WO2023133922A1 - Complexe lipidique à l'échelle micrométrique, son procédé de préparation et son utilisation - Google Patents

Complexe lipidique à l'échelle micrométrique, son procédé de préparation et son utilisation Download PDF

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WO2023133922A1
WO2023133922A1 PCT/CN2022/073143 CN2022073143W WO2023133922A1 WO 2023133922 A1 WO2023133922 A1 WO 2023133922A1 CN 2022073143 W CN2022073143 W CN 2022073143W WO 2023133922 A1 WO2023133922 A1 WO 2023133922A1
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lipid
micron
lipoplex
scale
sized
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PCT/CN2022/073143
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English (en)
Chinese (zh)
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刘密
程军平
潘韵芝
季阿芳
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苏州尔生生物医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a micron-scale lipid complex and its preparation and application.
  • mRNA vaccines have high efficacy, high production efficiency, low cost and safe injection, and have broad application prospects, and their research and development and clinical application are also advancing rapidly. Compared with other nucleic acid vaccines, mRNA vaccines are more efficient and safer, but their application is limited by low stability and delivery efficiency. To address this issue, a variety of delivery systems have been developed, the most commonly used of which are lipid carriers.
  • the classic lipid carrier formulation consists of: ionizable or cationic lipids, neutral phospholipids, structured lipids and polyethylene glycol lipids.
  • Ionizable lipids or cationic lipids are used to adsorb mRNA, neutral phospholipids and structural lipids help to stabilize the lipid bilayer of the carrier to improve the delivery efficiency of mRNA, polyethylene glycol lipids reduce plasma.
  • the specific adsorption of proteins and the formation of a hydration layer on the nanoparticles improve the stability of the colloid in the biological environment.
  • the particle size of the lipid carrier commonly used to load mRNA is about 100nm.
  • the lipid membrane can fuse with the cell membrane to enter the cell, and release the drug through endosome escape to play a role.
  • lipid carriers with small particle sizes are easy to be cleared quickly, cannot stay at the injection site for a long time to be acquired by antigen-presenting cells, and are widely distributed in various tissues and organs of the body, and cannot be effectively concentrated in the target tissues and organs.
  • the nucleic acid is encapsulated inside the lipid carrier, whether endosomal escape can occur and the release efficiency seriously affects the activation of the immune response.
  • the present invention provides a micron-scale lipid complex and its preparation and application.
  • the micron-scale lipoplex includes lipid carrier, nucleic acid and adjuvant, the nucleic acid and adjuvant are loaded inside and outside of the lipid carrier, and the particle size of the micron-scale lipoplex is 0.8 ⁇ m ⁇ 500 ⁇ m.
  • the particle size of the micron-sized lipoplex is 0.85 ⁇ m-100 ⁇ m.
  • the particle size of the micron-sized lipoplex is 0.9 ⁇ m-50 ⁇ m.
  • the particle size of the micron-sized lipoplex is 0.95 ⁇ m-10 ⁇ m.
  • the particle size of the micron-sized lipoplex is 1.0 ⁇ m-5.0 ⁇ m.
  • the particle size of the micron-sized lipoplex is 1.1 ⁇ m-3.0 ⁇ m.
  • the lipid complex with large particle size of the present invention has a long residence time at the injection site, and is more likely to be taken up by antigen-presenting cells at the injection site to trigger the body's immune response, and both the inside and the outside of the micron-sized lipoplex are loaded with nucleic acids And adjuvants, can simultaneously achieve the effect of immediate release and sustained release.
  • the adjuvant has almost no effect on the particle size of the micron lipoplex.
  • nucleic acid is selected from one or more of ribonucleic acid (RNA), nucleic acid aptamer (Aptamer) and deoxyribonucleic acid (DNA).
  • RNA ribonucleic acid
  • Aptamer nucleic acid aptamer
  • DNA deoxyribonucleic acid
  • the RNA is selected from one or more of mRNA, siRNA and microRNA.
  • the adjuvant is selected from immunopotentiators derived from microorganisms, products of the human or animal immune system, innate immune stimulants, adaptive immune stimulants, chemically synthesized drugs, fungal polysaccharides, and active ingredients of traditional Chinese medicines. one or more.
  • the adjuvant is selected from pattern recognition receptor agonists, Bacillus Calmette-Guerin (BCG), BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyclonal anti-A, mineral oil , virus-like particles, immune-enhanced reengineered influenza virions, cholera enterotoxin, saponins and their derivatives, immune response modifier (Resiquimod), thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharides, curcumin , immune adjuvant CpG, polyinosinic acid (Poly(I:C)), immune adjuvant poly ICLC, Corynebacterium brevis vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukin Interferon, interferon, polyinosinic acid, polyaden
  • the lipid carrier includes ionizable lipids, neutral lipids and cholesterol.
  • the molar ratio of the ionizable lipid, neutral lipid and cholesterol is 2-5:2-5:2-5.
  • the lipid carrier also includes PEGylated phospholipids (DMG-PEG2000), based on the molar mass of the lipid carrier, the molar percentage of the PEGylated phospholipids is no more than 10%, preferably Not more than 5%.
  • DMG-PEG2000 PEGylated phospholipids
  • the molar ratio of the ionizable lipids, neutral lipids, cholesterol and pegylated phospholipids is 20-50:20-50:20-50:0.1-10, preferably 35:16:46.5: 2.5.
  • the ionizable lipids include cationic lipids and anionic lipids.
  • the anionic lipid is selected from one or more of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, DOPG and dimyristoylphosphatidylglycerol.
  • the ionizable lipid is a cationic lipid selected from (2,3-dioleoyl-propyl)-trimethylammonium-chloride (DOTAP), dioctadecane Ammonium bromide (DDAB), 1,2-dioctadecenyloxy-3-methylammonium propane (DOTMA), 4-(N,N-dimethylamino)butanoic acid (6Z,9Z,28Z, 31Z)-heptanthic-6,9,28,31-tetraen-19-yl lipid (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1, 3]-dioxolane (DLin-K-DMA), 4-(N,N-dimethylamino)butanoic acid (diolinoleyl)methyl ester (DLin-MC3-DMA) and 1,2-di One or more of oleyl alcohol-3-dimethylamino
  • the mass ratio of the nucleic acid to the cationic lipid is 1:0.01-20000, preferably 1:10-20.
  • the neutral lipid is selected from (dioleoylphosphatidylcholine) DOPC, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine
  • DOPC dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • DOPE ethanolamine
  • DMPC dimyristoylphosphatidylcholine
  • DLPC dilauroyl lecithin
  • POPC 1-palmitoyl-2-oleoyl phosphatidylcholine
  • the second object of the present invention is to provide a method for preparing the micron-scale lipoplex, comprising the following steps:
  • the volume ratio of the organic phase to the aqueous phase is 1:0.2-50, preferably 1:0.5-10.
  • step (1) other pharmaceutical ingredients can also be added, which can be dissolved in the organic phase or the aqueous phase according to their polarity.
  • the potential of the micron-sized lipoplex is +10mV ⁇ +50mV.
  • the preparation method of the micron-scale lipoplex comprises the following steps:
  • aqueous phase is dropped into a rotary evaporator round bottom flask containing the lipid film, and a rotor is added to dissolve the lipid film to form the micron-sized lipid complex.
  • micron-scale lipid complex of the present invention contains an internal water phase, and negatively charged drugs are stably encapsulated in the internal water phase or adsorbed on the surface of the lipid membrane, and nucleic acids and adjuvants are tightly bound to cationic lipids through electrostatic interaction. adsorption.
  • the third object of the present invention is to provide a drug delivery system comprising the micron-sized lipoplex.
  • the micron-sized lipoplex is mixed with the adjuvant and incubated for 30-35 minutes before use. This step allows the negatively charged adjuvant to adsorb on the outside of the lipoplex and neutralize the positive surface charge, reducing the surface charge, thereby effectively reducing or avoiding cytotoxicity, and cells with a cell diameter of less than 20 ⁇ m can phagocytize it well, Preferably, the cell diameter is 7-20 ⁇ m.
  • micron-scale lipoplex loaded with nucleic acid and adjuvant inside and outside of the present invention can be enriched in the lipid carrier by selecting lipid compounds of different structures or modifying the structure of the lipid carrier or by other means. target tissue or cell.
  • the micron-scale lipid complex loaded with nucleic acid and adjuvant inside and outside of the present invention after the positively charged lipid carrier and the negatively charged nucleic acid and adjuvant form a lipid complex through electrostatic interaction, part of the drug can be further adsorbed on the complex Surface, reduce surface charge, under neutral conditions in vivo, can effectively reduce or avoid cytotoxicity, increase drug load and have high encapsulation efficiency.
  • the negatively charged adjuvant can neutralize the surface potential of the nucleic acid-lipid complex, making the lipoplex more tightly adsorbed, with higher biological safety and stronger stability.
  • the micron-scale lipoplex of the present invention can simultaneously deliver the nucleic acid and adjuvant that trigger the immune response and obtain a synergistic effect.
  • the micron-scale lipoplex can induce the production of Strong cellular and humoral immunity.
  • part of the drug is encapsulated inside the lipid membrane, which reduces the degradation of RNase and avoids its release through endosome escape.
  • the lipid complex with large particle size stays at the injection site for a long time and is easier to be injected
  • the antigen-presenting cells at the site will take it up and trigger the body's immune response; on the other hand, the electrostatic adsorption principle is used to load part of the drug on the lipid membrane, which can be released quickly, thereby triggering the body's immune response, achieving immediate and sustained release effects.
  • the preparation process of the invention is simple, and the required equipment is simple and easy to obtain and easy to operate.
  • Fig. 1 is a schematic diagram of the structure of the micron-scale nucleic acid-lipid complex loaded with nucleic acid and adjuvant inside and outside of the present invention.
  • Fig. 2 is the particle size and electric potential characterization figure of three kinds of lipoplexes of Small group, Medium group and Large group of the embodiment of the present invention; The particle sizes are 98nm, 328nm and 1.2 ⁇ m, respectively.
  • Figure 2d, Figure 2e and Figure 2f are the potential representation diagrams of the three lipoplexes respectively, and the potentials are +7.8mV, +7.5mV and +43.7mV respectively.
  • Fig. 3 is a graph of in vitro uptake of micron-sized nucleic acid-lipid complexes according to an embodiment of the present invention.
  • Figure 4 is a diagram of the in vivo uptake of three lipid complexes in the Small group, Medium group and Large group of the embodiment of the present invention
  • Figure 4a and Figure 4b are DC cells and CD8a + DC cells in the lymph nodes complexing the three lipid complexes
  • Figure 4c and Figure 4d are the uptake of the three groups of lipid complexes by B cells and macrophages in the lymph nodes;
  • Figure 5 is a diagram of the in vitro transfection of three lipoplexes in the Small group, Medium group and Large group according to the embodiment of the present invention
  • Figure 5a and Figure 5b are the percentage of transfected cells and the fluorescence intensity of GFP in the cells, respectively.
  • Fig. 6 is the in vivo of three kinds of lipoplexes of Small nanoparticle (Poly (I: C)) group, Medium nanoparticle (Poly (I: C)) group and Large microparticle (Poly (I: C)) group of the embodiment of the present invention
  • Figure 6a is the timeline of prevention and inoculation of tumors in mice
  • Figure 6b is the growth curve of tumors in mice
  • Figure 6c is the result of survival period of mice.
  • Fig. 7 is the antigen of three kinds of lipid complexes of Small nanoparticle (Poly (I: C)) group, Medium nanoparticle (Poly (I: C)) group and Large particle (Poly (I: C)) group of the embodiment of the present invention
  • Figure 7a and Figure 7b are the results of the specific T cell detection experiment;
  • Figure 7a and Figure 7b are the analysis of the CD4 + T cells and CD8 + T cells that synthesize IFN- ⁇ after the spleen cells of the mouse are activated after being stimulated by the antigen polypeptide in OVA.
  • Fig. 8 is the immunity of three kinds of lipid complexes of Small nanoparticle (Poly (I: C)) group, Medium nanoparticle (Poly (I: C)) group and Large microparticle (Poly (I: C)) group of the embodiment of the present invention Diagram of originality test results.
  • FIG. 1 The schematic diagram of the structure of the micron-sized nucleic acid-lipid complex loaded with nucleic acid and adjuvant inside and outside prepared in the embodiment of the present invention is shown in FIG. 1 .
  • nanoscale lipid complexes were prepared by water solvent diffusion method and microfluidic technology, which were marked as Small group and Medium group respectively.
  • Poly(I:C) in this embodiment can also be replaced by pattern recognition receptor agonist, Bacillus Calmette-Guerin (BCG), BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, poly Anti-A, mineral oil, virus-like particles, immune-boosting reconstituted influenza virion, cholera enterotoxin, saponins and their derivatives, immune response modifier (Resiquimod), thymosin, neonatal bovine liver active peptide, imiquimod Special, polysaccharide, curcumin, immune adjuvant CpG, polyinosinic acid (Poly(I:C)), immune adjuvant poly ICLC, Corynebacterium pumilus vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, poly Cytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum,
  • Example 1 Referring to Example 1 to prepare micron-sized lipoplexes, an equal volume of sodium citrate buffered saline solution was used to replace Poly(I:C);
  • the encapsulation efficiency of the micron-sized lipoplex prepared in the embodiment of the present invention is 83%-88%.
  • Reagent and lipid stock solution Weigh DOTAP (3.6mg), DOPE (1.8mg), cholesterol (2.6mg) and FITC-DSPE-PEG2000 (1.1 mg) into a beaker, add 0.6mL of absolute ethanol, and heat to 55°C to dissolve as a stock solution;
  • Fig. 3 show that the micron-sized lipoplex prepared in the embodiment of the present invention can be almost completely taken up by DC2.4 cells.
  • Figure 4a and Figure 4b respectively show the uptake of three lipoplexes by DC cells and CD8a + DC cells in lymph nodes;
  • Figure 4c and Figure 4d show the uptake of three lipoplexes by B cells and macrophages in lymph nodes intake situation.
  • micron-sized lipoplexes were prepared using EGFP-mRNA, and an equal volume of sodium citrate buffered saline solution was used to replace Poly(I:C), labeled (Large group);
  • DC2.4 cells were plated in a 24-well plate at a density of 5 ⁇ 10 4 /well 24 hours in advance, and 1.5 mL of DMEM complete medium containing 10% FBS was added to each well and cultured in a 37°C incubator;
  • nucleic acid-lipid complexes Three kinds of nucleic acid-lipid complexes (mRNA concentration about 1.2 ⁇ g/mL) were added to each well, mixed evenly, and cultured in a 37°C incubator;
  • Example 6 In vivo prevention experiment of micron-sized lipoplex loaded with nucleic acid and adjuvant inside and outside
  • OVA-mRNA was used to prepare micron-scale lipoplexes loaded with nucleic acid and adjuvants inside and outside, labeled as Large particle (Poly(I:C)) group;
  • the tumor grows to a measurable size in about 8 days, and the tumor volume and survival period are monitored;
  • Figure 6a The timeline of the prevention and inoculation of tumors in mice in this example is shown in Figure 6a
  • Figure 6b is a graph of tumor growth curves in mice
  • Figure 6c is a graph of the survival period of mice.
  • Figure 7a and Figure 7b are the results of CD4 + T cells and CD8 + T cells that synthesize IFN- ⁇ after the spleen cells of the mice are stimulated and activated by the antigen polypeptide in OVA, respectively.
  • Figure 7 shows that, compared with the PBS group, after the spleen was stimulated with polypeptides in the Large particle (Poly(I:C)) group, the CD4 + T cells that can produce IFN- ⁇ increased significantly (P ⁇ 0.05), while the other two groups did not Significant changes; CD8 + T cells that can produce IFN- ⁇ in the three groups all increased significantly, and the results indicated that micron-sized lipoplexes can effectively activate antigen-specific immune responses.
  • Figure 8 shows that, compared with the PBS group, both nanoscale and micron-scale nucleic acid-lipid complexes can make mice produce OVA protein-specific IgG antibodies, while there is no significant difference among the three groups, the results show that micron-scale nucleic acid lipids The complex can effectively activate the body's immune response.

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Abstract

L'invention concerne un complexe lipidique à l'échelle micrométrique, son procédé de préparation et son utilisation. Le complexe lipidique à l'échelle micrométrique comprend un support lipidique, un acide nucléique et un adjuvant, l'acide nucléique et l'adjuvant étant tous deux chargés à l'intérieur et à l'extérieur du support lipidique, et la taille des particules étant comprise entre 0,8 μm et 500 μm. Par rapport aux transporteurs lipidiques à l'échelle nanométrique, le complexe lipidique peut induire une forte immunité cellulaire et humorale, et présente des effets de libération rapide et lente. L'adjuvant chargé négativement peut neutraliser le potentiel de surface du complexe acide nucléique-lipide, ce qui permet au complexe de mieux s'adsorber et d'avoir une plus grande sécurité biologique et une plus grande stabilité.
PCT/CN2022/073143 2022-01-14 2022-01-21 Complexe lipidique à l'échelle micrométrique, son procédé de préparation et son utilisation WO2023133922A1 (fr)

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CN116196404A (zh) * 2022-07-19 2023-06-02 苏州尔生生物医药有限公司 基于激活的抗原提呈细胞的核酸递送粒子、核酸递送系统及制备方法

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