WO2023109242A1 - 可离子化脂质化合物在核酸药物递送系统中的应用 - Google Patents

可离子化脂质化合物在核酸药物递送系统中的应用 Download PDF

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WO2023109242A1
WO2023109242A1 PCT/CN2022/121378 CN2022121378W WO2023109242A1 WO 2023109242 A1 WO2023109242 A1 WO 2023109242A1 CN 2022121378 W CN2022121378 W CN 2022121378W WO 2023109242 A1 WO2023109242 A1 WO 2023109242A1
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nucleic acid
delivery system
drug delivery
acid drug
ionizable lipid
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French (fr)
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崔艳芳
吉帅洁
张宝倩
刘滨磊
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武汉滨会生物科技股份有限公司
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    • 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
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the disclosure belongs to the technical field of nucleic acid drug delivery, and in particular relates to the application of an ionizable lipid compound in a nucleic acid drug delivery system.
  • the active molecules of nucleic acid drugs include messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide (ASO) or plasmid DNA (pDNA), which have great application potential in vaccines and gene therapy in vitro or in vivo .
  • mRNA messenger RNA
  • siRNA small interfering RNA
  • ASO antisense oligonucleotide
  • pDNA plasmid DNA
  • the purpose of the present disclosure is to provide an application of an ionizable lipid compound in a nucleic acid drug delivery system, which can efficiently deliver nucleic acid drug molecules into the body and achieve efficient expression of nucleic acid, while in the liver
  • the nucleic acid expression level is low, which can reduce toxicity.
  • An embodiment of the present disclosure provides an application of an ionizable lipid compound in a nucleic acid drug delivery system.
  • the nucleic acid drug delivery system is a nucleic acid drug delivery system capable of delivering nucleic acid drug molecules to the spleen and/or lung.
  • the ionizable lipid compound is one or more of the compounds shown in general formula (I), general formula (II), and general formula (III):
  • R 1 is hydrogen, methyl, ethyl or isopropyl.
  • n is an integer between 1 and 10, for example, m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • n is an integer between 1 and 3, for example n is 1, 2 or 3.
  • f is an integer between 1 and 5, for example f is 1, 2, 3, 4 or 5.
  • x is an integer between 1 and 8, for example x is 1, 2, 3, 4, 5, 6, 7 or 8.
  • y is an integer between 1 and 9, for example, y is 1, 2, 3, 4, 5, 6, 7, 8 or 9.
  • R2 and R3 are independently hydrogen, methyl, ethyl or isopropyl.
  • p is an integer between 1 and 5, for example, p is 1, 2, 3, 4 or 5.
  • q is an integer between 1 and 3, for example, q is 1, 2 or 3.
  • said R 1 is hydrogen
  • one of said R2 and said R3 is hydrogen, and the other is methyl, ethyl or isopropyl.
  • one of said R2 and said R3 is hydrogen, and the other is methyl.
  • the m is an integer between 3-8; in other embodiments, m is an integer between 4-6.
  • f is an integer between 1 and 4; in other embodiments, f is 2 or 3.
  • the x is an integer between 2-5; in other embodiments, x is an integer between 2-4.
  • y is an integer between 3 and 9; in other embodiments, y is an integer between 5 and 9.
  • R 1 is hydrogen, m is an integer between 4 and 6, n is 2, and f is an integer between 1 and 3.
  • x is 3, and y is an integer between 6-9.
  • R 2 is methyl
  • R 3 is hydrogen
  • p is 5
  • q is an integer between 1 and 3.
  • the ionizable lipid compound is one or more of the compounds shown in the following structural formula:
  • the nucleic acid drug molecule is one or more of mRNA, siRNA, ASO or pDNA (plasmid DNA).
  • the mass ratio of the nucleic acid drug molecule to the nucleic acid drug delivery system is 1:(5-50); in some embodiments, it is 1:(5-40); in other embodiments In an example, it is 1:(5-30); in other embodiments, it is 1:(5-20).
  • the delivery system and the delivered nucleic acid drug form nano-lipid particles, and the average size of the nano-lipid particles is 50nm-200nm.
  • the polydispersity index of the lipid nanoparticles is ⁇ 0.4.
  • the average size of the nano-lipid particles is 50nm-150nm.
  • the polydispersity index of the lipid nanoparticles is ⁇ 0.3.
  • the ionizable lipid compound can optionally be modified with a targeting substance, and the targeting substance is one or more of folic acid, single-chain antibody or targeting polypeptide.
  • the nucleic acid drug delivery system further includes auxiliary molecules selectively modified with targeting substances, and the molar ratio of the ionizable lipid compound and the auxiliary molecules is ( 0.1-1): (0.1-1); in some embodiments (0.5-1): (0.5-1).
  • the auxiliary molecules include auxiliary lipids or lipid molecules commonly used in the art.
  • the auxiliary molecules include but are not limited to cholesterol, calcipotriol, stigmasterol, ⁇ -sitosterol, betulin, lupeol, ursolic acid, oleanolic acid, dioleic acid Acylphosphatidylcholine, Distearoylphosphatidylcholine, 1-Stearoyl-2-oleoyl lecithin, Dioleoylphosphatidylethanolamine, (1,2-Dioleoxypropyl)trimethyl Ammonium Chloride, Didecyl Dimethyl Ammonium Bromide, 1,2-Dimyristoyl-sn-Glycero-3-Ethylphosphocholine, Dipalmitoyl Phosphatidylethanolamine-Methoxypolyethylene Glycol 5000, one or more of distearoylphosphatidylethanolamine-polyethylene glycol 2000.
  • the targeting substance is one or more of folic acid, single-chain antibody or targeting polypeptide.
  • the Mal-PEG2000-DSPE and the polypeptide can be dissolved in ultrapure water, stirred and reacted for 48 hours, and concentrated by dialysis to obtain the polypeptide-PEG2000-DSPE, so as to obtain the targeting auxiliary molecule.
  • ionizable lipid molecules cholesterol: DOPE: polypeptide-PEG2000-DSPE are dissolved in absolute ethanol, and mixed with DNA or RNA to form polypeptide-modified liposomes.
  • the nucleic acid drug delivery system is an injection.
  • the nucleic acid drug delivery system further includes additives, and the additives include stabilizers and/or diluents.
  • the added amount of the additive is 1%-20% of the total mass of the injection.
  • the additives may be commonly used additives in this field.
  • the stabilizer includes but not limited to sucrose or trehalose.
  • the diluent includes but is not limited to buffers commonly used in the art, including but not limited to one or more of phosphate buffer, acetate buffer, tris hydrochloride buffer kind.
  • the nucleic acid drug delivery system is administered through local muscle, subcutaneous, endothelial, intratumoral injection or perfusion, or administered through intravenous injection.
  • the nucleic acid drug delivery system of the embodiments of the present disclosure can deliver nucleic acid drug molecules such as mRNA, siRNA, or pDNA through local muscle, subcutaneous, endothelial, intratumoral, and perfusion methods, and systemic drug delivery methods such as intravenous injection. Delivered into the body, it can even target the spleen/lung, and effectively express therapeutic protein drugs or antigens in cells in the body to prevent or treat diseases.
  • the nucleic acid drug delivery system of the embodiments of the present disclosure can significantly reduce the hepatic accumulation of liposomes. By selecting an appropriate administration method, nucleic acids can be efficiently delivered to the spleen or lungs and effectively translated into target molecules. Therefore, the implementation of the present disclosure
  • the potential of the example nucleic acid drug delivery system is suitable for delivering nucleic acid drugs into the body to function as prophylactic or therapeutic vaccines.
  • the present disclosure has the following advantages:
  • the nucleic acid drug delivery system of the embodiments of the present disclosure can efficiently deliver nucleic acid drug molecules to the spleen and/or lungs and effectively translate them into target molecules, and at the same time reduce the accumulation of side effects of liposomes in the liver, which is beneficial to the development and application of nucleic acid drugs is of great significance.
  • Fig. 1 is the hydrogen spectrogram of compound 1-1
  • Fig. 2 is the hydrogen spectrogram of 2-(bis(2-aminoethyl)amino)ethan-1-alcohol);
  • Fig. 3 is the hydrogen spectrogram of compound 1
  • Fig. 4 is the mass spectrogram of compound 1;
  • Fig. 5 is the hydrogen spectrogram of compound 2-1;
  • Fig. 6 is the hydrogen spectrogram of compound 2
  • Fig. 7 is the mass spectrogram of compound 2;
  • Fig. 8 is the hydrogen spectrogram of compound 3-1
  • Fig. 9 is the hydrogen spectrogram of 1,3-diamino-2-propanol
  • Figure 10 is the hydrogen spectrogram of compound 3
  • Fig. 11 is the mass spectrogram of compound 3.
  • Fig. 12 is the particle size distribution figure of Lipid-1, Lipid-2 and Lipid-3 in embodiment 4;
  • Figure 13 shows the fluorescence expression at different time points after intramuscular injection of Lipid-1 in mice
  • Figure 14 is the fluorescence expression of each organ in mice after intravenous injection and intramuscular injection of Lipid-1 6h;
  • Figure 15 shows the fluorescence expression at different time points after intramuscular injection of Lipid-2 in mice
  • Figure 16 shows the fluorescence expression of each organ in mice after intravenous injection and intramuscular injection of Lipid-2 for 6 hours;
  • Figure 17 shows the fluorescence expression of mice at different time points after intramuscular injection of Lipid-3
  • Figure 18 shows the fluorescence expression of each organ in mice after intravenous injection and intramuscular injection of Lipid-3 for 6 hours;
  • Figure 19 shows the in vivo fluorescence expression of mice after intravenous injection of Lipid-1, Lipid-2, and Lipid-3 for 6 hours, compared with SM-102.
  • the inventors conducted a lot of research on the main lipid compounds of the nucleic acid drug delivery system, and finally invented a new A nucleic acid drug delivery system, which can deliver nucleic acid drug molecules to specific organs or tissues and can achieve high-efficiency nucleic acid expression.
  • the expression level of the target nucleic acid drug molecule is similar or higher, and the degree of liver accumulation is significantly reduced.
  • the drug delivery system can deliver nucleic acid drug molecules to the spleen and/or lung, and the accumulation in the liver is low, and the drug delivery system includes an ionizable lipid compound, so
  • the ionizable lipid compound is one or more of the compounds shown in general formula (I), general formula (II), and general formula (III):
  • R1 is hydrogen, methyl, ethyl or isopropyl;
  • m is an integer between 1 and 10;
  • n is between 1 and 3 f is an integer between 1 and 5;
  • x is an integer between 1 and 8;
  • y is an integer between 1 and 9;
  • R 2 and R 3 are independently hydrogen, methyl, ethyl or Isopropyl;
  • p is an integer between 1 and 5;
  • q is an integer between 1 and 3.
  • the nucleic acid drug delivery system is nano-lipid particles.
  • nucleic acid drug delivery system of the embodiments of the present disclosure is that it can selectively target the spleen and/or lung even if no targeting group is attached, and there is only a small amount or only negligible distribution in the liver, thereby having a low of liver toxicity. This is the most notable feature of the nucleic acid drug delivery system in the embodiments of the present disclosure compared with most existing nucleic acid drug delivery systems. An advantage over the property of being degraded by nucleases.
  • Lipid-1 only refers to the use of Compound 1 prepared in Example 1 as a nano-lipid particle formed by an ionizable lipid compound and other components
  • Lipid-2 only refers to Compound 2 prepared in Example 2 As a nano-lipid particle formed by an ionizable lipid compound and other components
  • Lipid-3 only refers to the use of compound 3 prepared in Example 3 as a nano-lipid particle formed by an ionizable lipid compound and other components.
  • Encapsulated mRNA is not limited.
  • SM102 was used as a control, and the existing liposome SM-102 was used to encapsulate the nano-lipid particles formed by mRNA.
  • Lipo2000 was used as a control, and the existing liposome Lipo2000 was used to encapsulate the nano-lipid particles formed by the plasmid with the green fluorescent GPF tag.
  • Embodiment 4 the preparation of LNP-mRNA nano-lipid particle, the mensuration of particle diameter and electric potential:
  • the mRNA was dissolved in 25 mM sodium acetate buffer solution with a pH of about 5.2 to prepare a nucleic acid preparation with a final concentration of about 0.1 mg/mL.
  • the above prepared liposome raw material solution and nucleic acid preparation are prepared at a two-phase volume ratio of about 4:1, and the total rate of the two-phase solution is 12mL/min, through the Nano Assemblr microfluidic system, or by vortexing Quickly mix the two-phase solution to form uniform and stable nano-liposome particles, and then quickly change the environment of the nano-liposome particles from pH 5.2 to 7.0-7.4.
  • the specific operation is to dilute it to 20 times the volume with PBS buffer solution with pH 7.2 or sodium acetate buffer solution with pH 7.4, and then concentrate it with a 10KD ultrafiltration tube.
  • the speed of the centrifuge should not exceed the speed of the ultrafiltration tube.
  • Embodiment 5 In vivo transfection experiments of nano lipid particles in animals:
  • Adopt nano lipid particle intramuscular injection mode prepare nano lipid particle according to the preparation method of embodiment 4, wherein mRNA is the mRNA expressing Luciferase fluorescent protein, and the consumption of mRNA is 60 ⁇ g, ionizable liposome compound, DSPC, DMG-
  • the total amount of PEG2000 and cholesterol is 600 ⁇ g, use 200 ⁇ L of neutral PBS buffer to quickly change the liposome environment, and quickly inject it into the hindlimb inner muscle of 6-8 week female Babl/c mice, control the injection of 30 ⁇ g in the left and right hindlimbs mRNA, or by intravenous injection into 6-8 week female Babl/c mice, control intravenous injection of 50 ⁇ g mRNA.
  • the fluorescence intensity in the figure is closely related to the delivery efficiency of liposomes, and the stronger the fluorescence, the higher the delivery efficiency of liposomes.
  • Figure 14 shows the fluorescence expression of various organs in mice injected intravenously and intramuscularly with Lipid-1 for 6 hours.
  • IV intravenous injection
  • IM intramuscular injection
  • Figure 16 shows the fluorescence expression of various organs in mice after intravenous injection of Lipid-2 for 6 hours. The results showed that after intravenous injection (IV) of Lipid-2 liposomes, the fluorescence expression levels in each organ of the mouse were about 92% in the liver and 8% in the spleen.
  • mice containing Lipid-1, Lipid-2, Lipid-3 and SM102 were injected into the mice through the tail vein respectively. After 6 hours, obvious fluorescence distribution appeared in each mouse body, as shown in Figure 19. From the fluorescence distribution, it can be seen that The fluorescence of SM-102 and lipid-2 liposomes is mainly distributed in the liver, and the fluorescence of Lipid-1 and Lipid-3 liposomes is mainly distributed in the spleen, which is consistent with the fluorescence distribution of dissected organs of mice.
  • Lipid-1 and Lipid-3 liposomes have obvious spleen-targeting effects, are suitable for local intramuscular injection or intravenous systemic administration, and have low liver toxicity, while Lipid-2 is more suitable for intramuscular injection.

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Abstract

提供一种可离子化脂质化合物在核酸药物递送系统中的应用和药物递送系统，其中所述的核酸药物递送系统为能够将核酸药物分子递送至脾脏和/或肺脏的核酸药物递送系统。

Description

可离子化脂质化合物在核酸药物递送系统中的应用
相关申请的交叉引用
本申请基于申请号为202111534587.0、申请日为2021年12月15日的中国专利申请提出，并要求该中国专利申请的优先权，该中国专利申请的全部内容在此引入本申请作为参考。
技术领域
本公开属于核酸药物递送技术领域，具体涉及一种可离子化脂质化合物在核酸药物递送系统中的应用。
背景技术
核酸药物的活性分子包括信使RNA(mRNA)、小干扰RNA(siRNA)、反义寡核苷酸(ASO)或质粒DNA(pDNA)，在疫苗以及体外或体内的基因治疗中具有很大应用潜力。如何将核酸药物分子成功递送至体内细胞并高效表达，实现预防或治疗目的是目前急需的技术之一。多数现有核酸药物载体存在进入体内之后容易累积在肝脏部位，增加肝脏的代谢负担，引起明显的毒副作用的问题。又或存在由于递送系统的递送效率不高易于导致用药剂量增大引起毒副作用的现象。因此，开发毒副作用小且核酸递送效率高的递送系统具有重要的现实意义。
发明内容
本公开的目的是提供一种可离子化脂质化合物在核酸药物递送系统中的应用，所述的核酸药物递送系统能够将核酸药物分子高效递送至体内并能够实现核酸高效表达，同时在肝脏中核酸表达量较低，从而能够降低毒性。
本公开实施例提供了一种可离子化脂质化合物在核酸药物递送系统中的应用，所述的核酸药物递送系统为能够将核酸药物分子递送至脾脏和/或肺脏的核酸药物递送系统，所述的可离子化脂质化合物为通式(Ⅰ)、通式(Ⅱ)、通式(Ⅲ)所示的化合物中的一种或多种：
Figure PCTCN2022121378-appb-000001
Figure PCTCN2022121378-appb-000002
其中，
R为-OC(＝O)-或-C(＝O)O-。
本公开实施例中，R的结构以其所在链的靠近N的一端向另一端的方向进行限定的，即当R为-OC(＝O)-，靠近N的是酯基中的O，当R为-C(＝O)O-时，靠近N的是酯基中的C。
R 1为氢、甲基、乙基或异丙基。
m为1～10之间的整数，例如m为1、2、3、4、5、6、7、8、9或10。
n为1～3之间的整数，例如n为1、2或3。
f为1～5之间的整数，例如f为1、2、3、4或5。
x为1～8之间的整数，例如x为1、2、3、4、5、6、7或8。
y为1～9之间的整数，例如y为1、2、3、4、5、6、7、8或9。
R 2和R 3独立地为氢、甲基、乙基或异丙基。
p为1～5之间的整数，例如p为1、2、3、4或5。
q为1～3之间的整数，例如q为1、2或3。
在一些实施例中，所述的R为-C(＝O)O-。
在一些实施例中，所述的R 1为氢。
在一些实施例中，所述的R 2和所述的R 3中一个为氢，另一个为甲基、乙基或异丙基。
在一些实施例中，所述的R 2和所述的R 3中一个为氢，另一个为甲基。
在一些实施例中，所述的m为3～8之间的整数；在另一些实施例中m为4～6之间的整数。
在一些实施例中，所述的f为1～4之间的整数；在另一些实施例中f为2或3。
在一些实施例中，所述的x为2～5之间的整数；在另一些实施例中x为2～4之间的整数。
在一些实施例中，所述的y为3～9之间的整数；在另一些实施例中y为5～9之间的整数。
在一些实施例中，所述的R为-C(＝O)O-，所述的R 1为氢，所述的n为2，所述的R 2和所述的R 3中一个为氢，另一个为甲基，所述的p为5。
根据一些实施例，通式(I)中，R 1为氢，m为4～6之间的整数，n为2，f为1～3之间的整数。
根据一些实施例，通式(Ⅱ)中，x为3，y为6～9之间的整数。
根据一些实施例，通式(Ⅲ)中，R 2为甲基，R 3为氢，p为5，q为1～3之间的整数。
根据一些具体实施方式，所述的可离子化脂质化合物为如下结构式所示化合物中的一种或多种：
Figure PCTCN2022121378-appb-000003
在一些实施例中，所述的核酸药物分子为mRNA、siRNA、ASO或pDNA(质粒DNA)中的一种或多种。
在一些实施例中，所述的核酸药物分子和所述的核酸药物递送系统的质量比为1:(5～50)；在一些实施例中为1:(5～40)；在另一些实施例中为1:(5～30)；在另一些实施例中为1:(5～20)。
在一些实施例中，所述的递送系统与所递送的核酸药物形成纳米脂质颗粒，所述的纳米脂质颗粒的平均尺寸为50nm～200nm。
在一些实施例中，所述的纳米脂质颗粒的多分散指数≤0.4。
在一些实施例中，所述的纳米脂质颗粒的平均尺寸为50nm～150nm。
在一些实施例中，所述的纳米脂质颗粒的多分散指数≤0.3。
在一些实施例中，所述的可离子化脂质化合物可选择性地带有靶向物质修饰，所述的靶向物质为叶酸、单链抗体或靶向多肽中的一种或多种。
在一些实施例中，所述的核酸药物递送系统还包括选择性地带有靶向物质修饰的辅助性分子，所述的可离子化脂质化合物和所述的辅助性分子的投料摩尔比为(0.1～1)：(0.1～1)；在一些实施例中为(0.5～1)：(0.5～1)。
所述的辅助性分子包括本领域中常用的辅助脂质或类脂分子。
在一些实施例中，所述的辅助性分子包括但不限于胆固醇、卡泊三醇、豆甾醇、β-谷甾醇、白桦脂醇、羽扇豆醇、熊果酸、齐墩果酸、二油酰基磷脂酰胆碱、二硬脂酰磷脂酰胆碱、1-硬脂酰基-2-油酰基卵磷脂、二油酰磷脂酰乙醇胺、(1,2-二油氧基丙基)三甲基氯化铵、双十烷基二甲基溴化铵、1,2-二肉豆蔻酰基-sn-甘油-3-乙基磷酸胆碱、二棕榈酰磷脂酰乙醇胺-甲氧基聚乙二醇5000、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000中的一种或多种。
在一些实施例中，所述的靶向物质为叶酸、单链抗体或靶向多肽中的一种或多种。
本公开实施例中，可使用本领域中常规的方法进行靶向物质修饰可离子化脂质化合物或辅助性分子。
根据一些实施方式，可将Mal-PEG2000-DSPE与多肽溶于超纯水中，搅拌反应48h，透析浓缩得到多肽-PEG2000-DSPE，即可得到具有靶向性的辅助性分子。
进一步地，将可电离脂质分子：胆固醇：DOPE：多肽-PEG2000-DSPE溶解于无水乙醇中，与DNA或RNA混合形成多肽修饰的脂质体。
在一些实施例中，所述的核酸药物递送系统为注射剂。
在一些实施例中，所述的核酸药物递送系统还包括添加剂，所述的添加剂为包括稳定剂和/或稀释剂。
在一些实施例中，所述的添加剂的添加量为所述的注射剂的总质量的1％～20％。
所述的添加剂可以是本领域中常用的添加剂。
在一些实施例中，所述的稳定剂包括但不限于蔗糖或海藻糖。
在一些实施例中，所述的稀释剂包括但不限于本领域中常用的缓冲液，包括但不限于磷酸盐缓冲液、醋酸盐缓冲液、tris盐酸盐缓冲液中的一种或多种。
在一些实施例中，所述的核酸药物递送系统通过局部肌肉、皮下、内皮、瘤内注射或灌注方式给药，或通过静脉注射方式给药。
本公开实施例的核酸药物递送系统可通过局部肌肉、皮下、内皮、瘤内以及灌注等多种给药方式，还可通过静脉注射等全身给药方式，将mRNA、siRNA或pDNA等核酸药物分子递送到体内，甚至可以靶向脾脏/肺脏，在体内细胞中有效表达治疗性蛋白药物或抗原，起到预防或治疗疾病的作用。本公开实施例的核酸药物递送系统可明显降低脂质体的肝脏累积作用，通过选择适宜的给药方式，可以高效地将核酸送至脾脏或肺脏中并有效翻译成目标分子，因此本公开实施例的核酸药物递送系统的潜力在于适合于递送核酸药物进入体内行使预防性或治疗性疫苗的功能。
本公开与相关技术相比具有如下优势：
本公开实施例的核酸药物递送系统，可以高效地将核酸药物分子递送至脾脏和/或肺脏中并有效翻译成目标分子，同时可降低脂质体在肝脏累积副作用，对核酸药物的发展和应用具有重要的意义。
附图说明
图1为化合物1-1的氢谱图；
图2为2-(双(2-氨基乙基)氨基)乙-1-醇)的氢谱图；
图3为化合物1的氢谱图；
图4为化合物1的质谱图；
图5为化合物2-1的氢谱图；
图6为化合物2的氢谱图；
图7为化合物2的质谱图；
图8为化合物3-1的氢谱图；
图9为1,3-二氨基-2-丙醇的氢谱图；
图10为化合物3的氢谱图；
图11为化合物3的质谱图；
图12为实施例4中的Lipid-1、Lipid-2及Lipid-3的粒径分布图；
图13为小鼠肌肉注射Lipid-1后不同时间点的荧光表达情况；
图14为小鼠通过静脉注射和肌肉注射Lipid-1 6h后各器官的荧光表达情况；
图15为小鼠肌肉注射Lipid-2后不同时间点的荧光表达情况；
图16为小鼠通过静脉注射和肌肉注射Lipid-2 6h后各器官的荧光表达情况；
图17为小鼠肌肉注射Lipid-3后不同时间点的荧光表达情况；
图18为小鼠通过静脉注射和肌肉注射Lipid-3 6h后各器官的荧光表达情况；
图19为小鼠通过静脉注射Lipid-1、Lipid-2、Lipid-3 6h后的体内荧光表达情况与SM-102进行对照。
具体实施方式
下面结合实施例对本公开作进一步描述。但本公开并不限于以下实施例。实施例中采用的实施条件可以根据具体使用的不同要求做进一步调整，未注明的实施条件为本行业中的常规条件。本公开各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
为了能够将核酸药物分子有效包装及递送外源核酸药物靶向进入体内特定的器官、组织并高效表达，发明人对核酸药物递送系统的主要脂质化合物进行了大量的研究，最终发明一种新型核酸药物递送系统，其能够将核酸药物分子递送至特定器官或组织并能够实现核酸高 效表达。与该领域内常用的MC3系统相比，目标核酸药物分子的表达水平相近或更高，同时肝脏累积程度明显降低。
根据本公开实施例，所述的药物递送系统能够将核酸药物分子递送至脾脏和/或肺脏，且在肝脏中的积累量较低，所述的药物递送系统包括可离子化脂质化合物，所述的可离子化脂质化合物为通式(Ⅰ)、通式(Ⅱ)、通式(Ⅲ)所示的化合物中的一种或多种：
Figure PCTCN2022121378-appb-000004
其中，
R为-OC(＝O)-或-C(＝O)O-；R 1为氢、甲基、乙基或异丙基；m为1～10之间的整数；n为1～3之间的整数；f为1～5之间的整数；x为1～8之间的整数；y为1～9之间的整数；R 2和R 3独立地为氢、甲基、乙基或异丙基；p为1～5之间的整数；q为1～3之间的整数。
本公开实施例中，所述的核酸药物递送系统为纳米脂质颗粒。
体内实验显示，通过局部肌肉、皮下、内皮、瘤内以及灌注等多种给药方式，或通过全身给药，都可以将mRNA、siRNA或pDNA核酸分子大部分递送到脾脏或肺脏，而其在肝脏的分布比例明显降低。本公开实施例的核酸药物递送系统的优势在于，即使不连接靶向基团也可以选择性靶向脾脏和/或肺脏，而在肝脏中只有少量分布或只有可忽略的分布，从而具有较低的肝脏毒性。这是本公开实施例的核酸药物递送系统相比现有多数核酸药物递送系统的最显著的特点，是在其具备一般递送载体的可避免所载核酸药物分子(尤其是mRNA和siRNA)在体内被核酸酶降解这一属性之外的优势属性。
下面根据一些具体实施例，进一步说明本公开的技术方案和有益效果。
下述实施例中的实验方法，如无特殊说明，均为常规方法；所用的实验材料，如无特殊说明，均为常规生化试剂厂商购买得到的。
以下实施例中，Lipid-1仅是指使用实施例1制备的化合物1作为可电离脂质化合物与其他组分形成的纳米脂质颗粒，Lipid-2仅是指使用实施例2制备的化合物2作为可电离脂质化合物与其他组分形成的纳米脂质颗粒，Lipid-3仅是指使用实施例3制备的化合物3作为可电离脂质化合物与其他组分形成的纳米脂质颗粒，对其包封的mRNA没有限制。
SM102作为对照，使用现有脂质体SM-102包封mRNA形成的纳米脂质颗粒。
Lipo2000作为对照，使用现有脂质体Lipo2000包封带有绿色荧光GPF标签的质粒形成的纳米脂质颗粒。
实施例1
化合物1的合成路线：
Figure PCTCN2022121378-appb-000005
步骤1：化合物1-1的合成：
将亚麻醇(0.267g，1mmol)和三乙胺(0.133g，1.3mmol)加入反应瓶中冰水浴，加入二氯甲烷(6mL)，将丙烯酰氯(0.11g，1.2mmol)溶于二氯甲烷(2.2mL)，缓慢滴加入反应瓶中，反应持续10分钟，该反应保持在10℃以下，最后冰浴移除，反应液在室温条件下反应2小时。用饱和食盐水洗涤，得到粗产物，将粗产品通过色谱法(硅胶柱，洗脱剂为含有0.5％EA(体积百分比)的石油醚)纯化，并将纯产物蒸发，得到浅黄色油状化合物1-1(2-烯丙酸(9Z，12Z)- 十八碳二烯酯)(0.173g，收率：50％)化合物1-1的氢谱见图1。
1H NMR(400MHz,CDCl 3)δ:6.41(dd,J＝17.3,1.5Hz,1H),6.13(dd,J＝17.3,10.4Hz,1H),5.82(dd,J＝10.4,1.5Hz,1H),5.47-5.26(m,4H),4.16(t,J＝6.7Hz,2H),2.78(t,J＝6.5Hz,2H),2.06(dd,J＝13.6,6.7Hz,4H),1.75-1.60(m,2H),1.39-1.17(m,16H),0.88(dt,J＝10.4,5.3Hz,3H).
步骤2：化合物1的合成：
将2-(双(2-氨基乙基)氨基)乙-1-醇)(0.0735g，0.50mmol，氢谱见图2)和2-烯丙酸(9Z，12Z)-十八碳二烯酯(0.64g，2mmol)加入反应瓶中，在80℃下反应48小时。反应冷却至室温后，真空除去溶剂，得到粗产品，将粗产品通过色谱法(硅胶柱，洗脱剂为含有0.5％甲醇(体积百分比)二氯甲烷纯化，并将纯产物蒸发，得到黄色油状化合物1(25.7mg，收率：3.6％)。化合物的氢谱见图3，质谱见图4。
1H NMR(400MHz,CDCl 3)δ5.45-5.30(m,16H),4.07(t,J＝6.8Hz,8H),3.52(s,1H),2.80(dd,J＝12.5,6.4Hz,16H),2.63(s,4H),2.54(s,3H),2.48(t,J＝7.2Hz,8H),2.07(q,J＝6.7Hz,16H),1.62(dd,J＝13.4,6.6Hz,8H),1.42-1.25(m,68H),0.91(t,J＝6.8Hz,12H),0.91(t,J＝6.8Hz,1H)。
实施例2
化合物2的合成路线
Figure PCTCN2022121378-appb-000006
步骤1：化合物2-1的合成
将6-溴己酸(1.0g，5.13mmol)和十一醇(1.77g，10.25mmol)溶于二氯甲烷中(60mL)，加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC盐酸盐，0.98g，5.13mmol)和DMAP(0.125g，1.03mmol)。混合物在常温条件下搅拌18小时。反应结束后，用DCM(200mL)稀释，并用饱和的NaHCO 3(100mL)和盐水(100mL)洗涤。合并有机层用无水Na 2SO 4干燥，真空除去溶剂，得到粗产品，将粗产品通过色谱法(硅胶柱，洗脱剂为含有0.5％EA(体积百分比)的石油醚)纯化，并将纯产物蒸发，得到浅黄色油状化合物2-1(6-溴己酸十一酯)(0.69g，收率38.6％)。化合物2-1的氢谱见图5。
1H NMR(400MHz,CDCl 3)δ:4.10(t,J＝6.6Hz,2H),3.45(t,J＝6.7Hz,2H),2.36(t,J＝7.3Hz,2H),1.97-1.88(m,2H),1.68(tt,J＝14.5,7.3Hz,4H),1.53(dd,J＝15.1,7.9Hz,2H),1.33(d,J＝16.9Hz,16H),0.92(t,J＝6.5Hz,3H).
步骤2：化合物2的合成
将2-(双(2-氨基乙基)氨基)乙-1-醇)(0.044g，0.30mmol)和6-溴己酸十一酯(0.417g，1.20mmol)溶于THF/CH 3CN(1:1，6mL)，之后再加入DIPEA(0.155g，1.20mmol)。反应物在63℃条件下搅拌72h，反应冷却至室温后，真空除去溶剂。粗产品用乙酸乙酯和饱和的NaHCO 3萃取，合并有机层用无水Na 2SO 4干燥，真空除去溶剂，得到粗产品，将粗产品通过色谱法(硅胶柱，洗脱剂为含有1-2％甲醇(体积百分比)二氯甲烷)纯化，并将纯产物蒸发，得到黄色油状化合物2(14.64mg，收率4％)。化合物2的氢谱见图6，质谱见图7。
1H NMR(400MHz,CDCl 3)δ4.10-3.98(m,8H),3.63(d,J＝16.5Hz,2H),3.46(s,1H),3.22(d,J＝45.3Hz,3H),3.05(d,J＝4.0Hz,2H),3.01-2.73(m,9H),2.66(d,J＝14.3Hz,2H),2.33(dd,J＝16.6,7.4Hz,8H),1.98(s,2H),1.78(s,2H),1.64(dt,J＝23.5,7.0Hz,20H),1.41-1.20(m,74H),0.88(t,J＝6.7Hz,12H)。
实施例3
化合物3的合成路线
Figure PCTCN2022121378-appb-000007
步骤1：化合物3-1的合成
将8-溴辛酸(1.139g，5.13mmol)和3,7-二甲基辛-6-烯-1-醇(香茅醇，1.599g，10.25mmol)溶于二氯甲烷(60mL)，充分溶解后，加入EDC盐酸盐(0.98g，5.13mmol)和DMAP(0.125g，1.03mmol)。混合物在常温条件下搅拌18小时。反应结束后，用DCM(200mL)稀释，并用饱和的NaHCO 3(100mL)和盐水(100mL)洗涤。合并有机层用无水Na 2SO 4干燥，真空除去溶剂，得到粗产品,将粗产品通过色谱法(硅胶柱，洗脱剂为含有0.5％EA(体积百分比)的石油醚)纯化，并将纯产物蒸发，得到浅黄色油状化合物3-1(6-溴己酸3,7-二甲基辛-6-烯酯)(0.648g，35％)化合物3-1的氢谱见图8。
1H NMR(400MHz,CDCl 3)δ:5.09(s,1H),4.18-4.01(m,2H),3.40(t,J＝6.8Hz,2H),2.29 (t,J＝7.4Hz,2H),1.98(s,2H),1.84(dd,J＝14.3,7.0Hz,2H),1.70-1.60(m,9H),1.38(d,J＝37.7Hz,9H),0.89(t,J＝12.9Hz,4H).
步骤2：化合物3的合成
将1,3-二氨基-2-丙醇(0.027g，0.30mmol，氢谱见图9)和6-溴己酸3,7-二甲基辛-6-烯酯(0.398g，1.2mmol)加入反应瓶中溶于THF/CH 3CN(1:1，6mL)，之后再加入DIPEA(0.155g，1.20mmol)。反应物在63℃条件下搅拌72h，反应冷却至室温后，真空除去溶剂。粗产品用乙酸乙酯和饱和的NaHCO 3萃取，合并有机层用无水Na 2SO 4干燥，真空除去溶剂，得到粗产品，将粗产品通过色谱法将粗产品通过色谱法(硅胶柱，洗脱剂为含有1％甲醇(体积百分比)二氯甲烷)纯化，并将纯产物蒸发，得到浅黄色油状化合物3(11.63mg，收率3.2％)。化合物3的氢谱见图10，质谱见图11。
1H NMR(400MHz,CDCl 3)δ5.30(s,1H),5.08(t,J＝6.4Hz,4H),4.10(h,J＝10.9Hz,8H),3.67(s,1H),2.46(s,10H),2.28(t,J＝7.3Hz,8H),1.97(d,J＝9.2Hz,8H),1.74-1.51(m,41H),1.49-1.37(m,12H),1.37-1.16(m,33H),0.91(d,J＝5.9Hz,12H)。
实施例4：LNP-mRNA纳米脂质颗粒的制备、粒径及电位的测定：
使用无水乙醇作为溶剂，将实施例1至3中制备的化合物1、化合物2以及化合物3分别与DSPC、DMG-PEG2000及胆固醇按照摩尔比为50:10:1.5:38.5溶解混合得到脂质体原料溶液，控制各组分浓度之和为50mM，完全溶解混匀后放置-20℃保存。
将mRNA溶解在pH为5.2左右的25mM的醋酸钠缓冲液中，制得终浓度约为0.1mg/mL的核酸制剂。
将上述配制的脂质体原料溶液和核酸制剂以两相体积比约为4:1，两相溶液总速率为12mL/min条件下，通过Nano Assemblr微流控系统，也可通过涡旋的方法快速地将两相溶液混匀，形成均一稳定的纳米脂质体颗粒，然后快速地将纳米脂质体颗粒环境从pH为5.2转变成7.0～7.4。具体操作为，用pH为7.2的PBS缓冲液或pH为7.4的醋酸钠缓冲液将其稀释20倍体积后，利用10KD的超滤管进行浓缩，离心机的转速不bj要超过超滤管的最高转速限制，经过2～3次换液之后，纳米脂质体颗粒的溶液环境的pH在7.2～7.4左右，将其浓缩至终浓度为200mM左右，放置在4℃环境下备用，分别标记为Lipid-1、Lipid-2及Lipid-3。
利用Zetasizer Nano ZS(Malvern,Worcestershire,UK)检测纳米脂质体颗粒的粒径，PDI。粒径的测量通过将纳米脂质体颗粒溶液用1×PBS稀释50倍之后测量，Zeta电位的测定是通过将纳米脂质体颗粒稀释到15mM的PBS中进行测定，包封率的测定利用Quant-It RiboGreenRNA定量检测试剂盒在Modulus微孔型多功能检测仪上进行。粒径、PDI、包封率及电位的检测结果如表1和图12所示。
表1.代表性LNP-mRNA纳米脂质颗粒的物理参数
Figure PCTCN2022121378-appb-000008
Figure PCTCN2022121378-appb-000009
结果显示，Lipid-1、Lipid-2和Lipid-3的平均粒径在100-150nm，粒径分布均匀，包封效率均在90％以上，电位为-20.8mV～-10.2mV。
实施例5：纳米脂质颗粒动物体内转染实验：
采用纳米脂质颗粒肌肉注射方式，按照实施例4的制备方法制备纳米脂质颗粒，其中mRNA为表达Luciferase荧光蛋白的mRNA，mRNA的用量为60μg，可电离化脂质体化合物、DSPC、DMG-PEG2000及胆固醇的总量为600μg，采用200μL的中性PBS缓冲液快速转换脂质体环境，并迅速注射到6-8周雌性Babl/c小鼠的后肢内侧肌肉中，控制左右后肢分别注射30μg mRNA，或通过静脉注射到6-8周雌性Babl/c小鼠，控制静脉注射50μg mRNA。
小鼠腿部肌肉注射Lipid-1、Lipid-2、Lipid-3后不同时间点mRNA在小鼠体内4h、24h、48h及72h时的荧光表达情况，并对圈内部的荧光强度进行积分并得到荧光强度数值，用于定量表征荧光素酶的表达量并绘制柱形图。图中的荧光强度与脂质体的递送效率密切相关，荧光越强，代表脂质体的递送效率越高。
小鼠肌肉注射Lipid-1后不同时间点的荧光表达情况见图13。Lipid-1通过肌肉注射到小鼠的腿部之后，4h之后的平均荧光表达量为3.5×10 7，与该领域内的参照标准MC3以及Moderna公司的SM-102相比，Lipid-1在肝脏部位的荧光量明显降低，因此肝脏需要承受的代谢负担较低。而且荧光表达能够持续72h，说明脂质体对mRNA的保护效果好。
小鼠通过静脉注射和肌肉注射Lipid-1 6h后各器官的荧光表达情况见图14。结果显示，在静脉注射(IV)Lipid-1的脂质体后，小鼠各个器官中的荧光表达量分别约为脾脏56％，肝脏34％，肺部10％；肌肉注射(IM)后，小鼠各个器官中的荧光表达量分别约为脾脏60％，肝脏30％，肺部10％。
小鼠肌肉注射Lipid-2后不同时间点的荧光表达情况见图15。Lipid-2通过肌肉注射到小鼠的腿部之后，4h之后的平均荧光表达量为4.0×10 7，并且24h后肌肉组织的荧光表达量仍然高达1.0×10 6。与MC3及SM102相比，荧光在肝脏部位的分布显著降低。
小鼠通过静脉注射Lipid-2 6h后各器官的荧光表达情况见图16。结果显示，在静脉注射(IV)Lipid-2的脂质体后，小鼠各个器官中的荧光表达量分别约为肝脏92％，脾脏8％。
小鼠肌肉注射Lipid-3后不同时间点的荧光表达情况见图17。Lipid-3通过肌肉注射到小 鼠的腿部之后，4h之后的平均荧光表达量为1.0×10 6，并且持续48h之后荧光表达仍然能够大致保持在这一水平。与MC3及SM102相比，荧光在肝脏部位的分布显著降低。
小鼠通过静脉注射Lipid-3 6h后各器官的荧光表达情况见图18。结果显示，在静脉注射(IV)Lipid-3脂质体后，小鼠各个器官中的荧光表达量分别约为脾脏88％，肝脏12％，而采用肌肉注射后，脾脏中荧光表达量数值相对静脉注射低一点。
将含有Lipid-1、Lipid-2、Lipid-3以及SM102分别通过尾静脉注射到小鼠体内，6h后各小鼠体内都出现了明显的荧光分布，见图19，从荧光分布可以看出，SM-102和lipid-2脂质体的荧光主要分布在肝脏部位，Lipid-1和Lipid-3脂质体的荧光主要分布在脾脏部位，这与小鼠解剖的器官的荧光分布一致。Lipid-1和Lipid-3脂质体具有明显的脾脏部位靶向的作用，适合肌肉注射局部给药或静脉注射全身给药，肝脏毒性较低，而Lipid-2更适合肌肉注射给药方式。
以上对本公开做了详尽的描述，其目的在于让熟悉此领域技术的人士能够了解本公开的内容并加以实施，并不能以此限制本公开的保护范围，凡根据本公开的精神实质所作的等效变化或修饰，都应涵盖在本公开的保护范围内。

Claims (8)

  1. 一种可离子化脂质化合物在制备核酸药物递送系统中的应用，其中所述的核酸药物递送系统为能够将核酸药物分子递送至脾脏和/或肺脏的核酸药物递送系统，所述的可离子化脂质化合物为如下结构式所示化合物：
    Figure PCTCN2022121378-appb-100001
  2. 根据权利要求1所述的可离子化脂质化合物在制备核酸药物递送系统中的应用，其中所述的核酸药物分子为mRNA、siRNA、ASO或pDNA中的一种或多种；
    和/或，所述的核酸药物分子和所述的核酸药物递送系统的质量比为1:(5～50)。
  3. 根据权利要求1或2所述的可离子化脂质化合物在制备核酸药物递送系统中的应用，其中所述的核酸药物递送系统为纳米脂质颗粒，所述的纳米脂质颗粒的平均尺寸为50nm～200nm。
  4. 根据权利要求1至3中任一项所述的可离子化脂质化合物在制备核酸药物递送系统中的应用，其中所述的可离子化脂质化合物可选择性地带有靶向物质修饰，所述的靶向物质为叶酸、单链抗体或靶向多肽中的一种或多种。
  5. 根据权利要求1至4中任一项所述的可离子化脂质化合物在制备核酸药物递送系统中的应用，其中所述的核酸药物递送系统还包括选择性地带有靶向物质修饰的辅助性分子，所述的可离子化脂质化合物和所述的辅助性分子的投料摩尔比为(0.1～1)：(0.1～1)；所述的辅助性分子包括胆固醇、卡泊三醇、豆甾醇、β-谷甾醇、白桦脂醇、羽扇豆醇、熊果酸、齐墩果酸、二油酰基磷脂酰胆碱、二硬脂酰磷脂酰胆碱、1-硬脂酰基-2-油酰基卵磷脂、二油酰磷脂酰乙醇胺、(1,2-二油氧基丙基)三甲基氯化铵、双十烷基二甲基溴化铵、1,2-二肉豆蔻酰基-sn-甘油-3-乙基磷酸胆碱、二棕榈酰磷脂酰乙醇胺-甲氧基聚乙二醇5000、二硬脂酰基磷脂 酰乙醇胺-聚乙二醇2000中的一种或多种；所述的靶向物质为叶酸、单链抗体或靶向多肽中的一种或多种。
  6. 一种核酸药物递送系统，包括可离子化脂质化合物，所述可离子化脂质化合物为：
    Figure PCTCN2022121378-appb-100002
    其中所述的核酸药物递送系统为注射剂，所述的核酸药物递送系统还包括添加剂，所述的添加剂为包括稳定剂和/或稀释剂，所述的添加剂的添加量为所述的注射剂的总质量的1％～20％。
  7. 根据权利要求6所述的核酸药物递送系统，其中所述的稳定剂包括蔗糖或海藻糖；所述的稀释剂包括磷酸盐缓冲液、醋酸盐缓冲液、柠檬酸盐、tris盐酸盐缓冲液中的一种或多种。
  8. 根据权利要求6或7所述的核酸药物递送系统，其中所述的核酸药物递送系统通过局部肌肉、皮下、内皮、瘤内注射或灌注方式给药，或通过静脉注射方式给药。
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CN114262275B (zh) * 2021-12-15 2023-09-29 华中师范大学 一种高效低毒性dna及rna脂质递送载体
CN114191561B (zh) * 2021-12-15 2022-08-02 武汉滨会生物科技股份有限公司 一种可离子化脂质化合物在核酸药物递送系统中的应用
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CN114716355B (zh) * 2022-04-02 2023-09-05 华南理工大学 一种脂质化合物、包含其的组合物及应用
CN115957329A (zh) * 2022-10-25 2023-04-14 中南大学 可离子化脂质化合物在制备核酸药物递送系统中的应用
CN115925563B (zh) * 2023-02-28 2023-05-16 清华大学 一种可用于靶向肺部递送核酸的脂质分子及其制备方法与应用
CN116199646B (zh) * 2023-03-07 2024-01-26 山东大学 一种基于Tris的可电离脂质及其制备方法与应用
CN116284006B (zh) * 2023-05-10 2023-08-25 北京因诺惟康医药科技有限公司 可电离脂质化合物、包含其的脂质载体及应用

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130072543A1 (en) * 2011-07-13 2013-03-21 Arrowhead Research Corporation Compounds and Compositions for Nucleic Acid Formulation and Delivery
CN111315359A (zh) * 2017-08-31 2020-06-19 摩登纳特斯有限公司 制备脂质纳米颗粒的方法
CN111954542A (zh) * 2018-02-01 2020-11-17 塔夫茨大学信托人 脂质样纳米复合物及其用途
CN112961065A (zh) * 2021-02-05 2021-06-15 嘉晨西海(杭州)生物技术有限公司 一种可电离脂质分子及其制备方法及其在制备脂质纳米颗粒的应用
CN112979483A (zh) * 2021-05-14 2021-06-18 苏州艾博生物科技有限公司 一种阳离子脂质化合物、包含其的组合物及应用
CN113121381A (zh) * 2021-04-19 2021-07-16 浙江大学 一种神经酰胺类化合物及其阳离子脂质体、制备方法和应用
CN114163345A (zh) * 2021-12-15 2022-03-11 武汉滨会生物科技股份有限公司 一种可离子化脂质化合物及核酸体外细胞转染试剂
CN114191561A (zh) * 2021-12-15 2022-03-18 武汉滨会生物科技股份有限公司 一种可离子化脂质化合物在核酸药物递送系统中的应用
CN114262275A (zh) * 2021-12-15 2022-04-01 华中师范大学 一种高效低毒性dna及rna脂质递送载体

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3315125A1 (en) * 2016-10-31 2018-05-02 Silence Therapeutics (London) Ltd Lipid nanoparticle formulation

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130072543A1 (en) * 2011-07-13 2013-03-21 Arrowhead Research Corporation Compounds and Compositions for Nucleic Acid Formulation and Delivery
CN111315359A (zh) * 2017-08-31 2020-06-19 摩登纳特斯有限公司 制备脂质纳米颗粒的方法
CN111954542A (zh) * 2018-02-01 2020-11-17 塔夫茨大学信托人 脂质样纳米复合物及其用途
CN112961065A (zh) * 2021-02-05 2021-06-15 嘉晨西海(杭州)生物技术有限公司 一种可电离脂质分子及其制备方法及其在制备脂质纳米颗粒的应用
CN113121381A (zh) * 2021-04-19 2021-07-16 浙江大学 一种神经酰胺类化合物及其阳离子脂质体、制备方法和应用
CN112979483A (zh) * 2021-05-14 2021-06-18 苏州艾博生物科技有限公司 一种阳离子脂质化合物、包含其的组合物及应用
CN114163345A (zh) * 2021-12-15 2022-03-11 武汉滨会生物科技股份有限公司 一种可离子化脂质化合物及核酸体外细胞转染试剂
CN114191561A (zh) * 2021-12-15 2022-03-18 武汉滨会生物科技股份有限公司 一种可离子化脂质化合物在核酸药物递送系统中的应用
CN114262275A (zh) * 2021-12-15 2022-04-01 华中师范大学 一种高效低毒性dna及rna脂质递送载体

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