CN115417778B - Ionizable cationic lipid C5 and nanoliposome particles composed of same - Google Patents

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

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CN115417778B
CN115417778B CN202211357819.4A CN202211357819A CN115417778B CN 115417778 B CN115417778 B CN 115417778B CN 202211357819 A CN202211357819 A CN 202211357819A CN 115417778 B CN115417778 B CN 115417778B
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glycero
lipid
phosphorylcholine
rna
nanoliposome
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CN115417778A (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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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

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

Description

Ionizable cationic lipid C5 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 particles also include phospholipids, PEG lipids, structural lipids and therapeutic or prophylactic agents in specific proportions. Such nanoliposome particle carriers can deliver one or more prophylactic or therapeutic agents to cells or organs of a mammal.
Background
The nucleic acid vaccine, especially mRNA vaccine, has the advantages of short research and development period, strong flexibility, high safety and the like, and has remarkable advantages in coping with sudden diseases such as new crown pneumonia. However, nucleic acid drugs have many challenges in the pharmaceutical process, especially mRNA as a biological macromolecule is unstable and is easily degraded by ubiquitous ribozymes. Meanwhile, mRNA is rich in phosphate groups and is negatively charged, and phospholipid composing cell membranes is negatively charged, so that naked mRNA and the cell membranes are mutually repelled and are difficult to enter cells. The nucleic acid medicine needs to enter cells to exert corresponding biological functions, so that corresponding vectors are needed for the nucleic acid medicine, on one hand, the nucleic acid is delivered into the cells, and on the other hand, the nucleic acid medicine is protected from being rapidly degraded by organisms. Currently common nucleic acids fall into two categories: viral vectors and non-viral vectors, such as virus-like particles, often face problems of high immunogenicity, large side reactions, etc., and are not much used clinically; non-viral vectors such as polymers, nanoemulsions, liposomes, etc., are currently most clinically mature and use most of nanoliposome particles (LNP). The nano liposome particle carrier has the advantages of high delivery efficiency, good safety and the like, and the delivery carrier used by the two new crown mRNA vaccines on the market at present is the nano liposome particle. Such efficient and safe delivery vehicles are also widely studied, and in particular, in terms of improving the delivery efficiency of nanoliposome particle vehicles, the safety aspect is the focus of the current related research.
Although the nano liposome particle carrier is successfully applied to clinic, compared with other carriers, the nano liposome particle carrier has obvious advantages, but has strong patent barriers, the technical effect is still to be improved, so that the nano liposome particle carrier is suitable for the increasing application requirements, and the design of a new nano liposome carrier avoids the patent limitation and improves the technical effect, so that the nano liposome particle carrier is an important subject for the research of the nano liposome 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 lipids in the four lipids has an important impact on the delivery efficiency and safety of the nanoliposome particle carrier. Design and screening of high-efficiency and safe ionizable cationic lipid becomes a focus problem of nano liposome particle carriers. The invention aims to provide a novel ionizable cationic lipid, and further provides a nano liposome particle carrier consisting of 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 an ionizable cationic ester, which is 2-hexylsunflower-based 3- [3- [3- [ [3- (2-hexylsunflower-oxy) -3-oxo-propyl ] - (2-hydroxy-16-methyl-heptadecyl) amino ] propyl-methyl-amino ] propylamino ] propionate, having a chemical structural formula shown in formula (i):
Figure 402822DEST_PATH_IMAGE001
（Ⅰ）。
In the present invention, the ionizable cationic lipid is named "C5".
In a preferred embodiment, the ionizable cationic lipid has a structure obtained by nuclear magnetic resonance spectroscopy (1H NMR, (400 MHz CDCl) 3 ) As shown in fig. 1, C5-A2 is characterized by: delta 3.98 (dd, j=5.82, 3.81 Hz, 4H), 3.53-3.65 (m, 1H), 2.86-2.97 (m, 4H), 2.67-2.80 (m, 4H) 2.59, (brdd, j=13.13, 7.13 Hz, 4H), 2.33-2.51 (m, 9H), 2.22 (s, 3H), 1.57-1.84 (m, 7H), 1.15-1.54 (m, 81H), 0.81-0.98 (m, 18H), so that C5 contains CH 3 , CH 2 , -RH 2 -C00-RH 2 -, -RH(CH 3 ) 2 , -CH(OH)-,-N(CH 3 ) -an isopcharacteristic group.
Secondly, the invention provides a nano liposome particle carrier which is a composition containing the ionizable cationic lipid, phospholipid, structural lipid and PEGylated lipid.
In a preferred embodiment, the ionizable cationic lipid and phospholipid, structural lipid, PEGylated lipid is as described (40-65%): (5-25%): (25-50%): (0.5-15%) and the ionic lipid is prepared by mixing the components according to a molar ratio, wherein the molar ratio of nitrogen atoms in the ionic lipid to phosphate groups in RNA or DNA serving as a drug molecule is (N: P) is 6:1, the total lipid concentration of the ionizable cationic lipid and phospholipid, structural lipid, and PEGylated lipid is 6mM.
In a more preferred embodiment, the ionizable cationic lipid and phospholipid, structural lipid, pegylated lipid is present in an amount of 50:10:38.5:1.5 by mole ratio.
The phospholipid disclosed by the invention is an amphoteric lipid, contains a hydrophilic head part and a hydrophobic tail part, is an important component part of the spherical outer layer of the nano liposome particle, and can promote the fusion of the nano liposome particle and cells. In a preferred embodiment, wherein said phospholipid is selected from one or more phospholipid compounds from the group consisting of: 1, 2-Dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-di (undecanoyl) -sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 DietherPC), 1-oleoyl-2-sterolyl hemisuccinyl-sn-glycero-3-phosphorylcholine (OChemspp PC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (C), 1, 2-dioleoyl-glycero-3-phosphorylcholine (Lyocene-phosphorylcholine), 1, 2-dioleoyl-2-glycero-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-glycero-2-glycero-3-phosphorylcholine (POsoyl-phosphorylcholine (POP), 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), 1, 2-di-phytanoyl-sn-glycero-3-phosphate ethanolamine (ME16.0PE), 1, 2-di-stearoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-linoleoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di (docosahexaenoic acyl) -sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate-rac- (1-glycero) sodium salt (DOPG), sphingomyelin.
The structural lipid in the invention refers to a lipid which can increase the stability of nano-liposome particles, reduce the mobility of phospholipid in the nano-liposome particles, reduce the leakage of nucleic acid wrapped by the nano-liposome particles and increase the escape efficiency of endosomes of the nano-liposome particles, and in a preferred embodiment of the invention, the structural lipid is selected from any one or more of the following substances: cholesterol, stigmasterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycorine, tomato glycoside, ursolic acid, alpha-tocopherol. In another preferred embodiment, the structural lipid is any structural lipid compound selected from the group consisting of: cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherol, corticosteroids.
In another preferred embodiment, the pegylated lipid is selected from any one of the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.
In a specific embodiment of the present invention, the phospholipid, structural lipid, and PEGylated lipid are 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, cholesterol, and PEG2000-DMG, respectively.
Finally, the invention also provides application of the nano liposome particle carrier in preparation of RNA or DNA delivery drugs.
In a preferred embodiment, the method of preparing the RNA or DNA delivery drug comprises the steps of:
(1) Dissolving RNA or DNA as a drug molecule in sodium acetate buffer to form an aqueous phase;
(2) The molar ratio (N: P) of nitrogen atoms in the ionizable cationic lipid to phosphate groups in RNA or DNA as a drug molecule was (2-30) to 1, and the molar ratio of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, cholesterol, PEG2000-DMG was 50:10:38.5: mixing the four components uniformly in a ratio of 1.5 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 mRNA or DNA loaded nano-liposome particles in a microfluidic mixed mode, wherein the volume of the nucleic acid aqueous phase is 3 times that of the organic phase, and the mass ratio wt/wt ratio of lipid and nucleic acid in the nano-liposome particle carrier is 3:1 to about 100:1.
Preferably, the ratio of N to P is 5-20:1.
More preferably, the ratio of N to P is 8:1.
It is particularly preferred that the ratio of N to P is 6:1.
Preferably, the mass ratio wt/wt ratio of lipid to nucleic acid (mRNA/DNA) is about 20:1.
More preferably, the mass to wt/wt ratio of lipid to nucleic acid (mRNA/DNA) is about 6:1.
In a more preferred embodiment, the mass ratio of the carrier to the nucleic acid is (5-50): 1.
More preferably, the RNA is short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecule, microrna (miRNA), antisense RNA (antagomir), small hairpin RNA (shRNA) or messenger RNA (mRNA).
The ionizable cationic lipid has an ionizable amine head, and can be combined with hydrogen ions to be positively charged in an acidic environment, so that RNA molecules with negative charges are coupled, and the mRNA molecules are wrapped in small spheres formed by the ionizable cationic lipid to form nano liposome particles. The average diameter of the nano liposome particles is 50-150nm. The ionizable cationic lipid has a plurality of amino groups at the head, can carry a plurality of positive charges to better couple mRNA molecules, and achieves higher encapsulation efficiency; the lipid also has a branching structure and an ester bond at the tail, so that the degradability of the lipid is greatly improved, the retention time of the lipid in the organism is reduced, the probability of side reaction is reduced, and the safety and tolerance are improved. 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) and cholesterol are used as auxiliary lipids, which are helpful for improving the membrane fusion property of nano liposome particles, maintaining the structure and stability of the nano liposome particles, and increasing the escape efficiency of endosomes of the nano liposome particles, thereby improving the delivery efficiency, and the 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 is a nuclear magnetic resonance spectrum of an ionizable cationic lipid compound C5;
FIG. 2. Nanoliposome particle delivery Luc-DNA transfection efficiency assay;
FIG. 3 fluorescence microscopy images of delivery of eGFP-mRNA from nanoliposome particles;
FIG. 4 is a fluorescent microscopy image of delivery of eGFP-DNA from nanoliposome particles;
FIG. 5 is an in vivo imaging view of the delivery of LNPs-Luc-mRNA in nanoliposome particles;
FIG. 6. In vivo delivery of LNPs-Luc-mRNA luminescence intensity assay by nanoliposome particles.
Detailed Description
Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. 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. Unless otherwise indicated, techniques employed or contemplated herein are standard methods well known to those of ordinary skill in the art or are performed under conditions suggested by the manufacturer. 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 intended to be 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 the 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. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily caused by the standard deviation found in their respective test measurements. Recitation of ranges of values herein are 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 the application (including the 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, the numbers used to describe and claim the amounts, properties (e.g., molecular weight), reaction conditions, results, etc. of expressed components of certain embodiments of the disclosure are understood to be modified in some instances by the term "about". The meaning of the term "about" will be understood by one of ordinary skill in the art 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 devices or methods 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 the 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. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily caused by the standard deviation found in their respective test measurements.
The term "compound" includes all isotopes and isomers of the structures shown. "isotope" refers to atoms of the same atomic number but different mass numbers, which are produced by different numbers of neutrons in the core. Isotopes of hydrogen include, for example, tritium and deuterium. In addition, the compounds, salts, or complexes of the present disclosure may be prepared in combination with a solvent or water molecule to form solvates or hydrates by conventional methods. "isomer" means any geometric isomer, tautomer, zwitterionic, 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, such as racemates. The enantiomers and stereoisomers of the compounds and methods of decomposing them 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 "comprising," "having," "including," 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 cover 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 features not listed. 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" means the compositions, methods, and corresponding components as described herein, excluding any elements not described in this specification 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 lipid bilayers. In some embodiments, the average diameter (e.g., equivalent diameter) of the nanoparticle by DLS (dynamic light scattering) is between about 70 nm and about 150 nm, such as between about 80 nm and about 120 nm, 90 nm and 110nm. In some embodiments, the nanoparticle has an average kinetic diameter of 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, the "polydispersity index (PDI)" is a measure of the nanoparticle size distribution 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 lipids" are all lipids previously dissolved and mixed in ethanol, including ionizable cationic lipids, phospholipids, structural lipids, helper polymers, and PEG lipids, when the liposomal nanoparticles are synthesized by a nano-coprecipitation method. In one embodiment, the total lipid comprises an ionizable cationic lipid that is 2-hexylsunflower-based 3- [3- [3- [ [3- (2-hexylsunflower-oxy) -3-oxo-propyl ] - (2-hydroxy-16-methyl-heptadecyl) amino ] propyl-methyl-amino ] propylamino ] propionate having the formula (I):
Figure 166378DEST_PATH_IMAGE002
(I) A. The invention relates to a method for producing a fibre-reinforced plastic composite In the present invention, the ionizable cationic lipid is designated as C5.
1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) having a formula shown in formula (II):
Figure 734763DEST_PATH_IMAGE003
（II）。
cholesterol has a molecular formula shown in a formula (III):
Figure 419822DEST_PATH_IMAGE004
（III）。
DMG-PEG2000 (1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000) having a molecular formula represented by formula (IV):
Figure 833486DEST_PATH_IMAGE005
（IV）。
Wherein the number 44 in formula (IV) refers to: the number of ethylene glycol repeat units in brackets is 44.
As used herein, N: P (N/P 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 liposome volume ratio "is the volume ratio of mRNA when mixed with lipid components (including ionizable cationic lipids, structural lipids, helper lipids, PEG lipids) during lipid synthesis. In one embodiment mRNA: the volume ratio of total fat is 3:1.
as used herein, "mRNA: the total lipid flow rate ratio "," total flow rate "," Start wave "," End wave "are parameters set by the apparatus when nanoliposome particles are prepared on the Precision Nanosystems Inc microfluidic nanoparticle preparation system Ignite, the definition of which corresponds to the basic definition of Precision Nanosystems pairs, and the result is experimental adjustment. In one embodiment mRNA: the ratio of total lipid flow rate is 3:1, a total flow rate of 12 mL/min, a Start wash of 0.15 mL,End waste of 0.05 mL.
As used herein, the term "nucleic acid" in its broadest sense includes any compound and/or substance comprising a polymer of nucleotides linked by phosphodiester bonds. These polymers are commonly 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 "polypeptides" are generally used to refer to relatively larger polypeptides, and "peptides" are generally used to refer to small polypeptides, the use of these terms in the art overlaps and is varied. The terms "peptide," "protein," and "polypeptide" are sometimes used interchangeably herein.
In one embodiment of the invention, the ionizable cationic lipid, DSPC, cholesterol, PEG are dissolved in absolute ethanol at a molar ratio of 50%,10%,38.5%,1.5%, respectively; the total lipid concentration of the organic phase was 6mM. To form a homogeneous organic phase, all solutions were thoroughly vortexed to give good encapsulation. Wherein, when preparing the mRNA and DNA of firefly luciferase (luciferase) and green fluorescent protein (eGFP) reporter genes coated by nano liposome particles, N is P=6. Nucleic acids (mRNA and DNA) were dissolved in 25mM sodium acetate solution (pH 5.2) with a volume ratio of organic phase consisting of lipid mixture to aqueous phase consisting of mRNA sodium acetate buffer of 1:3.
LNPs solutions were prepared on a Ignite (Precision Nanosystems Inc) nanoliposome particle preparation system using the principle of nano-co-precipitation, with a total flow rate of 12 mL/min, a Start and End wash of 0.15. 0.15 mL and 0.05 mL, respectively, and the embodiments shown in Table 1.
TABLE 1 technical parameters for the preparation of ionizable cationic lipids
Figure 733440DEST_PATH_IMAGE006
。
After preparation, the LNPs solution was transferred to a 50mL ultrafilter tube (Millipore, 100 KD) and diluted 10-fold with 1 XPBS, centrifuged at 1300g (Thermo) for 30 min at 4℃and finally the ultrafiltered LNPs solution was collected and sized to a concentration of 150ug/mL with 1 XPBS.
After the prepared LNPs solution is diluted, the effective particle size, the polydispersity index (PDI) and the Zeta potential of the LNPs are measured by using a nanoBrook Omni (Brookhaven) multi-angle particle size and high-sensitivity Zeta potential analyzer, and meanwhile, the encapsulation efficiency of the LNPs and the concentration of nucleic acid in the LNPs are precisely measured by using a Quant-iT ™ riboGreen ™ RNA quantitative detection kit (Life Technology).
EXAMPLE 1 Synthesis route to ionizable cationic lipid C5 (chemical Structure shown in formula I)
The synthesis principle of the ionizable cationic lipid C5 is that the amino group of the compound 1 and acyl chloride are subjected to substitution reaction, then the amide bond is reduced into secondary amine through reduction reaction, the reduced product and acrylic ester are subjected to addition reaction, the product is subjected to debenzylation, and finally the product is added with alkyl ethylene oxide to generate the target product. The synthetic route is that compound 1 starts to synthesize compound 2, compound 2 starts to synthesize compound 3, compound 6a starts to synthesize compound 6b, compound 3 and compound 6b synthesize compound C5-1, compound C5-1 synthesizes compound C5-2; in addition, the compound A-2 is synthesized by the compound A-1, the compound A-3 is synthesized by the compound A-2, the compound A-4 is synthesized by the compound A-3, the compound A-5 is synthesized by the compound A-4, and finally the target compound ionizable cationic lipid C5 is synthesized by the compound C5-2 and the compound A-5, which comprises the following specific steps:
To a solution of compound 1 (N, N-bis (3-aminopropyl) methylamine, 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 reaction was complete, the reaction mixture was poured into water (50 mL), extracted with ethyl acetate (100 mL, 80 mL, sigma-Aldrich), the extracted organic layer was washed with brine 20mL, then dried over sodium sulfate, filtered, concentrated under reduced pressure to give compound 2 (N- [3- [ 3-benzoylaminopropyl (methyl) amino ] propyl ] benzamide 50.0 g) as a white solid.
Figure 410409DEST_PATH_IMAGE007
。
Lithium aluminum hydride (LAH, 6.44 g, 170 mmol, 6.00 eq, sigma-Aldrich) was added to tetrahydrofuran (THF, 200 mL,Sigma-Aldrich) and mixed well at 25℃followed by a solution of compound 2 (N- [3- [ 3-benzoylaminopropyl (methyl) amino ] propyl ] benzamide, 10.0 g, 28.3 mmol, 1.00 eq) in tetrahydrofuran (THF, 50mL, sigma-Aldrich). The mixture was stirred at 80 ℃ for 48 hours until the liquid chromatography mass spectrometry showed complete consumption of compound 2 and a major peak of satisfactory quality was detected. To the reaction mixture was slowly added water (6.44 mL), 15% sodium hydroxide (6.44 mL, sigma-Aldrich), water (19.4 mL) and sodium sulfate at 0 ℃ and then after stirring for 20 min at 20 ℃, 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 645081DEST_PATH_IMAGE008
。
To a solution of compound 6a (2-hexyl-1-decanol, 30.0 g, 124 mmol, 1.00 eq, sigma-Aldrich) in methylene chloride (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. 50 mL) was added to the reaction solution, and after extraction reaction with ethyl acetate (100 mL, 80mL, sigma-Aldrich), the organic phase was separated, washed with 30mL of brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. By column chromatography (SiO 2 Petroleum ether/ethyl acetate=1/0 to 0/1) gives compound 6b (2-hexylsunflower acrylate, 33.6 g, 113 mmol, 91.5%) as a colourless oil.
Figure 596857DEST_PATH_IMAGE009
。
To compound 3 (N-benzyl-N' - [3- (benzylamino) propyl)]-N' -methyl-propane-1, 3-diamine, 5.00 g, 15.4 To an ethanol solution (EtOH, 50mL,Sigma-Aldrich) of mmol, 1.00-eq) 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, and a main peak meeting the required quality was detected. The reaction mixture was poured into water (50 mL), extracted with ethyl acetate (30 mL, 20 mL, sigma-Aldrich), the extracted organic layer was washed with 30mL brine, dried over sodium sulfate, filtered, 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 yellowish oily compound C5-1 (2-hexyl sunflower radical 3- [ benzyl- [3- [3- [ benzyl- [3- (2-hexyl sunflower radical) -3-oxo-propyl radical)]Amino group]Propyl-methyl-amino group]Propyl group]Amino group]Propionate, 9.8 g, 10.7 mmol).
Figure 803847DEST_PATH_IMAGE010
。
Under argon, the compound C5-1 (2-hexylsunflower radical 3- [ benzyl- [3- (2-hexylsunflower radical) -3-oxo-propyl)]Amino group]Propyl-methyl-amino group]Propyl group]Amino group]To a solution of 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 then degassed and after 3 hydrogen purges the mixture was stirred at 25℃under hydrogen (50 Psi) for 12 hours until the LC-MS reaction was complete and a major peak of satisfactory quality was detected. The reaction product was filtered and concentrated under reduced pressure to give the dark brown oily compound C5-2 (2-hexylsunflower-based 3- [3- [3- [ [3- (2-hexylsunflower-oxy) -3-oxo-propyl)]Amino group]Propyl-methyl-amino group]Propylamino group]Propionate, 12.8 g).
Figure 714034DEST_PATH_IMAGE011
。
To tetrahydrofuran (THF, 100 mL,Sigma-Aldrich) of magnesium (9.48 g, 390 mmol, 5.17 eq, sigma-Aldrich) and iodine (191 mg, 754umol, 151uL, 0.01eq, sigma-Aldrich) was added 1-bromo-3-methylbutane (56.9 g, 377 After mmol, 47.4 mL, 5.00 eq, sigma-Aldrich) in tetrahydrofuran (THF 50 mL, 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 (ii) tetrachlorocuprate (0.1M, 45.2 mL, 0.06 eq, sigma-Aldrich) in tetrahydrofuran (THF, 50 mL, sigma-Aldrich). The mixture was subjected to nitrogen overnight at room temperature until thin layer chromatography showed complete consumption of reactant A-1. After completion of the reaction, diluted hydrochloric acid (1M, 500mL, sigma-Aldric) was added to the reaction mixture, diluted with water (300 mL), extracted with ethyl acetate (EtOAc, 300mL, 200 mL, sigma-Aldric), and the combined organic layers were washed with brine (300 mL), dried over sodium sulfate (Na 2 SO 4 Sigma-Aldric), filtering, concentrating under reduced pressure, and subjecting to column chromatography (SiO) 2 Petroleum ether/ethyl acetate=0 to 1) gives compound A-2 (15-methyl-hexadecanol 15.7, g, 61.2 mmol, 81.1%) as a white solid.
Figure 373686DEST_PATH_IMAGE012
。
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 reaction was complete by thin layer chromatography. 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 145464DEST_PATH_IMAGE013
。
To a solution of methyltriphenylphosphorous bromide (109 g, 305 mmol, 5.18 eq, sigma-Aldrich) in tetrahydrofuran (THF, 300ml, sigma-Aldrich) at-20deg.C was added potassium tert-butoxide (t-BuOK, 20.5 g, 183 mmol, 3.10 eq, sigma-Aldrich), the mixture was stirred at 25deg.C for 1 h, and Compound A-3 (15-methyl-16 alkanal, 15.0 g, 58.9 mmol, 1.00 eq) was added at-20deg.CHydrofuran (THF, 30 mL, sigma-Aldrich) solution. The mixture was stirred under nitrogen at 25 ℃ for 2 hours until thin layer chromatography showed clean reaction. The reaction mixture was poured into ammonium chloride (NH 4 Cl, 200 mL, sigma-Aldrich) was extracted with ethyl acetate (500 mL, 300mL, sigma-Aldrich), 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 2 Petroleum ether/ethyl acetate=1/0 to 50/1) gives compound A-4 (16-methyl-17-alkyl-1-ene, 14 g, 55.45 mmol, 94%) as a colourless oil.
Figure 941382DEST_PATH_IMAGE014
。
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 showed complete reaction. The reaction mixture was added sodium thiosulfate (Na 2 S 2 O 3 30 mL, sigma-Aldrich) quenched, then diluted with water (20 mL), extracted with ethyl acetate (50 mL, 30 mL, sigma-Aldrich) and the organic layer washed with brine (8 mL), dried over sodium sulfate, filtered, concentrated under reduced pressure, then purified by column chromatography (SiO 2 Petroleum ether/ethyl acetate=1/0 to 0/1) to give compound A-5 (2- (14-methyl-pentadecyl) oxirane, 13.3 g, 49.5 mmol, 89.3%) as a colourless oil.
Figure 22470DEST_PATH_IMAGE015
。
To compound C5-2 (2-hexylsunflower radical 3- [3- [3- [ [3- (2-hexylsunflower oxy) -3-oxo-propyl)]Amino group]Propyl-methyl-amino group]Propylamino group]To a solution of propionate, 4.00 g, 5.42 mmol, 1.00 eq) in potassium tert-butoxide (t-BuOH, 40 mL,Sigma-Aldrich) was added compound A-5 (2- (14-methylpentadecyl) oxirane, 1.45 g, 5.42 mmol, 1.00 eq), and the mixture was stirred at 90℃for 48 hours to liquid chromatographyIndicating that the reaction was complete. The reaction mixture was taken up in water (15 mL), extracted with ethyl acetate (50 mL, 30 ml, sigma-Aldrich), 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 to 0/1) to obtain butter-like compound C5 (2-hexylsunflower-based 3- [3- [3- [ [3- (2-hexylsunflower-oxy) -3-oxo-propyl) ]- (2-hydroxy-16-methyl-heptadecyl) amino group]Propyl-methyl-amino group]Propylamino group]Propionate, 1.63 g, 1.62 mmol).
Figure 966155DEST_PATH_IMAGE016
。
The nuclear magnetic pattern of C5 is shown in FIG. 1: from the nuclear magnetic resonance spectrum (1H NMR: (400 MHz CDCl) 3 ) C5-A2 is characterized by: delta 3.98 (dd, j=5.82, 3.81 Hz, 4H), 3.53-3.65 (m, 1H), 2.86-2.97 (m, 4H), 2.67-2.80 (m, 4H) 2.59, (brdd, j=13.13, 7.13 Hz, 4H), 2.33-2.51 (m, 9H), 2.22 (s, 3H), 1.57-1.84 (m, 7H), 1.15-1.54 (m, 81H), 0.81-0.98 (m, 18H), so that C5 contains CH 3 , CH 2 , -RH 2 -C00-RH 2 -, -RH(CH 3 ) 2 , -CH(OH)-,-N(CH 3 ) -an isopcharacteristic group.
The ionizable cationic lipid C5 is a multi-nitrogen structure, can combine hydrogen ions under an acidic condition to be positively charged and be electrostatically combined with mRNA which is negatively charged, so that mRNA is well wrapped, therefore, the encapsulation rate of nano liposome particles consisting of the ionizable cationic lipid to the mRNA reaches over ninety percent, and the encapsulation rate is good. On the other hand, the compound contains ester bonds, and can be hydrolyzed under the action of in-vivo related enzymes, so that the degradation of the compound in vivo is accelerated, the half-life of the compound in vivo is reduced, and the side reaction possibly induced by the compound in vivo is reduced. The compound is also a multi-branched structure, and the branched chains enable the nano liposome particles formed by the compound to have a lower phase transition temperature, so that the nano liposome particles are easy to form a cone-shaped structure, thereby destroying the membrane of an endosome and the structure of the endosome and releasing the packed mRNA. From the aspect of the integral structure, the structures such as the polynitrogen, the ester bond, the branched chain and the like of the compound are favorable for coupling mRNA in an acidic environment, so that the nano liposome particles formed by the compound can wrap the mRNA well, and higher encapsulation rate is achieved; on the other hand, the conformation of the nano liposome particles in cells is promoted to be changed, and the structural integrity of the nano liposome particles and endosomes is destroyed, so that mRNA release into cytoplasm is promoted, and the translation efficiency is improved.
EXAMPLE 2 preparation of nanoliposome particles and encapsulation of Luc-mRNA/DNA
The ionizable cationic lipid C5 was synthesized and identified by the pharmaceutical Minkand synthesis following the synthetic route of embodiment one, three lipids, DSPC (distearoyl phosphatidylcholine), cholesterol, DMG-PEG 2000 (1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000) were all from Avanti Polar Lipids (a Mo Di polar lipid company). These four lipids were dissolved in absolute ethanol (purity >99.9%, sigma) to prepare a 10mg/mL stock solution. The molar ratio of the four kinds of grease is 50:10:38.5:1.5, the total lipid concentration of the four lipids was 6mM, and the nitrogen-to-phosphorus ratio was 6 (N: P=6) at the time of preparation.
mRNA/DNA was dissolved in 25mM sodium acetate buffer (pH 5.2) at a volume ratio of 3:1 between aqueous and organic phases, and LNPs solution was prepared using Ignite (Precision Nanosystem Inc) nm manufacturing system with a flow rate ratio of 3:1, the total flow rate was 12mL/min, the pre-waste liquid (start waste) was 0.15mL, and the post-waste liquid (end waste) was 0.05mL.
Nanoliposome particles were prepared using Ignit and coated with Luc-mRNA/DNA according to the above preparation parameters to obtain corresponding LNPs-Luc-mRNA/DNA solutions (Luc-mRNA is firefly luciferase mRNA, 1929 nucleotides in length, a common reporter gene, luc-mRNA was supplied by TriLink Biotechnologies Co.). The resulting LNPs-Luc-mRNA/DNA solution was rapidly transferred to a 50mL ultrafiltration tube (Millipore, 100 KD) and the LNPs solution was 10-fold diluted with 1 XPBS, centrifuged at 1300rpf (Thermo) at 4℃for 30 minutes, and finally the ultrafiltered LNPs solution was collected and fixed to a concentration of 150ug/mL with 1 XPBS. The final LNPs-Luc-mRNA solution was assayed for physicochemical parameters of LNPs-Luc-mRNA including particle size, polydispersity index (PDI), zeta potential using a nanoT ™ riboGreen ™ RNA quantitative assay kit (Life Technology) using a nanoBrook Omni, brookhaven, as follows:
TABLE 2 physicochemical parameters and encapsulation efficiency of LNPs-Luc-mRNA/DNA groups
Figure 728575DEST_PATH_IMAGE017
. The delivery efficiency was expressed in units of firefly luciferase expression Luminescence Units in table 2.
From the physicochemical parameters, good nanoliposome particles can be formed from the new lipid C5 and the other three lipids and encapsulate Luc-mRNA/DNA. 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 nano liposome particle nucleic acid drug carrier is ideal. Thus, further testing of in vitro delivery efficiency was performed on a cell model. Using HEK293 cells, 96-well plates were plated at 20000 cells per well, after overnight, transfection experiments were performed at 500ng of LNPs-Luc-mRNA/DNA per group of five multiplex wells, and after 24 hours the transfection efficiency was measured using the firefly luciferase assay kit (Promega) (see FIG. 2).
The in vitro transfection result shows that the expression quantity of firefly luciferase is very high (see table 2), and the nano liposome particle carrier composed of the ionizable cationic lipid has very high delivery efficiency on the mRNA and DNA of the firefly luciferase, and is a very good nucleic acid drug delivery carrier.
Example 3: preparation of nanoliposome particles and delivery of eGFP-mRNA/DNA
To further verify that the novel nano-liposome particle delivery system composed of ionizable cationic lipid C5 is capable of well encapsulating and delivering mRNA and DNA, and that the high efficiency is universal, rather than having high delivery efficiency only for a specific mRNA/DNA. Preparation of nanoliposome particles using the novel ionizable cationic lipid C5 according to the same conditions and preparation parameters above encapsulates eGFP-mRNA/DNA (eGFP-mRNA is a green fluorescent protein capable of emitting green light upon fluorescence excitation, a common reporter gene, 996 nucleotides in length, also supplied by trilink biotechnology company) and the physicochemical parameters and delivery efficiency of the nanoliposome particle carrier were examined according to the same technical means.
TABLE 3 physicochemical parameters and encapsulation efficiency of LNPs-eGFP-mRNA/DNA groups
Figure 706895DEST_PATH_IMAGE018
。
From the physicochemical parameters, it is known that: the nano liposome particle composed of the novel lipid C5 can well wrap green fluorescent protein mRNA and DNA (eGFP-mRNA/DNA), and has good particle size and Polydispersity (PDI), and the encapsulation 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 The density of individual cells was plated in 24-well plates overnight, and transfection experiments were performed with 500ng of LNPs-eGFP-mRNA/DNA, 3 wells per group, and 24 hours later, the expression level of green fluorescent protein was observed using a fluorescent microscope, and fluorescent pictures showed high expression levels of eGFP-mRNA (FIG. 3)/DNA (FIG. 4).
The novel nano liposome particle composed of the ionizable cationic lipid C5 is used for wrapping and delivering green fluorescent protein eGFP-mRNA/DNA, and has good delivery efficiency, so that the nano liposome particle composed of the ionizable cationic lipid C5 can be used for efficiently delivering nucleic acid medicaments such as mRNA and DNA, and has the potential of a good nucleic acid medicament delivery carrier.
Example 3: in vivo experiments with nanoliposome particle vector delivery of firefly luciferase mRNA consisting of neolipo-C5
To further examine the in vivo delivery efficiency of the nanoliposome particle vector of the new lipid C5 composition, the examination of the delivery efficiency after the nanoliposome particle of the new lipid C5 composition encapsulates firefly luciferase mRNA was performed on a mouse animal model. The experimental protocol was also consistent with the cell model, with the ionizable cationic lipid C5, DSPC (distearoyl phosphatidylcholine), cholesterol, DMG-PEG 2000 (1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000) at a molar ratio of 50:10:38.5: 1.5. The total lipid concentration of the four lipids was 6mM and the nitrogen to phosphorus ratio was 6 (N: P=6) at the time of preparation. mRNA was dissolved in 25mM sodium acetate buffer pH5.2 at a volume ratio of aqueous phase to organic phase of 3:1, and LNPs solution was prepared using Ignite (Precision Nanosystem Inc) nm manufacturing system with a flow rate ratio of aqueous phase to organic phase of 3:1, total flow rate was 12mL/min, pre-waste (start waste) was 0.15mL, post-waste (end waste) was 0.05mL, and the same parameters wrapped Luc-mRNA. The prepared LNPs-Luc-mRNA was transferred to a 50mL ultrafiltration tube (Millipore, 100 KD) and the LNPs solution was 10-fold diluted with 1 xPS, centrifuged at 1300rpf (Thermo) at 4℃for 30 minutes, and finally the LNPs solution after ultrafiltration was collected and fixed to a concentration of 150ug/mL with 1 xPBS. The physicochemical parameters of LNPs-Luc-mRNA including particle size, polydispersity index (PDI), zeta potential were determined using a nanoBrook Omni, brookhaven, nanoliposome particle size analyzer, and encapsulation efficiency was determined using a Quant-iT ™ riboGreen ™ RNA quantitative detection kit (Life Technology) as follows:
TABLE 4 physicochemical parameters and encapsulation efficiency of LNPs-Luc-mRNA
Figure 630989DEST_PATH_IMAGE019
。
The delivery efficiency units in table 4 are: avg radius [ p/s/cm2/sr ].
The results of the physicochemical parameters can be seen as follows: the nano liposome particles composed of the novel lipid C5 have good physical and chemical parameters, the particle size is 80-120nm, the polydispersity index (PDI) is good, the encapsulation rate is more than 90%, and the encapsulation effect is good after the mRNA is encapsulated by the nano liposome particles. Further testing of delivery efficiency was performed at the animal level by intramuscular injection of LNPs-Luc-mRNA into mice (BALB/c, 8 weeks old) at a dose of 10 μg per mouse and four mice per group. Six hours later, mice were subjected to in vivo imaging using a living imager (PerkinElmer), the luminescence intensity of the mice was measured, and the in vivo delivery efficiency of the nanoliposome particle carrier was examined (see fig. 5, 6).
From the in vivo delivery efficiency results, it can be seen that: the nano liposome particle carrier composed of the novel lipid C5 can well wrap firefly fluorescein mRNA, and has the advantages of good physical and chemical parameters, high delivery efficiency, good survival state of mice and good safety.
The nano liposome particle carrier composed of the novel lipid C5 has good physical and chemical parameters, can well wrap DNA and mRNA, and the result of the implementation case shows that the novel lipid C5 is a good ionizable cationic lipid, and the nano liposome particle carrier composed of the novel lipid C5 can well deliver mRNA and DNA into a body, and simultaneously, the expression quantity of protein is also 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 (aiRNA), micro RNA (miRNA), antisense RNA, small hairpin RNA and the like, and has great prospect in the aspect of nucleic acid drug delivery.

Claims (11)

1. An ionizable cationic lipid, characterized in that the chemical structural formula is shown in formula (I):
Figure QLYQS_1
（Ⅰ）。
2. a nanoliposome particle carrier, wherein the nanoliposome particle carrier is a composition comprising the ionizable cationic lipid of claim 1 and a phospholipid, a structured lipid, and a pegylated lipid.
3. The nanoliposome particle carrier of claim 2, wherein the ionizable cationic lipid and phospholipid, structural lipid, pegylated lipid is present in an amount of (40-65%): (5-25%): (25-50%): (0.5-15%) and the ionic lipid is prepared by mixing the components according to a molar ratio of 6:1, the total lipid concentration of the ionizable cationic lipid and phospholipid, structural lipid, and PEGylated lipid is 6mM.
4. The nanoliposome particle carrier of claim 3, wherein the ionizable cationic lipid and phospholipid, structural lipid, pegylated lipid is present at a ratio of 50:10:38.5:1.5 by mole ratio.
5. The nanoliposome particle carrier of claim 2, wherein said phospholipid is selected from one or more phospholipid compounds from the group consisting of: 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dimyristoyl-sn-glycero-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, 1, 2-di (undecanoyl) -sn-glycero-phosphorylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine, 1, 2-di-O-octadecenyl-sn-glycero-3-phosphorylcholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphorylcholine, 1-hexadecyl-sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphorylcholine, 1, 2-di (docosahexaenoic acid) -sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di-phytanoyl-sn-glycero-3-phosphoethanolamine, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di (docosahexaenoic acid) -sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycero) sodium salt, sphingomyelin.
6. The nanoliposome particle carrier of claim 2, wherein the structural lipid is any structural lipid compound selected from the group consisting of: cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherol, corticosteroids.
7. The nanoliposome particle carrier of claim 2, wherein said pegylated lipid is selected from any one of the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.
8. The nanoliposome particle carrier of claim 2, wherein the phospholipid, structural lipid and pegylated lipid are 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine, cholesterol and PEG2000-DMG, respectively.
9. Use of the nanoliposome particle carrier of any of claims 2-8 for the preparation of a medicament for RNA or DNA delivery.
10. The use according to claim 9, wherein the method of preparation of the RNA or DNA delivery drug comprises the steps of:
(1) Dissolving RNA or DNA as a drug molecule in sodium acetate buffer to form an aqueous phase;
(2) Determining the mole number of the ionizable cationic lipid with the mole ratio of nitrogen atoms in the ionizable cationic lipid to phosphate groups in RNA or DNA as a drug molecule of (2-30) to 1, and further determining the mole ratio of 50 according to the mole ratio of the ionizable cationic lipid, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, cholesterol, PEG 2000-DMG: 10:38.5: mixing the four components uniformly in a ratio of 1.5 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 or DNA loaded nano liposome particles in a microfluidic mixed mode, wherein the volume of the nucleic acid aqueous phase is 3 times that of the organic phase, and the mass ratio of lipid to nucleic acid in the nano liposome particle carrier is 3:1 to 100:1.
11. The use according to claim 10, wherein the RNA is 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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