1.53(m,4H),1.41-1.35(m,18H),1.24(s,44H),0.86(t,J＝6.6Hz,6H).
2.2 Synthesis of intermediate 2
1.88G (2.0 mmol) of intermediate 1 was weighed into a reaction flask, and 20mL of HCl/EA was added thereto, followed by stirring at room temperature for 4 hours. After completion of the reaction, the solvent was pumped down by an oil pump to obtain 1.13g of a white solid powder in a yield of 80.7%.
2.3 Synthesis of intermediate 3
2.34G (5 mmol) of N, N' -bis (t-butoxycarbonyl) -L-homocysteine, 2.68g (10 mmol) of oleyl alcohol, 1.22g (10 mmol) of DMAP and 3.87g (30 mmol) of DIPEA are weighed into a 100mL eggplant-shaped bottle, 15mL of DCM is taken as solvent and stirred at room temperature for 20min. Subsequently, 15mL of a 1.91g (10 mmol) solution of EDCI in DCM was added dropwise to the reaction flask. The reaction was carried out overnight at room temperature. After completion of the reaction, column chromatography purification was performed to obtain 1.98g of a white solid powder in a yield of 49.8%.
1H NMR(400MHz,DMSO)δ7.31(d,J＝7.8Hz,2H),5.32(t,J＝4.8Hz,4H),4.05(d,J＝5.8Hz,4H),4.02-3.97(m,2H),2.72(s,4H),2.03-1.92(m,12H),1.54(d,J＝6.0Hz,4H),1.38(s,18H),1.24(s,44H),0.86(t,J＝6.7Hz,6H).
2.4 Synthesis of intermediate 4
1.94G (2.0 mmol) of intermediate 3 was weighed into a reaction flask, and 20mL of HCl/EA was added thereto, followed by stirring at room temperature for 4 hours. After completion of the reaction, the solvent was pumped down by an oil pump to obtain 1.01g of a white solid powder in a yield of 70.0%.
2.5 Synthesis of Compounds H1-H12
2.5.1 Synthesis of Compound H1
206Mg (2.0 mmol) of dimethylglycine, 230mg (2.0 mmol) of NHS and 282mg (2.0 mmol) of EDCI are weighed into a 100mL eggplant-shaped bottle, 15mL of DCM is taken as solvent and stirred for 30min at room temperature. 740mg of intermediate 2 was then added to the reaction flask and reacted overnight at room temperature. Column chromatography purification was performed after completion of the reaction, and 457mg of a pale yellow transparent oily liquid was obtained in a yield of 50.3%.
1H NMR(400MHz,DMSO)δ8.18(d,J＝8.3Hz,2H),5.40-5.26(m,4H),4.63(td,J＝8.3,5.2Hz,2H),4.04(dt,J＝8.0,5.4Hz,4H),3.21-3.08(m,4H),2.91(q,J＝15.4Hz,4H),2.24(s,12H),2.11-1.80(m,8H),1.55(dd,J＝13.3,6.4Hz,4H),1.24(s,48H),0.86(t,J＝6.8Hz,6H). The specific nuclear magnetism and mass spectrum are shown in figures 2 and 3.
2.5.2 Synthesis of Compound H2
306Mg (2.0 mmol) of 3-dimethylaminopropionic acid hydrochloride, 230mg (2.0 mmol) of NHS and 282mg (2.0 mmol) of EDCI are taken in a 100mL eggplant-shaped bottle, 15mL of DCM is taken as solvent and stirred at room temperature for 30min. 740mg of intermediate 2 was then added to the reaction flask and reacted overnight at room temperature. Column chromatography purification is carried out after the reaction is completed, 656mg of light yellow transparent oily liquid is obtained, and the yield is 70.1%.
1H NMR(400MHz,DMSO)δ8.18(d,J＝8.3Hz,2H),5.40-5.26(m,4H),4.63(td,J＝8.3,5.2Hz,2H),4.04(dt,J＝8.0,5.4Hz,4H),3.21-3.08(m,4H),2.91(q,J＝15.4Hz,4H),2.24(s,12H),2.11-1.80(m,8H),1.55(dd,J＝13.3,6.4Hz,4H),1.24(s,48H),0.86(t,J＝6.8Hz,6H).
2.5.3 Synthesis of Compound H3
262Mg (2.0 mmol) of 4- (dimethylamino) butanoic acid, 230mg (2.0 mmol) of NHS and 282mg (2.0 mmol) of EDCI are taken in a 100mL eggplant-shaped bottle, 15mL of DCM are taken as solvent and stirred at room temperature for 30min. 740mg of intermediate 2 was then added to the reaction flask and reacted overnight at room temperature. Column chromatography purification was performed after the reaction was completed to obtain 544mg of pale yellow transparent oily liquid, with a yield of 56.3%.
1H NMR(400MHz,DMSO)δ8.43(d,J＝7.9Hz,2H),5.32(s,4H),4.53(s,2H),4.05(s,4H),3.37(d,J＝6.9Hz,4H),3.10(d,J＝12.8Hz,2H),2.98-2.90(m,2H),2.28(s,12H),2.16(s,4H),1.99(s,8H),1.69(d,J＝5.9Hz,4H),1.56(s,4H),1.24(s,48H),0.86(s,6H).
2.5.4 Synthesis of Compound H4
286Mg (2.0 mmol) of 3-pyrrolidin-1-yl-propionic acid, 230mg (2.0 mmol) of NHS and 282mg (2.0 mmol) of EDCI were taken in a 100mL eggplant-shaped bottle and 15mL DCM was taken as solvent and stirred at room temperature for 30min. 740mg of intermediate 2 was then added to the reaction flask and reacted overnight at room temperature. After the reaction was completed, column chromatography purification was performed to obtain 420mg of a pale yellow transparent oily liquid, and the yield was 42.4%.
1H NMR(400MHz,DMSO)δ8.18(d,J＝8.3Hz,2H),5.40-5.26(m,4H),4.63(td,J＝8.3,5.2Hz,2H),4.04(dt,J＝8.0,5.4Hz,4H),3.21-3.08(m,4H),2.91(q,J＝15.4Hz,4H),2.24(s,12H),2.11-1.80(m,8H),1.55(dd,J＝13.3,6.4Hz,4H),1.24(s,48H),0.86(t,J＝6.8Hz,6H).
Synthesis of 2.5.5 Compound H5
286Mg (2.0 mmol) of 1-methylpiperidine-4-carboxylic acid, 230mg (2.0 mmol) of NHS and 282mg (2.0 mmol) of EDCI are taken in a 100mL eggplant-shaped bottle, 15mL of DCM are taken as solvent and stirred at room temperature for 30min. 740mg of intermediate 2 was then added to the reaction flask and reacted overnight at room temperature. Column chromatography purification was performed after completion of the reaction to give 448mg of a pale yellow transparent oily liquid with a yield of 45.3%.
1H NMR(400MHz,DMSO)δ8.38(d,J＝7.8Hz,2H),5.36-5.30(m,4H),4.54-4.49(m,2H),4.04(dd,J＝10.3,6.3Hz,4H),3.10(dd,J＝13.8,5.2Hz,4H),2.33(s,7H),2.22(s,8H),2.02-1.93(m,8H),1.63(dd,J＝36.5,24.3Hz,12H),1.24(s,48H),0.86(t,J＝4.8Hz,6H).
Synthesis of 2.5.6 Compound H6
344Mg (1.0 mmol) of 3- (4-methyl-1-piperazinyl) propionic acid, 230mg (2.0 mmol) of NHS and 282mg (2.0 mmol) of EDCI were taken in a 100mL eggplant-shaped bottle, 15mL DCM as solvent and stirred at room temperature for 30min. 740mg of intermediate 2 was then added to the reaction flask and reacted overnight at room temperature. After the reaction was completed, column chromatography purification was performed to obtain 650mg of a pale yellow transparent oily liquid, and the yield was 62.1%.
1H NMR(400MHz,DMSO)δ8.18(d,J＝8.3Hz,2H),5.40-5.26(m,4H),4.63(td,J＝8.3,5.2Hz,2H),4.04(dt,J＝8.0,5.4Hz,4H),3.21-3.08(m,4H),2.91(q,J＝15.4Hz,4H),2.24(s,12H),2.11-1.80(m,8H),1.55(dd,J＝13.3,6.4Hz,4H),1.24(s,48H),0.86(t,J＝6.8Hz,6H).
Synthesis of 2.5.7 Compound H7
206Mg (2.0 mmol) of dimethylglycine, 230mg (2.0 mmol) of NHS and 282mg (2.0 mmol) of EDCI are taken in a 100mL eggplant-shaped bottle, 15mL of DCM is taken as solvent and stirred at room temperature for 30min. 768mg of intermediate 4 was then added to the reaction flask and reacted overnight at room temperature. After the reaction was completed, column chromatography purification was performed to obtain 284mg of a pale yellow transparent oily liquid, with a yield of 30.3%.
1H NMR(400MHz,DMSO)δ8.11(d,J＝7.9Hz,2H),5.32(s,4H),4.42(s,2H),4.04(d,J＝12.0Hz,5H),2.91(dd,J＝47.0,14.9Hz,5H),2.78-2.63(m,5H),2.22(s,12H),2.15-1.92(m,12H),1.55(s,4H),1.24(s,48H),0.86(s,6H).
Synthesis of 2.5.8 Compound H8
306Mg (2.0 mmol) of 3-dimethylaminopropionic acid hydrochloride, 230mg (2.0 mmol) of NHS and 282mg (2.0 mmol) of EDCI are taken in a 100mL eggplant-shaped bottle, 15mL of DCM is taken as solvent and stirred at room temperature for 30min. 768mg of intermediate 4 was then added to the reaction flask and reacted overnight at room temperature. Column chromatography purification is carried out after the reaction is completed, 391mg of light yellow transparent oily liquid is obtained, and the yield is 40.5%.
1H NMR(400MHz,DMSO)δ8.58(s,2H),5.32(s,4H),4.36(s,2H),4.03(s,5H),3.02(d,J＝19.2Hz,8H),2.75(s,5H),2.53(d,J＝9.6Hz,12H),2.03(d,J＝42.1Hz,13H),1.56(s,4H),1.24(s,48H),0.86(s,6H).
Synthesis of 2.5.9 Compound H9
262Mg (2.0 mmol) of 4- (dimethylamino) butanoic acid, 230mg (2.0 mmol) of NHS and 282mg (2.0 mmol) of EDCI are taken in a 100mL eggplant-shaped bottle, 15mL of DCM are taken as solvent and stirred at room temperature for 30min. 768mg of intermediate 4 was then added to the reaction flask and reacted overnight at room temperature. Column chromatography purification was performed after the reaction was completed to give 498mg of a pale yellow transparent oily liquid in a yield of 50.1%.
1H NMR(400MHz,DMSO)δ8.30(d,J＝7.3Hz,2H),5.32(s,5H),4.35(s,2H),4.09-3.97(m,4H),2.29(d,J＝44.3Hz,12H),2.16(s,4H),1.98(s,8H),1.66(s,4H),1.55(s,4H),1.24(s,48H),0.86(s,6H).
Synthesis of 2.5.10 Compound H10
286Mg (2.0 mmol) of 3-pyrrolidin-1-yl-propionic acid, 230mg (2.0 mmol) of NHS and 282mg (2.0 mmol) of EDCI were taken in a 100mL eggplant-shaped bottle and 15mL DCM was taken as solvent and stirred at room temperature for 30min. 768mg of intermediate 4 was then added to the reaction flask and reacted overnight at room temperature. After the completion of the reaction, column chromatography purification was performed to obtain 321mg of a pale yellow transparent oily liquid, the yield was 31.6%.
1H NMR(400MHz,DMSO)δ8.18(d,J＝8.3Hz,2H),5.40-5.26(m,4H),4.63(td,J＝8.3,5.2Hz,2H),4.04(dt,J＝8.0,5.4Hz,4H),3.21-3.08(m,4H),2.91(q,J＝15.4Hz,4H),2.24(s,12H),2.11-1.80(m,8H),1.55(dd,J＝13.3,6.4Hz,4H),1.24(s,48H),0.86(t,J＝6.8Hz,6H).
Synthesis of 2.5.11 Compound H11
286Mg (2.0 mmol) of 1-methylpiperidine-4-carboxylic acid, 230mg (2.0 mmol) of NHS and 282mg (2.0 mmol) of EDCI are taken in a 100mL eggplant-shaped bottle, 15mL of DCM are taken as solvent and stirred at room temperature for 30min. 768mg of intermediate 4 was then added to the reaction flask and reacted overnight at room temperature. Column chromatography purification is carried out after the reaction is completed, 634mg of light yellow transparent oily liquid is obtained, and the yield is 62.3%.
1H NMR(400MHz,DMSO)δ8.24(d,J＝6.6Hz,2H),5.32(s,4H),4.32(s,2H),4.07-3.98(m,4H),2.90(d,J＝9.6Hz,4H),2.71(s,4H),2.28(s,8H),2.20(s,2H),2.10(s,8H),1.98(s,6H),1.68-1.53(m,12H),1.24(s,48H),0.85(s,6H).
2.5.12 Synthesis of Compound H12
344Mg (1.0 mmol) of 3- (4-methyl-1-piperazinyl) propionic acid, 230mg (2.0 mmol) of NHS and 282mg (2.0 mmol) of EDCI were taken in a 100mL eggplant-shaped bottle, 15mL DCM as solvent and stirred at room temperature for 30min. 768mg of intermediate 4 was then added to the reaction flask and reacted overnight at room temperature. Column chromatography purification is carried out after the reaction is completed, thus 429mg of light yellow transparent oily liquid is obtained, and the yield is 39.9%.
1H NMR(400MHz,CDCl3)δ8.86(s,2H),5.35(d,J＝16.7Hz,4H),4.68(d,J＝5.3Hz,3H),4.14(d,J＝3.8Hz,4H),3.65(dd,J＝14.5,7.1Hz,4H),2.86-2.74(m,24H),2.53(s,14H),2.03(d,J＝5.4Hz,8H),1.66(d,J＝6.7Hz,4H),1.28(s,49H),0.89(d,J＝7.0Hz,6H).
It should be noted that the examples of the present application are not fully listed, and those skilled in the art can implement the preparation and synthesis of H1 to H36 under the teachings of the present application; meanwhile, under the teaching of the application, the compounds in the structures I to V can be easily obtained by means of R 2a、R2b fatty chains, deprotection, condensation and the like in a similar synthetic route.
Example 3
The present invention provides lipid materials, wherein the lipid material further comprises one or more of a neutral lipid, a steroid, and/or a polymer conjugated lipid. Wherein the neutral lipid is selected from one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and the molar ratio of the compound to the neutral lipid is 1:1 to 10:1; the steroid is selected from the group consisting of sterols, preferably from cholesterol, in a molar ratio of 1:1 to 10:1; the polymer conjugated lipid is selected from pegylated lipids, and the molar ratio of the compound to the polymer conjugated lipid is 100:1 to 5:1.
In one embodiment, the lipid material is used in combination with a nucleic acid drug to effect nucleic acid drug delivery; the combination comprises preparing one or more pharmaceutical compositions; the nucleic acid drug is selected from one or more of ASO, siRNA, mRNA, miRNA and pDNA. The lipid material and the nucleic acid medicine are prepared into lipid nano particles containing the nucleic acid medicine, and the lipid nano particles are prepared by mixing an aqueous solution of the nucleic acid medicine with an ethanol solution of the lipid material; preferably by microfluidic devices, high pressure microfluidic homogenizers, high pressure homogenizers and/or T-tube mixers.
In another embodiment, the lipid material for nucleic acid delivery is used for the preparation of a therapeutic agent selected from one or more of infectious diseases, neoplastic diseases, congenital genetic diseases, and immune diseases.
This example mainly illustrates the nucleic acid drug delivery vehicle used for nucleic acid delivery such as ASO, siRNA, mRNA, miRNA and pDNA, and the preparation method of lipid nanoparticles containing the nucleic acid drug, and is not fully exemplified by the particle size, polydispersity, zeta potential, encapsulation efficiency and pKa measurement method of mRNA delivery vehicle.
3.1 Preparation of lipid nanoparticles
ASO, siRNA, mRNA, miRNA and/or pDNA were dissolved in 40mM citric acid buffer at ph=4, lipid materials as in example 1 or example 2 were mixed according to the prescription composition in table 2 and dissolved in ethanol; the lipid nanoparticles were prepared by rapid mixing with a microfluidic device, a high-pressure microfluidic homogenizer, a high-pressure homogenizer and/or a T-tube mixer according to a flow rate ratio of aqueous phase to alcoholic phase=1:3. The lipid nanoparticles thus prepared were then placed in dialysis bags with molecular weight cut-off of 3500, dialyzed overnight in PBS buffer with ph=7.4, free small molecules, ethanol were removed and pH was adjusted and the lipid nanoparticles obtained were stored at 4 ℃.
3.2 Determination of the particle size, polydispersity index (PDI) and Zeta potential of lipid nanoparticles
The particle size, polydispersity index (PDI) and Zeta potential of the lipid nanoparticles were determined using Malvem Zetasizer Pro using dynamic light scattering, wherein the particle size, PDI and Zeta potential of the lipid nanoparticles prepared from representative lipid materials encompassed by the present invention are shown in table 3 and fig. 4.
3.3 Determination of lipid nanoparticle encapsulation efficiency
The encapsulation efficiency of lipid nanoparticles was determined using a Quant-iT RiboGreen RNA ASSAY KIT RNA quantitative detection kit: samples were diluted to a concentration of about 5. Mu.g/mL in TE buffer, and the RiboGreen reagent was diluted 1:200 in TE buffer, 100. Mu.L of the diluted samples were transferred to 96-well plates and 100. Mu.L of the diluted RiboGreen solution was added. Incubate at 37℃for 15 min. Fluorescence intensity (excitation wavelength 480nm, emission wavelength 520 nm) was measured using a fluorescence plate reader, and free RNA concentration was calculated. The lipid nanoparticles prepared from representative lipid materials encompassed by the present invention are shown in table 3.
3.4 PKa determination of lipid nanoparticles
The apparent pKa of the lipid nanoparticle was determined by fluorescence of 2- (p-toluidinyl) -6-naphthalene sulfonic acid (TNS). Buffers were formulated at pH (ph=2.5-11.0) containing 150mM sodium chloride, 10mM sodium phosphate, 10mM sodium citrate, 10mM sodium borate. Mixing the lipid nanoparticle with buffers with different pH values, and adding TNS. The fluorescence intensity of the excitation wavelength 321nm and the emission wavelength 445nm was detected at room temperature using a fluorescence microplate reader. The fluorescence data were analyzed by fitting, with pKa being the pH that produced half maximum fluorescence intensity, wherein the pKa data for representative lipid nanoparticles encompassed by the present invention are shown in table 4.
Table 2 lipid nanoparticle formulations comprising the lipid materials of the present invention
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Table 3 characterization of lipid nanoparticles comprising the lipid material of the present invention
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Remarks: dipalmitoyl choline (DPPC), distearoyl choline (DSPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl lecithin (DOPC), dioleoyl phosphatidylethanolamine (DOPE), phosphatidylcholine (POPC), dimyristoyl glycerol-polyethylene glycol 2000 (DMG-PEG 2000).
TABLE 4 pka value of lipid nanoparticles comprising the lipid material of the present invention
| Lipid material | pka |
| L1 | 6.80 |
| L2 | 8.97 |
| L3 | 10.07 |
| L4 | 6.60 |
| L5 | 9.01 |
| L6 | 8.30 |
| L7 | 6.93 |
| L8 | 9.65 |
| L9 | 9.66 |
| L10 | 7.00 |
| L11 | 10.52 |
| L12 | 8.55 |
| L13 | 6.56 |
| L14 | 6.18 |
| L15 | 5.78 |
| L16 | 5.99 |
| L17 | 5.37 |
| L18 | 5.81 |
| L19 | 6.14 |
| L20 | 6.42 |
Conclusion: most of lipid nano particles prepared by the method are 100-250m, have good assembly capacity, PDI is 0.1-0.3, uniform particle size distribution and good stability; the encapsulation rate is 75-95%, and the nucleic acid encapsulation capacity is good; the Zeta potential is-15 mV- +10mV, the pka is 5.5-7.0, and the pH value is near neutral under the physiological environment of pH7.4, and the safety is good. These excellent nanoparticle properties make it applicable in the treatment of diseases in vivo and facilitate subsequent mass production.
Example 4
In this example, the mRNA cell transfection efficiency of lipid nanoparticles formed from each H-type lipid material was examined using commercially available cationic lipids (MC 3, ALC-0315 and SM-102) as a positive control.
HEK239T, U MG and Hela cells were inoculated into 96-well plates with a density of 10000 cells per well, cultured overnight, and lipid nanoparticles containing luciferase mRNA were added per well until the cell density reached 80% or more. After 6 hours, the fluorescence intensity of the expressed luciferase protein was detected using a luciferase detection kit and a chemiluminescent instrument. The data are shown in fig. 5, 6 and 7.
Conclusion: by not fully listing, the lipid nanoparticles formed by the compounds H1, H4, H7, H8 and H10 have obvious advantages over lipid nanoparticles prepared from commercially available cationic lipid materials (MC 3, ALC-0315 and SM-102) in terms of mRNA delivery effect in one or more cells.
Example 5
In this example, the gene silencing efficiency of lipid nanoparticles formed from each H-type lipid material on 293T cells was examined by qRT-PCR experiments using commercially available cationic lipid (MC 3) as a positive control.
293T cells were seeded at a density of 50 ten thousand per well in six well plates and after 24 hours of incubation, each well was charged with lipid particles comprising EGFR SIRNA, with the final concentration of siRNA set at 100nM. Subsequently, cells were transfected in incubator for 24 hours. After transfection was completed, the old medium was discarded and ready for RNA extraction. The six-well plate was removed from the incubator, 1mL of TRIZOL reagent was added to each well, followed by standing at 4 ℃ for 30 minutes to promote cell lysis. Then, 200. Mu.L of chloroform was added thereto, and the mixture was vigorously vortexed and mixed for 30 seconds, and left standing at room temperature for 15 minutes. Subsequently, RNA was extracted by centrifugation at 12000g for 15 minutes at 4℃and dissolved in an appropriate amount of DEPC-treated water. And taking a proper amount of RNA solution, measuring the A280 and A260 values of the RNA solution by using a Nanodrop ultramicro spectrophotometer, and accurately reading the concentration of RNA. Setting up internal reference group and target genome for reverse transcription and amplification. The parameters were set as follows: pre-denaturation at 95 ℃ for 60 seconds; the PCR cycle included denaturation at 95℃for 15 seconds and extension at 60℃for 60 seconds; and finally, carrying out melting curve analysis. Data processing was performed using MX3005P instrument.
Conclusion: by way of non-exhaustive list, lipid nanoparticles formed from lipid materials H1, H4, H10 have better gene silencing efficiency in 293T cells than lipid particles formed from commercial cationic lipid material (MC 3).
Example 6
Fluorescence intensity was used to assess the ability of lipid nanoparticles to deliver mRNA in vivo:
To evaluate lipid nanoparticles mRNA was efficiently delivered in vivo and the corresponding encoded protein was expressed, 6-8 week old female BALB/c tail intravenous injection was at a dose of 0.5mg/kg with mRNA liposome nanoparticles encapsulated with luciferase-expressing mRNA. After 6 hours, each mouse was intraperitoneally injected with a luciferase substrate, and a fluorescence image of the mouse was taken using an IVIS small animal optical in vivo imaging instrument (PERKINELME), and the fluorescence intensity of the whole body of the mouse was counted. The level of fluorescence intensity represents the level of expression of luciferase protein, i.e., reflects the efficiency of in vivo delivery of mRNA by the lipid nanoparticle, and the data is shown in fig. 10.
Conclusion: lipid nanoparticles prepared from H-type lipid materials exhibit specific spleen-targeting accumulation capacity compared to the large accumulation at liver sites exhibited by the cationic lipid materials marketed in bulk.
Example 7
To investigate the transfection of lipid nanoparticles prepared from type H lipids into cells in spleen tissue, mRNA lipid nanoparticles Encapsulating Green Fluorescent Protein (EGFP) were injected intravenously at a dose of 1.5mg/kg into 6-8 week old male C57BL/6J tails. After 24 hours, the spleen was isolated, minced by surgical scissors, and digested in 1mg/mL collagenase A and 10mg/mL DNAse I in RPMI 1640 medium to give a tissue suspension. Subsequently, the tissue suspension was passed through a 70mm nylon cell filter, centrifuged for 7min at 500g, lysed for 5min using red blood cell lysate, washed with PBS, and then cell counted, 6X 10 5 cells were taken, centrifuged for 5min at 500g, resuspended in PBS containing 2% BSA, and the antibodies were stained according to the designed flow staining protocol, analyzed using flow cytometry, the analysis results are shown in FIG. 10.
Conclusion: lipid nanoparticles prepared with H-type lipids were transfected mainly in DC cells in the spleen.
Example 8
Each of 4-6 week old C57BL/6 mice was injected with 2X 10 5 B16-OVA cells and randomly divided into 4 groups of 8 male and female halves. On the sixth and tenth days of tumor inoculation, PBS solution, OVA protein solution (15 mg/kg), MC3-LNP coated with OVA mRNA (15 mg/kg) and lipid nanoparticles prepared with H-type lipid coated with OVA mRNA (15 mg/kg) were administered twice in total, and tumor size was measured daily using a digital vernier caliper (tumor volume calculation formula=0.5×long diameter×short diameter 2), and tumor inhibition curves were plotted as shown in FIG. 11.
Conclusion: mRNA vaccines prepared from the H-type lipid material exhibit excellent tumor suppression effects.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. A lipid material for nucleic acid delivery, characterized in that the lipid material comprises a compound having the structure I:
wherein C nH2n comprises a straight or branched alkyl carbon, n is an integer between 0 and 10;
R 1a、R1b、R1c、R1d is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl; and/or R 1a、R1b、R1c、R1d is selected from a multi-membered carbocyclic ring, a nitrogen containing multi-membered ring, an oxygen containing multi-membered ring; and/or R 1a、R1b and R 1c、R1d each form a closed loop with N;
L 1a,L2a,L1b,L2b is selected from one or more of-C-, - (c=o) -O-, -O- (c=o) -, - (c=o) -NH-, -NH- (c=o) -, -O-, -S-;
-S-S-, -S-S-S-, -S-S-, -S-S-S-, -Se-, -one or more of Se-, -S-C (CH 3) 2 -S-, -O-;
r 2a、R2b is a saturated or unsaturated fatty chain structure containing 10-24 carbons, including cholesterol derivatives and/or tocopherol derivatives.
2. The lipid material of claim 1, wherein the compound has one or more of the following structures:
3. The lipid material of claim 1, wherein the compound has one or more of the following structures:
Wherein the method comprises the steps of
Wherein the method comprises the steps of
Wherein the method comprises the steps of
4. The lipid material of claim 1, wherein the compound has one or more of the following structures:
5. the lipid material of any one of claims 1 to 4, wherein the lipid material is prepared by a method comprising attaching an R 2a、R2b fatty chain, deprotecting and/or condensing.
6. The lipid material of claim 1, further comprising one or more of a neutral lipid, a steroid, and/or a polymer conjugated lipid.
7. The lipid material of claim 6, wherein the neutral lipid is selected from one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and the molar ratio of the compound to the neutral lipid is 1:1 to 10:1; the steroid is selected from the group consisting of sterols, preferably from cholesterol, in a molar ratio of 1:1 to 10:1; the polymer conjugated lipid is selected from pegylated lipids, and the molar ratio of the compound to the polymer conjugated lipid is 100:1 to 5:1.
8. The lipid material of claim 1, wherein the lipid material achieves nucleic acid drug delivery by use in combination with a nucleic acid drug; the combination comprises the preparation of one or more pharmaceutical compositions; the nucleic acid drug is selected from one or more of ASO, siRNA, mRNA, miRNA and pDNA.
9. The lipid material of claim 8, wherein the lipid material is prepared with a nucleic acid drug to form lipid nanoparticles comprising the nucleic acid drug, comprising mixing an aqueous solution of the nucleic acid drug with an ethanol solution of the lipid material; preferably by microfluidic devices, high pressure microfluidic homogenizers, high pressure homogenizers and/or T-tube mixers.
10. The lipid material of claim 1, wherein the lipid material for nucleic acid delivery is used for the preparation of a therapeutic drug selected from one or more of infectious diseases, neoplastic diseases, congenital genetic diseases, and immune diseases.
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