CN117137885A - Lipid nanoparticle based on mildronate derivative and application thereof - Google Patents
Lipid nanoparticle based on mildronate derivative and application thereof Download PDFInfo
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- CN117137885A CN117137885A CN202311096587.6A CN202311096587A CN117137885A CN 117137885 A CN117137885 A CN 117137885A CN 202311096587 A CN202311096587 A CN 202311096587A CN 117137885 A CN117137885 A CN 117137885A
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- mildronate
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- 150000002632 lipids Chemical class 0.000 title claims abstract description 100
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 96
- PVBQYTCFVWZSJK-UHFFFAOYSA-N meldonium Chemical class C[N+](C)(C)NCCC([O-])=O PVBQYTCFVWZSJK-UHFFFAOYSA-N 0.000 title claims abstract description 81
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 71
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 40
- 239000003814 drug Substances 0.000 claims abstract description 25
- 229940079593 drug Drugs 0.000 claims abstract description 18
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 18
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- 238000006482 condensation reaction Methods 0.000 claims abstract description 9
- 239000002994 raw material Substances 0.000 claims abstract description 4
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- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical group CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 24
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- LEACJMVNYZDSKR-UHFFFAOYSA-N 2-octyldodecan-1-ol Chemical compound CCCCCCCCCCC(CO)CCCCCCCC LEACJMVNYZDSKR-UHFFFAOYSA-N 0.000 claims description 5
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- XULHFMYCBKQGEE-MRXNPFEDSA-N 2-Hexyl-1-decanol Natural products CCCCCCCC[C@H](CO)CCCCCC XULHFMYCBKQGEE-MRXNPFEDSA-N 0.000 claims description 4
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- 230000035484 reaction time Effects 0.000 claims description 2
- SPWDQSXYMZXSIV-UHFFFAOYSA-N 11,12-dimethyldocosane Chemical group CCCCCCCCCCC(C)C(C)CCCCCCCCCC SPWDQSXYMZXSIV-UHFFFAOYSA-N 0.000 claims 1
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- 125000002091 cationic group Chemical group 0.000 description 14
- VLKZOEOYAKHREP-UHFFFAOYSA-N hexane Substances CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 12
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 11
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- RPROHCOBMVQVIV-UHFFFAOYSA-N 2,3,4,5-tetrahydro-1h-pyrido[4,3-b]indole Chemical compound N1C2=CC=CC=C2C2=C1CCNC2 RPROHCOBMVQVIV-UHFFFAOYSA-N 0.000 description 3
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- DEMBLPGWNXUBIQ-UHFFFAOYSA-N 2-dodecylhexadecan-1-ol Chemical compound CCCCCCCCCCCCCCC(CO)CCCCCCCCCCCC DEMBLPGWNXUBIQ-UHFFFAOYSA-N 0.000 description 2
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- QGWBEETXHOVFQS-UHFFFAOYSA-N 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate Chemical compound CCCCCCCCC(CCCCCC)C(=O)OCCCCCCN(CCCCO)CCCCCCOC(=O)C(CCCCCC)CCCCCCCC QGWBEETXHOVFQS-UHFFFAOYSA-N 0.000 description 1
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- PPQFUDZMTNGHBJ-UHFFFAOYSA-N propanoic acid;dihydrate Chemical compound O.O.CCC(O)=O PPQFUDZMTNGHBJ-UHFFFAOYSA-N 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
Abstract
The invention discloses a lipid nanoparticle based on mildronate derivatives and application thereof, and belongs to the technical field of medicines. The lipid nanoparticle comprises the following raw materials in percentage by weight: the meldonium derivative is prepared by condensation reaction of meldonium and fatty chain-containing alcohol or amine. The lipid nanoparticle based on mildronate derivatives provided by the invention can effectively deliver nucleic acid medicines such as mRNA and the like in animal bodies, and has higher transfection efficiency. Meanwhile, under the degradation of in vivo esterase, the head group of the mildronate derivative with strong electropositivity is decomposed into the electrically neutral small molecular mildronate, so that the cytotoxicity caused by electropositivity is reduced. The lipid nanoparticle provided by the invention has good biological safety, and is beneficial to improving the clinical application effects of nucleic acid medicaments such as mRNA and the like.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a lipid nanoparticle based on mildronate derivatives and application thereof in preparing mRNA vaccine and gene therapy products as a delivery carrier material.
Background
mRNA drugs are an emerging technology combining molecular biology with immunology. The mRNA synthesized in vitro can be used for encoding protein by using the expression system of the human body, and preventing and treating diseases. Compared with the traditional vaccine, the mRNA vaccine has the advantage of higher efficiency, in addition, mRNA can encode the whole protein structure, presents a plurality of antigen epitopes, and has unique advantages in the design of preventive and therapeutic vaccines. In terms of production, the production flow of mRNA is more stable than DNA or protein cultured on a medium, and large-scale production is easy to realize.
Despite the many advantages of mRNA, there are still many issues with mRNA vaccine design that need to be addressed. Among these, the lack of a safe and efficient delivery system is one of the main reasons limiting its application. How to efficiently deliver mRNA into cells is one of the problems that need to be addressed in mRNA vaccine research.
For the characteristics of mRNA that is unstable, negatively charged, and difficult to be taken up by cells, scientists have developed a range of delivery systems including lipid-based delivery systems (e.g., patent document CN 112961065A, US20220378701 A1), peptide-based delivery systems (e.g., patent document WO2021133931 A1), polymer-based delivery systems (e.g., patent documents EP3106177B1, WO2022125713 A1), and the like. Among them, lipid Nanoparticles (LNPs) are one of the most potential carrier materials due to their good biosafety and efficient delivery capacity, and many drugs based on Lipid Nanoparticles (LNPs) are currently available in the FDA.
At present, the structure-activity relationship among the components of Lipid Nanoparticles (LNPs) is not completely defined. Lipid Nanoparticles (LNPs) are typically composed of cationic lipids (ionizable lipids), phospholipids, PEG-modified lipids, and cholesterol. Among these, cationic lipids are core and soul, the properties of which can affect the overall formulation and biological properties of LNP, and a great deal of systematic research is currently devoted to designing ideal cationic lipids.
The overall structure of cationic lipids can be divided into three parts: (1) The head, the head group, is usually positively charged and is primarily involved in the process of encapsulating nucleic acids, stabilizing LNP, interacting with cell membranes, and facilitating endosomal escape. Clinically used ionizable lipids (DLin-MC 3-DMA, SM-102, ALC-0315) contain tertiary amine heads, and pH-dependent ionization can occur; (2) A linker segment which connects the head and tail, with biodegradable linkers (e.g., esters, amides, and thiols) being preferred, which are generally rapidly cleared in vivo, and which are useful in multiple doses and reduce side effects; (3) Tail, hydrophobic tail, affects pKa, lipophilicity, fluidity and fusogenicity, thereby affecting LNP formation and potency.
Because cationic lipids play a decisive role in the delivery of mRNA, screening for efficient, safe cationic lipids is of great clinical significance. Mildronate is a commercially available cardioprotective agent, the chemical name of which is 3- (2, 2-trimethylhydrazine) propionate dihydrate, and no related research report on the development of cationic lipids by modifying mildronate is currently seen.
Disclosure of Invention
The invention aims to provide a novel cationic lipid material with high biosafety, which is used for preparing lipid nanoparticles and is used as a drug delivery carrier to realize effective delivery of mRNA.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a lipid nanoparticle based on mildronate derivatives, which comprises the following raw materials in parts by weight: mildronate derivatives, auxiliary lipids, cholesterol, the raw materials may form lipid nanoparticles by self-assembly. The mildronate derivative is prepared by condensation reaction of mildronate and fatty chain-containing alcohol or amine, namely, cationic lipid obtained by reacting mildronate with fatty chain-containing alcohol or amine through condensation reaction.
The invention utilizes the condensation of carboxyl and hydroxyl of alcohol containing fatty chain to generate ester bond or the condensation of carboxyl and amino of amine containing fatty chain to generate amide bond to construct the mildronate derivative. The amine-linked group of mildronate forms a positively charged hydrophilic end, which can bind negatively charged drugs via electrostatic interactions.
The lipid material self-assembles due to the hydrophilic and hydrophobic supermolecular forces, thereby preparing the lipid nanoparticle. The nanomaterial can be used as a drug delivery carrier for encapsulating negatively charged drugs such as mRNA. The lipid material wraps the negatively charged drug by using the supermolecular acting force and the electrostatic acting force to form nano particles.
The research of the invention shows that the lipid nanoparticle formed by self-assembling the mildronate derivative and other lipid materials has high cell transfection efficiency as a drug delivery carrier, and can effectively deliver drugs such as mRNA in vivo so as to translate the mRNA into protein. After the lipid nanoparticle enters a body, under the action of in-vivo esterase, an ester bond is broken, and a head group with strong positive electricity of the mildronate derivative is decomposed into electrically neutral small molecular mildronate, so that cytotoxicity caused by electropositivity is reduced, and the supported drug and the mildronate derivative are facilitated to be deconstructed, so that the mildronate is conveniently released to cytoplasm, and the lipid nanoparticle plays a role.
Further, the preparation method of the mildronate derivative comprises the following steps: and (3) dissolving mildronate and fatty chain-containing alcohol or amine in an organic solvent, performing condensation reaction under the catalysis of a catalyst, and purifying the product to obtain the mildronate derivative.
Preferably, the fatty chain-containing alcohol may be any one of, but not limited to, 2-n-octyl-1-dodecanol, 2-decyl-1-tetradecanol, 2-dodecyl hexadec-1-ol, 2-hexyl-1-decanol, 8-hexadecanol, 7-tetradecanol, castor oil. More preferably, the fatty chain-containing alcohol is 2-decyl-1-tetradecanol.
Preferably, the aliphatic chain-containing amine may be, but is not limited to, bis-tetra-amine, bis-dodecyl-amine.
Preferably, the molar ratio of mildronate to fatty chain containing alcohol or amine is 1:1-3.
Preferably, the organic solvent may be, but is not limited to, N-dimethylformamide.
Preferably, the catalyst may employ, but is not limited to, an EDC/DMAP system.
Preferably, the condensation reaction conditions are: the temperature is 40-90 ℃, and the reaction time is 6-72h. More preferably, the heating is carried out at 50℃for 12 hours with stirring.
After the reaction is finished, removing the solvent by rotary evaporation, separating to obtain an initial product, and purifying by using a silica gel chromatography to obtain the mildronate derivative, wherein the volume ratio of an eluent of the silica gel chromatography is 1:1 with ethyl acetate.
Further, the lipid nanoparticle is formed by self-assembly after the mildronate derivative is mixed with auxiliary lipid and cholesterol. The preparation method of the lipid nanoparticle can be used, but is not limited to: ethanol injection, thin film, and ultrasonic. Specifically, when the lipid nanoparticle for encapsulating the drug is prepared, the lipid material and the drug to be encapsulated are subjected to self-assembly in a buffer solution through the interaction of supermolecular acting force and static electricity by adopting the method to form the nanoparticle.
Wherein, the ethanol injection method is to dissolve mildronate derivatives, auxiliary lipid and cholesterol in proper ethanol according to a certain proportion. And then injecting the ethanol solution containing the lipid material into a buffer solution containing the drug to be entrapped, self-assembling to form nano particles, and removing ethanol through dialysis to obtain stable nano particles.
Preferably, the helper lipids include phospholipids and polyethylene glycol functionalized lipids; the phospholipid may be, but is not limited to, distearoyl phosphatidylcholine (DSPC) or dioleoyl phosphatidylethanolamine (DOPE), and the polyethylene glycol functionalized lipid may be, but is not limited to, dimyristoyl glycerol-polyethylene glycol 2000 (DMG-PEG 2000).
Preferably, the molar ratio of mildronate derivative, phospholipid, cholesterol and polyethylene glycol functionalized lipid is 20-70:5-25:15-55:1-2. Further preferably, the molar ratio of the above 4 components is 40 to 55:7-22:21-47:1-2. More preferably, the molar ratio of the above 4 components is 55:15:29:1.
the invention also provides application of the lipid nanoparticle based on mildronate derivatives as a carrier in preparation of nucleic acid drug delivery.
The lipid nanoparticle based on mildronate derivatives provided by the invention can effectively deliver nucleic acid drugs such as mRNA and the like, and the nanomaterial has potential application value in nucleic acid drug development.
Specifically, the application includes: adding mildronate derivatives, auxiliary lipid and cholesterol into an acidic buffer solution containing nucleic acid, and self-assembling to form lipid nanoparticles encapsulating nucleic acid to prepare the nucleic acid drug.
Preferably, the mildronate derivative, the auxiliary lipid and the cholesterol are self-assembled in a buffer containing nucleic acid by an ethanol injection method to form lipid nanoparticles.
Further, the delivery nucleic acid drug is an mRNA vaccine.
Preferably, the ratio of the total mass of the lipid material to the mass of the mRNA is 20-160:1.mRNA molecules are larger, and when lipid materials are too few, mRNA is difficult to effectively wrap and protect, so that transfection efficiency is reduced, and when the lipid materials are too many, endosome escape efficiency is possibly reduced, so that transfection efficiency is reduced. In a proper mass ratio range, the better encapsulation efficiency and transfection efficiency can be ensured. More preferably, the ratio of the total mass of lipid material to the mass of mRNA is 80:1.
the invention has the beneficial effects that:
(1) According to the invention, mildronate derivatives are constructed by condensation reaction of mildronate and fatty chain-containing alcohol or amine, and are used as cationic lipids for preparing lipid nanoparticles. One end of the molecular structure of mildronate is trimethyl ammonia, the other end is carboxyl, and the mildronate derivative is constructed through one-step reaction of alcohol or amine with fatty chain and the carboxyl; its amine-binding group forms a positively charged hydrophilic end that can bind negatively charged drugs via electrostatic interactions.
(2) The lipid nanoparticle based on mildronate derivatives provided by the invention can effectively deliver nucleic acid medicines such as mRNA and the like in animal bodies, and has higher transfection efficiency. Meanwhile, under the degradation of in vivo esterase, the head group of the mildronate derivative with strong electropositivity is decomposed into the electrically neutral small molecular mildronate, so that the cytotoxicity caused by electropositivity is reduced. Compared with the commercially available ionizable lipid SM-102, the lipid nanoparticle prepared from the mildronate derivative provided by the invention has better biological safety, and is beneficial to improving the clinical application effects of nucleic acid medicaments such as mRNA and the like.
Drawings
FIG. 1 is a graph showing transfection effect of LNPs prepared in examples 1-7 in mice.
FIG. 2 shows the quantitative analysis results of the transfection effect in FIG. 1.
FIG. 3 is a graph showing the transfection effect of LNPs prepared by the lipid material of example 6 under different ratios in mice.
FIG. 4 shows the quantitative analysis results of the transfection effect shown in FIG. 3.
FIG. 5 shows the results of a lipid nanoparticle cell safety assay, wherein LNP is a SM-102 based lipid nanoparticle and mLNP-69 is a mildronate derivative based lipid nanoparticle of formulation No. 9.
Fig. 6 is a physical diagram of the in vivo safety test results of lipid nanoparticles, wherein PBS is a negative control, LNP is a SM-102 based lipid nanoparticle, mLNP-69 is a mildronate derivative based lipid nanoparticle of formulation No. 9, and the following is true.
FIG. 7 shows the results of the index of inflammation in the skin tissue of the mouse in FIG. 6, with the leukocyte fraction, the neutrophil fraction, and the neutrophil number in this order from left to right.
Fig. 8 is a diagram showing the cancer suppressing effect of the preparation of a tumor drug using lipid nanoparticles.
Fig. 9 is a graph showing comparison of cancer suppressing effects.
Detailed Description
The invention will be further illustrated with reference to specific examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The compounds and english abbreviations involved in the examples are as follows:
EDC: 1-ethyl- (3-dimethylaminopropyl) carbodiimide, CAS number: 1892-57-5; DMAP: 4-dimethylaminopyridine, CAS no: 1122-58-3;
DMF: n, N-dimethylformamide, CAS no: 68-12-2;
mildronate, CAS number: 76144-81-5;
didodecyl amine, CAS number: 3007-31-6;
n, N-ditetradecylamine, CAS number: 17361-44-3;
8-hexadecanol, CAS number: 19781-83-0;
2-hexyl-1-decanol, CAS number: 2425-77-6;
2-n-octyl-1-dodecanol, CAS number: 5333-42-6;
2-decyl-1-tetradecanol, CAS number: 58670-89-6;
2-dodecyl hexadecan-1-ol, CAS number: 72388-18-2;
d-luciferin potassium salt, CAS number: 115144-35-9;
DSPC phospholipid, CAS number: 816-94-4, the structural formula is as follows:
DMG-PEG2000 lipid, CAS number: 160743-62-4, the structural formula is as follows:
cholesterol, CAS number: 57-88-5, the structural formula is as follows:
example 1
The lipid nanoparticle based on mildronate derivatives is prepared by the following specific steps:
(1) Mitrezide (200 mg,1.37 mmol) and N, N-didodecylamine (403 mg,1.14 mmol) were dissolved in 5mL of DMF under catalysis of EDC (314 mg,1.64 mmol) and DMAP (33 mg,0.27 mmol), heated with stirring at 50℃for 12h, the solvent was removed by rotary evaporation, the initial product was isolated and purified by silica gel chromatography (product eluent: N-hexane: ethyl acetate=1:1 (volume ratio)) and the product THP-12A was obtained after vacuum drying. The reaction process is as follows:
(2) Firstly, mildronate derivative THP-12A, DSPC, DMG-PEG2000 and cholesterol are mixed according to a molar ratio of 50:10:1.5:38.5, the total mass is 400 micrograms of the mixture is dissolved in 20 microliters of ethanol; the ethanol solution was rapidly injected into 60. Mu.L of 20mM sodium acetate buffer containing 5. Mu.g of luciferase mRNA (purchased from offshore protein) under vortexing, vigorously stirred for 20s, and then allowed to stand for 10 minutes to prepare nanoparticles.
(3) The nanoparticle-containing sodium ethylacetate mixed solution prepared in (2) was dialyzed with 10mM PBS solution (dialysis bag Mw=100 kDa) having a neutral pH for 2 to 4 hours to remove ethanol, thereby obtaining the final product.
Example 2
The lipid nanoparticle based on mildronate derivatives is prepared by the following specific steps:
(1) Mitrezide (200 mg,1.37 mmol) and N, N-ditetradecylamine (467 mg,1.14 mmol) are dissolved in 5mL of DMF under catalysis of EDC (314 mg,1.64 mmol) and DMAP (33 mg,0.27 mmol), heated with stirring at 50℃for 12h, the solvent is removed by rotary evaporation, the initial product is isolated and purified by silica gel chromatography (product eluent: N-hexane: ethyl acetate=1:1 (volume ratio)) and the product THP-14A is obtained after vacuum drying. The reaction process is as follows:
(2) Firstly, mildronate derivative THP-14A, DSPC, DMG-PEG2000 and cholesterol are mixed according to a molar ratio of 50:10:1.5:38.5, the total mass is 400 micrograms of the mixture is dissolved in 20 microliters of ethanol; the ethanol solution was rapidly poured into 60. Mu.L of 20mM sodium acetate buffer containing 5. Mu.g of luciferase mRNA under vortexing, vigorously stirred for 20s, and then allowed to stand for 10 minutes to prepare nanoparticles.
(3) The nanoparticle-containing sodium ethylacetate mixed solution prepared in (2) was dialyzed with 10mM PBS solution (dialysis bag Mw=100 kDa) having a neutral pH for 2 to 4 hours to remove ethanol, thereby obtaining the final product.
Example 3
The lipid nanoparticle based on mildronate derivatives is prepared by the following specific steps:
(1) Mitrezide (200 mg,1.37 mmol) and 8-hexadecanol (276 mg,1.14 mmol) were dissolved in 5mL of DMF under catalysis of EDC (314 mg,1.64 mmol) and DMAP (33 mg,0.27 mmol), heated with stirring at 50deg.C for 12h, the solvent was removed by rotary evaporation, the initial product was isolated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate=1:1 (volume ratio)) and dried in vacuo to give the product THP-88. The reaction process is as follows:
(2) Firstly, mildronate derivatives THP-88, DSPC, DMG-PEG2000 and cholesterol are mixed according to a molar ratio of 50:10:1.5:38.5, the total mass is 400 micrograms of the mixture is dissolved in 20 microliters of ethanol; the ethanol solution was rapidly poured into 60. Mu.L of 20mM sodium acetate buffer containing 5. Mu.g of luciferase mRNA under vortexing, vigorously stirred for 20s, and then allowed to stand for 10 minutes to prepare nanoparticles.
(3) The nanoparticle-containing sodium ethylacetate mixed solution prepared in (2) was dialyzed with 10mM PBS solution (dialysis bag Mw=100 kDa) having a neutral pH for 2 to 4 hours to remove ethanol, thereby obtaining the final product.
Example 4
The lipid nanoparticle based on mildronate derivatives is prepared by the following specific steps:
(1) Mitrezide (200 mg,1.37 mmol) and 2-hexyl-1-decanol (276 mg,1.14 mmol) were dissolved in 5mL of DMF under catalysis of EDC (314 mg,1.64 mmol) and DMAP (33 mg,0.27 mmol), heated with stirring at 50℃for 12h, the solvent was removed by rotary evaporation, the initial product was isolated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate=1:1 (volume ratio)) and the product THP-710 was obtained after vacuum drying. The reaction process is as follows:
(2) Firstly, mildronate derivatives THP-710, DSPC, DMG-PEG2000 and cholesterol are mixed according to a molar ratio of 50:10:1.5:38.5, the total mass is 400 micrograms of the mixture is dissolved in 20 microliters of ethanol; the ethanol solution was rapidly poured into 60. Mu.L of 20mM sodium acetate buffer containing 5. Mu.g of luciferase mRNA under vortexing, vigorously stirred for 20s, and then allowed to stand for 10 minutes to prepare nanoparticles.
(3) The nanoparticle-containing sodium ethylacetate mixed solution prepared in (2) was dialyzed with 10mM PBS solution (dialysis bag Mw=100 kDa) having a neutral pH for 2 to 4 hours to remove ethanol, thereby obtaining the final product.
Example 5
The lipid nanoparticle based on mildronate derivatives is prepared by the following specific steps:
(1) Midrozide (200 mg,1.37 mmol) and 2-n-octyl-1-dodecanol (340 mg,1.14 mmol) were dissolved in 5mL of DMF under catalysis of EDC (314 mg,1.64 mmol) and DMAP (33 mg,0.27 mmol), heated with stirring at 50℃for 12h, the solvent was removed by rotary evaporation, the initial product was isolated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate=1:1 (volume ratio)) and the product THP-812 was obtained after drying in vacuo. The reaction process is as follows:
(2) Firstly, mildronate derivatives THP-812, DSPC, DMG-PEG2000 and cholesterol are mixed according to a molar ratio of 50:10:1.5:38.5, the total mass is 400 micrograms of the mixture is dissolved in 20 microliters of ethanol; the ethanol solution was rapidly poured into 60. Mu.L of 20mM sodium acetate buffer containing 5. Mu.g of luciferase mRNA under vortexing, vigorously stirred for 20s, and then allowed to stand for 10 minutes to prepare nanoparticles.
(3) The nanoparticle-containing sodium ethylacetate mixed solution prepared in (2) was dialyzed with 10mM PBS solution (dialysis bag Mw=100 kDa) having a neutral pH for 2 to 4 hours to remove ethanol, thereby obtaining the final product.
Example 6
The lipid nanoparticle based on mildronate derivatives is prepared by the following specific steps:
(1) Mitrezide (200 mg,1.37 mmol) and 2-decyl-1-tetradecanol (404 mg,1.14 mmol) are dissolved in 5mL of DMF under catalysis of EDC (314 mg,1.64 mmol) and DMAP (33 mg,0.27 mmol), the solvent is removed by rotary evaporation under stirring at 50℃for 12h, the initial product is isolated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate=1:1 (volume ratio)) and the product THP-1014 is obtained after drying in vacuo. The reaction process is as follows:
(2) Firstly, mildronate derivatives THP-1014, DSPC, DMG-PEG2000 and cholesterol are mixed according to a molar ratio of 50:10:1.5:38.5, the total mass is 400 micrograms of the mixture is dissolved in 20 microliters of ethanol; the ethanol solution was rapidly poured into 60. Mu.L of 20mM sodium acetate buffer containing 5. Mu.g of luciferase mRNA under vortexing, vigorously stirred for 20s, and then allowed to stand for 10 minutes to prepare nanoparticles.
(3) The nanoparticle-containing sodium ethylacetate mixed solution prepared in (2) was dialyzed with 10mM PBS solution (dialysis bag Mw=100 kDa) having a neutral pH for 2 to 4 hours to remove ethanol, thereby obtaining the final product.
Example 7
The lipid nanoparticle based on mildronate derivatives is prepared by the following specific steps:
(1) Mitrezide (200 mg,1.37 mmol) and 2-dodecyl hexadec-1-ol (268 mg,1.14 mmol) were dissolved in 5mL of DMF under catalysis of EDC (314 mg,1.64 mmol) and DMAP (33 mg,0.27 mmol), the solvent was removed by rotary evaporation under stirring at 50℃for 12h, the initial product was isolated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate=1:1 (volume ratio)) and the product THP-1216 was obtained after vacuum drying. The reaction process is as follows:
(2) Firstly, mildronate derivatives THP-1216, DSPC, DMG-PEG2000 and cholesterol are mixed according to a molar ratio of 50:10:1.5:38.5, the total mass is 400 micrograms of the mixture is dissolved in 20 microliters of ethanol; the ethanol solution was rapidly injected into 60. Mu.L of 20mM sodium acetate buffer containing 5. Mu.g of luciferase mRNA (near shore protein) under vortexing, vigorously stirred for 20s, and then allowed to stand for 10 minutes to prepare nanoparticles.
(3) The nanoparticle-containing sodium ethylacetate mixed solution prepared in (2) was dialyzed with 10mM PBS solution (dialysis bag Mw=100 kDa) having a neutral pH for 2 to 4 hours to remove ethanol, thereby obtaining the final product.
Test example 1: particle size and surface potential analysis of lipid nanoparticles
The lipid nanoparticles prepared in examples 1-7 were tested using a malvern particle sizer and the results were as follows:
the average particle diameter of the nanomaterial LNP prepared in example 1 was 255.3nm, and the distribution coefficient pdi=0.479; zeta potential was-0.211 mv and the nanoparticles were seen to be electrically neutral.
The average particle diameter of the nanomaterial LNP prepared in example 2 is 243.2nm, and the distribution coefficient pdi=0.333; zeta potential was-0.227 mv and it was seen that the nanoparticle was electrically neutral.
The average particle diameter of the nanomaterial LNP prepared in example 3 was 310.0nm, and the distribution coefficient pdi=0.168; zeta potential was-0.387 mv and it was seen that the nanoparticle was charge neutral.
The average particle diameter of the nanomaterial LNP prepared in example 4 was 198.3nm, and the distribution coefficient pdi=0.081; zeta potential was-0.408 mv and it was seen that the nanoparticle was electrically neutral.
The average particle diameter of the nanomaterial LNP prepared in example 5 is 221.6nm, and the distribution coefficient pdi=0.138; zeta potential was 0.105mv and the nanoparticle was seen to be electrically neutral.
The average particle diameter of the nanomaterial LNP prepared in example 6 is 231.4nm, and the distribution coefficient pdi=0.128; zeta potential was-0.054 mv and it was seen that the nanoparticle was electrically neutral.
The average particle diameter of the nanomaterial LNP prepared in example 7 is 241.7nm, and the distribution coefficient pdi=0.164; zeta potential was 0.009mv and the nanoparticle was seen to be electrically neutral.
Test example 2: in vivo transfection experiments of lipid nanoparticles
1. The dialyzed LNP in PBS was injected into mice by subcutaneous injection, 6 hours later, the luciferase substrate D-luciferin potassium salt (10 mg/mL, 200. Mu.L) was injected intraperitoneally, and a fluorescence signal was observed by a small animal in vivo imager.
As shown in fig. 1, we observed that there was a clear fluorescent signal in the mice at 6h, and it was seen that the lipid nanoparticle was successfully taken up by the cells at and around the injection site, while the vector successfully delivered the luciferase mRNA into the cytoplasm of the mice and successfully translated into a large amount of protein.
The transfection effect of the nanomaterial LNP prepared in each example in mice was quantitatively analyzed by a small animal living body imager. As shown in FIG. 2, it can be seen that the effect of the tail on liposome performance is enormous, with the liposomes of example 6 having the best transfection effect, to be used in subsequent experiments.
Therefore, the lipid nanoparticle can effectively deliver mRNA in animal bodies and has potential clinical application value.
2. Optimization of lipid nanoparticle formulation and in vivo transfection experiments
To increase the delivery efficiency of lipid nanoparticles, we designed nine different formulations, as shown in table 1, to explore the optimal molar ratio formulation to maximize the mRNA delivery efficiency of lipid nanoparticles.
TABLE 1
Meldonium derivatives THP-1014, DSPC, cholesterol, DMG-PEG2000 were dissolved in 20. Mu.L ethanol at a ratio of 240. Mu.g total mass, according to the molar ratios shown in Table 1; the ethanol solution was rapidly poured into 60. Mu.L of 20mM sodium acetate buffer containing 3. Mu.g of luciferase mRNA under vortexing, vigorously stirred for 20s, and then allowed to stand for 10 minutes to prepare nanoparticles.
The dialyzed LNP in PBS was injected into mice by subcutaneous injection, 6 hours later, the luciferase substrate D-luciferin potassium salt (10 mg/mL, 200. Mu.L) was injected intraperitoneally, and a fluorescence signal was observed by a small animal in vivo imager.
As shown in fig. 3, we observed that there was a distinct fluorescent signal in mice at 6h and a distinct difference between the different groups. It is demonstrated that the delivery efficiency of lipid nanoparticles to mRNA can be significantly affected and improved by adjusting the four components.
By using a small animal living body imager, quantitative analysis is carried out on the in-vivo transfection effect of mice with the formula, and as shown in fig. 4, the lipid nanoparticle delivery efficiency can be remarkably improved by adjusting the proportion and optimizing the formula. Subsequent experiments will be completed using formulation No. 9.
Test example 3: lipid nanoparticle safety experiments
1. Cellular level detection of lipid nanoparticle biosafety
Midrozide derivatives THP-1014, DSPC, cholesterol and DMG-PEG2000 were prepared according to No. 9 formulation under the conditions of 40 mug, 200 mug, 400 mug and 4000 mug of total lipid material mass respectively (the amount of the corresponding entrapped mRNA is 0.5 mug, 2.5 mug, 5 mug and 50 mug respectively based on 80 mass ratio of liposome to mRNA). A control group was also set up and commercially available SM-102 was used to replace empty LNP prepared from THP-1014.
Inoculating kidney cells of mice into colorless and transparent 96-well plate, and allowing cells to adhere and grow to 5×10 4 /well. The empty LNP prepared as described above was then added, corresponding to a deliverable mRNA mass of 0.5. Mu.g/mL, 2.5. Mu.g/mL, 5. Mu.g/mL, 50. Mu.g/mL. After 24h incubation, the medium was changed and cytotoxicity was assessed by CCK8 cytotoxicity assay.
As shown in fig. 5, at high concentration, compared with the commercial cationic liposome based on SM-102, the cationic liposome based on mildronate derivative has better biosafety, which is beneficial to widening the application field of the cationic liposome.
2. Lipid nanoparticle in vivo safety experiments
On days 1 and 4, empty lipid nanoparticles (corresponding to an amount of entrapped mRNA of 25 μg based on 80% by mass of liposome to mRNA) prepared at 2mg of total mass of lipid material according to formulation No. 9 were injected subcutaneously into the abdomen of mice (n=5) and on day 7, the mice were sacrificed and the skin of 1cm×1cm at the injection site was dissected and analyzed for leukocyte and neutrophil content in the tissues by flow cytometry.
As shown in fig. 6, it was seen with naked eyes that after injection of cationic liposomes based on SM-102, the abdomen of mice showed significant skin hyperplasia and hardening, and some mice also developed vesicles, while mice injected with liposomes based on mildronate derivatives had skin conditions closer to those of the PBS group in the natural state.
As shown in fig. 7, after injection of cationic liposomes based on SM-102, mice had a higher proportion of leukocytes and neutrophils and a higher number of neutrophils in skin tissue, suggesting a strong inflammatory response here, whereas cationic liposomes based on mildronate derivatives showed better biosafety.
Application example 1: application of lipid nanoparticle in tumor vaccine
On day 1, mice were vaccinated with 100 wan of B16-OVA tumor cells by subcutaneous injection, and on days 5, 8, and 11, lipid nanoparticles containing 5 μg of OVA mRNA prepared according to formulation 9 above were injected into the mice by subcutaneous injection.
As shown in fig. 8 and 9, the cationic lipid nanoparticle based on mildronate derivative has the same tumor inhibiting effect as the cationic lipid nanoparticle of SM-102.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.
Claims (10)
1. A lipid nanoparticle based on mildronate derivatives, characterized in that the raw material composition of the lipid nanoparticle comprises: the meldonium derivative is prepared by condensation reaction of meldonium and fatty chain-containing alcohol or amine.
2. The lipid nanoparticle based on mildronate derivatives according to claim 1, wherein the preparation method of the mildronate derivatives comprises: and (3) dissolving mildronate and fatty chain-containing alcohol or amine in an organic solvent, performing condensation reaction under the catalysis of a catalyst, and purifying the product to obtain the mildronate derivative.
3. The mildronate derivative-based lipid nanoparticle according to claim 1 or 2, wherein the fatty chain-containing alcohol is any one of 2-n-octyl-1-dodecanol, 2-decyl-1-tetradecanol, 2-dodecyl hexadec-1-ol, 2-hexyl-1-decanol, 8-hexadecanol, 7-tetradecanol, castor oil; the amine containing fatty chains is bis-tetraamine or bis-dodecyl amine.
4. The mildronate derivative-based lipid nanoparticle according to claim 2, wherein the organic solvent is N, N-dimethylformamide and the catalyst is EDC/DMAP system.
5. The mildronate derivative-based lipid nanoparticle of claim 2, wherein the condensation reaction conditions are: the temperature is 40-90 ℃, and the reaction time is 6-72h.
6. The mildronate derivative-based lipid nanoparticle of claim 1, wherein the helper lipid comprises a phospholipid and a polyethylene glycol functionalized lipid; the phospholipid is distearoyl phosphatidylcholine or dioleoyl phosphatidylethanolamine, and the polyethylene glycol functionalized lipid is dimyristoyl glycerol-polyethylene glycol 2000.
7. The mildronate derivative-based lipid nanoparticle of claim 6, wherein the molar ratio of mildronate derivative, phospholipid, cholesterol, polyethylene glycol functionalized lipid is 20-70:5-25:15-55:1-2.
8. Use of lipid nanoparticles based on mildronate derivatives according to any one of claims 1-7 as a carrier for the preparation of a drug for delivery of nucleic acids.
9. The application of claim 8, wherein the application comprises: adding mildronate derivatives, auxiliary lipid and cholesterol into an acidic buffer solution containing nucleic acid, and self-assembling to form lipid nanoparticles encapsulating nucleic acid to prepare the nucleic acid drug.
10. The use of claim 9, wherein the delivered nucleic acid agent is an mRNA vaccine and the ratio of the total mass of lipid material to the mass of mRNA is 20 to 160:1.
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