WO2023109881A1 - 一种高效低毒性dna及rna脂质递送载体 - Google Patents

一种高效低毒性dna及rna脂质递送载体 Download PDF

Info

Publication number
WO2023109881A1
WO2023109881A1 PCT/CN2022/139186 CN2022139186W WO2023109881A1 WO 2023109881 A1 WO2023109881 A1 WO 2023109881A1 CN 2022139186 W CN2022139186 W CN 2022139186W WO 2023109881 A1 WO2023109881 A1 WO 2023109881A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
delivery carrier
molecules
delivery
integer
Prior art date
Application number
PCT/CN2022/139186
Other languages
English (en)
French (fr)
Inventor
崔艳芳
吉帅洁
张宝倩
Original Assignee
华中师范大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 华中师范大学 filed Critical 华中师范大学
Publication of WO2023109881A1 publication Critical patent/WO2023109881A1/zh

Links

Images

Classifications

    • 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
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids

Definitions

  • the invention belongs to the technical field of nucleic acid drug delivery carrier, and in particular relates to a high-efficiency and low-toxicity DNA and RNA lipid delivery carrier.
  • lipid delivery vehicles have been widely used for drug delivery, especially with the development of nucleic acid drugs, the application of lipid delivery vehicles has expanded rapidly. Since the nucleic acid molecule itself has negative charge, it is not conducive to the function of the cell membrane, and the cell permeability is poor, and the nucleic acid in the naked state entering the tissue or cell is easily degraded by nucleases. Nucleic acid transfection not only requires the nucleic acid with biological functions To be successfully transported into the cell, it is also necessary to ensure that the nucleic acid can maintain its biological function in the cell. Carriers commonly used in research and clinical applications include viral vectors and non-viral vectors. Liposome vectors are non-viral vectors. Compared with viral vectors, they have higher nucleic acid transfection efficiency, lower cytotoxicity, and more convenient preparation process. It has higher practicality, so it has received more and more attention.
  • the carrier formed by the most common cationic lipid compound in the early stage is: a liposome-nucleic acid drug complex is formed by a positively charged cationic lipid compound and a negatively charged nucleic acid, and the cationic liposome-nucleic acid drug complex The entire surface of the object is positively charged, adsorbed to the negatively charged cell surface through electrostatic interaction, enters the cell through endocytosis, and forms endosomes.
  • the cationic lipid in the cationic liposome interacts electrostatically with the negatively charged lipid in the endosome, and the negatively charged lipid flips from the lumen of the endosome to the lumen, forming neutral ions with the positively charged lipid Yes, nucleic acid drugs are separated from cationic lipids.
  • the FDA approved the first siRNA drug (patisiran [Onpattro]), which uses Dlin-MC3-DMA liposome as a delivery vehicle.
  • ionizable lipids have attracted attention due to their ability to change their electrical charge in response to the pH in the environment.
  • ionizable lipids have made recent progress in drug delivery, and have shown obvious advantages over viral vectors and other types of non-viral vectors in terms of encapsulation efficiency, nucleic acid expression, and cytotoxicity, they are currently available for use. There are still very few ionizable lipid molecules, and most of them are easily distributed to the liver, which increases the metabolic burden of the liver and produces toxic and side effects. Therefore, it is still necessary to explore more ionizable lipid compounds suitable for the application of nucleic acid drugs, and to develop nucleic acid drug delivery vehicles that truly take into account high transfection efficiency, high expression effect and low toxicity.
  • the purpose of the present invention is to provide an ionizable lipid compound that has a simple preparation method, is easy to combine with nucleic acid, and is easy to degrade, which enriches the types of ionizable lipid compounds and provides more options for nucleic acid drug delivery.
  • Another object of the present invention is to provide a delivery carrier, which is a highly efficient and low-toxic lipid delivery carrier for delivering DNA and RNA.
  • the delivery carrier and the nucleic acid molecule encapsulated in the delivery carrier form a pharmaceutical composition, which ensures the activity of the nucleic acid drug and high expression efficiency of the nucleic acid molecule, and at the same time significantly reduces the distribution of the drug in the liver.
  • G 1 is a C 1 -C 10 linear alkylene group
  • R is C 5 -C 20 straight chain alkyl or R1 is hydrogen, methyl, ethyl or isopropyl
  • m is an integer between 1 and 10
  • n is an integer between 1 and 5
  • f is an integer between 1 and 5;
  • G 3 is a C 1 -C 10 linear alkylene group
  • R' is R 2 and R 3 are independently hydrogen, methyl, ethyl or isopropyl
  • q is an integer between 1 and 3.
  • said G 1 is a C 2 -C 8 linear alkylene group.
  • said G 3 is a C 5 -C 10 linear alkylene group.
  • said R 1 is hydrogen
  • said m is an integer between 3-8, more preferably an integer between 4-6.
  • said f is an integer between 1 and 4, more preferably 2 or 3.
  • said R is C 5 -C 15 linear alkyl or
  • one of said R2 and said R3 is hydrogen, and the other is methyl, ethyl or isopropyl.
  • one of said R 2 and said R 3 is hydrogen, and the other is methyl.
  • said R 2 is methyl, and said R 3 is hydrogen.
  • the ionizable lipid compound is one or more of the following compounds:
  • the present invention also provides a delivery carrier, which includes one or more of the ionizable lipid compounds represented by the general formula (I) and the general formula (II).
  • the delivery vehicle further includes auxiliary molecules.
  • the molar ratio of the ionizable lipid compound and the auxiliary molecule is (0.1-1): (0.1-1), more preferably (0.5-1): (0.5-1 ).
  • the auxiliary molecule may be an auxiliary molecule commonly used in the art.
  • the auxiliary molecules include artificially synthesized or naturally derived auxiliary lipids or lipid molecules, any species of animal sources, and any types of cells or vesicles (including exosomes) or their components, One or more of polypeptide molecules, polymer molecules, sugar molecules or inorganic substances.
  • the auxiliary molecules include cholesterol, calcipotriol, stigmasterol, ⁇ -sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, dioleoylphosphatidylcholine , Distearoylphosphatidylcholine, 1-stearyl-2-oleoyl lecithin, dioleoylphosphatidylethanolamine, (1,2-dioleoxypropyl) trimethylammonium chloride, bis Decyldimethylammonium bromide, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, distearyl One or more of acylphosphatidylethanolamine-polyethylene glycol 2000, activated carbon, silicon dioxide, and calcium phosphate.
  • the ionizable lipid compound and/or the auxiliary molecule is modified with a targeting substance.
  • the targeting substance includes one or more of folic acid, single-chain antibody or targeting polypeptide.
  • the delivery carrier is nano-lipid particles.
  • the average size of the nanoparticle preparation is 50nm-200nm.
  • the average size of the nanoparticle preparation is 50nm-150nm.
  • the polydispersity index of the nanoparticle preparation is ⁇ 0.4.
  • the polydispersity index of the nanoparticle preparation is ⁇ 0.3.
  • the nucleic acid molecule is one or more of pDNA, siRNA, ASO or mRNA.
  • the mass ratio of the nucleic acid molecule to the delivery carrier is 1:(5-50); further 1:(5-40), and still more preferably 1:(5-30).
  • the nucleic acid pharmaceutical composition includes pharmaceutically acceptable additives, and the additives include one or more of excipients, stabilizers or diluents.
  • additives include but not limited to sucrose, trehalose or other stabilizers.
  • the added amount of the additive is 1%-20% of the total mass of the pharmaceutical composition.
  • the nucleic acid pharmaceutical composition can be a freeze-dried powder or an injection, and the injection can be administered locally through a microneedle, injection or perfusion through the muscle, subcutaneous, endothelium, intratumoral, or intravenous injection. medicine.
  • the delivery vector or the nucleic acid pharmaceutical composition is used to deliver nucleic acid molecules to mammalian cells.
  • said mammal is human.
  • the present invention also provides a method for delivering nucleic acid molecules in vivo, using the delivery carrier to deliver the nucleic acid molecules into the body of a subject.
  • said subject is a mammal.
  • the subject is human.
  • the present invention has the following advantages:
  • the invention provides a new ionizable lipid compound, which enriches the types of ionizable lipid compounds, and the delivery carrier formed by it has the advantages of high encapsulation efficiency and low toxicity, and can efficiently deliver nucleic acid drugs in vivo and Efficient expression provides more options for nucleic acid drug delivery, which is of great significance to the development and application of nucleic acid drugs.
  • Fig. 1 is the hydrogen spectrogram of compound a
  • Fig. 2 is the hydrogen spectrogram of compound b
  • Fig. 3 is the hydrogen spectrogram of compound 1-1
  • Fig. 4 is the hydrogen spectrogram of compound 2-1;
  • Fig. 5 is the hydrogen spectrogram of compound 3-1
  • Fig. 6 is the hydrogen spectrogram of compound 1
  • Fig. 7 is the high-resolution mass spectrogram of compound 1;
  • Fig. 8 is the hydrogen spectrogram of compound 2
  • Fig. 9 is the high-resolution mass spectrum of compound 2;
  • Figure 10 is the hydrogen spectrogram of compound 3
  • Figure 11 is the high resolution mass spectrum of compound 3
  • Fig. 12 is the particle size distribution figure of the nano lipid particle prepared in embodiment 4.
  • Figure 13 is the in vivo delivery effect of Lipid-01 liposome mice in Example 5.
  • Figure 14 is the in vivo delivery effect of Lipid-02 liposome mice in Example 5.
  • Figure 15 is the in vivo delivery effect of Lipid-03 liposome in mice in Example 5.
  • nucleic acid drug delivery vectors In order to reduce the toxic and side effects caused by the accumulation and expression of drug components in the liver, reduce the production cost of nucleic acid drug delivery vectors, and improve the delivery and expression effect of nucleic acid drugs in vivo, the inventors conducted a lot of research and experimental verification, and developed a new The ionizable lipid compound can form a nucleic acid drug delivery vehicle that truly combines high transfection efficiency, high expression effect and low toxicity.
  • the ionizable lipid compound is a compound represented by general formula (I) and general formula (II):
  • G 1 is a C 1 -C 10 linear alkylene group
  • R is C 5 -C 20 straight chain alkyl or R1 is hydrogen, methyl, ethyl or isopropyl
  • m is an integer between 1 and 10
  • n is an integer between 1 and 5
  • f is an integer between 1 and 5;
  • G 3 is a C 1 -C 10 linear alkylene group
  • R' is R 2 and R 3 are independently hydrogen, methyl, ethyl or isopropyl
  • q is an integer between 1 and 3.
  • the ionizable lipid compound with a special structure can improve its binding ability to negatively charged nucleic acids, prevent nucleic acids from being degraded by nucleases prematurely in cells, and facilitate the liposomes loaded with nucleic acids to pass through the cell membrane. Effective degradation and rapid clearance in the body are achieved, and the toxic and side effects of nucleic acid drugs are reduced.
  • the delivery carrier includes one or more of the ionizable lipid compounds represented by the general formula (I) and the general formula (II), and optionally includes auxiliary molecules.
  • the delivery carrier is a lipid nanoparticle with a particle diameter of 50nm-200nm.
  • the delivery vector can be used to deliver one or more of pDNA, siRNA, ASO or mRNA.
  • the delivery carrier and the nucleic acid molecule encapsulated in the delivery carrier constitute a nucleic acid pharmaceutical composition
  • the nucleic acid molecule is one or more of pDNA, siRNA, ASO or mRNA.
  • the nucleic acid pharmaceutical composition can be a lyophilized powder or an injection, and the injection can be locally administered by microneedle, injection or perfusion through muscle, subcutaneous, endothelial, or intratumoral, or through intravenous injection. medication.
  • the delivery vector or the nucleic acid pharmaceutical composition is used to deliver nucleic acid molecules to mammalian cells, preferably the mammal is a human.
  • 6-Bromohexanoic acid (1.0g, 5.13mmol) and undecyl alcohol (1.77g, 10.25mmol) were dissolved in dichloromethane (60mL), EDC hydrochloride (0.98g, 5.13mmol) and DMAP (0.125 g, 1.03 mmol). The mixture was stirred at room temperature for 18 hours. After the reaction was completed, it was diluted with DCM (200 mL), and washed with saturated NaHCO 3 (100 mL) and brine (100 mL).
  • 1,3-Diamino-2-propanol (0.027 g, 0.30 mmol) and undecyl 6-bromohexanoate (0.417 g, 1.20 mmol) were dissolved in THF/CH 3 CN (1:1, 6 mL), Afterwards additional DIPEA (0.155 g, 1.20 mmol) was added. The reactant was stirred at 63 °C for 72 h. After the reaction was cooled to room temperature, the solvent was removed in vacuo.
  • the crude product was extracted with ethyl acetate and saturated NaHCO 3 , the combined organic layers were dried over anhydrous Na 2 SO 4 , the solvent was removed in vacuo to give the crude product, the crude product was passed through chromatography, the crude product was passed through chromatography Chromatography (silica gel column, the eluent is dichloromethane containing 1% methanol (volume percentage)) purification, and the purified product was evaporated to remove the eluent to obtain light yellow oily compound 3 (47.72mg, yield 4.1%) .
  • the hydrogen spectrum of compound 3 is shown in Figure 10, and the mass spectrum is shown in Figure 11.
  • DSPC DMG-PEG2000: cholesterol is 50:10:1.5:38.5 (molar ratio), with absolute ethanol as solvent, prepare liposome solution, control the sum of the concentration of each component to be 50mM, dissolve and mix well and store at -20°C for later use.
  • the liposome solution and the nucleic acid preparation are mixed with a two-phase volume ratio of about 4:1, and the total rate of the two-phase solution is 12mL/min, and the two-phase solution is mixed by manual vortexing to form nano-lipids
  • the particle solution is diluted with PBS buffer solution with pH 7.2 or sodium acetate buffer solution with pH 7.4 to dilute it 20 times in volume, use a 10KD ultrafiltration tube to concentrate, and the speed of the centrifuge should not exceed the speed of the ultrafiltration tube.
  • the pH of the nano-liposome particle solution is changed from 5.2 to 7.2, and finally the nano-liposome particle solution is concentrated to a final concentration of about 200mM, and stored at 4°C Download the spare.
  • the particle size, PDI, of the nanoliposome particle was detected by Zetasizer Nano ZS (Malvern, Worcestershire, UK).
  • Zeta potential was determined by diluting nanoliposome particles into 15 mM PBS.
  • Quant-It RiboGreen RNA Quantitative Detection Kit was used to measure the encapsulation efficiency on Modulus microporous multifunctional detector.
  • Embodiment 5 liposome animal transfection experiment in vivo:
  • nano-lipid particles according to the preparation method of Example 4, wherein the amount of mRNA is 120 ⁇ g, and the total amount of ionizable liposome compound, DSPC, DMG-PEG2000 and cholesterol is 1200 ⁇ g, using 400 ⁇ L of neutral PBS buffer quickly switches the liposome environment.
  • the above-prepared nano-lipid particles were rapidly injected intramuscularly (IM) into the hindlimb medial muscles of 6-8 week female Babl/c mice, and 30 ⁇ g of mRNA were injected into the left and right hindlimbs respectively.
  • IM intramuscularly
  • mRNA mRNA
  • the above-prepared nano-lipid particles were rapidly injected (IV) into 6-8 week old female Babl/c mice through the tail vein, and the amount of mRNA injected was 60 ⁇ g. At different time periods after injection, the expression of luciferase in the mice after injection was observed by a small animal imager.
  • mice After 4 hours, the heart, liver, spleen, lung, and kidney of the mice were subjected to fluorescence imaging.
  • the delivery effect of Lipid-01 liposome in mice is shown in Figure 13.
  • the results show that the fluorescence expression can reach 7 ⁇ 10 7 after 4 hours of intramuscular injection. It can be seen from the imaging of various organs of the mouse that the fluorescence in the organs after intramuscular injection The expression is mainly concentrated in the spleen (80%). After intravenous injection, the fluorescence is distributed in the liver (63%), spleen (31%) and lungs (6%), indicating that Lipid-01 intramuscular injection has good spleen targeting sex.
  • the delivery effect of Lipid-02 liposome in mice is shown in Figure 14.
  • the results show that the fluorescence expression is about 10 7 after 6 hours of intramuscular injection. It can be seen from the imaging of various organs of the mouse that the fluorescence in the organs after intravenous injection is mainly It is distributed in the liver, and the expression level of fluorescence in the liver of mice after intramuscular injection is significantly lower, indicating that Lipid-02 is more suitable for intramuscular injection.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

本发明提供一种高效低毒性DNA及RNA脂质递送载体。为了丰富核酸药物递送载体的种类以及提高核酸药物体内递送和表达效果，降低毒副作用，本发明提供一种新的特殊结构的可离子化脂质化合物，其与带负电荷的核酸的结合效果好，有效防止核酸在细胞内过早的被核酸酶降解，有利于装载核酸的脂质体穿越细胞膜，能够实现有效降解、体内快速清除，降低了核酸药物的毒副作用，可形成真正兼顾高转染效率、高表达效果以及低毒性的核酸药物递送载体。

Description

一种高效低毒性DNA及RNA脂质递送载体 技术领域
本发明属于核酸药物递送载体技术领域，具体涉及一种高效低毒性DNA及RNA脂质递送载体。
背景技术
目前，脂质递送载体已被广泛用于药物递送，尤其是随着核酸药物的发展，脂质递送载体的应用迅速扩大。由于核酸分子自身带有负电荷，不利于细胞膜作用，细胞渗透能力差，而进入组织或细胞中的处于裸露状态的核酸又极易被核酸酶降解，核酸转染不仅要求将具有生物功能的核酸成功转运至细胞内，还需保证核酸在细胞内能够维持其生物功能。研究和临床应用中常用的载体包括病毒载体和非病毒载体，脂质体载体属于非病毒载体，相比病毒载体具有更高的核酸转染效率，更低的细胞毒性，更便捷的制备工艺，实用性更高，因此受到越来越多的关注。
随着研究的深入，相关研究人员积极开发了多种脂质体载体，不断提升安全性和高效性。早期最常见的阳离子脂质化合物形成的载体，其过程是：通过带正电荷的阳离子脂质化合物与带有负电荷的核酸形成脂质体-核酸药物复合物，阳离子脂质体-核酸药物复合物表面的整体带有正电荷，通过静电相互作用吸附到带负电荷的细胞表面，通过细胞内吞作用进入细胞，形成内涵体。阳离子脂质体中的阳离子脂质与内涵体中带负电荷的脂质发生静电相互作用，带负电荷的脂质由内涵体的腔外翻转到腔内，与正电荷脂质形成中性离子对，核酸类药物脱离阳离子脂。2018年，FDA批准了第一种siRNA药物(patisiran[Onpattro])，该核酸药物采用的递送载体为Dlin-MC3-DMA脂质体。最近，随着研究发现可离子化脂质因其能够响应环境中的pH，更改其所带的电性，因而备受关注。
尽管可离子化脂质用于药物输送取得了最新的进展，在包封效率、核酸表达、细胞毒性等方面相比病毒载体和其他类型的非病毒载体表现出明显的优势，但是目前可以供应用的可离子化脂质分子依然很少，而且其中多数存在容易分布到肝脏器官中而增加肝脏的代谢负担因而产生毒副作用等问题。因此，仍然需要探索更多的适合于核酸药物应用的可离子化脂质化合物，开发真正兼顾高转染效率、高表达效果以及低毒性的核酸药物递送载体。
发明内容
本发明的目的是提供一种制备方法简单、易与核酸结合且易降解的可离子化脂质化合物，其丰富了可离子化脂质化合物的种类，为核酸药物递送提供了更多的选择。
本发明的另一目的是提供一种递送载体，该递送载体为高效且低毒性的用于递送DNA及RNA的脂质递送载体。该递送载体以及包封在所述的递送载体中的核酸分子组成药物组合物，保证了核酸药物的活性而且核酸分子的表达效率高，同时显著降低药物在肝脏中的分布。
为解决上述技术问题，本发明采用如下技术方案：
通式(I)、通式(Ⅱ)所示的可离子化脂质化合物，
Figure PCTCN2022139186-appb-000001
其中，
通式(I)中，G 1为C 1-C 10直链亚烷基，G 2为-OC(＝O)R、-C(＝O)OR或-C(＝O)NHR，R为C 5-C 20直链烷基或
Figure PCTCN2022139186-appb-000002
R 1为氢、甲基、乙基或异丙基，m为1～10之间的整数，n为1～5之间的整数，f为1～5之间的整数；
通式(Ⅱ)中，G 3为C 1-C 10直链亚烷基，G 4为-OC(＝O)R’、-C(＝O)OR’或-C(＝O)NHR’，R’为
Figure PCTCN2022139186-appb-000003
R 2和R 3独立地为氢、甲基、乙基或异丙基，q为1～3之间的整数。
优选地，所述的G 1为C 2-C 8直链亚烷基。
优选地，所述的G 3为C 5-C 10直链亚烷基。
优选地，所述的R 1为氢。
优选地，所述的m为3～8之间的整数，进一步优选为4～6之间的整数。
优选地，所述的f为1～4之间的整数，进一步优选为2或3。
优选地，所述的R为C 5-C 15直链烷基或
Figure PCTCN2022139186-appb-000004
优选地，所述的G 2为-C(＝O)OR。
优选地，所述的G 4为-C(＝O)OR’。
优选地，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。
进一步优选地，所述的R 2和所述的R 3中一个为氢，另一个为甲基。
再进一步优选地，所述的R 2为甲基，所述的R 3为氢。
根据一些具体且优选地实施方式，所述的可离子化脂质化合物为以下化合物中的一种或多种：
Figure PCTCN2022139186-appb-000005
本发明还提供一种递送载体，所述的递送载体包括所述的通式(I)和通式(Ⅱ)所示的可离子化脂质化合物中的一种或多种。
优选地，所述的递送载体还包括辅助性分子。
进一步优选地，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)，进一步优选为(0.5～1)：(0.5～1)。
所述的辅助性分子可以是本领域中常用的辅助性分子。
优选地，所述的辅助性分子包括人工合成的或自然来源的辅助脂质或类脂分子、任何种属的动物来源以及任何种类的细胞或囊泡(包含外泌体)或其组成部分、多肽分子、聚合物分子、糖类分子或无机物中的一种或多种。
进一步优选地，所述的辅助性分子包括胆固醇、卡泊三醇、豆甾醇、β-谷甾醇、羽扇豆醇、白桦脂醇、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、活性炭、二氧化硅以及磷酸钙中的一种或多种。
优选地，所述的可离子化脂质化合物和/或所述的辅助性分子带有靶向物质修饰。
再进一步优选地，所述的靶向物质包括叶酸、单链抗体或靶向多肽中的一种或多种。
优选地，所述的递送载体为纳米脂质颗粒。
进一步优选地，所述的纳米颗粒制剂的平均尺寸为50nm～200nm。
再进一步优选地，所述的纳米颗粒制剂的平均尺寸为50nm～150nm。
进一步地，所述的纳米颗粒制剂的多分散指数≤0.4。
再进一步地，所述的纳米颗粒制剂的多分散指数≤0.3。
本发明所述的递送载体以及包封在所述的递送载体中的核酸分子一起组成核酸药物组合物。
优选地，所述的核酸分子为pDNA、siRNA、ASO或mRNA中的一种或多种。
优选地，所述的核酸分子和所述的递送载体的质量比为1:(5～50)；进一步为1:(5～40)，再进一步优选为1:(5～30)。
优选地，所述的核酸药物组合物包括药物可用的添加剂，所述的添加剂包括赋形剂、稳定剂或稀释剂中的一种或多种。
进一步地，所述的添加剂包括但不限于蔗糖、海藻糖或其他稳定剂。
具体地，所述的添加剂的添加量为所述的药物组合物的总质量的1％～20％。
优选地，所述的核酸药物组合物可以为冻干粉或为注射剂，所述的注射剂通过肌肉、皮下、内皮、瘤内以微针、注射或灌注方式局部给药，或通过静脉注射方式给药。
本发明中，所述的递送载体或所述的核酸药物组合物用于向哺乳动物细胞中递送核酸分子。
优选地，所述的哺乳动物为人。
本发明还提供一种体内递送核酸分子的方法，采用所述的递送载体将所述的核酸分子递送至受试者的体内。
优选地，所述的受试者为哺乳动物。
进一步优选地，所述的受试者为人。
本发明与现有技术相比具有如下优势：
本发明提供了一种新的可离子化脂质化合物，丰富了可离子化脂质化合物种类，其形成的递送载体具有包封效率高且毒性低的优点，能够将核酸药物在体内高效递送并高效表达，从而为核酸药物递送提供了更多选择，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a的氢谱图；
图2为化合物b的氢谱图；
图3为化合物1-1的氢谱图；
图4为化合物2-1的氢谱图；
图5为化合物3-1的氢谱图；
图6为化合物1的氢谱图；
图7为化合物1的高分辨质谱图；
图8为化合物2的氢谱图；
图9为化合物2的高分辨质谱图；
图10为化合物3的氢谱图；
图11为化合物3的高分辨质谱图；
图12为实施例4中制备的纳米脂质颗粒的粒径分布图；
图13为实施例5中Lipid-01脂质体小鼠体内递送效果；
图14为实施例5中Lipid-02脂质体小鼠体内递送效果；
图15为实施例5中Lipid-03脂质体小鼠体内递送效果。
具体实施方式
下面结合实施例对本发明作进一步描述。但本发明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。本发明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。
为了降低药物成分累积在肝脏器官中表达而产生的毒副作用、降低核酸药物递送载体的生产成本以及提高核酸药物体内递送和表达效果，发明人进行了大量研究和实验验证，开发了一种新的可离子化脂质化合物，其能够形成真正兼顾高转染效率、高表达效果以及低毒性的核酸药物递送载体。
具体地，在本发明中，所述的可离子化脂质化合物为通式(I)、通式(Ⅱ)所示的化合物：
Figure PCTCN2022139186-appb-000006
其中，
通式(I)中，G 1为C 1-C 10直链亚烷基，G 2为-OC(＝O)R、-C(＝O)OR或-C(＝O)NHR，R为C 5-C 20直链烷基或
Figure PCTCN2022139186-appb-000007
R 1为氢、甲基、乙基或异丙基，m为1～10之间的整数，n为1～5之间的整数，f为1～5之间的整数；
通式(Ⅱ)中，G 3为C 1-C 10直链亚烷基，G 4为-OC(＝O)R’、-C(＝O)OR’或-C(＝O)NHR’，R’为
Figure PCTCN2022139186-appb-000008
R 2和R 3独立地为氢、甲基、乙基或异丙基，q为1～3之间的整数。
通过特殊结构的可离子化脂质化合物，能够提高其与带负电荷的核酸的结合能力，可以防止核酸在细胞内过早的被核酸酶降解，有利于装载核酸的脂质体穿越细胞膜，能够实现有效降解、体内快速清除，降低了核酸药物的毒副作用。
根据本发明，递送载体包括所述的通式(I)和通式(Ⅱ)所示的可离子化脂质化合物中的一种或多种，以及选择性地包括辅助性分子。
根据本发明，递送载体为粒径为50nm～200nm的脂质纳米颗粒。
根据本发明，所述的递送载体可用于pDNA、siRNA、ASO或mRNA中的一种或多种的递送。
根据本发明，递送载体以及包封在所述的递送载体中的核酸分子组成核酸药物组合物，所述的核酸分子为pDNA、siRNA、ASO或mRNA中的一种或多种。
根据本发明，所述的核酸药物组合物可以为冻干粉或为注射剂，所述的注射剂通过肌肉、皮下、内皮、瘤内以微针、注射或灌注方式局部给药，或通过静脉注射方式给药。
本发明中，所述的递送载体或所述的核酸药物组合物用于向哺乳动物细胞中递送核酸分子，优选所述的哺乳动物为人。
下面结合具体实施例进一步阐述本发明的技术方案和技术效果。
以下实施例中，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。
实施例1
化合物1的合成路线：
Figure PCTCN2022139186-appb-000009
步骤1：化合物1-1的合成
将8-溴辛酸(1.139g，5.13mmol)和香茅醇(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，并将纯化产物蒸发去除洗脱剂，得到浅黄色油状化合物(0.648g，35％)，化合物1-1的氢谱见图3。
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1的合成
将2-(双(2-氨基乙基)氨基)乙-1-醇)(化合物a，0.044g，0.30mmol，氢谱见图1)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(硅胶柱，洗脱剂为含有2％甲醇(体积百分比)的二氯甲烷)纯化，并将纯化产物蒸发去除洗脱剂，得到黄色油状化合1(25.7mg，收3.6％)。化合物1的氢谱见图6，质谱见图7。
1H NMR(400MHz,CDCl 3)δ5.09(s,4H),4.10(dd,J＝12.6,6.5Hz,8H),3.61(d,J＝21.6Hz,2H),3.19(s,1H),3.03(s,1H),2.98-2.92(m,1H),2.82(s,4H),2.67(s,4H),2.28(t,J＝7.5Hz,8H),2.03-1.93(m,8H),1.74-1.50(m,49H),1.36(ddd,J＝44.4,23.9,10.0Hz,39H),1.23-1.13(m,4H),0.91(d,J＝6.5Hz,12H).
实施例2
化合物2的合成路线
Figure PCTCN2022139186-appb-000010
步骤1：化合物2-1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，最后将冰浴移除，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，并将纯化产物蒸发去除洗脱剂，得到浅黄色油状化合物2-1(0.173g，收率：50％)，化合物2-1的氢谱见图4。
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2的合成
将1,3-二氨基-2-丙醇(化合物b，0.04504g，0.50mmol，氢谱见图2)和2-烯丙酸(9Z，12Z)-十八碳二烯酯(0.64g，2mmol)加入反应瓶中溶于THF/CH 3CN(1:1，6mL)，之后再加入DIPEA(0.155g，1.20mmol)，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，将粗产品通过色谱法(硅胶柱，洗脱剂为含有0.3％甲醇(体积百分比)的二氯甲烷)纯化，并将纯化产物蒸发去除洗脱剂，得到浅黄色油状化合2(20.58mg，收率3％)。化合物2的氢谱见图8，质谱见图9。
1H NMR(400MHz,CDCl 3)δ5.45-5.32(m,16H),4.09(t,J＝6.8Hz,8H),3.78(d,J＝20.7Hz,1H),3.51(d,J＝27.2Hz,1H),2.81(t,J＝6.4Hz,16H),2.49(s,10H),2.09(q,J＝6.8Hz,16H),1.69-1.60(m,8H),1.41-1.30(m,66H),0.93(t,J＝6.8Hz,12H).
实施例3
化合物3的合成路线
Figure PCTCN2022139186-appb-000011
步骤1：化合物3-1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，并将纯化产物蒸发去除洗脱剂，得到浅黄色油状化合物3-1(0.69g，收率38.6％)。化合物3-1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3的合成
将1,3-二氨基-2-丙醇(0.027g，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，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)的二氯甲烷)纯化，并将纯化产物蒸发去除洗脱剂，得到浅黄色油状化合3(47.72mg，收率4.1％)。化合物3的氢谱见图10，质谱见图11。
1H NMR(400MHz,CDCl 3)δ5.30(s,1H),4.05(t,J＝6.8Hz,8H),3.68(s,1H),2.47(s,8H),2.30(t,J＝7.5Hz,8H),1.62(dd,J＝15.1,7.7Hz,16H),1.47(s,6H),1.28(d,J＝16.8Hz,78H),0.88(t,J＝6.8Hz,12H).
实施例4：
制备纳米脂质颗粒并测试其粒径和电位。
纳米脂质颗粒的制备方法：
(1)按照可离子化化合物：DSPC：DMG-PEG2000：胆固醇为50:10:1.5:38.5(摩尔比)，以无水乙醇作为溶剂，配制脂质体溶液，控制各组分浓度之和为50mM，溶解混匀后放置-20℃保存、备用。
(2)使用pH为5.2左右的25mM的醋酸钠缓冲液溶解mRNA，配制终浓度约为0.1mg/mL的核酸制 剂。
(3)将脂质体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，通过手工涡旋的方式将两相溶液混匀形成纳米脂质体颗粒溶液后，立即用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由pH为5.2转变成7.2，最后将纳米脂质体颗粒溶液浓缩至终浓度为200mM左右，保存于4℃环境下备用。
将纳米脂质体颗粒溶液用1×PBS稀释50倍之后，利用Zetasizer Nano ZS(Malvern,Worcestershire,UK)检测纳米脂质体颗粒的粒径，PDI。
将纳米脂质体颗粒稀释到15mM的PBS中测定Zeta电位。
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包封率的测定。
粒径、PDI、包封率及电位的检测结果如表1和图12所示。
表1
Figure PCTCN2022139186-appb-000012
实施例5：脂质体动物体内转染实验：
使用表达Luciferase荧光蛋白的mRNA，按照实施例4的制备方法制备纳米脂质颗粒，其中mRNA的用量为120μg，可电离化脂质体化合物、DSPC、DMG-PEG2000及胆固醇的总量为1200μg，采用400μL的中性PBS缓冲液快速转换脂质体环境。
将上述制备的纳米脂质颗粒迅速通过肌肉注射(IM)到6-8周雌性Babl/c小鼠的后肢内侧肌肉中，左右后肢分别注射30μg的mRNA。注射后不同时间段，分别通过小动物成像仪观察注射后小鼠体内荧光素酶的表达情况。
将上述制备的纳米脂质颗粒迅速通过尾静脉注射(IV)到6-8周雌性Babl/c小鼠的体内，mRNA注射量为60μg。注射后不同时间段，分别通过小动物成像仪观察注射后小鼠体内荧光素酶的表达情况。
在4h后，将小鼠的心、肝、脾、肺、肾分别进行荧光成像。
Lipid-01脂质体小鼠体内递送效果见图13，结果显示：肌肉注射4小时后荧光表达能够达到7×10 7，小鼠的各个器官成像可以看出，肌肉注射后在器官中的荧光表达主要集中在脾脏(80％)部位，静脉注射之后，荧光分布在肝脏(63％)、脾脏(31％)和肺部(6％)，表明Lipid-01肌肉注射具有较好的脾脏靶向性。
Lipid-02脂质体小鼠体内递送效果见图14，结果显示：肌肉注射6小时后荧光表达约为10 7，通过小鼠的各个器官成像可以看出，静脉注射之后在器官中的荧光主要分布在肝脏部位，而肌肉注射之后小鼠的肝脏荧光表达量明显较低，表明，Lipid-02更适合用于肌肉注射。
Lipid-03脂质体小鼠体内递送效果见图15，结果显示：肌肉注射4小时后荧光表达约为10 7，通过小鼠的各个器官成像可以看出，肌肉注射和静脉注射之后在器官中的荧光都集中在脾脏部位，表明Lipid-03肌肉注射和静脉注射都具有较好的脾脏靶向性。
以上对本发明做了详尽的描述，其目的在于让熟悉此领域技术的人士能够了解本发明的内容并加以实施，并不能以此限制本发明的保护范围，凡根据本发明的精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围内。

Claims (16)

  1. 通式(I)、通式(Ⅱ)所示的可离子化脂质化合物，
    Figure PCTCN2022139186-appb-100001
    其中，
    通式(I)中，G 1为C 1-C 10直链亚烷基，G 2为-OC(＝O)R、-C(＝O)OR或-C(＝O)NHR，R为C 5-C 20直链烷基或
    Figure PCTCN2022139186-appb-100002
    R 1为氢、甲基、乙基或异丙基，m为1～10之间的整数，n为1～5之间的整数，f为1～5之间的整数；
    通式(Ⅱ)中，G 3为C 1-C 10直链亚烷基，G 4为-OC(＝O)R’、-C(＝O)OR’或-C(＝O)NHR’，R’为
    Figure PCTCN2022139186-appb-100003
    R 2和R 3独立地为氢、甲基、乙基或异丙基，q为1～3之间的整数。
  2. 根据权利要求1所述的可离子化脂质化合物，其特征在于，所述的G 1为C 2-C 8直链亚烷基；
    和/或，所述的G 3为C 5-C 10直链亚烷基；
    和/或，所述的R 1为氢；
    和/或，所述的m为3～8之间的整数，
    和/或，所述的f为1～4之间的整数；
    和/或，所述的R为C 5-C 15直链烷基或
    Figure PCTCN2022139186-appb-100004
  3. 根据权利要求2所述的可离子化脂质化合物，其特征在于，所述的m为4～6之间的整数；
    和/或，所述的f为2或3。4.根据权利要求1所述的可离子化脂质化合物，其特征在于，所述的G 2为-C(＝O)OR；
    和/或，所述的G 4为-C(＝O)OR’；
    和/或，所述的R 2为甲基，所述的R 3为氢。
  4. 根据权利要求1所述的可离子化脂质化合物，其特征在于，所述的可离子化脂质化合物为以下化合物中的一种或多种：
    Figure PCTCN2022139186-appb-100005
    Figure PCTCN2022139186-appb-100006
  5. 一种递送载体，其特征在于，所述的递送载体包括权利要求1至5中任一项所述的通式(I)和通式(Ⅱ)所示的可离子化脂质化合物中的一种或多种。
  6. 根据权利要求6所述的递送载体，其特征在于，所述的递送载体还包括辅助性分子，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)。
  7. 根据权利要求7所述的递送载体，其特征在于，所述的辅助性分子包括人工合成的或自然来源的辅助脂质或类脂分子、任何种属的动物来源以及任何种类的细胞或囊泡、多肽分子、聚合物分子、糖类分子或无机物中的一种或多种。
  8. 根据权利要求8所述的递送载体，其特征在于，所述的辅助性分子包括胆固醇、卡泊三醇、豆甾醇、羽扇豆醇、β-谷甾醇、白桦脂醇、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、活性炭、二氧化硅以及磷酸钙中的一种或多种。
  9. 根据权利要求7所述的递送载体，其特征在于，所述的可离子化脂质化合物和/或所述的辅助性分子带有靶向物质修饰，所述的靶向物质包括叶酸、单链抗体或靶向多肽中的一种或多种。
  10. 根据权利要求6所述的递送载体，其特征在于，所述的递送载体为纳米脂质颗粒，所述的纳米脂质颗粒的粒径为50nm～200nm；和/或，所述的纳米颗粒制剂的多分散指数≤0.4。12.根据权利要求11所述的递送载体，其特征在于，所述的递送载体能够递送核酸分子，所述的核酸分子包括但不限于质粒DNA(pDNA)、siRNA、ASO或mRNA中的一种或多种；和/或，所述的核酸分子和所述的递送载体的质量比为1:(5～50)。
  11. 根据权利要求9所述的递送载体，其特征在于，所述的递送载体和所述的核酸分子组成核酸药物组合物，所述的核酸药物组合物还包括药物可用的添加剂，所述的添加剂包括赋形剂、稳定剂或稀释剂中的一种或多种。
  12. 根据权利要求13所述的递送载体，其特征在于，所述的添加剂的添加量为所述的药物组合物的总质量的1％～20％。
  13. 根据权利要求6或13所述的递送载体，其特征在于，所述的递送载体或所述的核酸药物组合物为冻干粉剂或注射剂，通过肌肉、皮下、内皮、瘤内以微针、注射或灌注方式局部给药，或通过静脉注射方式给药。
  14. 一种体内递送核酸分子的方法，其特征在于，采用权利要求6至15中任一项所述的递送载体将所述的核酸分子递送至受试者的体内。
  15. 根据权利要求16所述的体内递送核酸分子的方法，其特征在于，所述的受试者为哺乳动物。
  16. 根据权利要求16或17所述的体内递送核酸分子的方法，其特征在于，所述的受试者为人。
PCT/CN2022/139186 2021-12-15 2022-12-15 一种高效低毒性dna及rna脂质递送载体 WO2023109881A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111533616.1 2021-12-15
CN202111533616.1A CN114262275B (zh) 2021-12-15 2021-12-15 一种高效低毒性dna及rna脂质递送载体

Publications (1)

Publication Number Publication Date
WO2023109881A1 true WO2023109881A1 (zh) 2023-06-22

Family

ID=80827296

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/139186 WO2023109881A1 (zh) 2021-12-15 2022-12-15 一种高效低毒性dna及rna脂质递送载体

Country Status (2)

Country Link
CN (1) CN114262275B (zh)
WO (1) WO2023109881A1 (zh)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023092242A1 (en) * 2021-11-29 2023-06-01 The Governing Council Of The University Of Toronto Multi-motif dendrons and their supramolecular structures and uses thereof
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
CN114163345B (zh) * 2021-12-15 2022-07-26 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
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
CN114306279A (zh) * 2021-12-30 2022-04-12 复旦大学 基于科罗索酸或其类似物的脂质纳米颗粒系统及其制备方法和应用
CN116813493A (zh) * 2022-03-21 2023-09-29 苏州科锐迈德生物医药科技有限公司 一种脂质化合物及基于其的脂质载体、核酸脂质纳米粒组合物和药物制剂
CN115626983A (zh) * 2022-04-27 2023-01-20 北京清科胜因生物科技有限公司 一种聚(2-噁唑啉)脂质及脂质纳米颗粒
CN116554046B (zh) * 2023-04-17 2024-05-28 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其脂质纳米颗粒

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006006196A2 (en) * 2004-07-15 2006-01-19 Universita' Degli Studi Di Milano Synthesis of organometallic molecules that can be used as markers of organic substances
CN103380113A (zh) * 2010-11-15 2013-10-30 生命科技公司 含胺的转染试剂及其制备和使用方法
CN106661138A (zh) * 2014-07-18 2017-05-10 利物浦大学 包含支化聚合物的颗粒
WO2017118842A1 (en) * 2016-01-07 2017-07-13 The University Of Liverpool Branched polyester carrying dendrons
WO2021156469A1 (en) * 2020-02-07 2021-08-12 Domino Printing Sciences Plc Ink compositions
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
CN113402404B (zh) * 2021-04-30 2022-03-11 苏州科锐迈德生物医药科技有限公司 脂质化合物及包含其的脂质载体、核酸脂质纳米粒组合物和药物制剂

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006006196A2 (en) * 2004-07-15 2006-01-19 Universita' Degli Studi Di Milano Synthesis of organometallic molecules that can be used as markers of organic substances
CN103380113A (zh) * 2010-11-15 2013-10-30 生命科技公司 含胺的转染试剂及其制备和使用方法
CN106661138A (zh) * 2014-07-18 2017-05-10 利物浦大学 包含支化聚合物的颗粒
WO2017118842A1 (en) * 2016-01-07 2017-07-13 The University Of Liverpool Branched polyester carrying dendrons
WO2021156469A1 (en) * 2020-02-07 2021-08-12 Domino Printing Sciences Plc Ink compositions
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TWYMAN LANCE J., ANTHONY E. BEEZER AND JOHN C. MITCHEL: "An Approach for the Rapid Synthesis of Moderately Sized Dendritic MacromoIecules", J. CHEM. SOC. PERKIN TRANS. 1, vol. 1994, no. 4, 1 January 1994 (1994-01-01), pages 407 - 411, XP093070972 *

Also Published As

Publication number Publication date
CN114262275A (zh) 2022-04-01
CN114262275B (zh) 2023-09-29

Similar Documents

Publication Publication Date Title
WO2023109881A1 (zh) 一种高效低毒性dna及rna脂质递送载体
WO2023109242A1 (zh) 可离子化脂质化合物在核酸药物递送系统中的应用
Mével et al. DODAG; a versatile new cationic lipid that mediates efficient delivery of pDNA and siRNA
Liu et al. Novel imidazole-functionalized cyclen cationic lipids: synthesis and application as non-viral gene vectors
EP4282855A1 (en) Ionizable lipid molecule, preparation method therefor, and application thereof in preparation of lipid nanoparticle
Habrant et al. Design of ionizable lipids to overcome the limiting step of endosomal escape: application in the intracellular delivery of mRNA, DNA, and siRNA
JP6997862B2 (ja) 生分解性化合物、脂質粒子、脂質粒子を含む組成物、およびキット
WO2023186149A1 (zh) 一种脂质化合物、包含其的组合物及应用
CN111087317B (zh) 不饱和阳离子脂质衍生物、制备方法以及在质粒递送系统中的应用
WO2023109243A1 (zh) 可离子化脂质化合物及核酸体外细胞转染试剂
US20210170046A1 (en) Improved lipid-peptide nanocomplex formulation for mrna delivery to cells
JP2001515060A (ja) 新リポポリアミン、その調製および適用
WO2023024513A1 (zh) 新型阳离子脂质化合物
EP3184506B1 (en) Cationic lipid for nucleic acid delivery
WO2024032753A1 (zh) 含氮链状化合物、其制备方法、包含其的组合物和应用
EP2802556B1 (en) Lipopolyamines of spermine type for construction of liposomal transfection systems
Neuberg et al. Design and evaluation of ionizable peptide amphiphiles for siRNA delivery
JP2005515990A (ja) 化合物
WO2023142167A1 (zh) 一种阳离子脂质类似物、其组合物及应用
WO2023029599A1 (zh) 新型阳离子脂质化合物
WO2023024515A1 (zh) 新型阳离子脂质化合物
WO2023190170A1 (ja) 核酸内封脂質ナノ粒子の製造方法及びこれを含む医薬品組成物の製造方法、並びに、核酸を細胞内又は標的細胞内に導入する方法
WO2023241314A1 (zh) 一类新的脂质化合物及其用途
WO2024041372A1 (zh) 一种用于有效递送核酸的分支链结构多肽载体及其变化形式
CN116143707B (zh) 一种碱基可电离脂质及其制备方法与应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22906626

Country of ref document: EP

Kind code of ref document: A1