CN116712408B - Lipid nanoparticle and preparation method and application thereof - Google Patents

Lipid nanoparticle and preparation method and application thereof Download PDF

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CN116712408B
CN116712408B CN202310782644.XA CN202310782644A CN116712408B CN 116712408 B CN116712408 B CN 116712408B CN 202310782644 A CN202310782644 A CN 202310782644A CN 116712408 B CN116712408 B CN 116712408B
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CN116712408A (en
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张炜
陈琬东
黄莎莎
李桃宇
梁万里
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Guangzhou National Laboratory
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Abstract

The invention belongs to the technical field of biological medicines, and discloses a lipid nanoparticle, a preparation method and application thereof. The present invention provides a lipid carrier comprising: neutral lipids, amphiphilic lipids, cationic lipids, steroidal lipids and divalent salts; by employing a divalent salt, the transfection efficiency of lipid nanoparticles comprising the lipid carrier can be improved, i.e., the delivery properties of the lipid nanoparticles can be improved, compared to monovalent salt (sodium acetate); at the same time, toxicity of the lipid nanoparticle is reduced.

Description

Lipid nanoparticle and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a lipid nanoparticle, and a preparation method and application thereof.
Background
Many mutations in genes that alter cell proliferation, angiogenesis, metastasis and tumor immunogenicity lead to human cancers, and have become one of the greatest threats to human life safety and health. Despite the increasing understanding of the molecular mechanisms of cancer, many malignant tumors remain resistant to established traditional therapies. However, the definition of tumor-associated gene mutations has increased interest in cancer therapy as a target for gene therapy.
Nucleic acid drug delivery systems can be divided into viral vectors and non-viral vectors. Good nucleic acid vectors such as viral vectors have high transfection efficiency and are at risk of increased immunogenicity and adverse reactions. Nucleic acid vectors using viruses may infect more cells than expected, transgenes may be inserted into the wrong location of the DNA strand, inserted genes may be overexpressed, the modified virus may cause inflammation or immune response, and the virus may be transmitted to other people or the environment. Non-viral delivery systems have several advantages over viral vectors, such as no immune response to the vector, and their ability to transport more nucleic acid. Furthermore, non-viral vectors generally have high reproducibility, acceptable chemical and physical stability, the possibility of surface modification and the possibility of targeting specific cells or tissues. Despite therapeutic benefits in animal models, poor nucleic acid transfer efficiency and low gene expression were observed in animal models.
Several promising nucleic acid candidate drugs are antisense oligonucleotide (ASO), small interfering RNA (siRNA), micro RNA (miRNA), messenger RNA (mRNA), cpG oligodeoxynucleotide, plasma DNA (pDNA). All of these biological macromolecules function in the intracellular environment: a nucleus (DNA) or a cytoplasm (RNA). Since all these nucleic acid molecules have negative charges and polyelectrolyte-like behavior, this makes them impermeable through the lipid bilayer of the plasma membrane. Typically, DNA molecules are double-stranded helical three-dimensional structures, while RNA is single-stranded and adopts three-dimensional structures. The former has a median size of about 50nm, while the latter has a size of 1-2 nm. On the other hand, the ability of nucleic acid molecules to form three-dimensional folded structures has been explored as drug delivery vehicles, forming scaffold structures. However, these systems also have some drawbacks because they require molecular modification to prevent rapid environmental degradation. Nucleic acid molecules alone are not easily accessible to the nucleus due to their hydrophilic, negatively charged, large size, etc. molecular characteristics. In addition, DNA and RNA molecules have short blood circulation times, must cross physiological barriers before reaching the cells, and are susceptible to extracellular and intracellular enzymatic degradation. Therefore, it is important to develop new delivery systems that can improve nucleic acid nuclear targeting.
In order to increase the efficiency of nucleic acid transfection, it is important to understand the process required for nucleic acid to reach the nucleus. Cell membranes are a dynamic and powerful barrier to intracellular delivery. The double-layer phospholipid mainly comprises a zwitterionic lipid double-layer and a negatively charged phospholipid, wherein the polar head of the phospholipid points to the water environment, and the hydrophobic tail forms a hydrophobic core. To achieve therapeutic goals, the non-viral nucleic acid delivery system should first be internalized by the cell, endocytosis occurs and degradation of the endosome or lysosome is avoided. Thereafter, the nucleic acid is released in the cytoplasm and enters the nucleus for corresponding gene expression.
Lipid Nanoparticles (LNPs) are submicron particles that contain ionizable cationic lipids, or other types of cationic lipids, while encapsulating nucleic acid drugs. The nucleic acid delivery capsule is used as a protecting capsule of nucleic acid cargoes, forms a multifunctional nucleic acid delivery platform and overcomes the degradation of nucleic acid and limited cellular uptake. However, naked mRNA has inherent instability and is readily degraded rapidly by nucleases and autohydrolases. The encapsulation of messenger mRNA in LNPs protects the mRNA from extracellular ribonucleases and facilitates the intracellular delivery of mRNA.
LNP preparation can be performed in a variety of ways, such as thin film hydration and reduction techniques, ethanol injection, microfluidic hydrodynamic focusing, T-junction mixing, staggered chevron mixing, and the like.
Thin film hydration is a common manufacturing method for producing liposomes, in which high energy size reduction methods are used to reform large lipid vesicles into small vesicles. Extrusion is a process of repeatedly forcing a heterogeneous suspension of particles through a polycarbonate or inorganic filter of a specified pore size (e.g., 0.1 μm), so the size of the unilamellar vesicles is within the size of the pores. Ethanol injection methods were developed as an improved alternative to membrane hydration methods in combination with sonication. The lipid ethanol solution was injected by syringe into KCl solution diluted to a concentration of 7.5% (v/v) ethanol to form a relatively uniform particle solution with an average size of ≡ 27nm (measured by electron microscopy). When ethanol is rapidly diluted in aqueous buffer, lipid vesicles self-assemble due to the increase in solvent polarity. For the preparation of lipid nanoparticles with this equipment, the main drawbacks of these two traditional production techniques are the large specific gravity of labor consumption, the small specific gravity of physical and chemical labor consumption, high cost and difficulty in scale-up, the easy scalability problem of the size reduction method and the small reproducibility of certain steps, and the large-scale time consumption of multiple steps. In the case of ethanol injection, reproducibility is difficult to achieve on a stirred batch scale. And sample contamination and degradation are also potential problems with some of the methods described above.
Microfluidic hydrodynamic focusing is a microfluidic mixing technique for manufacturing liposomes in a reproducible and scalable manner. But the equipment is expensive in manufacturing cost and limited in volume. In this method, mixing is slow at low FRR (water-organic flow rate), high FRR results in low particle concentration, parallelization is required to scale up, and most importantly, its temperature is difficult to control flexibly.
When the lipid nanoparticle is prepared by using a microfluidic mixing technology, the preparation temperature cannot be higher than the continuous phase transition temperature (more than 50 ℃), the lipid phase and the water phase solidify for many times in the mixing process, a liposome with uniform and stable morphology cannot be formed, part of pipelines need to be continuously heated by an external tool, the final lipid nanoparticle is failed to be prepared, and the particle size potential and uniformity of a compound cannot be measured.
In contrast to macroscopic mixing methods (e.g., vortexing or pipetting), the T-junction mixer provides a controlled mixing environment, enabling reproducible production of lipid complexes. While staggered chevron mixers are produced with poly (dimethylsiloxane) or cyclic olefin copolymers, solvent incompatibilities may occur, microchannel plugging may occur, and parallelization is required to extend longitudinally.
All the above-mentioned rapid mixing techniques have the disadvantage that they add large amounts of organic solvents during the manufacturing process, which may be present in the final product and present an explosion risk on the manufacturing scale. In this regard, ethanol is preferred as the solvent can be easily removed, but some lipids have limited solubility in ethanol, and have high requirements on system temperature, and some liposome preparation methods cannot reach the required temperature, so that lipid nanoparticle preparation fails, and the concentration of LNP in the mixed solution is low. There is a need for a lipid nanoparticle for delivering nucleic acids that is inexpensive, easy to operate, and flexible and easy to reach the desired temperature.
Disclosure of Invention
The object of the first aspect of the present invention is to provide a lipid carrier.
The object of the second aspect of the present invention is to provide a lipid nanoparticle.
The third aspect of the present invention is directed to a method for preparing the lipid nanoparticle according to the second aspect of the present invention.
The object of the fourth aspect of the present invention is to provide the use of the lipid carrier of the first aspect of the present invention, the lipid nanoparticle of the second aspect of the present invention for the preparation of a pharmaceutical composition, and/or the preparation method of the third aspect of the present invention for the preparation of a pharmaceutical composition.
The object of the fifth aspect of the present invention is to provide a pharmaceutical composition.
The object of a sixth aspect of the present invention is to provide the use of the device of the third aspect of the present invention for the preparation of lipid nanoparticles.
The seventh aspect of the present invention is directed to an apparatus for preparing lipid nanoparticles.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided a lipid carrier comprising: neutral lipids, amphiphilic lipids, cationic lipids, steroidal lipids and divalent salts.
Preferably, the cationic lipid is selected from: (4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315), octadecan-9-yl 8- ((2-hydroxyethyl) (6-oxO-6- (undecyloxy) hexyl) amino) octanoate (SM-102), 1, 2-dioleoyloxy-3- (trimethylammonio) propane (DOTAP), 313- [ N- (N, N-dimethylaminoethane) -carbamoyl ] cholesterol (DC cholesterol), dimethyl Dioctadecyl Ammonium (DDA), 1, 2-dimyristoyl-3-trimethylammoniopropane (DMTAP), dipalmitoyl (C16:0) trimethylammoniopropane (DPTAP), distearoyl trimethylammoniopropane (DSTAP), N- [1- (2, 3-dienropoxy) propyl ] -N, N, N-trimethylammonio-N, N-dioleoyl-N-dimethylammonium chloride (DODACs), 1, 2-dioleoyl-3-dioleoyl-trimethylammonio (DOEPC), 1, 2-dioleoyl-trimethylammonio-propane (DOTAP), one or more combinations of heptadeca-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA), 1, 2-diiodoxy-3-dimethylaminopropane (DLinDMA); further preferably, the cationic lipid is selected from the group consisting of: a combination of one or more of octadecane-9-yl 8- ((2-hydroxyethyl) (6-oxO-6- (undecyloxy) hexyl) amino) octanoate (SM-102), 1, 2-dioleoyloxy-3- (trimethylammonio) propane (DOTAP), heptadecan-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA); still more preferably, the cationic lipid is selected from the group consisting of: one or two of octadecane-9-yl 8- ((2-hydroxyethyl) (6-oxO-6- (undecyloxy) hexyl) amino) octanoate (SM-102), and heptadeca-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA); still further preferably, the cationic lipid is thirty-seven carbon-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA).
Preferably, the divalent salt is an organic divalent salt.
Preferably, the divalent salt is a soluble divalent salt.
Preferably, the divalent salt is selected from: one or more of calcium salt and magnesium salt; further preferably, the divalent salt is a calcium salt.
Preferably, the calcium salt is selected from: one or more of calcium acetate, calcium gluconate and calcium lactate; further preferably, the calcium salt is selected from the group consisting of: one or two of calcium acetate and calcium gluconate; still more preferably, the calcium salt is calcium acetate.
Preferably, the magnesium salt is selected from: magnesium acetate.
Preferably, the neutral lipid is selected from: one or more combinations of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (distearoyl phosphorylcholine, DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho- (1-rac-glycero) (DOPG), oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE); further preferred are: DSPC.
Preferably, the amphiphilic lipid is selected from: one or more combinations of PEG-DMG, PEG-C-DMG, PEG-C14, PEG-C-DMA, PEG-DSPE, PEG-PE, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, tween-20, tween-80, PEG-DPG, PEG-s-DMG, DAA, PEG-C-DOMG, and GalNAc-PEG-DSG; further preferred are: DMG-PEG2000.
Preferably, the steroid lipid is selected from: oat sterols, beta-sitosterols, campesterols, ergocalcitols, campesterols, cholestanol, cholesterol, stigmasterols, dehydrocholesterol, desmosterols, dihydroergocalcitols, dihydrocholesterol, dihydroergosterols, black-sea sterols, episterols, ergosterols, fucosterol, hexahydrophotosterol, hydroxycholesterols, lanosterols, photosterol, algae sterols, sitostanol, sitosterol, stigmastanol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid; further preferred are: cholesterol.
Preferably, the mass ratio of the neutral lipid, the amphiphilic lipid, the cationic lipid, the steroid lipid and the divalent salt is (1320-1980): 800: (5200-7800): (2480-3720): (5000-25000); further (1500-1800): 800: (6000-7100): (2800 to 3400): (6000-22000); further (1600-1700): 800: (6300-6700): (3000-3200): (7100 to 22000); still further 1650:800:6500:3100: (7112.38-21496.98).
Preferably, the neutral lipid, amphiphilic lipid, cationic lipid and/or steroid lipid has a purity of not less than 95wt%.
In a second aspect of the invention there is provided a lipid nanoparticle comprising a drug and a lipid carrier of the first aspect of the invention.
Preferably, the drug is a nucleic acid.
Preferably, the nucleic acid is selected from: DNA, RNA, DNA analog, RNA analog, or combinations of one or more thereof.
Further preferably, the nucleic acid is selected from the group consisting of: ssDNA, dsDNA, mRNA, lncRNA, siRNA, saRNA, shRNA, ASO, plasmid, circRNA, circDNA, miRNA, CRISPR-Cas, samRNA, antagomir, microrna inhibitor, microrna activator, and immunostimulatory nucleic acid.
Preferably, the nucleic acid is a modified nucleic acid.
Preferably, the concentration of the nucleic acid is not less than 100. Mu.g/mL.
Preferably, when the nucleic acid molecule is DNA, the OD value of the nucleic acid molecule is 260/280 equal to 1.8; when the nucleic acid molecule is RNA, the OD value of the nucleic acid molecule is 260/280 equal to 2.0.
Preferably, the medicament is a vaccine.
Preferably, the vaccine is a therapeutic or prophylactic vaccine.
Preferably, the vaccine is for the prevention and treatment of tumors, bacterial infections, viral infections, and/or fungal infections.
Preferably, the tumor comprises at least one of a solid tumor, a hematological tumor.
Preferably, the method comprises the steps of, the solid tumor comprises liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, gastric cancer, adrenocortical cancer, pararenal cortical cancer, anal cancer, appendiceal cancer, astrocytoma, atypical teratoma, rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumor, bronchogenic tumor, burkitt lymphoma, carcinoid tumor, cardiac tumor, cholangiocarcinoma, chordoma, carcinoma of large intestine, craniopharyngeal tumor, in situ breast cancer, germ tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial embryonal tumor, extragonadal germ cell tumor, eye cancer, oval tube cancer, gallbladder cancer, head and neck cancer, hypopharynx cancer, kaposi's sarcoma, renal cancer, langerhans cell cytopenia laryngeal carcinoma, lip carcinoma, oral carcinoma, merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplastic syndrome, mycosis fungoides, nasal sinus carcinoma, neuroblastoma, non-small cell lung carcinoma, ovarian carcinoma, pancreatic neuroendocrine tumor, islet cell tumor, papillomatosis, paraganglioma, nasal sinus cavity carcinoma, parathyroid carcinoma, penile carcinoma, throat carcinoma, pituitary tumor, pleural pneumoblastoma, primary peritoneal carcinoma, retinoblastoma, salivary gland tumor, sarcoma, cerclage syndrome, skin carcinoma, small cell lung carcinoma, small intestine carcinoma, soft tissue sarcoma, squamous cell carcinoma, testicular carcinoma, thymoma and thymus carcinoma, thyroid carcinoma, urethra carcinoma, uterine carcinoma, endometrial and uterine sarcoma, vaginal carcinoma, vascular tumor, vulval carcinoma, and single myeloma.
Preferably, the hematological neoplasm is selected from at least one of B-cell acute lymphoid leukemia, T-cell acute lymphoid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B-cell promyelocytic leukemia, blast plasmacytoid dendritic cell tumor, burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-hodgkin's lymphoma, plasmablastoid lymphoma, plasmacytoid dendritic cell tumor, waldenstrom macroglobulinemia, and pre-leukemia stage.
Preferably, the bacteria comprise at least one of gram negative bacteria, gram positive bacteria.
Preferably, the gram-positive bacteria comprise at least one of staphylococcus (Staphylococcus spp), streptococcus (Streptococci), enterococcus (Enterococcus spp), leuconostoc (Leuconostoc spp), corynebacteria (Corynebacterium spp), cryptobacter (Arcanobacteria spp), cryptococcus (Trueperella spp), rhodococcus (Rhodococcus spp), bacillus spp, anaerobacter (Anaerobic Cocci), anaerobic gram-positive non-spore-forming Bacillus (Nonsporulating Bacilli), actinomycetes (Actinomyces spp), clostridium (Clostridium spp), nocardia spp (Nocardia spp), erysipelas spp (Erysipelothrix spp), listeria spp, apostichopus japonicus co-producing actinomycetes (Kococcus spp), mycobacteria (Mycobacterium spp).
Preferably, the gram-negative bacteria comprises at least one of Neisseria (Neisseria spp), moraxella (Moraxella spp), escherichia (Escherichia spp), klebsiella (Klebsiella spp), proteus (Proteus spp), pseudomonas spp, salmonella (Salmonella spp), shigella spp, campylobacter (Campylobacter spp), helicobacter spp, bacteroides spp, yersinia spp, vibrio (Vibrio spp), legionella spp.
Preferably, the fungus comprises Acremonium (Acremonium), absidia (Absidia) (e.g., absidia umbrella (Absidia corymbifera)), alternaria (Alternaria), aspergillus (Aspergillus) (e.g., aspergillus clavatus (Aspergillus clavatus), aspergillus flavus (Aspergillus flavus), aspergillus fumigatus (Aspergillus fumigatus), aspergillus nidulans (Aspergillus nidulans), aspergillus niger (Aspergillus niger), aspergillus terreus (Aspergillus terreus) and Aspergillus variabilis (Aspergillus versicolor)), helminthosporium (Bipolaris), blastomyces (Blastomyces) (e.g., acremonium dermatitis (Blastomyces dermatitidis)), blastomyces (Blastoschizomyces) (e.g., rhizopus cephalosporans (Blastoschizomyces capitatus)), candida (Candida) (e.g., candida albicans (Candida albicans), candida glabra (Candida guilliermondii), candida albicans (Candida glabra), candida mongolica (Candida guilliermondii), candida lactis (Candida kefyr), candida krusei (Candida krusei), candida vinosa (Candida lusitaniae), candida parapsilosis (Candida parapsilosis), candida tropicalis (Candida pseudotropicalis), candida stellati (Candida glabra), candida tropicalis (Candida tropicalis), candida rue (Candida lutea utilis), candida lipolytica (Candida lipolytica), candida innominate (Candida famata) and Candida rugosa), candida Cladosporium (Cladosporium) (e.g., the genus Trichosporon (Cladosporium carrionii) and Trichosporon (Cladosporium trichoides)), the genus Sporoborium (Coccidioides) (e.g., cryptococcus (Coccidioides immitis)), the genus Cryptococcus (e.g., cryptococcus neoformans), the genus Curvularia (Curvularia), the genus Vermiliaria (Cunninghamella) (e.g., agroborium elegans (Cunninghamella elegans)), the genus Trichosporon (Dermatophyte), the genus Aphyllum (Exophiala) (e.g., lepidermatis (Exophiala dermatitidis) and Sporothecium spinosa (Exophiala spinifera)), the genus Epidermomyces (e.g., epidermomycetocercoston (Epidermophyton floccosum)), the genus Arisaema (Foensecaea) (e.g., pei Shifang Serrata (Fonsecaea pedrosoi)), the genus Fusarium (e.g., fusarium candidum), the genus Geotrichum (Geotrichum) and the genus Leucopia (e.g., geotrichum candidum) and the genus Leucopia (e.g., leucopia parvularia (25), the genus Leucopia (e.g., leucopia parvularia (e.g., leucopia) and the genus Leucopia (e.25) (e.g., leucopiaum parvulgare.25) and the genus Leucopia (e.g., leucopia) of the strain) (p.25, leucopiaum) and the genus Leucopiaum (p.p.p.25) and the strain (p.p.p.p.p.p.5) of the strain, leucopiaum (p.p.p.p.p.p.5) and the strain), penicillium (Penicillium) (e.g., penicillium marneffei), trichosporon (Phosphorora), pityrosporum (Pityrosporum ovale), pneumocystis (Pneumocystis) (e.g., pneumocystis carinii (Pneumocystis carinii)), armillariella pseudolaris (Pseudomonas pseudolaris) (e.g., pseudomonas borteus Li Shenmei (Pseudallescheria boydii)), rhizopus (Rhizopus) (e.g., rhizopus microsporum variation (Rhizopus microsporus var. Rhizosporidium) and Rhizopus oryzae (Rhizopus oryza)), saccharomyces (Saccharomyces) (e.g., saccharomyces cerevisiae (Saccharomyces cerevisiae)), saccharomyces (Scedosporium) (e.g., trichosporon tip (Scedosporium apiosperum)), trichosporon (Scolopendra), trichosporon (Sporoteium) (e.g., trichosporon tip (Scorpium) (Scedosporium apiosperum)), trichosporon (Sporoteium sp)), trichosporon (Trichosporon sp) (e.g., trichosporon rubrum (67)), trichosporon (Trichosporon rubrum (nucifolium) and Trichosporon (nux sp) (e.g., trichosporon sp) (Rhizopus microsporus) and Trichosporon (nux sp) (67)), trichosporon (Trichosporon sp) (e.g., trichosporon sp).
Preferably, the virus comprises at least one of a DNA virus and an RNA virus.
Preferably, the virus is selected from one or more of the Alphaviridae (Alphaviridae), flaviviridae (flavoviridae), hepadnaviridae (Hepadnaviridae), herpesviridae (Herpesviridae), orthomyxoviridae (Orthomyxoviridae), papovaviridae (Papovaviridae), paramyxoviridae (Paramyxoviridae), picornaviridae (Picornaviridae), polyomaviridae (Polyomaviridae), reoviridae (Reoviridae), retroviridae (Retroviridae), rhabdoviridae (Rhabdoviridae).
Preferably, the mass ratio of the lipid carrier to the drug is (15000-35000): 334.53; further, (19162.38-33546.98): 334.53; further, (19950.59 to 33546.98): 334.53.
preferably, the average particle size of the lipid nanoparticle is 100-300 nm; further 180-290 nm; further 220 to 270nm.
Preferably, the Zeta potential of the lipid nanoparticle is 32-63 mV; further 43 to 63mV.
Preferably, the lipid nanoparticle has a PDI of 0.08 to 0.25; further 0.16 to 0.22.
In a third aspect of the present invention, there is provided a method for preparing lipid nanoparticles of the second aspect of the present invention, comprising the steps of:
Mixing neutral lipid, amphiphilic lipid, cationic lipid and steroid lipid with ethanol to obtain oil phase;
mixing divalent salt with the medicine to obtain water phase;
mixing the oil phase and the water phase to obtain the lipid nanoparticle.
Preferably, the ethanol is absolute ethanol.
Preferably, the temperature at which the neutral lipid, the amphiphilic lipid, the cationic lipid and the steroid lipid are mixed with the ethanol is 55-75 ℃; more preferably 60 to 70 ℃.
Preferably, the mixing of the neutral lipid, amphiphilic lipid, cationic lipid, steroid lipid with ethanol is performed under water bath conditions.
Preferably, the volume ratio of the oil phase to the water phase is 1: (5-15); further 1: (8-12); still further 1:10.
preferably, the oil phase and the water phase are mixed by a preparation device of lipid nano particles.
Preferably, the preparation device comprises:
a three-way valve member including an a port and a b port, the a port being provided in two;
the two cavity members are provided with a push-pull plunger member, and the two cavity members are respectively connected with the two ports a;
a filter member connected to the port b;
Wherein the two ports a and b can be opened and closed respectively so that any two or three of the filter member and the two chamber members are communicated.
Preferably, the chamber member is detachably connected to the side wall of the port a.
Preferably, the chamber member is detachably connected to the side wall of the port a by a screw structure.
Preferably, the chamber member is a syringe.
Preferably, the filter member is detachably connected to the side wall of the port b.
Preferably, the filter member is detachably connected to the side wall of the port b through a screw structure.
Preferably, the filter member comprises a disposable aqueous phase needle filter head.
Preferably, the specific method for mixing the oil phase and the water phase is as follows:
one of the chamber components draws in an oil phase and the other chamber component draws in an aqueous phase;
closing the port b, respectively opening the two ports a, and pushing and pulling the plunger component to mix the oil phase and the water phase in the two cavity components;
pushing the solution in one chamber component to the other chamber component, wherein the opening a corresponding to the empty chamber component is closed, and the other opening a is opened;
Opening the port b to push the solution out through the filter member.
Preferably, the oil phase and the water phase are mixed in both of said chamber members by pushing and pulling said plunger member a plurality of times.
In a fourth aspect, the invention provides a lipid carrier according to the first aspect of the invention, a lipid nanoparticle according to the second aspect of the invention, and the use thereof in the manufacture of a pharmaceutical composition.
In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising any one of a 1) to a 2):
a1 A lipid carrier according to the first aspect of the invention;
a2 Lipid nanoparticles of the second aspect of the invention.
Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.
Preferably, the pharmaceutically acceptable auxiliary materials comprise at least one of diluents, excipients, binders, wetting agents, surfactants, lubricants and disintegrants.
Preferably, the pharmaceutical composition is an inhaled formulation, an injectable formulation or an oral formulation.
Preferably, the inhalation formulation is an aerosol inhalation or a dry powder inhalation.
Preferably, the injection formulation is a liquid formulation.
Preferably, the oral formulation is a tablet, pill, powder, granule, capsule, sustained release formulation, solution, emulsion, suspension, syrup or drop.
In a sixth aspect of the invention there is provided the use of a preparation device according to the third aspect of the invention for the preparation of lipid nanoparticles.
Preferably, the lipid nanoparticle is a lipid nanoparticle of the second aspect of the invention.
In a seventh aspect of the present invention, there is provided a production apparatus for lipid nanoparticle particles, which is the production apparatus in the third aspect of the present invention.
The beneficial effects of the invention are as follows:
the present invention provides a lipid carrier comprising: neutral lipids, amphiphilic lipids, cationic lipids, steroidal lipids and divalent salts; by employing a divalent salt, the transfection efficiency of lipid nanoparticles comprising the lipid carrier can be improved, i.e., the delivery properties of the lipid nanoparticles can be improved, compared to monovalent salt (sodium acetate); at the same time, toxicity of the lipid nanoparticle is reduced.
Further, the invention provides a lipid nanoparticle, which has uniform particle size, average particle size of 100-300 nm, PDI of 0.14-0.25 and zeta potential of 37-63 mV; the lipid nanoparticle has long shelf life, convenient storage, high stability, stable two-month storage at 4 ℃, good delivery effect, and solves the problems that the conventional transfection reagents (PEI, lipo 8000) need to be prepared at present and have large batch-to-batch difference when in use; in addition, the lipid nanoparticle has good delivery performance, and especially, the transfection efficiency of the lipid nanoparticle can reach about 80% at most by adopting SM-102 and DLin-MC3-DMA as cationic lipids; finally, the lipid nanoparticle has little toxicity and almost no toxicity.
Further, the present invention provides a method for preparing lipid nanoparticles, comprising the steps of using a preparation device for lipid nanoparticles, which is easily available in source and has a low equipment manufacturing threshold; the preparation method is simple to operate, convenient to prepare, and free of professional training for users, solves the problems of high manufacturing and using cost, high labor intensity, huge equipment, difficulty in large scale, low repeatability, sample degradation and pollution of other delivery devices, and the prepared lipid nanoparticle has uniform particle size, high stability and good delivery effect.
Drawings
Fig. 1 is a schematic structural view of a preparation apparatus of lipid nanoparticles.
Fig. 2 is an average particle diameter and Zeta potential map of lipid nanoparticles of example 1: wherein a is the average particle size map of the lipid nanoparticle of example 1 and B is the Zeta potential map of the lipid nanoparticle of example 1.
Fig. 3 is an average particle diameter and Zeta potential map of lipid nanoparticles of example 2: wherein a is the average particle size map of the lipid nanoparticle of example 2 and B is the Zeta potential map of the lipid nanoparticle of example 2.
Fig. 4 is an average particle diameter and Zeta potential chart of lipid nanoparticles of examples 3 to 5: wherein a is the average particle size map of the lipid nanoparticle of example 3, B is the Zeta potential map of the lipid nanoparticle of example 3, C is the average particle size map of the lipid nanoparticle of example 4, D is the Zeta potential map of the lipid nanoparticle of example 4, E is the average particle size map of the lipid nanoparticle of example 5, and F is the Zeta potential map of the lipid nanoparticle of example 5.
Fig. 5 is a graph showing the results of the stability test of lipid nanoparticles of examples 1 to 5 and comparative examples 2 and 3: wherein a is a graph showing a change in particle diameter after the lipid nanoparticles of examples 1 to 2 and comparative example 2 were left for two months, B is a graph showing a change in potential after the lipid nanoparticles of examples 1, 2 and comparative example 2 were left for two months, C is a graph showing a change in PDI after the lipid nanoparticles of examples 1, 2 and comparative example 2 were left for two months, D is a graph showing a change in particle diameter after the lipid nanoparticles of examples 1, 3 to 5 and comparative example 3 were left for two months, E is a graph showing a change in potential after the lipid nanoparticles of examples 1, 3 to 5 and comparative example 3 were left for two months, and F is a graph showing a change in PDI after the lipid nanoparticles of examples 1, 3 to 5 and comparative example 3 were left for two months.
FIG. 6 is a graph of the results (in 1 of Effect example 3) of the lipid nanoparticles of example 1 after transfection of HEK-293T cells after various gradient fold dilutions: wherein, A is a fluorescent effect graph of the lipid nanoparticle of the example 1 after being transfected into HEK-293T cells after being diluted by different gradient factors, and B is a bright field graph of the lipid nanoparticle of the example 1 after being transfected into HEK-293T cells after being diluted by different gradient factors; the scale bar is 50 μm.
FIG. 7 is a graph of the results of transfection of HEK-293T cells after various fold gradient dilutions of lipid nanoparticles of example 2: wherein, A is a fluorescent effect graph of the lipid nanoparticle of the example 2 after being transfected into HEK-293T cells after being diluted by different gradient factors, and B is a bright field graph of the lipid nanoparticle of the example 2 after being transfected into HEK-293T cells after being diluted by different gradient factors; the scale bar is 50 μm.
FIG. 8 is a flow chart of the lipid nanoparticle of example 1 after transfection of HEK-293T cells (in effect example 3, 2) after various gradient fold dilutions.
FIG. 9 is a flow chart of the lipid nanoparticles of example 2 after transfection of HEK-293T cells at different fold gradients.
FIG. 10 is a graph of results (in 2) of effect example 3) of transfected HEK-293T cells after lipid nanoparticles of examples 1, 3 were subjected to different gradient fold dilutions: wherein, A is a fluorescent effect graph of the lipid nanoparticle of the example 1 after being transfected into HEK-293T cells after being diluted by different gradient factors, and B is a bright field graph of the lipid nanoparticle of the example 1 after being transfected into HEK-293T cells after being diluted by different gradient factors; c is a graph of the fluorescence effect of the lipid nanoparticle of example 3 after transfection of HEK-293T cells after dilution with different gradient factors; d is a bright field plot of the lipid nanoparticles of example 3 after transfection of HEK-293T cells with different fold gradients; the scale bar is 50 μm.
FIG. 11 is a graph of the results of transfection of HEK-293T cells after various fold gradient dilutions of lipid nanoparticles of example 4, comparative example 1: wherein, A is a fluorescent effect graph of the lipid nanoparticle of the example 4 after being subjected to different gradient times of dilution and transfected into HEK-293T cells, and B is a bright field graph of the lipid nanoparticle of the example 4 after being subjected to different gradient times of dilution and transfected into HEK-293T cells; c is a fluorescent effect graph of the lipid nanoparticle of the comparative example 1 after being subjected to different gradient multiple dilutions and transfected into HEK-293T cells, and D is a bright field graph of the lipid nanoparticle of the comparative example 1 after being subjected to different gradient multiple dilutions and transfected into HEK-293T cells; the scale bar is 50 μm.
FIG. 12 is a graph of the results of transfection of HEK-293T cells after various fold gradient dilutions of lipid nanoparticles of example 5, comparative example 3: wherein, A is a fluorescent effect graph of the lipid nanoparticle of the example 5 after being subjected to different gradient times of dilution and transfected into HEK-293T cells, and B is a bright field graph of the lipid nanoparticle of the example 5 after being subjected to different gradient times of dilution and transfected into HEK-293T cells; c is a fluorescent effect graph of the lipid nanoparticle of comparative example 3 after being transfected into HEK-293T cells after being diluted by different gradient factors, and D is a bright field graph of the lipid nanoparticle of comparative example 3 after being transfected into HEK-293T cells after being diluted by different gradient factors; the scale bar is 50 μm.
FIG. 13 is a flow chart of the lipid nanoparticle of example 1 after transfection of HEK-293T cells (in effect example 3, 2) after various gradient fold dilutions.
FIG. 14 is a flow chart of the lipid nanoparticles of example 3 after transfection of HEK-293T cells at different fold gradients.
FIG. 15 is a flow chart of the lipid nanoparticles of example 4 after different fold gradient dilutions of transfected HEK-293T cells.
FIG. 16 is a flow chart of the lipid nanoparticles of example 5 after transfection of HEK-293T cells at different fold gradients.
FIG. 17 is a flow chart of transfected HEK-293T cells after differential fold dilutions of lipid nanoparticles of comparative example 1.
FIG. 18 is a flow chart of transfected HEK-293T cells after differential fold dilutions of lipid nanoparticles of comparative example 3.
Fig. 19 is a graph showing the results of toxicity analysis of lipid nanoparticles of comparative example 2 and examples 1 and 2: wherein a is a toxicity analysis result graph of the lipid nanoparticle of comparative example 2, B is a toxicity analysis result graph of the lipid nanoparticle of example 1, and C is a toxicity analysis result graph of the lipid nanoparticle of example 2.
FIG. 20 is a graph showing toxicity analysis results of lipid nanoparticles of examples 1, 3 to 5 and comparative example 1: wherein a is a toxicity analysis result graph of the lipid nanoparticle of example 1, B is a toxicity analysis result graph of the lipid nanoparticle of example 3, C is a toxicity analysis result graph of the lipid nanoparticle of example 4, D is a toxicity analysis result graph of the lipid nanoparticle of example 5, and E is a toxicity analysis result graph of the lipid nanoparticle of comparative example 1.
FIG. 21 is a graph showing the results of toxicity analysis of lipid nanoparticles of comparative example 3.
Fig. 22 is a graph of the results of transfection of H1299 cells after various gradient fold dilutions of lipid nanoparticles of examples 1, 2: wherein a is a graph of the fluorescent effect of the lipid nanoparticle of example 1 after being subjected to different gradient multiple dilutions and transfected into H1299 cells, B is a graph of the bright field of the lipid nanoparticle of example 1 after being subjected to different gradient multiple dilutions and transfected into H1299 cells, C is a graph of the fluorescent effect of the lipid nanoparticle of example 2 after being subjected to different gradient multiple dilutions and D is a graph of the bright field of the lipid nanoparticle of example 2 after being subjected to different gradient multiple dilutions and transfected into H1299 cells; the scale bar is 50 μm.
FIG. 23 is a flow chart of the lipid nanoparticle of example 1 after transfection of H1299 cells with various gradient fold dilutions.
FIG. 24 is a flow chart of the lipid nanoparticle of example 2 after transfection of H1299 cells with various gradient fold dilutions.
Reference numerals in fig. 1: 101. a three-way valve member; 102. a chamber member; 103. a plunger member; 104. a filter member.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents and the like used in this example are commercially available ones unless otherwise specified.
The following examples were purchased from Ai Weita (Shanghai) pharmaceutical technologies Inc., product number: o02006; 1-octyl nonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecyloxy) hexyl ] amino ] -octanoate (SM-102) was purchased from Ai Weita (Shanghai) pharmaceutical technologies Co., ltd., cat#: o02010; (2, 3-dioleoyl-propyl) -trimethylammonium-chloride salt; calcium acetate was purchased from Ai Weita (Shanghai) pharmaceutical technologies, inc., cat: 931280;1, 2-distearoyl-sn-glycero-3-phosphorylcholine (distearoyl phosphorylcholine, DSPC) was purchased from Ai Weita (Shanghai) pharmaceutical technologies, inc., cat: s01005; DMG-PEG2000 was purchased from Ai Weita (Shanghai) pharmaceutical technologies limited; cholesterol was purchased from Ai Weita (Shanghai) pharmaceutical technologies, inc., cat: o01001.
The schematic structural diagram of the preparation device of the lipid nanoparticle used in the following examples is shown in fig. 1, the preparation device comprises a three-way valve member 101, a filtering member 104 and two chamber members 102, the chamber members 102 are provided with a plunger member 103 capable of pushing and pulling, the plunger member 103 can enable a solution to enter and exit the chamber members 102, and the filtering member 104 and the chamber members 102 are respectively connected with the three-way valve member 101. Specifically, the three-way valve member 101 includes an a port and a b port, the a port is provided in two, the two chamber members 102 are connected to the two a ports, respectively, and the filter member 104 is connected to the b port.
It can be understood that the three-way valve member 101 can be controlled to realize on-off of different components in the preparation device, so as to change the flow direction of the medium. Specifically, the two ports a and b can be opened and closed, respectively, so that any two or three of the filter member 104, the two chamber members 102 communicate.
If port b is closed and both ports a are open, then both chamber members 102 communicate. If port b is open and one of ports a is open and the other is closed, the filter member 104 communicates with one of the chamber members 102. If both ports a and b are open, the three members 102, 104 are all in communication.
In some examples, the three-way valve member 101 includes a valve body and a valve core, where the port a and the port b are provided on a side wall of the valve body, respectively, and the opening and closing of the port a and the opening and closing of the port b can be switched by rotating the valve core. The structure of the valve body and the valve core is described in detail in the related art, and will not be described here.
The three-way valve member 101 is convenient and easy to obtain, the flow direction of the medium can be flexibly adjusted through the valve core, and further, the repeatable operation and production of the lipid compound are realized, when the control system for proportioning the fluid is used, the three-way valve can be used for simultaneously operating, so that the installation cost is reduced, the installation space is reduced, the manufacturing cost is low, the structure is light and easy to operate, and the three-way valve member is suitable for large-scale laboratory application. On the other hand, the medical three-way valve is adopted, the material is nontoxic and harmless, the valve body is made of PC material, the valve core is made of PE material, the chemical stability is good, the acid and alkali corrosion resistance is realized, and the valve core is insoluble in common solvents at normal temperature, so that the problem that solvent incompatibility can be generated in staggered herringbone equipment is solved.
As one embodiment, the chamber member 102 is detachably connected to the side wall of the port a, so that the chamber member 102 is detachable from the three-way valve member 101. It will be appreciated that after the chamber member 102 has been imbibed with solution, it is necessary to connect the chamber member 102 to the three-way valve member 101. And after the operation is completed, the chamber component 102 is conveniently removed, replaced or cleaned. On the other hand, the chamber member 102 is detachable, so that the chamber member 102 with different specifications can be conveniently replaced, and the device is suitable for operations with different sample amounts.
The chamber member 102 is detachably connected to the sidewall of the port a by a screw structure. Specifically, the port inner wall of the chamber member 102 is provided with internal threads and the outer wall of the port a side wall is provided with external threads. Alternatively, the outer wall of the port of the chamber member 102 is provided with external threads and the inner wall of the a-port sidewall is provided with internal threads.
Of course, as an alternative, it is also possible to design: in some examples, the port of the chamber member 102 is plugged with the port a and secured by a snap-fit structure. In some examples, the port of the chamber member 102 is plugged with the a-port and secured in an interference fit.
In some examples, the chamber member 102 is provided as a syringe.
As one embodiment, the filter member 104 is detachably connected to the side wall of the port b, so that the filter member 104 is detachable from the three-way valve member 101. It will be appreciated that removal of the filter element 104 after the operation is complete facilitates replacement or cleaning.
The filter member 104 is removably attached to the sidewall of the port b by a threaded configuration. Specifically, the port inner wall of the filter member 104 is provided with internal threads and the outer wall of the port side wall is provided with external threads. Alternatively, the outer wall of the port of the filter member 104 is provided with external threads and the inner wall of the b-port sidewall is provided with internal threads.
Of course, as an alternative, it is also possible to design: in some examples, the ports of the filter member 104 are plugged with the b-ports and secured by a snap-fit structure. In some examples, the port of the filter member 104 is plugged with the b-port and secured in an interference fit.
In some examples, the filter member 104 comprises a disposable aqueous phase needle filter head, which is widely used in the laboratory, is convenient and easy to obtain, does not need to change membranes and clean filters, saves complex and time-consuming preparation work, is mainly applied to sample prefiltering, clarification and particle removal, liquid and gas sterilization filtration, and is suitable for filtering small amounts of samples by HPLC and GC. Further, the filter membrane of the filter member 104 adopts a high-performance filter membrane, so that the product quality is stable and the reproducibility is good.
In some examples, the disposable water phase needle type filter head is a 0.22 μm disposable water phase needle type filter head with a screw, so that the disposable water phase needle type filter head can be better connected with various chamber components with screw, can also be directly and tightly connected with a valve port of a three-way valve, and can be directly used for collecting samples after sterilization and filtration.
The inlet of the filter member 104 is connected to the b port of the three-way valve member 101, and the outlet of the filter member 104 is connected to a container (e.g., a centrifuge tube) that collects the solution after the filtration treatment.
The following describes the details of the preparation process of the present invention in specific examples, and it should be noted that the following description is illustrative only and not limiting in any way.
The invention relates to a preparation method of lipid nano particles, which is implemented by a preparation device.
Specifically, the preparation method comprises the following workflow.
S1, mixing neutral lipid, amphiphilic lipid, cationic lipid and steroid lipid with ethanol to obtain an oil phase; mixing divalent salt and medicine to obtain water phase.
S2, one chamber component 102 sucks in the oil phase and the other chamber component 102 sucks in the water phase.
S3, closing the port b, respectively opening the two ports a, and pushing and pulling the plunger member 103 to mix the oil phase and the water phase in the two chamber members 102.
And S4, pushing the solution in one chamber component 102 to the other chamber component 102, wherein the opening a corresponding to the empty chamber component 102 is closed, and the other opening a is opened.
S5, opening the port b, pushing the solution out through the filtering component 104 and collecting the solution by using a centrifuge tube.
Further, in step S3, by pushing and pulling the plunger member 103 a plurality of times to mix the oil phase and the water phase in the two chamber members 102, the two phases are sufficiently mixed by striking back and forth in the two chamber members 102. In some examples, in step S3, the plunger member 103 is repeatedly pushed and pulled eighty times.
It will be appreciated that the three-way valve member 101 and the chamber member 102, which are required before the preparation process is performed, require preheating treatment. Specifically, the three-way valve member 101 and the chamber member 102 are preheated at 55-75 ℃ for 20-40 min; further preheating at 65deg.C for 30min.
Plasmid pEF-GFP in the following examples was purchased from the cloud organism, product number QP1560, of Shenzhen Baishun technology Co., ltd.
Example 1 preparation of lipid nanoparticles
A lipid nanoparticle comprising: distearylphosphocholine (DSPC), 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), thirty-seventeen carbon-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA), cholesterol, calcium acetate (CaAc) 2 ) And plasmid pEF-GFP; wherein DSPC, DMG-PEG2000, DLin-MC3-DMA, cholesterol, caAc 2 And pEF-GFP at a mass ratio of 1650:800:6500:3100:7900.59:334.53 (i.e., 1650. Mu.g, 800. Mu.g, 6500. Mu.g, 3100. Mu.g, 7900.59. Mu.g, 334.53. Mu.g, in that order).
The preparation method of the lipid nanoparticle comprises the following steps:
1) 1650 μg DSPC, 800 μg DMG-PEG2000, 3100 μg cholesterol, 6500 μg DLin-MC-DMA are dissolved in 100 μl absolute ethanol, and heated in water bath at 65deg.C until lipid phase is completely dissolved, to obtain oil phase;
2) 7900.59 μg CaAc 2 (equivalent to CaAc at a concentration of 1.5M and a volume of 33.3. Mu.L) 2 Solution) and 334.53. Mu.g of pEF-GFP (concentration: 886.528. Mu.g/mL, volume: 377.35. Mu.L) were mixed, and ultrapure water was added to a volume of 1mL to prepare an aqueous phase;
3) Preheating a three-way valve component and two cavity components in a preparation device of lipid nano particles for 30min at 65 ℃;
4) Sucking the oil phase in step 1) and the water phase in step 2) with the preheated two chamber components respectively (i.e. one chamber component sucks the oil phase in step 1) and the other chamber component sucks the water phase in step 2); to facilitate distinguishing between two chamber components: the chamber component filled with the oil phase is named as a chamber A, the chamber component filled with the water phase is named as a chamber B), and the three-way valve is adjusted to enable two ports a to be opened and two ports B to be closed;
5) The solution of the A chamber is completely injected into the B chamber through two ports a, and then the solution of the B chamber is completely injected into the A chamber through two ports a;
6) Repeating the step 5) forty times to uniformly mix the oil phase and the water phase, wherein all the mixed liquid of the oil phase and the water phase is in the chamber A;
7) Opening an opening a and an opening B connected with the chamber A, closing the opening a connected with the chamber B, passing the mixed solution through a filter component, and collecting the mixed solution by using a centrifuge tube to obtain the lipid nano particles.
Example 2 preparation of lipid nanoparticles
A lipid nanoparticle comprising: distearylphosphocholine (DSPC), 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), octadecan-9-yl 8- ((2-hydroxyethyl) (6-oxO-6- (undecyloxy) hexyl) amino) octanoate (SM-102), cholesterol, calcium acetate (CaAc) 2 ) And plasmid pEF-GFP; wherein DSPC, DMG-PEG2000, SM-102, cholesterol, caAc 2 And pEF-GFP at a mass ratio of 1650:800:6500:3100:7900.59:334.53 (i.e., 1650. Mu.g, 800. Mu.g, 6500. Mu.g, 3100. Mu.g, 7900.59. Mu.g, 334.53. Mu.g, in that order).
The preparation method of the lipid nanoparticle was the same as in example 1, except that DLin-MC3-DMA was replaced with SM-102.
Example 3 preparation of lipid nanoparticles
A lipid nanoparticle comprising: distearylphosphocholine (DSPC), 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), thirty-seventeen carbon-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA), cholesterol, magnesium acetate (MgAc) 2 ) And plasmid pEF-GFP; wherein DSPC, DMG-PEG2000, DLin-MC3-DMA, cholesterol, mgAc 2 And pEF-GFP at a mass ratio of 1650:800:6500:3100:7112.38:334.53 (i.e., 1650. Mu.g, 800. Mu.g, 6500. Mu.g, 3100. Mu.g, 7112.38. Mu.g, 334.53. Mu.g, in that order).
The preparation method of the lipid nanoparticle is the same as in example 1, except that CaAc is used 2 (mass 7900.59. Mu.g) replaced by MgAc 2 (mass 7112.38. Mu.g).
Example 4 preparation of lipid nanoparticles
A lipid nanoparticle comprising: distearylphosphine esterAcylcholine (DSPC), 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), heptadeca-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA), cholesterol, calcium gluconate (Ca (C) 6 H 11 O 7 ) 2 ) And plasmid pEF-GFP; wherein DSPC, DMG-PEG2000, DLin-MC3-DMA, cholesterol, ca (C) 6 H 11 O 7 ) 2 And pEF-GFP at a mass ratio of 1650:800:6500:3100:21496.98:334.53 (i.e., 1650. Mu.g, 800. Mu.g, 6500. Mu.g, 3100. Mu.g, 21496.98. Mu.g, 334.53. Mu.g, in that order).
The preparation method of the lipid nanoparticle is the same as in example 1, except that CaAc is used 2 (mass 7900.59. Mu.g) replaced with Ca (C) 6 H 11 O 7 ) 2 (mass 21496.98. Mu.g), ca (C) in step 2) 6 H 11 O 7 ) 2 Is 0.223M and has a volume of 223.991. Mu.L.
Example 5 preparation of lipid nanoparticles
A lipid nanoparticle comprising: distearylphosphocholine (DSPC), 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), thirty-seventeen carbon-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA), cholesterol, calcium lactate (C) 6 H 10 CaO 6 ) And plasmid pEF-GFP; wherein DSPC, DMG-PEG2000, DLin-MC3-DMA, cholesterol, C 6 H 10 CaO 6 And pEF-GFP at a mass ratio of 1650:800:6500:3100:10900:334.53 (i.e., 1650. Mu.g, 800. Mu.g, 6500. Mu.g, 3100. Mu.g, 10900. Mu.g, 334.53. Mu.g, in that order).
The preparation method of the lipid nanoparticle is the same as in example 1, except that CaAc is used 2 (mass 7900.59. Mu.g) is replaced by C 6 H 10 CaO 6 (mass 10900. Mu.g).
Comparative example 1 preparation of lipid nanoparticles
A lipid nanoparticle comprising: distearylphosphocholine (DSPC), 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), thirty-seventeen carbon-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA), cholesterol, sodium acetate (NaAc), and plasmid pEF-GFP; wherein, the mass ratio of DSPC, DMG-PEG2000, DLin-MC3-DMA, cholesterol, naAc and pEF-GFP is 1650:800:6500:3100:4097.40:334.53 (i.e., 1650. Mu.g, 800. Mu.g, 6500. Mu.g, 3100. Mu.g, 4097.40. Mu.g, 334.53. Mu.g, in that order).
The preparation method of the lipid nanoparticle is the same as in example 1, except that CaAc is used 2 (mass 7900.59. Mu.g) was replaced with NaAc (mass 4097.40. Mu.g).
Comparative example 2 preparation of lipid nanoparticles
A lipid nanoparticle comprising: distearylphosphocholine (DSPC), 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), cholesterol, calcium acetate (CaAc) 2 ) And plasmid pEF-GFP; wherein DSPC, DMG-PEG2000, cholesterol, caAc 2 And pEF-GFP at a mass ratio of 1650:800:3100:7900.59:334.53 (i.e., 1650. Mu.g, 800. Mu.g, 3100. Mu.g, 7900.59. Mu.g, 334.53. Mu.g, in that order).
The preparation method of the above lipid nanoparticle was the same as in example 1, except that the thirty-seven carbon-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA) was not included.
Comparative example 3 preparation of lipid nanoparticles
A lipid nanoparticle comprising: distearylphosphocholine (DSPC), 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), thirty-seventeen carbon-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA), cholesterol, calcium chloride (CaCl) 2 ) And plasmid pEF-GFP; wherein DSPC, DMG-PEG2000, DLin-MC3-DMA, cholesterol, caCl 2 And pEF-GFP at a mass ratio of 1650:800:6500:3100:4097.40:334.53 (i.e., 1650. Mu.g, 800. Mu.g, 6500. Mu.g, 3100. Mu.g, 4097.40. Mu.g, 334.53. Mu.g, in that order).
The preparation method of the lipid nanoparticle is the same as in example 1, except that CaAc is used 2 (mass 7900.59. Mu.g) replaced by CaCl 2 (mass 3695.6. Mu.g).
Effect example 1 average particle diameter of lipid nanoparticle, PDI and Zeta potential
1) Average particle size, PDI and Zeta potential of lipid nanoparticles containing different cationic lipids
The average particle size, PDI and Zeta potential of the lipid nanoparticles prepared in examples 1 to 3 were measured using a Malvern particle size potentiometer (serial No. MAL 1251109), and the relevant parameters were set in the ZS xrlor system:
(1) detecting particle size: cell was selected as DTS0012, using the corresponding dish, add 1:400 diluted samples to be detected are 1mL in total, material is selected as lipomes, dispersont is selected as Water, size is executed, balance time is selected as 30s, and the whole is repeatedly executed for 3 times, so that detection can be started to be executed;
(2) detecting potential: select Cell as DTS1070, add 1:400 diluted samples to be detected are 1mL in total, material is selected as lipomes, dispersont is selected as Water, zeta is executed, balance time is selected as 30s, and the whole is repeatedly executed for 3 times, so that detection can be started to be executed;
the results are shown in fig. 2 and 3: the lipid nanoparticles prepared in examples 1 and 2 were all milky white in color, the average particle size and Zeta potential of the lipid nanoparticles in example 1 were 236nm and 43.92mv, respectively, and the pdi was 0.1694; the lipid nanoparticle of example 2 had an average particle diameter and Zeta potential of 230nm and 62.73mv, respectively, and a pdi of 0.2426; it can be seen that using DLin-MC3-DMA as cationic lipid can result in a narrower particle size distribution of lipid nanoparticles (smaller PDI).
2) Average particle size, PDI and Zeta potential of lipid nanoparticles containing different ionic solutions
The average particle diameter, PDI and Zeta potential of the lipid nanoparticles prepared in examples 1 and 3 to 5 were measured by the same method as 1), and the results are shown in FIGS. 2 and 4: the lipid nanoparticle of example 1 had an average particle diameter and Zeta potential of 236nm and 43.92mv, respectively, and a pdi of 0.1694; the lipid nanoparticle of example 3 had an average particle diameter and Zeta potential of 246.1nm and 55.61mv, respectively, and a pdi of 0.1941; the lipid nanoparticle of example 4 had an average particle size and Zeta potential of 221.5nm and 43.45mV, respectively, and a PDI of 0.2109; the lipid nanoparticle of example 5 had an average particle size and Zeta potential of 222.7nm and 46.71mV, respectively, and a PDI of 0.08192.
Effect example 2 stability of lipid nanoparticles
1) Stability of lipid nanoparticles containing different cationic lipids
The lipid nanoparticles prepared in examples 1, 2 and comparative example 2 (corresponding to DLin-MC3-DMA, SM-102, DOTAP, DSPC, respectively) were stored at 4 ℃ for 2 months, the average particle diameter, PDI and Zeta potential before and after the storage of the lipid nanoparticles were detected, respectively (the method was the same as that of effect example 1), and the change values of the average particle diameter, PDI and Zeta potential (the values after the storage were subtracted from the values before the storage) were calculated, and each treatment was repeated at least twice, and the results are shown in fig. 5: the stability of the lipid nanoparticle can be improved by adding the cationic lipid, and the lipid nanoparticle prepared in the embodiment 1 and the embodiment 2 has better stability.
2) Stability of lipid nanoparticles containing different ionic solutions
The lipid nanoparticles prepared in examples 1, 3 to 5 (corresponding to calcium acetate, magnesium acetate, calcium gluconate, and calcium lactate, respectively) and comparative example 3 (calcium chloride) were stored at 4 ℃ for 2 months, the average particle diameter, PDI, and Zeta potential before and after the storage of the lipid nanoparticles were detected, respectively (the method was the same as that of effect example 1), and the change values of the average particle diameter, PDI, and Zeta potential (the values after the storage were subtracted from the values before the storage), and each treatment was repeated at least twice, and the results are shown in fig. 5: the lipid nanoparticles prepared in examples 1, 3-5 have better stability.
Therefore, the lipid nanoparticle provided by the application has long shelf life, convenient storage, high stability and stable two-month-uniform property when stored at 4 ℃, and solves the problems that the conventional transfection reagent (PEI, lipo 8000) needs to be prepared and used at present and has large batch-to-batch difference when used.
Effect example 3 transfection Effect of lipid nanoparticles
1) Transfection effect of lipid nanoparticles containing different cationic lipids
The lipid nanoparticles prepared in examples 1 and 2 were diluted with different gradient factors (1:50, 1:100, 1:200, 1:500, 1:1000) and transfected into HEK-293T cells as follows: appropriate amount of HEK-293T cells were seeded overnight in six well plates one day in advance, cells were waited for cell attachment, old medium was removed the next day, and 900. Mu.L of new DMEM high-sugar medium was added to each well, respectively. The lipid nanoparticles prepared in examples 1 and 2 were mixed with an appropriate amount of opti-MEM in an amount of 1. Mu.L, 2. Mu.L, 5. Mu.L, 10. Mu.L, and 20. Mu.L to form a mixed system of lipid nanoparticles and opti-MEM with a total volume of 100. Mu.L and different dilution factors (1:1000, 1:500, 1:200, 1:100, and 1:50), and the lipid nanoparticles were not added to the control group, and the whole 100. Mu.L was opti-MEM. The system can be directly added into the corresponding position of the six-hole plate without waiting for incubation, and after 24 hours, the system is respectively replaced by 1mL of fresh DMEM high-sugar culture medium, and the incubation is continued for 24 hours at 37 ℃. After 48 hours, the fluorescent appearance was photographed by a fluorescent microscope, and the exposure time was adjusted to 1000ms. After shooting, respectively digesting cells in the six-hole plate, re-suspending the cells by 500 mu L of PBS, fully dispersing the cells, detecting the duty ratio of positive fluorescent cells by using a FITC channel of a flow cytometer, sucking 30 mu L of cells each time, determining the range of detected cells according to a logarithmic scatter diagram, dividing a gate by taking a control group as 100% standard, and synchronizing the gate to the result of an experimental group to obtain the duty ratio of positive fluorescent cells after 48 hours of dilution transfection with different gradient multiples; the results are shown in FIGS. 6 to 9: the positive fluorescent cell ratios were in turn as different gradient multiples (1:50, 1:100, 1:200, 1:500, 1:1000) after 48h transfection of the lipid nanoparticle of example 1: 87.94%, 88.97%, 78.01%, 47.72%, 28.55%; the positive fluorescent cell ratios were in turn as different gradient multiples (1:50, 1:100, 1:200, 1:500, 1:1000) after 48h transfection of the lipid nanoparticle of example 2: 40.52%, 47.54%, 50.44%, 52.64%, 30.12%; therefore, the selection of the cationic lipid can influence the transfection effect of the lipid nanoparticle, and the SM-102 and DLin-MC3-DMA are adopted as the cationic lipid, so that the transfection efficiency of the lipid nanoparticle can be improved, namely the delivery performance of the lipid nanoparticle is improved.
The lipid nanoparticles prepared in examples 1 and 2 were diluted with different gradient factors (1:50, 1:100, 1:200, 1:500, 1:1000) and transfected into H1299 cells, and the specific operation steps were the same as those described above for HEK-293T cells; the results are shown in FIGS. 22 to 24: the positive fluorescent cell ratios were in turn as different gradient multiples (1:50, 1:100, 1:200, 1:500, 1:1000) after 48h transfection of the lipid nanoparticle of example 1: 53.42%, 63.56%, 54.42%, 32.28%, 26.98%; the positive fluorescent cell ratios were in turn as different gradient multiples (1:50, 1:100, 1:200, 1:500, 1:1000) after 48h transfection of the lipid nanoparticle of example 2: 57.34%, 73.28%, 79.36%, 77.84%, 72.82%; therefore, the selection of the cationic lipid can influence the transfection effect of the lipid nanoparticle, and the SM-102 and DLin-MC3-DMA are adopted as the cationic lipid, so that the transfection efficiency of the lipid nanoparticle can be improved, namely the delivery performance of the lipid nanoparticle is improved.
2) Stability of lipid nanoparticles containing different ionic solutions
The lipid nanoparticles prepared in examples 1, 3-5 and comparative examples 1 and 3 were subjected to dilution with different gradient factors (1:50, 1:100, 1:200, 1:500 and 1:1000) and then transfected into HEK-293T cells, and the specific operation steps were the same as those of the transfection of lipid nanoparticles containing different cationic lipids; the results are shown in FIGS. 10 to 18: the positive fluorescent cell ratios were in turn as different gradient multiples (1:50, 1:100, 1:200, 1:500, 1:1000) after 48h transfection of the lipid nanoparticle of example 1: 87.94%, 88.97%, 78.01%, 47.72%, 28.55%; the positive fluorescent cell ratios in terms of different gradient factors (1:50, 1:100, 1:200, 1:500, 1:1000) were sequentially: 55.52%, 67.16%, 68.70%, 53.62%, 28.36%; the positive fluorescent cell ratios in terms of different gradient factors (1:50, 1:100, 1:200, 1:500, 1:1000) were sequentially: 73.02%, 79.28%, 77.28%, 62.30%, 43.86%; the positive fluorescent cell ratios in terms of different gradient factors (1:50, 1:100, 1:200, 1:500, 1:1000) were sequentially: 38.22%, 32.60%, 26.95%, 21.58%, 13.39%; the positive fluorescent cell ratios of the lipid nanoparticle of comparative example 1 after 48h transfection were sequentially as follows according to different gradient factors (1:50, 1:100, 1:200, 1:500, 1:1000): 2.74%, 2.04%, 1.92%, 0.68%, 0.44%; the positive fluorescent cells were counted in different gradient multiples (1:50, 1:100) at a ratio of the lipid nanoparticle of comparative example 3 after 48h transfection: 0.81% and 0.52%; it can be seen that the choice of ionic solution affects the transfection effect of the lipid nanoparticle, and the transfection effect of the lipid nanoparticle using divalent salts (calcium acetate, magnesium acetate, calcium gluconate, calcium lactate, calcium chloride) as the ionic solution is significantly better than that of monovalent salt (sodium acetate), but as can be seen from comparative example 3, the transfection effect of inorganic divalent salt (calcium chloride) is not good, and in conclusion, the transfection effect of the lipid nanoparticle using divalent salt (such as calcium salt, magnesium salt) and especially organic divalent salt as the ionic solution is further improved.
Effect example 4 cytotoxicity assay of lipid nanoparticles
1) Cytotoxicity assay of lipid nanoparticles containing different cationic lipids
The lipid nanoparticles prepared in examples 1 and 2 and comparative example 2 were subjected to CCK8 experiments, and the lipid nanoparticles were specifically as follows:
(1) 3X 10 seed per well in 96 well plate 3 -5×10 3 The cells were plated in an incubator overnight (37 ℃,5% co) 2 )。
(2) Lipid nanoparticles prepared in examples 1 and 2 and comparative example 2 were added to the culture plates at different concentrations as shown in fig. 19.
(3) Cell viability was measured after incubation of the plates in the incubator for an appropriate 24 hours.
(4) Add 10. Mu.L of CCK-8 solution to each well (note that bubbles do not form in the wells, which can affect OD readings).
(5) Incubating the plates in the incubator for 1.5 hours.
(6) Measuring absorbance at 450nm with a multifunctional enzyme-labeled instrument. Cell viability (%) = [ a (dosing) -a (blank)/[ a (dosing) -a (blank) ×100, wherein a (dosing): absorbance of wells with cells, CCK-8 solution and drug solution; a (blank): absorbance of wells with medium and CCK-8 solution without cells; a (0 dosing): absorbance of wells with cells, CCK-8 solution without drug solution.
(7) Calculation of the concentration (IC) of the different examples at 50% inhibition of cell viability over the range of concentrations administered by statistical analysis of GrapfPad prism-7.00 50 )。
The results are shown in FIG. 19: the lipid nanoparticles obtained in example 1, example 2 (when used at a concentration of less than 30. Mu.L/mL, for example 10. Mu.L/mL) and comparative example 2 were not significantly cytotoxic.
2) Cytotoxicity analysis of lipid nanoparticles containing different ion solutions
The lipid nanoparticles prepared in examples 1, 3 to 5 and comparative examples 1 and 3 were subjected to CCK8 experiments, specifically as follows:
(1) 3X 10 seed per well in 96 well plate 3 -5×10 3 The cells were plated in an incubator overnight (37 ℃,5% co) 2 )。
(2) Lipid nanoparticles prepared in examples 1, 3 to 5 and comparative examples 1 and 3 were added to the culture plates at different concentrations as shown in FIGS. 20 and 21.
(3) Cell viability was measured after incubation of the plates in the incubator for an appropriate 24 hours.
(4) Add 10. Mu.L of CCK-8 solution to each well (note that bubbles do not form in the wells, which can affect OD readings).
(5) Incubating the plates in the incubator for 1.5 hours.
(6) Measuring absorbance at 450nm with a multifunctional enzyme-labeled instrument. Cell viability (%) = [ a (dosing) -a (blank)/[ a (dosing) -a (blank) ×100, wherein a (dosing): absorbance of wells with cells, CCK-8 solution and drug solution; a (blank): absorbance of wells with medium and CCK-8 solution without cells; a (0 dosing): absorbance of wells with cells, CCK-8 solution without drug solution.
(7) Calculation of the different instances in GrapfPad prism-7.00 statistical analysisConcentration within the concentration range administered at which cell viability was inhibited by 50% (IC 50 )。
The results are shown in fig. 20 and 21: the lipid nanoparticle obtained in example 1 had no significant cytotoxicity, whereas the lipid nanoparticle obtained in comparative example 1 had toxicity, and the semi-lethal concentration was 62.95. Mu.L/mL. The semi-lethal concentrations of examples 3 to 5 were 113.9. Mu.L/mL, 92.63. Mu.L/mL, and 154.6. Mu.L/mL, respectively, each of which was less toxic than comparative example 1.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (20)

1. A lipid carrier consisting of the following components: neutral lipids, amphiphilic lipids, cationic lipids, steroidal lipids and divalent salts;
the divalent salt is selected from: one or more of calcium salt and magnesium salt;
the calcium salt is selected from: one or two of calcium acetate and calcium gluconate;
the magnesium salt is selected from: magnesium acetate;
The cationic lipid is selected from: octadecane-9-yl 8- ((2-hydroxyethyl) (6-oxO-6- (undecyloxy) hexyl) amino) octanoate, and one or two of heptadeca-6,9,28,31-tetraen-19-yl esters of 4- (dimethylamino) butanoic acid;
the neutral lipid is 1, 2-distearoyl-sn-glycero-3-phosphorylcholine;
the amphiphilic lipid is DMG-PEG2000;
the steroid lipid is cholesterol;
the mass ratio of the neutral lipid, the amphiphilic lipid, the cationic lipid, the steroid lipid and the divalent salt is (1600-1700): 800: (6300-6700): (3000-3200): (7100 to 22000).
2. The lipid carrier according to claim 1, wherein:
the cationic lipid is heptadeca-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate.
3. A lipid nanoparticle comprising a drug and the lipid carrier of any one of claims 1-2;
the drug is a nucleic acid.
4. A lipid nanoparticle according to claim 3, wherein:
the mass ratio of the lipid carrier to the medicine is (15000-35000): 334.53.
5. the lipid nanoparticle of claim 4, wherein:
the mass ratio of the lipid carrier to the drug is (19162.38-33546.98): 334.53.
6. The lipid nanoparticle of claim 5, wherein:
the nucleic acid is selected from: one or more combinations of DNA and RNA; or (b)
The mass ratio of the lipid carrier to the drug is (19950.59-33546.98): 334.53.
7. the lipid nanoparticle of claim 6, wherein:
the nucleic acid is selected from one or more combinations of ssDNA, dsDNA, mRNA, lncRNA, siRNA, saRNA, shRNA, ASO, plasmid, circRNA, circDNA, miRNA, CRISPR-Cas, samna, and antagomir.
8. The lipid nanoparticle according to any one of claims 3 to 7, wherein: the medicament is a vaccine.
9. The lipid nanoparticle of claim 8, wherein: the vaccine is a therapeutic or prophylactic vaccine.
10. The lipid nanoparticle of claim 9, wherein: the vaccine is used for preventing and treating tumors, bacterial infections, viral infections, and/or fungal infections.
11. A method of preparing the lipid nanoparticle according to any one of claims 3 to 10, comprising the steps of:
mixing neutral lipid, amphiphilic lipid, cationic lipid and steroid lipid with ethanol to obtain oil phase;
Mixing divalent salt with the medicine to obtain water phase;
mixing the oil phase and the water phase to obtain the lipid nanoparticle.
12. The method of manufacturing according to claim 11, wherein:
the volume ratio of the oil phase to the water phase is 1: (5-15); or (b)
The oil phase and the water phase are mixed by a preparation device of lipid nano particles.
13. The method of manufacturing according to claim 12, wherein:
the preparation device comprises:
a three-way valve member including an a port and a b port, the a port being provided in two;
the two cavity members are provided with a push-pull plunger member, and the two cavity members are respectively connected with the two ports a;
a filter member connected to the port b;
wherein the two ports a and b can be opened and closed respectively so as to enable any two or three of the filtering component and the two cavity components to be communicated;
the specific method for mixing the oil phase and the water phase comprises the following steps:
one of the chamber components draws in an oil phase and the other chamber component draws in an aqueous phase;
closing the port b, respectively opening the two ports a, and pushing and pulling the plunger component to mix the oil phase and the water phase in the two cavity components;
Pushing the solution in one chamber component to the other chamber component, wherein the opening a corresponding to the empty chamber component is closed, and the other opening a is opened;
opening the port b to push the solution out through the filter member.
14. A pharmaceutical composition comprising any one of a 1) to a 2):
a1 A lipid carrier according to any one of claims 1-2;
a2 A lipid nanoparticle according to any one of claims 3 to 10.
15. The pharmaceutical composition according to claim 14, wherein:
the pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials.
16. The pharmaceutical composition according to claim 15, wherein:
the pharmaceutically acceptable auxiliary materials comprise at least one of a diluent, an adhesive, a wetting agent, a surfactant, a lubricant and a disintegrating agent; or (b)
The pharmaceutical composition is an inhalation preparation, an injection preparation or an oral preparation.
17. The pharmaceutical composition according to claim 15, wherein: the pharmaceutically acceptable auxiliary materials comprise excipients.
18. The pharmaceutical composition according to claim 16, wherein:
the inhalation preparation is an atomized inhalation or a dry powder inhalation; or (b)
The injection preparation is a liquid preparation; or (b)
The oral preparation is tablet, pill, powder, granule, capsule, solution, emulsion, suspension or syrup.
19. The pharmaceutical composition according to claim 16, wherein:
the oral preparation is a sustained release agent or drop.
20. Use of the lipid carrier of any one of claims 1-2, the lipid nanoparticle of any one of claims 3-10, and/or the method of preparation of any one of claims 11-13 in the preparation of a pharmaceutical composition.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2014057432A2 (en) * 2012-10-09 2014-04-17 Universita' Degli Studi Di Roma "La Sapienza" Multicomponent lipid nanoparticles and processes for the preparation thereof
CN209253666U (en) * 2018-04-18 2019-08-16 北京添医医学技术有限公司 It is a kind of can at any time sampling monitoring emulsified state and have Cryo Equipment trace antigen emulsifier
CN217449686U (en) * 2022-05-24 2022-09-20 杭州广科安德生物科技有限公司 Antigen emulsifying device

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014057432A2 (en) * 2012-10-09 2014-04-17 Universita' Degli Studi Di Roma "La Sapienza" Multicomponent lipid nanoparticles and processes for the preparation thereof
CN209253666U (en) * 2018-04-18 2019-08-16 北京添医医学技术有限公司 It is a kind of can at any time sampling monitoring emulsified state and have Cryo Equipment trace antigen emulsifier
CN217449686U (en) * 2022-05-24 2022-09-20 杭州广科安德生物科技有限公司 Antigen emulsifying device

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