CN115417780A - Ionizable cationic lipid C5-A2 and nanoliposome particles composed of same - Google Patents

Ionizable cationic lipid C5-A2 and nanoliposome particles composed of same Download PDF

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CN115417780A
CN115417780A CN202211373375.3A CN202211373375A CN115417780A CN 115417780 A CN115417780 A CN 115417780A CN 202211373375 A CN202211373375 A CN 202211373375A CN 115417780 A CN115417780 A CN 115417780A
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lipid
glycero
rna
phosphocholine
ionizable cationic
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CN115417780B (en
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赵兴卉
翟俊辉
王轲珑
陈波
王致远
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Huaxi Biotechnology Qingdao Co ltd
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Beijing Huapeng Biotechnology Co ltd
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Abstract

The invention provides an ionizable cationic lipid compound C5-A2 and application of a nano liposome particle delivery system (LNP) consisting of the compound, auxiliary lipid, structural lipid and PEGylated lipid. The nanoliposome particles can be used to deliver RNA or small molecule drugs to cells or organs of mammals for prophylactic or therapeutic effects.

Description

Ionizable cationic lipid C5-A2 and nanoliposome particles composed of same
Technical Field
The invention discloses an ionizable cationic lipid and a nano liposome particle carrier containing the compound. The nanoliposome particle also includes phospholipids, PEG lipids, structural lipids and therapeutic or prophylactic agents including unsaturated lipids in specific ratios. Such nanoliposome particle carriers can deliver one or more prophylactic or therapeutic agents to a cell or organ of a mammal.
Background
The nucleic acid vaccine, particularly the mRNA vaccine, has the advantages of short development cycle, strong flexibility, high safety and the like, and has remarkable advantages when dealing with sudden diseases such as new coronary disease. However, nucleic acid drugs have many challenges in drug development, and particularly mRNA is unstable as a biomacromolecule and is easily degraded by ubiquitous ribozymes. Meanwhile, mRNA is rich in phosphate groups and is in a negative charge state, and phospholipid forming a cell membrane is also in a negative charge state, so that naked mRNA and the cell membrane repel each other and are difficult to enter cells. Nucleic acid drugs need to enter cells to play corresponding biological functions, so that corresponding carriers are needed for the nucleic acid drugs, on one hand, the nucleic acid drugs are delivered into the cells, and on the other hand, the nucleic acid drugs are protected from being rapidly degraded by organisms. Nucleic acids that are currently common fall into two categories: viral vectors and non-viral vectors, and viral vectors such as virus-like particles often face the problems of high immunogenicity, large side effects and the like, and are not used frequently in clinic; non-viral vectors such as polymers, nanoemulsions, liposomes, etc., are currently the most mature clinically and the most used are the nanoliposomal particles (LNPs). The nano liposome particle carrier has the advantages of high delivery efficiency, good safety and the like, and the delivery carriers used by two new coronary mRNA vaccines which are on the market at present are nano liposome particles. The efficient and safe delivery carrier is also widely researched, and particularly, the safety aspect is the focus of related research in the aspect of improving the delivery efficiency of the nano liposome particle carrier.
Although the nanoliposome particle carrier is successfully applied to clinic and has obvious advantages compared with other carriers, the nanoliposome particle carrier has strong patent barriers and needs to be improved in technical effect, and the design of a new nanoliposome carrier avoids patent limitations and improves the technical effect so as to adapt to the continuously increasing application requirements, so that the nanoliposome particle carrier is an important subject of the research on the nanoliposome carrier at present. Common nanoliposome particles consist of four components, ionizable cationic lipids, phospholipids, structural lipids and pegylated lipids. The chemical structure of the ionizable cationic lipid of the four lipids has a significant impact on the delivery efficiency and safety of the nanoliposome particle carriers. The design and screening of high-efficiency and safe ionizable cationic lipid become the focus of the nano liposome particle carrier. The invention aims to provide a novel ionizable cationic lipid, and further provides a nano liposome particle carrier formed by the ionizable cationic lipid, so as to solve the problems in the prior art.
Disclosure of Invention
In view of the above, the present invention provides, in a first aspect, an ionizable cationic lipid which is 2-hexyldecanoyl 3- [3- [3- [ [3- (2-hexyldecanoyloxy) -3-oxo-propyl ] - (2-hydroxy-16-methyl-heptadecyl) amino ] propyl-methyl-amino ] propyl- (2-hydroxy-16-methyl-heptadecyl) amino ] propionate, said ionizable cationic lipid having a chemical structure represented by formula (i):
Figure 478523DEST_PATH_IMAGE001
（Ⅰ）。
in the present invention, the ionizable cationic lipid is designated as C5-A2.
In a preferred embodiment, the ionizable cationic lipid has a nuclear magnetic spectrum (1H NMR (400 MHz CDCl3)) as shown in FIG. 1, where C5-A2 is characterized as: δ 3.93-4.00 (m, 4H), 3.58 (br d, J = 8.00 Hz, 2H), 2.86-2.98 (m, 2H), 2.67-2.84 (m, 4H), 2.54-2.65 (m, 3H), 2.36-2.52 (m, 10H), 2.12-2.35 (m, 6H), 1.09-1.80 (m, 118H), 0.82-1.04 (m, 24H), thus C5-A2 contains CH 3 , CH 2 , -RH 2 -C00-RH 2 -, -RH(CH 3 ) 2 , -CH(OH)-,-N(CH 3 ) -iso-characteristic groups.
Secondly, the invention provides a nano liposome particle carrier, wherein the nano liposome particle carrier is a composition containing the ionizable cationic lipid and phospholipid, a structural lipid and a PEGylated lipid.
In a preferred embodiment, the ionizable cationic lipid and phospholipid, structural lipid, pegylated lipid are present in (40-65%): (5-25%): (25-50%): (0.5-15%) in a molar ratio of nitrogen atoms in the ionizable cationic lipid to phosphate groups in RNA as drug molecules, i.e., a nitrogen-to-phosphorus ratio of 6 (N: P = 6.
In a more preferred embodiment, the ionizable cationic lipid and phospholipid, structural lipid, pegylated lipid are present in a ratio of 50:10:38.5:1.5 according to the molar ratio.
The phospholipid is an amphiprotic lipid, contains a hydrophilic head and a hydrophobic tail, is an important component of the spherical outer layer of the nano liposome particles, and can promote the fusion of the nano liposome particles and cells. In a preferred embodiment, wherein said phospholipid is selected from one or more phospholipid compounds of the group consisting of: 1, 2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-di (undecanoyl) -sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18: 0DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16. SoLyPC), 1, 2-dilinolyl-sn-glycero-3-phosphocholine, 1, 2-dineoyl-sn-glycero-3-phosphocholine, 1, 2-di (docosahexenoyl) -sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dineoyltetraallyl-sn-glycero-3-phosphoethanolamine, 1, 2-di (docosahexenoyl) -sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycero) sodium salt (DOPG), sphingomyelin.
The structural lipid of the present invention refers to a lipid that can increase the stability of nanoliposome particles, reduce the mobility of phospholipids in nanoliposome particles, reduce leakage of nucleic acid encapsulated by nanoliposome particles, and increase the efficiency of endosome escape of nanoliposome particles, and in a preferred embodiment of the present invention, the structural lipid is selected from any one or more of the following substances: cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, lycoside, ursolic acid, and alpha-tocopherol. In another preferred embodiment, the structural lipid is selected from any of the structural lipid compounds in the group consisting of: cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycopersine, ursolic acid, alpha-tocopherol, corticosteroids.
In another preferred embodiment, the pegylated lipid is selected from any pegylated lipid compound selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.
In a specific embodiment of the invention, the phospholipids, structural lipids and PEGylated lipids are 1, 2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, PEG2000-DMG, respectively.
Finally, the invention also provides the application of the nano liposome particle carrier in the preparation of nucleic acid delivery drugs.
In a preferred embodiment, the nucleic acid is RNA.
In a preferred embodiment, the method for preparing the nucleic acid-delivered drug comprises the steps of:
(1) Dissolving RNA serving as a drug molecule in a sodium acetate buffer solution to form a water phase;
(2) Determining the mole number of the ionizable cationic lipid according to the mole ratio (N: P) of nitrogen atoms in the ionizable cationic lipid to phosphate groups in RNA as a drug molecule of (2-30) to 1, and then determining the mole ratio of 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine, cholesterol, PEG2000-DMG according to the mole ratio of the ionizable cationic lipid to 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine to 50:10:38.5:1.5, uniformly mixing the four components to form a uniform organic phase;
(3) Preparing the aqueous phase prepared in the step (1) and the organic phase prepared in the step (2) into nano liposome particles loaded with mRNA or DNA in a microfluidic mixing mode, wherein the volume of the nucleic acid aqueous phase is 3 times that of the organic phase, and the mass ratio wt/wt of the lipid and the nucleic acid in the nano liposome particle carrier is 3:1 to 100: 1.
Preferably, the ratio of N to P is (5-20) to 1.
More preferably, the ratio of N to P is 8: 1.
More preferably, the ratio of N to P is 6: 1.
Preferably, the mass ratio wt/wt ratio of lipid to nucleic acid (mRNA) is about 20: 1.
More preferably, the mass ratio wt/wt ratio of lipid to nucleic acid (mRNA) is about 6: 1.
In a more preferred embodiment, the mass ratio of the vector to the nucleic acid is (5-50): 1.
More preferably, the RNA is a short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecule, microrna (miRNA), antisense RNA (antagomir), ribozyme, dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), or messenger RNA (mRNA).
The ionizable cationic lipid has an ionizable amine group head, and can be combined with hydrogen ions to be positively charged under an acidic environment, so that RNA molecules with negative charges are coupled, and mRNA molecules are wrapped in small balls formed by the ionizable cationic lipid to form nano liposome particles. The average diameter of the nano liposome particles is 50-150nm. Multiple amino groups on the head of the ionizable cationic lipid can carry multiple positive charges, so that mRNA molecules can be better coupled, and higher encapsulation efficiency is achieved; the lipid also has a branch structure and an ester bond at the tail part, so that the degradability of the lipid is greatly increased, the retention time of the lipid in a body is reduced, the probability of side reaction is reduced, and the safety and the tolerance are improved. 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol are used as auxiliary lipids, which are helpful for improving the membrane fusion of the nano liposome particles, maintaining the structure and stability of the nano liposome particles and increasing the endosome escape efficiency of the nano liposome particles so as to improve the delivery efficiency, and 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (PEG 2000-DMG) can enable the nano liposome particles to form a compact spherical structure, prolong the circulation time of the nano liposome particles in vivo, reduce the aggregation of the nano liposome particles and increase the stability of the nano liposome particles.
Drawings
FIG. 1 NMR spectra of ionizable cationic lipid compounds C5-A2;
FIG. 2 detection of transfection efficiency of nano-liposome particle delivery Luc-mRNA;
FIG. 3 is a fluorescent microscope image of eGFP-mRNA delivered by nanoliposome particles;
FIG. 4 is an in vivo imaging view of the delivery of LNPs-Luc-mRNA in vivo by nanoliposome particles.
Detailed Description
Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Unless otherwise indicated, techniques employed or contemplated herein are standard procedures well known to those of ordinary skill in the art, or are performed according to conditions suggested by the manufacturer. The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA techniques within the skill of the art. These materials, methods, and examples are illustrative only and not limiting. The following is presented by way of illustration and is not intended to limit the scope of the present disclosure.
In some embodiments, the numerical parameters set forth in the specification are approximations that may vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, these numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Numerical values presented in some embodiments of the disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of numerical ranges herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein.
For convenience, certain terms used throughout this application (including this specification, examples, and appended claims) are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
In some embodiments, numbers expressing quantities of ingredients, properties (e.g., molecular weight), reaction conditions, and results, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in certain instances by the term "about". One of ordinary skill in the art will understand the meaning of the term "about" in the context of a defined value. In some embodiments, the term "about" is used to indicate that a value includes the standard deviation of the mean of the device or method used to determine the value. In some embodiments, the numerical parameters set forth in the specification are approximations that may vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, these numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Numerical values presented in some embodiments of the disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
The term "compound" includes all isotopes and isomers of the structure shown. "isotope" refers to atoms having the same atomic number but different mass numbers, which are produced by different numbers of neutrons in the core. For example, isotopes of hydrogen include tritium and deuterium. In addition, the compounds, salts, or complexes of the present disclosure can be prepared in combination with solvent or water molecules to form solvates or hydrates by conventional methods. "isomers" means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer or diastereomer of a compound. The compounds may include one or more chiral centers and/or double bonds and thus may exist as stereoisomers (e.g., double bond isomers or diastereomers). The present disclosure encompasses any and all isomers of the compounds described herein, including stereoisomerically pure forms as well as enantiomers and stereoisomeric mixtures, e.g., racemates. Enantiomers and stereoisomeric mixtures of compounds and methods for their decomposition into their component enantiomers or stereoisomers are well known in the art.
The terms "comprising," "having," and "including" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprises," "having," "includes," is also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only that one or more steps, and may also encompass other steps not listed. Similarly, any composition that "comprises," "has," or "includes" one or more features is not limited to having only that one or more features and may encompass other non-listed features. The use of any and all examples, or exemplary language (e.g., "such as") provided herein with respect to certain embodiments is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.
The term "consisting of" refers to the compositions, methods, and their corresponding components as described herein, excluding any elements not described in this description of the example.
As used herein, the term "delivery" means providing an entity to a destination. For example, delivering a therapeutic agent to a cell may involve administering to the cell a pharmaceutical composition comprising at least one nanoparticle comprising mRNA.
As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) Generating an RNA template from the DNA sequence (e.g., by transcription); (2) Processing of RNA transcripts (e.g., by splicing, editing, 5 'cap formation, and/or 3' end processing); (3) translating the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.
As used herein, the term "lipid component" is a component of a nanoparticle that includes one or more lipids. For example, the lipid component may include one or more ionizable cationic lipids, phospholipids, structural lipids, PEG lipids.
As used herein, a "nanoparticle" is a particle comprising one or more lipids and one or more therapeutic agents. The nanoparticles are typically on the order of microns or less in size and may include a lipid bilayer. In some embodiments, the nanoparticles have an average diameter (e.g., equivalent diameter) by DLS (dynamic light scattering) of between about 70 nm and about 150nm, such as between about 80 nm and about 120nm, 90 nm and 110nm in diameter. In some embodiments, the average kinetic diameter of the nanoparticle is about 90 nm, 91 nm, 92 nm, 93 nm, 94 nm, 95 nm, 96 nm, 97 nm, 98 nm, 99 nm, 100 nm, 101 nm, 102 nm, 103 nm, 104 nm, 105 nm, 106 nm, 107 nm, 108 nm, 109 nm, or 110nm. In some embodiments, the therapeutic agent is mRNA or DNA.
As used herein, "polydispersity index (PDI)" is a measure of the size distribution of nanoparticles in a nanoparticle sample. In some embodiments, the polydispersity index is between about 0.10 and 0.20, for example about 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20.
As used herein, "total lipid" is all lipids that are first dissolved and mixed in ethanol when liposomal nanoparticles are synthesized by the nano-coprecipitation method, including ionizable cationic lipids, phospholipids, structural lipids, helper polymers, and PEG lipids. In one embodiment, the total lipid comprises ionizable cationic lipids that are 2-hexyldecanoyl 3- [3- [3- [ [3- (2-hexyldecanoyloxy) -3-oxo-propyl ] - (2-hydroxy-16-methyl-heptadecyl) amino ] propyl-methyl-amino ] propyl- (2-hydroxy-16-methyl-heptadecyl) amino ] propionate, said ionizable cationic lipids having the chemical structure shown in formula (i):
Figure 461522DEST_PATH_IMAGE001
（Ⅰ）。
in the present invention, the ionizable cationic lipid is designated as C5-A2.
1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) having the formula (II):
Figure 325573DEST_PATH_IMAGE002
（II）。
cholesterol, the molecular formula is shown as formula (III):
Figure 608787DEST_PATH_IMAGE003
（III）。
DMG-PEG2000 (1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000) has the formula (IV):
Figure 900091DEST_PATH_IMAGE004
（IV）。
wherein the number 44 in formula (IV) means: the number of repeating units of ethylene glycol in the parentheses was 44.
As used herein, N: P (N/P ratio), i.e., nitrogen to phosphorus ratio, is the molar ratio of nitrogen atoms in the ionizable cationic lipid to phosphate groups in the therapeutic agent mRNA or DNA.
As used herein, "mRNA: total lipid volume ratio "," mRNA: the "total lipid volume ratio" is the ratio of the volume of the mRNA to the volume of the lipid component (including ionizable cationic lipid, structural lipid, helper lipid, PEG lipid) when they are mixed during lipid synthesis. In one embodiment, the mRNA: total lipid volume ratio was 3.
As used herein, "mRNA: the total lipid flow ratio "," total flow rate "," Start water "," End water "are set parameters of the apparatus when preparing nanoliposome particles on Precision Nanosystems Inc microfluidic nanoparticle preparation system Ignite, the definition of which conforms to the basic definition of Precision Nanosystems, and the results are obtained from experimental adjustments. In one embodiment, the mRNA: the total lipid flow ratio was 3.
As used herein, the term "nucleic acid" as used herein includes in its broadest sense any compound and/or substance comprising a polymer of nucleotides linked by phosphodiester bonds. These polymers are often referred to as oligonucleotides or polynucleotides, depending on size. The terms "polynucleotide sequence" and "nucleotide sequence" are also used interchangeably herein.
As used herein, a "protein" is a polymer consisting essentially of any of the 20 amino acids. Although "polypeptide" is generally used to refer to relatively larger polypeptides and "peptide" is generally used to refer to small polypeptides, the use of these terms in the art overlaps and varies. The terms "peptide", "protein" and "polypeptide" are sometimes used interchangeably herein.
In the implementation case of the invention, ionizable cationic lipid, DSPC, cholesterol and PEG are dissolved in absolute ethyl alcohol, and the molar ratios of the four components are respectively 50%,10%,38.5% and 1.5%; the total lipid concentration of the organic phase was 6mM. To form a homogeneous organic phase, all solutions were vortexed thoroughly to produce good encapsulation. Wherein, in the preparation of the mRNA of the firefly luciferase (luciferase) and the green fluorescent protein (eGFP) reporter gene encapsulated by the nano liposome particles, the ratio of N: P =6 (mass ratio of cationic lipid to mRNA is 6. Nucleic acids (mRNA) were dissolved in 25mM sodium acetate solution (pH 5.2) at a volume ratio of 1:3.
LNPs solutions were prepared on an Ignite (Precision Nanosystems Inc) nanoliposome particle preparation system using the principle of the nano-coprecipitation method, with a total flow rate of 12mL/min, and with a Start waste and End waste of 0.15mL and 0.05mL, respectively, and the embodiments are shown in Table 1.
TABLE 1 technical parameters for the preparation of ionizable cationic lipids
Figure 635966DEST_PATH_IMAGE005
。
Upon completion of the preparation, the LNPs solution was quickly transferred to a 50mL ultrafiltration tube (Millipore, 100 KD) and diluted 10-fold with 1XPBS, centrifuged at 1300g (Thermo) for 30 minutes at 4 ℃ and the ultrafiltered LNPs solution was collected and made up to a concentration of 150ug/mL with 1 XPBS.
After preparing a dilution of the finished LNPs solution, the effective particle size, polydispersity index (PDI) and Zeta potential were measured using a NanoBrook Omni (Brookhaven) multi-angle particle size and high sensitivity Zeta potential analyzer, while precisely measuring the encapsulation efficiency of LNPs and the concentration of nucleic acids in LNPs using a Quant-iT cell RiboGreen ™ RNA quantitative assay kit (Life Technology).
Example 1 Synthesis route to ionizable cationic lipid C5-A2
The synthesis principle of the ionizable cationic lipid C5-A2 is that diamine and halogen are subjected to substitution reaction, then reduction reaction is carried out to reduce amido bond into secondary amine, addition reaction is carried out on a reduction product and acrylic ester, debenzylation is carried out on the product, and finally, addition reaction is carried out on the product and alkyl ethylene oxide to generate a target product. The synthesis route is that a compound 2 is synthesized from a compound 1, a compound 3 is synthesized at the same time, a compound 6b is synthesized from a compound 6a, a compound C5-1 is synthesized from the compound 3 and the compound 6b, and a compound C5-2 is synthesized from the compound C5-1; in addition, compound A-2 was synthesized from compound A-1, compound A-3 was synthesized from compound A-2, compound A-4 was synthesized from compound A-3, compound A-5 was synthesized from compound A-4, and finally the objective compound ionizable cationic lipid C5-A2 was synthesized from compound C5-2 and compound A-5.
The preparation method of the ionizable cationic lipid C5-A2 comprises the following specific steps:
to a solution of compound 1 (N- (3-aminopropyl) -N-methyl-1, 3-propanediamine 20.0 g, 138 mmol, 1.00 eq, sigma-Aldrich) in dichloromethane (DCM, 140 mL, sigma-Aldrich) was added triethylamine (TEA, 41.8 g, 413 mmol, 57.5 mL, 3.00 eq, sigma-Aldrich) and benzoyl chloride (42.6 g, 303 mmol, 35.2 mL, 2.20 eq, sigma-Aldrich) at 0 ℃. The mixture was stirred at 25 ℃ for 3 hours until the reaction was complete. The reaction mixture was poured into water (50 mL), extracted with ethyl acetate (100 mL, 80 mL, sigma-Aldrich), and the extracted organic layer was washed with 20 mL of brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give Compound 2 (N- [3- [ 3-benzamidopropyl (methyl) amino ] propyl ] benzamide 50.0 g, crude) as a white solid.
Figure 38128DEST_PATH_IMAGE006
。
Lithium aluminum hydride (LAH, 6.44 g, 170 mmol, 6.00 eq) was added to tetrahydrofuran (THF, 200 mL) at 25 deg.C and mixed well, followed by the addition of a solution of compound 2 (N- [3- [ benzamidopropyl (methyl) amino ] propyl ] benzamide, 10.0 g, 28.3 mmol, 1.00 eq) in tetrahydrofuran (THF 50mL, sigma-Aldrich) and mixed well. The mixture was stirred at 80 ℃ for 48 hours. Liquid chromatography-mass spectrometry (Rt =0.487, MS +1 = 326) showed that compound 1 was completely consumed and one main peak of satisfactory mass was detected. After water (6.44 mL), 15% sodium hydroxide (6.44mL, sigma-Aldrich), water (19.4 mL) and sodium sulfate were slowly added to the reaction mixture at 0 ℃ and then stirred at 20 ℃ for 20 min, the mixture was filtered and concentrated under reduced pressure to give compound 3 (N-benzyl-N '- [3- (benzylamino) propyl ] -N' -methyl-propane-1, 3-diamine, 37.0 g) as a yellow oil.
Figure 175849DEST_PATH_IMAGE007
。
To a solution of compound 6a (2-hexyl-1-decanol, 30.0 g, 124 mmol, 1.00 eq) in dichloromethane (DCM, 210 mL, sigma-Aldrich) was added triethylamine (TEA, 25.0 g, 247 mmol, 34.5 mL, 2.00 eq, sigma-Aldrich) and acryloyl chloride (12.3 g, 136 mmol, 11.1 mL, 1.10 eq, sigma-Aldrich) at 0 ℃. After the mixture was stirred at 25 ℃ for 3 hours, water (50 mL) was added to the reaction solution, and the reaction was extracted with ethyl acetate (100 mL, 80 mL, sigma-Aldrich). The organic phase was separated, washed with 30ml brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue. Column chromatography (SiO 2, petroleum ether/ethyl acetate = 1/0-0/1) gave compound 6b (2-hexyldecyl acrylate, 33.6 g, 113 mmol, 91.5%) as a colorless oil.
Figure 372475DEST_PATH_IMAGE008
。
To the compound 3 (N-benzyl-N' - [3- (benzylamino) propyl)]-N' -methyl-propane-1, 3-diamine, 5.00 g, 15.4 mmol, 1.00 eq) in ethanol (EtOH, 50mL, sigma-Aldrich) was added compound 6b (2-hexylsunflower acrylate, 10.0 g, 33.8 mmol, 2.20 eq). The mixture was stirred at 70 ℃ for 12 hours. LC-MS (Rt = 0.918, MS +1 = 918.8) showed complete reaction, detecting a main peak of satisfactory mass. The reaction mixture was poured into water (50 mL), extracted with ethyl acetate (30 mL, 20 mL, sigma-Aldrich), and the extracted organic layer was washed with 30mL brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. By column chromatography (SiO) 2 Petroleum ether/ethyl acetate = 50/1 to 0/1) to obtain a yellow oily compound C5-1 (2-hexylsunflower-3- [ benzyl- [3- [3- [ benzyl- [3- (2-hexylsunflower) -3-oxo-propyl)]Amino group]Propyl-methyl-amino]Propyl radical]Amino group]Propionate, 9.8 g, 10.7 mmol).
Figure 595646DEST_PATH_IMAGE009
。
To the compound C5-1 (2-hexyldecanoyl 3- [ benzyl- [3- [3- [ benzyl- [3- (2-hexyldecanoyl) -3-oxo-propyl) under argon]Amino group]Propyl-methyl-amino]Propyl radical]Amino group]Propionate, 19.3 g, 21.0 mmol, 1.00 eq) in tetrahydrofuran (THF, 105 mL, sigma-Aldrich) was added palladium hydroxide (Pd (OH) 2 3.86 g, 5.50 mmol, 20%). The suspension was degassed and purged 3 times with hydrogen. The mixture being at 25 DEG CThe mixture was stirred under hydrogen (50 Psi) for 12 hours. LC-MS (product Rt = 0.791, MS +1 = 738.7) showed that the reaction was complete and a main peak of satisfactory mass was detected. The reaction was filtered and concentrated under reduced pressure to give a dark brown oily compound C5-2 (2-hexyldecanoyl 3- [3- [3- [ [3- (2-hexyldecanoyloxy) -3-oxo-propyl ] -propyl]Amino group]Propyl-methyl-amino]Propylamino group]Propionate, 12.8 g).
Figure 801499DEST_PATH_IMAGE010
。
After adding a solution of 1-bromo-3-methylbutane (56.9 g, 377 mmol, 47.4 mL, 5.00 eq, sigma-Aldrich) in tetrahydrofuran (THF 50mL, sigma-Aldrich) at 30 ℃ to magnesium (9.48 g, 390 mmol, 5.17 eq, sigma-Aldrich) and iodine (191 mg, 754umol, 151uL, 0.01eq, sigma-Aldrich), the mixture was stirred at 45 ℃ for 2 hours. The mixture was added to a solution of compound A-1 (12-bromo-1-dodecanol, 20 g, 75.4 mmol, 1.00 eq, sigma-Aldrich) and dilithium tetrachlorocuprate (II) (0.1M, 45.2 mL, 0.06 eq, sigma-Aldrich) in tetrahydrofuran (THF, 50mL, sigma-Aldrich). The mixture was allowed to stand overnight at room temperature under nitrogen to thin layer chromatography indicating complete consumption of reactant A-1. After completion of the reaction, dilute hydrochloric acid (1M, 500mL, sigma-Aldric) was added to the reaction solution, diluted with water (300 mL), extracted with ethyl acetate (EtOAc, 300mL, 200 mL, sigma-Aldric), and the combined organic layer was washed with brine (300 mL), and sodium sulfate (Na) (Na 2 SO 4 Sigma-Aldric) were dried, filtered, concentrated under reduced pressure, and then purified by column chromatography (SiO) 2 Petroleum ether/ethyl acetate = 0-1) gave compound a-2 (15-methyl-hexadecanol 15.7 g, 61.2 mmol, 81.1%) as a white solid.
Figure 548654DEST_PATH_IMAGE011
。
To a solution of compound a-2 (15-methyl-hexadecanol, 15.7 g, 61.2 mmol, 1.00 eq) in acetonitrile (ACN, 310mL, sigma-Aldric) was added 2-iodoxybenzoic acid (IBX, 25.7 g, 91.8 mmol, 1.50 eq, sigma-Aldric), and the mixture was stirred at 85 ℃ for 2 hours until the thin layer chromatography showed completion of the reaction. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound A-3 (15-methyl-16 alkanal, 15 g) as a white solid.
Figure 447340DEST_PATH_IMAGE012
。
To a solution of methyltriphenylphosphonium bromide (109 g, 305 mmol, 5.18 eq, sigma-Aldrich) in tetrahydrofuran (THF, 300mL, sigma-Aldrich) at-20 deg.C was added potassium tert-butoxide (t-BuOK, 20.5 g, 183 mmol, 3.10 eq, sigma-Aldrich), the mixture was stirred at 25 deg.C for 1h, and a solution of compound A-3 (15-methyl-16 alkanal, 15.0 g, 58.9 mmol, 1.00 eq) in tetrahydrofuran (THF, 30mL, sigma-Aldrich) was added at-20 deg.C. The mixture was stirred under nitrogen at 25 ℃ for 2 h until the thin layer chromatography showed a clean reaction. The reaction mixture was poured into ammonium chloride (NH) 4 Cl, 200 mL, sigma-Aldrich), extracted with ethyl acetate (500 mL, 300mL, sigma-Aldrich), and the combined organic layers were washed with 150 mL brine, dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (SiO. RTM.)) 2 Petroleum ether/ethyl acetate =1/0 to 50/1) gave compound a-4 (16-methyl-17-alkyl-1-ene, 14 g, 55.45 mmol, 94%) as a colorless oil.
Figure 423387DEST_PATH_IMAGE013
。
To a solution of compound A-4 (16-methyl-17-alkyl-1-ene, 14.0 g, 55.4 mmol, 1.00 eq) in dichloromethane (DCM, 98 mL, sigma-Aldrich) was added 3-chloroperoxybenzoic acid (m-CPBA, 17.9 g, 83.2 mmol, 80%,1.50 eq) at 0 ℃. The mixture was stirred at 25 ℃ for 3 hours until thin layer chromatography indicated that the reaction was complete. The reaction mixture was quenched by addition of sodium thiosulfate (Na 2S2O3 mL, sigma-Aldrich) at 0 deg.C, then diluted with water (20 mL), extracted with ethyl acetate (50 mL, 30mL, sigma-Aldrich) and the organic layer washed with brine (8 mL), dried over sodium sulfate, filtered, concentrated under reduced pressure, and then purified by column chromatography(SiO 2 Petroleum ether/ethyl acetate =1/0 to 0/1), yielding compound a-5 (2- (14-methyl-pentadecyl) oxirane, 13.3 g, 49.5 mmol, 89.3%) as a colorless oil.
Figure 167352DEST_PATH_IMAGE014
。
To the compound C5-2 (2-hexyldecanoyl 3- [3- [3- [ [3- (2-hexyldecanoyloxy) -3-oxo-propyl)]Amino group]Propyl-methyl-amino]Propylamino group]Propionate, 4.00 g, 5.42 mmol, 1.00 eq) in t-butanol (t-BuOH, 40 mL, sigma-Aldrich) A-5 (2- (14-methylpentadecyl) ethylene oxide, 1.45 g, 5.42 mmol, 1.00 eq) was added and the mixture was stirred at 90 ℃ for 48 hours until LC-MS indicated completion of the reaction. The reaction mixture was added with water (15 mL), extracted with ethyl acetate (50 mL, 30mL, sigma-Aldrich), and the extracted organic layer was washed with 10mL brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. By column chromatography (SiO) 2 Petroleum ether/ethyl acetate to dichloro/methanol = 10/1-0/1) to obtain a target compound C5-A2 (2-hexyldecanoyl 3- [3- [3- [ [3- (2-hexyldecanoyl) -3-oxo-propyl ] yellow oil]- (2-hydroxy-16-methyl-heptadecyl) amino]Propyl-methyl-amino]Propyl- (2-hydroxy-16-methyl-heptadecyl) amino]Propionate) 1.5 g, 1.18 mmol).
Figure 279664DEST_PATH_IMAGE015
。
The nuclear magnetic spectrum of C5-A2 is shown in figure 1: from the nuclear magnetic spectrum (1H NMR (400 MHz CDCl3)), it was found that: C5-A2 is characterized in that: δ 3.93-4.00 (m, 4H), 3.58 (br d, J = 8.00 Hz, 2H), 2.86-2.98 (m, 2H), 2.67-2.84 (m, 4H), 2.54-2.65 (m, 3H), 2.36-2.52 (m, 10H), 2.12-2.35 (m, 6H), 1.09-1.80 (m, 118H), 0.82-1.04 (m, 24H), so C5-A2 contains CH 3 , CH 2 , -RH 2 -C00-RH 2 -, -RH(CH 3 ) 2 , -CH(OH)-,-N(CH 3 ) -and the like.
The ionizable cationic lipid C5-A2 is a multi-nitrogen structure, can be combined with hydrogen ions under an acidic condition so as to be positively charged, and is electrostatically combined with mRNA which is negatively charged so as to well wrap the mRNA, so that the entrapment rate of the nano liposome particles consisting of the ionizable cationic lipid on the mRNA reaches more than ninety percent, and the entrapment rate is good. On the other hand, the compound contains ester bonds and can be hydrolyzed under the action of related enzymes in vivo, so that the degradation of the compound in vivo is accelerated, the half-life period in vivo is reduced, and side reactions possibly induced by the compound in vivo are reduced. The compound is also a multi-branched structure, and the branched chains enable the nano liposome particles consisting of the compound to have lower phase transition temperature, so that the nano liposome particles are easy to form a conical structure, and the membrane of an endosome and the structure of the nano liposome particles are damaged, so that the encapsulated mRNA is released. From the overall structure, the structures of polyazotic acid, ester bond, branched chain and the like of the compound are beneficial to the good coupling of mRNA in an acid environment, so that the formed nano liposome particles can wrap the mRNA well to achieve higher entrapment rate; on the other hand, the change of the conformation of the nano liposome particles in the cells is promoted, the structural integrity of the nano liposome particles and the endosome is destroyed, so that the mRNA is promoted to be released into cytoplasm, and the translation efficiency is improved.
Example 2 preparation of Nanosomal particles and encapsulation of Luc-mRNA
Ionizable cationic lipid C5-A2 was synthesized by Yammaglandin synthesis following the synthetic route of example 1 and identified, DSPC (distearoylphosphatidylcholine), cholesterol, DMG-PEG2000 (1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000) three Lipids were all from Avanti Polar Lipids (Avanta Polar Lipids, inc.). The four lipids were dissolved in absolute ethanol (purity >99.9%, sigma) to make up a stock solution of 10 mg/mL. The molar ratio of the four lipids is 50:10:38.5:1.5, 6mM total lipid concentration of the four lipids, prepared with a nitrogen to phosphorus ratio of 6 (N: P = 6).
mRNA was dissolved in 25mM sodium acetate buffer (ph 5.2) at a volume ratio of aqueous phase to organic phase of 3, LNPs solution was prepared using Ignite (Precision Nanosystem Inc) nano manufacturing system with preparation parameters of flow rate ratio of aqueous phase to organic phase of 3:1, total flow rate 12mL/min, pre-waste (start waste) 0.15mL, post-waste (end waste) 0.05mL.
Nanoliposome particles were prepared using Ignite according to the above preparation parameters and coated with Luc-mRNA to obtain the corresponding LNPs-Luc-mRNA solution (Luc-mRNA is firefly luciferase mRNA, 1929 nucleotides in length, which is a common reporter gene, and Luc-mRNA is supplied by TriLink Biotechnologies). The resulting LNPs-Luc-mRNA solution was quickly transferred to a 50mL ultrafiltration tube (Millipore, 100 KD) and the LNPs solution was diluted 10-fold with 1XPBS, centrifuged at 1300rpf (Thermo) for 30 minutes at 4 ℃ and finally the ultrafiltered LNPs solution was collected and made up to 150ug/mL with 1 XPBS. The physicochemical parameters of LNPs-Luc-mRNA including particle size, polydispersity index (PDI), zeta potential, determined from the final LNPs-Luc-mRNA solution using a nanoparticle size Analyzer (NanoBrook Omni, brookhaven), encapsulation efficiency using a Quant-iT cell RiboGreen ™ RNA quantitative detection kit (Life Technology) are shown in Table 2:
TABLE 2 physicochemical parameters and encapsulation efficiency of LNPs-mRNA
Figure 83672DEST_PATH_IMAGE016
。
Delivery efficiency in Table 2 was calculated as firefly luciferase expression levels Luminence Units).
From the view of physicochemical parameters, the ionizable cationic lipid C5-A2. And the other three lipids can form good nanoliposome particles and encapsulate mRNA. The particle size, the polydispersity index (PDI), the potential and other key parameters are good, the encapsulation rate reaches more than ninety percent, and the carrier is an ideal nucleic acid drug carrier of the nano liposome particles. Therefore, further in vitro delivery efficiency measurements were performed on cell models. The transfection experiments were performed in five wells per group at 500ng of LNPs-mRNA transfection amount overnight using HEK293 cells at 20000 cells per well, and after 24 hours the transfection efficiency was measured using the firefly luciferase assay kit (Promega).
In vitro transfection results show that the expression level of the firefly luciferase is very high, and the nano liposome particle carrier consisting of the ionizable cationic lipid C5-A2 has very high delivery efficiency on the firefly luciferase mRNA, so that the nano liposome particle carrier is a good nucleic acid drug delivery carrier.
Example 3: preparation of nanoliposome particles and delivery of eGFP-mRNA
To further verify that the nano liposome particle delivery system composed of the novel ionizable cationic lipid C5-A2 can well wrap and deliver mRNA, and that the high efficiency is universal, but not high in delivery efficiency only for a specific mRNA. Nanoliposome particle-encapsulated eGFP-mRNA (eGFP-mRNA is green fluorescent protein, emits green light under fluorescent excitation, is a common reporter gene, and is 996 nucleotides in length) was prepared using the new ionizable cationic lipid according to the same conditions and preparation parameters as above and the physicochemical parameters and delivery efficiency of the nanoliposome particle carrier were measured according to the same technical means.
TABLE 3 physicochemical parameters and encapsulation efficiency of LNPs-mRNA
Figure 750277DEST_PATH_IMAGE017
。
From the physicochemical parameters: the nano liposome particle composed of ionizable cationic lipid C5-A2 can well wrap green fluorescent protein mRNA (eGFP-mRNA), the particle size and the Polydispersity (PDI) are good, and the entrapment rate is more than ninety percent. Further testing of delivery efficiency was performed on a cell model. HEK293 cells were used at 1X 10 per well 5 After the cells are densely paved on a 24-well plate and are overnight, transfection experiments are carried out on 3 groups of multiple wells with the transfection quantity of 500ng of LNPs-mRNA, the expression quantity of green fluorescent protein is observed by using a fluorescence microscope after 24 hours, and fluorescence pictures show that eGFP-mRNA has high expression quantity (shown in figure 3).
The green fluorescent protein eGFP-mRNA is wrapped and delivered by the nano liposome particle consisting of the new ionizable cationic lipid C5-A2, and the good delivery efficiency is also achieved, so that the nano liposome particle consisting of the ionizable cationic lipid can be used for efficiently delivering nucleic acid drugs such as mRNA and the like, and has the potential of a good nucleic acid drug delivery carrier.
Example 4: in vivo experiment of delivering firefly luciferase mRNA by using nano liposome particle carrier consisting of ionizable cationic lipid C5-A2
In order to further improve the in vivo delivery efficiency of the ionizable cationic lipid C5-A2 composed nano liposome particle carrier, the delivery efficiency of the C5-A2 composed nano liposome particle carrier after being wrapped by firefly luciferase mRNA is tested on a mouse animal model. The protocol also remains consistent with the cellular model, ionizable cationic lipid C5-A2, DSPC (distearoylphosphatidylcholine), cholesterol, DMG-PEG2000 (1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000) in a molar ratio of 50:10:38.5: 1.5. The total lipid concentration of the four lipids was 6mM, prepared at a nitrogen to phosphorus ratio of 6 (N: P = 6. mRNA was dissolved in 25mM sodium acetate buffer pH5.2 at a volume ratio of aqueous phase to organic phase of 3:1, total flow rate 12mL/min, 0.15mL of pre-waste (start waste) and 0.05mL of post-waste (end waste) were wrapped with Luc-mRNA using the same parameters. The prepared LNPs-Luc-mRNA was transferred to a 50mL ultrafiltration tube (Millipore, 100 KD) and the LNPs solution was diluted 10-fold with 1xPBS, centrifuged at 1300rpf (Thermo) for 30 minutes at 4 ℃ and finally the ultrafiltered LNPs solution was collected and made up to 150ug/mL with 1 XPBS. Liposomal nanoparticles physicochemical parameters of LNPs-Luc-mRNA including particle size, polydispersity number (PDI), zeta potential were determined using a NanoBrook Omni, brookhaven nanoparticle size Analyzer (NanoBrook Omni), encapsulation efficiency was determined using Quant-iT ™ RiboGreen ™ RNA quantitative assay kit (Life Technology) as follows:
TABLE 4 measurement of physicochemical parameters and delivery efficiency of LNPs-Luc-mRNA
Figure 297933DEST_PATH_IMAGE018
。
Delivery efficiency is reported in Avg Radiance [ p/s/cm2/sr ] in Table 4.
From the results of the physicochemical parameters: after mRNA is wrapped by nano liposome particles consisting of ionizable cationic lipid C5-A2, the physical and chemical parameters are good, the particle size is 80-120nm, the polydispersity index (PDI) is good, the encapsulation rate is over 90 percent, and the encapsulation effect is excellent. Further, the delivery efficiency was examined at the animal level by injecting mice (BALB/c, 8 weeks old) intramuscularly with LNPs-Luc-mRNA at an amount of 10. Mu.g per mouse, four mice per group. The mice were imaged in vivo using a living body imager (PerkinElmer) six hours later, the luminescence intensity of the mice was measured, and the in vivo delivery efficiency of the nanoliposome particle carrier was examined (see fig. 4).
From the in vivo delivery efficiency results: the nano liposome particle carrier consisting of C5-A2 can well wrap firefly fluorescein mRNA, has good physicochemical parameters, high delivery efficiency, good survival state of mice and good safety.
The nano liposome particle carrier consisting of ionizable cationic lipid C5-A2 has good physicochemical parameters and can well wrap mRNA. The results of the implementation case show that the ionizable cationic lipid C5-A2 is a good ionizable cationic lipid, the nanoliposome particle carrier formed by the ionizable cationic lipid can well deliver mRNA to the body, the expression level of protein is high, and the in-vitro and in-vivo delivery efficiency is high. Meanwhile, the nano liposome particle carrier composed of the ionizable cationic lipid has the potential of delivering other RNAs such as SiRNA, asymmetric interfering RNA (airRNA), micro RNA (miRNA), antisense RNA, small hairpin RNA and other nucleic acids, and has great prospect in the aspect of nucleic acid drug delivery.

Claims (12)

1. An ionizable cationic lipid, wherein said ionizable cationic lipid is 2-hexyldecanoyl 3- [3- [3- [ [3- (2-hexyldecanoyloxy) -3-oxo-propyl ] - (2-hydroxy-16-methyl-heptadecyl) amino ] propyl-methyl-amino ] propyl- (2-hydroxy-16-methyl-heptadecyl) amino ] propionate, and wherein said ionizable cationic lipid has the chemical formula shown in formula (i):
Figure 585370DEST_PATH_IMAGE001
（Ⅰ）。
2. the ionizable cationic lipid of claim 1, having a nuclear magnetic spectrum of δ 3.93-4.00 (m, 4H), 3.58 (br d, J = 8.00 Hz, 2H), 2.86-2.98 (m, 2H), 2.67-2.84 (m, 4H), 2.54-2.65 (m, 3H), 2.36-2.52 (m, 10H), 2.12-2.35 (m, 6H), 1.09-1.80 (m, 118H), 0.82-1.04 (m, 24H).
3. A nanoliposome particle carrier, wherein the nanoliposome particle carrier is a composition comprising the ionizable cationic lipid of claim 2 and a phospholipid, a structural lipid, a pegylated lipid.
4. The nanoliposome particle carrier of claim 3, wherein the ionizable cationic lipid and phospholipid, structural lipid, PEGylated lipid are present in (40-65%): (5-25%): (25-50%): (0.5-15%) in a molar ratio of 6 to 1, wherein the total lipid concentration of the ionizable cationic lipid, the phospholipid, the structured lipid and the pegylated lipid is 6mM.
5. The nanoliposome particle carrier according to claim 3, wherein the ionizable cationic lipid and phospholipid, structural lipid, PEGylated lipid are present in a ratio of 50:10:38.5:1.5 of the total weight of the components.
6. The nanoliposome particle carrier of claim 5, wherein the phospholipid is selected from one or more phospholipid compounds selected from the group consisting of: 1, 2-dioleoyl-sn-glycero-3-phosphocholine, 1, 2-dimyristoyl-sn-glycero-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine, 1, 2-distearoyl-sn-glycero-3-phosphocholine, 1, 2-di (undecanoyl) -sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1, 2-dilinonyl-sn-glycero-3-phosphocholine, 1, 2-dithianoyl-sn-glycero-3-phosphocholine, 1, 2-di (docosahexenoyl) -sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, ethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di (docosahexaenoyl) -sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate-rac- (1-glycerol) sodium salt, sphingomyelin.
7. The nanoliposome particle carrier according to claim 5, wherein the structural lipid is selected from any structural lipid compound selected from the group consisting of: cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycopersine, ursolic acid, alpha-tocopherol, corticosteroids.
8. The nanoliposome particle carrier of claim 5, wherein the PEGylated lipid is selected from any PEGylated lipid compound selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.
9. The nanoliposome particle carrier according to any one of claims 3 to 8, wherein the phospholipid, structured lipid and PEGylated lipid are 1, 2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, PEG2000-DMG, respectively.
10. Use of the nanoliposome particle carrier of any one of claims 3 to 8 in the preparation of a medicament for RNA delivery.
11. The use of claim 10, wherein the method of preparing the RNA-delivered medicament comprises the steps of:
(1) Dissolving RNA serving as a drug molecule in a sodium acetate buffer solution to form a water phase;
(2) Determining the mole number of the ionizable cationic lipid according to the mole ratio of nitrogen atoms in the ionizable cationic lipid to phosphate groups in RNA as a drug molecule of (2-30) to 1, and then determining the mole number of the ionizable cationic lipid according to the mole ratio of the ionizable cationic lipid, 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine, cholesterol and PEG2000-DMG of 50:10:38.5:1.5, uniformly mixing the four components to form a uniform organic phase;
(3) Preparing the aqueous phase prepared in the step (1) and the organic phase prepared in the step (2) into RNA-loaded nano liposome particles in a microfluidic mixing mode, wherein the volume of the RNA aqueous phase is 3 times that of the organic phase, and the mass ratio wt/wt of lipid to RNA in the nano liposome particle carrier is 3:1 to 100: 1.
12. The use of claim 11, wherein the RNA is a short interfering RNA, an asymmetric interfering RNA, an RNA interfering molecule, a microrna, an antisense RNA, a ribozyme, a Dicer-substrate RNA, a small hairpin RNA, or a messenger RNA.
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