CN113372226B - Lipid molecule, lipid nanoparticle, and preparation methods and application thereof - Google Patents
Lipid molecule, lipid nanoparticle, and preparation methods and application thereof Download PDFInfo
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- CN113372226B CN113372226B CN202110637314.2A CN202110637314A CN113372226B CN 113372226 B CN113372226 B CN 113372226B CN 202110637314 A CN202110637314 A CN 202110637314A CN 113372226 B CN113372226 B CN 113372226B
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- FHQVHHIBKUMWTI-ZCXUNETKSA-N 1-palmitoyl-2-oleoyl phosphatidylethanolamine Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC FHQVHHIBKUMWTI-ZCXUNETKSA-N 0.000 claims 1
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Abstract
The invention provides a lipid molecule, a lipid nanoparticle, a preparation method and application thereof, wherein the lipid molecule has a structure shown in a formula I, and can be constructed into nano-scale to micro-scale lipid nanoparticles. The lipid nanoparticle can realize safe and efficient delivery of nucleic acid drugs, small molecule drugs, peptide drugs and protein drugs, and has the advantages of large drug loading, high stability and good safety. The lipid nanoparticle has simple components, the prepared nano-drug has good batch stability and high transfection efficiency when mRNA drugs are loaded, and provides a new idea for realizing safe and efficient drug delivery in vitro and in vivo.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a lipid molecule, a lipid nanoparticle, a preparation method and application thereof.
Background
Nucleic acid drugs are a generic term for oligoribonucleotides (RNAs) or oligodeoxyribonucleotides (DNAs) with different functions that function at the genetic level, including but not limited to: siRNA, miRNA, antisense nucleic acids, aptamers, DNA, messenger RNA (mRNA), and the like. Wherein mRNA is a single-stranded ribonucleic acid which is transcribed from one strand of DNA as a template, carries genetic information and can guide the synthesis of proteins. In recent years, in view of the excellent characteristics of translation in cytoplasm, simple production process, rapid synthesis, low cost, mass production and the like of mRNA when used as a drug, mRNA has been applied to prevention and treatment of various diseases, particularly in the field of vaccines. However, mRNA has disadvantages of easy degradation, instability, difficult cell entry, and the like, so that mRNA is required to be a suitable delivery system to compensate for the above-described disadvantages in addition to optimization of its own chemical structure. At present, mRNA delivery system is one of the most critical technologies restricting mRNA patent medicine.
mRNA delivery systems mainly include viral vectors, which are at risk of integration into the genome and produce various degrees of humoral or cellular immune responses, and non-viral vectors, and therefore have limited clinical use. Non-viral vectors are primarily focused on lipid nanoparticles, polymers, and proteins. Lipid nanoparticles are currently the most interesting and studied delivery system. For example CN112543639a discloses a lipid nanoparticle composition for delivery of mRNA and long nucleic acids comprising, in molar ratio, 5 to 50 cationically ionizable lipids, 10 to 45 phospholipids, 15 to 50 steroids, 0.5 to 10 pegylated lipids. CN111467321a discloses a mRNA nucleic acid drug delivery system, which comprises lipid nanoparticles loaded with one or more mrnas, wherein the lipid nanoparticles are prepared from raw materials comprising ionizable cationic lipids, phospholipid auxiliary lipids, cholesterol and phospholipid polyethylene glycol derivatives, and have better intracellular delivery efficiency of mRNA drugs.
From the existing research results, more related lipid nanoparticles have been applied to mRNA delivery, for example, mRNA-1273 vaccine of Moderna company in the United states and BNT162b2 vaccine of Biontech company in Germany all adopt lipid nanoparticle delivery technology; wherein, the lipid molecules mainly involved in the preparation of the lipid nanoparticle comprise MC3, ALC-0315, SM-102 and the like. However, in the long term, the lipid nanoparticle delivery technology of mRNA still faces many challenges, such as excessive lipid nanoparticle components, unfavorable production, low safety and efficiency of delivery, low specificity, and complex synthesis of a part of specific lipid molecules, which is difficult to realize in large scale application.
Therefore, the development of the lipid nanoparticle which has the advantages of good safety, high efficiency, simple components and easy preparation and realizes safe and efficient drug delivery in vitro and in vivo is the research focus in the field.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a lipid molecule, a lipid nanoparticle, a preparation method and application thereof, wherein the lipid nanoparticle constructed by the lipid molecule has the characteristics of large drug loading capacity, high stability and good safety, and can realize the efficient and safe delivery of nucleic acid drugs, small molecular drugs, peptide drugs and protein drugs.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a lipid molecule having a structure according to formula I:
in the formula I, R 1 Is selected from any one of C1-C50 straight-chain or branched-chain alkyl, C2-C50 straight-chain or branched-chain unsaturated alkyl, C3-C50 alicyclic alkyl, C2-C50 alicyclic heterocyclic group, C6-C50 aromatic alkyl or C6-C50 alicyclic alkyl containing aromatic ring.
In the formula I, R 2 Any one of hydrogen, C1-C50 straight-chain or branched-chain alkyl, C2-C50 straight-chain or branched-chain unsaturated alkyl, C3-C50 alicyclic alkyl, C2-C50 alicyclic heterocyclic group, C6-C50 aromatic alkyl or C6-C50 alicyclic alkyl containing aromatic ring.
The lipid molecule provided by the invention has a chemical structure shown in a formula I, can construct nanoscale to micron-scale lipid nanoparticles, can realize safe and efficient delivery of nucleic acid drugs, small-molecule drugs, peptide drugs and protein drugs, has large drug loading capacity, high stability and good safety, and has high transfection efficiency and good batch stability.
In the present invention, the C1 to C50 linear or branched alkyl group may be a linear or branched alkyl group such as C2, C5, C10, C12, C15, C18, C20, C22, C25, C28, C30, C32, C35, C38, C40, C42, C45 or C48, and is preferably a linear or branched alkyl group of C6 or more.
The C2 to C50 linear or branched unsaturated hydrocarbon group may be a linear or branched unsaturated hydrocarbon group of C3, C5, C10, C12, C15, C18, C20, C22, C25, C28, C30, C32, C35, C38, C40, C42, C45 or C48, etc., and the "unsaturated hydrocarbon group" means that the group contains at least one c=c double bond or at least one c≡c triple bond.
The C3-C50 alicyclic hydrocarbon group can be an alicyclic hydrocarbon group of C3, C5, C10, C12, C15, C18, C20, C22, C25, C28, C30, C32, C35, C38, C40, C42, C45 or C48, etc., and the "alicyclic hydrocarbon group" means a non-aromatic carbocyclic group including a saturated alicyclic hydrocarbon group or an unsaturated alicyclic hydrocarbon group; the saturated alicyclic hydrocarbon group includes a single ring, multiple rings (condensed rings containing 2,3 or 4 rings), spiro ring, and exemplary includes but is not limited to: cyclohexenyl, cycloheptyl, adamantyl, bicyclo [2.2.1] hept-2-yl, or bicyclo [3.1.0] hex-3-yl, and the like; by "unsaturated alicyclic hydrocarbon group" is meant an alicyclic hydrocarbon group containing at least one c=c double bond or at least one c≡c triple bond, illustratively including but not limited to: cyclohexenyl, cycloheptenyl, and the like.
The C2 to C50 alicyclic group may be an alicyclic group of C3, C5, C10, C12, C15, C18, C20, C22, C25, C28, C30, C32, C35, C38, C40, C42, C45, or C48, etc., that is, a group formed by introducing a heteroatom into an alicyclic hydrocarbon group, and the meaning of the alicyclic hydrocarbon group is as described above, and is not repeated herein.
The C6-C50 aromatic hydrocarbon group may be an aromatic hydrocarbon group of C6, C9, C10, C12, C15, C18, C20, C22, C25, C28, C30, C32, C35, C38, C40, C42, C45, or C48, etc., exemplary including but not limited to: phenyl, naphthyl, biphenyl, anthryl, phenanthryl, and the like.
The alicyclic hydrocarbon group having an aromatic ring of C6 to C50 (e.g., C6, C9, C10, C12, C15, C18, C20, C22, C25, C28, C30, C32, C35, C38, C40, C42, C45, or C48, etc.), that is, a group formed by introducing an aromatic ring to the alicyclic hydrocarbon group, and the meaning of the alicyclic hydrocarbon group is as described above and will not be repeated herein.
In the present invention, the heteroatoms include, but are not limited to N, P, O or S.
Preferably, said R 1 C8-C20 straight-chain alkyl, for example C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20 straight-chain alkyl.
Preferably, said R 2 Any one of hydrogen, C8-C20 (e.g., C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20) straight-chain alkyl.
Preferably, said R 1 、R 2 At least one of the following groups:
wherein the wavy line marks represent the bond of the group.
Preferably, the lipid molecule has any one of the following structures:
in a second aspect, the present invention provides a method of preparing a lipid molecule according to the first aspect, the method of preparing comprising method I or method II;
the method I comprises the following steps: r is R 2 Is hydrogen, oleylamine and compound R 1 -X 1 And (3) carrying out a reaction to obtain the lipid molecule, wherein the reaction formula is as follows:
the method II comprises the following steps: r is R 2 Any one of C1-C50 straight-chain or branched-chain alkyl, C2-C50 straight-chain or branched-chain unsaturated alkyl, C3-C50 alicyclic alkyl, C2-C50 alicyclic heterocyclic group, C6-C50 aromatic alkyl or C6-C50 alicyclic alkyl containing aromatic ring; oil (oil)Amine and compound R 1 -X 1 Compound R 2 -X 2 And (3) carrying out a reaction to obtain the lipid molecule, wherein the reaction formula is as follows:
wherein R is 1 Having the same defined ranges as in formula I;
X 1 、X 2 each independently selected from any one of Cl, br or I.
Preferably, the oleylamine is combined with compound R in Process I 1 -X 1 The molar ratio of (2) is 1 (1-1.2), and may be, for example, 1:1.02, 1:1.05, 1:1.08, 1:1.1, 1:1.12, 1:1.15, or 1:1.18.
Preferably, the reaction in process I is carried out in the presence of an alkaline substance, more preferably Cs 2 CO 3 In the presence of a catalyst.
Preferably, the reaction in process I is carried out in the presence of a solvent.
Preferably, the solvent comprises tetrahydrofuran.
Preferably, the temperature of the reaction in process I is 30 to 50℃and may be, for example, 31 ℃, 33 ℃, 35 ℃, 37 ℃, 39 ℃, 40 ℃, 41 ℃, 43 ℃, 45 ℃, 47 ℃ or 49 ℃ and specific values between the above values, the present invention is not exhaustive of the specific values included in the range for reasons of space and for reasons of simplicity.
Preferably, the reaction time in method I is from 12 to 72 hours, for example, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 40, 44, 48, 52, 56, 60, 64 or 68 hours, and the specific point values between the above points are limited in length and for brevity, the invention is not intended to be exhaustive.
Preferably, in process II, the compound R is present in an amount of 1 mole based on the amount of the oleylamine 1 -X 1 With compound R 2 -X 2 The sum of the amounts of (2) to (2.5) mol, for exampleSpecific point values for 2.05mol, 2.1mol, 2.15mol, 2.2mol, 2.25mol, 2.3mol, 2.35mol, 2.4mol or 2.45mol, and between the above point values, are for brevity and for brevity, the invention is not intended to be exhaustive.
Preferably, the reaction in method II is carried out in the presence of an alkaline substance, more preferably Cs 2 CO 3 In the presence of a catalyst.
Preferably, the reaction in process II is carried out in the presence of a solvent.
Preferably, the solvent comprises tetrahydrofuran.
Preferably, the temperature of the reaction in method II is 30 to 50℃and may be, for example, 31 ℃, 33 ℃, 35 ℃, 37 ℃, 39 ℃, 40 ℃, 41 ℃, 43 ℃, 45 ℃, 47 ℃ or 49 ℃ and specific values between the above values, the present invention is not exhaustive of the specific values included in the range for reasons of space and for reasons of simplicity.
Preferably, the reaction time in method II is from 12 to 72 hours, for example, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 40, 44, 48, 52, 56, 60, 64 or 68 hours, and the specific point values between the above points are limited in length and for brevity, the invention is not intended to be exhaustive.
Preferably, the reaction of the method I and the method II further comprises a post-treatment step after the reaction is completed.
Preferably, the post-processing method comprises: and extracting, purifying by chromatography, concentrating and drying the product obtained by the reaction in sequence to obtain the lipid molecule.
Preferably, the extracted reagent comprises chloroform.
Preferably, the system parameters of the chromatographic purification are: lambda (lambda) 1 ＝206nm，λ 2 ＝216nm。
Preferably, the chromatographically purified mobile phase comprises a combination of methylene chloride and methanol.
In a third aspect, the present invention provides a lipid nanoparticle comprising a lipid molecule according to the first aspect.
The lipid nanoparticle may contain one lipid molecule with a structure shown in formula I, or may contain a plurality of lipid molecules with structures shown in formula I.
Preferably, the lipid nanoparticle further comprises a second lipid, the second lipid being different in molecular structure from the lipid molecule.
Preferably, the molar ratio of the lipid molecule to the second lipid is 1 (0.125-8), for example, may be 1:0.2, 1:0.5, 1:0.8, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, etc., further preferably 1:0.5.
Preferably, the second lipid comprises any one or a combination of at least two of a non-cationic lipid, a cationic lipid or a polyethylene glycol modified lipid.
Preferably, the non-cationic lipid comprises 1, 2-dioleyl-sn-glycero-3-phosphorylethanolamine (DOPE), cholesterol (Chol), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE), 1, 2-myristoyl-sn-glycero-3-phosphorylethanolamine (DMPE), 1, 2-dioleoyl-rac- (1-glycero-sodium salt (DOPG), 1, 2-palmitoyl-phosphatidylglycerol (DPPG), 1-palmitoyl-2-oleoyl Phosphatidylcholine (PC), 1-acyl-2-oleoyl phosphatidylethanolamine (POPE), stearoyl-glycero-3-phosphorylethanolamine (DPPE), stearoyl-glycero-3-phosphorylethanolamine (POPE), stearoyl-phosphatidylethanolamine (POacyl-2-Phosphatidylethanolamine (PSE), stearoyl-2-phosphatidylethanolamine (POacyl-2-Phosphatidylethanolamine (PSE), any one or a combination of at least two of sphingomyelin or sterols.
Preferably, the cationic lipid comprises N, N-dioleyl-N, N-dimethyl ammonium chloride (DODAC), N, N-distearyl-N, N-dimethyl ammonium bromide (DDAB), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethyl ammonium chloride (DOTAP), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethyl ammonium chloride (DOTMA), N, N-dimethyl-2, 3-dioleyloxy propylamine (DODMA), 1, 2-dioleyloxy-3- (dimethylamino) acetoxypropane (DLin-DAC), 1, 2-dioleyloxy-3-dimethylaminopropane (DLin), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1, 2-dioleyloxy-3- (N-Methylpiperazine) Propane (MPZ), 1, 2-dioleyloxy-3- (DLOoxy-DAP), any one or a combination of at least two of N-dimethylaminopropane (DLinDMA) or 2, 2-diimine-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA).
Preferably, the polyethylene glycol modified lipid comprises PEG-phosphatidylethanolamine, PEG-phosphatidic acid, PEG-ceramide, PEG-dihydrocarbylamine, PEG-diglyceride, most typically DMG-PEG, DLPE-PEG, DMPE-PEG, DPPC-PEG, DSPE-PEG.
Preferably, the weight average molecular weight Mw of polyethylene glycol in the polyethylene glycol modified lipid is 1000-10000.
The lipid nanoparticle provided by the invention is obtained through self-assembly of a lipid molecule with a structure shown in a formula I, optionally a second lipid, and has the following advantages when used for drug delivery:
(1) Excellent transfection efficiency
Taking UC18-C16 constructed lipid nanoparticle as an example, the transfection efficiency of the lipid nanoparticle is better than Lipofectamine 2000 of Siemens under the same condition after the lipid nanoparticle is subjected to multi-round formula optimization.
(2) Good stability between batches
The lipid nanoparticle disclosed in the prior art is generally composed of four (or more than four) components, the components are more, the formula is complex, and the lipid nanoparticle provided by the invention can obtain high-efficiency transfection efficiency through the adjustment and optimization of single components or simple two components (lipid molecules and second lipid), so that the quality stability among batches is favorably maintained.
(3) High drug loading rate
When lipid nanoparticles disclosed in the prior art are used to deliver mRNA (nucleic acid-based drug), the mass ratio (wt: wt) of cationic or ionizable lipid to mRNA is typically maintained at 10:1. The lipid nanoparticle provided by the invention is optimized to have a lipid molecule to mRNA mass ratio of about (5-7): 1, which indicates that the mass of lipid molecules required to deliver an equivalent amount of mRNA is at least 1.4 times lower than other cationic or ionizable lipids. Therefore, the lipid nanoparticle provided by the invention has higher drug loading rate, and is beneficial to reducing the dosage of a delivery carrier.
(4) Good biocompatibility
The nano-drugs (lipid nanoparticle loaded mRNA drugs) with different concentrations have weak hemolytic capability on red blood cells under the condition of pH of 7.4, which indicates that the biocompatibility of the nano-drugs and the lipid nanoparticles is good in the test range.
In a fourth aspect, the present invention provides a nano-drug comprising the lipid nanoparticle according to the third aspect, and a drug supported on the lipid nanoparticle.
Preferably, the drug comprises any one or a combination of at least two of nucleic acid drugs, small molecule drugs, peptide drugs or protein drugs, and further preferably nucleic acid drugs.
Preferably, the nucleic acid drug comprises any one or a combination of at least two of an mRNA drug, an siRNA drug, an miRNA drug, an antisense nucleic acid drug, an aptamer drug or a DNA drug.
Preferably, the drug is a nucleic acid drug, the molar ratio of the N atom in the lipid molecule to the P atom in the nucleic acid drug is (0.125-32): 1, for example, may be 0.2:1, 0.5:1, 0.8:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1 or 30:1, and more preferably (3-5): 1).
Preferably, the hydration particle size of the nano-drug is 250-350 nm, for example, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm or 340nm, and specific point values between the above point values, which are limited in space and for the sake of brevity, the present invention is not exhaustive.
In a fifth aspect, the present invention provides a method for preparing a nano-drug according to the fourth aspect, the method comprising: mixing the lipid molecule according to the first aspect, optionally a second lipid, with an organic solvent to obtain a lipid molecule organic phase; mixing the medicine with an aqueous solvent to obtain a medicine solution; mixing the lipid molecular organic phase with a drug solution to obtain the nano-drug.
Preferably, the organic solvent comprises ethanol.
Preferably, the aqueous solvent is a buffer solution, more preferably a PBS buffer solution.
Preferably, the molar ratio of lipid molecules to second lipid is 1 (0.125-8), e.g. may be 1:0.2, 1:0.5, 1:0.8, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, etc.
Preferably, the drug is a nucleic acid drug, the molar ratio of the N atom in the lipid molecule to the P atom in the nucleic acid drug is (0.125-32): 1, for example, may be 0.2:1, 0.5:1, 0.8:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1 or 30:1, and more preferably (3-5): 1).
Preferably, the concentration of the drug in the drug solution is 20-25 ng/μl, for example, 20.5ng/μl, 21ng/μl, 21.5ng/μl, 22ng/μl, 22.5ng/μl, 23ng/μl, 23.5ng/μl, 24ng/μl, or 24.5ng/μl, and specific point values between the above point values are limited in length and for brevity, the present invention is not exhaustive of the specific point values included in the range.
Preferably, the volume ratio of the lipid molecule organic phase to the drug solution is 1 (1-10), for example, may be 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9 or 1:9.5, etc.
Preferably, the method of mixing the lipid molecular organic phase with the drug solution comprises microfluidic methods.
Preferably, the lipid molecular organic phase further comprises a step of standing after mixing with the drug solution.
Preferably, the standing time is 10-30 min, for example, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min or 19min, and specific point values among the above point values, which are limited in space and for simplicity, the present invention is not exhaustive.
Preferably, after the lipid molecular organic phase is mixed with the drug solution and is kept stand, the obtained product is a preparation comprising the nano-drug, and the preparation also comprises a solvent, wherein the solvent comprises a combination of an organic solvent and an aqueous solvent.
Preferably, the volume percent of organic solvent (ethanol) in the formulation is 5-30%, such as 6%, 8%, 10%, 12%, 15%, 18%, 20%, 22%, 25% or 28%, and the specific point values between the above point values, are for brevity and for simplicity, the invention is not intended to be exhaustive of the specific point values included in the range.
Compared with the prior art, the invention has the following beneficial effects:
the lipid molecule provided by the invention has a specific structure shown in the formula I, and can be constructed into nano-scale to micro-scale lipid nanoparticles, so that safe and efficient delivery of nucleic acid drugs, small-molecule drugs, peptide drugs and protein drugs is realized, and the lipid molecule has the advantages of large drug loading capacity, high stability and good safety. The lipid nanoparticle has simple components, the prepared nano-drug has good batch stability, and the prepared nano-drug has high transfection efficiency when the mRNA drug is loaded, thus providing a new idea for realizing safe and efficient drug delivery in vitro and in vivo.
Drawings
FIG. 1 is a mass spectrum of lipid molecules UC 18-C16;
FIG. 2 is a graph of screening for delivery efficiency of lipid nanoparticle-loaded drugs constructed from different lipid molecules;
FIG. 3 is a graph of optimized delivery efficiency of nanomedicines of different ratios;
FIG. 4 is a nucleic acid electrophoretogram of a nano-drug of different N/P ratios;
FIG. 5 is a transmission electron microscope image of the nano-drug provided in example 18;
FIG. 6 is a graph showing the hydration particle size distribution of the nano-drug provided in example 18;
FIG. 7 is a potential diagram of a nano-drug provided in example 18;
FIG. 8 is a graph showing the absorbance (660 nm) measurement of the nanopharmaceuticals provided in example 18 after incubation in 10% serum at room temperature;
FIG. 9 is a graph showing the stability of the nano-drug and free mRNA provided in example 18 against serum of different concentrations;
fig. 10 is a graph of biocompatibility tests for lipid nanoparticles at different concentrations.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Preparation example
A lipid molecule UC18-C16, having the structure:
the preparation method comprises the following steps:
by mixing oleylamine
And (3) with
Mixed in a molar ratio of 1:1 and dissolved in Tetrahydrofuran (THF), 1.5 molar equivalents Cs are added 2 CO 3 After reaction for 24 hours at 40℃based on 1mol of oleylamine, the product obtained is washed with water and with CHCl 3 Extraction was performed three times using a medium pressure preparative chromatography purification system (. Lamda.) 1 ＝206nm，λ 2 =216 nm), in CH 2 Cl 2 And methanol (volume ratio 10)1) purifying by taking the mixture as a mobile phase, concentrating and drying under reduced pressure to obtain a target product.
Characterization data: the mass spectrum of the lipid molecules UC18-C16 is shown in FIG. 1, and MS (m/z) can be seen from FIG. 1: 492.5482 (M+H) + 。
The synthesis method of other lipid molecules in the present invention is the same as the preparation examples described above, except that different kinds or amounts of bromine-containing compounds are replaced, and the details are not repeated here.
In the following examples of the present invention, the mRNA drugs involved were all Luciferase mRNA (accession number R1018-1, APExBIO).
Example 1
A lipid nanoparticle comprising UC18-2C10 (lipid molecule) and DOPE (second lipid) in a molar ratio of 1:1; the nano-drug comprises the lipid nano-particles and mRNA drugs loaded on the lipid nano-particles; the molar ratio of N atoms in UC18-2C10 to P atoms in the mRNA drug (hereinafter referred to as N/P ratio) is 2:1.
The preparation method of the nano-drug comprises the following steps:
(1) Mixing lipid molecules UC18-2C10 and DOPE in a molar ratio of 1:1 and dissolving in ethanol to obtain a lipid molecule organic phase; dissolving mRNA medicine in PBS buffer solution with pH value of 7.4 to obtain medicine solution;
(2) Mixing the lipid molecular organic phase obtained in the step (1) with a drug solution according to a volume ratio of 1:9, so that the molar ratio (N/P ratio) of N atoms in UC18-2C10 to P atoms in the mRNA drug is 2:1, mixing for 30s, standing at room temperature, and incubating for 15min to obtain a preparation containing the nano drug.
Examples 2 to 10
A lipid nanoparticle and a nano-drug comprising the same are different from example 1 only in the kind of lipid molecules in the lipid nanoparticle; other components, amounts and preparation methods are the same as in example 1; the specific components are shown in Table 1.
TABLE 1
Example 11
A lipid nanoparticle comprising UC18-C16 and DOPE (second lipid) in a molar ratio of 1:2; the nano-drug comprises the lipid nano-particles and mRNA drugs loaded on the lipid nano-particles; the molar ratio of N atoms in UC18-C16 to P atoms in the mRNA drug (hereinafter referred to as N/P ratio) is 2:1.
The preparation method of the nano-drug comprises the following steps:
(1) Mixing lipid molecules UC18-C16 and DOPE in a molar ratio of 1:2 and dissolving in ethanol to obtain a lipid molecule organic phase; dissolving mRNA medicine in PBS buffer solution with pH value of 7.4 to obtain medicine solution;
(2) Mixing the lipid molecular organic phase obtained in the step (1) with a drug solution according to a volume ratio of 1:9, so that the molar ratio (N/P ratio) of N atoms in UC18-C16 to P atoms in the mRNA drug is 2:1, mixing for 30s, standing and incubating at room temperature for 15min, and obtaining a preparation containing the nano drug, wherein the volume percentage of ethanol in the preparation is 10%.
Examples 12 to 21
The lipid nanoparticle and the nano-drug comprising the same, wherein examples 12-14 differ from example 11 only in the molar ratio of UC18-C16 to DOPE in the lipid nanoparticle; examples 15-17 differ from example 12 only in the N/P ratio; examples 18, 20 differ from example 16 only in the amount of ethanol in the formulation; examples 19, 21 differ from example 12 only in the ethanol content of the formulations; the specific components are shown in Table 2.
Examples 22 to 25
The lipid nanoparticle and the nano-drug comprising the same, wherein examples 22-25 differ from examples 18 and 19 only in N/P ratio, and specific components are shown in Table 2.
TABLE 2
Performance testing
Performance test and analysis are performed on the nano-drug obtained in the above embodiment, and the specific steps are as follows:
(1) Evaluation of different lipid molecules: 293T cells were spread in 96-well plates, each well contained 90. Mu.L of whole cell culture broth, 10. Mu.L of a preparation of the nano-drug to be tested (lipid nanoparticle-loaded drug, examples 1-10) was added to the wells, placed in a cell incubator for culture, and protein expression was examined after 24 hours.
FIG. 2 is a screening chart of the delivery efficiency of lipid nanoparticle-loaded drugs constructed by different lipid molecules, and as can be seen from FIG. 2, the lipid nanoparticle constructed by the lipid molecules provided by the invention has a molar ratio of 1:1 of lipid molecules to DOPE, an N/P ratio of 2:1 of lipid molecules to mRNA, and the UC18-C16 has the best delivery efficiency of mRNA.
(2) Evaluation of nano-drugs with different proportions: paving 293T cells in a 96-well plate, taking 10 mu L of a preparation of nano-drugs to be detected (lipid nano-particle loaded drugs, examples 11-22) from each well containing 90 mu L of whole cell culture solution, adding the preparation into the well, placing the well in a cell incubator for culture, and detecting the expression of protein after 24 hours; lipofectamine 2000 (noted lipo 2K, 0.2. Mu.L/well) from Siemens was used as a comparative example.
FIG. 3 is a graph of optimized delivery efficiency of nanomedicines at different ratios, wherein lipid nanoparticles constructed based on UC18-C16 are subjected to component ratio optimization after the lipid molecules UC18-C16 with optimal transfection efficiency are screened out in FIG. 2. First, when the ratio of UC18-C16 to mRNA N/P was 2:1, the molar ratio of UC18-C16 to DOPE was changed (examples 9 and 11-14), and the transfection efficiency of the lipid nanoparticle was highest when the ratio of UC18-C16 to mRNA N/P was 2:1 and the ratio of UC18-C16 to DOPE was also 2:1 (example 12).
Secondly, when the molar ratio of UC18-C16 to DOPE is 2:1, the N/P ratio of UC18-C16 to mRNA is changed (examples 12 and 15-17), so that the transfection efficiency of the lipid nanoparticle is the best when the N/P ratio of UC18-C16 to mRNA is 4:1; the results of the above screening and optimization show that: the efficiency of lipid nanoparticle delivery of mRNA is optimal when the molar ratio of UC18-C16 to DOPE is 2:1 and the N/P ratio of UC18-C16 to mRNA is 4:1.
Finally, from examining the effect of ethanol content in the preparation on the delivery efficiency, it was found that the preparation of nano-drug having 20%, 10% and 5% ethanol content (examples 16, 18, 20, examples 12, 19, and 21) was the most effective in delivering mRNA when the N/P ratio of UC18 to C16 to mRNA was the same and the molar ratio of UC18 to C16 to DOPE was the same.
(3) Load assessment of nanomedicine at different N/P ratios
FIG. 4 is a nucleic acid electrophoretogram of a nano-drug of different N/P ratios (examples 18-19, 22-25); as can be seen from FIG. 4, mRNA was already entrapped when the molar ratio of UC18-16 to DOPE was 2:1 and the N/P ratio of UC18-C16 to mRNA was 1.5:1 (example 23). Indicating that UC18-C16 can successfully encapsulate mRNA under the condition of low nitrogen-phosphorus ratio.
(4) Morphological characterization of nanomedicines
Morphology characterization of the nanomaterials using Hitachi TEM System transmission electron microscopy, an exemplary transmission electron microscopy of the nanomaterials provided in example 18 is shown in fig. 5, and the results show that the particle size of the nanomaterials is between 100 and 150 nm.
(5) Hydrated particle size and potential evaluation of nanomedicine
Analysis of hydration particle size and potential of the nano-drug (lipid nanoparticle-loaded mRNA) provided by the present invention using DLS (dynamic light scattering) is exemplified by fig. 6 which is a graph of hydration particle size distribution of the nano-drug provided in example 18, and fig. 7 which is a Zeta potential graph of the nano-drug provided in example 18, as can be seen from fig. 6 and 7, the hydration particle size of the nano-drug (lipid nanoparticle-loaded mRNA) is about 300nm, and the Zeta potential is about 30 mV.
(6) Stability evaluation of nano-drug
The stability of the nanopharmaceutical was monitored by the change in serum absorbance (660 nm) after incubation of the nanopharmaceutical in 10% serum at room temperature. As an example, the graph of the absorbance value (660 nm) of the nano-drug incubated at room temperature in 10% serum provided in example 18 is shown in fig. 8, and the absorbance value at 660nm does not change significantly after the nano-drug is incubated with 10% serum for different times (3 h, 6h, 24h, 48 h) at room temperature, which indicates that the nano-drug does not significantly aggregate in the measurement range.
(7) Antisera test of nano-drugs
The capacity of the nano-drug to resist serum degradation was evaluated by agarose gel electrophoresis, and the stability test chart of the nano-drug and free mRNA provided in example 18 against serum of different concentrations is shown in FIG. 9, lane 1 is a 48 hour incubation of the nano-drug in 10% serum at room temperature; lane 2 is free mRNA; lane 3 is the nano-drug incubated in 10% serum for 10 minutes at room temperature; lane 4 is the nano-drug incubated in 20% serum for 10 minutes at room temperature; lane 5 is the nano-drug incubated in 40% serum for 10 minutes at room temperature. As shown in fig. 9, the nano-drug provided in example 18 can exist stably in serum with different concentrations at different times, which indicates that the nano-drug has strong resistance to serum degradation in the measurement range.
(8) Biocompatibility evaluation:
the biocompatibility of the lipid nanoparticle (empty) provided by the invention is evaluated through a hemolysis rate test experiment of erythrocytes, 1% triton is used as a comparison, and a biocompatibility test chart (a hemolysis rate test chart of erythrocytes) of empty lipid nanoparticles (UC 18-C16 and DOPE with the molar ratio of 1:0.5) with different concentrations is obtained, as shown in fig. 10, and the chart shows that the hemolysis capability of erythrocytes is weaker under the condition of pH of 7.4, so that the lipid nanoparticle is proved to have better biocompatibility of carriers in the test range.
The applicant states that the present invention is illustrated by the above examples as a lipid molecule, lipid nanoparticle, and methods of making and using the same, but the present invention is not limited to, i.e., does not mean that the present invention must be practiced in dependence upon, the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.
Claims (12)
1. A nano-drug, characterized in that the nano-drug consists of lipid nanoparticles and a drug loaded on the lipid nanoparticles; the medicine is a nucleic acid medicine, and the nucleic acid medicine is an mRNA medicine;
the lipid nanoparticle is composed of a lipid molecule and a second lipid, the second lipid being different from the lipid molecule in molecular structure;
the mol ratio of the lipid molecules to the second lipid is 1:0.2-8, and the mol ratio of N atoms in the lipid molecules to P atoms in the nucleic acid medicines is 1.5-16:1;
the lipid molecule has any one of the following structures:
UC18-C10
UC18-C12
UC18-C14
UC18-C16
UC18-C18
UC18-2C14
UC18-2C18
2. the nano-drug of claim 1, wherein the second lipid is selected from any one or a combination of at least two of a non-cationic lipid, a cationic lipid, or a polyethylene glycol modified lipid.
3. The nanopharmaceutical of claim 2, wherein the non-cationic lipid comprises 1, 2-dioleyl-sn-glycero-3-phosphoethanolamine, cholesterol, 1, 2-dioleoyl-sn-glycero-3-phosphocholine, 1, 2-distearoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1, 2-myristoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, sodium salt of 1, 2-dioleoyl-rac- (1-glycero), 1, 2-palmitoyl-phosphatidylglycerol, 1-palmitoyl-2-oleoyl-lecithin, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, distearoyl-phosphatidylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine, stearoyl-2-phosphatidylethanolamine, sphingomyelin, or a combination of at least one, two of the sphingomyelin, or any of the two of the sphingomyelin, or the sphingomyelin.
4. The nanopharmaceutical of claim 2, wherein the cationic lipid comprises N, N-dioleyloxy-N, N-dimethyl ammonium chloride, N-distearyl-N, N-dimethyl ammonium bromide, N- (1- (2, 3-dioleyloxy) propyl) -N, N-trimethyl ammonium chloride, N- (1- (2, 3-dioleyloxy) propyl) -N, N-trimethyl ammonium chloride, N-dimethyl-2, 3-dioleyloxy-propylamine, 1, 2-dioleyloxy-3- (dimethylamino) acetoxypropane, 1, 2-dioleyloxy-3-dimethylaminopropane, 1-linoleyloxy-2-linoleyloxy-3-dimethylaminopropane, 1, 2-dioleyloxy-3- (N-methylpiperazinyl) propane, 3- (N, N-dioleyloxy) -1, 2-propanediol, 1, 2-dioleyloxy-3- (N-dimethylamino) acetoxypropane, 1, 2-dioleyloxy-3- (dimethylamino) acetoxypropane, or at least one of any combination of two of 1, 2-dioleyloxy-3- (dimethylamino) propane.
5. The nano-drug according to claim 1, wherein the molar ratio of N atoms in the lipid molecules to P atoms in the nucleic acid drugs is (3-5): 1.
6. The nano-drug according to claim 1, wherein the hydrated particle size of the nano-drug is 250-350 nm.
7. A method of preparing a nano-drug according to any one of claims 1 to 6, comprising: mixing the lipid molecules, the second lipid and an organic solvent to obtain a lipid molecule organic phase; mixing the medicine with an aqueous solvent to obtain a medicine solution; mixing the lipid molecular organic phase with a drug solution to obtain the nano-drug.
8. The method of claim 7, wherein the organic solvent comprises ethanol.
9. The method of claim 7, wherein the aqueous solvent is a buffer solution.
10. The method of claim 7, wherein the aqueous solvent is a PBS buffer solution.
11. The method according to claim 7, wherein the concentration of the drug in the drug solution is 20 to 25 ng/. Mu.L.
12. The method according to claim 7, wherein the volume ratio of the organic phase of the lipid molecule to the pharmaceutical solution is 1 (1-10).
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