WO2024032507A1 - 金属-磷脂复合物、金属-磷脂复合物颗粒和药物-脂质颗粒及其制备方法与应用 - Google Patents

金属-磷脂复合物、金属-磷脂复合物颗粒和药物-脂质颗粒及其制备方法与应用 Download PDF

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WO2024032507A1
WO2024032507A1 PCT/CN2023/111279 CN2023111279W WO2024032507A1 WO 2024032507 A1 WO2024032507 A1 WO 2024032507A1 CN 2023111279 W CN2023111279 W CN 2023111279W WO 2024032507 A1 WO2024032507 A1 WO 2024032507A1
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metal
phospholipid complex
phospholipid
lipid
formula
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English (en)
French (fr)
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王珊
胡敦
孙毅毅
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湖南健瑞医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the present disclosure relates to the field of biotechnology, and specifically relates to a metal-phospholipid complex, metal-phospholipid complex particles and drug-lipid particles and their preparation methods and applications.
  • Nucleic acid drugs refer to artificially synthesized DNA or RNA fragments with disease treatment functions. They have attracted much attention because of their huge application potential in disease diagnosis and treatment. Such drugs can directly act on disease-causing target genes or mRNA and exert therapeutic effects at the gene level. Compared with traditional small molecule drugs and antibody drugs, nucleic acid drugs are not limited by the druggability of target proteins, can treat a wider range of diseases, and can regulate the expression of disease-causing genes from the root. Nucleic acid drugs also have obvious advantages such as high efficiency, low toxicity and high specificity, and are expected to become the third largest type of drugs after small molecule drugs and antibody drugs.
  • nucleic acid drugs are easily degraded by nucleases in the body, and due to their large molecular weight and negative charge, they have difficulty in penetrating the cell membrane to work. Therefore, finding a safe and effective nucleic acid drug delivery system is an urgent bottleneck in the development of nucleic acid drugs.
  • vectors that can deliver nucleic acid drugs can be mainly divided into viral vectors and non-viral vectors. Viral vectors will cause an immune response after entering the human body and are rarely used now; the more commonly used non-viral vectors are mainly nanoparticles and small molecule conjugates.
  • nanoparticles are Particles can more effectively package nucleic acid drugs to prevent them from being rapidly degraded by nucleases in the body, thereby improving their circulation time in the body.
  • the mechanism of nanoparticles encapsulating nucleic acids relies on the adsorption of negatively charged nucleic acids by positively charged cationic lipids.
  • cationic lipids are highly cytotoxic, and their toxic mechanisms are: 1 causing cell atrophy, reducing the number of mitoses, and vacuolating the cytoplasm; 2 interacting with biological proteins such as protein kinase C, thereby destroying their activity; 3 through activation p38 mitogen-activated protein kinase and nuclear factor ⁇ B transcription factor trigger the secretion of a variety of pro-inflammatory cytokines and chemokines.
  • ionizable lipids can be used to partially or completely replace cationic lipids as the main component of nanoparticles responsible for the adsorption of nucleic acids.
  • ionizable lipids reduce the cytotoxic and highly pro-inflammatory effects of some permanently positively charged cationic lipids, their cytotoxicity and immunogenicity are still high.
  • Lipid Nanoparticles based on cationic lipids and/or ionizable lipids are nanoparticle nucleic acid drug delivery systems currently available for clinical use, in which cationic lipids and/or ionizable lipids serve as LNPs.
  • the main component is responsible for adsorbing nucleic acids and
  • cytotoxicity and immunogenicity mediated by cationic lipids and/or ionizable lipids are still one of the important reasons for the greater toxicity of LNPs.
  • nanoparticle delivery systems developed based on cationic lipids and/or ionizable lipids cannot fundamentally
  • cationic lipids and/or ionizable lipids cannot fundamentally to solve the toxicity problem of nanoparticle delivery systems, there is an urgent need for a low-toxic liposome delivery system that does not use cationic lipids and/or ionizable lipids.
  • the purpose of this disclosure is to provide a metal-phospholipid complex and a preparation method thereof, so as to alleviate at least one technical problem existing in the prior art.
  • the metal-phospholipid complex provided by the present disclosure is composed of a phospholipid molecule part, a linker molecule part and a metal ion part.
  • the phospholipid molecule part is connected to the linker molecule part, and the linker molecule part and the metal ion part are reacted through coordination. are bonded, and the metal-phospholipid complex is not a cationic lipid or an ionizable lipid.
  • the phospholipid molecule part is selected from lecithin (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), 1-phosphate ceramide (SP) , Phosphatidylinositol (PI), Phosphatidylthreonine (PT), Sphingomyelin (SM), Lysolecithin (LPC), Lysophosphatylethanolamine (LPE), Lysophosphatidylserine (LPS), Lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysosphingomyelin (LSM), sphingosine-1-phosphate (S1P), and A combination of one or more derivatives.
  • the phospholipid molecule part can be, for example, but not limited to, lecithin (PC), lecithin (PC) derivatives, phosphatidylethanolamine (PE), phosphatidylethanolamine (PE) derivatives, phosphatidylglycerol (PG), phosphatidyl Glycerol (PG) derivatives, phosphatidylglycerol (PG) and lecithin (PC), lecithin (PC) and lecithin (PC) derivatives, etc.
  • PC lecithin
  • PC phosphatidylethanolamine
  • PE phosphatidylethanolamine
  • PE phosphatidylglycerol
  • PG phosphatidyl Glycerol
  • PC phosphatidylglycerol
  • PC lecithin
  • PC lecithin
  • the phospholipid molecule part is selected from
  • R1 and R2 are independently:
  • the phospholipid molecule part is selected from lecithin (PC) (Formula 1), phosphatidylethanolamine (PE) (Formula 2), phosphatidic acid (PA) (Formula 4), phosphatidylglycerol (PG) (Formula 5) , and a combination of one or more of its derivatives.
  • PC lecithin
  • PE phosphatidylethanolamine
  • PA phosphatidic acid
  • PG phosphatidylglycerol
  • the phospholipid molecule part is selected from the group consisting of distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidic acid (DSPA), distearoylphosphatidylglycerol ( DSPG), and a combination of one or more of its derivatives.
  • DSPC distearoylphosphatidylcholine
  • DSPE distearoylphosphatidylethanolamine
  • DSPA distearoylphosphatidic acid
  • DSPG distearoylphosphatidylglycerol
  • the phospholipid molecule part is selected from and a combination of one or more of its derivatives.
  • the phospholipid molecule part is selected from DSPC (Formula 46), DSPE (Formula 47) or DSPA (Formula 48) or DSPG (Formula 49).
  • linker molecule part is selected from the group consisting of curcumin, chlorogenic acid, anthocyanins, quercetin, dihydromyricetin, hesperetin, naringenin, apigenin, catechins, tea polyphenols, and epigenate.
  • curcumin chlorogenic acid
  • anthocyanins quercetin
  • dihydromyricetin quercetin
  • dihydromyricetin hesperetin
  • naringenin apigenin
  • catechins catechins
  • tea polyphenols and epigenate.
  • epigallocatechin gallate ellagic acid, morin, epicatechin gallate, epigallocatechin gallate, epigallocatechin gallate, or hempine C, and one of its derivatives or a combination of multiple.
  • linker molecule part is selected from
  • R7 and R8 are H, OH or OCH 3 , R3 is H or sugar group, R4, R5 and R6 are OH or sugar group;
  • linker molecule part is selected from curcumin (formula 19), A combination of one or more.
  • linker molecule part is selected from the group consisting of curcumin (Formula 19), hesperetin (Formula 24), tea polyphenols (Formula 28), and one or more combinations of their derivatives.
  • linker molecule part is selected from curcumin (Formula 19), hesperetin (Formula 24) or tea polyphenols (Formula 28).
  • the metal ion part is selected from Fe 3+ , Ag + , Ba 2+ , Ca 2+ , Cd 2+ , Cu 2+ , Fe 2+ , Mn 2+ , Mg 2+ , Mo 2+ , Zn 2 + , Pt 2+ , Au 2+ , Al 3+ , Ce 3+ , Co 3+ , Cr 3+ , Eu 3+ , Gd 3+ , Ni 3+ , W 3+ , V 3+ , Zr 3+ One or a combination of more.
  • the metal ion part is selected from one or a combination of Fe 3+ , Ca 2+ , and Al 3+ .
  • the metal ion part is selected from Fe 3+ , Ca 2+ or Al 3+ .
  • the metal-phospholipid complex is made of a phospholipid molecule part, a linker molecule part and a metal ion part.
  • the phospholipid molecule part is selected from DSPC, DSPE or DSPA, and the linker molecule part is selected from curcumin and hesperetin. Or tea polyphenols, the metal ion part is selected from Fe 3+ , Ca 2+ or Al 3+ .
  • the metal-phospholipid complex is made of a phospholipid molecule part, a linker molecule part and a metal ion part, and the phospholipid molecule part is selected from DSPC (Formula 46), DSPE (Formula 47) or DSPA (Formula 48),
  • the linker molecule part is selected from curcumin (Formula 19), hesperetin (Formula 24) or tea polyphenols (Formula 28), and the metal ion part is selected from Fe 3+ , Ca 2+ or Al 3+ .
  • the molar ratio of the phospholipid molecule part, the linker molecule part and the metal ion part is 1:1: (0.5 ⁇ 2).
  • the phospholipid molecule part is DSPC (Formula 46), the linker molecule part is selected from curcumin (Formula 19), and the metal ion part is selected from Fe 3+ .
  • the molar ratio of the phospholipid molecule part, the linker molecule part and the metal ion part is 1:1:1.
  • the molar ratio of DSPC, curcumin and Fe 3+ /Al 3+ is 1:1:1.
  • the molar ratio of DSPC, hesperetin and Fe 3+ /Al 3+ is 1:1:1.
  • the molar ratio of DSPC, tea polyphenols and Fe 3+ /Al 3+ is 1:1:2.
  • Fe 3+ is selected from FeCl 3 and Al 3+ is selected from Al(NO 3 ) 3 ⁇ 9H 2 O.
  • the present disclosure provides a method for preparing a metal-phospholipid complex, which includes the following steps:
  • Step 1 React and connect phospholipid molecules and linker molecules to form a phospholipid complex
  • Step 2 React the phospholipid complex prepared in step 1 with metal ions through coordination bonds to form a metal-phospholipid complex.
  • step one the phospholipid molecule and the linker molecule are dissolved in ethanol and reacted, and then n-hexane is added to precipitate to obtain the phospholipid complex.
  • the reaction conditions include reaction at 65°C for 2 hours.
  • the molar ratio of the phospholipid molecule to the linker molecule is 1:1.
  • the metal-phospholipid complex is obtained after the phospholipid complex and the metal ion are dissolved in ethanol and reacted. Further, the reaction conditions include reaction at 60°C for 2 hours.
  • the molar ratio of the phospholipid complex to the metal ion is 1: (1 ⁇ 2).
  • the present disclosure provides a metal-chelated phospholipid complex nanoparticles (MPP), which contains (i) the above-mentioned metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, wherein the inhibition the conjugated lipid to which the particles aggregate is not a cationic lipid or an ionizable lipid; and (iii) a non-cationic lipid or a non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation Lipids.
  • MPP metal-chelated phospholipid complex nanoparticles
  • non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation may be simply referred to as "non-cationic lipids or non-ionizable lipids" quality.
  • conjugated lipids that inhibit particle aggregation include polyethylene glycol (PEG)-lipid conjugates and/or PEG-dialkoxypropyl (DAA).
  • PEG polyethylene glycol
  • DAA PEG-dialkoxypropyl
  • PEG-lipid conjugate is selected from
  • R1 and R2 are independently: oleoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucoyl, arachidoyl or phytanoyl.
  • the PEG-lipid conjugate is selected from one or more combinations of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000 or DSPE-PEG5000.
  • the PEG-lipid conjugate is selected from A combination of one or more.
  • the PEG-lipid conjugate is selected from DSPE-PEG2000 (formula 53), DSPE-PEG700 (Formula 50), DSPE-PEG1000 (Formula 51) or DSPE-PEG5000 (Formula 52).
  • the non-cationic lipid or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation is one or more of cholesterol and its derivatives. combination.
  • non-cationic lipid or non-ionizable lipid in (iii) other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation is cholesterol (Formula 40) and a combination of one or more of its derivatives.
  • non-cationic lipid or non-ionizable lipid in (iii) is cholesterol (Formula 40).
  • non-cationic lipids or non-ionizable lipids in (iii) other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation, in addition to cholesterol include selected from the group consisting of lecithin PC, phosphatidyl Ethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphate ceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysolecithin LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT
  • non-cationic lipids or non-ionizable lipids in (iii) other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation, in addition to cholesterol include: A combination of one or more of its derivatives;
  • R1 and R2 are: oleoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucoyl, arachidoyl or phytanoyl.
  • non-cationic lipids or non-ionizable lipids in (iii) other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation, including cholesterol are selected from DSPC, DSPE, DSPA or A combination of one or more types of DSPG.
  • non-cationic lipid or non-ionizable lipid described in (iii) includes cholesterol (Formula 40) and DSPC (Formula 46).
  • the metal-phospholipid complex particles are composed of (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) in addition to the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation.
  • the molar proportion of the metal-phospholipid complex in the raw material is 10% to 40%, and the conjugated lipid that inhibits particle aggregation is in the raw material
  • the molar proportion of cholesterol is 2% to 10%, the molar proportion of cholesterol in the raw material is 35% to 75%, and the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 0% to 40%.
  • the metal-phospholipid complex particles are composed of (i) metal-phospholipid complex, (ii) Made from conjugated lipids that inhibit particle aggregation and (iii) non-cationic lipids or non-ionizable lipids
  • the molar proportion of the metal-phospholipid complex in the raw material is 5% to less than 10%, which inhibits particle aggregation
  • the molar proportion of the conjugated lipid in the raw material is 2% to 10%
  • the molar proportion of the cholesterol in the raw material is 15% to less than 35%, 35% to 75%, or greater than 75% to 80%, except that
  • the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 0% to 40% or greater than 40% to 51%. or
  • the molar proportion of the metal-phospholipid complex in the raw material is greater than 40% to 50%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, and the molar proportion of cholesterol in the raw material is The ratio is 15% to less than 35%, 35% to 75%, or greater than 75% to 80%, and the molar proportion of the non-cationic lipids or non-ionizable lipids other than cholesterol in the raw materials is 0% to 40% or Greater than 40% to 51%. or
  • the molar proportion of the metal-phospholipid complex in the raw material is 10% to 40%, the molar proportion of the conjugated lipid that inhibits particle aggregation is 2% to 10% in the raw material, and the molar proportion of cholesterol in the raw material It is 15% to less than 35% or greater than 75% to 80%, and the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 0% to 40% or greater than 40% to 51%.
  • the molar proportion of the metal-phospholipid complex in the raw material is 7% to less than 10%, 10% to 30%, or 20% to 30%, preferably 25%.
  • the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10% or 5% to 10%, preferably 10%.
  • the molar proportion of cholesterol in the raw material is 15% to less than 35%, 35% to 56%, or 35% to 55%, preferably 40%.
  • the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 5% to 30%, 25% to 40%, greater than 40% to 45% or 20% to 25%.
  • the molar proportion of the metal-phospholipid complex in the raw material is 15% to 25%
  • the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 4% to 10%
  • the cholesterol is in the raw material.
  • the molar proportion is 40% to 46%
  • the molar proportion of the DSPC in the raw material is 25% to 35%
  • the metal ion part in the metal-phospholipid complex is selected from Fe 3+ .
  • the molar proportion of the metal-phospholipid complex (the metal ion part is Fe 3+ ) in the raw material is 15%, the molar proportion of the conjugated lipid that inhibits particle aggregation is 4% in the raw material, and the cholesterol
  • the molar proportion of the DSPC in the raw material is 46%, and the molar proportion of the DSPC in the raw material is 35%; or the molar proportion of the metal-phospholipid complex (the metal ion part is Fe 3+ ) in the raw material is 25%, so
  • the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 10%, the molar proportion of the cholesterol in the raw material is 40%, and the molar proportion of DSPC in the raw material is 25%.
  • the molar proportion of the metal-phospholipid complex in the raw material is 10% to 30%
  • the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%
  • the cholesterol is in the raw material.
  • the molar proportion of the DSPC in the raw material is 35% to 56%
  • the molar proportion of the DSPC in the raw material is 34% to 40%;
  • the metal ion part in the metal-phospholipid complex is selected from Al 3+ .
  • the molar proportion of the metal-phospholipid complex in the raw material is 10% to 30%
  • the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%
  • the cholesterol is in the raw material.
  • the molar proportion is 35% to 56%
  • the molar proportion of the DSPC in the raw material is 40% to 45%
  • the metal ion part in the metal-phospholipid complex is selected from Al 3+ ; or
  • the molar proportion of the metal-phospholipid complex in the raw material is 10% to 30%, the molar proportion of the conjugated lipid that inhibits particle aggregation is 3% to 10% in the raw material, and the molar proportion of cholesterol in the raw material is 15% to 35%, the DSPC molar proportion in the raw material is 34% to 40% or greater than 40% to 45%, and the metal ion part in the metal-phospholipid complex is selected from Al 3+ ; or
  • the molar proportion of the metal-phospholipid complex in the raw material is 7% to less than 10%
  • the molar proportion of the conjugated lipid that inhibits particle aggregation is 3% to 10% in the raw material
  • the molar proportion of cholesterol in the raw material is The ratio is 15% ⁇ smaller
  • the molar proportion of the DSPC in the raw material is 34% to 40% or greater than 40% to 45%
  • the metal ion part in the metal-phospholipid complex is selected from Al 3+ .
  • the molar proportion of the metal-phospholipid complex (the metal ion part is Al 3+ ) in the raw material is 7%
  • the molar proportion of the conjugated lipid that inhibits particle aggregation is 3% in the raw material
  • the cholesterol is 56%
  • the molar proportion of the DSPC in the raw material is 34%.
  • the metal-phospholipid complex is made of DSPC, curcumin and Fe 3+ in a molar ratio of 1:1:1, the conjugated lipid that inhibits particle aggregation is DSPE-PEG2000, and the non-cationic lipid or non-soluble lipid is The ionized lipids are cholesterol and DSPC.
  • the molar proportion of metal-phospholipid complex in the raw material is 15%, the molar proportion of DSPE-PEG2000 in the raw material is 4%, the molar proportion of cholesterol in the raw material is 46%, and the molar proportion of DSPC in the raw material is 15%.
  • the molar proportion is 35%.
  • the metal-phospholipid complex is made of DSPC, curcumin and Fe 3+ in a molar ratio of 1:1:1, the conjugated lipid that inhibits particle aggregation is DSPE-PEG2000, and the non-cationic lipid or non-soluble lipid is The ionized lipids are cholesterol and DSPC.
  • the molar proportion of metal-phospholipid complex in the raw material is 25%
  • the molar proportion of DSPE-PEG2000 in the raw material is 10%
  • the molar proportion of cholesterol in the raw material is 40%
  • the molar proportion of DSPC in the raw material is 25%.
  • the molar proportion is 25%.
  • the metal-phospholipid complex is made of DSPC, curcumin and Al 3+ in a molar ratio of 1:1:1, the conjugated lipid that inhibits particle aggregation is DSPE-PEG2000, and the non-cationic lipid or non-soluble lipid is DSPE-PEG2000.
  • the ionized lipids are cholesterol and DSPC.
  • the molar proportion of metal-phospholipid complex in the raw material is 7%
  • the molar proportion of DSPE-PEG2000 in the raw material is 3%
  • the molar proportion of cholesterol in the raw material is 56%
  • the molar proportion of DSPC in the raw material is 7%.
  • the molar proportion is 34%.
  • the present disclosure provides a method for preparing the above-mentioned metal-phospholipid complex particles, including (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) removing the metal-phospholipid complex and the
  • the metal-phospholipid complex particles are obtained by mixing non-cationic lipids other than conjugated lipids or non-ionizable lipids that inhibit particle aggregation.
  • the present disclosure provides a drug-lipid particle containing:
  • this metal-phospholipid complex particle is the metal-phospholipid complex particle of the present disclosure.
  • the drug is encapsulated in the metal-phospholipid complex particles.
  • the drug is selected from one or more combinations of nucleic acids, proteins, polypeptides, small molecules, nucleic acid analogs, protein analogs and polypeptide analogs.
  • the nucleic acid is selected from one or more combinations of mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, and artificial nucleic acids.
  • the nucleic acid is the mRNA sequence encoding eGFP (Enhanced Green Fluorescent Protein) shown in SEQ ID No. 1, and the receptor-binding protein encoding the S1 subunit of the new coronavirus shown in SEQ ID No. 2
  • the antisense strand is SEQ ID No.4
  • the sense strand is the siRNA sequence of the Bcl-2 gene (B-cell lymphoma/Leukemia-2 gene, B-cell lymphoma/Leukemia-2) shown in SEQ ID No.
  • the chains are the siRNA sequence of the PLK1 gene (polo-like Kinase 1, Polo-like Kinase 1) shown in SEQ ID No. 23, the siRNA sequence of the Gal-1 gene shown in SEQ ID No. 8, and the siRNA sequence of the Gal-1 gene shown in SEQ ID No. 10.
  • the ASO sequence of the STAT-3 gene shown in SEQ ID No. 12 the ASO sequence of the ⁇ -syn gene ( ⁇ -synuclein, ⁇ -synuclein) shown in SEQ ID No. 12, and the Bcl-2 gene shown in SEQ ID No.
  • the present disclosure provides a method for preparing the above-mentioned drug-lipid particles.
  • the drug is encapsulated in metal-phospholipid complex particles to obtain the drug-lipid particles.
  • metal-phospholipid complexes, conjugated lipids that inhibit particle aggregation, and non-cationic lipids or non-ionizable lipids are dissolved in organic compounds to form an organic phase, and drugs are dissolved in a buffer to form an aqueous phase.
  • the organic phase and the aqueous phase are mixed to obtain drug-lipid particles.
  • the organic compound is ethanol.
  • the buffer is enzyme-free PBS buffer.
  • the mixing method of the organic phase and the aqueous phase includes a microfluidic chip or ultrasound.
  • drug (i) metal-phospholipid complex, (ii) conjugated lipid that inhibits particle aggregation and (iii) non-cationic lipid or non-ionizable lipid are mixed to obtain the drug- lipid particles.
  • the present disclosure provides the use of the above-described metal-phospholipid complexes in nucleic acid delivery systems. Further, the nucleic acid delivery system is used to introduce nucleic acid into cells. Further, the nucleic acid is used to silence the expression of a target sequence in a mammalian subject, to deliver drugs in the body of a mammal, to deliver drugs from the body to mammalian cells, or to treat a disease in a mammal, or disease.
  • the present disclosure provides the use of the above-mentioned metal-phospholipid complex particles or the above-mentioned drug-lipid particles in a composition for delivery of drugs.
  • the composition is used to introduce drugs into cells.
  • the composition is a medicament.
  • the agent is used to silence the expression of a target sequence in a mammalian subject, to deliver drugs in the body of a mammal, to deliver drugs from the body to mammalian cells, or to treat a disease in a mammal, or disease.
  • the present disclosure provides the use of the above metal-phospholipid complex or metal-phospholipid complex particles or drug-lipid particles or medicaments in preventing/treating diseases or conditions in mammals.
  • the mammal is a human.
  • the disease or disorder is associated with the expression of a gene that contains the target sequence of the drug.
  • the disease or condition includes cancer, viral infection, autoimmune disease, diabetes or Alzheimer's disease.
  • the viral infection includes hepatitis A, hepatitis B, hepatitis C, SARS-Cov-2 (2019 new coronavirus), HIV (AIDS virus), HPV (human papillomavirus), influenza, smallpox or syphilis.
  • the cancer includes liver cancer, glioma, melanoma, lung cancer, pancreatic cancer or breast cancer.
  • the agent is a vaccine.
  • the administration route of the agent includes intrathecal injection, intramuscular administration, intracranial injection, intravenous injection or intratumoral injection.
  • a medicament containing the metal-phospholipid complex or metal-phospholipid complex particles or drug-lipid particles of the present disclosure is provided.
  • the medicament is a vaccine.
  • the vaccine is a new coronavirus vaccine.
  • the main function of the metal-phospholipid complex provided by the present disclosure is to adsorb negatively charged drugs, which self-assembles with other lipids into metal-phospholipid complex particles (MPP), ensuring that it is not inferior to those based on cationic lipids and
  • MPP metal-phospholipid complex particles
  • cationic lipids and ionizable lipids are not used. Therefore, compared with LNP, the toxicity is greatly reduced, the biological safety is significantly improved, and it is more conducive to the use of negatively charged drugs in the body. carry.
  • Figure 1-1 shows the percentage of eGFP-positive cells caused by transfection of 293T with eGFP-mRNA@MPP(Fe 3+ ) provided in Example 3.5.1 of the present disclosure
  • Figure 1-2 shows the expression level of RBD caused by transfection of 293T with RBD-mRNA@MPP(Fe 3+ ) provided in Example 3.5.1 of the present disclosure
  • Figures 1-3 show the ability of RBD-mRNA@MPP(Fe 3+ ) provided in Example 3.5.1 of the present disclosure to induce humoral immunity;
  • Figures 1-4 show the ability of NY-ESO-1-mRNA@MPP(Fe 3+ ) provided in Example 3.5.1 of the present disclosure to induce humoral immunity;
  • Figures 1-5 show the ability of RBD-mRNA@MPP(Fe 3+ ) to induce cellular immunity provided in Example 3.5.1 of the present disclosure
  • Figures 1-6 show the ability of NY-ESO-1-mRNA@MPP(Fe 3+ ) to induce cellular immunity provided in Example 3.5.1 of the present disclosure
  • Figures 1-7 show the percentage of eGFP-positive cells caused by transfection of 293T with eGFP-mRNA@MPP (Al 3+ ) provided in Example 3.5.2 of the present disclosure;
  • Figures 1-8 show the expression level of RBD caused by transfection of 293T with RBD-mRNA@MPP(Al 3+ ) provided in Example 3.5.2 of the present disclosure
  • Figures 1-9 show the ability of RBD-mRNA@MPP(Al 3+ ) provided in Example 3.5.2 of the present disclosure to induce humoral immunity;
  • Figures 1-10 show the ability of NY-ESO-1-mRNA@MPP(Al 3+ ) provided in Example 3.5.2 of the present disclosure to induce humoral immunity;
  • Figures 1-11 show the ability of RBD-mRNA@MPP(Al 3+ ) to induce cellular immunity provided in Example 3.5.2 of the present disclosure
  • Figures 1-12 show the ability of NY-ESO-1-mRNA@MPP(Al 3+ ) to induce cellular immunity provided in Example 3.5.2 of the present disclosure
  • Figures 1-13 show the ability of Bcl-2-siRNA@MPP(Fe 3+ ) to silence target genes provided in Example 3.6.1 of the present disclosure
  • Figures 1-14 show the ability of PLK1-siRNA@MPP(Fe 3+ ) provided in Example 3.6.1 of the present disclosure to silence target genes;
  • Figures 1-15 show the ability of Gal-1-siRNA@MPP(Fe 3+ ) to silence target genes provided in Example 3.6.1 of the present disclosure
  • Figures 1-16 show the ability of Bcl-2-siRNA@MPP(Al 3+ ) provided in Example 3.6.2 of the present disclosure to silence target genes;
  • Figure 1-17 shows the ability of PLK1-siRNA@MPP(Al 3+ ) provided in Example 3.6.2 of the present disclosure to silence target genes;
  • Figure 1-18 shows the ability of Gal-1-siRNA@MPP(Al 3+ ) to silence target genes provided in Example 3.6.2 of the present disclosure
  • Figure 1-19 shows the ability of STAT3-ASO@MPP(Fe 3+ ) provided in Example 3.7.1 of the present disclosure to silence cell target genes;
  • Figures 1-20 show the ability of ⁇ -syn-ASO@MPP(Fe 3+ ) provided in Example 3.7.1 of the present disclosure to silence cell target genes;
  • Figure 1-21 shows the ability of Bcl-2-ASO@MPP(Fe 3+ ) provided in Example 3.7.1 of the present disclosure to silence cell target genes;
  • Figure 1-22 shows the ability of STAT3-ASO@MPP(Al 3+ ) provided in Example 3.7.2 of the present disclosure to silence cell target genes;
  • Figure 1-23 shows the ability of ⁇ -syn-ASO@MPP(Al 3+ ) provided in Example 3.7.2 of the present disclosure to silence cell target genes;
  • Figure 1-24 shows the Bcl-2-ASO@MPP(Al 3+ ) silencing cell target provided in Example 3.7.2 of the present disclosure. genetic capabilities
  • Figure 1-25 shows the expression level of S protein caused by transfection of 293T with S-mRNA@MPP(Fe 3+ ) provided in Example 3.8.1 of the present disclosure
  • Figure 1-26 shows the functions of the drugs (dsDNA and ssDNA)-lipid particles (Fe 3+ ) provided in Embodiment 3.8.1 of the present disclosure
  • Figure 1-27 shows the expression level of S protein caused by transfection of 293T with S-mRNA@MPP(Al 3+ ) provided in Example 3.8.2 of the present disclosure
  • Figure 1-28 shows the function of the drug (dsDNA and ssDNA)-lipid particles (Al 3+ ) provided in Embodiment 3.8.2 of the present disclosure
  • Figure 2-1 is a differential scanning calorimetry diagram of the phospholipid complex provided in Example 4.1 of the present disclosure
  • Figure 2-2 is an ultraviolet absorption diagram of the metal-phospholipid complex (Fe 3+ ) provided in Example 4.1 of the present disclosure
  • Figure 2-3 is an ultraviolet absorption diagram of the metal-phospholipid complex (Al 3+ ) provided in Example 4.2 of the present disclosure
  • Figures 2-5 show the elemental analysis of the drug-metal-phospholipid complex particles (Fe 3+ ) provided in Example 6.1 of the present disclosure
  • Figures 2-6 are electron microscopy analysis of MPP in the drug-metal-phospholipid complex particles (Al 3+ ) provided in Example 6.2 of the present disclosure;
  • Figures 2-7 show the efficiency of encapsulating nucleic acids (mRNA and siRNA) in drug-lipid particles provided in Example 7 of the present disclosure
  • Figures 2-8 show the nucleic acid lysosome escape capabilities of siRNA/mRNA@MPP and siRNA/mRNA@LNP provided in Example 8 of the present disclosure
  • Figures 2-9 show the eGFP-positive cell rates of MPP and LNP provided in Example 9 of the present disclosure
  • Figure 2-10 is a comparison of the ability of MPP and LNP to promote mRNA expression provided in Example 10 of the present disclosure
  • Figure 2-11 is a comparison of the ability to promote humoral immunity between MPP and LNP provided in Embodiment 10 of the present disclosure
  • Figure 2-12 is a comparison of the ability to promote cellular immunity between MPP and LNP provided in Example 10 of the present disclosure
  • Figure 3-1 shows the effect of intratumoral injection of drug-metal-phospholipid complex particles provided in Example 13 of the present disclosure in the treatment of liver cancer.
  • lipid refers to a group of organic compounds that includes, but is not limited to, lipids of fatty acids. They are usually divided into three categories: “simple lipids”, “compound lipids”, and “derivatized lipids”. “Simple lipids” which include fats and oils as well as waxes; “compound lipids” which include phospholipids and glycolipids; “derivatized lipids” such as steroids.
  • lipid vesicle refers to any lipid composition useful for delivering a compound, including, but not limited to, liposomes, in which the aqueous volume is enclosed by an amphiphilic lipid bilayer; or in which lipids are coated Interiors that include macromolecular components, such as mRNA, with a reduced aqueous interior; or lipid aggregates or micelles, in which the encapsulated components are contained within a relatively chaotic lipid mixture.
  • MPP metal-phospholipid complex particles
  • Drugs such as nucleic acid mRNA, are encapsulated in MPP as encapsulated components. This "encapsulation" can be fully encapsulated and /or partially encapsulated.
  • phospholipid refers to lipids containing phosphate groups, which are complex lipids and are also called phospholipids and phospholipids.
  • Phospholipids are the main components of biological membranes and are divided into two categories: glycerophospholipids and sphingomyelin, which are composed of glycerol and sphingosine respectively.
  • Phospholipids are amphiphilic molecules with a hydrophilic nitrogen- or phosphorus-containing head at one end and a hydrophobic (lipophilic) long hydrocarbon chain at the other end. For this reason, the hydrophilic ends of phospholipid molecules are close to each other and the hydrophobic ends are close to each other. They often form a phospholipid bilayer with other molecules such as proteins, glycolipids, and cholesterol, which is the structure of the cell membrane.
  • part of a phospholipid molecule refers to the structure of the phospholipid molecule that is originally part of the phospholipid molecule after reacting with other substances.
  • linker molecule part refers to the structure that originally belongs to the linker molecule after the linker molecule reacts with other substances.
  • metal ion moiety refers to the structure of the metal ion originally belonging to the metal ion after the metal ion moiety reacts with other substances.
  • phospholipid complex refers to a complex formed by the reaction connection between the above-mentioned phospholipid molecule part having a phosphate group and the above-mentioned linker molecule part.
  • metal-phospholipid complex refers to a compound composed of the above-mentioned phospholipid molecule part having a phosphate group, the above-mentioned linker molecule part, and the above-mentioned metal ion part, the above-mentioned phospholipid molecule part is connected to the above-mentioned linker molecule part, and the above-mentioned The linker moiety is connected to the above-mentioned metal ion moiety through a coordination bond, and the metal-phospholipid complex is neither a cationic lipid nor an ionizable lipid.
  • ionizable lipid refers to a lipid containing positively charged ionizable amine groups that can be protonated to become positively charged at lower pH values but uncharged at physiological pH conditions.
  • neutral lipid refers to any of a number of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH.
  • lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol.
  • anionic lipid refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutaryl Phosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic groups attached to neutral lipids.
  • phosphatidylglycerol cardiolipin
  • diacylphosphatidylserine diacylphosphatidic acid
  • N-dodecanoylphosphatidylethanolamine N-succinylphosphatidylethanolamine
  • N-glutaryl Phosphatidylethanolamine N-glutaryl Phos
  • cationic lipid refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH. These lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”); N-(2,3-dioleyloxy)propyl)- N,N,N-trimethylammonium chloride (“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(2,3- Dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);3-(N-(N',N'-dimethylaminoethane)carbamoyl )Cholesterol (“DC-Chol”) and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylam
  • hydrophobic lipid refers to compounds having non-polar groups including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and which groups are optionally separated by one or more aromatic, alicyclic substituted by a cyclic or heterocyclic group. Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane and 1,2-dialkyl-3-amino Propane.
  • non-cationic lipid or non-ionizable lipid refers to a lipid that is neither a cationic lipid nor a non-ionizable lipid, for example, it can be an anionic lipid or a neutral lipid.
  • non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation means, exclude The remaining lipids in the metal-phospholipid complex particles after simultaneous elimination of conjugated lipids that inhibit particle aggregation.
  • the term "fusogenic” refers to the ability of liposomes, drug-lipid particles or other drug delivery systems to fuse with cell membranes.
  • the membrane may be a plasma membrane or a membrane surrounding cellular organelles such as endosomes, nuclei, etc.
  • non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation are present primarily as vesicle-forming lipids
  • the term "forming Vesicle lipids" tend to include any amphiphilic lipid that has a hydrophobic portion and a polar head group and that can itself spontaneously form bilayer vesicles in water, examples being most phospholipids.
  • the conjugated lipids that inhibit particle aggregation are present primarily as vesicle-adopting lipids, and the term "vesicle-adopting lipid” is intended to include any amphiphile that is stably associated with the lipid bilayer Hydrophilic lipids, as well as other amphipathic lipids, have their hydrophobic portions in contact with the inner, hydrophobic regions of the bilayer membrane, and their polar headgroup portions face toward the outer, polar surface of the membrane.
  • Lipids employing vesicles include lipids that can independently adapt to adopt a non-lamellar phase and can also adopt a bilayer structure in the presence of a bilayer stabilizing component.
  • Conjugated lipids that inhibit drug-lipid particle aggregation include, but are not limited to, polyamide oligomers (eg, ATTA-lipid derivatives), peptides, proteins, detergents, lipid derivatives, PEG- Lipid derivatives such as PEG coupled to dialkoxypropyl, PEG coupled to diacylglycerol, PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramide (see, U.S. Patent No. 5,885,613 , which is incorporated herein by reference).
  • polyamide oligomers eg, ATTA-lipid derivatives
  • peptides e.g, proteins
  • detergents lipid derivatives
  • PEG- Lipid derivatives such as PEG coupled to dialkoxypropyl, PEG coupled to diacylglycerol, PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramide (see, U.S. Patent No. 5,885,613 , which is incorporated herein
  • amphiphilic lipid refers to any suitable material in which the hydrophobic portion of the lipid material faces the hydrophobic phase and the hydrophilic portion faces the aqueous phase. Amphiphilic lipids are often the major components of lipid vesicles.
  • the hydrophilic nature results from the presence of polar or charged groups such as carbohydrates, phosphates, carboxyl, sulfate, amino, sulfhydryl, nitro, hydroxyl and other similar groups.
  • Hydrophobicity can be imparted by the inclusion of non-polar groups including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and by one or more aromatic, alicyclic or heterocyclic groups. Such a group is replaced by a group.
  • amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.
  • phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidyl Ethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine.
  • amphipathic lipids Other compounds lacking phosphorus, such as sphingomyelins, the glycosphingolipid family, diacylglycerols and beta-acyloxy acids are also in the group known as amphipathic lipids. Additionally, the amphiphilic lipids described above can be mixed with other lipids, including triglycerides and sterols.
  • diacylglycerol refers to a compound having a 2-fatty acyl chain in which R1 and R2 each independently have 2 to 30 carbon atoms bonded via ester bonds to the 1- and 2-positions of the glycerol.
  • the acyl group may be saturated or have varying degrees of unsaturation.
  • Diacylglycerol has the following formula 54:
  • the conjugated lipid that inhibits particle aggregation in the present disclosure may be a diacylglycerol-conjugated polyethylene glycol, i.e., a diacylglycerol-polyethylene glycol conjugate.
  • substance DAG-PEG conjugate or PEG-DAG conjugate.
  • the DAG-PEG conjugate is dilaurylglycerol (C12)-PEG conjugate, distetradecylglycerol (C14)-PEG conjugate (DMG), dipalmitoylglycerol (C16)-PEG conjugate or distearylglycerol (C18)-PEG conjugate (DSG).
  • DMG distetradecylglycerol
  • DSG distearylglycerol
  • Suitable DAG-PEG conjugates for use in the present disclosure and methods of making and using them are disclosed in U.S. Application No. 10/136,707, published as U.S.P.A. 2003/0077829, and PCT Patent Application No. CA 02/00669, The entire contents of each are incorporated by reference.
  • dialkoxypropyl refers to compounds having a 2-alkyl chain in which R1 and R2 each independently have 2 to 30 carbons. Alkyl groups may be saturated or have varying degrees of unsaturation. Dialkoxypropyl has the following formula 55:
  • dialkoxypropyl-coupled PEG the conjugated lipid that inhibits particle aggregation in the present disclosure may be a dialkoxypropyl-coupled PEG, i.e., a dialkoxypropyl conjugate ( PEG-DAA conjugate).
  • PEG-DAA conjugate a dialkoxypropyl conjugate
  • the PEG-DAA conjugate has the following formula 56:
  • R1 and R2 are independently selected and are long chain alkyl groups having from about 10 to about 22 carbon atoms. Long chain alkyl groups may be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, lauryl (C12), tetradecyl (C14), hexadecyl (C16), octadecyl (C18) and icosyl (C20). In a preferred embodiment, R1 and R2 are the same, that is, R1 and R2 are both tetradecyl (i.e., ditetradecyl), and R1 and R2 are both octadecyl (i.e., dioctadecyl) wait.
  • tetradecyl i.e., ditetradecyl
  • R1 and R2 are both octadecyl (i.e., dioctadecyl) wait.
  • PEG is a polyethylene glycol having an average molecular weight of about 550 to about 10,000 daltons and is optionally substituted at the terminal hydroxyl position with an alkyl, alkoxy, acyl, or aryl group.
  • the PEG has an average molecular weight of about 1,000 to about 5,000 Daltons, more preferably, an average molecular weight of about 1,000 to about 3,000 Daltons and even more preferably, an average molecular weight of about 2,000 Daltons. molecular weight.
  • PEG may be optionally substituted with alkyl, alkoxy, acyl or aryl groups.
  • L is the joint part.
  • linker moiety suitable for coupling PEG to the dialkoxypropyl backbone may be used.
  • Suitable linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbonate (OC(O)O-), Carbamate (-NHC(O)O-), urea (-NHC(O)NH-), succinyl (-(O)CCH 2 CH 2 C(O)-), ethers, disulfides and others Group combine.
  • Other suitable linkers are well known in the art.
  • Phosphatidylethanolamines with various acyl groups of varying chain lengths and degrees of saturation can be conjugated to polyethylene glycol to form a bilayer stabilizing component as a conjugated lipid to inhibit particle aggregation in the present disclosure. chain group.
  • These phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art.
  • Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths in the C10-C20 range are preferred. It is also possible to use phosphatidylethanolamines having mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids.
  • Suitable phosphatidylethanolamines include, but are not limited to, the following: dismyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoylphosphatidyl Ethanolamine (DSPE).
  • DMPE dismyristoylphosphatidylethanolamine
  • DPPE dipalmitoylphosphatidylethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • DSPE distearoylphosphatidyl Ethanolamine
  • ceramides with polyethylene glycols of varying chain lengths and saturation levels can be coupled to polyethylene glycol to serve as conjugated lipids that inhibit particle aggregation in the present disclosure, forming a bilayer stabilizing component.
  • acyl chain group It will be clear to those skilled in the art that, in contrast to phosphatidylethanolamine, ceramide has only one acyl group, which can be easily varied depending on its chain length and degree of saturation.
  • Ceramides suitable for use in accordance with the present disclosure are commercially available. Furthermore, ceramides can be isolated from eggs and brain, for example, using well-known isolation techniques, or synthesized using the methods and techniques disclosed in US Patent No. 5,820,873, which is incorporated herein by reference. Ceramides with saturated or unsaturated fatty acids having carbon chain lengths in the range C2-C31 can be prepared using the synthetic routes proposed in the aforementioned applications.
  • R is a member selected from the group consisting of hydrogen, alkyl and acyl
  • R1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R1 and the nitrogen atom to which they are bonded form Azide moiety
  • R2 is a member selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl and amino acid side chains
  • R3 is selected from the group consisting of hydrogen, halogen, hydroxyl, alkoxy, mercapto , a member of the group consisting of hydrazine, amino, and NR4R5, wherein R4 and R5 are independently hydrogen or alkyl
  • n is 4-80
  • m is 2-6
  • p is 1-4
  • q is 0 or 1.
  • mRNA or “messenger polyribonucleotide” or “messenger RNA” or “messenger RNA” are used interchangeably and mean that a strand of DNA is transcribed using a strand of DNA as a template, carrying a Genetic information, single-stranded polyribonucleotides that direct protein synthesis.
  • sgRNA small guide RNA
  • guide RNA guide RNA
  • gRNA small non-coding RNA that can pair with pre-mRNA.
  • gRNA edits RNA molecules, approximately 60-80 nucleotides in length, that are transcribed by individual genes.
  • RNA cyclic polyribonucleotide
  • circular RNA means having no free ends (i.e., no free 3' and/or 5' end) structure of the polyribonucleotide molecule, for example, a polyribonucleotide molecule that forms a cyclic or cyclic structure through covalent or non-covalent bonds.
  • microRNA or “miRNA” or “microRNA” are used interchangeably and mean a non-coding, single-stranded polyribose of approximately 22 nucleotides in length with free 3' and 5' ends. Nucleotides can inhibit the target gene protein by binding to the 3'-untranslated region (3'-UTR) of the target gene's mRNA. Translation in turn regulates the biological functions of cells.
  • 3'-UTR 3'-untranslated region
  • ASO antisense oligonucleotide
  • antisense oligonucleotide or “antisense oligonucleotide” are used interchangeably and mean a synthetic nucleic acid fragment that is complementary to a target gene or a certain segment of mRNA.
  • RNA small interfering or “short interfering” or “silencing RNA” or “small interfering RNA” or “silencing RNA” are used interchangeably and mean It is a type of double-stranded RNA molecule with a length of 20 to 25 nucleotides and can induce the degradation of target gene mRNA.
  • ecDNA or "extrachromosomal circular DNA” are used interchangeably and mean DNA that has been detached from a chromosome and exists in a circular structure outside the chromosome.
  • nucleic acid derivatives refers to modifications or substitutions of nucleic acid sequences, including but not limited to chemical modifications of residues, substitutions of nucleotides or deoxynucleotides, modifications of the sequence to increase half-life or stability, and labeling modifications .
  • chemical modifications include, but are not limited to, phosphorylation, methylation, amination, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopeation of any one or more bases.
  • substitutions for nucleotides or deoxynucleotides include, but are not limited to, nucleic acid analogs that replace the sugar-phosphate backbone with a polypeptide or other backbone (DNA or RNA replaced with PNA).
  • Modifications to the sequence to increase half-life or stability include, but are not limited to, PEG-linked modifications and fluorine modifications.
  • Label modifications include but are not limited to connecting fluorescent groups, amino groups, biotin, digoxigenin, small peptides, etc.
  • artificial nucleic acid a nucleic acid molecule that has been artificially modified, including but not limited to base modification, ribose modification, PNA, etc.
  • nucleic acid refers to a polymer containing at least two deoxynucleotides or nucleotides in single or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise stated, a specific nucleic acid sequence also implicitly encompasses its conservatively modified variants (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequences explicitly stated.
  • degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is composed of mixed bases and/or deoxyinosine residues. Substituted (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • "Nucleotide” includes the sugars deoxyribose (DNA) or ribose (RNA), bases, and phosphate groups.
  • Nucleotides are linked through phosphate groups.
  • “Base” includes purine and pyrimidine, which further includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include, but Modifications are not limited to substitution of new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.
  • DNA can be used as antisense, plasmid DNA, parts of plasmid DNA, pre-compressed DNA, polymerase chain reaction (PCR) products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, Chromosomal DNA or derivatives of these groups exist.
  • PCR polymerase chain reaction
  • P1, PAC, BAC, YAC, artificial chromosomes vectors
  • expression cassettes chimeric sequences, Chromosomal DNA or derivatives of these groups exist.
  • nucleic acid is used interchangeably with gene, cDNA, mRNA encoded by a gene, and interfering RNA molecules.
  • gene refers to a nucleic acid (e.g., DNA or RNA) sequence that includes a partial or full-length coding sequence for producing a polypeptide or polypeptide precursor (e.g., from A, B, C, D, E, Required for hepatitis G virus; or a polypeptide or polypeptide precursor of herpes simplex virus).
  • a nucleic acid e.g., DNA or RNA
  • a partial or full-length coding sequence for producing a polypeptide or polypeptide precursor e.g., from A, B, C, D, E, Required for hepatitis G virus; or a polypeptide or polypeptide precursor of herpes simplex virus.
  • gene product refers to the product of a gene such as a transcript including, for example, DNA, mRNA.
  • siRNAs of the present disclosure refers to the ability of the siRNAs of the present disclosure to initiate silencing of a target gene.
  • samples or assays from cells of a target organism or culture that express a particular construct are compared to control samples that do not express the construct.
  • Control samples (lacking expression of the construct) were set as a relative value of 100%. Inhibition of the expression of the target gene is successfully obtained when the test value relative to the control is about 90%, preferably 50%, more preferably 25%-0%.
  • Suitable assays include, for example, using techniques known to those skilled in the art such as spotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzymatic function, as well as phenotypic assays known to those skilled in the art to detect protein or mRNA levels.
  • a “therapeutically effective amount” or “effective amount” of siRNA is an amount sufficient to produce a desired effect, such as a reduction in target sequence expression compared to normal expression levels detected in the absence of siRNA.
  • aqueous solution refers to a composition containing, in whole or in part, water.
  • organic lipid solution refers to a composition containing in whole or in part an organic solvent with lipids.
  • systemic delivery refers to delivery that results in widespread biodistribution of a compound in an organism. Some administration techniques can result in systemic delivery of some compounds but not others. Systemic delivery means that an effective, preferably, therapeutic amount of the compound contacts a substantial portion of the body. To achieve broad biodistribution, blood survival is generally required such that the compound is not rapidly degraded or cleared (such as by primary passage through organs (liver, lungs, etc.)) or rapidly passes through before reaching disease sites distal to the site of administration. , non-specific cell binding).
  • Systemic delivery of drug-lipid particles can be performed in any manner known in the art, including, for example, intravenously, subcutaneously, intraperitoneally, and in a preferred embodiment, systemic delivery of drug-lipid particles It is delivered intravenously.
  • local delivery refers to the delivery of a compound directly to a target site within an organism.
  • compounds can be delivered locally by injection directly into the site of disease, such as a tumor, or other target site, such as a site of inflammation, or a target organ, such as the liver, heart, pancreas, kidneys, and the like.
  • the metal-phospholipid complex is composed of a phospholipid molecule moiety, a linker molecule moiety, and a metal ion moiety.
  • the cis-trans isomers of the phospholipid molecules of the present disclosure will not affect the effect to be achieved by the protection content of the present disclosure.
  • the linker molecule part is mainly derived from natural plant extracts, such as curcumin, which has a wide range of biological effects, including antibacterial, antiviral, antifungal, antioxidant and anti-inflammatory activities.
  • curcumin which has a wide range of biological effects, including antibacterial, antiviral, antifungal, antioxidant and anti-inflammatory activities.
  • it is also an effective immunomodulator, which can regulate the activity of T cells, B cells, macrophages, neutrophils, natural killer cells, dendritic cells and other immune cells to promote the balance of immunity. Enhance the body's immunity.
  • the curcumin molecule is extremely safe and has been listed in the catalog of food additives and pharmaceutical excipients. Its safety is conducive to the overall clinical drug registration of drug-lipids and shortens the length of clinical drug registration.
  • the proportion of each component in the metal-phospholipid complex can be adjusted according to the structure of the specific metal-phospholipid complex components.
  • the basis for adjusting the dosage ratio is: because the phospholipid molecules and the linker molecules are connected by hydrogen bonds, as long as the phospholipid molecules contain multiple phosphate groups, then when synthesizing the phospholipid complex, the dosage ratio of the phospholipid molecules and the linker molecules can be based on the phospholipid molecules.
  • the number of phosphate groups contained is adjusted, that is, when the phospholipid molecule contains two phosphate groups, the ratio of phospholipid molecules and linker molecules can be adjusted to 1:2; when the phospholipid molecule contains three phosphate groups, The ratio of phospholipid molecules and linker molecules can be adjusted to 1:3; because the hydroxyl groups of the linker molecules and metal ions are connected by coordination bonds, as long as the linker molecule contains multiple binding sites, then the linker molecules and metal ions The delivery ratio can be adjusted based on the number of binding sites contained in the linker.
  • phospholipid molecules with phosphate groups are connected to linker molecules to obtain phospholipid complexes, and then connected to metal ions to obtain metal-phospholipid complexes.
  • the phospholipid molecules and the linker molecules are dissolved in an appropriate amount of ethanol according to the molar ratio, reacted at about 65°C, and then n-hexane is added to precipitate the phospholipid complex; the phospholipid complex is then mixed with metal ions (such as FeCl 3, etc.) are dissolved in an appropriate amount of ethanol according to the molar ratio, and the metal-phospholipid complex can be obtained by reacting at about 60°C.
  • the principle of loading nucleic acids into metal-phospholipid complex particles assembled from metal-phospholipid complexes is that the linker molecules and phospholipid molecules are bonded together through hydrogen bonds, and at the same time, the linker molecules are connected to the metal ions through coordination bonds, forming a metal-phospholipid complex particle.
  • the metal ions of the metal-phospholipid complex are linked to the negatively charged drug through coordination bonds, thereby ensuring that the metal-phospholipid complex is connected with other components (conjugated lipids that inhibit particle aggregation, and in addition to Metal-phospholipid complexes and non-cationic lipids other than conjugated lipids that inhibit particle aggregation or non-ionizable lipids) self-assemble into MPP while loading negatively charged drugs into the nanoparticle MPP.
  • the "non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation” refers to the components in the metal-phospholipid complex particles.
  • a conjugated lipid that inhibits particle aggregation refers to a conjugated lipid that inhibits drug-lipid particle aggregation and whose primary function is to prevent drug-lipid particle aggregation, such as with dialkoxypropane PEG coupled to hydroxyl, PEG coupled to diacylglycerol, PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramide, preferably PEG-lipid conjugates.
  • dialkoxypropane PEG coupled to hydroxyl PEG coupled to diacylglycerol
  • PEG coupled to phosphatidylethanolamine PEG conjugated to ceramide, preferably PEG-lipid conjugates.
  • the cis-trans isomers of lipids will not affect the effect to be achieved by the protection content of this disclosure.
  • the molar proportion of the metal-phospholipid complex in the raw material is 5% to less than 10%, 10% to 40%, or greater than 40% to 50%, such as 5%, 6%, 7%, 8% , 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25 %, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%.
  • the molar proportion of the metal-phospholipid complex in the raw material is 7% to 40%, for example, it can be 10% to 40%, 7% to 30%, 15% to 25%, or 20% to 30%. %, more preferably 15%, 25%, 7% or 30%.
  • the molar proportion of the conjugated lipid that inhibits particle aggregation in the feedstock is 2% to 10%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.
  • the content may be 3% to 10%, 4% to 10%, or 5% to 10%, and more preferably 3%, 4%, or 10%.
  • the non-cationic lipid or non-ionizable lipid is cholesterol
  • the molar proportion of cholesterol in the raw material is 15% to less than 35%, 35% to 75%, or greater than 75% to 80%, such as 15% ,16%,17%,18%,19%,20%,21%,22%,23%,24%,25%,26%,27%,28%,29%,30%,31%,32 %, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65% , 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or
  • the metal-phospholipid complex particles may optionally contain other non-cationic lipids or non-ionizable lipids, whose molar proportion in the raw material is 0% to 40% or greater than 40%.
  • % ⁇ 51% such as 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% ,15%,16%,17%,18%,19%,20%,21%,22%,23%,24%,25%,26%,27%,28%,29%,30%,31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or 51%.
  • it is 5% to 30%, 25% to 35%, 34% to 45%, 20%-25%, preferably 25%, 35%,
  • Drug-lipid particles described herein typically include drugs (which are negatively charged molecules that may be selected from the group consisting of nucleic acids, proteins, polypeptides, small molecules, nucleic acid analogs, protein analogs, and polypeptide analogs).
  • the nucleic acid is a member of the group of one or more combinations selected from the group consisting of mRNA, siRNA, circular RNA, microRNA, DNA, ecDNA, artificial nucleic acids), metal-phospholipid complex substances, non-cationic lipids or non-ionizable Lipids, and bilayer stabilizing components, such as conjugated lipids that inhibit particle aggregation.
  • nucleic acids encapsulated in the drug-lipid particles of the present disclosure are resistant to degradation by nucleases in aqueous solution.
  • the drug is sufficiently encapsulated within the metal-phospholipid complex particles to avoid drug degradation and enable drug delivery into cells.
  • the drug-lipid particles provided by the present disclosure have a small diameter suitable for systemic delivery, with a particle size of 30 to 400 nm; a surface potential of -10 to 10 mV; and a stability of at least 3 days, preferably up to More than 7 days; cell delivery efficiency of at least 40%, such as at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95 %.
  • the drug of the drug-lipid particle is preferably a nucleic acid
  • the nucleic acid component typically includes mRNA, interfering RNA (i.e., siRNA), which may be provided in several forms, including, for example, one or more An isolated small interfering RNA (siRNA) duplex, a longer double-stranded RNA (dsRNA), or siRNA or dsRNA translated from a transcription cassette in a DNA plasmid.
  • siRNA interfering RNA
  • RNA populations can be used to provide long precursor RNAs, or long precursor RNAs that are substantially or completely identical to a selected target sequence that can be used to prepare siRNA.
  • the RNAs can be isolated from cells or tissues, synthesized, and/or cloned according to methods well known to those skilled in the art.
  • the RNA may be a mixed population (obtained from cells or tissues, transcribed from cDNA, etc.), or may represent a single target sequence.
  • the RNA may be naturally occurring, eg, isolated from a tissue or cell sample, synthesized in vitro, eg, using T7 or SP6 polymerase and a PCR product or cloned cDNA, or chemically synthesized.
  • the complement can also be transcribed in vitro and hybridized to form dsRNA.
  • RNA complements are also provided (e.g., to form dsRNA, which is cleaved by E. coli RNAse III or enzymes for digestion).
  • the precursor RNA then hybridizes to form double-stranded RNAs for digestion.
  • the dsRNAs can be directly encapsulated in SNALPs or can be digested in vitro before encapsulation.
  • one or more DNA plasmids encoding one or more siRNA templates can be encapsulated in nucleic acid-lipid particles.
  • siRNA can be transcribed from a DNA template in a plasmid with an RNA polymerase to a sequence that automatically folds into a duplex with a hairpin loop. III transcription unit (see, Brummelkamp, et al., Science 296:550 (2002); Donzé, et al., Nucleic Acids Res. 30:e46 (2002); Paddison, et al., Genes Dev.
  • the transcription unit or cassette will comprise an RNA transcription promoter sequence, such as an H1-RNA or U6 promoter, and a termination sequence operably linked to a template for the transcription of the siRNA sequence required, including 2-3 uridine residues and a polythymidine (T5) sequence (polyadenylation signal) (Brummelkamp, Science, supra).
  • Selected promoters can provide constitutive or inducible transcription.
  • Compositions and methods of DNA-guided transcription of RNA interference molecules are described in detail in U.S. Patent No. 6,573,099, which is incorporated herein by reference.
  • the synthetic or transcribed siRNA has a 3' overhang of about 1-4, preferably about 2-3 nucleotides and a 5' phosphate terminus (Elbashir, et al., Genes Dev. 15: 188(2001); et al., Cell 107:309(2001)).
  • the transcription unit is incorporated into a plasmid or DNA vector from which the interfering RNA is transcribed. Plasmids suitable for delivering genetic material in vivo for therapeutic purposes are described in detail in U.S. Patent Nos. 5,962,428 and 5,910,488, both of which are incorporated herein by reference. Selected plasmids can provide transient or stable delivery to target cells. It will be apparent to those skilled in the art that plasmids originally designed to express the desired gene sequence can be modified to contain a transcription unit cassette for transcribing siRNA.
  • RNA, synthesizing RNA, hybridizing nucleic acids, preparing and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al. al., supra), the same is true for PCR methods (see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)).
  • Expression libraries are also well known to those skilled in the art.
  • metal-phospholipid complexes Applications of metal-phospholipid complexes, metal-phospholipid complex particles, and drug-lipid particles
  • metal-phospholipid complexes and metal-phospholipid complex particles can be used to promote drug lysosomal escape and promote nucleic acid expression.
  • Metal-phospholipid complexes and metal-phospholipid complex particles can also be used to deliver drugs and introduce drugs into cells, thereby achieving the prevention and treatment of applicable diseases or conditions.
  • the present disclosure provides for use of such drug-lipid particles, for example, in compositions that enable the delivery of a drug or the introduction of a drug into a cell.
  • the composition is, for example, a medicament that can: silence the expression of a target sequence in a mammalian subject, deliver drugs (eg, drugs for treating tumors, imaging agents, etc.) in the body of a mammal, or deliver drugs from the body to lactation Animal cells or treatment of mammalian diseases or conditions, etc.
  • drug-lipid particles are the main active ingredients, which can be prepared into different dosage forms according to actual needs through different pharmaceutically acceptable excipients or preparation processes, such as solid dosage forms (powders, granules, pills, tablets, glue), semi-solid dosage forms (external ointments, pastes), liquid dosage forms (decoctions, mixtures, syrups, wines, injections), gas dosage forms (aerosols, smokes), etc.; for example, administration through the gastrointestinal tract
  • solid dosage forms pellets, granules, pills, tablets, glue
  • semi-solid dosage forms external ointments, pastes
  • liquid dosage forms decoctions, mixtures, syrups, wines, injections
  • gas dosage forms as aerosols, smokes
  • the present disclosure provides products prepared from the above metal-phospholipid complexes, metal-phospholipid complex particles, and drug-lipid particles, the product having metal-phospholipid complexes, metal-phospholipid complex particles, and
  • the above-mentioned functions and uses of drug-lipid particles can be of specific types, such as but not limited to kits, pharmaceuticals, etc.
  • the product optionally also contains other excipients.
  • target genes of drug-lipid particle action Generally, it is ideal to deliver drug-lipid particles such that the translation (ie, expression) of the target gene product is down-regulated or silenced.
  • Suitable classifications of gene products include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., diseases and disorders in which the liver is targeted, and liver diseases and disorders), and Genes related to tumorigenesis and cell transformation, angiogenesis genes, immune modulator genes such as those related to inflammation and autoimmune responses, ligand receptor genes and genes related to neurodegenerative disorders.
  • Genes associated with viral infection and survival include those expressed by the virus to bind to, enter, and replicate in cells.
  • viral sequences associated with chronic viral diseases include those of hepatitis viruses (Hamasaki, et al., FEBS Lett. 543:51 (2003); Yokota, et al., EMBO Rep. 4:602 (2003); Schlomai, et al., Hepatology 37:764 (2003); Wilson, et al., Proc. Natl. Acad. Sci. 100: 2783 (2003); Kapadia, et al., Proc. Natl. Acad. Sci.
  • Exemplary hepatitis virus nucleic acid sequences that can be silenced include, but are not limited to: nucleic acid sequences involved in transcription and translation (e.g., En1, En2, X, P), nucleic acid sequences encoding structural proteins (e.g., including C and C-related Core protein of a protein; capsid and envelope proteins including S, M, and/or L proteins, or fragments thereof) (see, eg, FIELDS VIROLOGY, 2001, supra).
  • Hepatitis C nucleic acid sequences that can be silenced include, but are not limited to: serine proteases (e.g., NS3/NS4), helicases (e.g., NS3), polymerases (e.g., NS5B), and envelope proteins (e.g., E1, E2 , and p7).
  • serine proteases e.g., NS3/NS4
  • helicases e.g., NS3
  • polymerases e.g., NS5B
  • envelope proteins e.g., E1, E2 , and p7.
  • the hepatitis A nucleic acid sequence is mentioned, for example, in the Genbank accession number NC_001489; the hepatitis B nucleic acid sequence is mentioned, for example, in the Genbank accession number NC_003977; the hepatitis C nucleic acid sequence is mentioned, for example, in the Genbank accession number NC_003977
  • the hepatitis D nucleic acid sequence is mentioned in, for example, Genbank accession number NC_001653; the hepatitis E nucleic acid sequence is mentioned, for example, in Genbank accession number NC_001434; and the hepatitis G nucleic acid sequence is mentioned, for example, in Genbank accession number NC_001710 mentioned in.
  • Silencing sequences encoding genes involved in viral infection and survival can be conveniently used in conjunction with the administration of conventional agents used to treat viral diseases.
  • Genes associated with metabolic diseases and disorders include, for example, in dyslipidemia (e.g., liver X receptors (e.g., LXR ⁇ and LXR ⁇ Genback accession number NM_007121)), Farnesoid X receptor (FXR) (Genbank accession number NM_005123), sterol regulatory element binding protein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl-CoA reductases (HMG coenzyme-A reductase, apolipoprotein (ApoB), and apolipoprotein (ApoE)) and genes expressed in diabetes (e.g., glucose-6-phosphate) (see, e.g., Forman et al ., Cell 81: 687 (1995); Seol et al., Mol.
  • dyslipidemia e.g., liver X receptors (e.g., LXR ⁇ and LXR ⁇ Genback accession number NM
  • genes associated with metabolic diseases and disorders include genes expressed in the liver itself as well as genes expressed in other organs and tissues Silencing of sequences encoding genes associated with metabolic diseases and disorders may be conveniently used in conjunction with the administration of conventional agents for the treatment of said diseases or disorders.
  • genes associated with tumorigenesis and cellular transformation include translocation sequences such as the MLL fusion gene, BCR-ABL (Wilda, et al., Oncogene, 21:5716 (2002); Scherr, et al., Blood 101:1566), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO, and AML1-MTG8 (Heidenreich, et al., Blood 101:3157 (2003)); overexpressed sequences such as multidrug Resistance genes (Nieth, et al., FEBS Lett. 545: 144 (2003); Wu, et al., Cancer Res.
  • Silencing sequences encoding DNA repair enzymes is used in conjunction with administration of chemotherapeutic agents (Collis, et al., Cancer Res. 63:1550 (2003)).
  • Genes encoding proteins associated with tumor migration are also target sequences of interest. Any complete or partial gene sequence that facilitates or promotes tumorigenesis or cell transformation, tumor growth or tumor migration can be included as a template sequence.
  • VEGF Vascular endothelial growth factor
  • Immunomodulator genes are genes that regulate one or more immune responses.
  • immunomodulator genes include cytokines such as growth factors (e.g., TGF-alpha, TGF-beta, EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., . , IL-2, IL-4, IL-12 (Hill, et al., J. Immunol. 171: 691 (2003)), IL-15, IL-18, IL-20, etc.), interferons (e.g. , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , etc.) and TNF.
  • growth factors e.g., TGF-alpha, TGF-beta, EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.
  • interleukins e.g., . IL-2, IL-4, IL
  • Fas and Fas ligand genes are also targeted immunomodulator target sequences (Song, et al., Nat. Med. 9:347 (2003)).
  • Genes encoding secondary signaling molecules in hematopoietic and lymphoid cells are also included in the present disclosure, for example, Tec family kinases such as Bruton's tyrosine kinase (Btk) (Heinonen, et al., FEBS Lett. 527:274 ( 2002)).
  • Btk Bruton's tyrosine kinase
  • Cellular receptor ligands include ligands that bind to cell surface receptors (e.g., insulin receptors, EPO receptors, G-protein coupled receptors, receptors with tyrosine kinase activity, cytokine receptors body, growth factor receptor, etc.) to regulate (e.g., inhibit, activate, etc.) physiological pathways in which the receptor is involved (e.g., glucose level regulation, blood cell development, mitogenesis, etc.).
  • cell receptor ligands include cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G-protein coupled receptor ligands, etc.).
  • Trinucleotide repeats e.g., CAG repeats
  • trinucleotide repeats such as spinobulbar muscular atrophy and Huntington's disease (Caplen, et al., Hum. Mol. Genet. 11:175 (2002)).
  • the drugs disclosed herein are delivered parenterally, intravenously, intramuscularly, subcutaneously, intradermally, or intraperitoneally, as described in U.S. Patent 5,543,158; U.S. Patent 5,641,515; and U.S. Patent 5,399,363- Lipid particles are ideal.
  • the drug-lipid particles can be injected locally into a target site (eg, a site of disease such as inflammation or tumor formation or into a target organ or tissue) or systemically for widespread distribution into the organism.
  • Solutions of the drug-lipid particles can be prepared in water, suitably mixed with a surfactant.
  • Dispersions can also be prepared in glycerin, liquid polyethylene glycols, and mixtures thereof, and in oils.
  • these preparations contain preservatives to prevent the growth of microorganisms.
  • the drug-lipid particle formulation is formulated with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier typically, ordinary buffered saline solution (135-150mM NaCl) will be used as the pharmaceutical carrier, but other suitable carriers will suffice. Additional suitable vectors are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, vehicle solutions, suspensions, Colloids etc.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce allergic or similar adverse effects when administered to humans.
  • the formulation of aqueous compositions containing a protein as an active ingredient is conventionally understood in the art. Alternatively, these compositions may be prepared as injections, liquid solutions, or suspensions; solid forms suitable for solution or suspension in liquid prior to injection may also be prepared. The formulations may also be emulsified.
  • Drug-lipid particles can be sterilized by conventional liposome sterilization techniques, such as filtration.
  • the drug-lipid particles may contain pharmaceutical auxiliary substances that are suitable for physiological conditions, such as pH adjusters and buffers, toxicity adjusters, wetting agents, and the like. These compositions can be sterilized using the techniques noted above, or alternatively they can be produced under sterile conditions.
  • the resulting aqueous solution can be packaged for use or filtered under sterile conditions and freeze-dried, and the freeze-dried formulation combined with the sterile aqueous solution prior to administration.
  • drug-lipid particles can be used for the prophylactic or therapeutic treatment of subjects (eg, mammalian subjects) suffering from a disease or disorder.
  • the disease or disorder is associated with the expression or overexpression of the target sequence.
  • the drug-lipid particles are administered to the subject in an amount sufficient to elicit a therapeutic response in the patient.
  • An amount sufficient to accomplish this is defined as a "therapeutically effective dose or amount” or an "effective dose or amount.”
  • the physician evaluates circulating plasma levels of the drug-lipid particle, Drug-lipid particle toxicity and progression of disease associated with expression or overexpression of target genes. Administration can be accomplished by single or divided doses.
  • the drug-lipid particles can be administered to a subject infected or at risk of infection by a pathogenic microorganism.
  • the drug should preferably correspond to a sequence that has a key role in the life history of the microorganism and should also be unique to the microorganism (or at least lacking in the native genome of the patient undergoing treatment).
  • the drug-lipid particles are introduced into target cells, tissues or organs at therapeutically effective doses by ex vivo or intravenous injection. Silencing sequences encoding genes associated with pathogenic infections can be conveniently used in conjunction with the administration of conventional agents used to treat pathogenic diseases.
  • the treatment may be administered prophylactically to persons who are at risk of becoming infected by, or already infected by, pathogenic microorganisms.
  • the drug-lipid particles of the present disclosure can be conveniently used to treat cancer, viral infections, autoimmune diseases, diabetes, and Alzheimer's disease.
  • Viral infections include hepatitis A, hepatitis B, hepatitis C, SARS-Cov-2, HIV, HPV, influenza, smallpox, and syphilis.
  • suitable sites for inhibition of hepatitis B virus include nucleic acid sequences encoding S, C, P and X proteins, PRE, EnI, and EnII (see, eg, FIELDSVIROLOGY, 2001, supra).
  • gene silencing associated with hepatitis infection can be combined with conventional treatments for hepatitis, such as, for example, immunoglobulins, interferons (e.g., PEGylated and non-PEGylated interferon a) (see, e.g., Medina et al., Antiviral Res. 60(2):135-143 (2003); ribavirin (see, e.g., Hugle and Cerny, Rev. Med. Virol.
  • immunoglobulins e.g., interferons (e.g., PEGylated and non-PEGylated interferon a)
  • interferons e.g., PEGylated and non-PEGylated interferon a
  • ribavirin see, e.g., Hugle and Cerny, Rev. Med. Virol.
  • the drug-lipid particles of the present disclosure may be conveniently used to treat diseases and conditions characterized by the expression or overexpression of a gene or group of genes.
  • the drug-lipid particles of the present disclosure can be used to treat metabolic diseases and disorders (eg, diseases and disorders in which the liver is a target and liver diseases and disorders) such as, for example, dyslipidemia and diabetes.
  • metabolic diseases and disorders eg, diseases and disorders in which the liver is a target and liver diseases and disorders
  • metabolic diseases and disorders eg, diseases and disorders in which the liver is a target and liver diseases and disorders
  • dyslipidemia and diabetes e.g, dyslipidemia and diabetes.
  • silencing of genes involved in dyslipidemia can be associated with the use of inhibins, bile acid chelators/resins, and cholesterol absorption inhibitors such as ezetimibe, plant stanols/sterols, polyphenols, and nutraceuticals such as oat bran, Flaxseed and soy protein, phytostanol analogs, squalene synthase inhibitor, bile acid transport inhibitor SREBP cleavage-activating protein (SCAP) activating ligand, nicotinic acid (nicotinic acid), acetimide Division, High Dose Fish Oil, Antioxidants and Sugar Cane Fatty Alcohols, Microsomal Triacylglycerol Transporter Protein (MTP) Inhibitor, Fatty Acyl-CoA:Cholesterol Acyltransferase (ACAT) Inhibitor, Gemcabene, Rifebello, Pantothenic Acid Similar niacin-receptor agonists,
  • Similar approaches are used to inhibit the expression of endogenous receptor cellular genes associated with tumorigenesis and cellular transformation, tumor growth and tumor migration; to inhibit the expression of angiogenic genes; to inhibit immune modulator genes such as Inhibition of expression of genes associated with inflammation and autoimmune responses; inhibition of expression of ligand receptor genes; inhibition of expression of genes associated with neurodegenerative disorders; and inhibition of expression of additional genes associated with viral infection and survival.
  • the target gene sequence of a specific target is as described above.
  • the drug-lipid particles herein are detected using any method known in the art.
  • the label is coupled directly or indirectly to components of the drug-lipid particle or other lipid-based carrier system using methods well known in the art.
  • a wide variety of labels can be used, with selection based on required sensitivity, ease of conjugation to drug-lipid particle components, stability requirements and availability of tools and preparation for processing.
  • Suitable labels include, but are not limited to, spectral labels such as fluorescent dyes (e.g., (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon GreenTM; rhodamine and derivatives such as Dekker Saas red, tetrarhodimine isothiocynate (TRITC), etc., digoxigenin, biotin, phycoerythrin, AMCA, CyDyesTM, etc.; radioactive labels, such as 3H, 125I, 35S, 14C, 32P, 33P, etc.; enzymes such as spicy Root peroxidase, alkaline phosphatase, etc.; spectroscopic colorimetric labels such as colloidal gold or colored glass or plastic beads such as polystyrene, polypropylene, latex, etc.). Use any means known in the art to label the Perform testing.
  • fluorescent dyes e.g., (e.g., flu
  • Detection of Nucleic Acids The nucleic acids herein are detected and quantified by any of a number of means well known to those skilled in the art. Detection of nucleic acids is performed by methods well known in the art such as Southern blot analysis, Northern blot analysis, gel electrophoresis, PCR, radioactive labeling, scintillation counting and affinity chromatography. Additional analytical biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography can also be applied.
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • the sensitivity of hybridization assays can be improved by using nucleic acid amplification systems that multiply the target nucleic acid that is detected.
  • nucleic acid amplification systems that multiply the target nucleic acid that is detected.
  • These in vitro amplification methods including polymerase chain reaction (PCR), ligase chain reaction (LCR), Q ⁇ -replicase amplification, and other RNA polymerase-mediated technologies (e.g., NASBATM), are sufficient to guide the skilled person in their techniques.
  • Solid phase phosphoramidites as described in Needham VanDevanter et al., Nucleic Acids Res., 12:6159 (1984), typically as described in Beaucage and Caruthers, Tetrahedron Letts., 22(20):1859 1862 (1981) Tryster methods, for example, use automated synthesizers to chemically synthesize oligonucleotides that are used as probes, for example, in in vitro amplification methods, as gene probes, or as inhibitor components. Purification of oligonucleotides is typically performed, if necessary, by native acrylamide gel electrophoresis or by anion exchange HPLC as described in Pearson and Regnier, J. Chrom., 255:137149 (1983). The sequence of synthetic oligonucleotides can be confirmed using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology, 65: 499.
  • the drug-lipid particles to be protected by this disclosure refer to drug-lipid particles other than containing cationic/ionizable lipids, that is, drug-loaded metal-chelated phospholipid complex nanoparticles, drug@ MPP).
  • phospholipid molecule with a phosphate group Connect the phospholipid molecule with a phosphate group to the linker molecule: add distearoylphosphatidylcholine (DSPC, formula 46) and curcumin (formula 19) into the reaction bottle at a molar ratio of 1:1, and add an appropriate amount of ethanol. Dissolve, react at 65°C for 2 hours, concentrate, and add n-hexane. The phospholipid complex is precipitated, filtered, and vacuum dried to obtain the phospholipid complex.
  • the structure of the phospholipid complex is as follows:
  • the phospholipid complex prepared in Example 1 is partially connected to the metal ion: add the phospholipid complex and FeCl 3 into the reaction bottle at a molar ratio of 1:1, add ethanol to dissolve, react at 60°C for 2 hours, and then suspend the reaction solution to dryness. It was then washed with ultrapure water and dried under vacuum to obtain the metal-phospholipid complex.
  • the metal-phospholipid complex structure is shown below.
  • Embodiment 2.1 The difference between this embodiment and Embodiment 2.1 is that FeCl 3 is replaced by Al(NO 3 ) 3 ⁇ 9H 2 O.
  • the structure of the prepared metal-phospholipid complex is shown below.
  • Example 3 Preparation of mRNA-metal-chelated phospholipid complex nanoparticles (mRNA-loaded metal-chelated phospholipid complex nanoparticles, mRNA@MPP)
  • the metal-phospholipid complex was prepared according to the method in Example 2.1, in which DSPC, curcumin, and FeCl were put in at a feeding ratio of 1:1:1, and the metal-phospholipid complex and distearoylphosphatidylcholine ( DSPC, Formula 46, as a non-cationic lipid or non-ionizable lipid), cholesterol (CHOL, Formula 40, as a non-cationic lipid or non-ionizable lipid), DSPE-PEG2000 (Formula 53, as a particle aggregation inhibitor Conjugated lipids) were dissolved in ethanol at different molar ratios as the organic phase.
  • DSPC Formula 46, as a non-cationic lipid or non-ionizable lipid
  • cholesterol cholesterol
  • DSPE-PEG2000 Formulamula 53, as a particle aggregation inhibitor Conjugated lipids
  • the proportions of metal-phospholipid complex, DSPC, CHOL and DSPE-PEG2000 are 15%, 35%, 46% and 4% respectively.
  • the mRNA was dissolved in enzyme-free PBS buffer as the aqueous phase at a concentration of 20 ⁇ g/mL (the components of PBS are 0.137M sodium chloride, 0.0027M potassium chloride, 0.01M disodium hydrogen phosphate and 0.0018M of potassium dihydrogen phosphate).
  • the mass of metal-phospholipid complex and the mass of mRNA were mixed in the microfluidic chip at a mass ratio of 40:1.
  • the volume ratio of aqueous phase to organic phase is 3:1.
  • the flow rate of the organic phase and aqueous phase in the microfluidic chip is 12ml/min.
  • the drug mRNA is the mRNA encoding the fluorescent protein eGFP, and its sequence is SEQ ID NO. 1 (720nt).
  • eGFP-mRNA@MPP was prepared.
  • eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2 ⁇ g/mL (concentration of mRNA).
  • the control group was incubated with MPP without drug loading. After 48 hours, the cell suspension was collected and eGFP positive was detected by flow cytometry. Cell percentage.
  • Example 3 The particle size, surface potential and stability of the eGFP-mRNA@MPP prepared in Example 3 were detected, and the efficiency of eGFP-mRNA@MPP in entrapping nucleic acids was calculated.
  • Methods for detecting particle size and results judgment criteria Use Malvern Zetasizer to test the particle size of nanoparticles. Particle sizes in the range of 30 to 400nm are considered acceptable.
  • Methods for detecting surface potential and criteria for judging results Use a Malvern laser particle size analyzer Zetasizer to test the surface potential of nanoparticles. A potential in the range of -10 to 10mV is considered acceptable.
  • Stability detection method and result judgment criteria Place the nanoparticles at 4°C for 7 days, and use a Malvern laser particle size analyzer Zetasizer to test the particle size and surface potential of the nanoparticles. When the particle size and surface potential have no obvious changes within 3-7 days Changes are seen as better stability.
  • Method for calculating nucleic acid encapsulation efficiency Specifically, agarose gel electrophoresis is used. First, the nucleic acid input amount of each group of lipid nanoparticles is set to 10 ⁇ g/mL. When the metal ion is Fe 3+ , the mass ratio of the metal-phospholipid complex to the mRNA is 40:1. Dissolve equal concentrations of nucleic acids in PBS. The buffer solution was used as a positive control, and the negative control was PBS buffer solution. The concentration of the agarose gel is 1.5%. At this time, the gaps in the gel only allow free nucleic acids to pass through but not lipid nanoparticles. The electrophoresis stops when the free nucleic acid bands can be clearly distinguished.
  • the positive control group is set as 100%.
  • the ratio of free nucleic acids in each group to the positive control is the relative amount of free nucleic acids.
  • the inclusion rate of each group is (100- Relative amount of free nucleic acid)%. A nucleic acid inclusion rate above 50% is considered an acceptable range.
  • Cell culture method Human embryonic kidney cell line 293T was cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2 .
  • Example 2.2 Prepare the metal-phospholipid complex according to the method in Example 2.2, wherein DSPC, curcumin, Al(NO 3 ) 3 ⁇ 9H 2 O are put in according to the feeding ratio of 1:1:1, and the metal-phospholipid complex and diamine are added.
  • Stearoylphosphatidylcholine (DSPC, formula 46, as a non-cationic lipid or non-ionizable lipid), cholesterol (CHOL, formula 40, as a non-cationic lipid or non-ionizable lipid), DSPE-PEG2000 ( Formula 53, as a conjugated lipid that inhibits particle aggregation) was dissolved in ethanol at different molar ratios as the organic phase.
  • the proportions of metal-phospholipid complex, DSPC, CHOL and DSPE-PEG2000 are 7%, 34%, 56% and 3% respectively.
  • the mRNA was dissolved in enzyme-free PBS buffer as the aqueous phase at a concentration of 20 ⁇ g/mL (the components of PBS are 0.137M sodium chloride, 0.0027M potassium chloride, 0.01M disodium hydrogen phosphate and 0.0018M phosphoric acid potassium dihydrogen).
  • the mass of metal-phospholipid complex and the mass of mRNA were mixed in the microfluidic chip at a mass ratio of 13.3:1.
  • the volume ratio of aqueous phase to organic phase is 3:1.
  • the flow rate of the organic phase and aqueous phase in the microfluidic chip is 12ml/min.
  • the drug mRNA is the mRNA encoding the fluorescent protein eGFP, and its sequence is SEQ ID NO. 1 (720nt).
  • eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2 ⁇ g/mL (concentration of mRNA).
  • the control group was incubated with MPP without drug loading. After 48 hours, the cell suspension was collected and eGFP positive was detected by flow cytometry. Cell percentage.
  • Example 3 The particle size, surface potential and stability of the eGFP-mRNA@MPP prepared in Example 3 were detected, and the efficiency of eGFP-mRNA@MPP in entrapping nucleic acids was calculated.
  • Methods for detecting particle size and results judgment criteria Use Malvern Zetasizer to test the particle size of nanoparticles. Particle sizes in the range of 30 to 400nm are considered acceptable.
  • Methods for detecting surface potential and criteria for judging results Use a Malvern laser particle size analyzer Zetasizer to test the surface potential of nanoparticles. A potential in the range of -10 to 10mV is considered acceptable.
  • Stability detection method and result judgment criteria Place the nanoparticles at 4°C for 7 days, and use a Malvern laser particle size analyzer Zetasizer to test the particle size and surface potential of the nanoparticles. When the particle size and surface potential have no obvious changes within 3-7 days Changes are seen as better stability.
  • Method for calculating nucleic acid encapsulation efficiency Specifically, agarose gel electrophoresis is used. First, the nucleic acid input amount of each group of lipid nanoparticles is set to 10 ⁇ g/mL. When the metal ion is Al 3+ , the mass ratio of the metal-phospholipid complex to the mRNA is 13.3:1. Dissolve equal concentrations of nucleic acids in PBS. The buffer solution was used as a positive control, and the negative control was PBS buffer solution. The concentration of the agarose gel is 1.5%. At this time, the gaps in the gel only allow free nucleic acids to pass through but not lipid nanoparticles. The electrophoresis stops when the free nucleic acid bands can be clearly distinguished.
  • the positive control group is set as 100%.
  • the ratio of free nucleic acids in each group to the positive control is the relative amount of free nucleic acids.
  • the inclusion rate of each group is (100- Relative amount of free nucleic acid)%. A nucleic acid inclusion rate above 50% is considered an acceptable range.
  • Cell culture method Human embryonic kidney cell line 293T was cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2 .
  • the principle of loading nucleic acids into metal-chelated phospholipid complex nanoparticles (MPP) assembled from metal-phospholipid complexes is that curcumin and DSPC are bonded together through hydrogen bonds, while curcumin binds to Fe through coordination bonds 3+ or Al 3+ are connected to form a metal-phospholipid complex.
  • the Fe 3+ or Al 3+ of the metal-phospholipid complex is connected to the nucleic acid through coordination bonds, thereby ensuring that the metal-phospholipid complex is connected with other lipid groups.
  • the particles self-assemble into MPP while loading nucleic acids into the nanoparticles.
  • Curcumin interacts with nucleic acids and assists MPP in loading nucleic acids. For example, curcumin assists in loading nucleic acids by inserting into the minor groove of nucleic acids; 2 Curcumin may not directly interact with nucleic acids. Nucleic acid interactions.
  • Example 3 DSPC, curcumin, and FeCl 3 in Example 3 were added according to different dosage ratios (1:1:1, 3:3:2, 2:2:1), and other steps were the same as in Example 3 to prepare different eGFP-mRNA@MPP, and their nucleic acid inclusion rates were measured respectively.
  • Result analysis As shown in the results table 1-1, when the dosage ratio of DSPC, curcumin, and FeCl 3 is 1:1:1, the eGFP-mRNA encapsulation efficiency of the prepared drug-lipid particles is 97%; When the dosage ratio of DSPC, curcumin, and FeCl 3 is 3:3:2, the eGFP-mRNA encapsulation efficiency of the prepared drug-lipid particles is 70%; when the dosage ratio of DSPC, curcumin, and FeCl 3 is At 2:2:1, the eGFP-mRNA entrapment efficiency of the prepared drug-lipid particles was 60%.
  • the function of Fe 3+ in drug-lipid particles is to connect phospholipid complexes and nucleic acids.
  • Each Fe 3+ has up to three complexing sites, so the delivery of DSPC, curcumin, and FeCl 3 in drug-lipid particles
  • the ratio should be 1:1:1 to ensure that the drug-lipid particles can encapsulate as much nucleic acid as possible.
  • the results of the experiment also confirmed that when the ratio of DSPC, curcumin, and FeCl 3 was 1:1:1, the drug-lipid particles prepared with it had the highest eGFP-mRNA encapsulation rate.
  • the dosage ratio of DSPC, curcumin, and FeCl 3 ranges from 1:1:1 to 2:2:1, the core of the drug-lipid particles
  • the acid inclusion rates are all above 60%.
  • Example 3 DSPC, curcumin, and Al(NO 3 ) 3 ⁇ 9H 2 O in Example 3 were added according to different dosage ratios (1:1:1, 3:3:2, 2:2:1), and other steps were the same.
  • different eGFP-mRNA@MPPs were prepared, and their nucleic acid inclusion rates were measured respectively.
  • the function of Al 3+ in metal-phospholipid complex particles is to connect the phospholipid complex and nucleic acid.
  • Each Al 3+ has up to three complexing sites, so DSPC, curcumin, Al(NO 3 ) 3 ⁇ 9H 2 O
  • the ratio of drug-lipid particles should be 1:1:1 to ensure that the metal-phospholipid complex particles can contain as much nucleic acid as possible.
  • the experimental results also confirmed that when the dosage ratio of DSPC, curcumin, and Al(NO 3 ) 3 ⁇ 9H 2 O is 1:1:1, the metal-phospholipid complex particles prepared with it have the highest eGFP-mRNA encapsulation rate. .
  • the nucleic acid inclusion rates of the metal-phospholipid complex particles are all at 58 %above.
  • Example 3.2 Preparation of drugs-proportions of metal-phospholipid complexes, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000 and cholesterol (CHOL) in lipid particles
  • Example 3 Compared with Example 3, the ratios of metal-phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) are as shown in Table 1-3 (the metal ion is Fe 3+ ) and As shown in Table 1-4 (the metal ion is Al 3+ ), the other conditions are the same.
  • the metal-phospholipid complex accounts for 15%, distearoylphosphatidylcholine (DSPC) accounts for 35%, cholesterol (CHOL) accounts for 46%, and DSPE-PEG2000 accounts for 4%, the drug-lipid complex accounts for 15%.
  • the performance of the plasmid particles is optimal, that is, the particle size is in the range of 110nm, the surface potential is in the range of -2.04mV, the in vitro stability is >7 days, the mRNA encapsulation rate is 87%, and the positive expression rate of eGFP protein is 97%.
  • mRNA@MPP mainly relies on metal-phospholipid complexes to absorb nucleic acids, the proportion of metal-phospholipid complexes cannot be too low; when the content of DSPC is in the range of 0-40%, the stability of its nanoparticles is within the acceptable range. , when the content of DSPC is 0%, because the metal-phospholipid complex contains DSPC, the stability of the nanoparticles is maintained; the function of DSPE-PEG2000 is to prevent the aggregation of nanoparticles and increase the circulation time in the body, and its content is 2 The performance will be better in the -10% range; the role of CHOL is to enhance nanoparticles The fluidity and maintaining a certain content are beneficial to the stability of nanoparticles.
  • the metal-phospholipid complex accounts for 7%
  • distearoylphosphatidylcholine (DSPC) accounts for 34%
  • cholesterol (CHOL) accounts for 56%
  • DSPE-PEG2000 accounts for 3%
  • the drug-lipid The performance of the plasmid particles is optimal, that is, the particle size is 100nm, the surface potential is -1.57mV, the in vitro stability is >7 days, the mRNA encapsulation rate is 92%, and the positive expression rate of eGFP protein is 98%.
  • mRNA@MPP mainly relies on metal-phospholipid complexes to absorb nucleic acids, the proportion of metal-phospholipid complexes cannot be too low; when the content of DSPC is in the range of 0-51%, the stability of its nanoparticles is within the acceptable range. , when the content of DSPC is 0%, because the metal-phospholipid complex contains DSPC, the stability of the nanoparticles is maintained; the function of DSPE-PEG2000 is to prevent the aggregation of nanoparticles and increase the circulation time in the body, and its content is 2 The performance will be better in the range of -10%; the function of CHOL is to enhance the fluidity of nanoparticles, and maintaining a certain content is beneficial to the stability of nanoparticles.
  • Example 3.3 Preparation of non-cationic lipids or non-ionizable lipid species in eGFP-mRNA@MPP
  • Example 3 Compared with Example 3, the substitution of distearoylphosphatidylcholine (DSPC) is as shown in Tables 1-5 and 1-6, and the remaining conditions are the same.
  • DSPC distearoylphosphatidylcholine
  • non-cationic lipids or non-ionizable lipids are shown below.
  • DSPE-PEG2000 (Formula 53) are as shown in Table 1-7 (the metal ion is Fe 3+ ) and Table 1-8 (the metal ion is Al 3+ ), and the remaining conditions are the same.
  • DSPE-PEG2000 in eGFP-mRNA@MPP can be Other conjugated lipids that inhibit particle aggregation were substituted and their efficacy was not affected.
  • the mRNA in Example 3 was replaced with the other two kinds of mRNA, and three kinds of mRNA@MPP containing different target protein mRNA sequences were prepared by referring to the method in Example 3.
  • the three different mRNA sequences are: 1
  • the mRNA sequence encoding the fluorescent protein eGFP is SEQ ID NO.1 (720nt); 2
  • the mRNA sequence encoding the receptor binding domain (RBD) of the S1 subunit of the new coronavirus It is SEQ ID NO.2 (669nt);
  • the mRNA sequence encoding the tumor antigen NY-ESO-1 is SEQ ID NO.3 (543nt).
  • the preparation process of the remaining drug (mRNA)-lipid particles was the same as in Example 3, and eGFP-mRNA@MPP, RBD-mRNA@MPP, and NY-ESO-1-mRNA@MPP were obtained respectively.
  • eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2 ⁇ g/mL (concentration of mRNA contained), and the control group was incubated with MPP. After 48 hours, the cell suspension was collected, and the percentage of eGFP-positive cells was detected by flow cytometry. The results are shown in Figure 1-1; RBD-mRNA@MPP was incubated with 293T cells at a concentration of 2 ⁇ g/mL (concentration of mRNA contained), and the control group was incubated with MPP. After 24 hours, the supernatant was centrifuged and frozen at -20°C for later use.
  • COVID-19 antigen RBD ELISA detection kit was used to detect the expression level of novel coronavirus antigen RBD protein on cells. The results are shown in Figure 1-2.
  • Sample collection Place the cell supernatant at room temperature for 2 hours, centrifuge at 1000 ⁇ g for 20 minutes, and take the supernatant;
  • Blank holes, standard holes, and sample holes to be tested are respectively set on the coating plate.
  • TMB substrate solution (TMB)
  • the experimental animals were randomly divided into 2 groups (experimental group and control group), with 5 animals in each group.
  • the RBD-mRNA@MPP animal model is BALB/c mice. Each mouse undergoes the first intramuscular administration on the 1st day and the second intramuscular administration on the 14th day.
  • the experimental group is injected with RBD-mRNA. @MPP, the control group was injected with metal-phospholipid complex particles (MPP) without loading mRNA.
  • the dose of each administration is 100 ⁇ L, and the RBD-mRNA@MPP preparation in the experimental group contains 30 mg of mRNA.
  • the animal model of NY-ESO-1-mRNA@MPP is C57BL/6 mice. Each mouse is administered intramuscularly four times on days 1, 7, 14, and 21.
  • the experimental group is injected with NY-ESO-1-mRNA. @MPP, the control group was injected with metal-phospholipid complex particles (MPP) without loading mRNA.
  • the dose of each administration is 100 ⁇ L, and the NY-ESO-1-mRNA@MPP preparation in the experimental group contains 30 mg of mRNA.
  • the blood of the mice was collected, the serum was separated and diluted in gradients, and the anti-NY-ESO-1 total IgG antibodies produced in the mice were detected by ELISA. The results are shown in Figures 1-4.
  • Coating solution Precisely weigh 8.4g NaHCO 3 and dissolve it in 1L distilled water (DDW). After all the solids are dissolved, use 1M NaOH solution to adjust the pH of the entire solution to 9.6. Store the prepared coating solution in Set aside at 4°C.
  • Washing solution Add 0.5mL Tween-20 to 1L 0.01M PBS solution, mix evenly and place at room temperature.
  • Blocking solution Precisely weigh 20g of BSA and add it to 1L of 0.01M PBS solution. Ultrasonicate the undissolved BSA powder in the solution. When all the solids in the solution are dissolved and the solution turns light yellow, place it in a refrigerator at 4°C. stand-by.
  • Antibody diluent Precisely weigh 2.5g of BSA and dissolve it in 250mL of 0.01M PBS solution. After the solid is completely dissolved, add 1.25mL of Tween-20 to it, mix evenly and store at 4°C for later use.
  • Chromogenic solution 0.1M citric acid: 19.2g citric acid, add DDW water to 1000mL (A) 0.2M disodium hydrogen phosphate: 28.4g anhydrous disodium hydrogen phosphate, add DDW water to 1000mL (B) 0.1M citric acid Solution (A) 24.3mL, 0.2M phosphate buffer (B) 25.7mL, add 50mL DDW water. Now add 50 mg of OPD (o-phenylenediamine) and 0.15 mL of 30% H 2 O 2 .
  • OPD o-phenylenediamine
  • Stop solution 2M H 2 SO 4 : 55.5 mL of concentrated sulfuric acid, add DDW to 500 mL.
  • Coating NY-ESO-1 antigen is diluted to 1 ⁇ g/mL with coating solution, added to a 96-well plate, 50 ⁇ L/well, and coated overnight at 4°C.
  • Blocking Dry the coating solution in the well plate, wash with blocking solution three times, once every 5 minutes and spin dry, add 150 ⁇ L blocking solution to each well, and incubate at 37°C for 2 hours.
  • Drying spin dry the blocking solution and incubate at 37°C for 1 to 2 hours until all the liquid at the bottom of the well plate is dry.
  • the spleens of normal mice were collected, prepared into a single cell suspension under sterile conditions, plated into cell well plates at 100,000 spleen cells/well, and added with a final concentration of 10 mg/mL.
  • the RBD protein was cultured for 48 hours, centrifuged to remove the supernatant, and the expression levels of IFN- ⁇ , IL-2, and IL-4 were measured using ELISA kits. The results are shown in Figure 1-5.
  • both RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP can effectively induce humoral immunity in mice and produce high levels of antigen-specific binding antibodies.
  • the IgG antibody titer of mice in the RBD-mRNA@MPP treated group reached 117268.8; the IgG antibody titer of mice in the NY-ESO-1-mRNA@MPP treated group reached 5319.52.
  • both RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP can effectively induce cellular immunity in mice, that is, activate immune cells and produce a large amount of cytokines.
  • RBD-mRNA@MPP increased the expression levels of cytokines IFN- ⁇ , IL-2, and IL-4 to 252.8pg/mL, 207.6pg/mL, and 56.6pg/mL respectively;
  • NY-ESO-1-mRNA@MPP increased The expression levels of cytokines IFN- ⁇ , IL-2, and TNF- ⁇ reached 70.79pg/mL, 75.29pg/mL, and 75.27pg/mL respectively.
  • mRNA@MPP can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), effectively inducing humoral immunity and cellular immunity in mice, producing high levels of antigen-specific binding antibodies and cytokines, and exerting The role of anti-COVID-19 mRNA vaccines and anti-tumor mRNA vaccines.
  • Example 3.5.1 The difference between this embodiment and Example 3.5.1 is that the metal ion Fe 3+ in Example 3.5.1 is replaced by Al 3+ .
  • the eGFP-positive cell rate in the eGFP-mRNA@MPP experimental group was 98.02%, while no eGFP signal was detected in the MPP control group; as shown in Figure 1-8, the MPP-encapsulated RBD-
  • the RBD protein encoded by the mRNA was 212.6ng/mL in the supernatant of 293T cells, while the RBD protein content in the supernatant of 293T cells transfected with the empty vector MPP was 0.
  • both RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP can effectively induce humoral immunity in mice and produce high levels of antigen-specific binding antibodies.
  • the IgG antibody titer of mice in the RBD-mRNA@MPP treated group reached 129113; the IgG antibody titer of mice in the NY-ESO-1-mRNA@MPP treated group reached 6507.4.
  • both RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP can effectively induce cellular immunity in mice, that is, activate immune cells and produce a large amount of cytokines.
  • RBD-mRNA@MPP increased the expression levels of cytokines IFN- ⁇ , IL-2, and IL-4 to 271.8pg/mL, 234.6pg/mL, and 68.4pg/mL respectively;
  • NY-ESO-1-mRNA@MPP increased The expression levels of cytokines IFN- ⁇ , IL-2, and TNF- ⁇ reached 83.8pg/mL, 98pg/mL, and 97.8pg/mL respectively.
  • mRNA@MPP can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), effectively inducing humoral immunity and cellular immunity in mice, producing high levels of antigen-specific binding antibodies and cytokines, and exerting The role of anti-COVID-19 mRNA vaccines and anti-tumor mRNA vaccines.
  • siRNA@MPP siRNA-loaded metal-chelated phospholipid complex nanoparticles
  • siRNA@MPP siRNA-loaded metal-chelated phospholipid complex nanoparticles
  • Example 3 Replace the mRNA in Example 3 with siRNA, and prepare three siRNA@MPPs containing different siRNAs by referring to the method in Example 3.
  • the genes and sequences targeted by three different siRNAs and their corresponding random control sequences are: 1
  • the sequence of siRNA targeting the Bcl-2 gene (Bcl-2-siRNA) is SEQ ID No.
  • siRNA targeting the PLK1 gene ( The sequence of PLK1-siRNA) is SEQ ID NO.6 (antisense strand) and SEQ ID No.23 (sense strand) (21bp), and its random control sequence is SEQ ID NO.7 (antisense strand) and SEQ ID No .24 (sense strand) (19bp); 3The sequence of siRNA targeting the Gal-1 gene (Gal-1-siRNA) is SEQ ID NO.8 (19bp); its random control sequence is SEQ ID NO.9 (19bp) ). The rest of the preparation process of siRNA@MPP is the same as in Example 3.
  • Bcl-2-siRNA The sequence of Bcl-2-siRNA is as follows:
  • Antisense 5′-CAGCUUAUAAUGGAUGUAC-3′(SEQ ID No.4);
  • the random control sequence of Bcl-2-siRNA is as follows:
  • Antisense 5’-ACGUGACACGUUCGGAGAA-3’(SEQ ID No.5);
  • PLK1-siRNA The sequence of PLK1-siRNA is as follows:
  • Antisense 5’-UAAGGAGGGGAUCUUCUUCA-3’(SEQ ID No.6);
  • the random control sequence of PLK1-siRNA is as follows:
  • Antisense 5’-CUUACGCUGAGUACUUCGA-3’(SEQ ID No.7);
  • Gal-1-siRNA The sequence of Gal-1-siRNA is as follows:
  • the random control sequence of Gal-1-siRNA is as follows:
  • U251 cells were seeded in a 6-well plate at a density of 1 ⁇ 10 6 cells per well for about 24 hours. After each well was incubated with siRNA@MPP containing the above siRNA (the concentration of siRNA was 2 ⁇ g/mL) for 72 hours, the cells were collected. , extract total cellular RNA, use RT-PCR technology to detect the mRNA expression of target genes (Bcl-2, PLK1, Gal-1), and count the ability of siRNA@MPP to silence cell target genes.
  • target genes Bcl-2, PLK1, Gal-1
  • Extraction of total RNA Discard the culture medium from the six-well plate, rinse it three times with PBS buffer, and add 1 mL Trizol to each well to lyse the cells. Add 200 ⁇ L chloroform, shake well, let stand at room temperature for 10 minutes, and centrifuge at 13000 rpm and 4°C for 15 minutes to obtain a layered three-phase liquid, in which RNA is dissolved in the upper water phase. Aspirate the upper aqueous phase and place it in a new enzyme-free 1.5ml centrifuge tube, add 500 ⁇ L isopropyl alcohol, let it stand at room temperature for 10min, and centrifuge at 13000rpm and 4°C for 15min to obtain RNA precipitation.
  • cDNA reverse transcription Use Ta Ka Ra Prime Script TM RT reagent Kit with g DNA Eraser kit to reverse transcribe RNA into cDNA respectively, and remove genomic DNA (gDNA) before the reverse transcription step. The results are more accurate and trustworthy.
  • RT-PCR operation This detection method is SYBR Green dye method and does not require a probe.
  • the details are as follows, Real-time PCR reactions are performed based on the cDNA of different samples as templates. Prepare the reaction solution on ice: 5 ⁇ L SYBR Premix Dimer Eraser (2 ⁇ ), 0.3 ⁇ L PCR Forward primer (10 ⁇ M), 0.3 ⁇ L PCR Reverse primer (10 ⁇ M), 0.2 ⁇ L ROX Reference DyeII (50 ⁇ ), 1 ⁇ L obtained in the previous step cDNA template and 3.2 ⁇ L dH 2 O. Add a sample of 10 ⁇ L to each well in the well plate.
  • the reaction program is 95°C, 30sec (1cycle) ⁇ 95°C, 5sec; 55°C, 30sec; 72°C, 30sec (40cycles) ⁇ 60°C-95°C ,2min(1cycle).
  • the experiment was repeated three times, the average value was taken to obtain the Ct value of each group, and the expression difference fold between the experimental group and the control group was calculated.
  • the control gene is GAPDH.
  • the RT-PCR primers are as follows: 1Bcl-2 primer: forward: 5'-AGGATTGTGGCCTTCTTTGAG-3', reverse: 5'-AGACAGCCAGGAGAAATC AAAC-3'; 2PLK1 primer: forward: 5'-ACCAGCACGTCGTAGGATTC-3', reverse: 5'- CAAGCAATTTGCCGTAGG-3'; 3Gal-1 primer: forward: 5'-CAATCAT GGCCTGTGGTCTG-3', reverse: 5'-GTG TAGGCACAGGTTGTTGCTG-3'. 4GAPDH primer: forward: 5'-TCAGGGGTTTCACATTTGGCA-3', reverse: 5'-GG AGCGGAA AACCA-3'.
  • the expression level of each target gene is expressed by RQ value (2 - ⁇ CT ).
  • ⁇ Ct ⁇ Ct experimental group – ⁇ Ct control group
  • ⁇ Ct Ct target gene – Ct internal reference gene
  • Calculation method of gene silencing efficiency Calculation method of gene silencing efficiency: 100% - gene expression level of experimental group/gene expression level of control group.
  • siRNA@MPP siRNA-loaded metal-chelated phospholipid complex nanoparticles
  • Example 3.6.1 The difference between this embodiment and Example 3.6.1 is that the metal ion Fe 3+ in Example 3.6.1 is replaced by Al 3+ .
  • siRNA@MPP can significantly interfere with their corresponding target genes.
  • the inhibition rate of Bcl-2-siRNA@MPP on the target gene Bcl-2 reaches 81%; the inhibition rate of PLK1-siRNA@MPP on the target gene PLK1 reaches 90%; the inhibition rate of Gal-1-siRNA@MPP on the target gene Gal-1 The inhibition rate reached 79%.
  • Example 3.7.1 Preparation and effect of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP) when the metal ion is Fe 3+
  • the genes and sequences targeted by three different ASOs and their corresponding random control sequences are: 1
  • the ASO (STAT3-ASO) sequence targeting the STAT3 gene is SEQ ID No.
  • ASO ( ⁇ -syn-ASO) sequence targeting ⁇ -syn gene is SEQ ID NO.12 (16nt), its random control sequence is SEQ ID NO.13 (16nt);
  • ASO (Bcl-2-ASO) sequence targeting the Bcl-2 gene is SEQ ID NO.14 (18nt), and its random control sequence is SEQ ID NO. 15(20nt).
  • the preparation process of the remaining ASO-metal-phospholipid composite particles is the same as in Example 3.
  • ASO@MPP targeting the STAT3 gene was incubated with U251 human brain glioblastoma cells; ASO@MPP targeting the ⁇ -syn gene was incubated with SH-SY5Y human neuroblastoma cells. ; Daudi human lymphoma cells were incubated with ASO@MPP targeting the Bcl-2 gene.
  • ASO@MPP targeting the STAT3 gene was incubated with U251 human brain glioblastoma cells
  • ASO@MPP targeting the ⁇ -syn gene was incubated with SH-SY5Y human neuroblastoma cells.
  • Daudi human lymphoma cells were incubated with ASO@MPP targeting the Bcl-2 gene.
  • About 24 hours after seeding in a 6-well plate at a density of 1 ⁇ 10 6 cells per well the cells in each well were incubated with ASO@MPP containing the above ASO (where the concentration of ASO is 1 ⁇ g/mL) for 48 hours, and then
  • SEQ ID No.10 sequence (sequence of STAT3-ASO) is as follows:
  • SEQ ID No.11 sequence (random control sequence of STAT3-ASO) is as follows:
  • SEQ ID No.12 sequence (sequence of ⁇ -syn-ASO) is as follows:
  • SEQ ID No.13 sequence (random control sequence of ⁇ -syn-ASO) is as follows:
  • SEQ ID No.14 sequence (sequence of Bcl-2-ASO) is as follows:
  • SEQ ID No.15 sequence (random control sequence of Bcl-2-ASO) is as follows:
  • Extraction of total RNA Discard the culture medium from the six-well plate, rinse it three times with PBS buffer, and add 1 mL Trizol to each well to lyse the cells. Add 200 ⁇ L chloroform, shake well, let stand at room temperature for 10 minutes, and centrifuge at 13000 rpm and 4°C for 15 minutes to obtain a layered three-phase liquid, in which RNA is dissolved in the upper water phase. Aspirate the upper aqueous phase and place it in a new enzyme-free 1.5ml centrifuge tube, add 500 ⁇ L isopropyl alcohol, let it stand at room temperature for 10min, and centrifuge at 13000rpm and 4°C for 15min to obtain RNA precipitation.
  • cDNA reverse transcription Use Ta Ka Ra Prime Script TM RT reagent Kit with g DNA Eraser kit to reverse transcribe RNA into cDNA respectively. Genomic DNA (gDNA) is removed before the reverse transcription step to make the results more accurate and credible.
  • gDNA Genomic DNA
  • RT-PCR operation This detection method is SYBR Green dye method and does not require a probe.
  • the details are as follows, Real-time PCR reactions are performed based on the cDNA of different samples as templates.
  • the reaction program is 95°C, 30sec (1cycle) ⁇ 95°C, 5sec; 55°C, 30sec; 72°C, 30sec (40cycles) ⁇ 60°C-95°C ,2min(1cycle).
  • the experiment was repeated three times, the average value was taken to obtain the Ct value of each group, and the expression difference fold between the experimental group and the control group was calculated.
  • the control gene is GAPDH.
  • the RT-PCR primer sequences are as follows: 1STAT3 primer: forward: 5'-TGATCACCTTTGAGACCGAGG-3', reverse: 5'-GATCACCACAACTGG CAA GG-3'; 2 ⁇ -syn primer: forward: 5'-TGACGGGTGTGACAGCAGTAG-3', reverse: 5 '-CAGTGGCTGCTGCAATG-3'; 3Bcl-2 primer: forward: 5'-AGGATT GTG GCCTTCTTTGAG-3', reverse: 5'-AGACAGCCAGGAGAAATCAAAC-3' 4GAPDH primer: forward: 5'-TCAGGGGG TTTCACATTTGGCA-3', reverse: 5 '-GGAGCGGAA AACCA-3'.
  • the expression level of each target gene is expressed by RQ value (2 - ⁇ CT ).
  • ⁇ Ct ⁇ Ct experimental group – ⁇ Ct control group
  • ⁇ Ct Ct target gene – Ct internal reference gene
  • the calculation method of gene silencing efficiency 100%-gene expression level of experimental group/gene expression level of control group.
  • Example 3.7.2 Preparation and effect of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP) when the metal ion is Al 3+
  • Example 3.7.1 The difference between this embodiment and Example 3.7.1 is that the metal ion Fe 3+ in Example 3.7.2 is replaced by Al 3+ .
  • Example 3.8 Preparation of drug (different types of nucleic acids)-metal-phospholipid complex particles and their effects
  • Example 3.8.1 Preparation of drug (different types of nucleic acids)-metal-phospholipid complex particles and their effects when the metal ion is Fe 3+
  • the mRNA in Example 3 was replaced with double-stranded RNA (siRNA), single-stranded DNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA respectively.
  • siRNA double-stranded RNA
  • ASO single-stranded DNA
  • mRNA double-stranded DNA
  • single-stranded DNA double-stranded DNA respectively.
  • Different types of nucleic acid sequences are: 1 The sequences of double-stranded RNA (Bcl-2-siRNA) are SEQ ID NO.4 (antisense strand) and SEQ ID No.21 (sense strand) (19bp), and its random control sequence It is SEQ ID NO.5 (antisense strand) and SEQ ID No.22 (sense strand) (19bp); 2
  • the sequence of single-stranded DNA STAT3-ASO
  • STAT3-ASO The sequence of single-stranded DNA
  • STAT3-ASO is
  • the drug-metal-phospholipid complex particles containing the above different types of nucleic acids (Bcl-2-siRNA@MPP, STAT3-ASO@MPP, S-mRNA@MPP, dsDNA@MPP, ssDNA@ MPP), and the preparation process of the remaining drug-lipid particles is the same as in Example 3.
  • sequence of double-stranded DNA is as follows:
  • Antisense 5’-TAGCTTATCAGACTGATGTTGA-3’(SEQ ID No.17);
  • SEQ ID No.18 sequence (sequence of single-stranded DNA) is as follows:
  • U251 cells were seeded in a 12-well plate at a density of 1 ⁇ 10 5 cells per well. About 24 hours later, the cells in each well were incubated with siRNA@MPP (where the concentration of siRNA was 2 ⁇ g/mL) or ASO@MPP (where the concentration of ASO was 2 ⁇ g/mL). mL) and incubated for 72 hours respectively, collect the cells, extract the total RNA of the cells, detect the mRNA expression of the target genes (Bcl-2, STAT3) using RT-PCR technology, and calculate the siRNA@MPP or ASO@MPP silencing cell target genes. Ability, the results are shown in Figure 1-13 in Example 3.6 and Figure 1-19 in Example 3.7.
  • S-mRNA@MPP was incubated with 293T cells at a concentration of 2 ⁇ g/mL (concentration of mRNA), and the control group was incubated with MPP. After 24 hours, the supernatant was centrifuged and stored at -20°C for later use; add 100 ⁇ L PBS to the cell pellet. The buffer solution was resuspended, frozen and thawed twice, and ultrasonicated for 10 minutes. The supernatant was centrifuged and the commercially available new coronavirus S protein ELISA detection kit was used to detect the expression levels of S protein in both the cell supernatant and cell lysate. The results are shown in Figure 1- 25 shown.
  • ss-DNA@MPP was incubated with HT22 mouse hippocampal neurons at a concentration of 200 nM (the concentration of DNA contained) for 2 hours. The remaining drug-lipid particles were removed. After washing the cells twice with PBS, the cells were observed with a high-content imaging system. , and calculate the efficiency of drug-lipid particle transfection into DNA. The results are shown in Figure 1-26.
  • the culture method of human brain glioblastoma U251 cells is the same as in Example 3.6.
  • the culture method of 293T cells is the same as in Example 3.
  • the RT-PCR method is the same as in Example 3.6.
  • Calculation method of transfection efficiency Use a high-content imaging system to randomly select 3-5 fields of view to obtain the cell morphology under ordinary light sources.
  • the excitation/emission light in the same field of view is 550nm/570nm (the excitation light of the fluorescent dye Cy3 of labeled DNA ), the fluorescence signal when the excitation/emission light is 352nm/461nm (the excitation light of the fluorescent dye Hoechst33342 that marks the cell nucleus), calculate the number of cells with Cy3 fluorescence signal in the cells in the randomly selected field of view, and the number of cells in the same field of view.
  • the proportion of cells with Hochest33342 fluorescent signal is the transfection efficiency.
  • the drug-metal-phospholipid complex particles can contain any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and achieve its function.
  • the length of the nucleic acid ranges from 16-3822nt. .
  • Example 3.8.2. Preparation of drug (different types of nucleic acids)-metal-phospholipid complex particles and their effects when the metal ion is Al 3+
  • Example 3.8.1 The difference between this embodiment and Example 3.8.1 is that the metal ion Fe 3+ in Example 3.8.1 is replaced by Al 3+ .
  • the drug-metal-phospholipid complex particles can contain any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and achieve its function.
  • the length of the nucleic acid ranges from 16-3822nt. .
  • Example 4.1 Synthesis characterization of metal-phospholipid complex when the metal ion is Fe 3+
  • the characterization method for the connection between DSPC and curcumin is differential scanning calorimetry.
  • the measurement conditions are: weigh 3 to 5 mg of the test substance, the heating rate is 10°C/min, and the temperature rise range is 30°C to 300°C.
  • Example 4.1 The difference between this example and Example 4.1 is that the connection between the phospholipid complex and Al 3+ is characterized by spectrophotometry: as shown in Figure 2-3, after the phospholipid complex (CUR-HSPC) is combined with Al 3+ , its maximum The absorption wavelength shifted from 420 nm to 433 nm, and the conjugated structure of the phospholipid complex changed, proving that Al 3+ was successfully complexed with curcumin.
  • Example 5 Characterization of the detachment of Fe 3+ from the metal-phospholipid complex under low pH conditions when the metal ion is Fe 3+
  • the phospholipid complex in the metal-phospholipid complex binds Fe 3+ through a coordination bond. Under the low pH value of the lysosome, the coordination bond between the phospholipid complex and Fe 3+ will be protonated (absorbing hydrogen ions) and break.
  • the coordination bond between the phospholipid complex and Fe 3+ will be protonated (absorbing hydrogen ions) and break.
  • the results suggest that Fe 3+ can be shed from the metal-phospholipid complex under the low pH conditions of lysosomes.
  • Example 6.1 Elemental composition of MPP in drug-metal-phospholipid complex particles when the metal ion is Fe 3+ analyze
  • Example 3 Replace the mRNA in Example 3 with thiol-modified siRNA, and prepare drug-metal-phospholipid complex particles siRNA@MPP by referring to the method in Example 3. Elemental analysis was performed using a transmission electron microscopy instrument. The results are shown in Figure 2-5: C, N, O, and P are common elements.
  • the Fe element analysis chart shows that Fe 3+ is evenly distributed on the lipid nanoparticles. Since siRNA is modified with sulfhydryl groups, the S element analysis chart The position of siRNA can be specifically expressed. It can be seen from the figure that siRNA is well complexed near Fe 3+ , proving that the drug-lipid nanoparticles successfully encapsulated siRNA.
  • Example 6.2 Electron microscopy analysis of MPP in drug-metal-phospholipid complex particles when the metal ion is Al 3+
  • Drug-metal-phospholipid complex particles MPP were prepared according to the method of Example 3. Morphological analysis was performed using a transmission electron microscopy instrument. The results are shown in Figure 2-6: The morphology of MPP in the drug-metal-phospholipid complex particles is a standard spherical shape with a uniform particle size of about 100 nm.
  • Example 7 The efficiency of encapsulating nucleic acids (siRNA and mRNA) in metal-phospholipid complex particles MPP when the metal ion is Fe 3+ or Al 3+ and its comparison with LNP.
  • Example 3 The mRNA in Example 3 was replaced with siRNA targeting the Bcl-2 gene (SEQ ID No. 4, 19 bp) and mRNA encoding the receptor binding domain (RBD) of the S1 subunit of the new coronavirus. (SEQ ID No. 2, 669nt), the nucleic acid-loaded drug-metal-phospholipid complex particles siRNA@MPP and mRNA@MPP were prepared respectively. The preparation process of the other drug-metal-phospholipid complex particles was the same as in Example 3.
  • siRNA@LNP and mRNA@LNP according to the same drug loading amount of siRNA@MPP in Example 3.6 and mRNA@MPP in Example 3.5.
  • the ratio of amino lipids to phosphate-containing nucleotides is 6:1.
  • the nucleic acid drug loading capacity is the same as that of the above-mentioned siRNA@MPP and mRNA@MPP.
  • Mix the aqueous phase and the organic phase Mix rapidly at a volume ratio of 3:1 at a flow rate of 14 mL/min.
  • dilute it ten times with enzyme-free PBS buffer solution and use a 100kDa ultrafiltration tube to concentrate the mixture to one-tenth.
  • the ethanol concentration in the mixture is reduced to less than 0.0005%.
  • the pH value of the solution is raised to the normal pH value of the PBS buffer solution (7.2 ⁇ 7.4), that is, siRNA@LNP and mRNA@LNP are produced respectively.
  • the agarose gel electrophoresis method was used to detect the encapsulation efficiency of nucleic acids (siRNA and mRNA) by siRNA@MPP, mRNA@MPP, siRNA@LNP and mRNA@LNP respectively.
  • the method for measuring the inclusion rate is as follows: the input amount of nucleic acid (siRNA and mRNA) in each group of lipid nanoparticles is set at 10 ⁇ g/mL, the mass ratio of lipid to nucleic acid is 40:1, and the nucleic acid is dissolved in PBS buffer solution , as the positive control group, and the negative control was nucleic acid-free PBS buffer solution.
  • the concentration of the agarose gel is 1.5%.
  • the gaps in the gel only allow free nucleic acids to pass through and do not allow lipid nanoparticles to pass.
  • the electrophoresis is stopped to prevent the nucleic acid from being degraded if the electrophoresis time is too long. .
  • the positive control group is set as 100%.
  • the ratio of free nucleic acids in each group to the positive control is the relative amount of free nucleic acids.
  • the inclusion rate of each group is (100- Relative amount of free nucleic acid)%.
  • Example 8 Nucleic acid lysosome escape ability of metal-phospholipid complex particle MPP when the metal ion is Fe 3+ or Al 3+ and its comparison with LNP
  • the Bcl-2-siRNA (SEQ ID No. 4) of Example 3.6 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@MPP (the concentration of siRNA contained was 100 nM).
  • the Bcl of Example 7 was -2-siRNA(SEQ ID No. 4) was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@LNP (the concentration of siRNA contained was 100 nM); the eGFP-mRNA (SEQ ID No.
  • Example 3.5 1) of Example 3.5 was replaced with Cy5 Labeled eGFP-mRNA, prepare Cy5-mRNA@MPP (containing mRNA concentration is 2 ⁇ g/mL), replace the RBD-mRNA of Example 7 with Cy5-labeled RBD-mRNA, prepare Cy5-mRNA@LNP (containing The concentration of mRNA was 2 ⁇ g/mL), and they were incubated with A549 cells for 3 hours with the cell lysosome probe Lysotracker Green. After that, a high-content imaging system was used to observe that the fluorescence signal of Cy5 (red) overlapped with the fluorescence signal of Lysotracker Green (green). In this case, the ability of the drug-lipid particles to promote nucleic acid lysosomal escape is determined and explored.
  • Determination criteria for the ability of drug-metal-phospholipid complex particles to promote nucleic acid lysosomal escape After incubating cells with drug-lipid nanoparticles for 3 hours, use a high-content imaging system to observe the fluorescence signal of Cy5 (red) and the fluorescence signal of Lysotracker Green ( Green) overlap, and use imageJ software to count the overlap rate of red fluorescence signal and green fluorescence signal.
  • the overlap rate of the red fluorescence signal and the green fluorescence signal was less than 50%, indicating that the nucleic acid can escape from the cell lysosome quickly, and its lipid nanoparticles have Better ability to promote nucleic acid lysosomal escape.
  • Example 9 The ability of metal-phospholipid complex particles MPP to promote nucleic acid expression when the metal ion is Fe 3+ or Al 3+ and its comparison with LNP
  • Example 7 The RBD-mRNA in Example 7 was replaced with mRNA encoding the fluorescent protein eGFP, and the remaining preparation methods were the same as in Example 7 to obtain eGFP-mRNA@LNP.
  • eGFP-mRNA@MPP prepared in Example 3 and the above-mentioned eGFP-mRNA@LNP (containing mRNA concentration is 2 ⁇ g/mL) were incubated with 293T cells respectively, and the control group was incubated with MPP or LNP, and the cell suspension was collected after 48 hours. The percentage of eGFP-positive cells was detected by flow cytometry.
  • Example 10 The ability of drug-metal-phospholipid complex particles MPP to promote humoral immunity and cellular immunity when the metal ion is Fe 3+ or Al 3+ and its comparison with LNP
  • the RBD-mRNA@MPP of Example 3.5 and the RBD-mRNA@LNP of Example 7 were incubated with 293T cells at a concentration of 2 ⁇ g/mL (concentration of mRNA contained), and the control group was incubated with MPP.
  • the method of detecting RBD expression level by ELISA is as described in Example 3.5.
  • the experimental animals were randomly divided into 3 groups (experimental group and control group), with 5 animals in each group.
  • the animal model was BALB/c mice. Each mouse received the first intramuscular administration on the 1st day and the second intramuscular administration on the 14th day.
  • the experimental group RBD-mRNA@MPP(Fe 3+ ), RBD-mRNA@MPP(Al 3+ ) or RBD-mRNA@LNP were injected respectively, and the control group was injected with MPP and LNP without mRNA.
  • the dose of each administration was 100 ⁇ L, and the RBD-mRNA@MPP(Fe 3+ ), RBD-mRNA@MPP(Al 3+ ) and RBD-mRNA@LNP preparations in the experimental group each contained 30 mg of mRNA.
  • the blood of mice was collected, the serum was separated and diluted in gradients, and the titers of RBD total IgG antibodies against the S1 subunit of the new coronavirus produced in the mice were detected by commercially available ELISA kits. The results As shown in Figure 2-11.
  • the method for detecting the titer of RBD total IgG antibodies against the S1 subunit of the new coronavirus by ELISA is as described in Example 3.5.
  • the method for detecting the expression levels of IFN- ⁇ , IL-2, and IL-4 by ELISA is as described in Example 3.5.
  • RBD-mRNA@MPP(Fe 3+ ), RBD-mRNA@MPP(Al 3+ ) and RBD-mRNA@LNP can all induce 293T cells to express a certain amount of RBD, but The ability of RBD-mRNA@MPP(Al 3+ ) to induce cells to express RBD is significantly stronger than that of RBD-mRNA@MPP(Fe 3+ ), and the ability of RBD-mRNA@MPP(Fe 3+ ) to induce cells to express RBD is significantly stronger than that of RBD.
  • the expression amount of RBD in the cell supernatant of the RBD-mRNA@MPP(Fe 3+ ) treated group is 205ng/mL, and the expression amount of RBD in the cell supernatant of the RBD-mRNA@MPP(Al 3+ ) treated group
  • the expression level of RBD in the cell supernatant of the RBD-mRNA@LNP treatment group was 115.7ng/mL.
  • RBD-mRNA@MPP effectively induced humoral immunity in mice and produced high levels of antigen-specific binding antibodies, and RBD-mRNA@MPP(Al 3+ ) induced humoral immunity in mice.
  • RBD-mRNA@MPP(Fe 3+ ) The ability is clearly better than RBD-mRNA@MPP(Fe 3+ ), and the ability of RBD-mRNA@MPP(Fe 3+ ) to induce humoral immunity in mice is clearly better than RBD-mRNA@LNP: RBD-mRNA@MPP(Fe 3+
  • the IgG antibody titer in the mice in the + ) treatment group reached 117233.8
  • the IgG antibody titer in the mice in the RBD-mRNA@MPP(Al 3+ ) treatment group reached 133116
  • the IgG antibody titer in the mice in the RBD-mRNA@LNP treatment group The degree reached only 67476.
  • RBD-mRNA@MPP(Al 3+ ) can effectively induce cellular immunity in mice, that is, activate immune cells and produce a large amount of cytokines, and mRNA@MPP(Al 3+ ) induces mouse
  • the ability of cellular immunity in mice is clearly better than RBD-mRNA@MPP(Fe 3+ ), and the ability of RBD-mRNA@MPP(Fe 3+ ) to induce cellular immunity in mice is clearly better than RBD-mRNA@LNP:
  • RBD-mRNA@ MPP(Fe 3+ ) increases the expression of cytokines IFN- ⁇ , IL-2, and IL-4 to 256.8pg/mL, 207.6pg/mL, and 61.8pg/mL respectively;
  • RBD-mRNA@MPP(Al 3+ ) The expression levels of cytokines IFN- ⁇ , IL-2, and IL-4 reached 298pg/mL, 249pg/mL, and 74.6
  • the expression levels of -4 are only 104.2pg/mL, 79.2pg/mL, and 27pg/mL.
  • the results suggest that the ability of mRNA@MPP(Al 3+ ) to deliver any mRNA and realize its function is significantly better than RBD-mRNA@MPP(Fe 3+ ), and RBD-mRNA@MPP(Fe 3+ ) induces cellular immunity in mice.
  • the ability is clearly better than that of RBD-mRNA@LNP: RBD-mRNA@MPP can more effectively promote the expression of target proteins by cells and can more effectively activate humoral immunity and cellular immunity in the body.
  • the drug (mRNA)-lipid particles are more effective in The effects of mRNA-loaded drugs, vaccines or other products are significantly better than the existing technology LNP.
  • MPP has a stronger ability to promote nucleic acid lysosomal escape
  • MPP has a stronger ability to promote nucleic acid expression into protein (antigen)
  • LNP ratio after the curcumin in MPP is separated from DSPC in the body, it serves as an immune adjuvant (also known as an immune modulator), which can activate humoral immunity and cellular immunity to enhance the effect of MPP delivered mRNA vaccine, and can also inhibit immune factors The storm thus suppresses excessive, harmful immune responses to the body.
  • Example 11 In vivo safety evaluation of metal-chelated phospholipid complex nanoparticles (MPP) when the metal ion is Fe 3+ or Al 3+
  • Half and half 14 animals in the medium-dose MPP group (16mg/kg) (including 8 in the experimental group and 6 in the recovery group), half and half male and half; 14 in the high-dose group (32mg/kg) (including 8 in the experimental group and 6 in the recovery group) 6), half male and half female.
  • the experimental group 32 animals in total
  • the recovery group 24 animals in total
  • the experimental animals were administered via tail vein injection, once every 2 days for a total of 20 days, and the body weight of SD rats was recorded once a week.
  • the prepared MPP was dissolved in DPBS, and the control group was injected with an equal amount of DPBS.
  • the low-dose MPP group, the medium-dose MPP group, and the high-dose MPP group were injected with 8 mg/kg, 16 mg/kg, and 32 mg/kg MPP respectively.
  • the basis for setting the dosage of the above-mentioned MPP when loading 200 ⁇ g/kg mRNA (the actual required amount for mRNA animal experiments), the required amount of empty carrier MPP is 8mg/kg.
  • the required amount of empty carrier MPP is 8mg/kg.
  • General indicator detection method After each administration, observe the general status of the animals in each group, including survival, diet, appearance characteristics, behavioral activities, weight, and whether there are any local reactions to administration.
  • a gross autopsy is performed, including timely weighing of the wet weight of major organs, such as the brain, heart, liver, spleen, lungs, and kidneys, calculation of organ-body ratios, and recording of pathological changes in each organ.
  • the organ-body ratio wet weight of rat organs/rat body weight ⁇ 100%.
  • the rats were dissected 20 days after administration and 20 days after the recovery period, and blood was collected from the abdominal aorta.
  • SD rats were anesthetized with isoflurane and fixed on the dissecting board with 75% ethanol.
  • Use a 5mL negative pressure ordinary blood collection tube to collect whole blood and let it stand at room temperature for 30 minutes. Centrifuge at 4°C and 1500rpm for 15 minutes. Take the supernatant in a 1.5mL centrifuge tube and store it at -20°C for analysis of blood biochemical indicators and immunology-related indicators. detection.
  • Routine blood testing methods Routine blood indicators include: number of white blood cells, number of lymphocytes, number of monocytes, number of neutrophils, percentage of lymphocytes, percentage of monocytes, percentage of neutrophils, number of red blood cells, hemoglobin, red blood cells Backlog, mean corpuscular volume, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, red blood cell distribution width coefficient of variation, platelet number, mean platelet volume, platelet distribution width, platelet packed volume. Gently mix the whole blood sample by inverting it, take a small amount of whole blood, and use a fully automatic blood cell analyzer to automatically analyze the results.
  • Blood biochemical indicators include inorganic ions (Fe 2+ , Na + , K + , Cl - , Ca 2+ ), liver function indicators (ALT, AST, ⁇ -GT, T-BIL, D- BIL, ALP, ALB), renal function indicators (BUN, UA, CR), cardiac function indicators (LDH, CK), glucose metabolism indicators (GSP, GLU, INS), lipid metabolism indicators (CHO, TG, LDL-C, HDL-C).
  • inorganic ions Fe 2+ , Na + , K + , Cl - , Ca 2+
  • liver function indicators ALT, AST, ⁇ -GT, T-BIL, D- BIL, ALP, ALB
  • renal function indicators BUN, UA, CR
  • cardiac function indicators LH, CK
  • glucose metabolism indicators GSP, GLU, INS
  • lipid metabolism indicators CHO, TG, LDL-C, HDL-C
  • Immunology-related indicators include thyroid function indicators (TT3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN- ⁇ , IFN- ⁇ , TNF- ⁇ ), immunoglobulins (IgG, IgA, IgM), serum complement (C3, CH50). Use ELISA method to detect the above indicators.
  • Methods for pathological examination of the main organs of SD rats At the end of the administration period and recovery period, rats in each group were anesthetized, and the main organs of the rats were removed by ophthalmology, including the whole brain, heart, liver, spleen, lungs, The kidneys were gently rinsed with 0.9% normal saline, fixed in 4% paraformaldehyde fixative, embedded in conventional paraffin, and stained with H&E. The histopathological changes of various organs of the rats in the control group and the experimental group were observed under an optical microscope.
  • the blood routine indicators (number of white blood cells, number of lymphocytes, and number of monocytes in the low, medium, and high dose MPP (Fe 3+ ) or MPP (Al 3+ ) groups , number of neutrophils, percentage of lymphocytes, percentage of monocytes, percentage of neutrophils, number of red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, coefficient of variation of red blood cell distribution width, There were no abnormalities in platelet number, average platelet volume, platelet distribution width, platelet packed volume); compared with the control group, the blood biochemical indicators of the low, medium and high dose MPP groups, including inorganic ions (Fe 2+ , Na + , K + , Cl - , Ca 2+ ), liver function indicators (ALT, AST, ⁇ -GT, T-BIL, D-BIL, ALP
  • the brain tissue structure of rats in the low, medium and high dose MPP (Fe 3+ ) or MPP (Al 3+ ) groups was intact, with normal tissue staining and intact cell morphology and structure.
  • the myocardial tissue structure is intact, the myocardial cells are arranged neatly, continuously, and tightly, the cell nuclei are clearly visible, and there is no obvious cell congestion, edema or necrosis; the liver cells are normal in shape, and there is no inflammatory cell aggregation and Necrosis; the structure of the spleen is normal, with clear boundaries between red and white pulp; the structure of the lung tissue is intact, the size of the alveoli is consistent, and there is no obvious accumulation and infiltration of inflammatory cells; the structure of the kidneys is normal.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ⁇ -GT glutamyl transpeptidase
  • T-BIL total bilirubin
  • D-BIL direct bilirubin
  • ALP alkaline phosphatase
  • ALB albumin
  • BUN urea nitrogen
  • UA uric acid
  • CR creatinine
  • LDH lactate dehydrogenase
  • CK creatine phosphokinase
  • GSP Fructosamine
  • GLU glucose
  • INS insulin
  • LDL-C low-density lipoprotein
  • HDL-C high-density lipoprotein
  • TT4 Tetraiodothyronine
  • TSH thyroid-stimulating hormone
  • IL-1 interleukin 1
  • Example 12 Comparison of the in vivo safety of metal-phospholipid composite particles (MPP) and LNP when the metal ion is Fe 3+ or Al 3+
  • the main toxicity of LNP comes from its main components - cationic lipids and/or ionizable lipids.
  • the 50% lethal dose (IC 50 ) of cationic lipids and/or ionizable lipids to biological cells is an important parameter for evaluating the toxicity of LNP to the body.
  • Metal-chelated phospholipid complex nanoparticles (MPP) replace cationic lipids/ionizable lipids in LNP with metal-phospholipid complexes. Therefore, we studied the relationship between metal-phospholipid complexes and cationic lipids. /The half lethal dose (IC 50 ) of ionizable lipids to biological cells, compare the differences in the toxicity of LNP and MPP (Fe 3+ ) or MPP (Al 3+ ).
  • DOTAP cationic lipid
  • ALC0315 ionizable lipid
  • Cell culture Cultivate cells in DMEM culture medium containing 10% FBS and 1% double antibody. Wait until the cell density reaches 80%-90% of the culture bottle before use;
  • Count Dilute the cell suspension to 10,0000 cells per 1mL according to the purpose, 100 ⁇ L per well in a 96-well plate, and at least 5 duplicate wells in each group. Incubate at 37°C, 5% CO 2 for 24 hours before adding medicine;
  • the calculation method of IC50 take the survival rate as the ordinate and the drug concentration as the abscissa. Use Graphpad to calculate IC 50 using the [Inhibitor] vs. normalized response--Variable slope analysis method.
  • MPP 8mg/kg
  • LNP 3.24mg/kg
  • nucleic acid 200 ⁇ g/kg mRNA
  • the IC 50 of the metal-phospholipid complex is significantly greater than the cationic lipid (DOTAP) and the ionizable lipid (ALC0315).
  • the results show that the toxicity of metal-phospholipid complexes is significantly less than that of cationic lipids and ionizable lipids.
  • the liver function (ALT, AST, ALP) and cytokines of the MPP (Fe 3+ ) or MPP (Al 3+ ) group (IL-6, IL-1 ⁇ ) showed no obvious abnormalities.
  • the liver function (ALT, AST, ALP) and cytokines (IL-6, IL-1 ⁇ ) in the LNP group were significantly increased. The results suggest that the in vivo safety of MPP (Fe 3+ ) or MPP (Al 3+ ) is higher than that of LNP.
  • the core component of LNP is synthetic "cationic lipid/ionizable lipid" and its cytotoxicity It has high immunogenicity, relatively stable structure, and is difficult to be catabolized in the body; while the core component of MPP (Fe 3+ ) or MPP (Al 3+ ) is a metal-phospholipid complex, and its metal-phospholipid complex is It is composed of phospholipid molecules, highly safe natural small molecule curcumin (an FDA-approved food additive and pharmaceutical excipient) and safe metal ions, and has been decomposed into natural molecules in the body after drug delivery.
  • MPP(Fe 3+ ) or MPP(Al 3+ ) because there are no cationic lipids/ionizable lipids in the components of MPP(Fe 3+ ) or MPP(Al 3+ ), it will not cause toxic side reactions related to cationic lipids/ionizable lipids, so MPP( Fe 3+ ) or MPP(Al 3+ ) are safer than LNP.
  • Table 2-2 IC 50 and comparison of metal-phospholipid complexes with cationic lipids (DOTAP) and ionizable lipids (ALC0315) when the metal ion is Fe 3+ or Al 3+
  • Example 13 Clinical application and administration route of drug-metal-phospholipid complex particles when the metal ion is Fe 3+ or Al 3+
  • Example 3 The mRNA in Example 3 was replaced with siRNA targeting the B7-H4 gene (B7-H4-siRNA) and its control (scr-siRNA), and the receptor binding domain encoding the S1 subunit of the new coronavirus. ,RBD) mRNA (RBD-mRNA).
  • sequences of the above different nucleic acids are: 1The sequences of B7-H4-siRNA are SEQ ID No. 19 (sense strand) and SEQ ID No. 26 (antisense strand) (25bp), and its random control sequence is SEQ ID No. 20 ( Sense strand) and SEQ ID No. 27 (antisense strand) (19bp); 2 The mRNA sequence encoding the receptor binding domain (RBD) of the S1 subunit of the new coronavirus is SEQ ID No. 2 (669nt) .
  • the drug-metal-phospholipid complex particles containing the above different types of nucleic acids (B7-H4-siRNA@MPP(Fe 3+ ), Preparation process and implementation of RBD-mRNA@MPP(Fe 3+ ), B7-H4-siRNA@MPP(Al 3+ ), RBD-mRNA@MPP(Al 3+ )), and other drug-metal-phospholipid complex particles Same as Example 3.
  • the above two different drug-metal-phospholipid complex particles (B7-H4-siRNA@MPP, RBD-mRNA@MPP) are respectively used to treat liver cancer and as an mRNA vaccine to prevent the new coronavirus.
  • B7-H4-siRNA The sequence of B7-H4-siRNA is as follows:
  • the random control sequence of B7-H4-siRNA is as follows:
  • HepG2 cells were used to create an animal model of liver cancer.
  • mice with liver cancer were randomly divided into 7 groups (5 mice in each group): PBS control group, blank vector MPP (Fe 3+ ) group, blank vector MPP (Al 3+ ) group, Scr-siRNA@MPP (Fe 3+ ) control group, B7-H4-siRNA@MPP(Fe 3+ ) treatment group, Scr-siRNA@MPP(Al 3+ ) control group, B7-H4-siRNA@MPP(Al 3+ ) treatment group.
  • mice in each group were intratumorally injected with PBS, MPP(Fe 3+ ), MPP(Al 3+ ), Scr-siRNA@MPP(Fe 3+ ), B7-H4 siRNA@MPP(Fe 3+ ), Scr-siRNA@MPP(Al 3+ ) and B7-H4 siRNA@MPP(Al 3+ ) were injected once at a dose of 200 ⁇ g siRNA/kg for 8 injections. Tumor volume was measured and recorded every 3 days. The results are shown in Figure 3-1.
  • the ELISA detection method is as described in Example 3.5.
  • liver cancer mouse model Collect HepG2 cells, re-suspend them in PBS at a density of 1 ⁇ 10 7 /mL, and store them on ice before inoculation. Then 100 ⁇ L of cell suspension was injected subcutaneously into the back area near the hind legs of female Balb/c nude mice to establish a liver cancer mouse model.
  • RBD-mRNA@MPP(Fe 3+ ) increased the expression level of mouse IgG antibodies to 117268.8 ( Figure 1-3), and increased the cytokine IFN- ⁇ , IL-2, and IL-4 expression levels reached 252.8pg/mL, 207.6pg/mL, and 56.6pg/mL respectively ( Figure 1-5).
  • RBD-mRNA@MPP(Al 3+ ) increased the expression level of mouse IgG antibodies to 129113 ( Figure 1-9), and increased the expression levels of cytokines IFN- ⁇ , IL-2, and IL-4 to 271.8pg/mL respectively.
  • RBD-mRNA@MPP can effectively induce humoral immunity in mice and produce high levels of antigen-specific binding antibodies; it can also effectively induce cellular immunity in mice, that is, activating immune cells and producing a large amount of cytokines. Therefore, RBD-mRNA@MPP can effectively prevent novel coronavirus infection.
  • B7-H4-siRNA@MPP can effectively treat liver cancer using the intratumoral injection route; as shown in Figure 1-3, Figure 1-5, Figure 1-9 and Figure 1-11 of Example 3.5 It is shown that RBD-mRNA@MPP can activate humoral immunity and cellular immunity by intramuscular injection route, thereby preventing new coronavirus infection. The results suggest that drug-metal-phospholipid complex particles can be administered through multiple routes.
  • Example 4 Functions of DSPC, curcumin, Fe 3+ or Al 3+ after being replaced by similar substances
  • Example 15 Functions of DSPC, curcumin, Fe 3+ or Al 3+ after being replaced by similar substances
  • Example 1 Example 2 and Example 3, DSPC, curcumin, and Fe 3+ were replaced with similar substances of DSPC, curcumin, and Fe 3+ respectively, and 28 different eGFP-mRNAs were prepared through different combinations.
  • @MPP The concentration of mRNA contained in each eGFP-mRNA@MPP is 2 ⁇ g/mL.
  • the names and structures of DSPC, curcumin, Fe 3+ and their congeners are shown in Table 4-1.
  • the combinations of DSPC, curcumin, Fe 3+ and their congeners in the 28 kinds of mRNA@MPP are shown in Table 4-2 shown.
  • the reaction temperature in Example 1 is 65°C and the reaction time is 2 hours.
  • the reaction temperature in Example 2 is 60°C and the reaction time is 2 hours. Other conditions remain unchanged.
  • the above 28 different eGFP-mRNA@MPP and the above-mentioned eGFP-mRNA@LNP were incubated with 293T cells respectively.
  • the control group was incubated with MPP or LNP, and the cell suspension was collected after 48 hours. , the percentage of eGFP-positive cells was detected by flow cytometry.
  • the main toxicity of LNP comes from its main component - cationic lipids/ionizable lipids.
  • IC 50 50% lethal dose
  • MPP Metal-chelated phospholipid complex nanoparticles
  • the IC 50 of 28 metal-phospholipid complexes are significantly greater than that of cationic lipids (DOTAP) and ionizable lipids (ALC0315). It is suggested that the toxicity of metal-phospholipid complexes is clearly less than that of cationic lipids and ionizable lipids, that is, lipid nanoparticles (MPP) composed of DSPC, curcumin, Fe 3+ and their congeners are safer than LNP.
  • MPP lipid nanoparticles
  • the core component of LNP is artificially synthesized "cationic lipid/ionizable lipid", which has high cytotoxicity and immunogenicity, and its structure is relatively stable and difficult to be catabolized in the body; while the core component of MPP It is a metal-phospholipid complex.
  • the metal-phospholipid complex is composed of phospholipid molecules, highly safe natural small molecule substances (of which curcumin is an FDA-approved food additive and pharmaceutical excipient) and safe metal ions. After drug delivery is complete, it has been broken down into natural molecules in the body.
  • lipid particles composed of DSPC, curcumin, Fe 3+ and their congeners do not contain cationic lipids/ionizable lipids and will not cause cationic lipids/ionizable lipids. toxic and side effects, so MPP is safer than LNP.
  • Table 4-2 List of combinations and functions of metal-phospholipid complexes in drug-lipid particles prepared from DSPC, curcumin, Fe 3+ and their congeners
  • Example 16 DSPC of different metal-phospholipid complexes, curcumin and its analogues, Fe 3+ component dosage ratio and the functions of the prepared drug-metal-phospholipid complex particles
  • Example 3 a metal-phospholipid complex was prepared, and curcumin was replaced with its analogue hesperetin. (1 molecule of hesperetin contains 4 hydroxyl groups), tea polyphenols (1 molecule of tea polyphenols contains 8 hydroxyl groups), and three metal-phospholipid complexes (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP29) were prepared.
  • the dosage ratios of DSPC, curcumin or its analogues, and FeCl 3 are respectively: 1:1:1, 1:1:1, and 1:1:2.
  • the corresponding drug-metal-phospholipid complex nanoparticles were prepared using these three metal-phospholipid complexes (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP29).
  • the mRNA is the mRNA encoding eGFP fluorescent protein, and its sequence is SEQ ID No. 1 (720nt).
  • SEQ ID No. 1 720nt
  • the basis for adjusting the dosage ratio is: because the congeners of DSPC and the congeners of curcumin are connected by hydrogen bonds, as long as the congeners of DSPC contain multiple phosphate groups, then when synthesizing the phospholipid complex, the congeners of DSPC and curcumin
  • the dosage ratio of the congeners of DSPC can be adjusted according to the number of phosphate groups contained in the congeners of DSPC, that is, when the congeners of DSPC contain two phosphate groups, the dosage ratio of the congeners of DSPC and the congeners of curcumin It can be adjusted to 1:2; when DSPC congeners contain three phosphate groups, the dosage ratio of DSPC congeners and curcumin congeners can be adjusted to 1:3; because the hydroxyl group and Fe of curcumin congeners 3+ congeners are connected by coordination bonds. As long as curcumin congeners contain multiple binding sites, the dosage ratio of curcumin congeners and Fe 3+ con
  • Example 17 DSPC, curcumin and its analogues, Al 3+ component dosage ratios of different metal-phospholipid complexes and the functions of the prepared drug-metal-phospholipid complex particles
  • Example 3 a metal-phospholipid complex was prepared, and curcumin was replaced with its analogs hesperetin (one molecule of hesperetin contains 4 hydroxyl groups) and tea polyphenols (one molecule of tea polyphenols contains 8 hydroxyl groups). ), three metal-phospholipid complexes (mRNA@MPP2, mRNA@MPP5, and mRNA@MPP30) were prepared. When preparing these three metal-phospholipid complexes, the dosage ratios of DSPC, curcumin or its analogues, and Al(NO 3 ) 3 ⁇ 9H 2 O are respectively: 1:1:1, 1:1:1, and 1:1. :2.
  • the corresponding drug-metal-phospholipid complex nanoparticles were prepared using these three metal-phospholipid complexes (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP29).
  • the mRNA is the mRNA encoding eGFP fluorescent protein, and its sequence is SEQ ID No. 1 (720nt).
  • SEQ ID No. 1 720nt
  • the dosage ratio of curcumin congeners can be adjusted based on the number of phosphate groups contained in DSPC congeners. That is, when DSPC congeners contain two phosphate groups, the proportion of DSPC congeners and curcumin congeners will The dosage ratio can be adjusted to 1:2; when DSPC congeners contain three phosphate groups, the dosage ratio of DSPC congeners and curcumin congeners can be adjusted to 1:3; because the hydroxyl group of curcumin congeners It is connected to Al 3+ congeners by coordination bonds. As long as curcumin congeners contain multiple binding sites, the dosage ratio of curcumin congeners and Al 3+ congeners can be based on the curcumin congeners. The number of binding sites contained is adjusted.
  • Example 3 The preparation of mRNA@MPP in Example 3 was completed by the research group of Professor Wang Shan from the Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Central South University.

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Abstract

一种金属-磷脂复合物、金属-磷脂复合物颗粒和药物-脂质颗粒及其制备方法与应用，涉及生物技术领域。金属-磷脂复合物由磷脂分子部分、连接物分子部分和金属离子部分反应组成。金属磷脂复合物颗粒含有金属-磷脂复合物、抑制颗粒聚集的缀合的脂质，以及非阳离子脂质或非可电离脂质。药物-脂质颗粒包含药物和金属-磷脂复合物颗粒。金属-磷脂复合物用于吸附带有负电荷的药物，其与其他组分自组装成金属-磷脂复合物颗粒(MPP)，在不低于基于阳离子脂质和/或可电离脂质的LNP的有效性情况下，未使用阳离子脂质和可电离脂质，较LNP，毒性大幅度降低，生物安全性显著提升，更利于在生物体内运载负电荷药物。

Description

金属-磷脂复合物、金属-磷脂复合物颗粒和药物-脂质颗粒及其制备方法与应用
相关申请的交叉引用
本公开要求于2022年08月09日提交中国专利局的申请号为202210951941.8、名称为“药物-脂质颗粒及其制备方法和应用”，2022年08月09日提交中国专利局的申请号为202210950392.2、名称为“金属-磷脂复合物颗粒及其制备方法和应用”，2022年08月09日提交中国专利局的申请号为202210950391.8、名称为“金属-磷脂复合物及其制备方法和应用”，3件中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及生物技术领域，具体涉及一种金属-磷脂复合物、金属-磷脂复合物颗粒和药物-脂质颗粒及其制备方法与应用。
背景技术
随着分子生物学技术的不断发展，基因与疾病之间的关联性认识也越来越深入。核酸药物是指人工合成的具有疾病治疗功能的DNA或RNA片段，因其在疾病诊断和治疗过程中显示出巨大的应用潜力而备受关注。此类药物能够直接作用于致病靶基因或mRNA，在基因水平上发挥治疗作用。与传统小分子药物和抗体药物相比，核酸药物不受靶点蛋白可成药性的限制，可治疗的疾病更广泛，并可从根源上调控致病基因的表达。核酸药物还具有高效性、低毒性和高特异性等明显优势，有望成为继小分子药物和抗体药物之后的第三大类型药物。
然而，核酸药物在体内易被核酸酶降解，并且由于其分子量较大以及其带负电荷的特性，导致其难以穿过细胞膜发挥作用。因此，寻找安全、有效的核酸药物递送系统是核酸药物开发亟待解决的瓶颈问题。目前，能递送核酸药物的载体主要可以分为病毒载体和非病毒载体。病毒载体进入人体后会引起免疫反应，现使用较少；非病毒载体中较为常用的主要是纳米颗粒和小分子缀合物，与直接与核酸药物缀合的小分子缀合物相比，纳米颗粒能更有效地将核酸药物包裹起来，防止其在体内被核酸酶快速降解，从而提高其体内循环时间。纳米颗粒包裹核酸的机制是靠带有正电荷的阳离子脂质吸附带负电荷的核酸。然而，阳离子脂质的细胞毒性较大，其毒性作用机制为：①使细胞萎缩、有丝分裂数量减少和细胞质空泡化；②与蛋白激酶C等生物蛋白质相互作用从而会破坏其活性；③通过激活p38丝裂原活化蛋白激酶和核因子κB转录因子触发多种促炎细胞因子和趋化因子的分泌。此外，可电离脂质是一种含有带正电荷的可电离胺基团的脂质，其在生理条件下(pH＝7.4)不带电，但在较低pH值时会被质子化而带上正电荷。因此，可电离的脂质可被用来部分或完全替代阳离子脂质作为纳米颗粒的主要成分，负责吸附核酸。当包含可电离脂质的纳米颗粒进入生物细胞溶酶体后，在溶酶体内的低pH值(pH＝4.0-6.5)环境中，可电离的脂质变为带正电的脂质。虽然可电离脂质降低了一些永久带正电荷的阳离子脂质的细胞毒性作用和高致炎症作用，但其细胞毒性及免疫原性依然较高。基于阳离子脂质和/或可电离脂质的脂质纳米粒(Lipid Nanoparticle,LNP)是目前可用于临床的纳米颗粒核酸药物递送系统，其中，阳离子脂质和/或可电离脂质作为LNP的主要成分，负责吸附核酸，同 时阳离子脂质和/或可电离脂质介导的细胞毒性及免疫原性依然是LNP毒性较大的重要原因之一。
因此，用递送系统递送带负电荷的药物(例如核酸药物、蛋白药物、多肽药物、小分子药物等)时，依赖阳离子脂质和/或可电离脂质开发的纳米颗粒递送系统均不能从根本上解决纳米颗粒递送系统的毒性问题，急需一种不使用阳离子脂质和/或可电离脂质的、毒性低的脂质体递送系统。
发明内容
本公开的目的在于提供一种金属-磷脂复合物及其制备方法，以至少缓解一个现有技术中存在的技术问题。
为了实现上述目的，本公开特采用如下技术方案：
本公开提供的金属-磷脂复合物由磷脂分子部分、连接物分子部分和金属离子部分反应组成，该磷脂分子部分与该连接物分子部分相连接，该连接物分子部分与该金属离子部分通过配位键连接，且该金属-磷脂复合物不是阳离子脂质或可电离脂质。
进一步地，该磷脂分子部分选自卵磷脂(PC)、磷脂酰乙醇胺(PE)、磷脂酰丝氨酸(PS)、磷脂酸(PA)、磷脂酰甘油(PG)、1-磷酸神经酰胺(SP)、磷脂酰肌醇(PI)、磷脂酰苏氨酸(PT)、鞘磷脂(SM)、溶血卵磷脂(LPC)、溶血磷酸酰乙醇胺(LPE)、溶血磷脂酰丝氨酸(LPS)、溶血磷脂酸(LPA)、溶血磷脂酰甘油(LPG)、溶血磷脂酰肌醇(LPI)、溶血磷脂酰苏氨酸(LPT)、溶血鞘磷脂(LSM)、1-磷酸鞘氨醇(S1P)，及其衍生物中一种或多种的组合。其中，“及其衍生物”中“其”是指“卵磷脂(PC)、磷脂酰乙醇胺(PE)、磷脂酰丝氨酸(PS)、磷脂酸(PA)、磷脂酰甘油(PG)、1-磷酸神经酰胺(SP)、磷脂酰肌醇(PI)、磷脂酰苏氨酸(PT)、鞘磷脂(SM)、溶血卵磷脂(LPC)、溶血磷酸酰乙醇胺(LPE)、溶血磷脂酰丝氨酸(LPS)、溶血磷脂酸(LPA)、溶血磷脂酰甘油(LPG)、溶血磷脂酰肌醇(LPI)、溶血磷脂酰苏氨酸(LPT)、溶血鞘磷脂(LSM)、1-磷酸鞘氨醇(S1P)”。本公开中，“及其衍生物”均为类似的含义。该磷脂分子部分，例如可以但不限于为卵磷脂(PC)，卵磷脂(PC)衍生物，磷脂酰乙醇胺(PE)，磷脂酰乙醇胺(PE)衍生物，磷脂酰甘油(PG)，磷脂酰甘油(PG)衍生物，磷脂酰甘油(PG)和卵磷脂(PC)，卵磷脂(PC)和卵磷脂(PC)衍生物，等。
进一步地，该磷脂分子部分选自
及其衍生物中一种或多种的组合；
其中，R1，R2均独立地为：

进一步地，该磷脂分子部分选自卵磷脂(PC)(式1)、磷脂酰乙醇胺(PE)(式2)、磷脂酸(PA)(式4)、磷脂酰甘油(PG)(式5)，及其衍生物中一种或多种的组合。
进一步地，该磷脂分子部分选自二硬脂酰基磷脂酰胆碱(DSPC)、二硬脂酰基磷脂酰乙醇胺(DSPE)、二硬脂酰基磷脂酸(DSPA)、二硬脂酰磷脂酰甘油(DSPG)，及其衍生物中一种或多种的组合。
进一步地，该磷脂分子部分选自 及其衍生物中一种或多种的组合。
进一步地，该磷脂分子部分选自DSPC(式46)、DSPE(式47)或DSPA(式48)或DSPG(式49)。
进一步地，该连接物分子部分选自姜黄素、绿原酸、花青素、槲皮素、二氢杨梅素、橙皮素、柚皮素、芹菜素、儿茶素、茶多酚、表没食子儿茶素没食子酸酯、鞣花酸、桑色素、表儿茶素没食子酸酯、儿茶素没食子酸酯、没食子儿茶素没食子酸酯或平贝碱C，及其衍生物中一种或多种的组合。
进一步地，该连接物分子部分选自
R7和R8是H、OH或OCH3，R3是H或糖基，R4、R5和R6是OH或糖基；
及其衍生物中一种或多种的组合。
进一步地，连接物分子部分选自姜黄素(式19)、 中一种或多种的组合。
进一步地，该连接物分子部分选自姜黄素(式19)、橙皮素(式24)、茶多酚(式28)，及其衍生物中一种或多种的组合。
进一步地，该连接物分子部分选自姜黄素(式19)、橙皮素(式24)或茶多酚(式28)。
进一步地，该金属离子部分选自Fe3+、Ag+、Ba2+、Ca2+、Cd2+、Cu2+、Fe2+、Mn2+、Mg2+、Mo2+、Zn2+、Pt2+、Au2+、Al3+、Ce3+、Co3+、Cr3+、Eu3+、Gd3+、Ni3+、W3+、V3+、Zr3+中一种或多种的组合。
进一步地，该金属离子部分选自Fe3+、Ca2+、Al3+中一种或多种的组合。
进一步地，该金属离子部分选自Fe3+、Ca2+或Al3+。
进一步地，该金属-磷脂复合物由磷脂分子部分、连接物分子部分和金属离子部分制成，该磷脂分子部分选自DSPC、DSPE或DSPA，该连接物分子部分选自姜黄素、橙皮素或茶多酚，该金属离子部分选自Fe3+、Ca2+或Al3+。
进一步地，所述金属-磷脂复合物由磷脂分子部分、连接物分子部分和金属离子部分制成，该磷脂分子部分选自DSPC(式46)、DSPE(式47)或DSPA(式48)，该连接物分子部分选自姜黄素(式19)、橙皮素(式24)或茶多酚(式28)，该金属离子部分选自Fe3+、Ca2+或Al3+。
进一步地，该磷脂分子部分、该连接物分子部分和该金属离子部分的摩尔比为1:1：(0.5～2)。
进一步地，该磷脂分子部分为DSPC(式46)，该连接物分子部分选自姜黄素(式19)，该金属离子部分选自Fe3+。
进一步地，该磷脂分子部分、该连接物分子部分和该金属离子部分的摩尔比为1:1:1。
进一步地，DSPC、姜黄素和Fe3+/Al3+的摩尔比为1:1:1。
进一步地，DSPC、橙皮素和Fe3+/Al3+的摩尔比为1:1:1。
进一步地，DSPC、茶多酚和Fe3+/Al3+的摩尔比为1:1:2。
进一步地，Fe3+选自FeCl3，Al3+选自Al(NO3)3·9H2O。
本公开提供金属-磷脂复合物的制备方法，包括以下步骤：
步骤一：将磷脂分子与连接物分子反应连接形成磷脂复合物；
步骤二：将步骤一中制备的所述磷脂复合物与金属离子通过配位键反应形成金属-磷脂复合物。
进一步地，该步骤一中，将该磷脂分子与该连接物分子溶于乙醇中反应，之后加入正己烷，沉淀得到该磷脂复合物。进一步地，反应的条件包括65℃反应2小时。
进一步地，该磷脂分子与该连接物分子的摩尔比为1:1。
进一步地，该步骤二中，该磷脂复合物与该金属离子溶于乙醇反应后，得到该金属-磷脂复合物。进一步地，反应的条件包括60℃反应2小时。
进一步地，该磷脂复合物与该金属离子的摩尔比为1：(1～2)。
本公开提供一种金属-磷脂复合物颗粒(Metal-chelated phospholipid complex nanoparticles，MPP)，其含有(i)上述金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质，其中该抑制颗粒聚集的缀合的脂质不是阳离子脂质或可电离脂质；以及(iii)除该金属-磷脂复合物和该抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质。其中，“(iii)除该金属-磷脂复合物和该抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质”可简称为“非阳离子脂质或非可电离脂质”。
进一步地，该抑制颗粒聚集的缀合的脂质包括聚乙二醇(PEG)-脂质缀合物和/或PEG-二烷氧基丙基(DAA)。
进一步地，该PEG-脂质缀合物选自
，及其衍生物中一种或多种的组合；
R1，R2均独立地为：葵酰基、月桂酰基、肉豆蔻酰基、棕榈酰基、硬脂酰基、油酰基、亚油酰基、芥酰基、花生酰基或植烷酰基。
进一步地，该PEG-脂质缀合物选自DSPE-PEG2000、DSPE-PEG700、DSPE-PEG1000或DSPE-PEG5000中一种或多种的组合。
进一步地，该PEG-脂质缀合物选自 中一种或多种的组合。
进一步地，PEG-脂质缀合物选自DSPE-PEG2000(式53)、DSPE-PEG700 (式50)、DSPE-PEG1000(式51)或DSPE-PEG5000(式52)。
进一步地，(iii)中除该金属-磷脂复合物和该抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质为胆固醇及其衍生物中一种或多种的组合。
进一步地，(iii)中除该金属-磷脂复合物和该抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质为胆固醇(式40)及其衍生物中一种或多种的组合。
进一步地，(iii)中的非阳离子脂质或非可电离脂质为胆固醇(式40)。
进一步地，(iii)中除该金属-磷脂复合物和该抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质，除胆固醇还包括选自卵磷脂PC、磷脂酰乙醇胺PE、磷脂酰丝氨酸PS、磷脂酸PA、磷脂酰甘油PG、1-磷酸神经酰胺SP、磷脂酰肌醇PI、磷脂酰苏氨酸PT、鞘磷脂SM、溶血卵磷脂LPC、溶血磷酸酰乙醇胺LPE、溶血磷脂酰丝氨酸LPS、溶血磷脂酸LPA、溶血磷脂酰甘油LPG、溶血磷脂酰肌醇LPI、溶血磷脂酰苏氨酸LPT、溶血鞘磷脂LSM、1-磷酸鞘氨醇S1P、胆固醇硫酸酯，及其衍生物中一种或多种的组合。
进一步地，(iii)中除该金属-磷脂复合物和该抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质，除胆固醇还包括选自： 及其衍生物中一种或多种的组合；
其中，R1，R2为：葵酰基、月桂酰基、肉豆蔻酰基、棕榈酰基、硬脂酰基、油酰基、亚油酰基、芥酰基、花生酰基或植烷酰基。
进一步地，(iii)中除该金属-磷脂复合物和该抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质，包括胆固醇，以及选自DSPC、DSPE、DSPA或DSPG中一种或多种的组合。
进一步地，(iii)中除该金属-磷脂复合物和该抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质，包括胆固醇(式40)，以及选自DSPC(式46)、DSPE(式47)、DSPA(式48)或DSPG(式49)中一种或多种的组合。
进一步地，(iii)中所述的非阳离子脂质或非可电离脂质包括胆固醇(式40)和DSPC(式46)。
进一步地，金属-磷脂复合物颗粒由(i)金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质和(iii)除该金属-磷脂复合物和该抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质制成，该金属-磷脂复合物在原料中摩尔占比为10％～40％，该抑制颗粒聚集的缀合的脂质在原料中摩尔占比为2％～10％，该胆固醇在原料中摩尔占比为35％～75％，该除胆固醇以外的非阳离子脂质或非可电离脂质在原料中摩尔占比为0％～40％。
进一步地，其中，该金属-磷脂复合物颗粒由(i)金属-磷脂复合物、(ii) 抑制颗粒聚集的缀合的脂质和(iii)非阳离子脂质或非可电离脂质制成，该金属-磷脂复合物在原料中摩尔占比为5％～小于10％，该抑制颗粒聚集的缀合的脂质在原料中摩尔占比为2％～10％，该胆固醇在原料中摩尔占比为15％～小于35％、35％～75％或大于75％～80％，该除胆固醇以外的非阳离子脂质或非可电离脂质在原料中摩尔占比为0％～40％或大于40％～51％。或
该金属-磷脂复合物在原料中摩尔占比为大于40％～50％，该抑制颗粒聚集的缀合的脂质在原料中摩尔占比为2％～10％，该胆固醇在原料中摩尔占比为15％～小于35％、35％～75％或大于75％～80％，该除胆固醇以外的非阳离子脂质或非可电离脂质在原料中摩尔占比为0％～40％或大于40％～51％。或
该金属-磷脂复合物在原料中摩尔占比为10％～40％，该抑制颗粒聚集的缀合的脂质在原料中摩尔占比为2％～10％，该胆固醇在原料中摩尔占比为15％～小于35％或大于75％～80％，该除胆固醇以外的非阳离子脂质或非可电离脂质在原料中摩尔占比为0％～40％或大于40％～51％。
进一步地，金属-磷脂复合物在原料中摩尔占比为7％～小于10％、10％～30％或20％～30％，优选为25％。
进一步地，抑制颗粒聚集的缀合的脂质在原料中摩尔占比为3％～10％或5％～10％，优选为10％。
进一步地，胆固醇在原料中摩尔占比为15％～小于35％、35％～56％或35％～55％，优选为40％。
进一步地，除胆固醇以外的非阳离子脂质或非可电离脂质在原料中摩尔占比为5％～30％、25％～40％、大于40％～45％或20％～25％。
进一步地，该金属-磷脂复合物在原料中摩尔占比为15％～25％，该抑制颗粒聚集的缀合的脂质在原料中摩尔占比为4％～10％，该胆固醇在原料中摩尔占比为40％～46％，该DSPC在原料中摩尔占比为25％～35％，该金属-磷脂复合物中金属离子部分选自Fe3+。
进一步地，该金属-磷脂复合物(金属离子部分为Fe3+)在原料中摩尔占比为15％，该抑制颗粒聚集的缀合的脂质在原料中摩尔占比为4％，该胆固醇在原料中摩尔占比为46％，该DSPC在原料中摩尔占比为35％；或所述金属-磷脂复合物(金属离子部分为Fe3+)在原料中摩尔占比为25％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为10％，所述胆固醇在原料中摩尔占比为40％，所述DSPC在原料中摩尔占比为25％。
进一步地，该金属-磷脂复合物在原料中摩尔占比为10％～30％，该抑制颗粒聚集的缀合的脂质在原料中摩尔占比为3％～10％，该胆固醇在原料中摩尔占比为35％～56％，该DSPC在原料中摩尔占比为34％～40％；该金属-磷脂复合物中金属离子部分选自Al3+。
进一步地，该金属-磷脂复合物在原料中摩尔占比为10％～30％，该抑制颗粒聚集的缀合的脂质在原料中摩尔占比为3％～10％，该胆固醇在原料中摩尔占比为35％～56％，该DSPC在原料中摩尔占比为40％～45％，该金属-磷脂复合物中金属离子部分选自Al3+；或
该金属-磷脂复合物在原料中摩尔占比为10％～30％，该抑制颗粒聚集的缀合的脂质在原料中摩尔占比为3％～10％，该胆固醇在原料中摩尔占比为15％～35％，该DSPC在原料中摩尔占比为34％～40％或大于40％～45％，该金属-磷脂复合物中金属离子部分选自Al3+；或
该金属-磷脂复合物在原料中摩尔占比为7％～小于10％，该抑制颗粒聚集的缀合的脂质在原料中摩尔占比为3％～10％，该胆固醇在原料中摩尔占比为15％～小 于35％或35％～56％，该DSPC在原料中摩尔占比为34％～40％或大于40％～45％，该金属-磷脂复合物中金属离子部分选自Al3+。
进一步地，该金属-磷脂复合物(金属离子部分为Al3+)在原料中摩尔占比为7％，该抑制颗粒聚集的缀合的脂质在原料中摩尔占比为3％，该胆固醇在原料中摩尔占比为56％，该DSPC在原料中摩尔占比为34％。
进一步地，金属-磷脂复合物为DSPC、姜黄素和Fe3+的摩尔比为1:1:1制成，抑制颗粒聚集的缀合的脂质为DSPE-PEG2000，非阳离子脂质或非可电离脂质为胆固醇和DSPC，金属-磷脂复合物在原料中摩尔占比为15％，DSPE-PEG2000在原料中摩尔占比为4％，胆固醇在原料中摩尔占比为46％，DSPC在原料中摩尔占比为35％。
进一步地，金属-磷脂复合物为DSPC、姜黄素和Fe3+的摩尔比为1:1:1制成，抑制颗粒聚集的缀合的脂质为DSPE-PEG2000，非阳离子脂质或非可电离脂质为胆固醇和DSPC，金属-磷脂复合物在原料中摩尔占比为25％，DSPE-PEG2000在原料中摩尔占比为10％，胆固醇在原料中摩尔占比为40％，DSPC在原料中摩尔占比为25％。
进一步地，金属-磷脂复合物为DSPC、姜黄素和Al3+的摩尔比为1:1:1制成，抑制颗粒聚集的缀合的脂质为DSPE-PEG2000，非阳离子脂质或非可电离脂质为胆固醇和DSPC，金属-磷脂复合物在原料中摩尔占比为7％，DSPE-PEG2000在原料中摩尔占比为3％，胆固醇在原料中摩尔占比为56％，DSPC在原料中摩尔占比为34％。
本公开提供上述金属-磷脂复合物颗粒的制备方法，包括将(i)金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质和(iii)除该金属-磷脂复合物和该抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质混合，得到该金属-磷脂复合物颗粒。
本公开提供一种药物-脂质颗粒，其中含有：
(a)药物，其中该药物为带有负电荷的分子；和
(b)金属-磷脂复合物颗粒(Metal-chelated phospholipid complex nanoparticles，MPP)，此金属-磷脂复合物颗粒为本公开的金属-磷脂复合物颗粒。
进一步地，该药物包封在该金属-磷脂复合物颗粒中。
进一步地，该药物选自核酸、蛋白、多肽、小分子、核酸类似物、蛋白类似物和多肽类似物中一种或多种的组合。
进一步地，该核酸选自mRNA、siRNA、sgRNA、ASO、circRNA、microRNA、DNA、ecDNA、人工核酸中一种或多种的组合。
进一步地，该核酸为SEQ ID No.1所示的编码eGFP(增强绿色荧光蛋白，Enhanced Green Fluorescent Protein)的mRNA序列、SEQ ID No.2所示的编码新型冠状病毒S1亚基的受体结合域RBD的mRNA序列、SEQ ID No.3所示的编码NY-ESO-1(纽约食管鳞状细胞1，New York esophageal squamous cell carcinoma 1)的mRNA序列、反义链为SEQ ID No.4和正义链为SEQ ID No.21所示的Bcl-2基因(B细胞淋巴瘤/白血病-2基因，B-cell lymphoma/Leukemia-2)的siRNA序列、反义链为SEQ ID No.6和正义链为SEQ ID No.23所示的PLK1基因(polo样激酶1，Polo-like Kinase 1)的siRNA序列、SEQ ID No.8所示的Gal-1基因的siRNA序列、SEQ ID No.10所示的STAT-3基因的ASO序列、SEQ ID No.12所示的α-syn基因(α-突触核蛋白，α-synuclein)的ASO序列、SEQ ID No.14所示的Bcl-2基因的ASO序列、SEQ ID No.16所示的编码野生型新型冠状病毒S蛋白的mRNA序列、反义链为SEQ ID No.17和正义链为SEQ ID NO.25所示的双链DNA序列、SEQ ID No.18所示的单链DNA、或正义链为SEQ ID No.19和反义链为SEQ ID No.26所示的B7-H4基因的siRNA序列。
本公开提供上述药物-脂质颗粒的制备方法，将药物包载于金属-磷脂复合物颗粒中，得到该药物-脂质颗粒。
进一步地，金属-磷脂复合物、抑制颗粒聚集的缀合的脂质，以及非阳离子脂质或非可电离脂质溶于有机化合物中形成有机相，药物溶于缓冲液中形成水相，将有机相与水相混匀得到药物-脂质颗粒。
进一步地，有机化合物为乙醇。
进一步地，缓冲液为无酶PBS缓冲液。
进一步地，有机相与水相的混匀方式包含微流控芯片或超声。
进一步地，将(a)药物、(i)金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质和(iii)非阳离子脂质或非可电离脂质混合，得到该药物-脂质颗粒。
本公开提供上述金属-磷脂复合物在核酸递送系统中的应用。进一步地，该核酸递送系统用于将核酸引入细胞。进一步地，该核酸用于在哺乳动物受试者中使靶序列的表达沉默、用于在哺乳动物体内传递药物、用于将药物从体内传递到哺乳动物细胞或用于治疗哺乳动物的疾病或病症。
本公开提供上述金属-磷脂复合物颗粒或上述药物-脂质颗粒在组合物中的应用，该组合物用于药物的递送。进一步地，该组合物用于将药物引入细胞。该组合物为药剂。进一步地，该药剂用于在哺乳动物受试者中使靶序列的表达沉默、用于在哺乳动物体内传递药物、用于将药物从体内传递到哺乳动物细胞或用于治疗哺乳动物的疾病或病症。
本公开提供上述金属-磷脂复合物或金属-磷脂复合物颗粒或药物-脂质颗粒或药剂在预防/治疗哺乳动物的疾病或病症中的应用。
在上述技术方案中，进一步地，哺乳动物为人。进一步地，该疾病或病症与基因的表达相关，该基因包含药物的靶序列。进一步地，该疾病或病症包括癌症、病毒感染、自身免疫性疾病、糖尿病或阿尔兹海默症。进一步地，该病毒感染包括甲肝、乙肝、丙肝、SARS-Cov-2(2019新型冠状病毒)、HIV(艾滋病病毒)、HPV(人乳头瘤病毒)、流感、天花或梅毒。进一步地，该癌症包括肝癌、胶质瘤、黑色素瘤、肺癌、胰腺癌或乳腺癌。
进一步地，该药剂为疫苗。进一步地，该药剂的给药途径包括鞘内注射、肌肉给药、颅内注射、静脉注射或瘤内注射。
一种含有本公开金属-磷脂复合物或金属-磷脂复合物颗粒或药物-脂质颗粒的药剂。
进一步地，药剂为疫苗。
进一步地，疫苗为新型冠状病毒疫苗。
与现有技术相比，本公开的有益效果如下：
本公开提供的金属-磷脂复合物主要作用在于吸附带有负电荷的药物，其与其他脂质共同自组装成金属-磷脂复合物颗粒(MPP)，在保证了不低于基于阳离子脂质和/或可电离脂质的LNP的有效性情况下，未使用阳离子脂质和可电离脂质，因此较LNP，毒性大幅度降低，生物安全性显著提升，更利于在生物体内进行负电荷药物的运载。
附图说明
为了更清楚地说明本公开具体实施方案或现有技术中的技术方案，下面将对具体实施方案或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本公开的一些实施方案，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1-1为本公开实施例3.5.1提供的eGFP-mRNA@MPP(Fe3+)转染293T所致eGFP阳性细胞百分比；
图1-2为本公开实施例3.5.1提供的RBD-mRNA@MPP(Fe3+)转染293T所致RBD的表达水平；
图1-3为本公开实施例3.5.1提供的RBD-mRNA@MPP(Fe3+)诱导体液免疫的能力；
图1-4为本公开实施例3.5.1提供的NY-ESO-1-mRNA@MPP(Fe3+)诱导体液免疫的能力；
图1-5为本公开实施例3.5.1提供的RBD-mRNA@MPP(Fe3+)诱导细胞免疫的能力；
图1-6为本公开实施例3.5.1提供的NY-ESO-1-mRNA@MPP(Fe3+)诱导细胞免疫的能力；
图1-7为本公开实施例实施例3.5.2提供的eGFP-mRNA@MPP(Al3+)转染293T所致eGFP阳性细胞百分比；
图1-8为本公开实施例3.5.2提供的RBD-mRNA@MPP(Al3+)转染293T所致RBD的表达水平；
图1-9为本公开实施例3.5.2提供的RBD-mRNA@MPP(Al3+)诱导体液免疫的能力；
图1-10为本公开实施例3.5.2提供的NY-ESO-1-mRNA@MPP(Al3+)诱导体液免疫的能力；
图1-11为本公开实施例3.5.2提供的RBD-mRNA@MPP(Al3+)诱导细胞免疫的能力；
图1-12为本公开实施例3.5.2提供的NY-ESO-1-mRNA@MPP(Al3+)诱导细胞免疫的能力；
图1-13为本公开实施例3.6.1提供的Bcl-2-siRNA@MPP(Fe3+)沉默靶基因的能力；
图1-14为本公开实施例3.6.1提供的PLK1-siRNA@MPP(Fe3+)沉默靶基因的能力；
图1-15为本公开实施例3.6.1提供的Gal-1-siRNA@MPP(Fe3+)沉默靶基因的能力；
图1-16为本公开实施例3.6.2提供的Bcl-2-siRNA@MPP(Al3+)沉默靶基因的能力；
图1-17为本公开实施例3.6.2提供的PLK1-siRNA@MPP(Al3+)沉默靶基因的能力；
图1-18为本公开实施例3.6.2提供的Gal-1-siRNA@MPP(Al3+)沉默靶基因的能力；
图1-19为本公开实施例3.7.1提供的STAT3-ASO@MPP(Fe3+)沉默细胞靶基因的能力；
图1-20为本公开实施例3.7.1提供的α-syn-ASO@MPP(Fe3+)沉默细胞靶基因的能力；
图1-21为本公开实施例3.7.1提供的Bcl-2-ASO@MPP(Fe3+)沉默细胞靶基因的能力；
图1-22为本公开实施例3.7.2提供的STAT3-ASO@MPP(Al3+)沉默细胞靶基因的能力；
图1-23为本公开实施例3.7.2提供的α-syn-ASO@MPP(Al3+)沉默细胞靶基因的能力；
图1-24为本公开实施例3.7.2提供的Bcl-2-ASO@MPP(Al3+)沉默细胞靶 基因的能力；
图1-25为本公开实施例3.8.1提供的S-mRNA@MPP(Fe3+)转染293T所致S蛋白的表达水平；
图1-26为本公开实施例3.8.1提供的药物(dsDNA及ssDNA)-脂质颗粒(Fe3+)的功能；
图1-27为本公开实施例3.8.2提供的S-mRNA@MPP(Al3+)转染293T所致S蛋白的表达水平；
图1-28为本公开实施例3.8.2提供的药物(dsDNA及ssDNA)-脂质颗粒(Al3+)的功能；
图2-1为本公开实施例4.1提供的磷脂复合物的差示扫描量热图；
图2-2为本公开实施例4.1提供的金属-磷脂复合物(Fe3+)的紫外吸收图；
图2-3为本公开实施例4.2提供的金属-磷脂复合物(Al3+)的紫外吸收图；
图2-4为本公开实施例5提供的在低pH值(pH＝5.0)条件下Fe3+从金属-磷脂复合物中脱落的表征；
图2-5为本公开实施例6.1提供的药物-金属-磷脂复合物颗粒(Fe3+)的元素分析；
图2-6为本公开实施例6.2提供的药物-金属-磷脂复合物颗粒(Al3+)中MPP的电镜分析；
图2-7为本公开实施例7提供的药物-脂质颗粒包载核酸(mRNA及siRNA)的效率；
图2-8为本公开实施例8提供的siRNA/mRNA@MPP和siRNA/mRNA@LNP的核酸溶酶体逃逸能力；
图2-9为本公开实施例9提供的MPP与LNP的eGFP阳性细胞率；
图2-10为本公开实施例10提供的MPP与LNP的促mRNA表达的能力对比；
图2-11为本公开实施例10提供的MPP与LNP的促体液免疫的能力对比；
图2-12为本公开实施例10提供的MPP与LNP的促细胞免疫的能力对比；
图3-1为本公开实施例13提供的药物-金属-磷脂复合物颗粒瘤内注射治疗肝癌的作用。
具体实施方式
定义
为了便于说明，此处统整性地说明本说明书、实施例以及后附的申请专利范围中所记载的特定术语。除非本说明书另有定义，此处所用的科学与技术词汇的含义与本公开所属技术领域中具有通常知识者所理解与惯用的意义相同。另外，除非上下文另外要求，否则应理解，单数术语应包括相同的复数形式，而复数术语应包括单数。具体来说，除非上下文另有明确说明，本文和后附的申请专利范围所使用的术语“至少一个(种)”和“一个(种)或多个”包括一个(种)，两个(种)，三个(种)或更多个(种)。
虽然用以界定本公开较广范围的数值范围与参数皆是约略的数值，此处已尽可能精确地呈现具体实施例中的相关数值。然而，任何数值本质上不可避免地含有因个别测试方法所致的标准偏差。在此处，“约”通常是指实际数值在一特定数值或范围的正负10％、5％、1％或0.5％之内。或者是，“约”一词代表实际数值落在平均值的可接受标准误差之内，视本公开所属技术领域中具有通常知识者的考虑而定。除了实验例之外，或除非另有明确的说明，当可理解此处所用的所有范围、数量、数值与百 分比(例如用以描述材料用量、时间长短、温度、操作条件、数量比例及其他相似者)均经过“约”的修饰。因此，除非另有相反的说明，本说明书与附随申请专利范围所公开的数值参数皆为约略的数值，且可视需求而更动。至少应将这些数值参数理解为所指出的有效位数与套用一般进位法所得到的数值。
术语“脂质”指一组有机化合物，其包括，但不限于脂肪酸的脂。通常将它们分成三类：“简单的脂质”、“化合物脂质”、“衍生的脂质”。“简单的脂质”其包括脂肪和油以及蜡；“化合物脂质”其包括磷脂和糖脂；“衍生的脂质”诸如类固醇。
术语“脂质小泡”指可用于传递化合物的任何脂质组合物，其包括，但不限于，脂质体，其中水体积被两亲性脂双层所包封；或其中脂质包被包括大分子组分的内部，诸如包括mRNA，伴随减少的水性内部；或脂质聚集体或胶团，其中被包封的成分包含在相对混乱的脂质混合物中。本文中，金属-磷脂复合物颗粒(MPP)即为“脂质小泡”，药物，例如核酸mRNA，作为被包封的成分包封于MPP中，该“包封”可为充分包封和/或部分包封。
术语中“磷脂”是指含有磷酸基团的脂类，属于复合脂，也称磷脂类、磷脂质。磷脂是组成生物膜的主要成分，分为甘油磷脂与鞘磷脂两大类，分别由甘油和鞘氨醇构成。磷脂为两性分子，一端为亲水的含氮或磷的头，另一端为疏水(亲油)的长烃基链。由于此原因，磷脂分子亲水端相互靠近，疏水端相互靠近，常与蛋白质、糖脂、胆固醇等其它分子共同构成磷脂双分子层，即细胞膜的结构。
本文中短语“磷脂分子部分”指磷脂分子与其他物质反应后原属于磷脂分子的结构。
本文中短语“连接物分子部分”指连接物分子与其他物质反应后原属于连接物分子的结构。
本文中短语“金属离子部分”指金属离子部分与其他物质反应后原属于金属离子的结构。
本文中短语“磷脂复合物”指通过具有磷酸基团的上述磷脂分子部分与上述连接物分子部分反应连接形成的复合物。
本文中短语“金属-磷脂复合物”指具有由具有磷酸基团的上述磷脂分子部分、上述连接物分子部分、上述金属离子部分反应组成，上述磷脂分子部分与上述连接物分子部分相连接，上述连接物部分与上述金属离子部分通过配位键连接，且所述金属-磷脂复合物既不是阳离子脂质也不是可电离脂质。
术语“可电离脂质”指一种含有正电荷可电离胺基团的脂质，可以在较低pH值时质子化带上正电荷而在生理pH值条件下不带电。
术语“中性脂质”指在选定的pH以未带电荷或中性两性离子形式存在的许多脂质种类中的任何一种。在生理pH，这样的脂质包括，例如，二酰基磷脂酰胆碱、二酰基磷脂酰乙醇胺、神经酰胺、神经鞘磷脂、脑磷脂、胆固醇、脑苷脂和二酰基甘油。
术语“阴离子脂质”指在生理pH带负电荷的任何脂质。这些脂质包括，但不限于，磷脂酰甘油、心磷脂、二酰基磷脂酰丝氨酸、二酰基磷脂酸、N-十二烷酰磷脂酰乙醇胺、N-琥珀酰磷脂酰乙醇胺、N-戊二酰磷脂酰乙醇胺，赖氨酰磷脂酰甘油、棕榈酰油酰磷脂酰甘油(POPG)，和其它与中性脂质连接的阴离子基团。
术语“阳离子脂质”指许多脂质种类中的任何一种，其在选定的pH，诸如生理pH携带净正电荷。这些脂质包括，但不限于，N，N-二油基-N，N-二甲基氯化铵(“DODAC”)；N-(2，3-二油基氧基)丙基)-N，N，N-三甲基氯化铵(“DOTMA”)；N，N-二硬脂基-N，N-二甲基溴化铵(“DDAB”)；N-(2，3-二油酰氧基)丙基)-N，N，N-三甲基氯化铵(“DOTAP”)；3-(N-(N’，N’-二甲基氨基乙烷)氨基甲酰基)胆固醇(“DC-Chol”) 和N-(1，2二肉豆蔻基氧基丙-3-基)-N，N-二甲基-N-羟乙基溴化铵(“DMRIE”)。如下的脂质是阳离子的并且在低于生理pH时具有正电荷：DODAP，DODMA，DMDMA等。
术语“疏水脂质”指具有非极性基团的化合物，其包括，但不限于，长链饱和和不饱和脂族烃基团并且这些基团任选地被一个或多个芳香族、脂环族或杂环族基团所取代。合适的实例包括，但不限于，二酰基甘油、二烷基甘油、N-N-二烷基氨基、1，2-二酰氧基-3-氨基丙烷和1，2-二烷基-3-氨基丙烷。
术语“非阳离子脂质或非可电离脂质”是指既不是阳离子脂质也不是非可电离脂质的脂质，例如可以为阴离子脂质、中性脂质。
在金属-磷脂复合物颗粒的组分中，(iii)中“除金属-磷脂复合物和抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质”是指，排除金属-磷脂复合物同时排除抑制颗粒聚集的缀合的脂质后，金属-磷脂复合物颗粒中剩余的脂质。
术语“融合性”指脂质体，药物-脂质颗粒或其它药物传递系统与细胞膜融合的能力。该膜可以是质膜或围绕细胞器，例如内体、核等的膜。
在金属-磷脂复合物颗粒中，除金属-磷脂复合物和抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质主要作为形成小泡的脂质存在，术语“形成小泡的脂质”倾向于包括任何具有疏水部分和极性头部基团的两亲性脂质并且其本身可以在水中自发形成双层小泡，示例为大多数磷脂。
在金属-磷脂复合物颗粒中，抑制颗粒聚集的缀合的脂质主要作为采用小泡的脂质存在，术语“采用小泡的脂质”倾向于包括稳定与脂双层结合的任何两亲性脂质，以及其它的两亲性脂质，其疏水部分与内部，双层膜的疏水区域接触，并且其极性头部基团部分朝向外部，膜的极性表面。采用小泡的脂质包括这样的脂质，其能够独立地适宜于采用非层状的相，还能够在存在双层稳定组分时，采取双层结构。抑制药物-脂质颗粒聚集的缀合的脂质包括，但不限于，聚酰胺低聚物(例如，ATTA-脂质衍生物)、肽、蛋白质、去污剂、脂质衍生物、PEG-脂质衍生物诸如与二烷氧基丙基偶联的PEG、与二酰基甘油偶联的PEG、与磷脂酰乙醇胺偶联的PEG，和与神经酰胺缀合的PEG(见，美国专利号5,885,613，将其并入本文作为参考)。
术语“两亲性脂质”指任何适合的材料，其中脂质材料的疏水部分朝向疏水相，而亲水部分朝向水相。两亲性脂质通常是脂质小泡的主要成分。亲水性质来自极性或带电基团诸如碳水化合物，磷酸盐(酯)，羧基、硫酸根合、氨基、巯基、硝基、羟基和其它类似基团的存在。疏水性可以通过非极性基团的包含来赋予，所述基团包括，但不限于，长链饱和和不饱和脂族烃基团和由一个或多个芳香族、脂环族或杂环基团取代的这样的基团。两亲性化合物的实例包括，但不限于，磷脂、氨脂质和神经鞘脂类。磷脂的代表性实例包括，但不限于，磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酸、棕榈酰油酰磷脂酰胆碱、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺、二棕榈酰磷脂酰胆碱、二油酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱或二亚油酰磷脂酰胆碱。缺乏磷的其它化合物，诸如鞘磷脂、鞘糖脂家族、二酰基甘油和β-酰氧基酸也在被称为两亲性脂质的组中。另外，上述的两亲性脂质可与其它脂质混和，该脂质包括甘油三酯和固醇。
术语“二酰基甘油”指具有2-脂肪酰基链的化合物，其R1和R2都独立地具有通过酯键与甘油的1-和2-位置键合的2-30个碳原子。该酰基基团可以是饱和的或具有不同程度的不饱和。二酰基甘油具有如下的式54：
术语“二酰基甘油偶联的聚乙二醇”，本公开中抑制颗粒聚集的缀合的脂质可以为二酰基甘油偶联的聚乙二醇，即二酰基甘油-聚乙二醇缀合物(DAG-PEG缀合物或PEG-DAG缀合物)。在一个优选的实施方案中，DAG-PEG缀合物是二月桂基甘油(C12)-PEG缀合物、双十四烷基甘油(C14)-PEG缀合物(DMG)，二棕榈酰甘油(C16)-PEG缀合物或二硬脂基甘油(C18)-PEG缀合物(DSG)。本领域技术人员将容易地理解其它二酰基甘油可以用在本公开的DAG-PEG缀合物中。用在本公开中的合适的DAG-PEG缀合物和制备及使用它们的方法公开于被公布作U.S.P.A 2003/0077829的美国申请号10/136,707，和PCT专利申请号CA 02/00669中，将其每个的全部内容并入作为参考。
术语“二烷氧基丙基”指具有2-烷基链的化合物，其R1和R2都独立地具有2-30个碳。烷基基团可以是饱和的或具有不同程度的不饱和。二烷氧基丙基具有如下的式55：
术语“二烷氧基丙基偶联的PEG”，本公开中抑制颗粒聚集的缀合的脂质可以为二烷氧基丙基偶联的PEG，即二烷氧基丙基缀合物(PEG-DAA缀合物)。在一个优选的实施方案中，PEG-DAA缀合物具有下式56：
在式56中，R1和R2被独立地进行选择并且是具有约10至约22个碳原子的长链烷基基团。长链烷基基团可以是饱和的或不饱和的。合适的烷基基团包括，但不限于，月桂基(C12)、十四烷基(C14)、十六烷基(C16)、十八烷基(C18)和icosyl(C20)。在优选的实施方案中，R1和R2是相同的，即R1和R2都是十四烷基(即双十四烷基)，R1和R2都是十八烷基(即双十八烷基)等。在式56中，PEG是具有约550到约10000道尔顿的平均分子量的聚乙二醇并且在末端羟基位置任选地被烷基，烷氧基，酰基，或芳基取代。在一个优选的实施方案中，PEG具有约1000-约5000道尔顿的平均分子量，更优选地，约1,000至约3,000道尔顿的平均分子量并且甚至更优选地，约2000道尔顿的平均分子量。PEG可以任选地被烷基、烷氧基、酰基或芳基基团所取代。在式56中，L是接头部分。可以使用任何适合于将PEG偶联到二烷氧基丙基主链的接头部分。合适的接头部分包括，但不限于，酰氨基(-C(O)NH-)、氨基(-NR-)、羰基(-C(O)-)、碳酸酯(O-C(O)O-)，氨基甲酸酯(-NHC(O)O-)，尿素(-NHC(O)NH-)，琥珀酰(-(O)CCH2CH2C(O)-)，醚，二硫化物和其组 合。其它合适的接头是本领域众所周知的。
可以将磷脂酰乙醇胺与聚乙二醇缀合从而作为本公开中抑制颗粒聚集的缀合的脂质，形成双层稳定组分，所述磷脂酰乙醇胺具有不同链长度和饱和程度的各种酰基链基团。这些磷脂酰乙醇胺是可商购的，或可以使用那些本领域技术人员已知的常规技术来分离或合成。包含饱和的或不饱和的脂肪酸的磷脂酰乙醇胺是优选的，其具有在C10-C20范围内的碳链长度。还可以使用这样的磷脂酰乙醇胺，其具有单或双不饱和脂肪酸及饱和和不饱和脂肪酸的混合物。合适的磷脂酰乙醇胺包括，但不限于，如下：双肉豆蔻酰磷脂酰乙醇胺(DMPE)，二棕榈酰磷脂酰乙醇胺(DPPE)，二油酰磷脂酰乙醇胺(DOPE)和二硬脂酰磷脂酰乙醇胺(DSPE)。
如磷脂酰乙醇胺，神经酰胺可以与聚乙二醇偶联从而作为本公开中抑制颗粒聚集的缀合的脂质，形成双层稳定组分，所述神经酰胺具有不同链长度和饱和程度的多个酰基链基团。本领域技术人员需清楚的是，与磷脂酰乙醇胺比较，神经酰胺仅具有一个酰基基团，所述酰基基团可以根据其链长度和饱和程度来容易地进行变化。适合于按照本公开应用的神经酰胺是可以商购的。此外，使用众所周知的分离技术可以例如从卵和脑中分离神经酰胺，或使用在美国专利号5,820,873中公开的方法和技术来合成它们，将美国专利号5,820,873并入本文作为参考。使用在前述申请中提出的合成途径，可以制备具有饱和或不饱和脂肪酸的神经酰胺，所述脂肪酸具有在C2-C31范围内的碳链长度。
术语“ATTA”或“聚酰胺”指，但不限于，在美国专利号6,320,017和6,586,559中公开的化合物，将它们都并入本文作为参考。这些化合物包括具有如下式57的化合物：
其中：R是选自由氢、烷基和酰基组成的组中的成员；R1是选自由氢和烷基组成的组中的成员；或任选地，R和R1和它们所结合的氮原子形成叠氮基部分；R2是选自氢、任选地取代的烷基、任选地取代的芳基和氨基酸侧链的组的成员；R3是选自由氢、卤素、羟基、烷氧基、巯基、肼基、氨基和NR4R5组成的组的成员，其中R4和R5独立地是氢或烷基；n是4-80；m是2-6；p是1-4；并且q是0或1。那些本领域技术人员将清楚的是其它聚酰胺可用在本公开的化合物中。
术语“同类物”指起到相同或相似作用的类似物，或起到相同或相似作用的相同母核的衍生物。
如本文中使用的，术语“mRNA”或“信使多核糖核苷酸”或“信使RNA”或“messenger RNA”可互换使用，并且意指以DNA的一条链作为模板转录而来、携带有遗传信息、可以指导蛋白质合成的单链多核糖核苷酸。
如本文中使用的，术语“sgRNA”或“small guide RNA”或“向导RNA”或“gRNA”可互换使用，在RNA编辑的过程中引导尿苷残基插入或缺失到动质体(kinetoplastid)中，属于一种小型非编码RNA，可与pre-mRNA配对。gRNA编辑RNA分子，长度大约60-80个核苷酸，由单独的基因转录。
如本文中使用的，术语“circRNA”或“circular RNA”或“环状多核糖核苷酸”或“环状RNA”可互换使用，并且意指具有无游离端(即，没有游离3’和/或5’端)结构的多核糖核苷酸分子，例如通过共价或非共价键形成环状或环形结构的多核糖核苷酸。
如本文中使用的，术语“microRNA”或“miRNA”或“微小RNA”可互换使用，并且意指长度约为22个核苷酸、有游离3’和5’端的非编码单链多核糖核苷酸，能通过与靶基因的mRNA 3'-非翻译区(3'-untranslated region，3’-UTR)结合，抑制靶基因蛋白 翻译进而调控细胞的生物学功能。
如本文中使用的，术语“ASO”或“antisense oligonucleotide”或“反义寡核苷酸”可互换使用，并且意指人工合成的，与靶基因或mRNA某一区段互补的核酸片断，可以通过碱基互补原则结合于靶基因/mRNA上，从而封闭基因的表达的单链多(脱氧)核糖核苷酸，包括反义DNA和反义RNA。
如本文中使用的，术语“siRNA”或“small interfering”或“short interfering”或“silencing RNA”或“小干扰RNA”或“短干扰RNA”或“沉默RNA”可互换使用，并且意指为一类长度为20到25个核苷酸，并能诱导靶基因mRNA降解的双链RNA分子。
如本文中使用的，术语“ecDNA”或“染色体外环状DNA”可互换使用，并且意指从染色体上脱落，脱离染色体以环状结构存在的DNA。
术语“核酸衍生物”指对核酸序列的修饰或替代，包括但不限于对残基的化学修饰、对核苷酸或脱氧核苷酸的替代、对序列提高半衰期或稳定性的修饰、标记修饰。例如，化学修饰包括但不限于磷酸化、甲基化、氨基化、巯基化、用硫取代氧、用硒取代氧或同位素化任一或多个碱基。对核苷酸或脱氧核苷酸的替代包括但不限于以多肽或其它骨架取代糖磷酸主链的核酸类似物(将DNA或RNA替换为PNA)。对序列提高半衰期或稳定性的修饰包括但不限于与PEG连接修饰、氟修饰。标记修饰包括但不限于连接荧光基团、氨基、生物素、地高辛、小肽等。
术语“人工核酸”：经过人工修饰的核酸分子，包括但不限于碱基修饰，核糖修饰，PNA等。
术语“核酸”指包含至少两个脱氧核苷酸或核苷酸的以单或双链形式存在的聚合物。除非具体限制，该术语涵盖包含天然核苷酸的已知类似物的核酸，其具有与参照核酸相似的结合特性并且以与天然存在的核苷酸相似的方式进行代谢。除非另外指出，具体的核酸序列还暗含涵盖其保守性修饰的变体(例如，简并密码子取代)，等位基因，直向同源物，SNPs和互补序列以及明显指出的序列。具体地，可通过产生序列来获得简并密码子取代，在所述序列中一个或多个选定(或所有)的密码子的第三个位置由混和的碱基和/或脱氧肌苷残基所取代(Batzer et al.，Nucleic Acid Res.19：5081(1991)；Ohtsuka et al.，J.Biol.Chem.260：2605-2608(1985)；和Cassol et al.(1992)；Rossolini et al.，Mol.Cell.Probes 8：91-98(1994))。“核苷酸”包含糖脱氧核糖(DNA)或核糖(RNA)，碱基，和磷酸基团。核苷酸通过磷酸基团进行连接。“碱基”包括嘌呤和嘧啶，其进一步包括天然化合物腺嘌呤、胸腺嘧啶、鸟嘌呤、胞嘧啶、尿嘧啶、肌苷，和天然类似物，以及嘌呤和嘧啶的合成衍生物，其包括，但不限于取代新的反应基的修饰，所述反应基诸如，但不限于，胺、醇、硫醇、羧酸盐(酯)和卤代烷。DNA可以以反义、质粒DNA、质粒DNA的部分、预压缩的DNA、聚合酶链反应(PCR)的产物、载体(P1，PAC，BAC，YAC，人工染色体)、表达盒、嵌合序列、染色体DNA或这些组的衍生物存在。术语核酸与基因、cDNA、由基因编码的mRNA和干扰RNA分子可替交地使用。
术语“基因”指包括部分长度或全长的编码序列的核酸(例如，DNA或RNA)序列，所述编码序列是产生多肽或多肽前体(例如，来自A，B，C，D，E，G型肝炎病毒；或单纯疱疹病毒的多肽或多肽前体)所必需的。
用于本文时，“基因产物”指基因的产物诸如包括，例如DNA的转录物，mRNA。
短语“靶基因的表达沉默”指本公开的siRNA启动使靶基因沉默的能力。为了确定基因沉默的程度，将目标生物或培养物中的细胞的样品或测定与对照样品进行比较，所述目标生物或培养物的细胞表达具体构建体，所述对照不表达所述构建体。将对照样品(缺乏构建体的表达)设定为100％的相对值。当相对于对照的测试值是约90％，优选地50％，更优选地25％-0％时，成功获得对靶基因的表达的抑制。合适的测定包括，例如，使用本领域技术人员已知的技术诸如点渍法、RNA印迹法、原位杂交、ELISA、免疫沉淀法、酶 作用、以及本领域技术人员已知的表型测定来检测蛋白质或mRNA水平。
siRNA的“治疗有效量”或“有效量”是足以产生理想效果的量，所述理想效果是例如与在缺乏siRNA时检测到的正常表达水平比较靶序列表达的减少。
用于本文时，术语“水溶液”指全部或部分包含水的组合物。
用于本文时，术语“有机脂质溶液”指全部或部分包含具有脂质的有机溶剂的组合物。
用于本文时，“全身传递”指导致化合物在生物体广泛生物分布的传递。一些施用的技术可以导致某些化合物的全身传递，但是不能导致其它化合物的全身传递。全身传递指有效的，优选地，治疗量的化合物与身体的大部分接触。为了获得广泛的生物分布，通常需要血液生存期从而使化合物在到达施用位点远端的疾病位点前，不被迅速降解或清除(诸如通过初次通过器官(肝、肺等))或通过迅速、非特异性的细胞结合)。药物-脂质颗粒的全身传递可以以本领域已知的任何方式进行，所述方式包括，例如，静脉内、皮下、腹膜内，在一个优选的实施方案中，药物-脂质颗粒的全身传递是通过静脉内的传递。
用于本文时，“局部传递”指在生物体内，化合物直接向靶位点的传递。例如，化合物可以通过直接注射到疾病位点诸如肿瘤或其它靶位点诸如炎症位点或靶器官诸如肝、心脏、胰腺、肾等来进行局部传递。
金属-磷脂复合物
在本公开中，金属-磷脂复合物由磷脂分子部分、连接物分子部分和金属离子部分构成。
对于磷脂分子部分，需要说明的是，本公开的磷脂分子的顺反异构体不会对本公开保护内容所要达到的效果产生影响。
对于连接物分子部分，其主要来源于天然植物提取物，例如姜黄素，具有广泛的生物作用，包括抗菌、抗病毒、抗真菌、抗氧化和抗炎活性。此外，它还是一种有效的免疫调节剂，能调节T细胞、B细胞、巨噬细胞、中性粒细胞、自然杀伤细胞以及树突细胞等多种免疫细胞的活性，促进免疫力的平衡，增强机体的免疫力。基于姜黄素分子潜在的免疫增强、抗炎症、抗氧化及抗sars-cov-2作用，其有望成为一种潜在的抗COVID-19的辅助治疗手段。并且，姜黄素分子的安全性极高，已被列举到食品添加剂和药用辅料的目录中，其安全性利于药物-脂质整体的临床药物注册，缩短了临床药物注册的时间长度。
对于金属离子部分，金属-磷脂复合物中连接物分子部分和金属离子部分之间的配位键在例如溶酶体的低pH值(pH＝5.0)条件下会发生断裂，金属离子从金属-磷脂复合物中脱落。
金属-磷脂复合物中各组分的投放比例可根据具体金属-磷脂复合物组分的结构进行调整。投放比例可以调整的依据是：因为磷脂分子与连接物分子靠氢键相连，只要磷脂分子含有多个磷酸基团，那么合成磷脂复合物时，磷脂分子和连接物分子的投放比例可以依据磷脂分子所含磷酸基团的个数进行调整，即当磷脂分子含两个磷酸基团时，磷脂分子和连接物分子的投放比例可调整为1：2；当磷脂分子含三个磷酸基团时，磷脂分子和连接物分子的投放比例可调整为1：3；因为连接物分子的羟基与金属离子靠配位键健连接，只要连接物分子含有多个结合位点，那么连接物分子和金属离子的投放比例可以依据连接物所含结合位点的数量进行调整。
先将具有磷酸基团的磷脂分子与连接物分子连接得到磷脂复合物，然后在与金属离子连接得到金属-磷脂复合物。具体地，磷脂分子与连接物分子按照摩尔比在适量的乙醇中溶解，在65℃左右的条件下进行反应，之后加入正己烷可以沉淀得到磷脂复合物；磷脂复合物再与金属离子(例如FeCl3等)按照摩尔比在适量的乙醇中溶解，在60℃左右的条件下进行反应，可以得到金属-磷脂复合物。
金属-磷脂复合物颗粒(Metal-chelated phospholipid complex nanoparticles， MPP)
由金属-磷脂复合物组装的金属-磷脂复合物颗粒装载核酸的原理是：连接物分子与磷脂分子通过氢键结合在一起，同时连接物分子通过配位键与金属离子相连，形成了金属-磷脂复合物，该金属-磷脂复合物的金属离子通过配位键与带有负电荷的药物连接，从而确保金属-磷脂复合物与其他组分(抑制颗粒聚集的缀合的脂质，以及除金属-磷脂复合物和抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质)自组装成MPP同时将带有负电荷的药物载入纳米颗粒MPP中。在本文中，所述的“除金属-磷脂复合物和抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质”即是指金属-磷脂复合物颗粒中的组分(iii)。
在一些实施方案中，抑制颗粒聚集的缀合的脂质是指抑制药物-脂质颗粒聚集的缀合的脂质，主要功能在于防止药物-脂质颗粒的聚集，例如与二烷氧基丙基偶联的PEG、与二酰基甘油偶联的PEG、与磷脂酰乙醇胺偶联的PEG，和与神经酰胺缀合的PEG，优选为PEG-脂质缀合物。其中，脂质的顺反异构体不会对本公开保护内容所要达到得效果产生影响。
在一些实施方案中，金属-磷脂复合物在原料中摩尔占比为5％～小于10％、10％～40％或大于40％～50％，例如5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、21％、22％、23％、24％、25％、26％、27％、28％、29％、30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％或50％。
在优选的实施方式中，金属-磷脂复合物在原料中摩尔占比为7％～40％，例如可以为10％～40％、7％～30％、15％～25％、20％～30％，进一步优选为15％、25％、7％或30％。
在一些实施方案中，抑制颗粒聚集的缀合的脂质在原料中摩尔占比为2％～10％，例如2％、3％、4％、5％、6％、7％、8％、9％或10％。例如可以为3％～10％、4％～10％、5％～10％，进一步优选为3％、4％或10％。
在一些实施方案中，非阳离子脂质或非可电离脂质为胆固醇，胆固醇在原料中摩尔占比为15％～小于35％、35％～75％或大于75％～80％，例如15％、16％、17％、18％、19％、20％、21％、22％、23％、24％、25％、26％、27％、28％、29％、30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％或80％。例如可以为35％～75％、15％～56％、40％～46％和35％～55％，优选为15％、40％、46％或56％。
在一些实施方案中，除胆固醇外，金属-磷脂复合物颗粒还可任选地含有其他非阳离子脂质或非可电离脂质，其在原料中摩尔占比为0％～40％或大于40％～51％，例如0％、1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、21％、22％、23％、24％、25％、26％、27％、28％、29％、30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％或51％。例如为5％～30％、25％～35％、34％～45％、20％-25％，优选为25％、35％、34％或45％。
药物-脂质颗粒
本文描述的药物-脂质颗粒典型地包括药物(其为带有负电荷的分子，所述分子可以选自核酸、蛋白、多肽、小分子、核酸类似物、蛋白类似物、多肽类似物中的一种或多种组合的组中的成员，核酸是选自mRNA、siRNA、环状RNA、microRNA、DNA、ecDNA、人工核酸中一种或多种组合的组中的成员)、金属-磷脂复合物、非阳离子脂质或非可电离 脂质、以及双层稳定组分，例如抑制颗粒聚集的缀合的脂质。此外，包封在本公开的药物-脂质颗粒中的核酸在水溶液中对于用核酸酶进行的降解是具有抗性的。
在一些实施方案中，药物被充分包封在金属-磷脂复合物颗粒的内部从而避免药物的降解，实现药物被递送进入细胞。
在一些实施方案中，本公开提供的药物-脂质颗粒具有适合于全身传递的小直径，粒径在30～400nm；表面电势为-10～10mV；稳定性至少在3天，较优可达7天以上；细胞递送效率至少40％，例如至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％或至少95％。
在一些实施方案中，药物-脂质颗粒的药物优选为核酸，核酸组分典型地包括可以以数种形式提供的mRNA、干扰RNA(即，siRNA)，所述形式包括，例如，一个或多个分离的小干扰RNA(siRNA)双链体，更长的双链RNA(dsRNA)或翻译自DNA质粒中转录盒的siRNA或dsRNA。
RNA群可以用于提供长的前体RNAs，或与可用于制备siRNA的选定靶序列具有基本或完全同一性的长前体RNAs。所述RNAs可以按照本领域技术人员中众所周知的方法来从细胞或组织中分离，合成，和/或克隆。所述RNA可以是混和的群(获自细胞或组织，转录自cDNA等)，或可以代表单一靶序列。RNA可以是天然存在的，例如分离自组织或细胞样品，例如使用T7或SP6聚合酶和PCR产物或克隆的cDNA在体外合成的；或以化学方法合成的。
为了形成长dsRNA，对于合成性RNAs，互补体还可以在体外转录并杂交以形成dsRNA。如果使用天然存在的RNA群，例如通过转录相应于RNA群的cDNAs，或通过使用RNA聚合酶，还提供了RNA互补体(例如，形成dsRNA，其通过大肠杆菌(E.coli)RNAse III或切酶进行消化)。接着前体RNA杂交以形成双链RNAs从而消化。所述dsRNAs可直接被包封在SNALPs中或可在包封前被体外消化。
或者，可以将一个或多个编码一个或多个siRNA模板的DNA质粒包封在核酸-脂质颗粒中。例如，基于小核RNAU6或人RNase P RNA H1的天然存在转录单位，可以从质粒中的DNA模板将siRNA转录为自动折叠为具有发夹环的双链体的序列，所述质粒具有RNA聚合酶III转录单位(见，Brummelkamp，et al.，Science 296：550(2002)；Donzé，et al，Nucleic AcidsRes.30：e46(2002)；Paddison，et al.，Genes Dev.16：948(2002)；Yu，et al.，Proc.Natl.Acad.Sci.99：6047(2002)；Lee，et al.，Nat.Biotech.20：500(2002)；Miyagishi，et al.，Nat.Biotech.20：497(2002)；Paul，et al.，Nat.Biotech.20：505(2002)；和Sui，et al.，Proc.Natl.Acad.Sci.99：5515(2002))。典型地，转录单位或盒将包含RNA转录启动子序列，诸如H1-RNA或U6启动子和终止序列，所述启动子序列可操作地与转录所需siRNA序列的模板连接，所述终止序列包括2-3个尿苷残基和聚胸苷(T5)序列(多腺苷酸化信号)(Brummelkamp，Science，同上)。选定的启动子可以提供组成性或可诱导的转录。将组合物和DNA-指导的RNA干扰分子的转录的方法详细描述于美国专利号6,573,099，将其并入本文作为参考。优选地，所述合成性或转录的siRNA具有约1-4个，优选地约2-3个核苷酸的3’突出端以及5’磷酸末端(Elbashir，et al.，Genes Dev.15：188(2001)；et al.，Cell 107：309(2001))。将转录单位结合到质粒或DNA载体中，干扰RNA转录自所述质粒或DNA载体。将适合于体内传递遗传物质用于治疗目的的质粒详细描述于美国专利号5,962,428和5,910,488中，它们两者都结合到本文作为参考。选定的质粒可以提供靶细胞的瞬时或稳定的传递。本领域那些技术人员将清楚的是起初被设计用于表达所需基因序列的质粒可以被进行修饰从而包含转录siRNA的转录单位盒。
分离RNA，合成RNA，杂交核酸，制备和筛选cDNA文库以及进行PCR的方法是本领域众所周知的(见，例如.，Gubler&Hoffman，Gene25：263-269(1983)；Sambrook et al.，同上；Ausubel et al.，同上)，PCR方法也是这样(见美国专利4,683,195和4,683,202； PCR Protocols：A GuidetoMethods and Applications(Innis et al.，eds，1990))。表达文库也是本领域技术人员众所周知的。公开用在本公开中的一般方法的另外的基本书籍包括Sambrook et al.，Molecular Cloning，A Laboratory Manual(2nd ed.1989)；Kriegler，Gene Transfer and Expression：A Laboratory Manual(1990)；和Current Protocols in Molecular Biology(Ausubel et al.，eds.，1994))。
金属-磷脂复合物、金属-磷脂复合物颗粒、药物-脂质颗粒的应用
在一些实施方案中，当药物为核酸时，金属-磷脂复合物、金属-磷脂复合物颗粒均可用于促药物溶酶体逃逸中，以及促进核酸表达中。金属-磷脂复合物、金属-磷脂复合物颗粒也可用于递送药物，将药物引入细胞中，从而实现药物对适用疾病或病症的预防与治疗。
在一些实施方案中，本公开提供该药物-脂质颗粒的应用，例如用于组合物，该组合物可实现药物的递送或将药物引入细胞。组合物例如为药剂，该药剂可实现：在哺乳动物受试者中使靶序列的表达沉默、在哺乳动物体内传递药物(药物例如为治疗肿瘤、显影剂等)、将药物从体内传递到哺乳动物细胞或治疗哺乳动物的疾病或病症等。药剂中，药物-脂质颗粒为主要有效成分，可根据实际需求，通过不同药学上可接受的辅料或制备工艺，制备成不同的剂型，例如固体剂型(散剂、颗粒剂、丸剂、片剂、胶剂)、半固体剂型(外用膏剂、糊剂)、液体剂型(汤剂、合剂、糖浆剂、酒剂、注射剂)、气体剂型(气雾剂、烟剂)等；例如经胃肠道给药的剂型、经直肠给药的剂型、不经胃肠给药的剂型等。
在一些实施方案中，本公开提供上述金属-磷脂复合物、金属-磷脂复合物颗粒，以及药物-脂质颗粒制备的产品，该产品具有金属-磷脂复合物、金属-磷脂复合物颗粒，以及药物-脂质颗粒的上述功能与用途，具体类型例如可以但不限于为试剂盒、药剂等，该产品可选地还含有其他辅料。
对于药物-脂质颗粒作用的靶基因：通常，传递药物-脂质颗粒从而使目标基因产物的翻译(即，表达)下调或沉默是理想的。基因产物的合适的分类包括，但不限于，与病毒感染和存活相关的基因，与代谢疾病和病症(例如，其中肝作为靶目标的疾病和病症，和肝疾病和病症)相关的基因，与肿瘤发生和细胞转化相关的基因，生血管基因，免疫调节剂基因诸如与炎症和自身免疫应答相关的那些，配体受体基因和与神经变性病症相关的基因。
与病毒感染和存活相关的基因包括通过病毒表达从而结合，进入并在细胞中复制的那些。特别是与慢性病毒疾病相关的病毒序列。例如病毒序列包括肝炎病毒的序列(Hamasaki，et al.，FEBS Lett.543：51(2003)；Yokota，et al，EMBO Rep.4：602(2003)；Schlomai，et al.，Hepatology 37：764(2003)；Wilson，et al.，Proc.Natl.Acad.Sci.100：2783(2003)；Kapadia，et al.，Proc.Natl.Acad.Sci.100：2014(2003)；和FIELDSVIROLOGY(Knipe et al.eds.2001))，人免疫缺陷病毒(HIV)(Banerjea，etal.，Mol Ther.8：62(2003)；Song，et al.，J.Virol.77：7174(2003)；StephensonJAMA 289：1494(2003)；Qin，et al.，Proc.Natl.Acad.Sci.100：183(2003))，疱疹病毒(Jia，et al.，J.Virol.77：3301(2003))，和人乳头状瘤病毒(HPV)(Hall，et al.，J.Virol.77：6066(2003)；Jiang，et al.，Oncogene 21：6041(2002))。可以被沉默的示例性肝炎病毒核酸序列包括，但不限于：涉及转录和翻译的核酸序列(例如，En1，En2，X，P)，编码结构蛋白质的核酸序列(例如，包括C和C相关的蛋白质的核心蛋白；包括S，M，和/或L蛋白质的衣壳和包膜蛋白质，或其片段)(见，例如，FIELDS VIROLOGY，2001，同上)。可以被沉默的丙型肝炎核酸序列包括但不限于：丝氨酸蛋白酶(例如，NS3/NS4)，解旋酶(例如，NS3)，聚合酶(例如，NS5B)和包膜蛋白(例如，E1，E2，和p7)。甲型肝炎核酸序列在例如Genbank登记号NC_001489中提及；乙型肝炎核酸序列在例如Genbank登记号NC_003977中提及；丙型肝炎核酸序列在例如 Genbank登记号NC_004102中提及；丁型肝炎核酸序列在例如Genbank登记号NC_001653中提及；戊型肝炎核酸序列在例如Genbank登记号NC_001434中提及；并且G型肝炎核酸序列在例如Genbank登记号NC_001710中提及。使编码与病毒感染和存活相关的基因的序列沉默可以方便地与用于治疗病毒疾病的常规药剂的施用结合来进行使用。
与代谢疾病和病症(例如，其中肝被靶向的病症和肝疾病以及病症)相关的基因包括，例如，在血脂异常(例如，肝X受体(例如，LXRα和LXRβGenback登记号NM_007121))，类法尼醇X受体(FXR)(Genbank登记号NM_005123)，固醇调节元件结合蛋白质(SREBP)，位点-1蛋白酶(S1P)，3-羟基-3-甲基戊二酰基辅酶-A还原酶(HMG辅酶-A还原酶)，载脂蛋白(ApoB)，和载脂蛋白(ApoE))和糖尿病(例如，葡糖-6-磷酸)中表达的基因(见，例如，Forman et al.，Cell 81：687(1995)；Seol et al.，Mol.Endocrinol.9：72(1995)，Zavacki et al.，PNAS USA 94：7909(1997)；Sakai，et al.，Cell85：1037-1046(1996)；Duncan，et al.，J.Biol.Chem.272：12778-12785(1997)；Willy，et al.，Genes Dev.9(9)：1033-45(1995)；Lehmann，et al.，J.Biol.Chem.272(6)：3137-3140(1997)；Janowski，et al.，Nature 383：728-731(199；Peet，etal.，Cell93：693-704(1998))。本领域技术人员将理解与代谢疾病和病症(例如其中肝被靶向的疾病和病症以及肝疾病和病症)相关的基因包括在肝本身中表达的基因以及在其它器官和组织中表达的基因。使编码与代谢疾病和病症相关的基因的序列沉默可以方便地与用于治疗所述疾病或病症的常规药剂的施用结合来进行使用。
与肿瘤发生和细胞转化相关的基因的实例包括易位序列诸如MLL融合基因，BCR-ABL(Wilda，et al.，Oncogene，21：5716(2002)；Scherr，et al，Blood 101：1566)，TEL-AML1，EWS-FLI1，TLS-FUS，PAX3-FKHR，BCL-2，AML1-ETO和AML1-MTG8(Heidenreich，et al.，Blood 101：3157(2003))；过度表达的序列诸如多药物抗性基因(Nieth，et al.，FEBS Lett.545：144(2003)；Wu，et al，Cancer Res.63：1515(2003))，细胞周期蛋白(Li，et al.，CancerRes.63：3593(2003)；Zou，et al.，Genes Dev.16：2923(2002))，β-联蛋白(Verma，et al.，Clin Cancer Res.9：1291(2003))，端粒末端转移酶基因(Kosciolek，et al.，Mol Cancer Ther.2：209(2003))，c-MYC，N-MYC，BCL-2，ERBB1和ERBB2(Nagy，et al.Exp.Cell Res.285：39(2003))；和突变序列诸如RAS(综述于Tuschl和Borkhardt，Mol.Interventions，2：158(2002))。使编码DNA修复酶的序列沉默与化疗剂的施用结合使用(Collis，et al.，CancerRes.63：1550(2003))。编码与肿瘤迁移相关的蛋白质的基因也是目标靶序列，所述蛋白质，例如整联蛋白、选择蛋白和金属蛋白水解酶。可以将有利于或促进肿瘤发生或细胞转化，肿瘤生长或肿瘤迁移的任何完整或部分基因序列包括进来作为模板序列。
生血管基因能够促进新血管的形成。血管内皮生长因子(VEGF)是重点研究方向(Reich，et al.，Mol.Vis.9：210(2003))。
免疫调节剂基因是调节一个或多个免疫应答的基因。免疫调节剂基因的实例包括细胞因子诸如生长因子(例如，TGF-α，TGF-β，EGF，FGF，IGF，NGF，PDGF，CGF，GM-CSF，SCF，等)，白细胞介素(例如.，IL-2，IL-4，IL-12(Hill，et al.，J.Immunol.171：691(2003))，IL-15，IL-18，IL-20，等)，干扰素(例如，IFN-α，IFN-β，IFN-γ，等)和TNF。Fas和Fas配体基因也是目标免疫调节剂靶序列(Song，et al.，Nat.Med.9：347(2003))。在造血和淋巴样细胞中编码次级信号分子的基因也包括在本公开中，例如，Tec家族激酶，诸如Bruton’s酪氨酸激酶(Btk)(Heinonen，et al.，FEBS Lett.527：274(2002))。
细胞受体配体包括这样的配体，其能结合细胞表面受体(例如，胰岛素受体、EPO受体、G-蛋白质偶联受体、具有酪氨酸激酶活性的受体、细胞因子受体、生长因子受体等)以调节(例如，抑制，激活等)受体涉及的生理途径(例如，葡萄糖水平调节、血液细胞发展、有丝分裂发生等)。细胞受体配体的实例包括细胞因子、生长因子、白细胞介素、干扰素、促红细胞生成素(EPO)、胰岛素、胰高血糖素、G-蛋白质偶联受体配体等)。编码 三核苷酸重复序列(例如，CAG重复序列)扩张的模板发现用于使在神经变性疾病中的病原序列沉默，所述疾病由三核苷酸重复序列的扩张所引起，诸如脊髓延髓肌肉萎缩和亨廷顿病(Caplen，et al.，Hum.Mol.Genet.11：175(2002))。
可注射的传递：在某些情形中，如美国专利5,543,158；美国专利5,641,515和美国专利5,399,363所述，通过肠胃外，静脉内，肌内，皮下，皮内或腹膜内来传递本文公开的药物-脂质颗粒是理想的。可以将所述药物-脂质颗粒局部注射到目标位点(例如，疾病位点诸如炎症或肿瘤形成或到靶器官或组织)或全身注射以广泛分布到生物体。可以在水中制备所述药物-脂质颗粒的溶液，所述水合适地混和以表面活性剂。还可以在甘油，液体聚乙二醇及其混合物中以及在油中制备分散体。可选的，这些制剂包含防腐剂以阻止微生物的生长。通常，当被静脉内施用时，将药物-脂质颗粒制剂与合适的药用载体一起进行配制。通常，普通缓冲盐溶液(135-150mM NaCl)将被用作药用载体，但是其它合适的载体将足够满足需求。另外的合适载体描述在例如REMINGTON’S PHARMACEUTICAL SCIENCES，Mack Publishing Company，Philadelphia，PA，17th ed.(1985)。用于本文时，“载体”包括任何和所有的溶剂，分散介质，媒介，包衣，稀释剂，抗菌剂和抗真菌剂，等渗和吸收延缓剂，缓冲剂，载体溶液，混悬液，胶体等。短语“药用”指分子实体和组合物，所述分子实体和组合物被施用给人时，不产生变态或相似的不良反应。水性组合物的制剂是本领域的常规理解，所述组合物包含作为活性成分的蛋白质。可选地，将这些组合物制备为注射液，液体溶液或混悬液；还可以制备在注射前适合于在液体中的溶液或混悬液的固体形式。还可以将所述制剂进行乳化。
可以通过常规脂质体灭菌技术，诸如过滤来对药物-脂质颗粒进行灭菌。所述药物-脂质颗粒可以包含药用辅助物质，所述药用辅助物质是合适的生理条件，诸如pH调节剂和缓冲剂，毒性调节剂，润湿剂等。使用上文所指的技术可以对这些组合物进行灭菌，或者替代地，它们可以在无菌条件下产生。可以对得到的水溶液包装以进行使用或在无菌条件下进行过滤并进行冷冻干燥，在施用前将冷冻干燥的制剂与无菌水溶液结合在一起。
预防性和治疗性的处理：在一些实施方案中，可以将药物-脂质颗粒用于对患有疾病或病症的受试者(例如，哺乳动物受试者)的预防性或治疗性的处理，所述疾病或病症与靶序列的表达或过表达相关。将所述药物-脂质颗粒以足够激发患者的治疗性应答的量施用给受试者。将足以完成此的量定义为“治疗上有效的剂量或量”或“有效剂量或量”。在确定待施用于疾病的治疗或预防中的药物-脂质颗粒的有效量中，所述疾病由于靶基因的表达或过表达导致，所述内科医师评价药物-脂质颗粒的循环血浆水平，药物-脂质颗粒毒性和与靶基因的表达或过表达相关的疾病的进展。施用可以通过单一或分剂量来完成。
例如，可以将所述药物-脂质颗粒施用给受试者，所述受试者被病原微生物感染或有被病原微生物感染的风险。所述药物应该优选地对应于序列，并还应该对于微生物是唯一的(或至少在经历治疗的患者的天然基因组中是缺乏的)，所述序列在微生物的生活史中具有关键作用。通过来自体内(ex vivo)或静脉内注射以治疗上有效的剂量将所述药物-脂质颗粒引入靶细胞，组织或器官。使编码与病原感染相关的基因的序列沉默可以方便地与用于治疗病原疾病的常规试剂的施用结合进行使用。所述治疗可以被预防性地施用给有风险被病原微生物感染或已经被病原微生物感染的人。
在一个优选的实施方案中，本公开药物-脂质颗粒可以方便地用于治疗癌症、病毒感染、自身免疫性疾病、糖尿病、阿尔兹海默症。病毒感染包括甲肝、乙肝、丙肝、SARS-Cov-2、HIV、HPV、流感、天花、梅毒。例如，对于抑制乙型肝炎病毒的合适位点包括编码S，C，P和X蛋白质，PRE，EnI，和EnII的核酸序列(见，例如，FIELDSVIROLOGY，2001，同上)。本领域的技术人员将理解与肝炎感染相关的基因沉默可以与对于肝炎的常规治疗，诸如，例如免疫球蛋白、干扰素(例如，PEG化和未PEG化的干扰素a)(见，例如，Medina et al.，AntiviralRes.60(2)：135-143(2003)；利巴韦林(见，例如，Hugle和Cerny， Rev.Med.Virol.13(6)：361-71(2003)；阿德福韦和拉米夫定(见，例如，Kock et al.，Hepatology 38(6)：1410-8(2003)；异戊二烯化抑制剂(见，例如Bordier et al.，J.Clin.Invest.112(3)：407-414(2003))；泛昔洛韦(见，例如.，Yurdaydin et al.，J Hepatol.37(2)：266-71(2002)；和柴胡皂苷c和d(见，例如.，Chiang et al.，Planta Med.69(8)：705-9(2003)。
在另一个实施方案中，本公开药物-脂质颗粒可以便利地用于治疗特征在于基因的或基因群的表达或过表达的疾病和病症。在一些方面中，本公开的药物-脂质颗粒可以用于治疗代谢性疾病和病症(例如，其中肝是靶目标的疾病和病症以及肝疾病和病症)诸如，例如，血脂异常和糖尿病。本领域技术人员将理解与代谢疾病和病症相关的基因的沉默可以与这些疾病的常规治疗结合。例如，涉及血脂异常的基因的沉默可以与用抑制素、胆汁酸鳌合剂/树脂和胆固醇吸收抑制剂诸如依泽替米贝，植物甾烷醇/甾醇，多元酚，以及营养制品诸如燕麦糠，亚麻籽和大豆蛋白，植物甾烷醇(phytostanol)类似物，角鲨烯合成酶抑制剂，胆汁酸转运抑制剂SREBP裂解激活蛋白质(SCAP)激活配体，烟酸(烟酸)，阿西莫司，高剂量鱼油，抗氧化剂和甘蔗脂肪醇，微粒体三酰甘油转运蛋白(MTP)抑制剂，脂酰辅酶A：胆固醇酰基转移酶(ACAT)抑制剂，gemcabene，利非贝罗，泛酸类似物，烟酸-受体激动剂，抗炎剂(诸如Lp-PLA(2)拮抗剂和AGI1067)功能油，PPAR-α，γ，δ激动剂，以及双重PPAR-α，/γ和‘pan’PPAR-α/γ，/δ激动剂，胆甾醇酯转移蛋白(CETP)抑制剂(诸如torcetrapib)，CETP疫苗，ATP-结合盒式转运蛋白(ABC)A1的上调剂，卵磷脂胆固醇酰基转移酶(LCAT)和清除剂受体类B类型1(SRB1)，以及合成性载脂蛋白(Apo)E-相关肽，延缓释放的烟酸/洛伐他汀，阿托伐他汀/氨氯地平，依泽替米贝/辛伐他汀，阿托伐他汀/CETP抑制剂，statin/PPAR激动剂，在开发中的延缓释放的烟酸/辛伐他汀和普伐他汀/阿司匹林，以及抗-肥胖症试剂进行的治疗结合(见，例如，Bays和Stein，Expert Opin.Pharmacother.4(11)：1901-38(2003))。同样地，涉及糖尿病的基因的沉默可以与用胰岛素进行的治疗以及饮食修改和锻炼结合。
类似的方法用于抑制内源受体细胞基因的表达，所述内源受体基因与肿瘤发生和细胞转化，肿瘤生长和肿瘤迁移相关；抑制生血管基因的表达；抑制免疫调节剂基因，诸如那些与炎症和自身免疫应答相关基因的表达；抑制配体受体基因的表达；抑制与神经变性病症相关的基因的表达；和抑制另外的与病毒感染和存活相关的另外的基因的表达。具体目标的靶基因序列同上描述。
检测所述颗粒：使用本领域已知的任何方法检测本文的药物-脂质颗粒。例如，使用本领域众所周知的方法将标记直接或间接与药物-脂质颗粒的成分或其它基于脂质的载体系统偶联。可以使用广泛种类的标记，其中基于需要的敏感性，与药物-脂质颗粒成分的缀合的容易性，稳定性要求和可获得的工具和处理的准备来进行选择。合适的标记包括，但不限于，光谱标记，诸如荧光染料(例如，(例如，荧光素和衍生物，诸如异硫氰酸荧光素(FITC)和Oregon GreenTM；若丹明和衍生物，诸如德克萨斯红，tetrarhodimine isothiocynate(TRITC)，等，洋地黄毒苷，生物素，藻红蛋白，AMCA，CyDyesTM等；放射性标记，诸如3H，125I，35S，14C，32P，33P，等；酶诸如辣根过氧化物酶，碱性磷酸酶等；光谱比色标记诸如胶态金或有色玻璃或塑料珠，诸如聚苯乙烯，聚丙烯，胶乳等)。使用本领域已知的任何方式来对标记进行检测。
核酸的检测：通过本领域那些技术人员众所周知的许多方式的任何一种来对本文的核酸进行检测和定量。通过本领域众所周知的方法诸如DNA印迹分析，RNA印迹分析，凝胶电泳，PCR，放射性标记，闪烁计数和亲和性层析法来进行核酸的检测。还可以应用另外的分析生化方法诸如分光光度测定法，X光线照相术，电泳，毛细管电泳，高效液相层析(HPLC)，薄层层析法(TLC)，hyperdiffusion层析法。
通过应用核酸扩增系统可以提高杂交测定的敏感性，所述核酸扩增系统使被检测的靶核酸成倍增加。已知适合于扩增用作分子探针的序列或产生核酸片段以进行随后的 亚克隆的体外扩增技术。通过这些体外扩增方法，包括聚合酶链反应(PCR)，连接酶链反应(LCR)，Qβ-复制酶扩增和其它的RNA聚合酶介导技术(例如，NASBATM)足以指导技术人员的技术实例见于Sambrook，et al.，Molecular Cloning：A Laboratory Manual，ColdSpring Harbor Laboratory Press，2000，和Ausubel et al.，SHORT PROTOCOLSIN MOLECULAR BIOLOGY，eds.，Current Protocols，ajoint venture between Greene Publishing Associates，Inc.and John Wiley&Sons，Inc.，(2002)，以及Mullis et al.(1987)，美国专利号4,683,202；PCR Protocols A Guide toMethods and Applications(Innis et al.eds)Academic Press Inc.San Di ego，CA(1990)(Innis)；Arnheim&Levinson(October 1，1990)，C&EN 36；TheJournal Of NIH Research，3：81(1991)；(Kwoh et al.，Proc.Natl.Acad.Sci.USA，86：1173(1989)；Guatelli et al.，Proc.Natl.Acad.Sci.USA，87：1874(1990)；Lomell et al.，J.Clin.Chem.，35：1826(1989)；Landegren et al.，Science，241：1077(1988)；Van Brunt，Biotechnology，8：291(1990)；Wu和Wallace，Gene，4：560(1989)；Barringer et al.，Gene，89：117(1990)，和Sooknanan和Malek，Biotechnology，13：563(1995)。克隆体外扩增核酸的改善的方法描述于Wallace et al.，美国专利号5,426,039。本领域描述的其它方法是基于核酸序列的扩增(NASBATM，Cangene，Mississauga，Ontario)和Qβ复制酶系统。
如Needham VanDevanter et al.，Nucleic Acids Res.，12：6159(1984)所述，典型地按照Beaucage和Caruthers，Tetrahedron Letts.，22(20)：1859 1862(1981)所述的固相亚磷酰胺三酯方法，例如使用自动合成仪来化学合成寡核苷酸，所述寡核苷酸用作例如在体外扩增方法的探针，用作基因探针，或作为抑制剂成分。如果必要，如Pearson和Regnier，J.Chrom.，255：137149(1983)所述，通过天然丙烯酰胺凝胶电泳或通过阴离子交换HPLC来典型地进行寡核苷酸的纯化。使用Maxam和Gilbert(1980)在Grossman和Moldave(eds.)Academic Press，New York，Methods in Enzymology，65：499中的化学降解方法可以证实合成性寡核苷酸的序列。
随后的实施例提供举例说明，但不限定被要求的本公开。本领域技术人员将容易地识别各种非关键性的参数，其可以在实质上产生相同的结果。
本公开要保护的药物-脂质颗粒是指除含阳离子/可电离脂质以外的药物-脂质颗粒，即药物-金属-磷脂复合物颗粒(drug-loaded metal-chelated phospholipid complex nanoparticles，drug@MPP)。
实验例一、制备药物-金属-磷脂复合物颗粒
实施例1、制备磷脂复合物
将具有磷酸基团的磷脂分子与连接物分子连接：将二硬脂酰基磷脂酰胆碱(DSPC，式46)和姜黄素(式19)按照摩尔比1：1加入反应瓶中，加入适量乙醇溶解，于65℃反应2h后，浓缩，加入正己烷。沉淀出来磷脂复合物，过滤，真空干燥得到磷脂复合物。磷脂复合物结构如下所示：
结果分析：姜黄素与DSPC在65℃的条件下反应2小时获得的目标产物的产率为94％。
实施例2、制备金属-磷脂复合物
实施例2.1制备金属离子为Fe3+时的金属-磷脂复合物
实施例1中制备的磷脂复合物与金属离子部分连接：将磷脂复合物和FeCl3按照摩尔比1：1加入反应瓶中，加入乙醇溶解，于60℃反应2h后，将反应液悬干，随后用超纯水洗涤，真空干燥得到金属-磷脂复合物。金属-磷脂复合物结构如下所示。
结果分析：磷脂复合物与FeCl3在60℃的条件下反应2小时，磷脂复合物投料浓度为4.5mg/mL以及磷脂复合物与FeCl3的投料比为1：1时获得的目标产物的产率为95％。
实施例2.2制备金属离子为Al3+时的金属-磷脂复合物
本实施例与实施例2.1的区别在于，FeCl3替换为Al(NO3)3·9H2O。制备得到的金属-磷脂复合物结构如下所示。
结果分析：磷脂复合物与Al(NO3)3·9H2O在60℃的条件下反应2小时，磷脂复合物投料浓度为4.5mg/mL以及磷脂复合物与Al(NO3)3·9H2O的投料比为1：1时获得的目标产物的产率为95％。
实施例3、制备mRNA-金属-磷脂复合物颗粒(mRNA-loaded metal-chelated phospholipid complex nanoparticles，mRNA@MPP)
制备金属离子为Fe3+时的mRNA-金属-磷脂复合物颗粒
按照实施例2.1中的方法制备金属-磷脂复合物，其中DSPC、姜黄素、FeCl3按照1:1:1的投料比投入，并将金属-磷脂复合物和二硬脂酰基磷脂酰胆碱(DSPC，式46，作为非阳离子脂质或非可电离脂质)、胆固醇(CHOL，式40，作为非阳离子脂质或非可电离脂质)、DSPE-PEG2000(式53，作为抑制颗粒聚集的缀合的脂质)按不同的摩尔占比溶于乙醇中作为有机相。其中金属-磷脂复合物、DSPC、CHOL及DSPE-PEG2000的占比分别为15％、35％、46％、4％。mRNA按20μg/mL的浓度溶于无酶PBS缓冲液中作为水相(PBS的组分为0.137M的氯化钠、0.0027M的氯化钾、0.01M的磷酸氢二钠和0.0018M 的磷酸二氢钾)。以金属-磷脂复合物质量与mRNA质量按40:1的质量比在微流控芯片中混合。水相与有机相的体积比为3:1。有机相和水相在微流控芯片中的流速为12ml/min。其中，药物mRNA为编码荧光蛋白eGFP的mRNA，其序列为SEQ ID NO.1(720nt)。制备得到eGFP-mRNA@MPP。将eGFP-mRNA@MPP按2μg/mL的浓度(所含mRNA的浓度)与293T细胞孵育，对照组用未进行载药的MPP孵育，48h后收取细胞悬液，通过流式细胞术检测eGFP阳性细胞百分比。
对实施例3中制备的eGFP-mRNA@MPP进行粒径、表面电势及稳定性的检测，并计算eGFP-mRNA@MPP包载核酸的效率。
检测粒径的方法及结果判断标准：使用马尔文激光粒度仪Zetasizer测试纳米粒子粒径，粒径在30～400nm范围内视为可接受。
检测表面电势的方法及结果判断标准：使用马尔文激光粒度仪Zetasizer测试纳米粒子表面电势，电势在-10～10mV范围视为可接受。
检测稳定性的方法及结果判断标准：将纳米粒子在4℃放置7天，使用马尔文激光粒度仪Zetasizer测试纳米粒子粒径以及表面电势，当其粒径以及表面电势在3-7天内无明显变化视为稳定性较好。
计算核酸包载效率的方法：具体采用琼脂糖凝胶电泳法。首先将每组脂质纳米颗粒的核酸投料量均定为10μg/mL，金属离子为Fe3+时的金属-磷脂复合物质量与mRNA质量比为40:1，将等浓度的核酸溶于PBS缓冲溶液作为阳性对照，阴性对照为PBS缓冲溶液。琼脂糖凝胶的浓度为1.5％，此时胶的空隙只允许游离核酸通过而不允许脂质纳米颗粒通过，当游离核酸条带电泳至可清晰分辨时停止电泳。用Image J软件统计不同组别游离核酸的灰度值，阳性对照组定为100％，每组的游离核酸相对于阳性对照的比值为游离核酸相对量，则每组包载率为(100-游离核酸相对量)％。核酸包载率在50％以上视为可接受范围。
细胞培养方法：人胚胎肾细胞系293T用含10％FBS和1％青霉素—链霉素的DMEM培基在37℃，5％CO2的条件下培养。
流式细胞术方法分析eGFP阳性细胞百分比的方法：将293T细胞接种于24孔板上，接种密度为5×105个细胞/孔，当细胞密度达到80％时，加入1mL MPP或eGFP-mRNA@MPP孵育细胞，其中eGFP-mRNA@MPP浓度为2μg/mL。48h后收集细胞悬液，用流式细胞仪FITC通道收集20000个细胞，并分析eGFP阳性细胞百分比，其计算公式为：eGFP阳性细胞率计算公式＝表达eGFP细胞数/细胞总数×100％。eGFP阳性细胞百分比达40％以上视为可接受范围。
制备金属离子为Al3+时的mRNA-金属-磷脂复合物颗粒
按照实施例2.2中的方法制备金属-磷脂复合物，其中DSPC、姜黄素、Al(NO3)3·9H2O按照1:1:1的投料比投入，并将金属-磷脂复合物和二硬脂酰基磷脂酰胆碱(DSPC，式46，作为非阳离子脂质或非可电离脂质)、胆固醇(CHOL，式40，作为非阳离子脂质或非可电离脂质)、DSPE-PEG2000(式53，作为抑制颗粒聚集的缀合的脂质)按不同的摩尔占比溶于乙醇中作为有机相。其中金属-磷脂复合物、DSPC、CHOL及DSPE-PEG2000的占比分别为7％、34％、56％、3％。mRNA按20μg/mL的浓度溶于无酶PBS缓冲液中作为水相(PBS的组分为0.137M的氯化钠、0.0027M的氯化钾、0.01M的磷酸氢二钠和0.0018M的磷酸二氢钾)。以金属磷脂复合物质量与mRNA质量按13.3:1的质量比在微流控芯片中混合。水相与有机相的体积比为3:1。有机相和水相在微流控芯片中的流速为12ml/min。其中，药物mRNA为编码荧光蛋白eGFP的mRNA，其序列为SEQ ID NO.1(720nt)。制备得到的eGFP-mRNA@MPP。将eGFP-mRNA@MPP按2μg/mL的浓度(所含mRNA的浓度)与293T细胞孵育，对照组用未进行载药的MPP孵育，48h后收取细胞悬液，通过流式细胞术检测eGFP阳性细胞百分比。
对实施例3中制备的eGFP-mRNA@MPP进行粒径、表面电势及稳定性的检测，并计算eGFP-mRNA@MPP包载核酸的效率。
检测粒径的方法及结果判断标准：使用马尔文激光粒度仪Zetasizer测试纳米粒子粒径，粒径在30～400nm范围内视为可接受。
检测表面电势的方法及结果判断标准：使用马尔文激光粒度仪Zetasizer测试纳米粒子表面电势，电势在-10～10mV范围视为可接受。
检测稳定性的方法及结果判断标准：将纳米粒子在4℃放置7天，使用马尔文激光粒度仪Zetasizer测试纳米粒子粒径以及表面电势，当其粒径以及表面电势在3-7天内无明显变化视为稳定性较好。
计算核酸包载效率的方法：具体采用琼脂糖凝胶电泳法。首先将每组脂质纳米颗粒的核酸投料量均定为10μg/mL，金属离子为Al3+时的金属-磷脂复合物质量与mRNA质量比为13.3:1，将等浓度的核酸溶于PBS缓冲溶液作为阳性对照，阴性对照为PBS缓冲溶液。琼脂糖凝胶的浓度为1.5％，此时胶的空隙只允许游离核酸通过而不允许脂质纳米颗粒通过，当游离核酸条带电泳至可清晰分辨时停止电泳。用Image J软件统计不同组别游离核酸的灰度值，阳性对照组定为100％，每组的游离核酸相对于阳性对照的比值为游离核酸相对量，则每组包载率为(100-游离核酸相对量)％。核酸包载率在50％以上视为可接受范围。
细胞培养方法：人胚胎肾细胞系293T用含10％FBS和1％青霉素—链霉素的DMEM培基在37℃，5％CO2的条件下培养。
流式细胞术方法分析eGFP阳性细胞百分比的方法：将293T细胞接种于24孔板上，接种密度为5×105个细胞/孔，当细胞密度达到80％时，加入1mL MPP或eGFP-mRNA@MPP孵育细胞，其中eGFP-mRNA@MPP浓度为2μg/mL。48h后收集细胞悬液，用流式细胞仪FITC通道收集20000个细胞，并分析eGFP阳性细胞百分比，其计算公式为：eGFP阳性细胞率计算公式＝表达eGFP细胞数/细胞总数×100％。eGFP阳性细胞百分比达40％以上视为可接受范围。
由金属-磷脂复合物组装的金属-磷脂复合物颗粒(Metal-chelated phospholipid complex nanoparticles，MPP)装载核酸的原理是：姜黄素与DSPC通过氢键结合在一起，同时姜黄素通过配位键与Fe3+或Al3+相连，形成了金属-磷脂复合物，该金属-磷脂复合物的Fe3+或Al3+通过配位键与核酸连接，从而确保金属-磷脂复合物与其他脂质组分自组装成MPP同时将核酸载入纳米颗粒中。姜黄素在MMP装载核酸中的贡献有两种可能性：①姜黄素与核酸相互作用，协助MPP装载核酸，例如姜黄素通过插入核酸的小沟中协助装载核酸；②姜黄素也可能不直接与核酸相互作用。
实施例3.1、金属-磷脂复合物的组分的投放比例
将实施例3中的DSPC、姜黄素、FeCl3按照不同的投放比例(1:1:1、3:3:2、2:2:1)投放，其他步骤同实施例3，制备得到不同的eGFP-mRNA@MPP，分别检测其核酸包载率。
结果分析：如结果表1-1所示，当DSPC、姜黄素、FeCl3的投放比例为1:1:1时，制备得到的药物-脂质颗粒的eGFP-mRNA包载效率为97％；当DSPC、姜黄素、FeCl3的投放比例为3:3:2时，制备得到的药物-脂质颗粒的eGFP-mRNA包载效率为70％；当DSPC、姜黄素、FeCl3的投放比例为2:2:1时，制备得到的药物-脂质颗粒的eGFP-mRNA包载效率为60％。在药物-脂质颗粒中Fe3+的功能是连接磷脂复合物与核酸，每个Fe3+最多有三个络合位点，所以DSPC、姜黄素、FeCl3在药物-脂质颗粒中的投放比例应该为1：1：1，才能保证药物-脂质颗粒能尽可能多地包载核酸。实验的结果也证实当DSPC、姜黄素、FeCl3的投放比例为1:1:1时，用其制备的药物-脂质颗粒的eGFP-mRNA包载率最高。当DSPC、姜黄素、FeCl3的投放比例从1:1:1到2:2:1范围为内，其药物-脂质颗粒的核 酸包载率均在60％以上。
表1-1金属-磷脂复合物的组分投放比例及其制备的药物-脂质颗粒的功能
将实施例3中的DSPC、姜黄素、Al(NO3)3·9H2O按照不同的投放比例(1:1:1、3:3:2、2:2:1)投放，其他步骤同实施例3，制备得到不同的eGFP-mRNA@MPP，分别检测其核酸包载率。
结果分析：如结果表1-2所示，当DSPC、姜黄素、Al(NO3)3·9H2O的投放比例为1:1:1时，制备得到的金属-磷脂复合物颗粒的eGFP-mRNA包载效率为98％；当DSPC、姜黄素、Al(NO3)3·9H2O的投放比例为3:3:2时，制备得到的金属-磷脂复合物颗粒的eGFP-mRNA包载效率为72％；当DSPC、姜黄素、Al(NO3)3·9H2O的投放比例为2:2:1时，制备得到的金属-磷脂复合物颗粒的eGFP-mRNA包载效率为58％。在金属-磷脂复合物颗粒中Al3+的功能是连接磷脂复合物与核酸，每个Al3+最多有三个络合位点，所以DSPC、姜黄素、Al(NO3)3·9H2O在药物-脂质颗粒中的投放比例应该为1：1：1，才能保证金属-磷脂复合物颗粒能尽可能多地包载核酸。实验的结果也证实当DSPC、姜黄素、Al(NO3)3·9H2O的投放比例为1:1:1时，用其制备的金属-磷脂复合物颗粒的eGFP-mRNA包载率最高。当DSPC、姜黄素、Al(NO3)3·9H2O的投放比例从1:1:1到2:2:1范围为内，其金属-磷脂复合物颗粒的核酸包载率均在58％以上。
表1-2金属-磷脂复合物的组分投放比例及其制备的金属-磷脂复合物颗粒的功能
实施例3.2、制备药物-脂质颗粒中金属-磷脂复合物、二硬脂酰磷脂酰胆碱(DSPC)、DSPE-PEG2000及胆固醇(CHOL)的比例
与实施例3相比，以金属-磷脂复合物、二硬脂酰磷脂酰胆碱(DSPC)、DSPE-PEG2000、胆固醇(CHOL)的比例如表1-3(金属离子为Fe3+)和表1-4(金属离子为Al3+)所示，其余条件相同。
结果分析：如结果表1-3所示，当金属-磷脂复合物(金属离子为Fe3+)占比在(10-40)％范围时、DSPC占比在(0-40)％范围时、CHOL占比在(35-75)％范围时，DSPE-PEG2000占比在(2-10)％范围时，药物-脂质颗粒的粒径在50～400nm范围、表面电势在-10～10mV范围、体外稳定性3天以上、mRNA包载率50％以上，eGFP蛋白的阳性表达率70％以上。其中，当金属-磷脂复合物占比15％、二硬脂酰磷脂酰胆碱(DSPC)占比35％、胆固醇(CHOL)占比46％及DSPE-PEG2000占比4％时，药物-脂质颗粒的性能最优，即粒径在110nm范围、表面电势在-2.04mV范围、体外稳定性>7天、mRNA包载率为87％，eGFP蛋白的阳性表达率97％。因为mRNA@MPP主要靠金属-磷脂复合物吸附核酸，所以金属-磷脂复合物的占比不能太低；DSPC的含量在0-40％范围内时，其纳米颗粒的稳定性在可接受范围内，当DSPC的含量为0％时，因为金属-磷脂复合物中含有DSPC，使其纳米颗粒的稳定性能得以维持；DSPE-PEG2000的作用是防止纳米颗粒聚集以及增长体内循环时间，其含量在2-10％范围性能会较优；CHOL的作用是增强纳米颗粒 的流动性，其维持一定的含量利于纳米颗粒的稳定性。
上述结果提示，金属-磷脂复合物(金属离子为Fe3+)占比在(10-40)％范围时、DSPC占比在(0-40)％范围时、CHOL占比在(35-75)％范围时，DSPE-PEG2000占比在(2-10)％范围时，mRNA@MPP具备较好的载药性能。
表1-3不同组分比例的eGFP-mRNA@MPP(Fe3+)性能检测
结果分析：如结果表1-4所示，当金属-磷脂复合物(金属离子为Al3+)占比在(5-50)％范围时、DSPC占比在(0-51)％范围时、CHOL占比在(15-80)％范围时，DSPE-PEG2000占比在(2-10)％范围时，药物-脂质颗粒的粒径在50～400nm范围、表面电势在-10～10mV范围、体外稳定性3天以上、mRNA包载率50％以上，eGFP蛋白的阳性表达率70％以上。其中，当金属-磷脂复合物占比7％、二硬脂酰磷脂酰胆碱(DSPC)占比34％、胆固醇(CHOL)占比56％及DSPE-PEG2000占比3％时，药物-脂质颗粒的性能最优，即粒径为100nm、表面电势为-1.57mV、体外稳定性>7天、mRNA包载率为92％，eGFP蛋白的阳性表达率98％。因为mRNA@MPP主要靠金属-磷脂复合物吸附核酸，所以金属-磷脂复合物的占比不能太低；DSPC的含量在0-51％范围内时，其纳米颗粒的稳定性在可接受范围内，当DSPC的含量为0％时，因为金属-磷脂复合物中含有DSPC，使其纳米颗粒的稳定性能得以维持；DSPE-PEG2000的作用是防止纳米颗粒聚集以及增长体内循环时间，其含量在2-10％范围性能会较优；CHOL的作用是增强纳米颗粒的流动性，其维持一定的含量利于纳米颗粒的稳定性。
上述结果提示，金属-磷脂复合物(金属离子为Al3+)占比在(5-50)％范围时、DSPC占比在(0-51)％范围时、CHOL占比在(15-80)％范围时，DSPE-PEG2000占比在(2-10)％范围时，mRNA@MPP具备较好的载药性能。
表1-4不同组分比例的eGFP-mRNA@MPP(Al3+)性能检测

实施例3.3、制备eGFP-mRNA@MPP中非阳离子脂质或非可电离脂质种类
与实施例3相比，二硬脂酰磷脂酰胆碱(DSPC)的替代如表1-5和表1-6所示，其余条件相同。
结果分析：为探索eGFP-mRNA@MPP中DSPC可以被其他非阳离子脂质或非可电离脂质替代，我们分别选用另外三种非阳离子脂质或非可电离脂质，即DSPE、DSPA及DSPG分别替代DSPC，并通过检测粒径、表面电势、稳定性及mRNA包载率，证明eGFP-mRNA@MPP中DSPC可以被其他非阳离子脂质或非可电离脂质所替代，替代后其功能等同于包含DSPC的eGFP-mRNA@MPP的功效(表1-5(金属离子为Fe3+)和表1-6(金属离子为Al3+))。因为非阳离子脂质或非可电离脂质DSPC在eGFP-mRNA@MPP中的主要作用是使脂质体膜融合更好、稳定性更高以及毒性更小，而其他非阳离子脂质或非可电离脂质也具有使脂质体膜融合更好、稳定性更高以及毒性更小的功能，所以，药物-脂质颗粒中的DSPC可以被除金属-磷脂复合物和抑制颗粒聚集的缀合的脂质以外的其他非阳离子脂质或非可电离脂质替代，且其功效不被影响。
三种非阳离子脂质或非可电离脂质(DSPE、DSPA及DSPG)的结构式如下所示。

表1-5含有不同种类除金属-磷脂复合物和抑制颗粒聚集的缀合的脂质以外的其他非阳离子脂质或非可电离脂质的eGFP-mRNA@MPP(Fe3+)的性能
表1-6含有不同种类除金属-磷脂复合物和抑制颗粒聚集的缀合的脂质以外的其他非阳离子脂质或非可电离脂质的eGFP-mRNA@MPP(Al3+)的性能
实施例3.4、制备eGFP-mRNA@MPP中抑制颗粒聚集的缀合的脂质种类
与实施例3相比，DSPE-PEG2000(式53)的替代如表1-7(金属离子为Fe3+)和表1-8(金属离子为Al3+)所示，其余条件相同。三种其他抑制颗粒聚集的缀合的脂质DSPE-PEG700(式50)及DSPE-PEG5000(式52)及DSPE-PEG1000(式51)。
结果分析：为探索eGFP-mRNA@MPP中DSPE-PEG2000可以被其他抑制颗粒聚集的缀合的脂质替代，我们分别选用三种其他抑制颗粒聚集的缀合的脂质，即DSPE-PEG700、DSPE-PEG5000及DSPE-PEG1000分别替代DSPE-PEG2000，并通过检测粒径、表面电势、稳定性及mRNA包载率，证明eGFP-mRNA@MPP中DSPE-PEG2000可以被其他抑制颗粒聚集的缀合的脂质所替代，替代后其功能等同于包含DSPE-PEG2000的eGFP-mRNA@MPP的功效(表1-7和表1-8)。因为DSPE-PEG2000在eGFP-mRNA@MPP中的主要作用是抑制聚集，而其他抑制颗粒聚集的缀合的脂质也具有抑制聚集的功能，所以，eGFP-mRNA@MPP中的DSPE-PEG2000可以被其他抑制颗粒聚集的缀合的脂质替代，且其功效不被影响。
表1-7含有不同种类抑制颗粒聚集的缀合的脂质的eGFP-mRNA@MPP(Fe3+)的性能

表1-8含有不同种类抑制颗粒聚集的缀合的脂质的eGFP-mRNA@MPP(Al3+)的性能
实施例3.5、mRNA@MPP的制备及效果表征
实施例3.5.1、金属离子为Fe3+的mRNA@MPP的制备及效果表征
将实施例3中的mRNA替换为其他两种mRNA，参照实施例3的方法分别制备三种含不同目的蛋白mRNA序列的mRNA@MPP。三种不同的mRNA序列分别为：①编码荧光蛋白eGFP的mRNA序列为SEQ ID NO.1(720nt)；②编码新型冠状病毒S1亚基的受体结合域(receptor binding domain，RBD)的mRNA序列为SEQ ID NO.2(669nt)；③编码肿瘤抗原NY-ESO-1的mRNA序列为SEQ ID NO.3(543nt)。其余药物(mRNA)-脂质颗粒的制备过程与实施例3相同，分别得到eGFP-mRNA@MPP、RBD-mRNA@MPP、NY-ESO-1-mRNA@MPP。
将eGFP-mRNA@MPP按2μg/mL的浓度(所含mRNA的浓度)与293T细胞孵育，对照组用MPP孵育，48h后收取细胞悬液，通过流式细胞术检测eGFP阳性细胞百分比，结果见图1-1；将RBD-mRNA@MPP按2μg/mL的浓度(所含mRNA的浓度)与293T细胞孵育，对照组用MPP孵育，24h后，离心后上清液-20℃冻存备用，利用市售新冠抗原RBD ELISA检测试剂盒检测细胞上清新冠抗原RBD蛋白的表达水平，结果见图1-2。
ELISA检测RBD表达水平的方法：
1.样品收集：细胞上清液室温放置2小时，于1000×g离心20min，取上清；
2.加样：包被板上分别设空白孔、标准品孔、待测样品孔。空白孔加样品稀释液100μL，标准品孔分别加入依次梯度稀释标准品，待测样品孔加待测样品100μL，37℃孵育60min；
3.弃去孔内液体，洗板3次，每次浸泡1-2min。每孔分别加入配好的生物素标记的抗RBD抗体工作液100μL，混匀，37℃孵育60min；
4.弃去孔内液体，洗板3次，每次浸泡1-2min；
5.每孔加入配好的链霉亲和素HRP工作液100μL，混匀，37℃孵育45min；
6.弃去孔内液体，洗板3次，每次浸泡1-2min；
7.每孔加TMB底物溶液(TMB)100μL，37℃避光孵育15min；
8.每孔加终止液100μL，终止反应；
9.在450nm波长测量各孔的光密度(OD值)。
数据分析：以标准品的浓度为横坐标，OD值为纵坐标，绘制标准曲线。
将实验动物被随机分成2组(实验组和对照组)，每组5只。其中，RBD-mRNA@MPP动物模型为BALB/c小鼠，每只小鼠在第1天进行第一次肌肉给药，在第14天进行第二次肌肉给药，实验组注射RBD-mRNA@MPP，对照组注射未装载mRNA的金属-磷脂复合物颗粒(MPP)。每次给药的剂量为100μL，其中实验组中RBD-mRNA@MPP制剂含30mg的mRNA。距第一次给药后第28天收集小鼠血液，分离血清梯度稀释，通过市售ELISA试剂盒检测小鼠体内所产生的抗新型冠状病毒S1亚基的RBD总IgG抗体，结果如图1-3所示。
NY-ESO-1-mRNA@MPP的动物模型为C57BL/6小鼠，每只小鼠在第1，7，14，21天进行四次肌肉给药，实验组注射NY-ESO-1-mRNA@MPP，对照组注射未装载mRNA的金属-磷脂复合物颗粒(MPP)。每次给药的剂量为100μL，其中实验组中NY-ESO-1-mRNA@MPP制剂含30mg的mRNA。距第一次给药后第28天收集小鼠血液，分离血清梯度稀释，用ELISA法检测小鼠体内所产生的抗NY-ESO-1总IgG抗体，结果如图1-4所示。
检测小鼠体内抗NY-ESO-1总IgG抗体的方法：
ELISA法所用试剂配制：
1.包被液：精密称取8.4g NaHCO3溶于1L蒸馏水(DDW)中，待固体全部溶解后，使用1M的NaOH溶液将整个溶液pH调至9.6，将配制好的包被液存放于4℃待用。
2.洗液：向1L 0.01M PBS溶液中加入0.5mL Tween-20，混合均匀后于室温放置。
3.封闭液：精密称取20g BSA加入1L 0.01M PBS溶液中，对溶液中未溶解的BSA粉末进行超声处理，待溶液中固体全部溶解且溶液呈淡黄色时，放置于4℃冰箱中保存待用。
4.抗体稀释液：精密称取2.5g BSA溶于250mL 0.01M PBS溶液中，待固体完全溶解后，向其中加入1.25mL Tween-20，混合均匀后于4℃保存待用。
5.显色液：0.1M柠檬酸：19.2g柠檬酸加入DDW水至1000mL(A)0.2M磷酸氢二钠：28.4g无水磷酸氢二钠加入DDW水至1000mL(B)0.1M柠檬酸溶液(A)24.3mL，0.2M的磷酸盐缓冲液(B)25.7mL，加DDW水50mL。现用现加OPD(邻苯二胺)50mg，加入30％H2O20.15mL。
6.终止液：2M H2SO4：浓硫酸55.5mL，加DDW至500mL。
ELISA法测定小鼠血清中抗体的滴度：
1.包被：NY-ESO-1抗原用包被液稀释至1μg/mL，加入96孔板中，50μL/孔，4℃包被过夜。
2.封闭：甩干孔板内的包被液，用封闭液洗涤3次，5min一次并甩干，每孔加入150μL封闭液，37℃孵育2h。
3.干燥：甩干封闭液，37℃孵育1～2h，待孔板底部液体全部干燥为止。
4.免疫：将血清样品用抗体稀释液初始稀释1：1000，再按1：2依次进行系列稀释，将稀释后的血清样品加入封闭的96孔板中，100μL/孔，于37℃孵育2h；甩干孔板内液体，加入洗液，300μL/孔，缓慢振荡40s，重复此步骤三次；向孔板中加入1：1000稀释的生物素化的羊抗鼠IgG抗体，100μL/孔，37℃孵育1h；甩干孔板内液体，加入洗液，重复上述洗板步骤；加入新鲜配制的链酶亲和素标记的辣根过氧化物酶HRP工作液，100μL/孔，37℃孵育1h；甩干孔板内液体，加入洗液，重复上述洗板步骤；于避光条件下加入显色液，100μL/孔，室温反应5min后，加入终止液终止显色，50μL/孔；使用酶标仪 测定450nm下的吸光度。
在给RBD-mRNA@MPP后第28天采集正常小鼠脾脏，在无菌条件下制备成单细胞悬液，按照100000脾细胞/孔铺板于细胞孔板中，加入终浓度为10mg/mL的RBD蛋白培养48h，离心去上清，通过ELISA试剂盒测定IFN-γ、IL-2、IL-4的表达水平，结果如图1-5所示。
在给NY-ESO-1-mRNA@MPP后第28天采集正常小鼠脾脏，在无菌条件下制备成单细胞悬液，按照100000脾细胞/孔铺板于细胞孔板中，加入终浓度为10mg/mL的NY-ESO-1蛋白培养48h，离心去上清，通过ELISA试剂盒测定IFN-γ、IL-2、TNF-α的表达水平，结果如图1-6所示。
结果分析：如图1-1所示，eGFP-mRNA@MPP实验组eGFP阳性细胞率为97％，而MPP对照组未检测到eGFP信号；如图1-2所示，MPP包载的RBD-mRNA所编码的RBD蛋白在293T细胞上清中为193.3ng/mL，而转染了空载体MPP的293T细胞上清RBD蛋白含量为0。结果提示，mRNA-MPP可以包载、递送任意mRNA，并在细胞内直接编码多肽。如图1-3，1-4结果所示，RBD-mRNA@MPP和NY-ESO-1-mRNA@MPP均能有效诱导小鼠的体液免疫，产生高水平的抗原特异性结合抗体。其中RBD-mRNA@MPP处理组小鼠体内IgG抗体滴度达117268.8；NY-ESO-1-mRNA@MPP处理组小鼠IgG抗体滴度达5319.52。如图1-5，1-6所示，RBD-mRNA@MPP和NY-ESO-1-mRNA@MPP均能有效诱导小鼠的细胞免疫，即激活免疫细胞并产生大量的细胞因子。其中RBD-mRNA@MPP使细胞因子IFN-γ、IL-2、IL-4的表达量分别达252.8pg/mL、207.6pg/mL、56.6pg/mL；NY-ESO-1-mRNA@MPP使细胞因子IFN-γ、IL-2、TNF-α的表达量分别达70.79pg/mL、75.29pg/mL、75.27pg/mL。结果提示，mRNA@MPP可以包载、递送任意mRNA，从而促进目标蛋白(抗原)的表达，进而有效诱导小鼠的体液免疫和细胞免疫，产生高水平的抗原特异性结合抗体和细胞因子，发挥抗新冠病毒mRNA疫苗及抗肿瘤mRNA疫苗的作用。
实施例3.5.2、金属离子为Al3+的mRNA@MPP的制备及效果表征
本实施例与实施例3.5.1的区别在于，将实施例3.5.1中的金属离子Fe3+替换为Al3+。
结果分析：如图1-7所示，eGFP-mRNA@MPP实验组eGFP阳性细胞率为98.02％，而MPP对照组未检测到eGFP信号；如图1-8所示，MPP包载的RBD-mRNA所编码的RBD蛋白在293T细胞上清中为212.6ng/mL，而转染了空载体MPP的293T细胞上清RBD蛋白含量为0。结果提示，mRNA-MPP可以包载、递送任意mRNA，并在细胞内直接编码多肽。如图1-9，1-10结果所示，RBD-mRNA@MPP和NY-ESO-1-mRNA@MPP均能有效诱导小鼠的体液免疫，产生高水平的抗原特异性结合抗体。其中RBD-mRNA@MPP处理组小鼠体内IgG抗体滴度达129113；NY-ESO-1-mRNA@MPP处理组小鼠IgG抗体滴度达6507.4。如图1-11，1-12所示，RBD-mRNA@MPP和NY-ESO-1-mRNA@MPP均能有效诱导小鼠的细胞免疫，即激活免疫细胞并产生大量的细胞因子。其中RBD-mRNA@MPP使细胞因子IFN-γ、IL-2、IL-4的表达量分别达271.8pg/mL、234.6pg/mL、68.4pg/mL；NY-ESO-1-mRNA@MPP使细胞因子IFN-γ、IL-2、TNF-α的表达量分别达83.8pg/mL、98pg/mL、97.8pg/mL。结果提示，mRNA@MPP可以包载、递送任意mRNA，从而促进目标蛋白(抗原)的表达，进而有效诱导小鼠的体液免疫和细胞免疫，产生高水平的抗原特异性结合抗体和细胞因子，发挥抗新冠病毒mRNA疫苗及抗肿瘤mRNA疫苗的作用。
实施例3.6、siRNA-金属-磷脂复合物颗粒(siRNA-loaded metal-chelated phospholipid complex nanoparticles，siRNA@MPP)的制备及效果
实施例3.6.1、金属离子为Fe3+时的siRNA-金属-磷脂复合物颗粒(siRNA-loaded metal-chelated phospholipid complex nanoparticles，siRNA@MPP)的制备及效果
将实施例3中的mRNA替换为siRNA，参照实施例3的方法分别制备三种含不同siRNA的siRNA@MPP。三种不同siRNA靶向的基因、序列及其对应的随机对照序列分别为：①靶向Bcl-2基因的siRNA(Bcl-2-siRNA)的序列为SEQ ID No.4(反义链)和SEQ ID No.21(正义链)(19bp)，其随机对照序列为SEQ ID No.5(反义链)和SEQ ID No.22(正义链)(19bp)；②靶向PLK1基因的siRNA(PLK1-siRNA)的序列为SEQ ID NO.6(反义链)和SEQ ID No.23(正义链)(21bp)，其随机对照序列为SEQ ID NO.7(反义链)和SEQ ID No.24(正义链)(19bp)；③靶向Gal-1基因的siRNA(Gal-1-siRNA)的序列为SEQ ID NO.8(19bp)；其随机对照序列为SEQ ID NO.9(19bp)。其余siRNA@MPP的制备过程与实施例3相同。
Bcl-2-siRNA的序列如下：
Antisense：5′-CAGCUUAUAAUGGAUGUAC-3′(SEQ ID No.4)；
Sense：5′-GUACAUCCAUUAUAAGCUG-3′(SEQ ID No.21)(19bp)。
Bcl-2-siRNA的随机对照序列如下：
Antisense：5’-ACGUGACACGUUCGGAGAA-3’(SEQ ID No.5)；
Sense：5’-UUCUCCGAACGUGUCACGU-3’(SEQ ID No.22)(19bp)。
PLK1-siRNA的序列如下：
Antisense：5’-UAAGGAGGGUGAUCUUCUUCA-3’(SEQ ID No.6)；
Sense：5’-UGAAGAAGAUCACCCUCCUUA-3’(SEQ ID No.23)(21bp)。
PLK1-siRNA的随机对照序列如下：
Antisense：5’-CUUACGCUGAGUACUUCGA-3’(SEQ ID No.7)；
Sense：5’-UCGAAGUACUCAGCGUAAG-3’(SEQ ID No.24)(19bp)。
Gal-1-siRNA的序列如下：
5’-GCUGCCAGAUGGAUACGAA-3’(SEQ ID No.8)(19bp)。
Gal-1-siRNA的随机对照序列如下：
5’-GGAAAUCCCCCAACAGUGA-3’(SEQ ID No.9)(19bp)。
细胞培养方法：U251人脑胶质母细胞瘤细胞在高糖(4.5g/L)DMEM+10％胎牛血清(FBS)、1％青霉素/链霉素和2mml-谷氨酰胺(Bio Industries)培养基中单层生长，且在37℃、5％CO2条件下培养，每周传代2次。
U251细胞以每孔1×106细胞密度接种于6孔板中约24h后，每孔细胞与含上述siRNA的siRNA@MPP(其中siRNA的浓度为2μg/mL)分别孵育72小时后，收集细胞，提取细胞总RNA，用RT-PCR技术分别检测目标基因(Bcl-2、PLK1、Gal-1)的mRNA表达量，统计siRNA@MPP沉默细胞靶基因的能力。
RT-PCR具体流程：
总RNA的提取：将六孔板弃去培养基，PBS缓冲液漂洗3遍，每孔加入1mL Trizol对细胞进行裂解处理。加入200μL氯仿，充分摇匀，室温静置10min，13000rpm 4℃离心15min，得到分层的三相液体，其中RNA溶解在上层的水相中。吸取上层水相置于新的无酶1.5ml离心管中，加入500μL异丙醇，室温静置10min，13000rpm 4℃离心15min，得到RNA沉淀。去上清，每管加入1mL以无RNA酶水新配置的75％(v/v)乙醇，小心吹打，吹起管底RNA白色沉淀，7500rpm 4℃离心10min，去上清，尽量吸干管底的液体。开盖室温晾干管底的RNA沉淀，加入50μL无酶水溶解，使用超微量紫外可见分光光度计检测RNA的纯度及浓度。
cDNA逆转录：使用Ta Ka Ra Prime ScriptTMRT reagent Kit with g DNA Eraser试剂盒分别将RNA反转录成cDNA，在反转录步骤之前去除基因组DNA(gDNA)使得 结果更加准确可信。在冰上配制总RNA反转录反应体系：1μL Prime Script RT Enzyme MixⅠ，1μL RT Primer Mix，4μL 5×Prime Script Buffer2，4μL RNase Free dH2O。配制好反应混合液后，置于37℃反应15min，之后置入85℃ 5sec终止反应，随后于4℃保存待用。
RT-PCR操作：该检测方法为SYBR Green染料法，无需探针。具体如下，根据不同样品的cDNA为模板进行Real-time PCR反应。在冰上配制反应液：5μL SYBR Premix Dimer Eraser(2×)，0.3μL PCR Forward引物(10μM)，0.3μL PCR Reverse引物(10μM)，0.2μL ROX Reference DyeⅡ(50×)，1μL上一步得到的cDNA模板和3.2μL dH2O。在孔板中进行加样，每孔10μL，加样完成后进行离心(1000rpm,5min)消除液体挂壁及反应液中的气泡。使用ABI ViiA7实时荧光定量PCR仪进行Real-time PCR反应检测，反应程序为95℃，30sec(1cycle)→95℃，5sec；55℃,30sec；72℃,30sec(40cycles)→60℃-95℃，2min(1cycle)。实验重复三次，取平均值得到每组Ct值，计算实验组与对照组的表达差异倍数。对照基因为GAPDH。RT-PCR引物如下：①Bcl-2引物：forward：5’-AGGATTGTGGCCTTCTTTGAG-3’，reverse：5’-AGACAGCCAGGAGAAATC AAAC-3’；②PLK1引物：forward：5’-ACCAGCACGTCGTAGGATTC-3’，reverse：5’-CAAGCAATTTGCCGTAGG-3’；③Gal-1引物：forward：5’-CAATCAT GGCCTGTGGTCTG-3’，reverse：5’-GTG TAGGCACAGGTTGTTGCTG-3’。④GAPDH引物：forward：5’-TCAGGGGTTTCACATTTGGCA-3’，reverse：5’-GG AGCGGAA AACCA-3’。各目的基因表达水平用RQ值(2-ΔΔCT)来表示。公式如下：
Fold Change＝2–ΔΔCt
其中，ΔΔCt＝ΔCt实验组–ΔCt对照组，ΔCt＝Ct目的基因-Ct内参基因
基因沉默效率的计算方法：基因沉默效率的计算方法：100％-实验组基因表达水平/对照组基因表达水平。
结果分析：如图1-13，1-14，1-15所示(其中，scr siRNA为随机对照序列)，三种siRNA@MPP均能明显干扰其对应的靶基因。Bcl-2-siRNA@MPP对靶基因Bcl-2的抑制率达到76％；PLK1-siRNA@MPP对靶基因PLK1的抑制率达到86％；Gal-1-siRNA@MPP对靶基因Gal-1的抑制率达到73％。结果提示，siRNA@MPP可以携带任意siRNA进行靶基因的干预治疗，发挥载siRNA药物、疫苗或其他产品的作用。
实施例3.6.2、金属离子为Al3+时的siRNA-金属-磷脂复合物颗粒(siRNA-loaded metal-chelated phospholipid complex nanoparticles，siRNA@MPP)的制备及效果
本实施例与实施例3.6.1的区别在于，将实施例3.6.1中的金属离子Fe3+替换为Al3+。
结果分析：如图1-16，1-17，1-18所示，三种siRNA@MPP均能明显干扰其对应的靶基因。Bcl-2-siRNA@MPP对靶基因Bcl-2的抑制率达到81％；PLK1-siRNA@MPP对靶基因PLK1的抑制率达到90％；Gal-1-siRNA@MPP对靶基因Gal-1的抑制率达到79％。结果提示，siRNA@MPP可以携带任意siRNA进行靶基因的干预治疗，发挥载siRNA药物、疫苗或其他产品的作用。
实施例3.7、ASO-金属-磷脂复合物颗粒(ASO-loaded metal-chelated phospholipid complex nanoparticles，ASO@MPP)的制备及效果
实施例3.7.1、金属离子为Fe3+时的ASO-金属-磷脂复合物颗粒(ASO-loaded metal-chelated phospholipid complex nanoparticles，ASO@MPP)的制备及效果
将实施例3中的mRNA替换为ASO，参照实施例3的方法分别制备三种含不同ASO的ASO@MPP。三种不同ASO靶向的基因、序列及其对应的随机对照序列分别为：①靶向STAT3基因的ASO(STAT3-ASO)序列为SEQ ID No.10(17nt)，其随机对照序列为SEQ ID NO.11(18nt)；②靶向α-syn基因的ASO(α-syn-ASO)序列为SEQ ID NO.12 (16nt)，其随机对照序列为SEQ ID NO.13(16nt)；③靶向Bcl-2基因的ASO(Bcl-2-ASO)序列为SEQ ID NO.14(18nt)，其随机对照序列为SEQ ID NO.15(20nt)。其余ASO-金属-磷脂复合物颗粒的制备过程与实施例3相同。将不同的ASO@MPP孵育不同的细胞：靶向STAT3基因的ASO@MPP孵育U251人脑胶质母细胞瘤细胞；靶向α-syn基因的ASO@MPP孵育SH-SY5Y人神经母细胞瘤细胞；靶向Bcl-2基因的ASO@MPP孵育Daudi人淋巴瘤细胞。以每孔1×106细胞密度接种于6孔板中约24h后，每孔细胞与含上述ASO的ASO@MPP(其中ASO的浓度为1μg/mL)分别孵育48小时后，收集细胞，提取细胞总RNA，用RT-PCR技术分别检测目标基因(STAT3、α-syn、Bcl-2)的mRNA表达量，计算ASO@MPP沉默细胞靶基因的能力。
SEQ ID No.10序列(STAT3-ASO的序列)如下：
5’-GCTCCAGCATCTGCTTC-3’(17nt)。
SEQ ID No.11序列(STAT3-ASO的随机对照序列)如下：
5’-GAAGCAGCAGATGCTGGA-3’(18nt)。
SEQ ID No.12序列(α-syn-ASO的序列)如下：
5’-GCTCCCTCCACTGTCT-3’(16nt)。
SEQ ID No.13序列(α-syn-ASO的随机对照序列)如下：
5’-ACTCCCGAACCTGTCT-3’(16nt)。
SEQ ID No.14序列(Bcl-2-ASO的序列)如下：
5’-TCTCCCAGCGTGCGCCAT-3’(18nt)。
SEQ ID No.15序列(Bcl-2-ASO的随机对照序列)如下：
5’-CAGCGTGCGCCATCCTTCCC-3’(20nt)。
细胞培养：①U251人脑胶质母细胞瘤细胞在高糖(4.5g/L)DMEM+10％胎牛血清(FBS)、1％青霉素/链霉素和2mml-谷氨酰胺(Bio Industries)的培养基中单层生长，且在37℃、5％CO2条件下培养，每周传代2次；②SH-SY5Y人神经母细胞瘤细胞在高糖(4.5g/L)DMEM+10％胎牛血清(FBS)、1％青霉素/链霉素和2mml-谷氨酰胺(Bio Industries)的培养基中单层生长，且在37℃、5％CO2条件下培养，每周传代2次；③Daudi人淋巴瘤细胞在RPMI 1640+10％胎牛血清(FBS)、1％青霉素/链霉素和2mml-谷氨酰胺(Bio Industries)的培养基中生长，且在37℃、5％CO2条件下培养，每周传代2次。
RT-PCR具体流程：
总RNA的提取：将六孔板弃去培养基，PBS缓冲液漂洗3遍，每孔加入1mL Trizol对细胞进行裂解处理。加入200μL氯仿，充分摇匀，室温静置10min，13000rpm 4℃离心15min，得到分层的三相液体，其中RNA溶解在上层的水相中。吸取上层水相置于新的无酶1.5ml离心管中，加入500μL异丙醇，室温静置10min，13000rpm 4℃离心15min，得到RNA沉淀。去上清，每管加入1mL以无RNA酶水新配置的75％(v/v)乙醇，小心吹打，吹起管底RNA白色沉淀，7500rpm 4℃离心10min，去上清，尽量吸干管底的液体。开盖室温晾干管底的RNA沉淀，加入50μL无酶水溶解，使用超微量紫外可见分光光度计检测RNA的纯度及浓度。
cDNA逆转录：使用Ta Ka Ra Prime ScriptTMRT reagent Kit with g DNA Eraser试剂盒分别将RNA反转录成cDNA，在反转录步骤之前去除基因组DNA(gDNA)使得结果更加准确可信。在冰上配制总RNA反转录反应体系：1μL Prime Script RT Enzyme MixⅠ，1μL RT Primer Mix，4μL 5×Prime Script Buffer 2，4μL RNase Free dH2O。配制好反应混合液后，置于37℃反应15min，之后置入85℃5sec终止反应，随后于4℃保存待用。
RT-PCR操作：该检测方法为SYBR Green染料法，无需探针。具体如下，根据不同样品的cDNA为模板进行Real-time PCR反应。在冰上配制反应液：5μL SYBR Premix Dimer Eraser(2×)，0.3μL PCR Forward引物(10μM)，0.3μL PCR Reverse引物(10μM)， 0.2μL ROX Reference DyeⅡ(50×)，1μL上一步得到的cDNA模板和3.2μL dH2O。在孔板中进行加样，每孔10μL，加样完成后进行离心(1000rpm,5min)消除液体挂壁及反应液中的气泡。使用ABI ViiA7实时荧光定量PCR仪进行Real-time PCR反应检测，反应程序为95℃，30sec(1cycle)→95℃，5sec；55℃,30sec；72℃,30sec(40cycles)→60℃-95℃，2min(1cycle)。实验重复三次，取平均值得到每组Ct值，计算实验组与对照组的表达差异倍数。对照基因为GAPDH。RT-PCR引物序列如下：①STAT3引物：forward：5’-TGATCACCTTTGAGACCGAGG-3’，reverse：5’-GATCACCACAACTGG CAA GG-3’；②α-syn引物：forward:5’-TGACGGGTGTGACAGCAGTAG-3’，reverse:5’-CAGTGGCTGCTGCAATG-3’；③Bcl-2引物：forward：5’-AGGATT GTG GCCTTCTTTGAG-3’，reverse：5’-AGACAGCCAGGAGAAATCAAAC-3’④GAPDH引物：forward：5’-TCAGGGG TTTCACATTTGGCA-3’，reverse：5’-GGAGCGGAA AACCA-3’。各目的基因表达水平用RQ值(2-ΔΔCT)来表示。公式如下：
Fold Change＝2–ΔΔCt
其中，ΔΔCt＝ΔCt实验组–ΔCt对照组，ΔCt＝Ct目的基因-Ct内参基因
基因沉默效率的计算方法：100％-实验组基因表达水平/对照组基因表达水平。
结果分析：如图1-19,1-20,1-21所示(其中，scr ASO为随机对照序列)，三种ASO@MPP均能明显干扰其对应的靶基因，其中STAT3-ASO@MPP对靶基因STAT3的抑制率达到75％；α-syn-ASO@MPP对靶基因α-syn的抑制率达到71％；Bcl-2-ASO@MPP对靶基因Bcl-2的抑制率达到66％。结果提示，ASO@MPP可以携带任意ASO进行靶基因的干预治疗，发挥载ASO药物、疫苗或其他产品的作用。
实施例3.7.2、金属离子为Al3+时的ASO-金属-磷脂复合物颗粒(ASO-loaded metal-chelated phospholipid complex nanoparticles，ASO@MPP)的制备及效果
本实施例与实施例3.7.1的区别在于，将实施例3.7.2中的金属离子Fe3+替换为Al3+。
结果分析：如图1-22,1-23,1-24所示，三种ASO@MPP均能明显干扰其对应的靶基因，其中STAT3-ASO@MPP对靶基因STAT3的抑制率达到79％；α-syn-ASO@MPP对靶基因α-syn的抑制率达到80％；Bcl-2-ASO@MPP对靶基因Bcl-2的抑制率达到74％。结果提示，ASO@MPP可以携带任意ASO进行靶基因的干预治疗，发挥载ASO药物、疫苗或其他产品的作用。
实施例3.8、制备药物(不同种类核酸)-金属-磷脂复合物颗粒及其效果
实施例3.8.1、制备金属离子为Fe3+时的药物(不同种类核酸)-金属-磷脂复合物颗粒及其效果
将实施例3中的mRNA分别替换为双链的RNA(siRNA)、单链的DNA(ASO)、单链的RNA(mRNA)、双链DNA、单链DNA。不同种类的核酸序列为：①双链的RNA(Bcl-2-siRNA)的序列为SEQ ID NO.4(反义链)和SEQ ID No.21(正义链)(19bp)，其随机对照序列为SEQ ID NO.5(反义链)和SEQ ID No.22(正义链)(19bp)；②单链的DNA(STAT3-ASO)的序列为SEQ ID NO.10(17nt)，其随机对照序列为SEQ ID NO.11(18nt)；③单链的RNA(编码野生型新冠病毒S蛋白的mRNA)序列为SEQ ID No.16(3822nt)；④双链DNA(dsDNA)的序列为SEQ ID NO.17(反义链)和SEQ ID NO.25(正义链)(22bp)(其序列3’端用荧光探针Cy3标记)；⑤单链DNA(ssDNA)的序列为SEQ ID NO.18(22nt)(其序列3’端用荧光探针Cy3标记)。参照实施例3的方法分别制备包载上述不同种类核酸的药物-金属-磷脂复合物颗粒(Bcl-2-siRNA@MPP、STAT3-ASO@MPP、S-mRNA@MPP、dsDNA@MPP、ssDNA@MPP)，其余药物-脂质颗粒的制备过程与实施例3相同。
双链DNA的序列如下：
Antisense：5’-TAGCTTATCAGACTGATGTTGA-3’(SEQ ID No.17)；
Sence:5’-TCAACATCAGTCTGATAAGCTA-3’(SEQ ID No.25)(22bp)。
SEQ ID No.18序列(单链DNA的序列)如下：
5’-TCAACATCAGTCTGATAAGCTA-3’(22nt)。
U251细胞以每孔1×105细胞密度接种于12孔板中约24h后，每孔细胞与siRNA@MPP(其中siRNA的浓度为2μg/mL)或ASO@MPP(其中ASO的浓度为2μg/mL)分别孵育72小时后，收集细胞，提取细胞总RNA，用RT-PCR技术分别检测目标基因(Bcl-2、STAT3)的mRNA表达量，计算siRNA@MPP或ASO@MPP沉默细胞靶基因的能力，结果如实施例3.6中图1-13及实施例3.7中图1-19所示。
将S-mRNA@MPP按2μg/mL的浓度(所含mRNA的浓度)与293T细胞孵育，对照组用MPP孵育，24h后，离心后上清液-20℃冻存备用；细胞沉淀加100μL PBS缓冲溶液重悬后冻融2次并超声10min后离心取上清液，利用市售新冠病毒S蛋白ELISA检测试剂盒检测细胞上清和细胞裂解液两者S蛋白的表达水平，结果如图1-25所示。
将ds-DNA@MPP按100nM的浓度(所含DNA的浓度)孵育A549肺癌细胞2小时后去掉剩余的药物-脂质颗粒，用PBS洗涤细胞两次后，用Hochest33342染料染细胞核3分钟后去染料，用PBS洗涤细胞2次，用高内涵成像系统观察细胞，并计算药物-脂质颗粒转染DNA的效率，结果见图1-26。
ss-DNA@MPP按200nM的浓度(所含DNA的浓度)孵育HT22小鼠海马神经元2小时后去掉剩余的药物-脂质颗粒，用PBS洗涤细胞两次后，用高内涵成像系统观察细胞，并计算药物-脂质颗粒转染DNA的效率，结果见图1-26。
人脑胶质母细胞瘤U251细胞的培养方法同实施例3.6。
293T细胞的培养方法同实施例3。
HT22小鼠海马神经元的培养方法：用含10％FBS和1％青霉素—链霉素的DMEM培基培养在37℃，5％CO2的条件下。
RT-PCR的方法同实施例3.6。
ELISA检测S蛋白的表达水平：将实施例3.5中ELISA检测RBD的方法中的“抗RBD抗体工作液”换成“抗S蛋白抗体工作液”，其余步骤同实施例3.5。
基因沉默效率的计算方法同实施例3.6。
转染效率的计算方法：用高内涵成像系统随机选取3-5个视野，获得普通光源下的细胞形态、同视野下激发/发射光为550nm/570nm(标记的DNA的荧光染料Cy3的激发光)时的荧光信号、激发/发射光为352nm/461nm(标记细胞核的荧光染Hoechst33342的激发光)时的荧光信号，计算随机选取的视野内细胞内有Cy3荧光信号的细胞数占相同视野内细胞内有Hochest33342荧光信号的细胞数的比例，即为转染效率。
结果分析：如实施例3.6.1中图1-13所示，药物(双链RNA)-金属-磷脂复合物颗粒(Bcl-2-siRNA@MPP)对靶基因Bcl-2的抑制率达到76％；如实施例3.7.1中图1-19药物(单链DNA)-金属-磷脂复合物颗粒(STAT3-ASO@MPP)对靶基因STAT3的抑制率达到75％；如图1-25所示，转染了药物(单链RNA)-金属-磷脂复合物颗粒(S-mRNA@MPP)的293T细胞的上清中S蛋白表达水平为161.3ng/mL，而转染了空载体MPP的293T细胞上清中S蛋白含量为0；药物(双链DNA)-金属-磷脂复合物颗粒(dsDNA@MPP)转染双链DNA入细胞的效率为100％(图1-26)；药物(单链DNA)-金属-磷脂复合物颗粒(ssDNA@MPP)转染单链DNA入细胞的效率为100％(图1-26)。结果提示，药物-金属-磷脂复合物颗粒可以包载任意核酸(双链的RNA、单链的RNA、双链DNA、单链DNA)并实现其功能，其中核酸的长度变化范围从16-3822nt。
实施例3.8.2、制备金属离子为Al3+时的药物(不同种类核酸)-金属-磷脂复合物颗粒及其效果
本实施例与实施例3.8.1的区别在于，将实施例3.8.1中的金属离子Fe3+替换为Al3+。
结果分析：如实施例3.6.2中图1-16所示，药物(双链RNA)-金属-磷脂复合物颗粒(Bcl-2-siRNA@MPP)对靶基因Bcl-2的抑制率达到81％；如实施例3.7.2中图1-22药物(单链DNA)-金属-磷脂复合物颗粒(STAT3-ASO@MPP)对靶基因STAT3的抑制率达到79％；如图1-27所示，转染了药物(单链RNA)-金属-磷脂复合物颗粒(S-mRNA@MPP)的293T细胞的上清中S蛋白表达水平为178.7ng/mL，而转染了空载体MPP的293T细胞上清中S蛋白含量为0；药物(双链DNA)-金属-磷脂复合物颗粒(dsDNA@MPP)转染双链DNA入细胞的效率为100％(图1-28)；药物(单链DNA)-金属-磷脂复合物颗粒(ssDNA@MPP)转染单链DNA入细胞的效率为100％(图1-28)。结果提示，药物-金属-磷脂复合物颗粒可以包载任意核酸(双链的RNA、单链的RNA、双链DNA、单链DNA)并实现其功能，其中核酸的长度变化范围从16-3822nt。
实验例二、药物-金属-磷脂复合物颗粒的性能表征
实施例4、金属-磷脂复合物的合成表征
实施例4.1、金属离子为Fe3+时的金属-磷脂复合物的合成表征
DSPC与姜黄素连接表征方法为差示扫描量热法，测定条件为：称取3～5mg的检测物，升温速率为10℃/min，升温范围30～300℃，分别对姜黄素、DSPC、磷脂复合物进行扫描，根据得到的数据绘制曲线，结果见图2-1。从图谱中可以看出，姜黄素具有晶格结构，在185℃时有明显的熔融峰出现；DSPC是一种混合物，曲线上有多处凹陷，可能是不同组分在不同温度条件下表现出热量变化；磷脂复合物中姜黄素熔变峰附近没有峰出现，基本上为一条直线，说明姜黄素与DSPC发生结合，以无定型形式存在，证明磷脂复合物制备成功。
磷脂复合物与Fe3+连接表征为分光光度法：如图2-2所示，磷脂复合物(CUR-HSPC)与Fe3+结合后，其最大吸收波长从420nm偏移至375nm，磷脂复合物的共轭结构发生改变，证明Fe3+成功与姜黄素络合。
实施例4.2、金属离子为Al3+时的金属-磷脂复合物的合成表征
本实施例与实施例4.1的区别在于，磷脂复合物与Al3+连接表征为分光光度法：如图2-3所示，磷脂复合物(CUR-HSPC)与Al3+结合后，其最大吸收波长从420nm偏移至433nm，磷脂复合物的共轭结构发生改变，证明Al3+成功与姜黄素络合。
实施例5、金属离子为Fe3+时的在低pH值条件下Fe3+从金属-磷脂复合物中脱落的表征
金属-磷脂复合物中的磷脂复合物通过配位键结合Fe3+，在溶酶体的低pH值条件下，磷脂复合物与Fe3+之间的配位键会发生质子化(吸收氢离子)而断裂。为证明金属-磷脂复合物中的Fe3+确实是通过上述机制从脂复合物中脱落，我们设计了以下实验：分别在生理pH值(pH＝7.4)及溶酶体低pH值(pH＝5.0)条件下，观察金属-磷脂复合物的颜色。如图2-4所示，金属-磷脂复合物在溶酶体低pH值(pH＝5.0)条件下从棕红色变成了亮黄色，提示Fe3+已从复合物上脱落。结果提示：在溶酶体的低pH值条件下，Fe3+可从金属-磷脂复合物中脱落。
在低pH值条件下Fe3+从金属-磷脂复合物中脱落的的原理是：姜黄素与Fe3+之间的配位键在低pH值条件下(pH＝5.0)发生质子化，即姜黄素会从溶液中结合大量质子(H+)，导致Fe3+与姜黄素之间的配位健断裂，从而使得Fe3+与姜黄素分离，最终导致Fe3+从金属-磷脂复合物中分离出来(图2-4)。
实施例6、药物-金属-磷脂复合物颗粒中MPP的元素分析
实施例6.1、金属离子为Fe3+时的药物-金属-磷脂复合物颗粒中MPP的元素分 析
将实施例3中的mRNA替换为巯基修饰的siRNA，参照实施例3的方法制备药物-金属-磷脂复合物颗粒siRNA@MPP。用透射电镜仪器进行元素分析。结果如图2-5所示：C、N、O、P为共有元素，Fe元素分析图显示Fe3+均匀地分布在脂质纳米颗粒上，因siRNA上修饰了巯基，所以S元素分析图可以特异性表示siRNA的位置，由图可知siRNA很好地络合在Fe3+的附近，证明药物-脂质纳米颗粒成功包载了siRNA。
实施例6.2、金属离子为Al3+时的药物-金属-磷脂复合物颗粒中MPP的电镜分析
参照实施例3的方法制备药物-金属-磷脂复合物颗粒MPP。用透射电镜仪器进行形态学分析。结果如图2-6所示：药物-金属-磷脂复合物颗粒中MPP的形态为标准球状，粒径均一，大小在100nm左右。
实施例7、金属离子为Fe3+或为Al3+时的金属-磷脂复合物颗粒MPP包载核酸(siRNA及mRNA)的效率及其与LNP的对比。
将实施例3中的mRNA分别替换为靶向Bcl-2基因的siRNA(SEQ ID No.4，19bp)，及编码新型冠状病毒S1亚基的受体结合域(receptor binding domain，RBD)的mRNA(SEQ ID No.2，669nt)，分别制备包载核酸的药物-金属-磷脂复合物颗粒siRNA@MPP和mRNA@MPP，其余药物-金属-磷脂复合物颗粒的制备过程与实施例3相同。
按实施例3.6中siRNA@MPP及实施例3.5中mRNA@MPP相同的载药量制备siRNA@LNP及mRNA@LNP，具体方法为：根据Onpattro脂质纳米颗粒配方配制有机相溶液，即将可电离脂质ALC0315、DSPE-PEG2000、DSPC和胆固醇按照50％：1.5％：10％：38.5％的摩尔比例溶于乙醇中。将Bcl-siRNA或RBD-mRNA加入水相(0.1M、pH＝4.0的醋酸-醋酸钠缓冲溶液)中。其中，氨基脂与含磷酸核苷酸的比例(N/P)为6：1，同时确保核酸载药量与上述siRNA@MPP和mRNA@MPP的核酸载药量相同，将水相与有机相按3:1的体积比在14mL/min的流速下快速混合。混合后，用无酶PBS缓冲溶液稀释十倍，并使用100kDa超滤管将混合液浓缩至十分之一，反复稀释、浓缩操作3次后，使混合液中乙醇浓度减少至0.0005％以下，溶液pH值升高至PBS缓冲溶液的正常pH值(7.2～7.4)，即分别制得siRNA@LNP及mRNA@LNP。
使用琼脂糖凝胶电泳法分别检测siRNA@MPP、mRNA@MPP、siRNA@LNP及mRNA@LNP对核酸(siRNA及mRNA)的包载率。包载率的测定方法为：每组脂质纳米颗粒的核酸(siRNA及mRNA)投料量均定为10μg/mL，脂质与核酸的质量比为40:1，将核酸溶于PBS缓冲溶液中，作为阳性对照组，阴性对照为无核酸的PBS缓冲溶液。琼脂糖凝胶的浓度为1.5％，此时胶的空隙只允许游离核酸通过而不允许脂质纳米颗粒通过，当游离核酸条带电泳至可清晰分辨时停止电泳，防止电泳时间过长核酸降解。用Image J软件统计不同组别游离核酸的灰度值，阳性对照组定为100％，每组的游离核酸相对于阳性对照的比值为游离核酸相对量，则每组包载率为(100-游离核酸相对量)％。
结果分析：如图2-7所示，MPP(Fe3+)包载siRNA及mRNA的效率分别为85.86％及87.11％；MPP(Al3+)包载siRNA及mRNA的效率分别为89.73％及92.23％；LNP包载siRNA及mRNA的效率分别为84.98％及79.12％。结果提示MPP与LNP包载核酸的效率无明显差异。
实施例8、金属离子为Fe3+或为Al3+时的金属-磷脂复合物颗粒MPP的核酸溶酶体逃逸能力及其与LNP的对比
将实施例3.6的Bcl-2-siRNA(SEQ ID No.4)替换为Cy5标记的Bcl-2-siRNA，制备Cy5-siRNA@MPP(所含siRNA的浓度为100nM)，将实施例7的Bcl-2-siRNA(SEQ  ID No.4)替换为Cy5标记的Bcl-2-siRNA，制备Cy5-siRNA@LNP(所含siRNA的浓度为100nM)；将实施例3.5的eGFP-mRNA(SEQ ID No.1)替换为Cy5标记的eGFP-mRNA，制备Cy5-mRNA@MPP(所含mRNA的浓度为2μg/mL)，将实施例7的RBD-mRNA替换为Cy5标记的RBD-mRNA，制备Cy5-mRNA@LNP(所含mRNA的浓度为2μg/mL)，将它们分别与细胞溶酶体探针Lysotracker Green共同孵育A549细胞3小时后使用高内涵成像系统观察Cy5的荧光信号(红色)与Lysotracker Green荧光信号(绿色)重叠的情况，判断探讨所述药物-脂质颗粒促核酸溶酶体逃逸的能力。
药物-金属-磷脂复合物颗粒促核酸溶酶体逃逸能力的判定标准：药物-脂质纳米颗粒孵育细胞3小时后，使用高内涵成像系统观察Cy5的荧光信号(红色)与Lysotracker Green荧光信号(绿色)重叠的情况，并使用imageJ软件统计红色荧光信号与绿色荧光信号的重叠率。当药物-金属-磷脂复合物颗粒孵育细胞3小时后，红色荧光信号与绿色荧光信号的重叠率低于50％提示核酸能较快地从细胞溶酶体中逃逸出来，其脂质纳米颗粒具有较好的促核酸溶酶体逃逸能力。
结果分析：如图2-8所示，当Cy5-siRNA@MPP(Fe3+)和Cy5-mRNA@MPP(Fe3+)孵育A549细胞3小时后，红色荧光信号与绿色荧光信号的重叠率分别为36.05％和43.07％，即溶酶体逃逸率分别为63.95％和56.93％；当Cy5-siRNA@MPP(Al3+)和Cy5-mRNA@MPP(Al3+)孵育A549细胞3小时后，红色荧光信号与绿色荧光信号的重叠率分别为32.39％和40.17％，即溶酶体逃逸率分别为67.61％和59.83％；而Cy5-siRNA@LNP和Cy5-mRNA@LNP孵育A549细胞3小时后，红色荧光信号与绿色荧光信号的重叠率分别为76.89％和86.87％，即溶酶体逃逸能力分别为23.11％和13.13％。提示所述药物-脂质纳米颗粒MPP具有较好的促核酸溶酶体逃逸能力，且MPP促溶酶体逃逸的能力明显强于LNP。
实施例9、金属离子为Fe3+或为Al3+时的金属-磷脂复合物颗粒MPP促进核酸表达的能力及其与LNP的对比
将实施例7中的RBD-mRNA替换为编码荧光蛋白eGFP的mRNA，其余制备方法同实施例7，获得eGFP-mRNA@LNP。
将实施例3制备的eGFP-mRNA@MPP和上述eGFP-mRNA@LNP(所含mRNA的浓度为2μg/mL)分别与293T细胞孵育，对照组用MPP或LNP孵育，48h后收取细胞悬液，通过流式细胞术检测eGFP阳性细胞百分比。
流式细胞术方法分析eGFP阳性细胞率的方法如实施例3所述。
结果分析：如图2-9所示，MPP(Fe3+)、MPP(Al3+)与LNP处理293T细胞后其eGFP阳性细胞百分比分别为97.2％、98.1％和63.03％％。结果提示：MPP促进核酸表达的功能优于LNP。其可能的原因是：如实施例8所述MPP的促核酸发生溶酶体逃逸的能力强于LNP，所以MPP装载的更多的核酸能被有效释放到胞质中，从而被翻译为蛋白质。
实施例10、金属离子为Fe3+或为Al3+时的药物-金属-磷脂复合物颗粒MPP促体液免疫及细胞免疫的能力及其与LNP的对比
将实施例3.5的RBD-mRNA@MPP和实施例7的RBD-mRNA@LNP按2μg/mL的浓度(所含mRNA的浓度)与293T细胞孵育，对照组用MPP孵育，24h后，离心后取上清液于-20℃冻存备用；细胞沉淀加100μL PBS缓冲溶液重悬后冻融2次并超声10min后离心取上清液，利用市售新冠病毒抗原RBD ELISA检测试剂盒检测细胞上清和细胞裂解液两者RBD蛋白的表达水平，结果见图2-10。
ELISA检测RBD表达水平的方法如实施例3.5所述。
将实验动物被随机分成3组(实验组和对照组)，每组5只。动物模型为BALB/c小鼠，每只小鼠在第1天进行第一次肌肉给药，在第14天进行第二次肌肉给药，实验组 分别注射RBD-mRNA@MPP(Fe3+)、RBD-mRNA@MPP(Al3+)或RBD-mRNA@LNP，对照组注射未装载mRNA的MPP和LNP。每次给药的剂量为100μL，其中实验组中RBD-mRNA@MPP(Fe3+)、RBD-mRNA@MPP(Al3+)和RBD-mRNA@LNP制剂各含30mg的mRNA。距第一次给药后第28天收集小鼠血液，分离血清梯度稀释，通过市售ELISA试剂盒检测小鼠体内所产生的抗新型冠状病毒S1亚基的RBD总IgG抗体的滴度，结果如图2-11所示。
ELISA检测抗新型冠状病毒S1亚基的RBD总IgG抗体的滴度的方法如实施例3.5所述。
在给RBD-mRNA@MPP(Fe3+)、RBD-mRNA@MPP(Al3+)和RBD-mRNA@LNP后第28天采集正常小鼠脾脏，在无菌条件下制备成单细胞悬液，按照100000脾细胞/孔铺板于细胞孔板中，加入终浓度为10mg/mL的RBD蛋白培养48h，离心去上清，通过ELISA试剂盒测定IFN-γ、IL-2、IL-4的表达水平，结果如图2-12所示。
ELISA检测IFN-γ、IL-2、IL-4表达水平的方法如实施例3.5所述。
结果分析：如图2-10所示，RBD-mRNA@MPP(Fe3+)、RBD-mRNA@MPP(Al3+)和RBD-mRNA@LNP均能诱导293T细胞表达一定量的RBD，但RBD-mRNA@MPP(Al3+)诱导细胞表达RBD的能力明显强于RBD-mRNA@MPP(Fe3+)、RBD-mRNA@MPP(Fe3+)诱导细胞表达RBD的能力明显强于RBD-mRNA@LNP：RBD-mRNA@MPP(Fe3+)处理组细胞上清中RBD的表达量为205ng/mL，RBD-mRNA@MPP(Al3+)处理组细胞上清中RBD的表达量为230ng/mL，RBD-mRNA@LNP处理组细胞上清中RBD的表达量为115.7ng/mL。如图2-11结果所示，RBD-mRNA@MPP有效诱导小鼠的体液免疫，产生高水平的抗原特异性结合抗体，且RBD-mRNA@MPP(Al3+)诱导小鼠的体液免疫的能力明确优于RBD-mRNA@MPP(Fe3+)，RBD-mRNA@MPP(Fe3+)诱导小鼠的体液免疫的能力明确优于RBD-mRNA@LNP：RBD-mRNA@MPP(Fe3+)处理组小鼠体内IgG抗体滴度达117233.8；RBD-mRNA@MPP(Al3+)处理组小鼠体内IgG抗体滴度达133116；而RBD-mRNA@LNP处理组小鼠体内IgG抗体滴度达仅为67476。如图2-12所示，RBD-mRNA@MPP(Al3+)能有效诱导小鼠的细胞免疫，即激活免疫细胞并产生大量的细胞因子，且mRNA@MPP(Al3+)诱导小鼠的细胞免疫的能力明确优于RBD-mRNA@MPP(Fe3+)，RBD-mRNA@MPP(Fe3+)诱导小鼠的细胞免疫的能力明确优于RBD-mRNA@LNP：RBD-mRNA@MPP(Fe3+)使细胞因子IFN-γ、IL-2、IL-4的表达量分别达256.8pg/mL、207.6pg/mL、61.8pg/mL；RBD-mRNA@MPP(Al3+)使细胞因子IFN-γ、IL-2、IL-4的表达量分别达298pg/mL、249pg/mL、74.6pg/mL；而RBD-mRNA@LNP使细胞因子IFN-γ、IL-2、IL-4的表达量仅为104.2pg/mL、79.2pg/mL、27pg/mL。结果提示，mRNA@MPP(Al3+)递送任意mRNA并实现其功能的能力明显优于RBD-mRNA@MPP(Fe3+)，RBD-mRNA@MPP(Fe3+)诱导小鼠的细胞免疫的能力明确优于RBD-mRNA@LNP：RBD-mRNA@MPP能更有效的促进细胞表达目的蛋白，能更有效的激活体内体液免疫和细胞免疫，因此所述药物(mRNA)-脂质颗粒在载mRNA的药物、疫苗或其他产品作用方面明显优于现有技术LNP。其可能的原因是：1)与LNP比，MPP具有更强的促核酸溶酶体逃逸能力；2)与LNP比，MPP具有更强的促核酸表达成蛋白(抗原)的能力；3)与LNP比，MPP中的姜黄素在体内与DSPC分离后，作为一个免疫佐剂(又称免疫调节剂)，即能激活体液免疫和细胞免疫从而增强MPP递送mRNA疫苗的效果，又能抑制免疫因子风暴从而抑制过度的、对机体有害的免疫反应。
实施例11、金属离子为Fe3+或为Al3+时的金属-磷脂复合物颗粒(Metal-chelated phospholipid complex nanoparticles，MPP)的体内安全性评价
以SD大鼠为研究对象，对MPP进行了为期20天的亚慢毒性研究，并设立了为期20天的恢复期。具体实验方法如下：
SPF级SD大鼠(220±20g)56只，雌雄各半，饲养于温度为25℃，湿度为45％ -55％，光照12h的环境中。适应性饲养3～5天后按性别随机分组：实验组32只，恢复组24只。空白对照组(Control)14只(含实验组8只和恢复组6只)，雌雄各半；低剂量MPP组(8mg/kg)14只(含实验组8只和恢复组6只)，雌雄各半；中等剂量MPP组(16mg/kg)14只(含实验组8只和恢复组6只)，雌雄各半；高剂量组(32mg/kg)14只(含实验组8只和恢复组6只)，雌雄各半。实验组(共32只)在给药结束后解剖取材，恢复组(共24只)在给药结束后，继续正常饲养20天后解剖取材。
给药方法：实验动物通过尾静脉注射给药，每2天给药一次，共给药20天，每周记录一次SD大鼠体重。将制备的MPP溶于DPBS，对照组注射等量DPBS，低剂量MPP组、中剂量MPP组、高剂量MPP组分别注射8mg/kg、16mg/kg、32mg/kg的MPP。
设置上述MPP给药剂量的依据：包载200μg/kg mRNA(mRNA动物实验的实际需要量)时，空载体MPP的需要量为8mg/kg。为充分证明MPP的安全性，我们选取了MPP动物实验实际需要量的1、2及4倍给药，即8mg/kg、16mg/kg及32mg/kg。
一般指标检测方法：每次给药后观察各组动物的一般状态，包括存活情况、饮食情况、外观特征、行为活动、体重以及是否有给药局部反应。解剖时进行大体尸检，包括及时称量主要脏器湿重，如脑、心、肝、脾、肺、肾，计算脏体比并记录各脏器的病理改变。其中脏体比＝大鼠脏器湿重/大鼠体重×100％。
SD大鼠全血及血清的获取及保存：在给药20天后以及20天恢复期后解剖大鼠，腹主动脉采血，即异氟烷麻醉SD大鼠并固定于解剖板上，75％乙醇消毒腹部，无菌眼科剪剪开大鼠腹部，用棉球轻轻拨开内脏暴露出腹主动脉，采用500μL负压EDTAK2抗凝采血管收集全血，4℃保存，用于血常规检测。采用5mL负压普通采血管收集全血于常温静置30min，于4℃，1500rpm离心15min，取上清于1.5mL离心管中，-20℃保存，用于血液生化指标及免疫学相关指标的检测。
血常规检测的方法：血常规指标包括：白细胞数目、淋巴细胞数目、单核细胞数目、中性粒细胞数目、淋巴细胞百分比、单核细胞百分比、中性粒细胞百分比、红细胞数目、血红蛋白、红细胞积压、平均红细胞体积、平均红细胞血红蛋白含量、平均红细胞血红蛋白浓度、红细胞分布宽度变异系数、血小板数目、平均血小板体积、血小板分布宽度、血小板压积。全血标本轻轻颠倒混匀，取少量全血，采用全自动血液细胞分析仪自动分析结果。
血液生化指标检测的方法：血液生化指标包括无机离子(Fe2+，Na+，K+，Cl-，Ca2+)、肝功能指标(ALT，AST，γ-GT，T-BIL，D-BIL，ALP，ALB)、肾功能指标(BUN，UA，CR)、心脏功能指标(LDH，CK)、糖代谢指标(GSP，GLU，INS)、脂代谢指标(CHO，TG，LDL-C，HDL-C)。解冻血清样品3000rpm离心15min取上清分装好待用，在全自动生化仪上设置好相应参数，加入配置好的工作液，再加入待测血清，全自动生化仪自动测定结果。
免疫学相关指标的检测方法：免疫学相关指标包括甲状腺功能指标(TT3,TT4,TSH)、细胞因子(IL-1，IL-2，IL-4，IFN-γ，IFN-α,TNF-α)、免疫球蛋白(IgG，IgA，IgM)、血清补体(C3,CH50)。用ELISA方法检测上述指标。
SD大鼠的主要脏器病理学检查方法：在给药期末和恢复期末，将各组大鼠麻醉后，眼科剪取下大鼠主要脏器，包括全脑、心、肝、脾、肺、肾，用0.9％生理盐水轻轻漂洗，置于4％多聚甲醛固定液中固定，常规石蜡包埋，H&E染色，光学显微镜观察对照组及实验组大鼠各个器官的组织病理学变化。
结果分析：如表2-1所示，在给药期末和恢复期末，与对照组相比，低、中、高剂量MPP(Fe3+)或MPP(Al3+)组大鼠存活良好，饮食情况正常，外观、行为活动正常，给药后未见明显不良反应；与对照组相比，低、中、高剂量MPP组雄性SD大鼠及雌性SD大鼠的体重增长值均无显著性差异；对照组相比，低、中、高剂量MPP组的脏体比均 无显著性差异。
在给药期末和恢复期末，与对照组相比，低、中、高剂量MPP(Fe3+)或MPP(Al3+)组的血常规指标(白细胞数目、淋巴细胞数目、单核细胞数目、中性粒细胞数目、淋巴细胞百分比、单核细胞百分比、中性粒细胞百分比、红细胞数目、血红蛋白、红细胞积压、平均红细胞体积、平均红细胞血红蛋白含量、平均红细胞血红蛋白浓度、红细胞分布宽度变异系数、血小板数目、平均血小板体积、血小板分布宽度、血小板压积)未见异常；与对照组相比，低、中、高剂量MPP组的血液生化指标，包括无机离子(Fe2+，Na+，K+，Cl-，Ca2+)、肝功能指标(ALT，AST，γ-GT，T-BIL，D-BIL，ALP，ALB)、肾功能指标(BUN，UA，CR)、心脏功能指标(LDH，CK)、糖代谢指标(GSP，GLU，INS)、脂代谢指标(CHO，TG，LDL-C，HDL-C)，均未见异常；与对照组相比，低、中、高剂量MPP组的免疫学相关指标包括甲状腺功能指标(TT3,TT4,TSH)、细胞因子(IL-1，IL-2，IL-4，IFN-γ，IFN-α,TNF-α)、免疫球蛋白(IgG，IgA，IgM)、血清补体(C3,CH50)，均未见异常。
在给药期末和恢复期末，与对照组相比，低、中、高剂量MPP(Fe3+)或MPP(Al3+)组大鼠脑组织结构完整，组织染色正常，细胞形态结构完整，无核固缩现象及炎性细胞浸润；心肌组织结构完整，心肌细胞排列整齐、连续、紧密，细胞核清晰可见，未见明显细胞充血，水肿或坏死；肝细胞形态正常，无炎性细胞聚集及坏死；脾脏结构正常，红白髓界限清楚；肺组织结构完整，肺泡大小一致，无明显炎症细胞聚集、浸润；肾脏结构正常。
以上结果提示，长期、大量给SD大鼠注射MPP(Fe3+)或MPP(Al3+)未发现明显的慢性毒性反应，提示MPP的安全性较高。
表2-1 MPP的体内安全性评价


注：ALT，谷丙转氨酶；AST，谷草转氨酶；γ-GT，谷氨酰转肽酶；T-BIL，总胆红素；
D-BIL，直接胆红素；ALP，碱性磷酸酶；ALB，白蛋白；BUN，尿素氮；UA，尿酸；CR，肌酐；LDH，乳酸脱氢酶；CK，肌酸磷酸激酶；GSP，果糖胺；GLU，葡萄糖；INS，胰岛素；CHO，胆固醇；TG，甘油三酯；LDL-C，低密度脂蛋白；HDL-C，高密度脂蛋白；TT3，三碘甲状腺原氨酸；TT4，四碘甲状腺原氨酸；TSH，促甲状腺激素；IL-1，白介素1；IL-2，白介素2；IL-4，白介素4；IFN-γ，干扰素γ；IFN-α，干扰素α；TNF-α，肿瘤坏死因子α；IgG，免疫球蛋白G；IgA，免疫球蛋白A；IgM，免疫球蛋白M；C3，补体C3；CH50总补体CH50
实施例12、金属离子为Fe3+或为Al3+时的金属-磷脂复合物颗粒(MPP)与LNP的体内安全性对比
LNP的主要毒性来自于其主要成分——阳离子脂质和/或可电离脂质。当LNP在体内代谢过程中，游离的阳离子脂质和/或可电离脂质会对机体产生明显的毒性。阳离子脂质和/或可电离脂质对生物细胞的半数致死量(IC50)是评估LNP对机体毒性大小的重要参数。金属-磷脂复合物颗粒(Metal-chelated phospholipid complex nanoparticles，MPP)用金属-磷脂复合物代替了LNP中的阳离子脂质/可电离脂质，因此，我们通过研究金属-磷脂复合物与阳离子脂质/可电离脂质对生物细胞的半数致死量(IC50)，比较LNP与MPP(Fe3+)或MPP(Al3+)的毒性的差异。
将不同浓度的所述金属-磷脂复合物(0、0.1、0.3、0.9、2.7、8.1、24.3、72.9、218.7μM)、阳离子脂质(DOTAP，0、0.1、0.3、0.9、2.7、8.1、24.3、72.9、218.7μM，结构式如下)及可电离脂质(ALC0315，0、0.1、0.3、0.9、2.7、8.1、24.3、72.9、218.7μM，结构式如下)均分别孵育293T细胞48小时后，用CCK8活性检测试剂盒检测细胞活性，分别计算所述金属-磷脂复合物、阳离子脂质(DOTAP)及可电离脂质(ALC0315)对293T细胞的半数致死量IC50。
CCK8的检测方法：
1.细胞培养：用含10％FBS，1％双抗的DMEM培养液培养细胞，待细胞密度至培养瓶的80％-90％待用；
2.用PBS洗净培养瓶中剩余培养基，加入胰酶，迅速将培养瓶转移至37℃含5％CO2的培养箱中。注意观察，待细胞稍稍变圆后，加入培养液终止消化。转移至离心管中，1500RPM离心5min，用新鲜培养基重悬细胞；
3.计数：按照目的将细胞悬液稀释成每1mL 10,0000个细胞，96孔板中每孔100μL，每组至少5个复孔。37℃，5％CO2培养24h后加药；
4.药物孵育48h后，加入含10％CCK8，孵育1-3h，酶标仪450nm测吸光度；
5.存活率(％)＝[A(加药)-A(空白)]/[A(0加药)-A(空白)]×100％。
IC50的计算方法：以存活率为纵坐标，药物浓度为横坐标，运用Graphpad采用[Inhibitor]vs.normalized response--Variable slope分析方法计算IC50。
为比较MPP及LNP的体内安全性，取能载等量核酸(200μg/kg mRNA)的MPP(8mg/kg)及LNP(3.24mg/kg)，按照实施例11方法进行体内实验，评估、对比MPP与LNP的体内毒性。
结果分析：如表2-2所示，金属-磷脂复合物的IC50明显大于阳离子脂质(DOTAP)及可电离脂质(ALC0315)。结果表明，金属-磷脂复合物的毒性明显小于阳离子脂质及可电离脂质。
如表结果2-3所示，在给药期末和恢复期末，与对照组相比，MPP(Fe3+)或MPP(Al3+)组的肝功能(ALT，AST，ALP)及细胞因子(IL-6，IL-1β)均未见明显异常。但与对照组相比，LNP组的肝功能(ALT，AST，ALP)及细胞因子(IL-6，IL-1β)均明显升高。结果提示，MPP(Fe3+)或MPP(Al3+)的体内安全性高于LNP，其原因是：LNP的核心成分为人工合成的“阳离子脂质/可电离脂质”，其细胞毒性及免疫原性较高，且其结构较为稳定，在体内难以分解代谢；而MPP(Fe3+)或MPP(Al3+)的核心成分为金属-磷脂复合物，其金属-磷脂复合物是由磷脂分子、安全性高的天然小分子物质姜黄素(为FDA批准的食品添加剂及药用辅料)及安全的金属离子组成，且其在完成药物递送后已在体内分解为天然分子。综上，因为MPP(Fe3+)或MPP(Al3+)组成成分中无阳离子脂质/可电离脂质，不会引起阳离子脂质/可电离脂质相关的毒副反应，所以MPP(Fe3+)或MPP(Al3+)的安全性高于LNP。
表2-2金属离子为Fe3+或为Al3+时的金属-磷脂复合物与阳离子脂质(DOTAP)及可电离脂质(ALC0315)的IC50与对比
表2-3 MPP及LNP的慢性毒性实验指标对比
实施例三、药物-金属-磷脂复合物颗粒的临床应用及给药途径
实施例13、金属离子为Fe3+或Al3+时的药物-金属-磷脂复合物颗粒的临床应用及给药途径
将实施例3中的mRNA分别替换为靶向B7-H4基因的siRNA(B7-H4-siRNA)及其对照(scr-siRNA)、编码新型冠状病毒S1亚基的受体结合域(receptor binding domain，RBD)的mRNA(RBD-mRNA)。
上述不同核酸的序列为：①B7-H4-siRNA的序列为SEQ ID No.19(正义链)和SEQ ID No.26(反义链)(25bp)，其随机对照序列为SEQ ID No.20(正义链)和SEQ ID No.27(反义链)(19bp)；②编码新型冠状病毒S1亚基的受体结合域(receptor binding domain，RBD)的mRNA序列为SEQ ID No.2(669nt)。参照实施例3的方法分别制备包载上述不同种类核酸的药物-金属-磷脂复合物颗粒(B7-H4-siRNA@MPP(Fe3+)、 RBD-mRNA@MPP(Fe3+)、B7-H4-siRNA@MPP(Al3+)、RBD-mRNA@MPP(Al3+))，其余药物-金属-磷脂复合物颗粒的制备过程与实施例3相同。上述2种不同的药物-金属-磷脂复合物颗粒(B7-H4-siRNA@MPP、RBD-mRNA@MPP)分别用于治疗肝癌作为mRNA疫苗用于预防新型冠状病毒。
B7-H4-siRNA的序列如下：
sense 5’-GGG AGA CAC UCC AUC ACA GUC ACU A-3’(SEQ ID No.19)。
antisense 5’-UAG UGA CUG UGA UGG AGU GUC UCC C-3’(SEQ ID No.26)(25bp)。
B7-H4-siRNA的随机对照序列如下：
sense 5’-UUCUCCGAACGUGUCACGU-3’(SEQ ID No.20)。
antisense 5’-ACGUGACACGUUCGGAGAA-3’(SEQ ID No.27)(19bp)。
为评价B7-H4-siRNA@MPP(Fe3+)和B7-H4-siRNA@MPP(Al3+)治疗肝癌的作用，用HepG2细胞制作肝癌动物模型，待肿瘤大小增加到约100mm3，将肝癌小鼠随机分为7组(每组5只)：PBS对照组、空白载体MPP(Fe3+)组、空白载体MPP(Al3+)组、Scr-siRNA@MPP(Fe3+)对照组、B7-H4-siRNA@MPP(Fe3+)治疗组、Scr-siRNA@MPP(Al3+)对照组、B7-H4-siRNA@MPP(Al3+)治疗组。每组小鼠每3天分别瘤内注射PBS、MPP(Fe3+)、MPP(Al3+)、Scr-siRNA@MPP(Fe3+)、B7-H4 siRNA@MPP(Fe3+)、Scr-siRNA@MPP(Al3+)、B7-H4 siRNA@MPP(Al3+)一次，剂量为200μg siRNA/kg，进行8次注射。每隔3天测量并记录肿瘤体积。结果如图3-1所示。
为评价RBD-mRNA@MPP作为mRNA疫苗预防新冠病毒的作用，实验过程及实验方法如前实施例3.5所示。
ELISA检测方法如实施例3.5所述。
肝癌小鼠模型的建立：收集将HepG2细胞，以1×107/mL的密度在PBS中重新悬浮，接种前置于冰上保存。然后将100μL的细胞悬液注射皮下注射到雌Balb/c裸鼠后腿附近的背部区域，建立肝癌小鼠模型。
结果分析：
如图3-1所示，Scr-siRNA@MPP(Fe3+)、Scr-siRNA@MPP(Al3+)对肝癌HepG2细胞的生长几乎没有抑制效果，而B7-H4-siRNA@MPP(Fe3+)和B7-H4 siRNA@MPP(Al3+)则显示出高效的治疗效果，能有效抑制肝癌肿瘤的生长。结果提示，药物-金属-磷脂复合物颗粒可以包载、递送B7-H4 siRNA，通过抑制目标基因的表达，从而抑制肝癌发展。
如前实施例3.5图1-3,图1-5所示，RBD-mRNA@MPP(Fe3+)使小鼠IgG抗体的表达水平达117268.8(图1-3)，使细胞因子IFN-γ、IL-2、IL-4的表达量分别达252.8pg/mL、207.6pg/mL、56.6pg/mL(图1-5)。RBD-mRNA@MPP(Al3+)使小鼠IgG抗体的表达水平达129113(图1-9)，使细胞因子IFN-γ、IL-2、IL-4的表达量分别达271.8pg/mL、234.6pg/mL、68.4pg/mL(图1-11)。结果提示，RBD-mRNA@MPP能有效诱导小鼠的体液免疫，产生高水平的抗原特异性结合抗体；同时能有效诱导小鼠的细胞免疫，即激活免疫细胞并产生大量的细胞因子。因此，RBD-mRNA@MPP能有效的预防新型冠状病毒的感染。
如图3-1所示，B7-H4-siRNA@MPP采用瘤内注射给药途径能有效治疗肝癌；如实施例3.5图1-3、图1-5、图1-9和图1-11所示，RBD-mRNA@MPP采用肌肉注射给药途径能激活体液免疫及细胞免疫，从而发挥预防新型冠状病毒感染。结果提示：药物-金属-磷脂复合物颗粒可通过多种途径给药。
实施例四：DSPC、姜黄素、Fe3+或Al3+被同类物替换后的功能
实施例15、DSPC、姜黄素、Fe3+或Al3+被同类物替换后的功能
参照实施例1、实施例2及实施例3用DSPC、姜黄素、Fe3+的同类物分别对DSPC、姜黄素、Fe3+进行替代，通过不同的组合分别制备28种不同的eGFP-mRNA@MPP， 其中每种eGFP-mRNA@MPP所含mRNA的浓度均为2μg/mL。DSPC、姜黄素、Fe3+及其同类物的名称及结构如表4-1所示，28种mRNA@MPP中DSPC、姜黄素、Fe3+及其同类物的组合方式如表4-2所示。其中，实施例1中的反应温度为65℃、反应时间为2h，实施例2中的反应温度为60℃、反应时间为2h，其他条件不变。
为对比所述28种不同的eGFP-mRNA@MPP与eGFP-mRNA@LNP的效果，我们参照实施例9制备了包载等量eGFP-mRNA的LNP，得到eGFP-mRNA@LNP。
将上述28种不同的eGFP-mRNA@MPP和上述eGFP-mRNA@LNP(所含mRNA的浓度均为2μg/mL)分别与293T细胞孵育，对照组用MPP或LNP孵育，48h后收取细胞悬液，通过流式细胞术检测eGFP阳性细胞百分比。
流式细胞术方法分析eGFP阳性细胞率的方法如实施例3所述。
LNP的主要毒性来自于其主要成分——阳离子脂质/可电离脂质。当LNP在体内代谢过程中，游离的阳离子脂质/可电离脂质会对机体产生明显的毒性。阳离子脂质/可电离脂质对生物细胞的半数致死量(IC50)是评估LNP对机体毒性大小的重要参数。金属-磷脂复合物颗粒(Metal-chelated phospholipid complex nanoparticles，MPP)用金属-磷脂复合物代替了LNP中的阳离子脂质/可电离脂质，因此，我们通过研究表4-2种28种金属-磷脂复合物与阳离子脂质(DOTAP)/可电离脂质(ALC0315)对生物细胞的半数致死量(IC50)，比较LNP与28种MPP的毒性的差异。
IC50的计算方法如实施例12所述。
结果分析：如表4-3所示，28种不同的eGFP-mRNA@MPP处理293T细胞后其eGFP阳性细胞百分比均明显高于eGFP-mRNA@LNP，其中由DSPC、姜黄素、Fe3+组成的mRNA@MPP的eGFP阳性细胞百分比最高。结果提示：DSPC、姜黄素、Fe3+被其同类物替代后形成的mRNA@MPP的功能不及由DSPC、姜黄素、Fe3+组成的mRNA@MPP，但优于mRNA@LNP的功能，其可能的原因是：如实施例8所述MPP的促核酸发生溶酶体逃逸的能力强于LNP，所以MPP装载的更多的核酸能被有效释放到胞质中，从而被翻译为蛋白质。
上述结果提示，只要满足以下条件，则DSPC、姜黄素、Fe3+被其同类物替代后组成的药物-金属-磷脂复合物颗粒的功能不受影响：①DSPC的同类物为两亲性磷脂分子；②Fe3+的同类物为金属离子；③姜黄素的同类物与DSPC的同类物形成磷脂复合物的同时，又能与金属离子络合；④姜黄素与Fe3+之间的配位键能响应溶酶体的低pH环境而发生断裂。
如表4-3所示，28种金属-磷脂复合物的IC50均明显大于阳离子脂质(DOTAP)及可电离脂质(ALC0315)。提示，金属-磷脂复合物的毒性明确小于阳离子脂质及可电离脂质，即由DSPC、姜黄素、Fe3+及其同类物组成的脂质纳米颗粒(MPP)的安全性高于LNP，其原因是：LNP的核心成分为人工合成的“阳离子脂质/可电离脂质”，其细胞毒性及免疫原性较高，且其结构较为稳定，在体内难以分解代谢；而MPP的核心成分为金属-磷脂复合物，其金属-磷脂复合物是由磷脂分子、安全性高的天然小分子物质(其中姜黄素为FDA批准的食品添加剂及药用辅料)及安全的金属离子组成，且其在完成药物递送后已经在体内分解为天然分子。综上，由DSPC、姜黄素、Fe3+及其同类物组成的脂质颗粒(MPP)的组成成分中无阳离子脂质/可电离脂质，不会引起阳离子脂质/可电离脂质相关的毒副反应，所以MPP的安全性高于LNP。
表4-1 DSPC、姜黄素、Fe3+及其同类物的名称及结构


表4-2 DSPC、姜黄素、Fe3+及其同类物制备的药物-脂质颗粒中金属-磷脂复合物的成组合方式列表及功能

表4-3 DSPC、姜黄素、Fe3+及其同类物制备的金属-磷脂复合物的IC50

实施例16、不同金属-磷脂复合物的DSPC、姜黄素及其同类物、Fe3+组分投放比例及其制备的药物-金属-磷脂复合物颗粒的功能
按照实施例3，制备金属-磷脂复合物，并将姜黄素分别替换为其同类物橙皮素 (1分子橙皮素含4个羟基)，茶多酚(1分子茶多酚含8个羟基)，制备得到三种金属-磷脂复合物(mRNA@MPP1、mRNA@MPP4、mRNA@MPP29)。制备这三种金属-磷脂复合物时DSPC、姜黄素或其同类物、FeCl3的投放比例分别为：1:1:1，1:1:1，1:1:2。并用这三种金属-磷脂复合物(mRNA@MPP1、mRNA@MPP4、mRNA@MPP29)制备的对应的药物-金属-磷脂复合物纳米颗粒。其中mRNA为编码eGFP荧光蛋白的mRNA，其序列为SEQ ID No.1(720nt)。按照实施例3.5所述的实验过程及实验方法检测这4种药物-脂质纳米颗粒的mRNA包载率及其处理293T细胞后促进eGFP荧光蛋白表达的能力。
结果分析：如表4-4显示，依据金属-磷脂复合物组分的化学结构使用不同的投放比例制备的药物-金属-磷脂复合物颗粒的mRNA包载效率及促目的蛋白表达的能力相当。结果提示，金属-磷脂复合物组分的投放比例可根据具体金属-磷脂复合物组分的结构进行调整。投放比例可以调整的依据是：因为DSPC的同类物与姜黄素的同类物靠氢键相连，只要DSPC的同类物含有多个磷酸基团，那么合成磷脂复合物时，DSPC的同类物和姜黄素的同类物的投放比例可以依据DSPC的同类物所含磷酸基团的个数进行调整，即当DSPC的同类物含两个磷酸基团时，DSPC的同类物和姜黄素的同类物的投放比例可调整为1：2；当DSPC的同类物含三个磷酸基团时，DSPC的同类物和姜黄素的同类物的投放比例可调整为1：3；因为姜黄素的同类物的羟基与Fe3+的同类物靠配位键连接，只要姜黄素的同类物含有多个结合位点，那么姜黄素的同类物和Fe3+的同类物的投放比例可以依据姜黄素的同类物所含结合位点的数量进行调整。
表4-4不同金属-磷脂复合物的组分投放比例及其制备的药物-脂质颗粒的功能
实施例17、不同金属-磷脂复合物的DSPC、姜黄素及其同类物、Al3+组分投放比例及其制备的药物-金属-磷脂复合物颗粒的功能
按照实施例3，制备金属-磷脂复合物，并将姜黄素分别替换为其同类物橙皮素(1分子橙皮素含4个羟基)，茶多酚(1分子茶多酚含8个羟基)，制备得到三种金属-磷脂复合物(mRNA@MPP2、mRNA@MPP5、mRNA@MPP30)。制备这三种金属-磷脂复合物时DSPC、姜黄素或其同类物、Al(NO3)3·9H2O的投放比例分别为：1:1:1，1:1:1，1:1:2。并用这三种金属-磷脂复合物(mRNA@MPP1、mRNA@MPP4、mRNA@MPP29)制备的对应的药物-金属-磷脂复合物纳米颗粒。其中mRNA为编码eGFP荧光蛋白的mRNA，其序列为SEQ ID No.1(720nt)。按照实施例3.5所述的实验过程及实验方法检测这4种药物-脂质纳米颗粒的mRNA包载率及其处理293T细胞后促进eGFP荧光蛋白表达的能力。
结果分析：如表4-5显示，依据金属-磷脂复合物组分的化学结构使用不同的投放比例制备的药物-金属-磷脂复合物颗粒的mRNA包载效率及促目的蛋白表达的能力相当。结果提示，金属-磷脂复合物组分的投放比例可根据具体金属-磷脂复合物组分的结构进行调整。投放比例可以调整的依据是：因为DSPC的同类物与姜黄素的同类物靠氢键相连，只要DSPC的同类物含有多个磷酸基团，那么合成磷脂复合物时，DSPC的同类物和 姜黄素的同类物的投放比例可以依据DSPC的同类物所含磷酸基团的个数进行调整，即当DSPC的同类物含两个磷酸基团时，DSPC的同类物和姜黄素的同类物的投放比例可调整为1：2；当DSPC的同类物含三个磷酸基团时，DSPC的同类物和姜黄素的同类物的投放比例可调整为1：3；因为姜黄素的同类物的羟基与Al3+的同类物靠配位键连接，只要姜黄素的同类物含有多个结合位点，那么姜黄素的同类物和Al3+的同类物的投放比例可以依据姜黄素的同类物所含结合位点的数量进行调整。
表4-5不同金属-磷脂复合物的组分投放比例及其制备的药物-脂质颗粒的功能
实施例3中mRNA@MPP制备为中南大学化学化工学院制药工程系王珊教授课题组完成。
除非另有定义，本公开全文所使用的所有技术和科学术语与本公开所属技术领域的技术人员通常理解的含义相同。如有不一致，以本公开全文中所说明的含义或者根据本公开全文中记载的内容得出的含义为准。另外，本说明中所使用的术语只是为了描述本公开实施例的目的，不是旨在限制本公开。
注意，上述仅为本公开的较佳实施例及所运用的技术原理。本领域技术人员会理解，本公开不限于这里所述的特定实施例，对本领域技术人员来说能够进行各种明显的变化、重新调整和替代而不会脱离本公开的保护范围。因此，虽然通过以上实施例对本公开进行了较为详细的说明，但是本公开不仅仅限于以上实施例，在不脱离本公开的技术构思的情况下，还可以包括更多其他等效实施例，均属于本公开的保护范畴。

Claims (82)

  1. 一种金属-磷脂复合物，其中，所述金属-磷脂复合物由磷脂分子部分、连接物分子部分和金属离子部分反应组成，所述磷脂分子部分与所述连接物分子部分相连接，所述连接物分子部分与所述金属离子部分通过配位键连接，且所述金属-磷脂复合物不是阳离子脂质或可电离脂质。
  2. 根据权利要求1所述的金属-磷脂复合物，其中，所述磷脂分子部分选自卵磷脂(PC)、磷脂酰乙醇胺(PE)、磷脂酰丝氨酸(PS)、磷脂酸(PA)、磷脂酰甘油(PG)、1-磷酸神经酰胺(SP)、磷脂酰肌醇(PI)、磷脂酰苏氨酸(PT)、鞘磷脂(SM)、溶血卵磷脂(LPC)、溶血磷酸酰乙醇胺(LPE)、溶血磷脂酰丝氨酸(LPS)、溶血磷脂酸(LPA)、溶血磷脂酰甘油(LPG)、溶血磷脂酰肌醇(LPI)、溶血磷脂酰苏氨酸(LPT)、溶血鞘磷脂(LSM)、1-磷酸鞘氨醇(S1P)，及其衍生物中一种或多种的组合。
  3. 根据权利要求2所述的金属-磷脂复合物，其中，所述磷脂分子部分选自
    卵磷脂(PC)
    磷脂酰乙醇胺(PE)
    磷脂酰丝氨酸(PS)
    磷脂酸(PA)
    磷脂酰甘油(PG)
    1-磷酸神经酰胺(SP)
    磷脂酰肌醇(PI)
    磷脂酰苏氨酸(PT)
    鞘磷脂(SM)
    溶血卵磷脂(LPC)
    溶血磷酸酰乙醇胺(LPE)
    溶血磷脂酰丝氨酸(LPS)
    溶血磷脂酸(LPA)
    溶血磷脂酰甘油(LPG)
    溶血磷脂酰肌醇(LPI)
    溶血磷脂酰苏氨酸(LPT)
    溶血鞘磷脂(LSM)
    1-磷酸鞘氨醇(S1P)及其衍生物中一种或多种的组合；
    其中，R1，R2均独立地为：
    葵酰基
    月桂酰基
    肉豆蔻酰基
    棕榈酰基
    硬脂酰基
    油酰基
    亚油酰基
    芥酰基
    花生酰基
    植烷酰基
  4. 根据权利要求3所述的金属-磷脂复合物，其中，所述磷脂分子部分选自卵磷脂(PC，式1)、磷脂酰乙醇胺(PE，式2)、磷脂酸(PA，式4)、磷脂酰甘油(PG，式5)，及其衍生物中一种或多种的组合。
  5. 根据权利要求4所述的金属-磷脂复合物，其中，所述磷脂分子部分选自DSPC、DSPE、DSPA、DSPG，及其衍生物中一种或多种的组合。
  6. 根据权利要求5所述的金属-磷脂复合物，其中，所述磷脂分子部分选自DSPC(式46)DSPE(式47)DSPA(式48)DSPG(式49) 及其衍生物中一种或多种的组合。
  7. 根据权利要求6所述的金属-磷脂复合物，其中，所述磷脂分子部分选自DSPC(式46)、DSPE(式47)或DSPA(式48)。
  8. 根据权利要求1至7任一项所述的金属-磷脂复合物，其中，所述连接物分子部分选自姜黄素、绿原酸、花青素、槲皮素、二氢杨梅素、橙皮素、柚皮素、芹菜素、儿茶素、茶多酚、表没食子儿茶素没食子酸酯、鞣花酸、桑色素、表儿茶素没食子酸酯、儿茶素没食子酸酯、没食子儿茶素没食子酸酯或平贝碱C，及其衍生物中一种或多种的组合。
  9. 根据权利要求8所述的金属-磷脂复合物，其中，所述连接物分子部分选自
    姜黄素
    绿原酸
    花青素其中，R7和R8是H、OH或OCH3，R3是H或糖基，R4、R5和R6是OH或糖基、
    槲皮素
    二氢杨梅素
    橙皮素
    柚皮素
    芹菜素
    儿茶素
    茶多酚
    表没食子儿茶素没食子酸酯
    鞣花酸
    桑色素
    表儿茶素没食子酸酯
    儿茶素没食子酸酯
    没食子儿茶素没食子酸酯
    平贝碱C及其衍生物中一种或多种的组合。
  10. 根据权利要求9所述的金属-磷脂复合物，其中，所述连接物分子部分选自姜黄素(式19)、二氢姜黄素(式36)六氢姜黄素(式37)硫酸姜黄素(式38)双去甲氧基姜黄素(式39)中一种或多种的组合。
  11. 根据权利要求9所述的金属-磷脂复合物，其中，所述连接物分子部分选自姜黄素(式19)、橙皮素(式24)、茶多酚(式28)，及其衍生物中一种或多种的组合。
  12. 根据权利要求11所述的金属-磷脂复合物，其中，所述连接物分子部分选自姜黄素(式19)、橙皮素(式24)或茶多酚(式28)。
  13. 根据权利要求1至12任一项所述的金属-磷脂复合物，其中，所述金属离子部分选自Fe3+、Ag+、Ba2+、Ca2+、Cd2+、Cu2+、Fe2+、Mn2+、Mg2+、Mo2+、Zn2+、Pt2+、Au2+、Al3+、Ce3+、Co3+、Cr3+、Eu3+、Gd3+、Ni3+、W3+、V3+、Zr3+中一种或多种的组合。
  14. 根据权利要求13所述的金属-磷脂复合物，其中，所述金属离子部分选自Fe3+、Ca2+、Al3+中一种或多种的组合。
  15. 根据权利要求14所述的金属-磷脂复合物，其中，所述金属离子部分选自Fe3+、Ca2+或Al3+。
  16. 根据权利要求1至15任一项所述的金属-磷脂复合物，其中，所述金属-磷脂复合物由磷脂分子部分、连接物分子部分和金属离子部分制成，所述磷脂分子部分选自DSPC、DSPE或DSPA，所述连接物分子部分选自姜黄素、橙皮素或茶多酚，所述金属离子部分选自Fe3+、Ca2+或Al3+。
  17. 根据权利要求16所述的金属-磷脂复合物，其中，所述金属-磷脂复合物由磷脂分子部分、连接物分子部分和金属离子部分制成，所述磷脂分子部分选自DSPC(式46)、DSPE(式47)或DSPA(式48)，所述连接物分子部分选自姜黄素(式19)、橙皮素(式24)或茶多酚(式28)，所述金属离子部分选自Fe3+、Ca2+或Al3+。
  18. 根据权利要求16或17所述的金属-磷脂复合物，其中，所述磷脂分子部分、所述连接物分子部分和所述金属离子部分的摩尔比为1:1:(0.5～2)。
  19. 根据权利要求18所述的金属-磷脂复合物，所述磷脂分子部分、所述连接物分子部分和所述金属离子部分的摩尔比为1:1:1。
  20. 权利要求1至19任一项所述金属-磷脂复合物的制备方法，其中，所述制备方法包括以下步骤：
    步骤一：将磷脂分子与连接物分子反应连接形成磷脂复合物；
    步骤二：将步骤一中制备的所述磷脂复合物与金属离子通过配位键反应形成金属-磷脂复合物。
  21. 根据权利要求20所述的制备方法，其中，步骤一中，将所述磷脂分子与所述连接物分子溶于乙醇中反应，之后加入正己烷，沉淀得到所述磷脂复合物。
  22. 根据权利要求21所述的制备方法，其中，所述磷脂分子与所述连接物分子的摩尔比为1:1。
  23. 根据权利要求21或22所述的制备方法，其中，所述反应的条件包括65℃反应2小时。
  24. 根据权利要求20至23任一项所述的制备方法，其中，步骤二中，磷脂复合物与金属离子溶于乙醇反应后，得到所述金属-磷脂复合物。
  25. 根据权利要求24所述的制备方法，其中，所述磷脂复合物与所述金属离子的摩尔比为1：(1～2)。
  26. 根据权利要求24或25所述的制备方法，其中，所述反应的条件包括60℃反应2小时。
  27. 一种金属-磷脂复合物颗粒，其中，所述金属-磷脂复合物颗粒含有：
    (i)金属-磷脂复合物，其中所述金属-磷脂复合物为权利要求1至19任一项所述的金属-磷脂复合物；
    (ii)抑制颗粒聚集的缀合的脂质，其中所述抑制颗粒聚集的缀合的脂质不是阳离子脂质或可电离脂质；以及
    (iii)除所述金属-磷脂复合物和所述抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质。
  28. 根据权利要求27所述的金属-磷脂复合物颗粒，其中，所述抑制颗粒聚集的缀合的脂质包括PEG-脂质缀合物和/或PEG-DAA。
  29. 根据权利要求28所述的金属-磷脂复合物颗粒，其中，所述PEG-脂质缀合物选自磷脂酰乙醇胺-聚乙二醇2000(式42)磷脂酰乙醇胺-聚乙二醇700(式43)磷脂酰乙醇胺-聚乙二醇1000(式44)磷脂酰乙醇胺-聚乙二醇5000(式45)，及其衍生物中一种或多种的组合；
    R1，R2均独立地为：葵酰基、月桂酰基、肉豆蔻酰基、棕榈酰基、硬脂酰基、油酰基、亚油酰基、芥酰基、花生酰基或植烷酰基。
  30. 根据权利要求29所述的金属-磷脂复合物颗粒，其中，所述PEG-脂质缀合物选自DSPE-PEG2000、DSPE-PEG700、DSPE-PEG1000或DSPE-PEG5000中一种或多种的组合。
  31. 根据权利要求30所述的金属-磷脂复合物颗粒，其中，所述PEG-脂质缀合物选自DSPE-PEG2000(式53) DSPE-PEG700(式50)DSPE-PEG1000(式51)或DSPE-PEG5000 (式52)
  32. 根据权利要求27至31任一项所述的金属-磷脂复合物颗粒，其中，(iii)中所述的非阳离子脂质或非可电离脂质为胆固醇及其衍生物中一种或多种的组合。
  33. 根据权利要求32所述的金属-磷脂复合物颗粒，其中，(iii)中所述的非阳离子脂质或非可电离脂质为胆固醇(式40)
  34. 根据权利要求32所述的金属-磷脂复合物颗粒，其中，(iii)中所述的非阳离子脂质或非可电离脂质还包括选自卵磷脂PC、磷脂酰乙醇胺PE、磷脂酰丝氨酸PS、磷脂酸PA、磷脂酰甘油PG、1-磷酸神经酰胺SP、磷脂酰肌醇PI、磷脂酰苏氨酸PT、鞘磷脂SM、溶血卵磷脂LPC、溶血磷酸酰乙醇胺LPE、溶血磷脂酰丝氨酸LPS、溶血磷脂酸LPA、溶血磷脂酰甘油LPG、溶血磷脂酰肌醇LPI、溶血磷脂酰苏氨酸LPT、溶血鞘磷脂LSM、1-磷酸鞘氨醇S1P、胆固醇硫酸酯，及其衍生物中一种或多种的组合。
  35. 根据权利要求34所述的金属-磷脂复合物颗粒，其中，(iii)中所述的非阳离子脂质或非可电离脂质还包括选自卵磷脂(PC，式1)、磷脂酰乙醇胺(PE，式2)、磷脂酰丝氨酸(PS，式3)、磷脂酸(PA，式4)、磷脂酰甘油(PG，式5)、1-磷酸神经酰胺(SP，式6)、磷脂酰肌醇(PI，式7)、磷脂酰苏氨酸(PT，式8)、鞘磷脂(SM，式9)、溶血卵磷脂(LPC，式10)、溶血磷酸酰乙醇胺(LPE，式11)、溶血磷脂酰丝氨酸(LPS，式12)、溶血磷脂酸(LPA，式13)、溶血磷脂酰甘油(LPG，式14)、溶血磷脂酰肌醇(LPI，式15)、溶血磷脂酰苏氨酸(LPT，式16)、溶血鞘磷脂(LSM，式17)、1-磷酸鞘氨醇(S1P，式18)、胆固醇硫酸酯 及其衍生物中一种或多种的组合。
  36. 根据权利要求34或35所述的金属-磷脂复合物颗粒，其中，(iii)中所述的非阳离子脂质或非可电离脂质包括胆固醇，以及选自DSPC、DSPE、DSPA或DSPG中一种或多种的组合。
  37. 根据权利要求36所述的金属-磷脂复合物颗粒，其中，(iii)中所述的非阳离子脂质或非可电离脂质包括胆固醇(式40)和DSPC(式46)。
  38. 根据权利要求34至37任一项所述的金属-磷脂复合物颗粒，其中，所述金属-磷脂复合物颗粒由(i)金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质和(iii)非阳离子脂质或非可电离脂质制成，所述金属-磷脂复合物在原料中摩尔占比为10％～40％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为2％～10％，所述胆固醇在原料中摩尔占比为35％～75％，所述除胆固醇以外的非阳离子脂质或非可电离脂质在原料中摩尔占比为0％～40％。
  39. 根据权利要求34或37任一项所述的金属-磷脂复合物颗粒，其中，所述金属-磷脂复合物颗粒由(i)金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质和(iii)非阳离子脂质或非可电离脂质制成，所述金属-磷脂复合物在原料中摩尔占比为5％～小于10％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为2％～10％，所述胆固醇在原料中摩尔占比为15％～小于35％、35％～75％或大于75％～80％，所述除胆固醇以外的非阳离子脂质或非可电离脂质在原料中摩尔占比为0％～40％或大于40％～51％；或
    所述金属-磷脂复合物在原料中摩尔占比为大于40％～50％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为2％～10％，所述胆固醇在原料中摩尔占比为15％～小于35％、35％～75％或大于75％～80％，所述除胆固醇以外的非阳离子脂质或非可电离脂质在原料中摩尔占比为0％～40％或大于40％～51％；或
    所述金属-磷脂复合物在原料中摩尔占比为10％～40％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为2％～10％，所述胆固醇在原料中摩尔占比为15％～小于35％或大于75％～80％，所述除胆固醇以外的非阳离子脂质或非可电离脂质在原料中摩尔占比为0％～40％或大于40％～51％。
  40. 根据权利要求38或39所述的金属-磷脂复合物颗粒，其中，所述金属-磷脂复合物在原料中摩尔占比为7％～小于10％、10％～30％或20％～30％，优选为25％。
  41. 根据权利要求38至40所述的金属-磷脂复合物颗粒，其中，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为3％～10％或5％～10％，优选为10％。
  42. 根据权利要求38至41任一项所述的金属-磷脂复合物颗粒，其中，所述胆固醇在原料中摩尔占比为15％～小于35％、35％～56％或35％～55％，优选为40％。
  43. 根据权利要求38至42任一项所述的金属-磷脂复合物颗粒，其中，除胆固醇以外的非阳离子脂质或非可电离脂质在原料中摩尔占比为5％～30％、25％～40％、大于40％～45％或20％～25％。
  44. 根据权利要求38所述的金属-磷脂复合物颗粒，其中，所述金属-磷脂复合物在原料中摩尔占比为15％～25％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占 比为4％～10％，所述胆固醇在原料中摩尔占比为40％～46％，所述DSPC在原料中摩尔占比为25％～35％，所述金属-磷脂复合物中金属离子部分选自Fe3+。
  45. 根据权利要求44所述的金属-磷脂复合物颗粒，其中，所述金属-磷脂复合物在原料中摩尔占比为15％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为4％，所述胆固醇在原料中摩尔占比为46％，所述DSPC在原料中摩尔占比为35％；或所述金属-磷脂复合物在原料中摩尔占比为25％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为10％，所述胆固醇在原料中摩尔占比为40％，所述DSPC在原料中摩尔占比为25％；所述金属-磷脂复合物中金属离子部分选自Fe3+。
  46. 根据权利要求38所述的金属-磷脂复合物颗粒，其中，所述金属-磷脂复合物在原料中摩尔占比为10％～30％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为3％～10％，所述胆固醇在原料中摩尔占比为35％～56％，所述DSPC在原料中摩尔占比为34％～40％；所述金属-磷脂复合物中金属离子部分选自Al3+。
  47. 根据权利要求39所述的金属-磷脂复合物颗粒，其中，所述金属-磷脂复合物在原料中摩尔占比为10％～30％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为3％～10％，所述胆固醇在原料中摩尔占比为35％～56％，所述DSPC在原料中摩尔占比为40％～45％；或
    所述金属-磷脂复合物在原料中摩尔占比为10％～30％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为3％～10％，所述胆固醇在原料中摩尔占比为15％～35％，所述DSPC在原料中摩尔占比为34％～40％或大于40％～45％；或
    所述金属-磷脂复合物在原料中摩尔占比为7％～小于10％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为3％～10％，所述胆固醇在原料中摩尔占比为15％～小于35％或35％～56％，所述DSPC在原料中摩尔占比为34％～40％或大于40％～45％；所述金属-磷脂复合物中金属离子部分选自Al3+。
  48. 根据权利要求47所述的金属-磷脂复合物颗粒，其中，所述金属-磷脂复合物在原料中摩尔占比为7％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为3％，所述胆固醇在原料中摩尔占比为56％，所述DSPC在原料中摩尔占比为34％；所述金属-磷脂复合物中金属离子部分选自Al3+。
  49. 权利要求27至48任一项所述金属-磷脂复合物颗粒的制备方法，其中，所述制备方法包括将(i)金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质和(iii)除所述金属-磷脂复合物和所述抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质混合，得到所述金属-磷脂复合物颗粒。
  50. 一种药物-脂质颗粒，其中，所述药物-脂质颗粒含有：
    (a)药物，其中所述药物为带有负电荷的分子；和
    (b)金属-磷脂复合物颗粒，所述金属-磷脂复合物颗粒为权利要求27至48任一项所述金属-磷脂复合物颗粒。
  51. 根据权利要求50所述的药物-脂质颗粒，其中，所述药物包封在所述金属-磷脂复合物颗粒中。
  52. 根据权利要求50或51所述的药物-脂质颗粒，其中所述药物选自核酸、蛋白、多肽、小分子、核酸类似物、蛋白类似物和多肽类似物中一种或多种的组合。
  53. 根据权利要求52所述的药物-磷脂颗粒，其中，所述核酸选自mRNA、siRNA、sgRNA、ASO、circRNA、microRNA、DNA、ecDNA、人工核酸中一种或多种的组合。
  54. 根据权利要求52或53所述的药物-磷脂颗粒，其中，所述核酸为SEQ ID No.1所示的编码eGFP的mRNA序列、SEQ ID No.2所示的编码新型冠状病毒S1亚基的受体结合域RBD的mRNA序列、SEQ ID No.3所示的编码NY-ESO-1的mRNA序列、反义链为SEQ ID No.4和正义链为SEQ ID No.21所示的Bcl-2基因的siRNA序列、反 义链为SEQ ID No.6和正义链为SEQ ID No.23所示的PLK1基因的siRNA序列、SEQ ID No.8所示的Gal-1基因的siRNA序列、SEQ ID No.10所示的STAT-3基因的ASO序列、SEQ ID No.12所示的α-syn基因的ASO序列、SEQ ID No.14所示的Bcl-2基因的ASO序列、SEQ ID No.16所示的编码野生型新型冠状病毒S蛋白的mRNA序列、反义链为SEQ ID No.17和正义链为SEQ ID NO.25所示的双链DNA序列、SEQ ID No.18所示的单链DNA、或正义链为SEQ ID No.19和反义链为SEQ ID No.26所示的B7-H4基因的siRNA序列。
  55. 权利要求50至54任一项所述药物-脂质颗粒的制备方法，其中，将药物包载于金属-磷脂复合物颗粒中，得到所述药物-脂质颗粒。
  56. 根据权利要求55所述的制备方法，其中，金属-磷脂复合物、抑制颗粒聚集的缀合的脂质，以及除所述金属-磷脂复合物和所述抑制颗粒聚集的缀合的脂质以外的非阳离子脂质或非可电离脂质溶于有机化合物中形成有机相，药物溶于缓冲液中形成水相，将有机相与水相混匀得到药物-脂质颗粒。
  57. 根据权利要求56所述的制备方法，其中，所述有机化合物为乙醇。
  58. 根据权利要求56或57所述的制备方法，其中，所述缓冲液为无酶PBS缓冲液。
  59. 根据权利要求56至58任一项所述的制备方法，其中，有机相与水相的混匀方式包含微流控芯片或超声。
  60. 权利要求1至19任一项所述金属-磷脂复合物在核酸递送系统中的应用。
  61. 根据权利要求60所述的应用，其中，所述核酸递送系统用于将核酸引入细胞。
  62. 根据权利要求61所述的应用，其中，所述核酸用于在哺乳动物受试者中使靶序列的表达沉默、用于在哺乳动物体内传递药物、用于将药物从体内传递到哺乳动物细胞或用于治疗哺乳动物的疾病或病症。
  63. 权利要求27至48任一项所述金属-磷脂复合物颗粒或权利要求47至51任一项所述药物-脂质颗粒在组合物中的应用，所述组合物用于药物的递送。
  64. 根据权利要求63所述的应用，其中，所述组合物用于将药物引入细胞。
  65. 根据权利要求63或64所述的应用，其中，所述组合物为药剂。
  66. 根据权利要求65所述的应用，其中，所述药剂用于在哺乳动物受试者中使靶序列的表达沉默、用于在哺乳动物体内传递药物、用于将药物从体内传递到哺乳动物细胞或用于治疗哺乳动物的疾病或病症。
  67. 根据权利要求62或66所述的应用，其中，所述哺乳动物为人。
  68. 根据权利要求62或66所述的应用，其中，所述疾病或病症与基因的表达相关，所述基因包含药物的靶序列。
  69. 根据权利要求62或66所述的应用，其中，所述疾病或病症包括癌症、病毒感染、自身免疫性疾病、糖尿病或阿尔兹海默症。
  70. 根据权利要求69所述的应用，其中，所述病毒感染包括甲肝、乙肝、丙肝、SARS-Cov-2、HIV、HPV、流感、天花或梅毒。
  71. 根据权利要求69所述的应用，其中，所述癌症包括肝癌、胶质瘤、黑色素瘤、肺癌、胰腺癌或乳腺癌。
  72. 根据权利要求65所述的应用，其中，所述药剂为疫苗。
  73. 根据权利要求65或72所述的应用，其中，所述药剂的给药途径包括鞘内注射、肌肉给药、颅内注射、静脉注射或瘤内注射。
  74. 一种含有权利要求1至19任一项所述金属-磷脂复合物或权利要求27至48任一项所述金属-磷脂复合物颗粒或权利要求50至54任一项所述药物-脂质颗粒的药 剂。
  75. 根据权利要求74所述的药剂，其中，所述药剂为疫苗。
  76. 根据权利要求75所述的药剂，其中，所述疫苗为新型冠状病毒疫苗。
  77. 权利要求1至19任一项所述金属-磷脂复合物或权利要求27至48任一项所述金属-磷脂复合物颗粒或权利要求50至54任一项所述药物-脂质颗粒或权利要求74至76任一项所述的药剂在预防/治疗哺乳动物的疾病或病症中的应用。
  78. 根据权利要求77所述的应用，其中，所述哺乳动物为人。
  79. 根据权利要求77所述的应用，其中，所述疾病或病症与基因的表达相关，所述基因包含药物的靶序列。
  80. 根据权利要求77所述的应用，其中，所述疾病或病症包括癌症、病毒感染、自身免疫性疾病、糖尿病或阿尔兹海默症。
  81. 根据权利要求80所述的应用，其中，所述病毒感染包括甲肝、乙肝、丙肝、SARS-Cov-2、HIV、HPV、流感、天花或梅毒。
  82. 根据权利要求80所述的应用，其中，所述癌症包括肝癌、胶质瘤、黑色素瘤、肺癌、胰腺癌或乳腺癌。
PCT/CN2023/111279 2022-08-09 2023-08-04 金属-磷脂复合物、金属-磷脂复合物颗粒和药物-脂质颗粒及其制备方法与应用 WO2024032507A1 (zh)

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