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WO2025167726A1 - 靶向载体、靶向药物及制备方法与应用 - Google Patents

靶向载体、靶向药物及制备方法与应用

Info

Publication number
WO2025167726A1
WO2025167726A1 PCT/CN2025/074622 CN2025074622W WO2025167726A1 WO 2025167726 A1 WO2025167726 A1 WO 2025167726A1 CN 2025074622 W CN2025074622 W CN 2025074622W WO 2025167726 A1 WO2025167726 A1 WO 2025167726A1
Authority
WO
WIPO (PCT)
Prior art keywords
formula
metal
phospholipid
phospholipid complex
drug
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/074622
Other languages
English (en)
French (fr)
Inventor
王珊
胡敦
任欢欢
刘喜荣
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hunan Lonstar Biotech Co Ltd
Original Assignee
Hunan Lonstar Biotech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hunan Lonstar Biotech Co Ltd filed Critical Hunan Lonstar Biotech Co Ltd
Publication of WO2025167726A1 publication Critical patent/WO2025167726A1/zh
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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Definitions

  • the present application relates to the field of biotechnology, and specifically provides a targeted carrier, a targeted drug, and a preparation method and application thereof.
  • nucleic acid drugs artificially synthesized DNA or RNA fragments with therapeutic potential, have attracted considerable attention due to their enormous potential for application in disease diagnosis and treatment. These drugs can act directly on pathogenic target genes or mRNAs, exerting therapeutic effects at the genetic level. Compared with traditional small molecule and antibody drugs, nucleic acid drugs are not limited by the druggability of their target proteins, can treat a wider range of diseases, and can regulate the expression of pathogenic genes at their root. Nucleic acid drugs also offer significant advantages such as high efficacy, low toxicity, and high specificity, and are expected to become the third largest type of drug after small molecule and antibody drugs.
  • nucleic acid drugs are easily degraded by nucleases in the body, and their large molecular weight and negative charge make them difficult to cross cell membranes to exert their effects. Therefore, finding safe and effective delivery systems for nucleic acid drugs is a critical bottleneck in their development.
  • vectors capable of delivering nucleic acid drugs can be divided into viral and non-viral vectors. Viral vectors induce an immune response upon entry into the human body and are therefore less commonly used. Among non-viral vectors, nanoparticles and small molecule conjugates are the most commonly used.
  • Nanoparticles can more effectively encapsulate nucleic acid drugs, preventing their rapid degradation by nucleases in the body and thereby increasing their circulation time. Nanoparticles encapsulate nucleic acids by adsorbing negatively charged nucleic acids onto positively charged cationic lipids.
  • cationic lipids are highly cytotoxic, with the following mechanisms of action: 1 cell atrophy, decreased mitotic activity, and cytoplasmic vacuolation; 2 interaction with biological proteins such as protein kinase C, thereby disrupting their activity; and 3 activation of p38 mitogen-activated protein kinase and nuclear factor- ⁇ B transcription factors, triggering the secretion of various proinflammatory cytokines and chemokines.
  • ionizable lipids can be used to partially or completely replace cationic lipids as the main component of nanoparticles, responsible for adsorbing nucleic acids.
  • Lipid nanoparticles based on cationic lipids and/or ionizable lipids are nanoparticle nucleic acid drug delivery systems that can be used in clinical practice.
  • Cationic lipids and/or ionizable lipids are the main components of LNPs and are responsible for the adsorption of nucleic acids.
  • the cytotoxicity and immunogenicity mediated by cationic lipids and/or ionizable lipids are still one of the important reasons for the high toxicity of LNPs.
  • nanoparticle delivery systems developed based on cationic lipids and/or ionizable lipids cannot fundamentally solve the toxicity problem of nanoparticle delivery systems.
  • a liposome delivery system with higher safety.
  • One of the purposes of this application is to provide a targeted carrier, a targeted drug, and a preparation method and application thereof, so as to provide a new drug carrier and an effective therapeutic drug.
  • PC Phosphatidylcholine
  • the phospholipid molecule is selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48), DSPG (Formula 49), and a combination of one or more of their derivatives;
  • the linker molecule is selected from the group consisting of curcumin, chlorogenic acid, anthocyanidin, quercetin, dihydromyricetin, hesperetin, naringenin, apigenin, catechin, tea polyphenols, epigallocatechin gallate, ellagic acid, morin, epicatechin gallate, catechin gallate, epigallocatechin gallate or picoflavine C, and combinations of one or more thereof.
  • the linker molecule is selected from
  • R1 and R2 are H, OH or OCH3, R3 is H or glycosyl, R4, R5 and R6 are OH or glycosyl,
  • Pingbei alkaloid C (Formula 35) and combinations of one or more of their derivatives;
  • the linker molecule is selected from one or more combinations of curcumin (Formula 19), dihydrocurcumin (Formula 36), hexahydrocurcumin (Formula 37), curcumin sulfate (Formula 38), and bisdemethoxycurcumin (Formula 39);
  • the linker molecule is selected from the group consisting of curcumin (Formula 19), hesperetin (Formula 24), tea polyphenols (Formula 28), and a combination of one or more of their derivatives;
  • the metal ion moiety is selected from a combination of one or more of 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+ , and Zr 3+ ;
  • the metal ion moiety is selected from Fe 3+ , Mg 2+ , Ca 2+ or Al 3+ .
  • conjugated lipid that inhibits particle aggregation comprises PEG-lipid conjugate and/or PEG-DAA;
  • the PEG-lipid conjugate is selected from:
  • R1 and R2 are independently: capryloyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytanoyl;
  • the PEG-lipid conjugate is selected from a combination of one or more of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000 or DSPE-PEG5000;
  • the PEG-lipid conjugate is selected from DSPE-PEG2000 (Formula 53), DSPE-PEG700 (Formula 50), DSPE-PEG1000 (Formula 51) or DSPE-PEG5000 (Formula 52);
  • non-cationic lipid or non-ionizable lipid in (iii) is a combination of one or more of cholesterol and its derivatives;
  • the non-cationic lipid or non-ionizable lipid in (iii) is cholesterol (Formula 40)
  • the non-cationic lipid or non-ionizable lipid described in (iii) further comprises at least one selected from phosphatidylcholine PC (Formula 1), phosphatidylethanolamine PE (Formula 2), phosphatidylserine PS (Formula 3), phosphatidic acid PA (Formula 4), phosphatidylglycerol PG (Formula 5), 1-phosphoceramide SP (Formula 6), phosphatidylinositol PI (Formula 7), phosphatidylthreonine PT (Formula 8), sphingomyelin SM (Formula 9), lysophosphatidylcholine LPC (Formula 10), lysophosphatidylethanolamine LPE (Formula 11), lysophosphatidylserine LPS (Formula 12), lysophosphatidic acid LPA (Formula 13), lysophosphatid
  • the non-cationic lipid or non-ionizable lipid in (iii) comprises cholesterol, and a combination of one or more selected from DSPC, DSPE, DSPA or DSPG;
  • the non-cationic lipid or non-ionizable lipid in (iii) comprises cholesterol (Formula 40) and DSPC (Formula 46).
  • the metal-phospholipid complex is made of a phospholipid molecule portion, a linker molecule portion, and a metal ion portion
  • the phospholipid molecule portion is selected from DSPC, DSPE, DSPA, or DSPG
  • the linker molecule portion is selected from curcumin, hesperetin, or tea polyphenols
  • the metal ion portion is selected from Fe 3+ , Mg 2+ , Ca 2+ , or Al 3+ ;
  • the metal-phospholipid complex is made of a phospholipid molecule portion, a linker molecule portion and a metal ion portion
  • the phospholipid molecule portion is selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48) or DSPG (Formula 49)
  • the linker molecule portion is selected from curcumin (Formula 19), hesperetin (Formula 24) or tea polyphenols (Formula 28)
  • the metal ion portion is selected from Fe 3+ , Mg 2+ , Ca 2+ or Al 3+ ;
  • the molar ratio of the phospholipid molecule portion, the linker molecule portion, and the metal ion portion is 1:1:(0.5-2);
  • the phospholipid molecule portion is DSPC (Formula 46), the linker molecule portion is selected from curcumin (Formula 19), the metal ion portion is selected from Fe 3+ , Mg 2+ or Al 3+ , and the molar ratio of the phospholipid molecule portion, the linker molecule portion and the metal ion portion is 1:1:1.
  • the metal-phospholipid complex particles are made of (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the metal-phospholipid complex accounts for 5% to 50% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 1% to 10% by mole in the raw material, the cholesterol accounts for 15% to 80% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 0% to 51% by mole in the raw material;
  • the metal-phospholipid complex accounts for 5% to 40% by mole in the raw material, preferably 10% to 40%;
  • the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%;
  • the molar proportion of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 0% to 50%, preferably 0% to 40%.
  • the (b) targeting structure comprises a hydrophobic region, a connecting region and a targeting binding region connected in sequence; the hydrophobic region is connected to the outer layer of the metal-phospholipid complex particle based on hydrophilicity and hydrophobicity;
  • the hydrophobic region comprises at least one or more of DSPE and its derivatives
  • the linker region comprises at least one or more of PEG-2000 and its derivatives
  • the targeting binding region can bind to at least one of CD62L, CD8, CD3, nucleolin protein, T cells, natural killer cells, macrophages, pancreatic cancer cells or liver cancer cells;
  • the target binding region comprises at least one of a nucleic acid, a polypeptide, a protein, and a small molecule;
  • the targeting binding region is an aptamer
  • the target of the targeting binding region is CD8, and the aptamer is preferably represented by SEQ ID NO.84; or,
  • the targeting binding region is an aptamer
  • the target of the targeting binding region is nucleolin protein
  • the aptamer is preferably represented by SEQ ID NO.86; or,
  • the targeting binding region is an aptamer
  • the target cell of the targeting binding region is a T cell
  • the aptamer is preferably represented by SEQ ID NO.44;
  • the targeting binding region is an aptamer
  • the target cell of the targeting binding region is a pancreatic cancer cell
  • the aptamer is preferably represented by SEQ ID NO.88; or,
  • the targeting structure is DSPE-PEG2000-aptamer, preferably DSPE-PEG2000-CD62L aptamer, and the CD62L aptamer sequence is shown in SEQ ID NO.44.
  • the method for preparing the above-mentioned targeting carrier comprises connecting the targeting structure to the outer surface of the metal-phospholipid complex particles to form the targeting carrier.
  • the metal-phospholipid complex (i) the metal-phospholipid complex, (ii) the conjugated lipid that inhibits particle aggregation, and (iii) the non-cationic lipid or the non-ionizable lipid are mixed to obtain the metal-phospholipid complex particles;
  • the targeting structure is DSPE-PEG2000-aptamer
  • the preparation process is as follows: DSPE-PEG2000 and the aptamer are connected through an intermediate pair reaction to obtain DSPE-PEG2000-aptamer;
  • the intermediate pair is selected from DSPE-PEG2000-MAL and aptamer-C6-SH, DSPE-PEG2000-NHS and aptamer-NH 2 , DSPE-PEG2000-COOH and aptamer-NH 2 , DSPE-PEG2000-NCO and aptamer-NH 2 , DSPE-PEG2000-N 3 and aptamer-DBCO, preferably DSPE-PEG2000-MAL and aptamer-C6-SH;
  • the DSPE-PEG2000-aptamer forms micelles and then connects to the outer surface of the metal-phospholipid complex particles to form the targeting carrier;
  • the preparation method of the micelles includes direct dissolution method, ethanol injection method, dialysis method or ultrasound method.
  • the preparation method comprises:
  • Step 1 reacting phospholipid molecules, linker molecules and metal ions to form a metal-phospholipid complex
  • Step 2 mixing the metal-phospholipid complex prepared in step 1, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid to prepare metal-phospholipid complex particles;
  • Step 3 mixing the metal-phospholipid complex particles prepared in step 2 with the targeting structure to prepare the targeting carrier;
  • the phospholipid molecules, linker molecules and metal ions are dissolved in ethanol for reaction, the molar ratio of the phospholipid molecules, linker molecules and metal ions is preferably 1:1:1, and the reaction conditions are preferably 40-60° C. for 1-5 hours;
  • the reaction conditions of the metal-phospholipid complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.
  • the preparation method comprises:
  • Step 1 Reaction and connection of phospholipid molecules with linker molecules to form phospholipid complexes
  • Step 2 reacting the phospholipid complex prepared in step 1 with metal ions through coordination bonds to form a metal-phospholipid complex
  • Step 3 mixing the metal-phospholipid complex prepared in step 2, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid to prepare metal-phospholipid complex particles;
  • Step 4 mixing the metal-phospholipid complex particles prepared in step 3 with the targeting structure to prepare the targeting carrier;
  • the phospholipid molecules and the linker molecules are dissolved in ethanol for reaction, and then n-hexane is added for precipitation to obtain the phospholipid complex.
  • the molar ratio of the phospholipid molecules to the linker molecules is preferably 1:1.
  • the reaction conditions are preferably 65° C. for 2 hours.
  • the phospholipid complex and the metal ion are dissolved in ethanol, and triethylamine is added to react to obtain the metal-phospholipid complex.
  • the molar ratio of the phospholipid complex to the metal ion is preferably 1:(1-2), and the molar ratio of the phospholipid complex to the triethylamine is preferably 1:1.
  • the reaction conditions are preferably 60° C. for 2 hours.
  • the reaction conditions of the metal-phospholipid complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.
  • the above-mentioned targeting carrier or preparation method is used for drug delivery, imaging agent or vaccine application.
  • a targeted drug comprises a drug and the above-mentioned targeting carrier, wherein the drug is encapsulated in the metal-phospholipid complex particles of the targeting carrier.
  • the drug is selected from a combination of one or more 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 acid;
  • the drug is an mRNA encoding a chimeric antigen receptor CAR, wherein the CAR comprises a transmembrane domain, a signaling domain, an antigen binding domain, a co-stimulatory signaling region, and a region connecting the antigen binding domain and the transmembrane domain;
  • the transmembrane domain is selected from at least one of SEQ ID No. 28, SEQ ID No. 29, and SEQ ID No. 30; and/or,
  • the signaling domain is selected from at least one of SEQ ID No. 31, SEQ ID No. 32, and SEQ ID No. 33; and/or,
  • the antigen binding domain is selected from at least one of SEQ ID No. 34, SEQ ID No. 35, and SEQ ID No. 36; and/or
  • the targeting carrier includes metal-phospholipid complex particles and a targeting structure, and the drug is encapsulated in the metal-phospholipid complex particles to obtain drug-metal-phospholipid complex particles; the targeting structure is connected to the outer surface of the drug-metal-phospholipid complex particles to form the targeted drug.
  • the drug (i) the metal-phospholipid complex, (ii) the conjugated lipid for inhibiting particle aggregation, and (iii) the non-cationic lipid or the non-ionizable lipid are mixed to obtain the drug-metal-phospholipid complex particles.
  • the preparation method comprises:
  • Step 1 reacting phospholipid molecules, linker molecules and metal ions to form a metal-phospholipid complex
  • Step 2 mixing the metal-phospholipid complex prepared in step 1, (ii) a conjugated lipid that inhibits particle aggregation, (iii) a non-cationic lipid or a non-ionizable lipid, and a drug to prepare drug-metal-phospholipid complex particles;
  • Step 3 mixing the drug-metal-phospholipid complex particles prepared in step 2 with the targeting structure to prepare the targeted drug;
  • the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer to form an aqueous phase, and the organic phase and the aqueous phase are mixed to obtain drug-metal-phospholipid complex particles, wherein the organic compound is preferably ethanol; preferably, the mixing method of the organic phase and the aqueous phase includes a microfluidic chip or ultrasound;
  • the preparation method comprises:
  • Step 2 reacting the phospholipid complex prepared in step 1 with metal ions through coordination bonds to form a metal-phospholipid complex
  • Step 3 mixing the metal-phospholipid complex prepared in step 2, (ii) a conjugated lipid that inhibits particle aggregation, (iii) a non-cationic lipid or a non-ionizable lipid, and a drug to prepare drug-metal-phospholipid complex particles;
  • Step 4 mixing the drug-metal-phospholipid complex particles prepared in step 3 with the targeting structure to prepare the targeted drug;
  • the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer to form an aqueous phase, and the organic phase and the aqueous phase are mixed to obtain drug-metal-phospholipid complex particles, wherein the organic compound is preferably ethanol; preferably, the mixing method of the organic phase and the aqueous phase includes a microfluidic chip or an ultrasonic
  • step 4 the reaction conditions of the drug-metal-phospholipid complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.
  • the intermediate pair is selected from DSPE-PEG2000-MAL and aptamer-C6-SH, DSPE-PEG2000-NHS and aptamer-NH 2 , DSPE-PEG2000-COOH and aptamer-NH 2 , DSPE-PEG2000-NCO and aptamer-NH 2 , DSPE-PEG2000-N 3 and aptamer-DBCO, preferably DSPE-PEG2000-MAL and aptamer-C6-SH;
  • the DSPE-PEG2000-aptamer forms micelles and then connects to the outer surface of the drug-metal-phospholipid complex particles to form the targeted drug;
  • the preparation method of the micelles includes direct dissolution method, ethanol injection method, dialysis method or ultrasound method.
  • the targeted drug is used for drug delivery, treatment and/or prevention
  • the targeted drug is used to introduce the drug into cells or blood;
  • the cells preferably include immune cells, tumor cells, germ cells, nerve cells, endocrine cells, blood cells, phagocytes, white blood cells, red blood cells, epithelial cells, myocardial cells or stem cells;
  • the mammal is a human
  • the treatment of the disease or condition is associated with the expression of a gene comprising a target sequence for the drug;
  • the disease or condition comprises cancer, viral infection, autoimmune disease, disease caused by overactivation of the immune system, metabolic disease, fibrotic disease, tissue fibrosis, cell senescence, atherosclerosis, diabetes or osteoarthritis;
  • the viral infection comprises hepatitis B virus, hepatitis C virus, SARS-Cov-2, human immunodeficiency virus, cytomegalovirus, invasive Aspergillus or conjugate virus;
  • the cancer includes hematological tumors and solid tumors
  • the hematological tumor comprises acute B-cell leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), multiple myeloma (MM), acute myeloid leukemia (AML) or T-cell lymphoma;
  • ALL acute B-cell leukemia
  • DLBCL diffuse large B-cell lymphoma
  • MM multiple myeloma
  • AML acute myeloid leukemia
  • T-cell lymphoma T-cell lymphoma
  • the diffuse large B-cell lymphoma includes Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL);
  • the autoimmune disease includes pemphigus vulgaris, systemic lupus erythematosus (SLE), hemophilia, myasthenia gravis, immune rejection caused by transplanted tissues and organs, type 1 diabetes (T1D), rheumatoid arthritis, systemic sclerosis, multiple sclerosis, idiopathic pulmonary fibrosis, Crohn's disease or colitis;
  • the disease caused by excessive activation of the immune system includes cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS) or graft-versus-host disease (GVHD);
  • CRS cytokine release syndrome
  • ICANS immune effector cell-associated neurotoxicity syndrome
  • GVHD graft-versus-host disease
  • the metabolic disease includes atherosclerosis, congenital hyperinsulinemia, non-alcoholic steatohepatitis or non-obese diabetes (NOD);
  • atherosclerosis congenital hyperinsulinemia
  • non-alcoholic steatohepatitis non-obese diabetes (NOD)
  • NOD non-obese diabetes
  • the fibrosis includes myocardial fibrosis, hypertrophic cardiomyopathy, COVID-19, COVID-19 myocarditis, ischemic cardiomyopathy, cirrhosis, liver fibrosis, primary cholangitis, pulmonary fibrosis, skeletal muscle fibrosis, skin diseases, myelofibrosis, sarcoidosis or Duchenne muscular dystrophy;
  • the skin disease includes keloid or wound healing
  • the administration route of the targeted drug includes intrathecal injection, intramuscular administration, intracranial injection, intravenous injection or intratumoral injection;
  • the targeted carrier provided by the present application is no less effective than LNPs based on cationic lipids and/or ionizable lipids, but does not use cationic lipids and ionizable lipids, significantly reduces toxicity, significantly improves biosafety, and is more conducive to carrying negatively charged drugs in vivo. It has a wide range of applications and can be used for drugs of different sizes. Targeted drugs can achieve high expression levels of nucleic acid drugs, significantly improve biosafety, have good targeting, and can achieve efficient treatment of various diseases.
  • Figure 1-1 shows the percentage of eGFP-positive cells resulting from transfection of 293T cells with eGFP-mRNA@MPP(Fe 3+ ) as provided in Example 1.3.5.1 of the present application;
  • Figure 1-2 shows the expression of RBD caused by transfection of 293T cells with RBD-mRNA@MPP(Fe 3+ ) provided in Example 1.3.5.1 of the present application;
  • Figures 1-4 show the ability of NY-ESO-1-mRNA@MPP(Fe 3+ ) to induce humoral immunity as provided in Example 1.3.5.1 of the present application;
  • Figures 1-5 show the ability of RBD-mRNA@MPP(Fe 3+ ) to induce cellular immunity as provided in Example 1.3.5.1 of this application;
  • Figures 1-6 show the ability of NY-ESO-1-mRNA@MPP(Fe 3+ ) to induce cellular immunity as provided in Example 1.3.5.1 of the present application;
  • Figure 1-1-2 shows the percentage of eGFP-positive cells caused by transfection of 293T cells with eGFP-mRNA@MPP(Al 3+ ) as provided in Example 1.3.5.2 of this application;
  • Figure 1-2-2 shows the expression of RBD caused by transfection of 293T cells with RBD-mRNA@MPP(Al 3+ ) provided in Example 1.3.5.2 of this application;
  • Figure 1-3-2 shows the ability of RBD-mRNA@MPP(Al 3+ ) to induce humoral immunity as provided in Example 1.3.5.2 of this application;
  • Figure 1-4-2 shows the ability of NY-ESO-1-mRNA@MPP(Al 3+ ) to induce humoral immunity provided in Example 1.3.5.2 of the present application;
  • Figure 1-5-2 shows the ability of RBD-mRNA@MPP(Al 3+ ) to induce cellular immunity provided in Example 1.3.5.2 of this application;
  • Figure 1-6-2 shows the ability of NY-ESO-1-mRNA@MPP(Al 3+ ) to induce cellular immunity provided in Example 1.3.5.2 of the present application;
  • Figure 1-1-3 shows the percentage of eGFP-positive cells caused by transfection of 293T cells with eGFP-mRNA@MPP(Mg 2+ ) as provided in Example 1.3.5.3 of the present application;
  • Figure 1-3-3 shows the ability of RBD-mRNA@MPP(Mg 2+ ) to induce humoral immunity provided in Example 1.3.5.3 of the present application;
  • Figure 1-4-3 shows the ability of NY-ESO-1-mRNA@MPP(Mg 2+ ) to induce humoral immunity provided in Example 1.3.5.3 of the present application;
  • Figure 1-5-3 shows the ability of RBD-mRNA@MPP(Mg 2+ ) to induce cellular immunity provided in Example 1.3.5.3 of this application;
  • Figure 1-6-3 shows the ability of NY-ESO-1-mRNA@MPP(Mg 2+ ) to induce cellular immunity provided in Example 1.3.5.3 of the present application;
  • Figures 1-7 show the ability of Bcl-2-siRNA@MPP(Fe 3+ ) to silence target genes provided in Example 1.3.6.1 of this application;
  • Figures 1-8 show the ability of PLK1-siRNA@MPP(Fe 3+ ) to silence target genes provided in Example 1.3.6.1 of the present application;
  • Figure 1-9 shows the ability of Gal-1-siRNA@MPP(Fe 3+ ) to silence target genes provided in Example 1.3.6.1 of the present application;
  • Figure 1-7-2 shows the ability of Bcl-2-siRNA@MPP(Al 3+ ) to silence target genes provided in Example 1.3.6.2 of this application;
  • Figure 1-8-2 shows the ability of PLK1-siRNA@MPP(Al 3+ ) to silence target genes provided in Example 1.3.6.2 of this application;
  • Figure 1-9-2 shows the ability of Gal-1-siRNA@MPP(Al 3+ ) to silence target genes provided in Example 1.3.6.2 of this application;
  • Figure 1-7-3 shows the ability of Bcl-2-siRNA@MPP(Mg 2+ ) to silence target genes provided in Example 1.3.6.3 of the present application;
  • Figure 1-8-3 shows the ability of PLK1-siRNA@MPP(Mg 2+ ) to silence target genes provided in Example 1.3.6.3 of the present application;
  • Figure 1-9-3 shows the ability of Gal-1-siRNA@MPP(Mg 2+ ) to silence target genes provided in Example 1.3.6.3 of the present application;
  • Figure 1-10 shows the ability of STAT3-ASO@MPP(Fe 3+ ) to silence cellular target genes as provided in Example 1.3.7.1 of the present application;
  • Figure 1-12 shows the ability of Bcl-2-ASO@MPP(Fe 3+ ) to silence cellular target genes as provided in Example 1.3.7.1 of this application;
  • Figure 1-10-2 shows the ability of STAT3-ASO@MPP(Al 3+ ) to silence cell target genes as provided in Example 1.3.7.2 of this application;
  • Figure 1-11-2 shows the ability of ⁇ -syn-ASO@MPP(Al 3+ ) to silence cell target genes as provided in Example 1.3.7.2 of this application;
  • Figure 1-12-2 shows the ability of Bcl-2-ASO@MPP(Al 3+ ) to silence cell target genes as provided in Example 1.3.7.2 of this application;
  • Figure 1-10-3 shows the ability of STAT3-ASO@MPP(Mg 2+ ) to silence cellular target genes as provided in Example 1.3.7.3 of the present application;
  • Figure 1-11-3 shows the ability of ⁇ -syn-ASO@MPP(Mg 2+ ) to silence cellular target genes as provided in Example 1.3.7.3 of the present application;
  • Figure 1-12-3 shows the ability of Bcl-2-ASO@MPP(Mg 2+ ) to silence cell target genes as provided in Example 1.3.7.3 of this application;
  • Figure 1-13 shows the expression of S protein caused by transfection of 293T cells with S-mRNA@MPP(Fe 3+ ) provided in Example 1.3.8.1 of the present application;
  • Figure 1-14 shows the functions of drug (dsDNA and ssDNA)@MPP(Fe 3+ ) provided in Example 1.3.8.1 of the present application;
  • Figure 1-13-2 shows the expression of S protein caused by transfection of 293T cells with S-mRNA@MPP(Al 3+ ) provided in Example 1.3.8.2 of this application;
  • Figure 1-14-2 shows the function of drug (dsDNA and ssDNA)@MPP(Al 3+ ) provided in Example 1.3.8.2 of the present application;
  • Figure 1-13-3 shows the expression of S protein caused by transfection of 293T cells with S-mRNA@MPP(Mg 2+ ) provided in Example 1.3.8.3 of the present application;
  • Figure 2-1 is a differential scanning calorimetry diagram of the phospholipid complex provided in Example 2.1.1 of the present application;
  • Figure 2-2 is a UV absorption graph of the metal-phospholipid complex (Fe 3+ ) provided in Example 2.1.1 of the present application;
  • Figure 2-2-3 is the UV absorption graph of the metal-phospholipid complex (Mg 2+ ) provided in Example 2.1.3 of the present application;
  • Figure 2-4 shows the elemental analysis of the drug-metal-phospholipid complex particles (Fe 3+ ) provided in Example 2.3 of the present application;
  • Figures 2-5 show the efficiency of nucleic acid (mRNA and siRNA) encapsulation by siRNA/mRNA@MPP (Fe 3+ , Al 3+ or Mg 2+ ) and siRNA/mRNA@LNP provided in Example 2.4 of the present application;
  • Figure 2-6 is a statistical graph of the nucleic acid lysosomal escape ability of siRNA/mRNA@MPP (Fe 3+ , Al 3+ or Mg 2+ ) and siRNA/mRNA@LNP provided in Example 2.5 of the present application;
  • Figure 2-7 shows the eGFP-positive cell rates of MPP (Fe 3+ , Al 3+ or Mg 2+ ) and LNP provided in Example 2.6 of the present application;
  • Figure 2-8 is a comparison of the ability of MPP (Fe 3+ , Al 3+ or Mg 2+ ) and LNP to promote RBD-mRNA expression provided in Example 2.7 of the present application;
  • Figure 2-9 is a comparison of the humoral immunity-promoting abilities of MPP (Fe 3+ , Al 3+ or Mg 2+ ) and LNP provided in Example 2.7 of the present application;
  • Figure 2-10 is a comparison of the ability of MPP (Fe 3+ , Al 3+ or Mg 2+ ) and LNP to promote cellular immunity provided in Example 2.7 of the present application;
  • Figure 2-11 is a diagram showing the results of the nucleic acid lysosomal escape-promoting function test of siRNA/mRNA@MPP(Fe 3+ ) and siRNA/mRNA@LNP provided in Example 2.5 of the present application;
  • FIG3-1 shows the effect of intratumoral injection of drug-metal-phospholipid complex particles (Fe 3+ ) in treating liver cancer provided in Example 3 of the present application;
  • FIG3-2 shows the effect of intratumoral injection of drug-metal-phospholipid complex particles (Al 3+ ) in treating liver cancer provided in Example 3 of the present application;
  • FIG3-3 shows the effect of intratumoral injection of drug-metal-phospholipid complex particles (Mg 2+ ) in treating liver cancer provided in Example 3 of the present application;
  • Figure 4-1 is a statistical graph showing the percentage of CD19-CAR-positive cells for the targeted drug (CD19 CAR mRNA@Apt-MPP)(Fe 3+ ) and drug-metal-phospholipid particles (CD19 CAR mRNA@MPP)(Fe 3+ ) in Example 6.3 of the present application;
  • Figure 4-1-2 is a statistical graph showing the percentage of CD19-CAR-positive cells for the targeted drug (CD19 CAR mRNA@Apt-MPP)(Al 3+ ) and drug-metal-phospholipid particles (CD19 CAR mRNA@MPP)(Al 3+ ) in Example 6.3 of the present application;
  • Figure 4-1-3 is a statistical graph showing the percentage of CD19-CAR-positive cells for the targeted drug (CD19 CAR mRNA@Apt-MPP)(Mg 2+ ) and drug-metal-phospholipid particles (CD19 CAR mRNA@MPP)(Mg 2+ ) in Example 6.3 of the present application;
  • Figure 4-2 is a comparative diagram of the therapeutic effects of the targeted drug (CD19 CAR mRNA@Apt-MPP)(Fe 3+ ) and drug-metal-phospholipid particles (CD19 CAR mRNA@MPP)(Fe 3+ ) on mice with acute B-lymphocytic leukemia in Example 6.4 of the present application;
  • Figure 4-2-2 is a comparative diagram of the therapeutic effects of the targeted drug (CD19 CAR mRNA@Apt-MPP)(Al 3+ ) and drug-metal-phospholipid particles (CD19 CAR mRNA@MPP)(Al 3+ ) on acute B-lymphocytic leukemia mice in Example 6.4 of the present application;
  • Figure 4-2-3 is a comparative diagram of the therapeutic effects of the targeted drug (CD19 CAR mRNA@Apt-MPP)(Mg 2+ ) and drug-metal-phospholipid particles (CD19 CAR mRNA@MPP)(Mg 2+ ) on mice with acute B-lymphocytic leukemia in Example 6.4 of the present application;
  • Figure 5-1 is a comparison of the overall survival rates of mice treated with the targeted drugs CD19 CAR mRNA@Apt(CD8)-MPP(Fe 3+ ) and CD19 CAR mRNA@MPP(Fe 3+ ) in Example 7.1 of the present application against acute B-lymphocytic leukemia;
  • Figure 5-1-2 is a comparison of the overall survival rates of mice treated with the targeted drugs CD19 CAR mRNA@Apt(CD8)-MPP(Al 3+ ) and CD19 CAR mRNA@MPP(Al 3+ ) in Example 7.1 of the present application against acute B-lymphocytic leukemia;
  • Figure 5-1-3 is a comparison of the overall survival rates of mice treated with the targeted drugs CD19 CAR mRNA@Apt(CD8)-MPP(Mg 2+ ) and CD19 CAR mRNA@MPP(Mg 2+ ) in Example 7.1 of the present application;
  • Figure 5-2 is a comparison of the overall survival rates of lung cancer mice treated with the targeted drugs ASO@Apt-MPP(Fe 3+ ) and ASO@MPP(Fe 3+ ) in Example 7.2 of the present application;
  • Figure 5-2-2 is a comparison of the overall survival rates of lung cancer mice treated with the targeted drugs ASO@Apt-MPP(Al 3+ ) and ASO@MPP(Al 3+ ) in Example 7.2 of the present application;
  • Figure 5-2-3 is a comparison of the overall survival rates of lung cancer mice treated with the targeted drugs ASO@Apt-MPP(Mg 2+ ) and ASO@MPP(Mg 2+ ) in Example 7.2 of this application;
  • Figure 5-3 is a comparison of the overall survival rates of pancreatic cancer mice treated with the targeted drugs siRNA@Apt-MPP(Fe 3+ ) and siRNA@MPP(Fe 3+ ) in Example 7.3 of the present application;
  • Figure 5-3-2 is a comparison of the overall survival rates of pancreatic cancer mice treated with the targeted drugs siRNA@Apt-MPP(Al 3+ ) and siRNA@MPP(Al 3+ ) in Example 7.3 of the present application;
  • Figure 5-3-3 is a comparison of the overall survival rates of pancreatic cancer mice treated with the targeted drugs siRNA@Apt-MPP(Mg 2+ ) and siRNA@MPP(Mg 2+ ) in Example 7.3 of the present application;
  • Figure 6-1 is a comparative graph of the survival rates of mice treated with CD19 CAR mRNA@Apt(CD62L)-MPP and CD19 CAR mRNA@Apt(CD62L)-LNP against acute B-lymphocytic leukemia in Example 8.2 of the present application, using Fe 3+ , CD19 CAR as the drug, and CD62L as the aptamer;
  • Figure 6-1-2 is a comparative graph of the survival rates of mice treated with CD19 CAR mRNA@Apt(CD62L)-MPP and CD19 CAR mRNA@Apt(CD62L)-LNP against acute B-lymphocytic leukemia in Example 8.2 of the present application, using Al 3+ , CD19 CAR as the drug, and CD62L as the aptamer;
  • Figure 6-1-3 is a comparative graph of the survival rates of mice treated with CD19 CAR mRNA@Apt(CD62L)-MPP and CD19 CAR mRNA@Apt(CD62L)-LNP against acute B-lymphocytic leukemia in Example 8.2 of the present application, using Mg 2+ , CD19 CAR as the drug, and CD62L as the aptamer;
  • Figure 6-2 is a comparative graph showing the survival rates of anti-lung cancer mice treated with ASO@Apt(AS1411)-MPP and ASO@Apt(AS1411)-LNP when Fe 3+ is used, the drug is ASO, and the aptamer is AS1411 in Example 8.2 of the present application;
  • Figure 6-2-2 is a comparison of the survival rates of anti-lung cancer mice treated with ASO@Apt(AS1411)-MPP and ASO@Apt(AS1411)-LNP when Al 3+ , the drug is ASO, and the aptamer is AS1411 in Example 8.2 of the present application;
  • Figure 6-2-3 is a comparison of the survival rates of anti-lung cancer mice treated with ASO@Apt(AS1411)-MPP and ASO@Apt(AS1411)-LNP in Example 8.2 of the present application, using Mg 2+ , ASO as the drug, and AS1411 as the aptamer;
  • Figure 6-3 is a comparative graph showing the survival rates of pancreatic cancer mice treated with siRNA@Apt(P19)-MPP and siRNA@Apt(P19)-LNP, using Fe 3+ , siRNA as the drug, and P19 as the aptamer in Example 8.2 of the present application;
  • Figure 6-3-2 is a comparative graph of the survival rates of pancreatic cancer mice treated with siRNA@Apt(P19)-MPP and siRNA@Apt(P19)-LNP, when Al 3+ is used, the drug is siRNA, and the aptamer is P19, in Example 8.2 of the present application;
  • Figure 6-3-3 is a comparative graph of the survival rates of pancreatic cancer mice treated with siRNA@Apt(P19)-MPP and siRNA@Apt(P19)-LNP, when Mg 2+ is used, the drug is siRNA, and the aptamer is P19, in Example 8.2 of the present application;
  • Figure 7-1 is a comparison of the overall survival rates of Fe 3+ -positive CD19 CAR mRNA@MPP, CD19 CAR mRNA@Apt(CD62L)-MPP, CAR mRNA@Apt(CD8)-MPP, and CD19 CAR mRNA@Apt(CD3)-MPP anti-acute B lymphoblastic leukemia mice in Examples 6.4, 9.1, and 9.2 of the present application;
  • Figure 7-1-2 is a comparison of the overall survival rates of mice treated with Al 3+ CD19 CAR mRNA@MPP, CD19 CAR mRNA@Apt(CD62L)-MPP, CAR mRNA@Apt(CD8)-MPP, and CD19 CAR mRNA@Apt(CD3)-MPP against acute B-lymphocytic leukemia in Examples 6.4, 9.1, and 9.2 of the present application;
  • Figure 7-1-3 is a comparison of the overall survival rates of mice treated with Mg2 + -containing CD19 CAR mRNA@MPP, CD19 CAR mRNA@Apt(CD62L)-MPP, CAR mRNA@Apt(CD8)-MPP, and CD19 CAR mRNA@Apt(CD3)-MPP against acute B-lymphocytic leukemia in Examples 6.4, 9.1, and 9.2 of the present application;
  • Figure 8-1 is a graph showing the percentage of CAR-positive cells in myeloid cells for CD19 CAR mRNA@MPP (Fe 3+ , Al 3+ or Mg 2+ ) and CD19 CAR mRNA@LNP in Example 2.8 of the present application;
  • Figure 8-2 is a graph showing the percentage of CAR-positive cells in peripheral blood T cells of CD19 CAR mRNA@Apt-MPP (Fe 3+ , Al 3+ or Mg 2+ ) and CD19 CAR mRNA@Apt-LNP in Example 6.5 of the present application;
  • Figure 8-3 shows the CAR positivity rates of CD19 CAR mRNA@Apt-MPP(Ca 2+ ) and CD19 CAR mRNA@Apt-LNP(Ca 2+ ) modified with different target head ratios in Example 6.6;
  • Figure 8-4 shows the CAR positivity rates of CD19 CAR mRNA@Apt-MPP(Mg 2+ ) and CD19 CAR mRNA@Apt-LNP(Mg 2+ ) modified with different target head ratios in Example 6.6;
  • Figure 8-5 is a graph showing the percentage of CAR-positive cells in peripheral blood myeloid cells of CD19 CAR mRNA@Apt-MPP (Fe 3+ , Al 3+ or Mg 2+ ) and CD19 CAR mRNA@Apt-LNP in Example 6.5 of the present application.
  • lipid refers to a group of organic compounds that include, but are not limited to, lipids of fatty acids. They are generally divided into three categories: “simple lipids,” “compound lipids,” and “derivative lipids.” “Simple lipids” include fats and oils, as well as waxes; “compound lipids” include phospholipids and glycolipids; and “derivative lipids” include steroids.
  • ionizable lipid refers to a lipid containing a positively charged ionizable amine group that can be protonated to become positively charged at lower pH values but is 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, cerebrosides, 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-lauroylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic groups attached to neutral lipids.
  • phosphatidylglycerol cardiolipin
  • diacylphosphatidylserine diacylphosphatidic acid
  • N-lauroylphosphatidylethanolamine N-succinylphosphatidylethanolamine
  • N-glutarylphosphatidylethanolamine N-glutarylphosphatidylethanolamine
  • 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-dioleyloxy)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-hydroxyethylammonium bromid
  • phospholipid molecule portion refers to the structure originally belonging to the phospholipid molecule after the phospholipid molecule reacts with other substances.
  • phospholipid refers to lipids containing phosphate groups. These complex lipids are also known as phospholipids or phospholipids. Phospholipids are the primary components of biological membranes and are divided into two main groups: glycerophospholipids and sphingomyelins, composed of glycerol and sphingosine, respectively. Phospholipids are amphiphilic molecules, with a hydrophilic, nitrogen- or phosphorus-containing head at one end and a long, hydrophobic (lipophilic) hydrocarbon chain at the other. Because of this, phospholipid molecules are positioned close together at their hydrophilic and hydrophobic ends, often forming the phospholipid bilayer, the structure of the cell membrane, along with other molecules such as proteins, glycolipids, and cholesterol.
  • linker molecule refers to a structure that originally belongs to the linker molecule after the linker molecule reacts with other substances.
  • metal ion portion refers to the structure originally belonging to the metal ion after the metal ion reacts with other substances.
  • metal-phospholipid complex refers to a complex formed by the reaction of the above-mentioned phospholipid molecule portion having a phosphate group, the above-mentioned linker molecule portion, and the above-mentioned metal ion portion, wherein the above-mentioned phospholipid molecule portion is connected to the above-mentioned linker molecule portion, and the above-mentioned linker portion is connected to the above-mentioned metal ion portion through a coordination bond, and the metal-phospholipid complex is neither a cationic lipid nor an ionizable lipid.
  • drug-metal-phospholipid complex particles refers to drug-loaded metal-phospholipid complex particles, where the drug is connected to the metal-phospholipid complex by encapsulation, loading, chemical bonds or non-chemical bonds.
  • CD8 refers to a dimeric coreceptor that enables T cells to recognize peptides presented by histocompatibility complex class I proteins and is a surface marker protein for cytotoxic T lymphocytes (CTLs).
  • CTLs cytotoxic T lymphocytes
  • Perforins are cytolytic proteins that form pores in the cell membrane of target cells. CTLs use these pores to direct the release of granzymes, a class of serine proteases that continue apoptosis within the cytoplasm. These proteases shut down target cells by cleaving viral and cellular proteins involved in normal cell maintenance. Targeted cells undergoing apoptosis are then cleared by nearby phagocytes.
  • CD8+ T cells can also indirectly kill target cells by releasing cytokines such as TNF- ⁇ .
  • CD62L also known as L-selectin, refers to a family of genes that form leukocyte-endothelial cell adhesion molecules. Its extracellular domain contains a C-type lectin-like domain, an EGF-like domain, and two CCP domains at the N-terminus. L-selectin is constitutively expressed by leukocytes and mediates binding to oligosaccharide ligands expressed by vascular endothelial cells. CD62L is a family of vascular adhesion molecules with close structural and functional relationships. Its primary function is to promote the rolling behavior of leukocytes along endothelial cells prior to firm adhesion and subsequent migration.
  • targeting vector refers to a system with specific targeting and drug loading capabilities. Targeting vectors have the functions of improving the metabolic kinetics of drugs in the body, increasing the targeted accumulation of drugs to specific treatment sites or even specific cell types, improving efficacy and reducing toxic side effects. It can be a nanoparticle or a composite macromolecule. In this application, the targeting vector can be used to encapsulate CAR nucleic acid drugs,
  • lipid delivery system refers to a delivery technology for small or large molecules. It primarily utilizes the properties of lipids (such as phospholipids) to encapsulate and transport small or large molecules, effectively delivering them to target cells or tissues, thereby improving their bioavailability and therapeutic efficacy.
  • exosomes refers to a nanoscale vesicle secreted by cells, which contains bioactive substances such as proteins and nucleic acids (such as mRNA and miRNA).
  • virus refers to an artificially constructed virus-like particle. It typically combines key viral structures (such as envelope proteins) with non-viral nucleic acids (such as reporter genes). While it lacks the ability to replicate like a full virus, it can mimic some of the virus's biological behaviors, such as cell entry. This allows for applications in a wide range of fields, including studying viral infection mechanisms, vaccine development, and drug screening.
  • small molecule refers to low-molecular-weight substances that can regulate physiological processes in organisms. These small molecules can be endogenous, such as hormones produced by metabolic processes in the body (e.g., adrenaline), or exogenous, such as small drug molecules extracted from plants (e.g., artemisinin). They can exert their effects by interacting with biological macromolecules (e.g., proteins, nucleic acids), participating in processes such as signal transduction and enzyme inhibition or activation.
  • biological macromolecules e.g., proteins, nucleic acids
  • phospholipid transport system refers to a transport system constructed using phospholipid components, which is mainly used as a mechanism to transport various substances (such as drugs, nutrients, biomolecules, etc.) from one location to another.
  • LNP refers to a nanoscale colloidal delivery system formed by self-assembly of components such as cationic lipids or ionizable lipids, auxiliary lipids, cholesterol and pegylated lipids; among them, cationic lipids play a key role in mediating the encapsulation and cellular uptake of nucleic acids, and the positive charge carried by cationic lipids or ionizable lipids after ionization can bind to negatively charged nucleic acids (such as mRNA, siRNA, ASO, etc.) through electrostatic interaction, thereby effectively protecting the nucleic acids and promoting their delivery to target cells.
  • negatively charged nucleic acids such as mRNA, siRNA, ASO, etc.
  • aptamer or “aptamer” is derived from the Latin word “aptus” and is a single-stranded oligonucleotide (RNA) or single-stranded oligodeoxynucleotide (DNA) composed of 20-60 bases.
  • RNA oligonucleotide
  • DNA oligodeoxynucleotide
  • Aptamer is used to refer to a polymeric form of nucleotides of any length, ribonucleotides or deoxyribonucleotides.
  • the term includes but is not limited to single-stranded, double-stranded or multi-stranded DNA or RNA, DNA genome, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. It can specifically bind to a variety of target molecules such as proteins, small molecules, ions and cells. Aptamers are obtained by screening through the SELEX technology, which can be used to screen out nucleic acid aptamers (Aptamers) that specifically bind to the target with high affinity from a random single-stranded nucleic acid sequence library.
  • Aptamers nucleic acid aptamers
  • GalNAc refers to a partial structure containing N-acetyl-D-galactosamine (GalNAc) that is capable of binding to the asialoglycoprotein receptor (ASGPR) on hepatocytes of the liver.
  • GalNAc contains a phosphate group or a phosphorothioate group and is used to bind a linear or branched linker structure to an oligonucleotide.
  • Such a structure containing a phosphate group or a phosphorothioate group for binding may be referred to as "GalNAc.”
  • the number of GalNAc groups contained in a GalNAc is not limited and may be those known and disclosed in this specification.
  • GalNAc The structure of GalNAc may be modified as long as the ability to bind to ASGPR is maintained. In addition, GalNAc with a protecting group introduced during the preparation process is also included.
  • the term "GalNAc” has its conventional scientific meaning and refers herein to N-acetylgalactosamine and its IUPAC name: 2-(acetylamino)-2-deoxy-D-galactose.
  • P19 refers to a nucleic acid sequence that can specifically recognize pancreatic cancer cells.
  • targeted drug also known as targeted preparation refers to a drug or its preparation that is endowed with targeting ability.
  • transmembrane domain is a membrane-spanning sequence of a CAR that can be designed to include a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain.
  • the transmembrane domain can be any protein structure that is thermodynamically stable in the membrane, typically an alpha helix containing several hydrophobic residues.
  • signal domain refers to a sequence encoding a signal peptide or a sequence encoding a substance that plays the same role as a signal peptide.
  • Signal domain refers to a functional part of a protein that plays a role by transmitting information within the cell, thereby regulating cell activity via a determined signaling pathway by producing a second messenger or by responding to such a messenger as an effector.
  • the signal peptide allows the nascent protein to be directed to the endoplasmic reticulum and then to the surface of the cell where it is expressed when the CAR is expressed in a cell such as a T cell.
  • the core of the signal peptide can contain a long stretch of hydrophobic amino acids that have a tendency to form a single ⁇ -helix.
  • antigen binding domain generally refers to a domain that can specifically bind to an antigen, a chimeric antigen receptor domain or fragment that has the ability to specifically bind to a target antigen.
  • the antigen binding domain is the antigen-recognizing portion of CAR.
  • Many antigen binding domains are known in the art, including those based on the antigen binding sites of antibodies, antibody mimics, and T cell receptors.
  • co-stimulatory signal transduction domain refers to the intracellular signal transduction domain from co-stimulatory protein receptors such as CD28, 41BB and ICOS that can enhance T cell activation through T cell receptors.
  • Co-stimulatory signal transduction domain refers to the domain in the CAR molecule that is responsible for providing a second activation signal to T cells, mainly including the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD27).
  • the main function of the co-stimulatory domain is to provide a second activation signal, synergistically stimulate molecules and activate intracellular signals, so that T cells continue to proliferate and release cytokines, thereby improving the anti-tumor ability of T cells.
  • antigen-binding domain-transmembrane domain linking region refers to the region responsible for connecting the antigen-binding domain and the transmembrane domain.
  • a spacer domain such as an oligopeptide or polypeptide that functions to connect the transmembrane domain to the extracellular domain or cytoplasmic domain in the polypeptide chain, may be incorporated between the extracellular domain and the transmembrane domain of the chimeric membrane protein, or between the cytoplasmic domain and the transmembrane domain of the chimeric membrane protein.
  • the spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.
  • CAR chimeric antigen receptor
  • CAR molecules consist of three main parts: the extracellular domain, the transmembrane domain, and the intracellular domain.
  • the antigen-binding domain in the extracellular domain is usually a single-chain fragment (scFv) molecule derived from an antibody. It is mainly composed of the variable light chain (VL) and variable heavy chain (VH) of the antibody, connected by the linker region in the middle, and then connected to the transmembrane domain through the hinge region, which is responsible for antigen recognition.
  • VL variable light chain
  • VH variable heavy chain
  • the main function of the transmembrane domain is to anchor the CAR molecule to the cell membrane, which plays an important role in the stability of CAR molecule expression.
  • the intracellular domain includes the costimulatory domain and the signal transduction domain, which work together to fully activate T cells.
  • T cell receptor refers to a complex of membrane proteins that participates in the activation of T cells in response to antigen presentation. It is composed of variable region (V region) and constant region (C region) domains, of which the variable regions of the ⁇ chain and ⁇ chain constitute the antigen recognition unit, and each variable region contains three complementarity determining regions (CDR1, CDR2 and CDR3).
  • V region variable region
  • C region constant region
  • CDR1, CDR2 and CDR3 three complementarity determining regions
  • CDR3 is the most critical sequence that determines the specificity of TCR recognition of antigens.
  • TCR stimulation is triggered by the major histocompatibility complex molecules (MHC) on antigen-presenting cells, which present the antigen peptide to the T cell and bind to the TCR complex to induce a series of intracellular signaling cascades.
  • MHC major histocompatibility complex molecules
  • targeted immune cell drugs refers to a class of drugs specifically designed to act on immune cells. These drugs include: gene editing drugs (such as Crisp cas9, CRISPR-Cas, etc.), antibody drugs (such as monoclonal antibodies, bispecific antibodies), immune bridging therapy drugs (such as TCE (T-cell Engager), ADC (Antibody-Drug Conjugate), APDC (Antibody-Peptide-Drug Conjugate), etc.), targeted therapy drugs (including chemotherapy or radiotherapy) (such as ADC (Antibody-Drug Conjugate), APDC (Antibody-Peptide-Drug Conjugate), etc.), aptamer drugs (such as bispecific aptamers), and cell therapy drugs (such as CAR-T (Chimeric Antigen Receptor T-cell), TCR-T (T-cell Receptor Engineered T-cell), etc.). These drugs can accurately identify specific targets on the surface or inside immune cells, and intervene in the body's immune response by genetically modifying immune cells or
  • CAR chimeric antigen receptor
  • CAR chimeric antigen receptor
  • CAR is a synthetic receptor whose structure includes a single-chain antibody variable region (scFv) that can recognize tumor antigens, a transmembrane region, and an intracellular signal transduction region.
  • CAR drugs are obtained by genetically engineering various immune cells (such as T cells, macrophages, myeloid cells, NK cells, etc.) to obtain CAR-immune cells (such as CAR-T cells, CAR-macrophages, CAR-myeloid cells, CAR-NK cells, etc.) to exert their therapeutic effects.
  • in situ CAR refers to the in vivo generation of CAR-expressing cells based on the use of mRNA technology to recode the T cell receptor (CCR) using a CAR nucleic acid sequence encoded by mRNA.
  • the CAR comprises an antigenic domain, a transmembrane domain, a co-stimulatory signaling region, and a signaling domain.
  • in situ CAR refers to the in vivo conversion of T cells into CAR-T cells using mRNA or a combination of mRNAs encoding the CAR gene as the antigen. Compared to traditional in vitro CAR-T, the production process is greatly simplified and the cost is significantly reduced.
  • CAR chimeric antigen receptor for tumor
  • messenger RNA as a carrier carrying the chimeric antigen receptor (CAR)
  • the CCR is transferred into the patient's T cells, where it is activated and equipped with a CAR (chimeric antigen receptor for tumor) targeting and navigation device, transforming these ordinary “soldiers" into “super soldiers,” or CAR-T cells.
  • CAR chimeric antigen receptor for tumor
  • These cells utilize their CAR "targeting and navigation device” to specifically identify tumor cells in the body and, through immune action, release a large number of multiple effector factors that effectively kill tumor cells, thereby achieving the goal of treating malignant tumors.
  • lipid vesicle refers to any lipid composition that can be used to deliver a compound, including, but not limited to, liposomes, in which an aqueous volume is encapsulated by an amphiphilic lipid bilayer; or in which lipids encapsulate an interior containing macromolecular components, such as mRNA, with a reduced aqueous interior; or lipid aggregates or micelles, in which the encapsulated components are contained in a relatively chaotic lipid mixture.
  • metal-phospholipid complex particles are "lipid vesicles," and drugs, such as nucleic acid mRNA, are encapsulated in MPPs as encapsulated components, and the “encapsulation” can be full encapsulation and/or partial encapsulation.
  • hydrophobic lipid refers to a compound having a non-polar group, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and these groups are optionally substituted with one or more aromatic, alicyclic or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerols, dialkylglycerols, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane and 1,2-dialkyl-3-aminopropane.
  • non-cationic lipid or non-ionizable lipid refers to a lipid that is neither a cationic lipid nor a non-ionizable lipid, and may be, for example, an anionic lipid or a neutral lipid.
  • the "non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation" in (iii) means that the non-cationic lipids or non-ionizable lipids in (iii) are the lipids remaining in the metal-phospholipid complex particles after excluding the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation.
  • the term "fusogenicity” refers to the ability of a liposome, drug-lipid particle, targeted drug or other drug delivery system to fuse with a cell membrane.
  • the membrane may be a plasma membrane or a membrane surrounding a cell organelle, such as an endosome, nucleus, or the like.
  • non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation are primarily present as vesicle-forming lipids, and the term "vesicle-forming lipid" is intended to include any amphiphilic lipid having a hydrophobic portion and a polar head group and which can spontaneously form bilayer vesicles in water, exemplified by most phospholipids.
  • vesicle-adopting lipid is intended to include any amphiphilic lipid that stably associates with the lipid bilayer, as well as other amphiphilic lipids whose hydrophobic portion contacts the inner, hydrophobic region of the bilayer membrane and whose polar head group portion faces the outer, polar surface of the membrane.
  • Vesicle-adopting lipids include lipids that are capable of independently adapting to adopt a non-lamellar phase and are also capable of adopting a bilayer structure in the presence of a bilayer-stabilizing component.
  • Conjugated lipids that inhibit particle aggregation include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid derivatives), peptides, proteins, detergents, lipid derivatives, PEG-lipid derivatives such as PEG conjugated to dialkoxypropyl groups, PEG conjugated to diacylglycerols, PEG conjugated to phosphatidylethanolamine, and PEG conjugated to ceramides (see, U.S. Patent No. 5,885,613, incorporated herein by reference).
  • polyamide oligomers e.g., ATTA-lipid derivatives
  • peptides e.g., proteins
  • detergents lipid derivatives
  • PEG-lipid derivatives such as PEG conjugated to dialkoxypropyl groups
  • PEG conjugated to diacylglycerols PEG conjugated to phosphatidylethanolamine
  • PEG conjugated to ceramides see, U.S. Patent No. 5,885,613,
  • amphiphilic lipid refers to any suitable material, wherein the hydrophobic portion of the lipid material is oriented toward the hydrophobic phase, while the hydrophilic portion is oriented toward the aqueous phase.
  • Amphiphilic lipids are typically the main components of lipid vesicles.
  • the hydrophilic nature comes from the presence of polar or charged groups such as carbohydrates, phosphates, carboxyls, sulfates, aminos, sulfhydryls, nitros, hydroxyls, 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 such groups substituted by one or more aromatic, alicyclic, or heterocyclic groups.
  • non-polar groups including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, alicyclic, or heterocyclic groups.
  • amphiphilic compounds include, but are not limited to, phospholipids, amino lipids, and sphingolipids.
  • phospholipid include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine.
  • amphipathic lipid such as sphingomyelin, sphingosylglycolipid family, diacylglycerol and beta-acyloxy acid.
  • amphipathic lipid can be mixed with other lipid, and this lipid comprises triglyceride and sterol.
  • diacylglycerol refers to a compound having a 2-fatty acyl chain, wherein R1 and R2 each independently have 2 to 30 carbon atoms bonded to the 1- and 2-positions of glycerol via ester bonds.
  • the acyl groups may be saturated or have varying degrees of unsaturation.
  • Diacylglycerol has the following formula 54:
  • diacylglycerol-coupled polyethylene glycol refers to the conjugated lipid that inhibits particle aggregation in the present application and can be diacylglycerol-coupled polyethylene glycol, i.e., a diacylglycerol-polyethylene glycol conjugate (DAG-PEG conjugate or PEG-DAG conjugate).
  • the DAG-PEG conjugate is a dilaurylglycerol (C12)-PEG conjugate, a ditetradecylglycerol (C14)-PEG conjugate (DMG), a dipalmitoylglycerol (C16)-PEG conjugate, or a distearylglycerol (C18)-PEG conjugate (DSG).
  • DMG ditetradecylglycerol
  • DMG dipalmitoylglycerol
  • C16 dipalmitoylglycerol
  • DSG distearylglycerol
  • Suitable DAG-PEG conjugates for use in this application 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 of which are incorporated by reference.
  • dialkoxypropyl refers to a compound having a 2-alkyl chain, wherein R1 and R2 each independently have 2 to 30 carbon atoms.
  • the alkyl group may be saturated or have varying degrees of unsaturation.
  • Dialkoxypropyl has the following formula 55:
  • dialkoxypropyl-coupled PEG refers to a lipid conjugated to inhibit particle aggregation in the present application, which can 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 can be saturated or unsaturated.
  • Suitable alkyl groups include, but are not limited to, lauryl (C12), tetradecyl (C14), hexadecyl (C16), octadecyl (C18) and icosyl (C20).
  • R1 and R2 are the same, i.e., R1 and R2 are both tetradecyl (i.e., dioctadecyl), R1 and R2 are both octadecyl (i.e., dioctadecyl), etc.
  • the PEG is a polyethylene glycol having an average molecular weight of from about 550 to about 10,000 daltons and is optionally substituted at the terminal hydroxyl position by an alkyl, alkoxy, acyl, or aryl group.
  • the PEG has an average molecular weight of from about 1,000 to about 5,000 daltons, more preferably, an average molecular weight of from about 1,000 to about 3,000 daltons and even more preferably, an average molecular weight of about 2,000 daltons.
  • the PEG can be optionally substituted with an alkyl, alkoxy, acyl, or aryl group.
  • L is a linker moiety. Any linker moiety suitable for coupling the PEG to the dialkoxypropyl backbone can be used.
  • Suitable linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbonate (O-C(O)O-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), succinyl (-(O)CCH2CH2C(O)-), ethers, disulfides, and combinations thereof.
  • Other suitable linkers are well known in the art.
  • Phosphatidylethanolamine can be conjugated to polyethylene glycol to form a bilayer stabilizing component as a conjugated lipid that inhibits particle aggregation in the present application.
  • the phosphatidylethanolamine has various acyl chain groups with different chain lengths and degrees of saturation. 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 are preferred, having a carbon chain length in the range of C10-C20. Phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used.
  • Suitable phosphatidylethanolamines include, but are not limited to, the following: dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidylethanolamine (DSPE).
  • DMPE dimyristoylphosphatidylethanolamine
  • DPPE dipalmitoylphosphatidylethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • DSPE distearoylphosphatidylethanolamine
  • ceramide can be coupled with polyethylene glycol to form a bilayer stabilizing component as a conjugated lipid to inhibit particle aggregation in the present application.
  • the ceramide has multiple acyl chain groups with different chain lengths and degrees of saturation. It should be clear to those skilled in the art that, compared to phosphatidylethanolamine, ceramide has only one acyl group, which can be easily varied according to its chain length and degree of saturation.
  • Ceramides suitable for use in accordance with the present application are commercially available.
  • ceramides can be isolated, for example, from eggs and brain using well-known separation techniques, or synthesized using the methods and techniques disclosed in U.S. Patent No.
  • ceramides with saturated or unsaturated fatty acids can be prepared, wherein the fatty acids have carbon chain lengths ranging from C2 to C31.
  • ATTA or "polyamide” refers to, but is not limited to, compounds disclosed in U.S. Patent Nos. 6,320,017 and 6,586,559, both of which are incorporated herein by reference. These compounds include compounds having the following formula 57:
  • 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 bound form an azido moiety
  • R2 is a member selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, and an amino acid side chain
  • R3 is a member selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, thiol, 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 refer to a single-stranded polyribonucleotide that is transcribed from a strand of DNA as a template, carries genetic information, and can guide protein synthesis.
  • sgRNA small guide RNA
  • guide RNA guide RNA
  • gRNA RNA molecules, approximately 60-80 nucleotides in length, that are transcribed from separate genes.
  • RNA refers to polyribonucleotide molecules having a structure without free ends (i.e., without free 3’ and/or 5’ ends), such as polyribonucleotides that form a ring or annular structure through covalent or non-covalent bonds.
  • microRNA or “miRNA” or “microRNA” are used interchangeably and refer to a non-coding single-stranded polyribonucleotide with a length of about 22 nucleotides and free 3' and 5' ends, which can inhibit the translation of target gene protein by binding to the 3'-untranslated region (3'-UTR) of the target gene mRNA and thus regulate the biological function of the cell.
  • 3'-UTR 3'-untranslated region
  • ASO antisense oligonucleotide
  • antisense oligonucleotide refers to artificially synthesized nucleic acid fragments that are complementary to a certain segment of the target gene or mRNA, and can bind to the target gene/mRNA through the principle of base complementarity, thereby blocking the expression of the gene. It is a single-stranded poly (deoxy) ribonucleotide, including antisense DNA and antisense RNA.
  • RNA small interfering or “short interfering” or “silencing RNA” or “small interfering RNA” or “short interfering RNA” or “silencing RNA” are used interchangeably and refer to a class of double-stranded RNA molecules with a length of 20 to 25 nucleotides that can induce the degradation of target gene mRNA.
  • ecDNA or "extrachromosomal circular DNA” are used interchangeably and refer to DNA that has fallen off chromosomes and exists in a circular structure.
  • nucleic acid derivative refers to a modification or replacement of a nucleic acid sequence, including but not limited to chemical modification of residues, replacement of nucleotides or deoxynucleotides, modification of the sequence to increase half-life or stability, and labeling modification.
  • chemical modification includes but is not limited to phosphorylation, methylation, amination, sulfhydrylation, replacement of oxygen with sulfur, replacement of oxygen with selenium, or isotopization of any one or more bases.
  • Replacement of nucleotides or deoxynucleotides includes but is not limited to nucleic acid analogs that replace the sugar-phosphate backbone with polypeptides or other backbones (replacing DNA or RNA with PNA).
  • Modifications to sequences that increase half-life or stability include but are not limited to modification with PEG linkage and fluorine modification.
  • Labeling modification includes but is not limited to connection to fluorescent groups, amino groups, biotin, digoxigenin, small peptides, etc.
  • artificial nucleic acid refers to a nucleic acid molecule that has been artificially modified, including but not limited to base modifications, ribose modifications, PNA, etc.
  • nucleic acid refers to a polymer that exists in a single or double-stranded form and comprises at least two deoxynucleotides or nucleotides. Unless specifically limited, the term encompasses nucleic acids comprising known analogs of natural nucleotides that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise noted, specific nucleic acid sequences also implicitly encompass variants (e.g., degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences, as well as sequences clearly indicated.
  • degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (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)).
  • a "nucleotide” comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group.
  • Bases include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that substitute new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.
  • DNA can be present as antisense, plasmid DNA, portions of plasmid DNA, pre-compressed DNA, products of polymerase chain reaction (PCR), vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups.
  • PCR polymerase chain reaction
  • P1, PAC, BAC, YAC, artificial chromosomes vectors
  • expression cassettes chimeric sequences, chromosomal DNA, or derivatives of these groups.
  • 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 necessary for the production of a polypeptide or polypeptide precursor (e.g., a polypeptide or polypeptide precursor from hepatitis A, B, C, D, E, G virus; or herpes simplex virus).
  • a polypeptide or polypeptide precursor e.g., a polypeptide or polypeptide precursor from hepatitis A, B, C, D, E, G virus; or herpes simplex virus.
  • gene product refers to the product of a gene such as, but not limited to, the transcript of DNA, mRNA.
  • expression silencing of target gene refers to the ability of the siRNA of the present application to start silencing the target gene.
  • a sample or assay of the cells in the target organism or culture is compared with a control sample, wherein the cells of the target organism or culture express a specific construct and the control does not express the construct.
  • the control sample (lack of construct expression) is set to a relative value of 100%. When the test value relative to the control is about 90%, preferably 50%, more preferably 25-0%, the inhibition of the expression of the target gene is successfully obtained.
  • Suitable assays include, for example, using techniques known to those skilled in the art such as dot blots, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme action, and 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 the desired effect, eg, a decrease in expression of the target sequence as compared to normal expression levels detected in the absence of the siRNA.
  • aqueous solution refers to a composition that comprises, in whole or in part, water.
  • organic lipid solution refers to a composition comprising 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 certain compounds, but not other compounds. Systemic delivery refers to contact of an effective, preferably therapeutic, amount of a compound with most of the body. In order to achieve widespread biodistribution, a blood survival period is generally required so that the compound is not rapidly degraded or cleared (such as by initial passage through organs (liver, lungs, etc.) or by rapid, non-specific cell binding) before reaching the disease site distal to the site of administration.
  • Systemic delivery of targeted drugs can be performed in any manner known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of targeted drugs is by intravenous delivery.
  • local delivery refers to the delivery of a compound directly to a target site in the body.
  • a compound can be delivered locally by direct injection into a disease site such as a tumor or other target site such as an inflammatory site or a target organ such as the liver, heart, pancreas, kidney, etc.
  • the RNA population can be used to provide long precursor RNAs, or long precursor RNAs having substantial or complete identity to a selected target sequence that can be used to prepare siRNA.
  • the RNAs can be isolated, synthesized, and/or cloned from cells or tissues according to methods well known to those skilled in the art.
  • the RNA can be a mixed population (obtained from cells or tissues, transcribed from cDNA, etc.), or can represent a single target sequence.
  • the RNA can be naturally occurring, for example, isolated from a tissue or cell sample, for example, synthesized in vitro using T7 or SP6 polymerase and PCR products or cloned cDNA; or chemically synthesized.
  • complements can also be transcribed in vitro and hybridized to form dsRNAs.
  • RNA complements are also provided (e.g., to form dsRNAs, which are digested by E. coli RNAse III or Dicer).
  • the precursor RNAs are then hybridized 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 within nucleic acid-lipid particles.
  • siRNAs based on the naturally occurring transcription units of small nuclear RNA U6 or human RNase P RNA H1 can be transcribed as sequences that automatically fold into duplexes with hairpin loops from DNA templates in plasmids harboring RNA polymerase III transcription units (see, Brummelkamp, et al., Science 296:550 (2002); Donzé, et al., Nucleic Acids Res. 30:e46 (2002); Paddison, et al., Genes Dev. 16:948 (2002); Yu, et al., Proc. Natl.
  • the transcription unit or cassette will contain an RNA transcription promoter sequence, such as the H1-RNA or U6 promoter, operably linked to a template for transcription of the desired siRNA sequence and a termination sequence comprising 2-3 uridine residues and a polythymidine (T5) sequence (polyadenylation signal) (Brummelkamp, Science, supra).
  • the selected promoter can provide constitutive or inducible transcription.
  • Compositions and methods for transcription of DNA-guided 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 in vivo delivery of genetic material 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 a plasmid initially designed to express a desired gene sequence can be modified to include 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., supra), as are 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.
  • the cis-trans isomers of the phospholipid molecule of the present application will not affect the effect to be achieved by the protection content of the present application.
  • the phospholipid molecule portion is selected from phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphoceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysophosphatidylcholine LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, 1-phosphate sphingosine S1P, and a combination of one or more of their derivatives.
  • the “their” in “and their derivatives” refers to “phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), 1-phosphoceramide (SP), phosphatidylinositol (PI), phosphatidylthreonine (PT), sphingomyelin (SM), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysosphingomyelin (LSM), and 1-sphingosine 1-phosphate (S1
  • the phospholipid molecule portion can be, for example, but not limited to, selected from phosphatidylcholine (PC), phosphatidylcholine (PC) derivatives, phosphatidylethanolamine (PE), phosphatidylethanolamine (PE) derivatives, phosphatidylglycerol (PG), phosphatidylglycerol (PG) derivatives, phosphatidylglycerol (PG) and phosphatidylcholine (PC), phosphatidylcholine (PC) and phosphatidylcholine (PC) derivatives, etc.
  • PC phosphatidylcholine
  • PC phosphatidylcholine
  • PC phosphatidylcholine
  • PC phosphatidylcholine
  • PC phosphatidylcholine
  • PC phosphatidylcholine
  • PC phosphatidylcholine
  • PC phosphatidylcholine
  • PC phosphatidylcholine
  • PC
  • the phospholipid molecule portion is selected from phosphatidylcholine (PC) (Formula 1), phosphatidylethanolamine (PE) (Formula 2), phosphatidylserine (PS) (Formula 3), phosphatidic acid (PA) (Formula 4), phosphatidylglycerol (PG) (Formula 5), 1-phosphoceramide (SP) (Formula 6), phosphatidylinositol (PI) (Formula 7), phosphatidylthreonine (PT) (Formula 8), sphingomyelin (SM) (Formula 9), hemolytic phosphatidylcholine (LPC) (Formula 10), hemolytic phosphatidylethanolamine (LPE) (Formula 11), hemolytic phosphatidylserine (LPS) (Formula 12), hemolytic phosphatidic acid (LPA) (Formula 13), hemolytic phosphatidyl
  • PC
  • the phospholipid molecule is selected from at least one of phosphatidylcholine (PC) (Formula 1), phosphatidylethanolamine (PE) (Formula 2), phosphatidic acid (PA) (Formula 4), phosphatidylglycerol (PG) (Formula 5), and derivatives thereof.
  • the phospholipid molecule is selected from at least one of DSPC, DSPE, DSPA, DSPG, and derivatives thereof.
  • the phospholipid molecule is selected from at least one of DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48), DSPG (Formula 49), and derivatives thereof.
  • the linker molecule As for the linker molecule, it 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. In addition, it is also an effective immunomodulator that can regulate the activity of various immune cells such as T cells, B cells, macrophages, neutrophils, natural killer cells and dendritic cells, promote the balance of immunity, and enhance the body's immunity. Based on the potential immune-enhancing, anti-inflammatory, antioxidant and anti-SARS-CoV-2 effects of curcumin molecules, it is expected to become a potential auxiliary treatment for COVID-19. In addition, the safety of curcumin molecules is extremely high and has been listed in the catalog of food additives and pharmaceutical excipients. Its safety is conducive to the overall clinical drug registration of targeted drugs and shortens the length of clinical drug registration.
  • the linker molecule portion is selected from curcumin, chlorogenic acid, anthocyanidin, quercetin, dihydromyricetin, hesperetin, naringenin, apigenin, catechin, tea polyphenols, epigallocatechin gallate, ellagic acid, morin, epicatechin gallate, catechin gallate, epigallocatechin gallate or picarapine C, and a combination of one or more of their derivatives.
  • the linker molecule is selected from at least one of curcumin (Formula 19), chlorogenic acid (Formula 20), anthocyanin (Formula 21), quercetin (Formula 22), dihydromyricetin (Formula 23), hesperetin (Formula 24), naringenin (Formula 25), apigenin (Formula 26), catechin (Formula 27), tea polyphenols (Formula 28), epigallocatechin gallate (Formula 29), ellagic acid (Formula 30), morin (Formula 31), epicatechin gallate (Formula 32), catechin gallate (Formula 33), gallocatechin gallate (Formula 34), flavonoid C (Formula 35), and derivatives thereof.
  • curcumin Formmula 19
  • chlorogenic acid Formula 20
  • anthocyanin Formmula 21
  • quercetin Formmula 22
  • dihydromyricetin Formmula 23
  • the linker molecule is selected from at least one of curcumin (Formula 19), dihydrocurcumin (Formula 36), hexahydrocurcumin (Formula 37), curcumin sulfate (Formula 38), and bisdemethoxycurcumin (Formula 39).
  • the linker molecule is selected from at least one of curcumin (Formula 19), hesperetin (Formula 24), tea polyphenols (Formula 28), and derivatives thereof.
  • the linker molecule is selected from curcumin (Formula 19), hesperetin (Formula 24), or tea polyphenols (Formula 28).
  • the metal ion moiety is selected from at least one of 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+ , and Zr 3+ .
  • the metal ion moiety is selected from at least one of Fe 3+ , Mg 2+ , Ca 2+ , and Al 3+ .
  • the metal ion moiety is selected from Fe 3+ , Mg 2+ , Ca 2+ , or Al 3+ .
  • the ratio of each component in the metal-phospholipid complex can be adjusted based on the structure of the specific metal-phospholipid complex components. This adjustment is based on the following: Because phospholipid molecules and linker molecules are linked by hydrogen bonds, as long as the phospholipid molecule contains multiple phosphate groups, the ratio of phospholipid to linker molecules can be adjusted based on the number of phosphate groups contained in the phospholipid molecule during phospholipid complex synthesis. For example, when the phospholipid molecule contains two phosphate groups, the ratio of phospholipid to linker molecules can be adjusted to 1:2; when the phospholipid molecule contains three phosphate groups, the ratio of phospholipid to linker molecules can be adjusted to 1:3.
  • the ratio of linker to metal ion can be adjusted based on the number of binding sites contained in the linker, as long as the linker molecule contains multiple binding sites.
  • the metal ion functions to connect the phospholipid complex to the nucleic acid. Therefore, when the linker molecule is attached to as few complexing sites as possible for the metal ion, the metal-phospholipid complex can maximize nucleic acid encapsulation.
  • the phospholipid molecule is selected from DSPC, DSPE, DSPA, or DSPG; the linker molecule is selected from curcumin, hesperetin, or tea polyphenols; and the metal ion is selected from Fe 3+ , Mg 2+ , Ca 2+ , or Al 3+ .
  • the metal-phospholipid complex is composed of a phospholipid molecule, a linker molecule, and a metal ion;
  • the phospholipid molecule is selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48), or DSPG (Formula 49);
  • the linker molecule is selected from curcumin (Formula 19), hesperetin (Formula 24), or tea polyphenols (Formula 28); and the metal ion is selected from Fe 3+ , Mg 2+ , Ca 2+ , or Al 3+ .
  • the molar ratio of the phospholipid molecule, the linker molecule, and the metal ion is 1:(1-3):(0.5-2).
  • the molar ratio of the phospholipid molecule portion, the linker molecule portion and the metal ion portion can be, but is not limited to, 1:1:0.5, 1:1:1, 1:1:1.5, 1:1:2, 1:2:1, 1:2:2, 1:2:0.5, 1:3:0.5, 1:3:1 or 1:3:2.
  • the phospholipid molecule portion is DSPC (Formula 46), the linker molecule portion is selected from curcumin (Formula 19), the metal ion portion is selected from Fe 3+ , Mg 2+ or Al 3+ , and the molar ratio of the phospholipid molecule portion, the linker molecule portion and the metal ion portion is 1:1:1.
  • the metal-phospholipid complex can be prepared by a "one-step method” or by a “step-by-step method”.
  • the "one-step method” and the “step-by-step method” have equivalent capabilities for constructing metal-phospholipid complexes.
  • the "one-step method” can significantly shorten the time required for the industrialization process, which is crucial in the scenario of industrial large-scale production.
  • the “one-step method” reduces the problems of process connection and time intervals brought about by step-by-step operations, greatly improving the continuity and compactness of production, thereby effectively reducing production costs and improving production efficiency. From the perspective of industrial application, the "one-step method” is more in line with the needs of large-scale production, and has broad application prospects and extremely high practical value.
  • phospholipid molecules, linker molecules, and metal ions react to form a metal-phospholipid complex.
  • the phospholipid molecules, linker molecules, and metal ions are dissolved in ethanol for reaction.
  • the molar ratio of the phospholipid molecules, linker molecules, and metal ions is preferably 1:1:(1-2).
  • the reaction conditions are preferably 40-60° C. for 1-5 hours.
  • the molar ratio of the phospholipid molecules, linker molecules, and metal ions can be, but is not limited to, 1:1:1, 1:1:1.5, or 1:1:2.
  • the reaction temperature can be, but is not limited to, 40° C., 42° C., 46° C., 48° C., 50° C., 52° C., 54° C., 56° C., 58° C., or 60° C.
  • the reaction time can be, but is not limited to, 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours.
  • the phospholipid molecules are first reacted with the linker molecules to form a phospholipid complex; then the prepared phospholipid complex is reacted with the metal ion through a coordination bond to form a metal-phospholipid complex.
  • the phospholipid molecules and the linker molecules are dissolved in ethanol for reaction, and then n-hexane is added for precipitation to obtain a phospholipid complex.
  • the molar ratio of the phospholipid molecules to the linker molecules is preferably 1:1; the reaction conditions are preferably 65° C. for 2 hours.
  • the phospholipid complex and the metal ion are dissolved in ethanol, and triethylamine is added to react to obtain a metal-phospholipid complex.
  • the molar ratio of the phospholipid complex to the metal ion is preferably 1: (1-2), and the molar ratio of the phospholipid complex to triethylamine is preferably 1: 1.
  • the reaction conditions are preferably 60 ° C for 2 hours.
  • MPP Metal-chelated phospholipid complex nanoparticles
  • the metal-phospholipid complex particles contain: (i) a metal-phospholipid complex; (ii) a conjugated lipid that inhibits particle aggregation, wherein the conjugated lipid that inhibits particle aggregation 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.
  • Conjugated lipids that inhibit particle aggregation refer to conjugated lipids that inhibit the aggregation of drug-metal-phospholipid complex particles or targeted drugs. Their primary function is to prevent the aggregation of drug-metal-phospholipid complex particles or targeted drugs. Examples include PEG conjugated to dialkoxypropyl groups, PEG conjugated to diacylglycerols, PEG conjugated to phosphatidylethanolamines, and PEG conjugated to ceramides, preferably PEG-lipid conjugates. Cis- and trans-isomers of the lipids do not affect the effects to be achieved in the present application.
  • the conjugated lipid that inhibits particle aggregation includes a PEG-lipid conjugate and/or PEG-DAA.
  • the PEG-lipid conjugate is selected from at least one of phosphatidylethanolamine-polyethylene glycol 2000 (Formula 42), phosphatidylethanolamine-polyethylene glycol 700 (Formula 43), phosphatidylethanolamine-polyethylene glycol 1000 (Formula 44), phosphatidylethanolamine-polyethylene glycol 5000 (Formula 45), and derivatives thereof.
  • the PEG-lipid conjugate is selected from at least one of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000, or DSPE-PEG5000.
  • 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 at least one of cholesterol (preferably cholesterol formula 40) and its derivatives.
  • it further comprises a combination of one or more selected from phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphoceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysophosphatidylcholine LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidy
  • the non-cationic lipid or non-ionizable lipid in (iii) includes cholesterol, and a combination of one or more selected from DSPC, DSPE, DSPA or DSPG, for example, the non-cationic lipid or non-ionizable lipid in (iii) includes cholesterol (Formula 40) and DSPC (Formula 46).
  • the metal-phospholipid complex as an integral component reacts with component (ii) and component (iii) to self-assemble to obtain metal-phospholipid complex particles.
  • the metal-phospholipid complex particles can be used as drug carriers, wherein the drug is a negatively charged drug, such as a nucleic acid.
  • the principle of loading the drug into the metal-phospholipid complex particles assembled by the metal-phospholipid complex is as follows: the linker molecule and the phospholipid molecule are bound together by hydrogen bonds, and the linker molecule is connected to the metal ion through a coordination bond to form a metal-phospholipid complex.
  • the metal ion of the metal-phospholipid complex is connected to the negatively charged drug through a coordination bond, thereby ensuring that the metal-phospholipid complex and other components (conjugated lipids that inhibit particle aggregation, and non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation) self-assemble into MPPs, while the negatively charged drug is loaded into the nanoparticle MPPs to obtain drug-metal-phospholipid complex particles.
  • non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation refers to component (iii) in the metal-phospholipid complex particles, which can be simply referred to as “non-cationic lipids or non-ionizable lipids”.
  • the proportions of the components in the metal-phospholipid complex particles can be as follows: the metal-phospholipid complex comprises 5% to 50% by mole of the raw material, the conjugated lipid that inhibits particle aggregation comprises 1% to 10% by mole of the raw material, the cholesterol comprises 15% to 80% by mole of the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol comprises 0% to 51% by mole of the raw material.
  • “raw material” refers to the sum of the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, cholesterol, and the non-cationic lipid or non-ionizable lipid other than cholesterol.
  • the molar proportion of the metal-phospholipid complex in the raw material can be, but is not limited to, 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 5% to 40%, preferably 10% to 40%.
  • the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material can be, but is not limited to, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In some embodiments, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%.
  • the non-cationic lipid or non-ionizable lipid is cholesterol, and the molar proportion of cholesterol in the raw material can be, but is not limited to, 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%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material can be, but is not limited to, 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%.
  • the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 0% to 50%, preferably 0% to 40%, preferably 5% to 30%, and further 20% to 25%.
  • the metal-phospholipid complex particles are prepared by mixing the metal-phospholipid complex, a conjugated lipid that inhibits particle aggregation, and a non-cationic lipid or a non-ionizable lipid to obtain the metal-phospholipid complex particles.
  • the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or the non-ionizable lipid are mixed in a homogeneous phase, such as an organic phase (in some embodiments, the solvent is ethanol).
  • the preparation method comprises mixing the metal-phospholipid complex, a conjugated lipid that inhibits particle aggregation, a non-cationic lipid or a non-ionizable lipid, and the drug to prepare the drug-metal-phospholipid complex particles.
  • the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or non-ionizable lipid are dissolved in an organic compound to form an organic phase
  • the drug is dissolved in a buffer to form an aqueous phase
  • the organic and aqueous phases are mixed to produce the drug-metal-phospholipid complex particles.
  • the buffer can be PBS or Tris-HCl.
  • the organic and aqueous phases can be mixed using a microfluidic chip or ultrasound.
  • Targeting vector (containing MPP)
  • the targeting vector comprises a carrier delivery system and a targeting structure.
  • the carrier delivery system includes at least one of a lipid delivery system, exosomes, pseudoviruses, small molecules, cells, or a carrier protein.
  • the lipid delivery system is a metal-phospholipid complex particle, LNP, or a phospholipid delivery system.
  • the pseudovirus is an adenovirus.
  • the cell is a red blood cell.
  • the carrier protein is apoferritin.
  • the LNP is a cationic lipid or an ionizable lipid.
  • the carrier delivery system is a metal-phospholipid complex particle (MPP), and the targeting structure is attached to the outer surface of the metal-phospholipid complex particle (MPP).
  • MPP metal-phospholipid complex particle
  • the targeting structure includes a hydrophobic region, a connecting region, and a targeting binding region that are sequentially connected, and the hydrophobic region is connected to the outer layer of the metal-phospholipid complex particle based on hydrophilicity and hydrophobicity.
  • the hydrophobic region comprises at least one or more of DSPE and its derivatives, and the targeting structure is connected to the outer layer of the metal-phospholipid complex particle through this portion.
  • the linking region comprises at least one or more of PEG-2000 and its derivatives.
  • the targeting binding region can guide the targeting carrier to a specific target (i.e., the target or target cell of the targeting binding region) through specific recognition, thereby enabling the drug-loaded LNP particles and metal-phospholipid complex particles to transport the drug and act on the target (i.e., the target or target cell of the drug).
  • the targeting binding region can bind to at least one of CD62L, CD8, CD3, nucleolin protein, T cells, natural killer cells, macrophages, pancreatic cancer cells, or liver cancer cells.
  • the targeting binding region includes at least one of a nucleic acid, a polypeptide, a protein, or a small molecule.
  • the targeting binding region includes one of an aptamer, an antibody, an antigen binding portion, or galnac.
  • the targeting binding region is an aptamer, the target of the targeting binding region is CD62L, and the aptamer is preferably as shown in SEQ ID NO.44.
  • the targeting binding region is an aptamer, the target of the targeting binding region is CD8, and the aptamer is preferably as shown in SEQ ID NO.84.
  • the targeting binding region is an aptamer, the target of the targeting binding region is CD3, and the aptamer is preferably shown in SEQ ID NO.95.
  • the targeting binding region is an aptamer, the target of the targeting binding region is nucleolin protein, and the aptamer is preferably shown in SEQ ID NO.86.
  • the targeting binding region is an aptamer, the target cell of the targeting binding region is a T cell, and the aptamer is preferably shown in SEQ ID NO.44.
  • the targeting binding region is an aptamer, the target cell of the targeting binding region is a pancreatic cancer cell, and the aptamer is preferably shown in SEQ ID NO.88 (e.g., P19).
  • the targeting binding region is an aptamer, and the target cell of the targeting binding region is a liver cancer cell.
  • the targeting structure is DSPE-PEG2000-aptamer, preferably DSPE-PEG2000-CD62L aptamer, and the CD62L aptamer sequence is shown in SEQ ID NO.44.
  • the preparation method of the targeting structure can first connect the hydrophobic region and the linker region, and then connect the hydrophobic region-linker region to the targeting binding region through an intermediate pair.
  • the preparation process is as follows: DSPE-PEG2000 and the CD62L aptamer are connected through the intermediate pair reaction to obtain the DSPE-PEG2000-CD62L aptamer.
  • the intermediate pair is selected from DSPE-PEG2000-MAL and CD62L aptamer-C6-SH, DSPE-PEG2000-NHS and CD62L aptamer-NH2, DSPE-PEG2000-COOH and CD62L aptamer-NH2, DSPE-PEG2000-NCO and CD62L aptamer-NH2, DSPE-PEG2000-N3 and CD62L aptamer-DBCO, preferably DSPE-PEG2000-MAL and CD62L aptamer-C6-SH.
  • the DSPE-PEG2000-aptamer is first prepared into micelles and then attached to the outer surface of a carrier delivery system (e.g., metal-phospholipid complex particles (MPP)) to form a targeted carrier.
  • a carrier delivery system e.g., metal-phospholipid complex particles (MPP)
  • the micelles are prepared by direct dissolution, ethanol infusion, dialysis, or ultrasound.
  • the targeting carrier When the targeting carrier is used for drug encapsulation, the drug, metal-phospholipid complex, conjugated lipids that inhibit particle aggregation, non-cationic lipids or non-ionizable lipids are first prepared into drug-lipid particles (i.e., drug-metal-phospholipid complex particles), and then the targeting structure is reacted and connected with the drug-lipid particles to obtain the targeted drug.
  • drug-lipid particles i.e., drug-metal-phospholipid complex particles
  • the reaction mass ratio of the targeting structure to the drug-lipid particle is 1:(14-84), and the mass ratio can be, but is not limited to, 1:14, 1:16, 1:16.8, 1:18, 1:20, 1:21, 1:22, 1:24, 1:26, 1:28, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:82 or 1:84.
  • the reaction conditions of the drug-lipid particle and the targeting structure are incubation at -10 to 10° C. for 0.2-12 hours.
  • the reaction temperature may be, but is not limited to, -10° C., -9° C., -8° C., -7° C., -6° C., -5° C., -4° C., -3° C., -2° C., -1° C., 0° C., 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C.
  • the reaction time may be, but is not limited to, 0.2 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or 12 h.
  • the targeted drug includes a drug and a targeting carrier, the drug is encapsulated in the targeting carrier, more specifically, the drug is encapsulated in metal-phospholipid complex particles.
  • a drug is a negatively charged molecule, which can be selected from the group consisting of 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 the group consisting of one or more combinations of mRNA, siRNA, circular RNA, microRNA, ASO, sgRNA, DNA, ecDNA, and artificial nucleic acids.
  • the drug is mRNA, which is an mRNA encoding a chimeric antigen receptor CAR or TCR.
  • the targeted drug realizes in situ CAR cell therapy or TCR cell therapy. After delivering this mRNA encoding the corresponding receptor to specific cells in the body, these cells can be prompted to express CAR or TCR in situ in the body, thereby enabling these cells to have related functions such as targeted recognition and killing of specific tumor cells.
  • the in situ CAR cell therapy achieved by targeted drugs provides a new strategy for the treatment of diseases (especially tumor diseases). This therapy has relatively stronger targeting and may also reduce the complex preparation and return process in traditional cell therapy, which has significant advantages.
  • the drug is an mRNA encoding a chimeric antigen receptor CAR, which includes a transmembrane domain, a signaling domain, an antigen binding domain, a co-stimulatory signaling region, and a region connecting the antigen binding domain and the transmembrane domain.
  • the transmembrane domain is selected from at least one of SEQ ID No. 28, SEQ ID No. 29, and SEQ ID No. 30; and/or the signaling domain is selected from at least one of SEQ ID No. 31, SEQ ID No. 32, and SEQ ID No. 33; and/or the antigen binding domain is selected from at least one of SEQ ID No. 34, SEQ ID No. 35, and SEQ ID No.
  • the co-stimulatory signaling region is selected from at least one of SEQ ID No. 37, SEQ ID No. 39, and SEQ ID No. 75; and/or the antigen binding domain and transmembrane domain connecting region is selected from at least one of SEQ ID No. 40, SEQ ID No. 41, and SEQ ID No. 42.
  • the drug is an mRNA having a nucleotide sequence as shown in SEQ ID No.43.
  • nucleic acid encapsulated in the targeted drug of the present application is resistant to degradation by nucleases in aqueous solution.
  • the drug is fully encapsulated inside the metal-phospholipid complex particles to avoid drug degradation, while the drug is delivered into specific cells through the action of the targeting binding region in the targeting structure.
  • the targeted drugs provided herein have a small diameter suitable for systemic delivery.
  • the drugs of the present application are preferably nucleic acids
  • the nucleic acid component typically includes mRNA, interfering RNA (i.e., siRNA), which can be provided in several forms, including, for example, one or more isolated small-interfering RNA (siRNA) duplexes, longer double-stranded RNA (dsRNA), or siRNA or dsRNA translated from a transcription cassette in a DNA plasmid.
  • siRNA interfering RNA
  • siRNA interfering RNA
  • the targeted drug or preparation method of the present application can be used for drug delivery, imaging drugs, and vaccines.
  • targeted drugs are used for drug delivery, treatment and/or prevention.
  • targeted drugs are used to introduce drugs into cells or blood; cells preferably include immune cells, tumor cells, germ cells, neural cells, endocrine cells, blood cells, phagocytes, white blood cells, red blood cells, epithelial cells, cardiomyocytes or stem cells.
  • the targeted drug is used to express or silence a target sequence in a mammalian subject, to deliver a drug within a mammal, to deliver a drug from within the body to a mammalian cell, to deliver a drug from within the body to a mammalian cell for expression or silencing of a target sequence, or to treat/or prevent a disease or condition in a mammal.
  • the mammal is a human.
  • the disease or condition being treated is associated with expression of a gene that contains the drug's target sequence.
  • the disease or condition comprises cancer, viral infection, autoimmune disease, disease caused by overactivation of the immune system, metabolic disease, fibrotic disease, tissue fibrosis, cellular senescence, atherosclerosis, diabetes, or osteoarthritis.
  • the cancer includes hematologic malignancies and solid tumors.
  • hematologic malignancies include acute B-cell leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), multiple myeloma (MM), acute myeloid leukemia (AML) or T-cell lymphoma, and diffuse large B-cell lymphoma (DLBCL) preferably includes Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL).
  • solid tumors include liver cancer, glioma, gastric cancer, pancreatic cancer, lung cancer, non-small cell lung cancer, renal cell carcinoma, prostate cancer, osteosarcoma, breast cancer, colorectal cancer, ovarian cancer, melanoma, neuroblastoma, hemangioblastoma, anaplastic meningioma, recurrent glioblastoma or differentiated thyroid cancer.
  • the viral infection comprises hepatitis B virus, hepatitis C virus, SARS-CoV-2, human immunodeficiency virus, cytomegalovirus, invasive Aspergillus, or a conjugate virus.
  • the autoimmune disease comprises pemphigus vulgaris, systemic lupus erythematosus (SLE), hemophilia, myasthenia gravis, immune rejection caused by transplanted tissues and organs, type 1 diabetes (T1D), rheumatoid arthritis, systemic sclerosis, multiple sclerosis, idiopathic pulmonary fibrosis, Crohn's disease, or colitis.
  • the disease caused by excessive activation of the immune system comprises cytokine release syndrome (CRS), immune effector cell associated neurotoxicity syndrome (ICANS), or graft-versus-host disease (GVHD).
  • CRS cytokine release syndrome
  • ICANS immune effector cell associated neurotoxicity syndrome
  • GVHD graft-versus-host disease
  • the metabolic disease comprises atherosclerosis, congenital hyperinsulinemia, nonalcoholic steatohepatitis, or non-obese diabetes mellitus (NOD).
  • atherosclerosis congenital hyperinsulinemia
  • nonalcoholic steatohepatitis nonalcoholic steatohepatitis
  • NOD non-obese diabetes mellitus
  • fibrosis comprises myocardial fibrosis, hypertrophic cardiomyopathy, COVID-19, COVID-19 myocarditis, ischemic cardiomyopathy, cirrhosis, liver fibrosis, primary cholangitis, pulmonary fibrosis, skeletal muscle fibrosis, skin diseases, myelofibrosis, sarcoidosis, or Duchenne muscular dystrophy.
  • skin diseases comprise keloids or wound healing.
  • the administration route of the targeted drug includes intrathecal injection, intramuscular administration, intracranial injection, intravenous injection, or intratumoral injection.
  • the targeted drug is used in combination therapy.
  • the targeted drug is an immune cell targeted drug
  • the immune cells are T cells and/or myeloid cells.
  • the immune cell targeted drug is a chimeric antigen receptor (CAR) drug.
  • CAR chimeric antigen receptor
  • the present application provides a pharmaceutical agent containing a targeted drug, which is preferably a vaccine, and more preferably a new coronavirus vaccine.
  • a targeting vector can be used to facilitate lysosomal escape and promote nucleic acid expression.
  • Targeting vectors can also be used to deliver the drug, introducing the drug into cells to prevent or treat the disease or condition for which the drug is intended.
  • the present application provides pharmaceutical agents that can achieve: silencing the expression of target sequences in mammalian subjects, delivering drugs in mammals (e.g., drugs for treating tumors, imaging agents, etc.), delivering drugs from the body to mammalian cells, or treating mammalian diseases or conditions, etc.
  • drugs in mammals e.g., drugs for treating tumors, imaging agents, etc.
  • delivering drugs from the body to mammalian cells e.g., drugs from the body to mammalian cells, or treating mammalian diseases or conditions, etc.
  • the targeted drug is the main active ingredient and 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, gels), semi-solid dosage forms (external ointments, pastes), liquid dosage forms (decoctions, mixtures, syrups, wines, injections), gaseous dosage forms (aerosols, smokes), etc.; for example, dosage forms for gastrointestinal administration, dosage forms for rectal administration, dosage forms for non-gastrointestinal administration, etc.
  • Products containing the pharmaceutical agent can be, for example, but not limited to, kits, pharmaceutical agents, etc., and the product can optionally contain other excipients.
  • target genes for targeted drug action Generally, it is desirable to deliver drug-lipid particles so that the translation (i.e., expression) of the target gene product is downregulated or silenced.
  • Suitable categories 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 a target, and liver diseases and disorders), genes associated with tumorigenesis and cell transformation, angiogenic genes, immunomodulator genes such as those associated with inflammation and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.
  • Genes associated with viral infection and survival include those expressed by viruses to bind, enter and replicate in cells.
  • viral sequences associated with chronic viral diseases include sequences of hepatitis viruses (Hamasaki, et al., FEBS Lett. 543: 51 (2003); Yokota, et al., EMBO Rep. 4: 602 (2003); Schlommai, 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); and FIELDSVIROLOGY (Knipe et al.
  • HIV human immunodeficiency virus
  • Bonerjea et al., Mol Ther. 8: 62 (2003); Song, et al., J. Virol. 77: 7174 (2003); Stephenson JAMA 289: 1494 (2003); Qin, et al., Proc. Natl. Acad. Sci. 100: 183 (2003)
  • herpes virus Jia, et al., J. Virol. 77: 3301 (2003)
  • HPV human papillomavirus
  • Exemplary hepadnavirus 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., core proteins including C and C-related proteins; capsid and envelope proteins including S, M, and/or L proteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY, 2001, supra).
  • structural proteins e.g., core proteins including C and C-related proteins; capsid and envelope proteins including S, M, and/or L proteins, or fragments thereof
  • 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).
  • Hepatitis A nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_001489; hepatitis B nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_003977; hepatitis C nucleic acid sequences are referenced, for example, in Genbank Accession No.
  • Silencing sequences encoding genes associated with viral infection and survival can be conveniently used in conjunction with the administration of conventional pharmaceutical agents for treating viral diseases.
  • Genes associated with metabolic diseases and disorders include, for example, those in dyslipidemia (e.g., liver X receptors (e.g., LXR ⁇ and LXR ⁇ Genbank Accession No. NM_007121)), farnesoid X receptor (FXR) (Genbank Accession No.
  • dyslipidemia e.g., liver X receptors (e.g., LXR ⁇ and LXR ⁇ Genbank Accession No. NM_007121)
  • FXR farnesoid X receptor
  • NM_005123 sterol regulatory element binding protein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA) -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. Endocrinol. 9: 72 (1995), Zavacki et al., PNAS USA 94: 7909 (1997); Sakai, ...
  • SREBP sterol regulatory element binding protein
  • S1P site-1 protease
  • HMG-CoA 3-hydroxy-3-methylglutaryl-CoA reductase
  • ApoB apolipoprotein
  • ApoE apolipoprotein
  • 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 sequences encoding genes associated with metabolic diseases and disorders can be conveniently used in conjunction with the administration of conventional agents for treating the disease or disorder.
  • genes associated with tumorigenesis and cell transformation include translocation sequences such as MLL fusion genes, 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.
  • MLL fusion genes such as MLL fusion genes, BCR-ABL (Wilda, et al., Oncogene, 21:5716 (2002); Scherr, et al., Blood 101:1566), TEL-AML1, EWS-FLI1, TLS-
  • Silencing sequences encoding DNA repair enzymes is used in conjunction with the 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- ⁇ , TGF- ⁇ , 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- ⁇ , TGF- ⁇ , EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.
  • interleukins e.g., IL-2, IL-4, IL-12 (Hill, et al., J.
  • Fas and Fas ligand genes are also target sequences for immunomodulators of interest (Song, et al., Nat. Med. 9: 347 (2003)).
  • Genes encoding secondary signaling molecules in hematopoietic and lymphoid cells are also included in the present application, for example, Tec family kinases, such as Bruton’s tyrosine kinase (Btk) (Heinonen, et al., FEBS Lett. 527: 274 (2002)).
  • Cell receptor ligands include ligands that can bind to cell surface receptors (e.g., insulin receptors, EPO receptors, G-protein coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) to regulate (e.g., inhibit, activate, etc.) physiological pathways involved in the receptors (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 repeat sequence e.g., CAG repeat sequence
  • Injectable delivery In certain circumstances, it may be desirable to deliver the targeted drugs disclosed herein parenterally, intravenously, intramuscularly, subcutaneously, intradermally, or intraperitoneally, as described in U.S. Patent No. 5,543,158; U.S. Patent No. 5,641,515; and U.S. Patent No. 5,399,363.
  • the targeted drug may be injected locally to the target site (e.g., a disease site such as inflammation or tumor formation or to a target organ or tissue) or systemically for widespread distribution to the organism.
  • Solutions of the targeted drug may be prepared in water suitably mixed with a surfactant.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oils.
  • these formulations contain a preservative to prevent the growth of microorganisms.
  • the targeted drug formulation is formulated with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier typically, a normal buffered saline solution (135-150 mM NaCl) will be used as a pharmaceutical carrier, but other suitable carriers will suffice. Additional suitable carriers 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, carrier solutions, suspensions, colloids, and the like.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a human.
  • the formulation of aqueous compositions comprising a protein as the active ingredient is conventionally understood in the art. Alternatively, these compositions are prepared as injectable solutions, liquid solutions, or suspensions; solid forms suitable for solution or suspension in liquid prior to injection can also be prepared. The formulations can also be emulsified.
  • Described targeted drug can comprise pharmaceutical auxiliary substance, and described pharmaceutical auxiliary substance is suitable physiological condition, such as pH adjusting agent and buffer, toxicity regulator, wetting agent etc.Use the technology indicated above to sterilize these compositions, or alternatively, they can produce under aseptic conditions.Can be packaged to use or filter under aseptic conditions and carry out freeze drying to the aqueous solution obtained, before using, freeze-dried preparation is combined with sterile aqueous solution.
  • targeted drugs can be used for the prophylactic or therapeutic treatment of a subject (e.g., a mammalian subject) having a disease or condition associated with expression or overexpression of a target sequence.
  • the targeted drug is administered to the subject in an amount sufficient to stimulate 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 targeted drug, targeted drug toxicity, and progression of the disease associated with expression or overexpression of the target gene.
  • Administration can be accomplished by single or divided doses.
  • the targeted drug can be administered to a subject who is infected with or at risk of being infected by a pathogenic microorganism.
  • the drug should preferably correspond to a sequence that plays a key role in the life history of the microorganism and should also be unique to the microorganism (or at least absent in the genome of the natural genome of the patient undergoing treatment).
  • the targeted drug is introduced into the target cell, tissue or organ by ex vivo or intravenous injection at a therapeutically effective dose.
  • Silencing sequences encoding genes associated with pathogenic infections can be conveniently used in combination with the administration of conventional agents for treating pathogenic diseases.
  • the treatment can be administered prophylactically to people who are at risk of being infected by or have been infected by pathogenic microorganisms.
  • the targeted drugs of the present application 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 inhibiting hepatitis B virus include nucleic acid sequences encoding the S, C, P, and X proteins, PRE, EnI, and EnII (see, for example, 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 ⁇ ) (see, for example, Medina et al., Antiviral Res. 60(2): 135-143 (2003); ribavirin (see, for example, Hugle and Cerny, Rev. Med. Virol.
  • immunoglobulins e.g., interferons (e.g., PEGylated and non-PEGylated interferon ⁇ )
  • interferons e.g., PEGylated and non-PEGylated interferon ⁇
  • ribavirin see, for example, Hugle and Cerny, Rev. Med. Virol.
  • the application's targeted drug can be used to treat easily and be characterized by gene or gene cluster expression or the disease and illness of overexpression.
  • the application's targeted drug can be used to treat metabolic diseases and illness (for example, wherein liver is the disease and illness and hepatic disease and illness of target object) such as, for example, dyslipidemia and diabetes.
  • metabolic diseases and illness for example, wherein liver is the disease and illness and hepatic disease and illness of target object
  • the silence of the gene relevant to metabolic diseases and illness can be combined with the conventional treatment of these diseases.
  • silencing of genes involved in dyslipidemia can be combined with statins, bile acid sequestrants/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 inhibitors, bile acid transport inhibitor SREBP cleavage activating protein (SCAP) activating ligand, nicotinic acid (niacin), acipimox, high-dose fish oil, antioxidants and policosanol, microsomal triglyceride transport protein (MTP) inhibitors, acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors, gemcabene, rifiberol, pantothenic acid analogs, niacin-receptor agonists, anti-inflammatory agents (such as Lp-PLA(2)
  • the targeted drug can be used to treat cancer, viral infection, autoimmune disease, disease caused by overactivation of the immune system, metabolic disease, fibrotic disease, tissue fibrosis, cell senescence, atherosclerosis, diabetes or osteoarthritis.
  • the cancer includes hematological tumors and solid tumors.
  • the hematological tumor includes acute B cell leukemia (ALL), diffuse large B cell lymphoma (DLBCL), multiple myeloma (MM), acute myeloid leukemia (AML) or T cell lymphoma.
  • the diffuse large B cell lymphoma (DLBCL) includes Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL).
  • the solid tumor includes liver cancer, brain glioma, gastric cancer, pancreatic cancer, lung cancer, non-small cell lung cancer, renal cell carcinoma, prostate cancer, osteosarcoma, breast cancer, colorectal cancer, ovarian cancer, melanoma, neuroblastoma, hemangioblastoma, anaplastic meningioma, recurrent glioblastoma or differentiated thyroid cancer.
  • the autoimmune diseases include pemphigus vulgaris, systemic lupus erythematosus (SLE), hemophilia, myasthenia gravis, immune rejection caused by transplanted tissues and organs, type 1 diabetes (T1D), rheumatoid arthritis, systemic sclerosis, idiopathic pulmonary fibrosis, Crohn's disease or colitis.
  • Diseases caused by excessive activation of the immune system include cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS) or graft-versus-host disease (GVHD).
  • CRS cytokine release syndrome
  • ICANS immune effector cell-associated neurotoxicity syndrome
  • GVHD graft-versus-host disease
  • the metabolic diseases include atherosclerosis, congenital hyperinsulinemia, non-alcoholic steatohepatitis or non-obese diabetes (NOD).
  • the fibrosis includes myocardial fibrosis, hypertrophic cardiomyopathy, COVID-19, COVID-19 myocarditis, ischemic cardiomyopathy, cirrhosis, liver fibrosis, primary cholangitis, pulmonary fibrosis, skeletal muscle fibrosis, skin diseases, myelofibrosis, sarcoidosis or Duchenne muscular dystrophy.
  • the skin diseases include keloids or wound healing.
  • Similar methods are used to inhibit the expression of endogenous receptor cellular genes that are associated with tumorigenesis and cell transformation, tumor growth and tumor migration; inhibit the expression of angiogenic genes; inhibit the expression of immunomodulator genes, such as those associated with inflammation and autoimmune responses; inhibit the expression of ligand receptor genes; inhibit the expression of genes associated with neurodegenerative disorders; and inhibit the expression of additional genes associated with viral infection and survival.
  • Specific target gene sequences are as described above.
  • the targeted drug herein can be detected using any method known in the art.
  • a label can be coupled directly or indirectly to the targeted drug component or other lipid-based carrier system using methods well known in the art.
  • a wide variety of labels can be used, with selection being based on desired sensitivity, ease of conjugation to the targeted drug component, stability requirements, and available tools and processing readiness.
  • Suitable labels include, but are not limited to, spectral labels, such as fluorescent dyes (e.g., fluorescein and derivatives, such as fluorescein isothiocyanate (FITC) and Oregon GreenTM; rhodamine and derivatives, such as Texas 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 horseradish peroxidase, alkaline phosphatase, etc.; spectral colorimetric labels such as colloidal gold or colored glass or plastic beads, such as polystyrene, polypropylene, latex, etc.).
  • the labels are detected using any means known in the art.
  • Nucleic acids herein can be detected and quantified by any of a number of methods well known to those skilled in the art. Detection of nucleic acids can be performed by methods well known in the art such as Southern blot analysis, Northern blot analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Additional analytical biochemical methods such as spectrophotometry, X-ray photography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography can also be used.
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • the sensitivity of hybridization assays can be increased by the use of nucleic acid amplification systems that multiply the target nucleic acid being detected.
  • In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known.
  • RNA polymerase-mediated techniques e.g., NASBATM
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Q ⁇ -replicase amplification e.g., NASBATM
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Q ⁇ -replicase amplification e.g., NASBATM
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Q ⁇ -replicase amplification e.g., Q ⁇ -replicase amplification
  • NASBATM RNA polymerase-mediated techniques
  • Oligonucleotides are chemically synthesized, for example, using an automated synthesizer, as described by Needham Van Devanter et al., Nucleic Acids Res., 12: 6159 (1984), typically according to the solid-phase phosphoramidite triester method described by Beaucage and Caruthers, Tetrahedron Letts., 22(20): 1859-1862 (1981).
  • the oligonucleotides are used, for example, as probes in in vitro amplification methods, as gene probes, or as inhibitor components. If necessary, the oligonucleotides are typically purified by native acrylamide gel electrophoresis or by anion exchange HPLC, as described by Pearson and Regnier, J.
  • CD62L as a target for immune cell drugs can increase the expression level of drugs and be more conducive to the treatment of diseases.
  • the drug-lipid particles herein refer to drug-lipid particles other than those containing cationic/ionizable lipids, namely drug-loaded metal-chelated phospholipid complex nanoparticles (drug@MPPs), which are linked to a targeting structure to form a targeted drug. After removing the drug from the targeted drug, the remaining components include the targeting carrier.
  • drug@MPPs drug-loaded metal-chelated phospholipid complex nanoparticles
  • a phospholipid molecule with a phosphate group to a linker molecule: Distearoylphosphatidylcholine (DSPC, Formula 46) and curcumin (Formula 19) were added to a reaction flask at a molar ratio of 1:1. An appropriate amount of ethanol was added to dissolve the mixture. After reacting at 65°C for 2 hours, the mixture was concentrated and n-hexane was added. The resulting phospholipid complex was precipitated, filtered, and vacuum dried to obtain the phospholipid complex.
  • the structure of the phospholipid complex is shown below:
  • Example 1.2.1 Step-by-step preparation of metal-phospholipid complexes when the metal ion is Fe 3+
  • the phospholipid complex prepared in Example 1.1 was partially linked to the metal ion: the phospholipid complex and FeCl3 were added to a reaction flask at a molar ratio of 1:1. Ethanol was added to dissolve the mixture. Triethylamine was then added at a molar ratio of 1:1 to the phospholipid complex. The mixture was reacted at 60°C for 2 h. The reaction solution was then suspended to dryness, washed with ultrapure water, and dried under vacuum to obtain a metal-phospholipid complex. The structure of the metal-phospholipid complex is shown below.
  • the yield of the target product obtained was 95% when the phospholipid complex and FeCl 3 were reacted at 60°C for 2 hours, the phospholipid complex feed concentration was 4.5 mg/mL, and the feed ratio of phospholipid complex to FeCl 3 was 1:1.
  • Example 1.2.3 Step-by-step preparation of metal-phospholipid complexes when the metal ion is Mg 2+
  • the yield of the target product was 95.5% when the phospholipid complex and MgCl2 were reacted at 60°C for 2 hours, the phospholipid complex concentration was 4.5 mg/mL, and the phospholipid complex to MgCl2 ratio was 1:1.
  • Phospholipid molecules with phosphate groups are linked to linker molecules and metal ions: Distearoylphosphatidylcholine (DSPC, Formula 46), curcumin (Formula 19), and FeCl3 are added to a reaction flask at a molar ratio of 1:1:1. An appropriate amount of ethanol is added to dissolve the mixture. The reaction is then carried out at 50 ⁇ 10°C for 2 hours to obtain a metal-phospholipid complex. This one-step reaction yields a 95.6% yield of the target Fe3+ metal-phospholipid complex.
  • the metal ion can be replaced with Mg2+ , Al3 + , or Ca2 + .
  • the reaction temperature is 50 ⁇ 10°C, and the reaction time is 1-5 hours, preferably 2 hours.
  • Phospholipid molecules with phosphate groups were linked to linker molecules and metal ions: Distearoylphosphatidylcholine (DSPC, Formula 46), curcumin, and Al( NO3 ) 3 ⁇ 9H2O were added to a reaction flask at a molar ratio of 1:1:1. Ethanol was added to dissolve the mixture, and the reaction was carried out at 50 ⁇ 10°C for 2 hours to obtain a metal-phospholipid complex. This one-step reaction yielded a 95.6% yield of the desired Al3+ metal-phospholipid complex.
  • DSPC Distearoylphosphatidylcholine
  • curcumin Curcumin
  • Al( NO3 ) 3 ⁇ 9H2O Al( NO3 ) 3 ⁇ 9H2O
  • Phospholipid molecules with phosphate groups were linked to linker molecules and metal ions: Distearoylphosphatidylcholine (DSPC, Formula 46), curcumin, and CaCl2 were added to a reaction flask at a molar ratio of 1:1:1. Ethanol was added for dissolution and the mixture was reacted at 50 ⁇ 10°C for 2 hours to obtain a metal-phospholipid complex. This "one-step" reaction yielded a 95% yield of the target Ca2 + metal-phospholipid complex product.
  • the step-by-step preparation protocol for mRNA-metal-phospholipid complex particles containing Ca2 + was similar to the step-by-step preparation protocol for mRNA-metal-phospholipid complex particles containing Fe3 + .
  • the phospholipid complex was reacted with CaCl2 at 60°C for 2 hours. When the phospholipid complex concentration was 4.5 mg/mL and the phospholipid complex to CaCl2 ratio was 1:1, the yield of the metal-phospholipid complex obtained when the target product metal ion was Ca2 + was 95%.
  • the performance comparison analysis of the metal-phospholipid complex prepared by the "one-step method" and the step-by-step method is as follows:
  • Phospholipid molecules with phosphate groups were linked to linker molecules and metal ions: Distearoylphosphatidylcholine (DSPC, Formula 46), curcumin, and MgCl2 were added to a reaction flask at a molar ratio of 1:1:1. Ethanol was added to dissolve the molecule and the reaction was continued at 50 ⁇ 10°C for 2 hours to obtain a metal-phospholipid complex. This one-step reaction yielded a 95% yield of the desired Mg2 + metal-phospholipid complex.
  • the characteristics and effectiveness of metal-phospholipid complexes prepared by the "one-step method” and the step-by-step method were compared.
  • the two methods had similar encapsulation efficiency values, and both showed stable performance in ensuring the effective encapsulation of metals in the complexes.
  • the data of the "one-step method” and the step-by-step method were also similar, proving that the "one-step method” and the step-by-step method have equivalent capabilities for constructing metal-phospholipid complexes.
  • the "one-step method” can significantly shorten the time required for the industrialization process. In the scenario of industrial large-scale production, time efficiency is crucial.
  • the "one-step method” reduces the problems of process connection and time intervals caused by step-by-step operations, greatly improving the continuity and compactness of production, thereby effectively reducing production costs and improving production efficiency. From the perspective of industrial application, compared with the step-by-step method, the "one-step method" is more in line with the needs of large-scale production, and has broad application prospects and extremely high practical value.
  • a metal-phospholipid complex was prepared according to the method described in Example 1.2.1.
  • DSPC, curcumin, and FeCl3 were added at a 1:1:1 ratio.
  • the metal-phospholipid complex, distearoylphosphatidylcholine (DSPC) as a non-cationic or non-ionizable lipid, cholesterol (CHOL) as a non-cationic or non-ionizable lipid, and DSPE-PEG2000 (a conjugated lipid to inhibit particle aggregation) were dissolved in ethanol at varying molar ratios as the organic phase.
  • the metal-phospholipid complex, DSPC, CHOL, and DSPE-PEG2000 accounted for 15%, 35%, 46%, and 4%, respectively.
  • mRNA was dissolved at a concentration of 20 ⁇ g/mL in PBS (PBS composed of 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.0018 M potassium dihydrogen phosphate) as the aqueous phase.
  • PBS PBS composed of 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.0018 M potassium dihydrogen phosphate
  • the total mass of the metal phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) was mixed with the mRNA mass at a mass ratio of 40:1 in a microfluidic chip.
  • the volume ratio of the aqueous phase to the organic phase was 3:1.
  • the flow rate of the organic phase and the aqueous phase in the microfluidic chip was 12 ml/min.
  • the drug mRNA was mRNA encoding the fluorescent protein eGFP, and its sequence was SEQ ID NO.1 (720 nt).
  • the prepared eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2 ⁇ g/mL (the concentration of the contained mRNA).
  • the control group was incubated with MPP without drug loading. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.
  • Example 1.4 Preparation of mRNA-loaded metal-chelated phospholipid complex nanoparticles (mRNA@MPP) containing Al 3+ metal ions
  • a metal-phospholipid complex was prepared according to the method described in Example 1.2.2.
  • DSPC, curcumin, and Al( NO3 ) 3 ⁇ 9H2O were added at a 1:1:1 ratio.
  • the metal-phospholipid complex, distearoylphosphatidylcholine (DSPC, a non-cationic or non-ionizable lipid), cholesterol (CHOL, a non-cationic or non-ionizable lipid), and DSPE-PEG2000 (a conjugated lipid to inhibit particle aggregation) were dissolved in ethanol at varying molar ratios as the organic phase.
  • the metal-phospholipid complex, DSPC, CHOL, and DSPE-PEG2000 accounted for 7%, 34%, 56%, and 3%, respectively.
  • mRNA was dissolved at a concentration of 20 ⁇ g/mL in PBS (PBS composed of 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.0018 M potassium dihydrogen phosphate) as the aqueous phase.
  • PBS PBS composed of 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.0018 M potassium dihydrogen phosphate
  • the total mass of the metal phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) was mixed with the mRNA mass at a mass ratio of 40:1 in a microfluidic chip.
  • the volume ratio of the aqueous phase to the organic phase was 3:1.
  • the flow rate of the organic phase and the aqueous phase in the microfluidic chip was 12 ml/min.
  • the drug mRNA was mRNA encoding the fluorescent protein eGFP, and its sequence was SEQ ID NO.1 (720 nt).
  • the prepared eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2 ⁇ g/mL (the concentration of the contained mRNA).
  • the control group was incubated with MPP without drug loading. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.
  • Example 1.5 Preparation of mRNA-loaded metal-chelated phospholipid complex nanoparticles (mRNA@MPP) containing Mg2 + as the metal ion
  • a metal-phospholipid complex was prepared using the one-step method described in Example 1.2.4.
  • DSPC, curcumin, and MgCl2 were added at a 1:1:1 ratio.
  • the metal-phospholipid complex, distearoylphosphatidylcholine (DSPC) as a non-cationic or non-ionizable lipid, cholesterol (CHOL) as a non-cationic or non-ionizable lipid, and DSPE-PEG2000 (a conjugated lipid to inhibit particle aggregation) were dissolved in ethanol at varying molar ratios as the organic phase.
  • the metal-phospholipid complex, DSPC, CHOL, and DSPE-PEG2000 accounted for 15%, 40%, 43.2%, and 1.8%, respectively.
  • mRNA was dissolved at a concentration of 20 ⁇ g/mL in PBS (PBS composed of 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.0018 M potassium dihydrogen phosphate) as the aqueous phase.
  • PBS PBS composed of 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.0018 M potassium dihydrogen phosphate
  • the total mass of the metal phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) was mixed with the mRNA mass at a mass ratio of 40:1 in a microfluidic chip.
  • the volume ratio of the aqueous phase to the organic phase was 3:1.
  • the flow rate of the organic phase and the aqueous phase in the microfluidic chip was 12 ml/min.
  • the drug mRNA was mRNA encoding the fluorescent protein eGFP, and its sequence was SEQ ID NO.1 (720 nt).
  • the prepared eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2 ⁇ g/mL (the concentration of the contained mRNA).
  • the control group was incubated with MPP without drug loading. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.
  • the particle size, surface potential, and stability of the eGFP-mRNA@MPP prepared in Examples 1.3-1.5 were tested, and the efficiency of nucleic acid encapsulation in the eGFP-mRNA@MPP was calculated.
  • Method for detecting particle size and result judgment standard The particle size of nanoparticles was tested using a Malvern Zetasizer laser particle size analyzer, and a particle size within the range of 50 to 400 nm was considered acceptable.
  • Stability testing method and result judgment criteria The nanoparticles were placed at 4°C for 7 days, and the particle size and surface potential of the nanoparticles were measured using a Malvern Zetasizer. If there was no significant change in the particle size and surface potential within 3-7 days, the stability was considered to be good.
  • Method for calculating nucleic acid encapsulation efficiency Specifically, agarose gel electrophoresis method is used. First, the nucleic acid feed amount of each group of lipid nanoparticles is set to 10 ⁇ g/mL, the mass ratio of lipid to nucleic acid is 40:1, and equal concentrations of nucleic acid are dissolved in PBS buffer solution as a positive control, and the negative control is PBS buffer solution. The concentration of 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. When the free nucleic acid bands are electrophoresed to a clearly distinguishable state, electrophoresis is stopped.
  • Image J software is used to count the grayscale values of free nucleic acids in different groups.
  • the positive control group is set to 100%.
  • the ratio of free nucleic acid in each group to the positive control is the relative amount of free nucleic acid.
  • the encapsulation rate of each group is (100-relative amount of free nucleic acid)%. A nucleic acid encapsulation rate of more than 50% is considered to be within the acceptable range.
  • the principle of nucleic acid loading onto metal-chelated phospholipid complex nanoparticles (MPPs) assembled from metal-phospholipid complexes is as follows: curcumin binds to DSPC via hydrogen bonds, while curcumin coordinates with Fe 3+ , Ca 2+ , Al 3+ , or Mg 2+ to form a metal-phospholipid complex.
  • the Fe 3+ , Ca 2+ , Al 3+ , or Mg 2+ of the metal-phospholipid complex connects to the nucleic acid via coordination bonds, thereby enabling the metal-phospholipid complex to self-assemble with other lipid components into the MPP and simultaneously loading the nucleic acid into the nanoparticle.
  • Curcumin interacts with nucleic acids, assisting MPP loading, for example, by inserting into the minor groove of nucleic acids; 2 Curcumin may not directly interact with nucleic acids.
  • Example 1.3.1 Dosage ratio of components of the metal-phospholipid complex when the metal ion is Fe 3+
  • Example 1.3 DSPC, curcumin, and FeCl3 in Example 1.3 were added at different molar ratios (1:1:1, 3:3:2, and 2:2:1). Other steps were the same as in Example 1.3 to prepare different eGFP-mRNA@MPPs, and their nucleic acid encapsulation efficiencies were tested.
  • the molar ratio of DSPC, curcumin, and FeCl3 in the drug-lipid particles should be 1:1:1 to ensure that the metal-phospholipid complex particles can encapsulate as many nucleic acids as possible.
  • Experimental results also confirmed that the metal-phospholipid complex particles prepared with a 1:1:1 molar ratio of DSPC, curcumin, and FeCl3 exhibited the highest eGFP-mRNA encapsulation efficiency. Nucleic acid encapsulation rates exceeded 60 % when the molar ratios of DSPC, curcumin, and FeCl3 ranged from 1:1:1 to 2:2:1.
  • Example 1.4.1 Dosage ratio of components of the metal-phospholipid complex when the metal ion is Al 3+
  • Example 1.4 DSPC, curcumin, and Al(NO 3 ) 3 ⁇ 9H 2 O in Example 1.4 were added at different molar ratios (1:1:1, 3:3:2, and 2:2:1). Other steps were the same as in Example 1.4 to prepare different eGFP-mRNA@MPPs, and their nucleic acid encapsulation efficiencies were tested.
  • the metal-phospholipid complex particles In the metal-phospholipid complex particles, Al3+ functions as a bridge between the phospholipid complex and the nucleic acid. Each Al+++ has a maximum of three complexation sites. Therefore, the molar ratio of DSPC, curcumin, and Al( NO3 ) ⁇ ⁇ 9H2O in the drug-lipid particles should be 1:1:1 to maximize nucleic acid loading. Experimental results confirmed that a 1:1:1 molar ratio of DSPC, curcumin, and Al( NO3 ) ⁇ ⁇ 9H2O resulted in the highest eGFP-mRNA loading efficiency in the metal-phospholipid complex particles. Nucleic acid loading efficiency exceeded 58% when the molar ratio of DSPC, curcumin, and Al( NO3 ) ⁇ ⁇ 9H2O ranged from 1:1:1 to 2:2:1.
  • Example 1.5.1 Dosage ratio of components of the metal-phospholipid complex when the metal ion is Mg 2+
  • Example 1.5 DSPC, curcumin, and MgCl2 in Example 1.5 were added at different molar ratios (1:1:1, 3:3:2, and 2:2:1). Other steps were the same as in Example 1.5 to prepare different eGFP-mRNA@MPPs, and their nucleic acid encapsulation efficiencies were tested.
  • the molar ratio of DSPC, curcumin, and MgCl2 in the drug-lipid particles should be 1:1:1 to maximize nucleic acid encapsulation.
  • the experimental results also confirmed that the metal-phospholipid complex particles prepared with a molar ratio of DSPC, curcumin, and MgCl2 exhibited the highest eGFP-mRNA encapsulation efficiency.
  • the nucleic acid encapsulation efficiency of the metal-phospholipid complex particles is above 72.6%.
  • Example 1.3.2 Proportions of metal-phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) in the metal-phospholipid complex particles prepared when the metal ion is Fe 3+
  • Example 1.3 Compared with Example 1.3, the ratios of metal-phospholipid complex (metal ion is Fe 3+ ), distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) are as shown in Table 1-4, and other conditions are the same.
  • metal ion is Fe 3+
  • DSPC distearoylphosphatidylcholine
  • DSPE-PEG2000 DSPE-PEG2000
  • cholesterol cholesterol
  • the performance of the metal-phospholipid complex 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 adsorb nucleic acids, the proportion of metal-phospholipid complexes cannot be too low; when the DSPC content is in the range of 0-40%, the stability of its nanoparticles is within an acceptable range.
  • the DSPC content is 0%, the stability of its nanoparticles can be maintained because the metal-phospholipid complex contains DSPC; the role of DSPE-PEG2000 is to prevent nanoparticle aggregation and increase circulation time in the body. Its performance will be better when its content is in the range of 2-10%; the role of CHOL is to enhance the fluidity of nanoparticles, and maintaining a certain content is beneficial to the stability of nanoparticles.
  • Example 1.4.2 Proportions of metal-phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) in the metal-phospholipid complex particles prepared when the metal ion is Al 3+
  • Example 1.4 Compared with Example 1.4, the ratios of metal-phospholipid complex (metal ion is Al 3+ ), distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) are as shown in Table 1-5, and other conditions are the same.
  • metal-phospholipid complex metal ion is Al 3+
  • DSPC distearoylphosphatidylcholine
  • DSPE-PEG2000 DSPE-PEG2000
  • CHOL cholesterol
  • the drug-lipid particles exhibited optimal performance when the metal-phospholipid complex (MPC) comprised 7%, distearoylphosphatidylcholine (DSPC) 34%, cholesterol (CHOL) 56%, and DSPE-PEG2000 3%. These results included a particle size of 100 nm, a surface potential of -1.57 mV, in vitro stability of >7 days, an mRNA loading efficiency of 92%, and eGFP protein expression of 98%. Because mRNA@MPP relies primarily on the metal-phospholipid complex for nucleic acid adsorption, the metal-phospholipid complex ratio must be maintained. Nanoparticle stability was acceptable when the DSPC content ranged from 0% to 51%.
  • DSPE-PEG2000 prevents nanoparticle aggregation and increases in vivo circulation time; its performance was optimal when its content ranged from 2% to 10%.
  • CHOL enhances nanoparticle mobility; maintaining a certain CHOL content contributes to nanoparticle stability.
  • Example 1.5.2 Proportions of metal-phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) in the metal-phospholipid complex particles prepared when the metal ion is Mg 2+
  • Example 1.5 Compared with Example 1.5, the ratios of metal-phospholipid complex (metal ion is Mg 2+ ), distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) are as shown in Table 1-6, and other conditions are the same.
  • metal ion is Mg 2+
  • DSPC distearoylphosphatidylcholine
  • DSPE-PEG2000 DSPE-PEG2000
  • cholesterol cholesterol
  • the performance of the metal-phospholipid complex particles was optimal, that is, the particle size was in the range of 115nm, the surface potential was in the range of -2.9mV, the in vitro stability was >7 days, the mRNA encapsulation rate was 93.9%, and the positive expression rate of eGFP protein was 98%.
  • mRNA@MPP mainly relies on metal-phospholipid complexes to adsorb nucleic acids, the proportion of metal-phospholipid complexes cannot be too low; when the DSPC content is in the range of 0-50%, the stability of its nanoparticles is within an acceptable range.
  • the DSPC content is 0%, the stability of its nanoparticles can be maintained because the metal-phospholipid complex contains DSPC; the role of DSPE-PEG2000 is to prevent nanoparticle aggregation and increase circulation time in the body. Its performance will be better when its content is in the range of 1-10%; the role of CHOL is to enhance the fluidity of nanoparticles, and maintaining a certain content is beneficial to the stability of nanoparticles.
  • Example 1.3.3.1 Preparation of non-cationic lipids or non-ionizable lipid species in eGFP-mRNA@MPP(Fe 3+ )
  • Example 1.3 Compared with Example 1.3, the replacement of distearoylphosphatidylcholine (DSPC) is shown in Table 1-7, and the other conditions are the same.
  • DSPC distearoylphosphatidylcholine
  • non-cationic lipid or non-ionizable lipid DSPC in eGFP-mRNA@MPP(Fe 3+ ) is to make the liposome membrane fusion better, more stable and less toxic
  • other non-cationic lipids or non-ionizable lipids also have the function of making the liposome membrane fusion better, more stable and less toxic
  • DSPC in the drug-lipid particles can be replaced by other non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation, and its efficacy is not affected.
  • Example 1.3.3.2 Preparation of non-cationic lipids or non-ionizable lipids in eGFP-mRNA@MPP(Al 3+ )
  • Example 1.4 Compared with Example 1.4, the replacement of distearoylphosphatidylcholine (DSPC) is shown in Table 1-8, and the other conditions are the same.
  • DSPC distearoylphosphatidylcholine
  • non-cationic lipid or non-ionizable lipid DSPC in eGFP-mRNA@MPP(Al 3+ ) is to make the liposome membrane fusion better, more stable and less toxic
  • other non-cationic lipids or non-ionizable lipids also have the function of making the liposome membrane fusion better, more stable and less toxic
  • DSPC in the drug-lipid particles can be replaced by other non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation, and its efficacy is not affected.
  • Example 1.3.3.3 Preparation of non-cationic lipids or non-ionizable lipid species in eGFP-mRNA@MPP(Mg 2+ )
  • Example 1.5 Compared with Example 1.5, the replacement of distearoylphosphatidylcholine (DSPC) is shown in Table 1-9, and the other conditions are the same.
  • DSPC distearoylphosphatidylcholine
  • DSPC in eGFP-mRNA@MPP(Mg 2+ ) can be replaced by other non-cationic lipids or non-ionizable lipids, and its function after replacement is equivalent to that of eGFP-mRNA@MPP(Mg 2+ ) containing DSPC (Table 1-9).
  • non-cationic lipid or non-ionizable lipid DSPC in eGFP-mRNA@MPP(Mg 2+ ) is to make the liposome membrane fusion better, more stable and less toxic
  • other non-cationic lipids or non-ionizable lipids also have the function of making the liposome membrane fusion better, more stable and less toxic
  • DSPC in the drug-lipid particles can be replaced by other non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation, and its efficacy is not affected.
  • Example 1.3.4 Preparation of conjugated lipid species for inhibiting particle aggregation in eGFP-mRNA@MPP
  • Example 1.3.4.1 Preparation of conjugated lipid species for inhibiting particle aggregation in eGFP-mRNA@MPP(Fe 3+ )
  • DSPE-PEG2000 Compared to Example 1.3, the substitutions for DSPE-PEG2000 (Formula 53) are shown in Table 1-10 (metal ion is Fe 3+ ), and all other conditions remain the same. Three other conjugated lipids that inhibit particle aggregation are DSPE-PEG700 (Formula 50), DSPE-PEG5000 (Formula 52), and DSPE-PEG1000 (Formula 51).
  • DSPE-PEG2000 in eGFP-mRNA@MPP(Fe 3+ ) is to inhibit aggregation, and other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation, DSPE-PEG2000 in eGFP-mRNA@MPP(Fe 3+ ) can be replaced by other conjugated lipids that inhibit particle aggregation without affecting its efficacy.
  • DSPE-PEG2000 Compared to Example 1.4, the substitutions for DSPE-PEG2000 (Formula 53) are shown in Table 1-11 (the metal ion is Al 3+ ), and all other conditions remain the same. Three other conjugated lipids that inhibit particle aggregation are DSPE-PEG700 (Formula 50), DSPE-PEG5000 (Formula 52), and DSPE-PEG1000 (Formula 51).
  • DSPE-PEG2000 in eGFP-mRNA@MPP(Al 3+ ) is to inhibit aggregation, and other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation, DSPE-PEG2000 in eGFP-mRNA@MPP(Al 3+ ) can be replaced by other conjugated lipids that inhibit particle aggregation without affecting its efficacy.
  • Example 1.3.4.3 Preparation of conjugated lipid species for inhibiting particle aggregation in eGFP-mRNA@MPP(Mg 2+ )
  • DSPE-PEG2000 (Formula 53) is shown in Table 1-12 (the metal ion is Mg 2+ ), and all other conditions are the same.
  • Three other conjugated lipids that inhibit particle aggregation are DSPE-PEG700 (Formula 50), DSPE-PEG5000 (Formula 52), and DSPE-PEG1000 (Formula 51).
  • DSPE-PEG2000 in eGFP-mRNA@MPP(Mg 2+ ) can be replaced by other conjugated lipids that inhibit particle aggregation, and after replacement, its function is equivalent to that of eGFP-mRNA@MPP(Mg 2+ ) containing DSPE-PEG2000 (Table 1-12).
  • DSPE-PEG2000 in eGFP-mRNA@MPP(Mg 2+ ) is to inhibit aggregation, and other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation, DSPE-PEG2000 in eGFP-mRNA@MPP(Mg 2+ ) can be replaced by other conjugated lipids that inhibit particle aggregation without affecting its efficacy.
  • Example 1.3 The mRNA in Example 1.3 was replaced with two other mRNAs, and three mRNA@MPP(Fe 3+ ) containing different target protein mRNA sequences were prepared according to the method of Example 1.3.
  • the three different mRNA sequences are: 1 the mRNA sequence encoding the fluorescent protein eGFP is SEQ ID NO.1 (720 nt); 2 the mRNA sequence encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit is SEQ ID NO.2 (669 nt); and 3 the mRNA sequence encoding the tumor antigen NY-ESO-1 is SEQ ID NO.3 (543 nt).
  • the preparation process of the remaining drug (mRNA)-metal-phospholipid complex particles was the same as in Example 1.3, and eGFP-mRNA@MPP(Fe 3+ ), RBD-mRNA@MPP(Fe 3+ ), and NY-ESO-1-mRNA@MPP(Fe 3+ ) were obtained, respectively.
  • eGFP-mRNA@MPP(Fe 3+ ) was incubated with 293T cells at a concentration of 2 ⁇ g/mL (the concentration of the contained mRNA), 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 (the concentration of the contained mRNA), and the control group was incubated with MPP. After 24 hours, the supernatant was centrifuged and frozen at -20°C for later use. The expression level of the new coronavirus antigen RBD protein on the cells was detected using a commercially available new coronavirus antigen RBD ELISA detection kit. The results are shown in Figure 1-2.
  • Sample collection Place whole blood samples at room temperature for 2 hours, centrifuge at 1000 ⁇ g for 20 minutes, and collect the supernatant;
  • Sample addition Set up blank wells, standard wells, and test sample wells on the coated plate. Add 100 ⁇ L of sample diluent to the blank wells, add serially diluted standards to the standard wells, and add 100 ⁇ L of the test sample wells. Incubate at 37°C for 60 minutes.
  • TMB substrate solution (TMB)
  • the experimental animals were randomly divided into two groups (experimental group and control group), with 5 mice in each group.
  • the RBD-mRNA@MPP(Fe 3+ ) animal model was BALB/c mice. Each mouse received the first intramuscular administration on the first day and the second intramuscular administration on the 14th day.
  • the experimental group was injected with RBD-mRNA@MPP(Fe 3+ ), and the control group was injected with metal-phospholipid complex particles (MPP) without mRNA loading.
  • the dose of each administration was 100 ⁇ L, and the RBD-mRNA@MPP(Fe 3+ ) preparation in the experimental group contained 30 mg of mRNA.
  • the NY-ESO-1-mRNA@MPP(Fe 3+ ) formulation was administered intramuscularly four times per mouse on days 1, 7, 14, and 21.
  • the experimental group received NY-ESO-1-mRNA@MPP(Fe 3+ ), while the control group received metal-phospholipid complex particles (MPP) without mRNA loading.
  • Each dose was 100 ⁇ L, with the experimental group receiving 30 mg of mRNA in the NY-ESO-1-mRNA@MPP(Fe 3+ ) formulation.
  • Blood was collected from mice 28 days after the first dose, and serum was isolated and serially diluted. The total anti-NY-ESO-1 IgG antibodies produced in the mice were detected by ELISA. The results are shown in Figures 1-4.
  • Coating solution Accurately weigh 8.4 g of NaHCO3 and dissolve it in 1 L of distilled water (DDW). After the solid is completely dissolved, use 1 M NaOH solution to adjust the pH of the entire solution to 9.6. Store the prepared coating solution at 4°C until use.
  • wash solution Add 0.5 mL of Tween-20 to 1 L of 0.01 M PBS solution, mix well and place at room temperature.
  • Blocking solution Accurately weigh 20 g of BSA and add it to 1 L of 0.01 M 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 for use.
  • Antibody diluent Accurately weigh 2.5 g of BSA and dissolve it in 250 mL of 0.01 M PBS solution. After the solid is completely dissolved, add 1.25 mL of Tween-20, mix well, and store at 4°C until use.
  • Color development solution 0.1M citric acid: Add 19.2g of citric acid to 1000mL of DDW water (A).
  • 0.2M disodium hydrogen phosphate Add 28.4g of anhydrous disodium hydrogen phosphate to 1000mL of DDW water (B). 24.3mL of 0.1M citric acid solution (A), 25.7mL of 0.2M phosphate buffer (B), and 50mL of DDW water. Add 50mg of OPD (o-phenylenediamine) and 0.15mL of 30% H2O2 immediately before use.
  • Stop solution 2M H 2 SO 4 : 55.5 mL of concentrated sulfuric acid, add DDW to 500 mL.
  • Blocking Dry the coating solution in the well plate, wash with blocking solution 3 times, 5 minutes each time and dry, add 150 ⁇ L blocking solution to each well, and incubate at 37°C for 2 hours.
  • the serum sample was initially diluted 1:1000 with antibody diluent, and then serially diluted 1:2.
  • the diluted serum sample was added to a closed 96-well plate at 100 ⁇ L/well and incubated at 37°C for 2 hours.
  • the liquid in the well plate was shaken dry, and the washing solution was added at 300 ⁇ L/well.
  • the plate was shaken slowly for 40 seconds and this step was repeated three times.
  • a 1:1000 diluted biotinylated goat anti-mouse IgG antibody was added at 100 ⁇ L/well and incubated at 37°C.
  • the eGFP-mRNA@MPP(Fe 3+ ) experimental group showed an eGFP-positive cell rate of 97.7%, while no eGFP signal was detected in the MPP(Fe 3+ ) control group.
  • the RBD protein encoded by the MPP-encapsulated RBD-mRNA was present at 193.3 ng/mL in the supernatant of 293T cells, while the RBD protein content in the supernatant of 293T cells transfected with the empty MPP(Fe 3+ ) vector was zero.
  • both RBD-mRNA@MPP(Fe 3+ ) and NY-ESO-1-mRNA@MPP(Fe 3+ ) effectively induced humoral immunity in mice, producing high levels of antigen-specific binding antibodies.
  • the IgG antibody titer in mice treated with RBD-mRNA@MPP(Fe 3+ ) reached 117,268.8, while that in mice treated with NY-ESO-1-mRNA@MPP(Fe 3+ ) reached 5,319.52.
  • both RBD-mRNA@MPP(Fe 3+ ) and NY-ESO-1-mRNA@MPP(Fe 3+ ) effectively induced cellular immunity in mice, activating immune cells and producing large amounts of cytokines.
  • RBD-mRNA@MPP(Fe 3+ ) increased the expression levels of cytokines IFN- ⁇ , IL-2, and IL-4 to 252.8 pg/mL, 207.6 pg/mL, and 56.6 pg/mL, respectively;
  • NY-ESO-1-mRNA@MPP(Fe 3+ ) increased the expression levels of cytokines IFN- ⁇ , IL-2, and TNF- ⁇ to 70.79 pg/mL, 75.29 pg/mL, and 75.27 pg/mL, respectively.
  • mRNA@MPP(Fe 3+ ) can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), effectively inducing humoral and cellular immunity in mice, producing high levels of antigen-specific binding antibodies and cytokines, and exerting the effects of anti-new coronavirus mRNA vaccines and anti-tumor mRNA vaccines.
  • Example 1.4 The mRNA in Example 1.4 was replaced with two other mRNAs, and three mRNA@MPP(Al 3+ ) containing different target protein mRNA sequences were prepared according to the method of Example 1.4. Specific mRNA information and experimental procedures were referred to Example 1.3.5.1.
  • results Analysis As shown in Figure 1-1-2, the eGFP-positive cell rate in the eGFP-mRNA@MPP(Al 3+ ) experimental group was 98.02%, while no eGFP signal was detected in the MPP(Al 3+ ) control group. As shown in Figure 1-2-2, the RBD protein encoded by the RBD-mRNA encapsulated by MPP(Al 3+ ) was 212.6 ng/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. The results suggest that mRNA-MPP(Al 3+ ) can encapsulate and deliver any mRNA and directly encode polypeptides in cells.
  • both RBD-mRNA@MPP(Al 3+ ) and NY-ESO-1-mRNA@MPP(Al 3+ ) can effectively induce humoral immunity in mice and produce high levels of antigen-specific binding antibodies.
  • the IgG antibody titer in mice treated with RBD-mRNA@MPP(Al 3+ ) reached 129113, while that in mice treated with NY-ESO-1-mRNA@MPP(Al 3+ ) reached 6507.4.
  • RBD-mRNA@MPP(Al 3+ ) increased the expression levels of cytokines IFN- ⁇ , IL-2, and IL-4 to 271.8 pg/mL, 234.6 pg/mL, and 68.4 pg/mL, respectively;
  • NY-ESO-1-mRNA@MPP(Al 3+ ) increased the expression levels of cytokines IFN- ⁇ , IL-2, and TNF- ⁇ to 83.8 pg/mL, 98 pg/mL, and 97.8 pg/mL, respectively.
  • mRNA@MPP(Al 3+ ) can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), effectively inducing humoral and cellular immunity in mice, producing high levels of antigen-specific binding antibodies and cytokines, and exerting the effects of anti-new coronavirus mRNA vaccines and anti-tumor mRNA vaccines.
  • Example 1.5 The mRNA in Example 1.5 was replaced with two other mRNAs, and three mRNA@MPP(Mg 2+ ) containing different target protein mRNA sequences were prepared according to the method of Example 1.5.
  • mRNA@MPP(Mg 2+ ) For specific mRNA information and experimental procedures, refer to Example 1.3.5.1.
  • both RBD-mRNA@MPP(Mg 2+ ) and NY-ESO-1-mRNA@MPP(Mg 2+ ) effectively induced humoral immunity in mice, producing high levels of antigen-specific binding antibodies.
  • the IgG antibody titer in mice treated with RBD-mRNA@MPP(Mg 2+ ) reached 130614.40, while that in mice treated with NY-ESO-1-mRNA@MPP(Mg 2+ ) reached 6842.78.
  • RBD-mRNA@MPP(Mg 2+ ) increased the expression levels of cytokines IFN- ⁇ , IL-2, and IL-4 to 296.40 pg/mL, 243.33 pg/mL, and 75.30 pg/mL, respectively;
  • NY-ESO-1-mRNA@MPP(Mg 2+ ) increased the expression levels of cytokines IFN- ⁇ , IL-2, and TNF- ⁇ to 85.35 pg/mL, 98.58 pg/mL, and 98.02 pg/mL, respectively.
  • mRNA@MPP(Mg 2+ ) can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), effectively inducing humoral and cellular immunity in mice, producing high levels of antigen-specific binding antibodies and cytokines, and exerting the effects of anti-COVID-19 mRNA vaccines and anti-tumor mRNA vaccines.
  • Example 1.3 The mRNA in Example 1.3 was replaced with siRNA, and three siRNA@MPP(Fe 3+ ) containing different siRNAs were prepared according to the method of Example 1.3.
  • the genes, sequences, and corresponding random control sequences of the three different siRNAs are as follows: 1
  • the sequence of the siRNA targeting the Bcl-2 gene (Bcl-2-siRNA) is SEQ ID No. 4 (antisense strand) and SEQ ID No. 21 (sense strand) (19 bp), and its random control sequence is SEQ ID No. 5 (antisense strand) and SEQ ID No. 22 (sense strand) (19 bp);
  • the sequence of the siRNA targeting the PLK1 gene (PLK1-siRNA) is SEQ ID NO.
  • siRNA targeting the Gal-1 gene is SEQ ID NO. 8 (19 bp); its random control sequence is SEQ ID NO. 9 (19 bp).
  • the preparation process of the remaining siRNA@MPP is the same as that in Example 1.3.
  • U251 cells were seeded in 6-well plates at a density of 1 ⁇ 10 6 cells per well for approximately 24 hours. After each well of cells was incubated with siRNA@MPP(Fe 3+ ) containing the above-mentioned siRNA (wherein the concentration of siRNA was 2 ⁇ g/mL) for 72 hours, the cells were collected, and total cellular RNA was extracted. The mRNA expression levels of the target genes (Bcl-2, PLK1, Gal-1) were detected by RT-PCR, and the ability of siRNA@MPP(Fe 3+ ) to silence the target genes in cells was calculated.
  • RNA precipitate Extraction of total RNA: discard the culture medium from the six-well plate, rinse three times with PBS buffer, and add 1 mL of Trizol to each well to lyse the cells. Add 200 ⁇ L of chloroform, shake well, let stand at room temperature for 10 minutes, and centrifuge at 13,000 rpm at 4°C for 15 minutes to obtain a layered three-phase liquid, in which RNA is dissolved in the upper aqueous phase. Pipette the upper aqueous phase into a new enzyme-free 1.5 ml centrifuge tube, add 500 ⁇ L of isopropanol, let stand at room temperature for 10 minutes, and centrifuge at 13,000 rpm at 4°C for 15 minutes to obtain RNA precipitate.
  • RT-PCR Procedure This detection method uses SYBR Green dye and does not require a probe. Specifically, real-time PCR reactions were performed using cDNA from different samples as templates. The reaction mixture was prepared 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 Dye II (50 ⁇ ), 1 ⁇ L cDNA template obtained in the previous step, and 3.2 ⁇ L dH2O . 10 ⁇ L of sample was added to each well of the plate. After addition, the plate was centrifuged (1000 rpm, 5 min) to remove any remaining liquid and air bubbles.
  • Real-time PCR was performed using an ABI ViiA7 real-time fluorescence quantitative PCR instrument.
  • the reaction procedure was: 95°C, 30 seconds (1 cycle) ⁇ 95°C, 5 seconds; 55°C, 30 seconds; 72°C, 30 seconds (40 cycles) ⁇ 60°C-95°C, 2 minutes (1 cycle).
  • the experiment was repeated three times, and the average Ct value was calculated for each group.
  • the fold difference in expression between the experimental and control groups was calculated.
  • the control gene was GAPDH.
  • RT-PCR primers were as follows: 1 Bcl-2 primer: forward: 5'-AGGATTGTGGCCTTCTTTGAG-3', reverse: 5'-AGACAGCCAGGAGAAATC AAAC-3'; 2 PLK1 primer: forward: 5'-ACCAGCACGTCGTAGGATTC-3', reverse: 5'-CAAGCAATTTGCCGTAGG-3'; 3 Gal-1 primer: forward: 5'-CAATCAT GGCCTGTGGTCTG-3', reverse: 5'-GTG TAGGCACAGGTTGTTGCTG-3'. 4 GAPDH primer: forward: 5'-TCAGGGGTTTCACATTTGGCA-3', reverse: 5'-GG AGCGGAA AACCA-3'.
  • Calculation method of gene silencing efficiency 100% - gene expression level of experimental group / gene expression level of control group.
  • siRNA@MPP(Fe 3+ ) As shown in Figures 1-7 ("scr siRNA” in the figures refers to "Scramble siRNA," a negative control, similarly below), 1-8, and 1-9, all three siRNA@MPP(Fe 3+ ) significantly interfered with their corresponding target genes.
  • Bcl-2-siRNA@MPP(Fe 3+ ) achieved a 76% inhibition rate against the target gene Bcl-2
  • PLK1-siRNA@MPP(Fe 3+ ) achieved an 86% inhibition rate against the target gene PLK1
  • Gal-1-siRNA@MPP(Fe 3+ ) achieved a 73% inhibition rate against the target gene Gal-1.
  • Example 1.4 The mRNA in Example 1.4 was replaced with siRNA, and three siRNA@MPP(Al 3+ ) containing different siRNA sequences were prepared according to the method of Example 1.4. Specific siRNA information and experimental procedures were referred to Example 1.3.6.1.
  • Example 1.5 The mRNA in Example 1.5 was replaced with siRNA, and three siRNA@MPP(Mg 2+ ) containing different siRNA sequences were prepared according to the method of Example 1.5. Specific siRNA information and experimental procedures were referred to Example 1.3.6.1.
  • Example 1.3 The mRNA in Example 1.3 was replaced with an ASO, and three ASO@MPP(Fe 3+ ) containing different ASOs were prepared according to the method of Example 1.3.
  • the genes, sequences, and corresponding random control sequences of the three different ASOs were as follows: 1
  • the ASO targeting the STAT3 gene (STAT3-ASO) had a sequence of SEQ ID No. 10 (17 nt), and its random control sequence was SEQ ID NO. 11 (18 nt);
  • the ASO targeting the ⁇ -syn gene ( ⁇ -syn-ASO) had a sequence of SEQ ID NO. 12 (16 nt), and its random control sequence was SEQ ID NO.
  • the ASO targeting the Bcl-2 gene had a sequence of SEQ ID NO. 14 (18 nt), and its random control sequence was SEQ ID NO. 15 (20 nt).
  • the preparation process of the remaining ASO-metal-phospholipid complex particles was the same as in Example 1.3.
  • ASO@MPP(Fe 3+ )s were incubated with different cells: U251 human glioblastoma cells were incubated with ASO@MPP(Fe 3+ ) targeting the STAT3 gene; SH-SY5Y human neuroblastoma cells were incubated with ASO@MPP(Fe 3+ ) targeting the ⁇ -syn gene; and Daudi human lymphoma cells were incubated with ASO@MPP(Fe 3+ ) targeting the Bcl-2 gene.
  • each well was incubated with ASO@MPP(Fe 3+ ) containing the aforementioned ASOs (at a concentration of 2 ⁇ g/mL) for 48 hours. Cells were then harvested, and total RNA was extracted. RT-PCR was used to measure the mRNA expression of the target genes (STAT3, ⁇ -syn, and Bcl-2). The ability of ASO@MPP(Fe 3+ ) to silence these target genes was calculated.
  • RNA precipitate Extraction of total RNA: discard the culture medium from the six-well plate, rinse three times with PBS buffer, and add 1 mL of Trizol to each well to lyse the cells. Add 200 ⁇ L of chloroform, shake well, let stand at room temperature for 10 minutes, and centrifuge at 13,000 rpm at 4°C for 15 minutes to obtain a layered three-phase liquid, in which RNA is dissolved in the upper aqueous phase. Pipette the upper aqueous phase into a new enzyme-free 1.5 ml centrifuge tube, add 500 ⁇ L of isopropanol, let stand at room temperature for 10 minutes, and centrifuge at 13,000 rpm at 4°C for 15 minutes to obtain RNA precipitate.
  • RT-PCR Procedure This detection method uses SYBR Green dye and does not require a probe. Specifically, real-time PCR reactions were performed using cDNA from different samples as templates. The reaction mixture was prepared 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 Dye II (50 ⁇ ), 1 ⁇ L cDNA template obtained in the previous step, and 3.2 ⁇ L dH2O . 10 ⁇ L of sample was added to each well of the plate. After addition, the plate was centrifuged (1000 rpm, 5 min) to remove any remaining liquid and air bubbles.
  • Real-time PCR was performed using an ABI ViiA7 real-time fluorescence quantitative PCR instrument.
  • the reaction procedure was: 95°C, 30 seconds (1 cycle) ⁇ 95°C, 5 seconds; 55°C, 30 seconds; 72°C, 30 seconds (40 cycles) ⁇ 60°C-95°C, 2 minutes (1 cycle).
  • the experiment was repeated three times, and the average Ct value was calculated for each group.
  • the fold difference in expression between the experimental and control groups was calculated.
  • the control gene was GAPDH.
  • RT-PCR primer sequences are as follows: 1 STAT3 primer: forward: 5'-TGATCACCTTTGAGACCGAGG-3', reverse: 5'-GATCACCACAACTGG CAA GG-3'; 2 ⁇ -syn primer: forward: 5'-TGACGGGTGTGACAGCAGTAG-3', reverse: 5'-CAGTGGCTGCTGCAATG-3'; 3 Bcl-2 primer: forward: 5'-AGGATT GTG GCCTTCTTTGAG-3', reverse: 5'-AGACAGCCAGGAGAAATCAAAC-3'; 4 GAPDH primer: forward: 5'-TCAGGGG TTTCACATTTGGCA-3', reverse: 5'-GGAGCGGAA AACCA-3'.
  • the calculation method of gene silencing efficiency is: 100% - gene expression level of experimental group/gene expression level of control group.
  • Example 1.4 The mRNA in Example 1.4 was replaced with ASO, and three ASO@MPP(Al 3+ ) containing different ASO sequences were prepared according to the method of Example 1.4. Specific ASO information and experimental procedures were referred to Example 1.3.7.1.
  • Example 1.5 The mRNA in Example 1.5 was replaced with ASO, and three ASO@MPP(Mg 2+ ) containing different ASO sequences were prepared according to the method of Example 1.5. Specific ASO information and experimental procedures were referred to Example 1.3.7.1.
  • Example 1.3.8.1 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 1.3 was replaced by double-stranded RNA (siRNA), single-stranded RNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively.
  • the different types of nucleic acid sequences are: 1 The sequence of double-stranded RNA (Bcl-2-siRNA) is SEQ ID NO.4 (antisense chain) and SEQ ID No.21 (sense chain) (19 bp), and its random control sequence is SEQ ID NO.5 (antisense chain) and SEQ ID No.22 (sense chain) (19 bp); 2 The sequence of single-stranded RNA (STAT3-ASO) is SEQ ID NO.10 (17 nt), and its random control sequence is SEQ ID NO.11 (18 nt); 3 The sequence of single-stranded RNA (mRNA encoding wild-type SARS-CoV-2 S protein) is SEQ ID No.16 (3822 nt); 4
  • Drug-metal-phospholipid complex particles encapsulating the above-mentioned different types of nucleic acids (Bcl-2-siRNA@MPP(Fe 3+ ), STAT3-ASO@MPP(Fe 3+ ), S-mRNA@MPP(Fe 3+ ), dsDNA@MPP(Fe 3+ ), ssDNA@MPP(Fe 3+ )) were prepared respectively according to the method of Example 1.3 . The preparation process of the remaining drug-lipid particles was the same as that of Example 1.3.
  • U251 cells were seeded in a 12-well plate at a density of 1 ⁇ 10 5 cells per well for approximately 24 hours. After that, each well was incubated with siRNA@MPP(Fe 3+ ) (wherein the concentration of siRNA was 2 ⁇ g/mL) or ASO@MPP(Fe 3+ ) (wherein the concentration of ASO was 2 ⁇ g/mL) for 72 hours, respectively.
  • the cells were collected, and total cellular RNA was extracted.
  • the mRNA expression levels of the target genes (Bcl-2, STAT3) were detected by RT-PCR, and the ability of siRNA@MPP(Fe 3+ ) or ASO@MPP(Fe 3+ ) to silence the target genes in the cells was calculated. The results are shown in Figures 1-7 of Example 1.3.6 and Figures 1-10 of Example 1.3.7.
  • S-mRNA@MPP(Fe 3+ ) was incubated with 293T cells at a concentration of 2 ⁇ g/mL (the concentration of the contained mRNA).
  • the control group was incubated with MPP(Fe 3+ ). After 24 hours, the supernatant was centrifuged and stored at -20°C for later use. The cell pellet was resuspended in 100 ⁇ L PBS buffer solution, frozen and thawed twice, and sonicated for 10 minutes before centrifugation to obtain the supernatant.
  • the expression levels of S protein in both the cell supernatant and cell lysate were detected using a commercially available SARS-CoV-2 S protein ELISA kit. The results are shown in Figures 1-13.
  • A549 lung cancer cells were incubated with ds-DNA@MPP(Fe 3+ ) at a concentration of 100 nM (the concentration of DNA contained) for 2 hours, and then the remaining drug-lipid particles were removed.
  • the cells were washed twice with PBS, and the cell nuclei were stained with Hochest33342 dye for 3 minutes. The dye was then removed and the cells were washed twice with PBS. The cells were observed using a high-content imaging system, and the efficiency of drug-lipid particle transfection with DNA was calculated. The results are shown in Figures 1-14.
  • HT22 mouse hippocampal neurons were incubated with ss-DNA@MPP(Fe 3+ ) at a concentration of 200 nM (the concentration of DNA contained). After 2 hours, the remaining drug-lipid particles were removed and the cells were washed twice with PBS. The cells were observed using a high-content imaging system, and the efficiency of drug-lipid particle transfection with DNA was calculated. The results are shown in Figures 1-14.
  • the culture method of human glioblastoma U251 cells is the same as that in Example 1.3.6.
  • the culture method of 293T cells is the same as that in Example 1.3.5.
  • the RT-PCR method is the same as in Example 1.3.6.
  • To calculate transfection efficiency Use a high-content imaging system to randomly select 3-5 fields of view. Obtain cell morphology under a standard light source, fluorescence signals under excitation/emission light of 550nm/570nm (excitation light of the fluorescent dye Cy3 that labels DNA), and fluorescence signals under excitation/emission light of 352nm/461nm (excitation light of the fluorescent dye Hoechst33342 that labels the cell nucleus) in the same field of view (results are shown in Figure 1-15). Calculate the ratio of cells with Cy3 fluorescence signals in the randomly selected field of view to the number of cells with Hoechst33342 fluorescence signals in the same field of view, which is the transfection efficiency.
  • the drug-metal-phospholipid complex particles can encapsulate and function with any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA), ranging in length from 16 to 3822 nt.
  • Example 1.4 The mRNA in Example 1.4 was replaced with double-stranded RNA (siRNA), single-stranded RNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively.
  • siRNA double-stranded RNA
  • ASO single-stranded RNA
  • mRNA single-stranded DNA
  • DNA double-stranded DNA
  • the drug-metal-phospholipid complex particles can encapsulate and function with any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA), ranging in length from 16 to 3822 nt.
  • Example 1.5 The mRNA in Example 1.5 was replaced with double-stranded RNA (siRNA), single-stranded RNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively.
  • siRNA double-stranded RNA
  • ASO single-stranded RNA
  • mRNA single-stranded DNA
  • DNA double-stranded DNA
  • the connection between DSPC and curcumin was characterized by differential scanning calorimetry. The following conditions were used: 3-5 mg of the test sample was weighed, and the temperature was raised at a rate of 10°C/min over a range of 30°C to 300°C. Curcumin, DSPC, and the curcumin-DSPC complex were scanned separately. Curves were plotted based on the data obtained, as shown in Figure 2-1. The plot shows that curcumin has a lattice structure, with a distinct melting peak at 185°C. DSPC is a mixture, with multiple depressions on the curve, likely due to thermal variations between the different components at different temperatures. The curcumin-DSPC complex exhibits no peaks near its melting peak, essentially forming a straight line. This indicates that curcumin and DSPC are bound together and exist in an amorphous form, confirming the successful preparation of the phospholipid complex.
  • Example 2.1.1 The difference between this example and Example 2.1.1 is that the connection between the phospholipid complex and Al 3+ is characterized by spectrophotometry: as shown in Figure 2-2-2, after the phospholipid complex (CUR-DSPC) is combined with Al 3+ , its maximum absorption wavelength shifts from 420nm to 433nm, and the conjugated structure of the phospholipid complex changes, proving that Al 3+ is successfully complexed with curcumin.
  • Example 2.1.1 The difference between this example and Example 2.1.1 is that the connection between the phospholipid complex and Mg 2+ is characterized by spectrophotometry: as shown in Figure 2-2-3, after the phospholipid complex (CUR-DSPC) binds to Mg 2+ , the maximum absorption peak shifts, and its maximum absorption wavelength shifts from 426nm to 420nm. The conjugated structure of the phospholipid complex changes, proving that Mg 2+ is successfully complexed with curcumin.
  • CUR-DSPC phospholipid complex
  • the phospholipid complex in the metal-phospholipid complex binds Fe 3+ through a coordination bond.
  • the coordination bond between the phospholipid complex and the Fe 3+ is protonated (absorbs a hydrogen ion) and cleaved.
  • Example 1.3 The mRNA in Example 1.3 was replaced with thiol-modified siRNA, and drug-metal-phospholipid complex particles, siRNA@MPP(Fe 3+ ), were prepared according to the method of Example 1.3. Elemental analysis was performed using transmission electron microscopy. The results are shown in Figures 2-4 : C, N, O, and P are common elements. The Fe elemental analysis shows that Fe 3+ is evenly distributed on the lipid nanoparticles. Because the siRNA is modified with thiol groups, the S elemental analysis specifically indicates the location of the siRNA. The figure shows that the siRNA is well complexed near the Fe 3+ ions, confirming that the drug-lipid nanoparticles successfully encapsulated the siRNA.
  • Example 2.4 Efficiency of Nucleic Acids (siRNA and mRNA) Encapsulated in Metal-Phospholipid Complex Particles (MPPs) Containing Fe 3+ , Al 3+ , or Mg 2+ and Comparison with LNPs
  • Example 1.3.5 The mRNA in Example 1.3.5 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 novel coronavirus S1 subunit (SEQ ID No. 2, 669 nt), respectively, to prepare nucleic acid-encapsulated drug-metal-phospholipid complex particles siRNA@MPP and mRNA@MPP, respectively.
  • the preparation process of the remaining drug-metal-phospholipid complex particles was the same as that in Example 1.3.5.
  • siRNA@LNPs and mRNA@LNPs were prepared using the same drug loading as described in Example 1.3.6 for siRNA@MPP and Example 1.3.5 for mRNA@MPP.
  • the organic phase solution was prepared according to the Onpattro lipid nanoparticle formulation, where the ionizable lipid ALC0315, DSPE-PEG2000, DSPC, and cholesterol were dissolved in ethanol at a molar ratio of 50%:1.5%:10%:38.5%.
  • Bcl-siRNA or RBD-mRNA was added to the aqueous phase (0.1 M acetic acid-sodium acetate buffer, pH 4.0).
  • the amino lipid to phosphate nucleotide ratio (N/P) was 6:1, ensuring the same nucleic acid loading as described for the siRNA@MPP and mRNA@MPP formulations.
  • the aqueous and organic phases were rapidly mixed at a volume ratio of 3:1 at a flow rate of 14 mL/min. After mixing, the mixture was diluted tenfold with enzyme-free PBS buffer solution and concentrated to one-tenth using a 100 kDa ultrafiltration tube. After repeating the dilution and concentration operation three times, the ethanol concentration in the mixture was reduced to below 0.0005% and the pH value of the solution was increased to the normal pH value of PBS buffer solution (7.2-7.4), thus producing siRNA@LNP and mRNA@LNP, respectively.
  • Agarose gel electrophoresis was used to detect the nucleic acid (siRNA and mRNA) encapsulation rates of siRNA@MPP, mRNA@MPP, siRNA@LNP and mRNA@LNP, respectively.
  • the encapsulation rate was determined as follows: the nucleic acid (siRNA and mRNA) feed amount of each group of lipid nanoparticles was set at 10 ⁇ g/mL, the mass ratio of lipid to nucleic acid was 40:1, the nucleic acid was dissolved in PBS buffer solution as the positive control group, and the negative control was a PBS buffer solution without nucleic acid.
  • the concentration of agarose gel was 1.5%.
  • the gaps in the gel only allowed free nucleic acids to pass through but not lipid nanoparticles.
  • electrophoresis was stopped to prevent nucleic acid degradation due to long electrophoresis time.
  • Image J software was used to count the grayscale values of free nucleic acids in different groups.
  • the positive control group was set at 100%.
  • the ratio of free nucleic acid in each group to the positive control was the relative amount of free nucleic acid.
  • the encapsulation rate of each group was (100-relative amount of free nucleic acid)%.
  • Example 2.5 Nucleic acid lysosomal escape ability of metal-phospholipid complex particles MPP containing Fe 3+ , Al 3+ or Mg 2+ and comparison with LNP
  • the Bcl-2-siRNA (SEQ ID No. 4) of Example 1.3.6 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@MPP (the concentration of the siRNA contained was 100 nM), and the Bcl-2-siRNA (SEQ ID No. 4) of Example 2.4 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@LNP (the concentration of the siRNA contained was 100 nM); the eGFP-mRNA (SEQ ID No.
  • Example 13.5 1) of Example 1.3.5 was replaced with Cy5-labeled eGFP-mRNA to prepare Cy5-mRNA@MPP (the concentration of the mRNA contained was 2 ⁇ g/mL), and the RBD-mRNA of Example 2.4 was replaced with Cy5-labeled RBD-mRNA to prepare Cy5-mRNA@LNP (the concentration of the mRNA contained was 2 ⁇ g/mL), and they were respectively mixed with the cell lysosome probe Lysotracker After co-incubating A549 cells with Cy5 and Lysotracker Green for 3 hours, a high-content imaging system was used to observe the overlap of the Cy5 fluorescence signal (red) and the Lysotracker Green fluorescence signal (green) (the results when the metal ion was Fe 3+ are shown in Figure 2-11) to determine the ability of the drug-lipid particles to promote nucleic acid lysosomal escape.
  • Cy5 fluorescence signal red
  • the Lysotracker Green fluorescence signal green
  • the ability of drug-metal-phospholipid complex particles to promote lysosomal escape of nucleic acids was assessed by incubating cells with the drug-lipid nanoparticles for 3 hours.
  • the overlap between the Cy5 fluorescence signal (red) and the Lysotracker Green fluorescence signal (green) was observed using a high-content imaging system, and the overlap ratio between the red and green fluorescence signals was calculated using imageJ software. If the overlap ratio between the red and green fluorescence signals was less than 50% after incubating cells with the drug-metal-phospholipid complex particles for 3 hours, this indicated that nucleic acids could escape from lysosomes more rapidly and that the lipid nanoparticles possessed a strong ability to promote lysosomal escape of nucleic acids.
  • Example 2.6 Ability of Metal-Phospholipid Complex Particles (MPPs) Containing Fe 3+ , Al 3+ , or Mg 2+ to Promote Nucleic Acid Expression and Comparison with LNPs
  • Example 2.4 The RBD-mRNA in Example 2.4 was replaced with mRNA encoding the fluorescent protein eGFP, and the rest of the preparation method was the same as Example 2.4 to obtain eGFP-mRNA@LNP.
  • the eGFP-mRNA@MPP prepared in Example 1.3 and the above-mentioned eGFP-mRNA@LNP (containing mRNA at a concentration of 2 ⁇ g/mL) were incubated with 293T cells, respectively.
  • the control group was incubated with MPP or LNP. After 48 h, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.
  • the method for analyzing the eGFP-positive cell rate by flow cytometry is as described in Example 1.3.
  • the eGFP-positive cell percentage method is used to characterize the nucleic acid expression ability of MPPs composed of different metal ions (Fe 3+ , Al 3+ or Mg 2+ ), such as MPP(Fe 3+ ), MPP(Al 3+ ) or MPP(Mg 2+ ).
  • MPP(Fe 3+ ), MPP(Al 3+ ) or MPP(Mg 2+ ) because the performance of the eGFP protein itself is insufficient to distinguish subtle differences, the differences in the ability to promote nucleic acid expression between MPPs composed of metal ions (Fe 3+ , Al 3+ or Mg 2+ ), such as MPP(Fe 3+ ), MPP(Al 3+ ) or MPP(Mg 2+ ) cannot be distinguished.
  • Example 2.7 Ability of Drug-Metal-Phospholipid Complex Particles (MPPs) Containing Fe 3+ , Al 3+ , or Mg 2+ Metal Ions in Promoting Humoral and Cellular Immunity and Comparison with LNPs
  • the RBD-mRNA@MPP of Example 1.3.5 and the RBD-mRNA@LNP of Example 2.4 were incubated with 293T cells at a concentration of 2 ⁇ g/mL (the concentration of the contained mRNA).
  • the control group was incubated with MPP. After 24 hours, the supernatant was centrifuged and frozen at -20°C for later use. The cell pellet was resuspended in 100 ⁇ L PBS buffer solution, frozen and thawed twice, and sonicated for 10 minutes before centrifugation to obtain the supernatant.
  • the expression levels of RBD protein in both the cell supernatant and the cell lysate were detected using a commercially available new coronavirus antigen RBD ELISA detection kit. The results are shown in Figure 2-8.
  • the ELISA method for detecting RBD expression levels is as described in Example 1.3.5.
  • the experimental animals were randomly divided into two groups (experimental group and control group), with 5 mice in each group.
  • the animal model was BALB/c mice. Each mouse received the first intramuscular administration on day 1 and the second intramuscular administration on day 14.
  • the experimental group was injected with RBD-mRNA@MPP and RBD-mRNA@LNP, respectively, while the control group was injected with MPP and LNP without mRNA.
  • the dose of each administration was 100 ⁇ L, of which the RBD-mRNA@MPP and RBD-mRNA@LNP preparations in the experimental group each contained 30 mg of mRNA.
  • the ELISA method for detecting the titer of total RBD IgG antibodies against the novel coronavirus S1 subunit is as described in Example 1.3.5.
  • the ELISA method for detecting the expression levels of IFN- ⁇ , IL-2, and IL-4 is as described in Example 1.3.5.
  • RBD-mRNA@MPP(Fe 3+ ), RBD-mRNA@MPP(Al 3+ ), RBD-mRNA@MPP(Mg 2+ ) and RBD-mRNA@LNP can all induce 293T cells to express a certain amount of RBD, but the ability of RBD-mRNA@MPP(Mg 2+ ) to induce cell expression of RBD is significantly stronger than that of RBD-mRNA@MPP(Al 3+ ) and RBD-mRNA@MPP(Fe 3+ ).
  • the ability of the three to induce cell expression of RBD is significantly stronger than that of RBD-mRNA@MPP(LNP): the expression level of RBD in the cell supernatant of the RBD-mRNA@MPP(Fe 3+ ) treatment group is 218.93 ng/mL, the expression level of RBD in the cell supernatant of the RBD-mRNA@MPP(Al 3+ ) treatment group is 239.93 ng/mL, and the expression level of RBD in the cell supernatant of the RBD-mRNA@MPP(Mg 2+ ) treatment group was 303.63 ng/mL, and the expression level of RBD in the cell supernatant of the RBD-mRNA@LNP treatment group was 126.67 ng/mL.
  • RBD-mRNA@MPP effectively induced humoral immunity in mice and produced high levels of antigen-specific binding antibodies.
  • the ability of RBD-mRNA@MPP(Mg 2+ ) to induce humoral immunity in mice was significantly better than that of RBD-mRNA@MPP(Al 3+ ) and RBD-mRNA@MPP(Fe 3+ ).
  • mice treated with RBD-mRNA@MPP(Mg 2+ ) reached 171782.00; the IgG antibody titer in mice treated with RBD-mRNA@MPP(Fe 3+ ) reached 122666.67; the IgG antibody titer in mice treated with RBD-mRNA@MPP(Al 3+ ) reached 134833.33; while the IgG antibody titer in mice treated with RBD-mRNA@LNP was only 73694.00.
  • RBD-mRNA@MPP(Mg 2+ ) can effectively induce cellular immunity in mice, i.e., activate immune cells and produce a large amount of cytokines. Moreover, the ability of RBD-mRNA@MPP(Mg 2+ ) to induce cellular immunity in mice is significantly better than that of RBD-mRNA@MPP(Fe 3+ ) and RBD-mRNA@MPP(Al 3+ ).
  • RBD-mRNA@MPP(Fe 3+ ) makes the expression levels of cytokines IFN- ⁇ , IL-2, and IL-4 reach 286.2 pg/mL, 209.67 pg/mL, and 58.02 pg/mL, respectively;
  • RBD-mRNA@MPP(Al 3+ ) increased the expression levels of cytokines IFN- ⁇ , IL-2, and IL-4 to 306 pg/mL, 239.67 pg/mL, and 71 pg/mL, respectively;
  • RBD-mRNA@MPP(Mg 2+ ) increased the expression levels of cytokines IFN- ⁇ , IL-2, and IL-4 to 333 pg/mL, 270.33 pg/mL, and 73 pg/mL, respectively; while RBD-m
  • RBD-mRNA@MPP(Mg 2+ ) has significantly better ability to deliver arbitrary mRNA and realize its function than RBD-mRNA@MPP(Fe 3+ ) and mRNA@MPP(Al 3+ ).
  • RBD-mRNA@MPP(Fe 3+ ) has significantly better ability to induce cellular immunity in mice than RBD-mRNA@LNP:
  • RBD-mRNA@MPP can more effectively promote cell expression of target proteins and can more effectively activate humoral immunity and cellular immunity in the body. Therefore, drug (mRNA)-lipid particles are significantly superior to existing technology LNPs in the effects of mRNA-carrying drugs, vaccines or other products.
  • MPP has a stronger ability to promote nucleic acid lysosomal escape
  • MPP has a stronger ability to promote the expression of nucleic acids into proteins (antigens)
  • curcumin in MPP is separated from DSPC in the body, and acts as an immune adjuvant (also known as an immunomodulator), which can activate humoral immunity and cellular immunity to enhance the effect of MPP in delivering mRNA vaccines, and can also inhibit the immune factor storm to inhibit excessive and harmful immune responses to the body.
  • an immune adjuvant also known as an immunomodulator
  • MPP Compared with MPPs composed of different metal ions (such as MPP (Fe 3+ ) and MPP (Al 3+ )), MPP (Mg 2+ ) can more effectively promote cell expression of target proteins.
  • Mg 2+ maintains a weak chelation effect with RNA, which can enhance the function of RNA, including increasing its thermodynamic stability, chemical stability and catalytic activity (Biochemistry. 2021 Aug 10; 60(31): 2374-2386.), Mg 2+ can maintain the structural stability and translation activity of ribosomes, partially supplement the function of ribosomal proteins (Biosci Biotechnol Biochem. 2021 Jun 24; 85(7): 1582-1593.) and enhance the immune response.
  • Mg2 + can regulate LFA-1 downstream signal transduction by inducing conformational changes in the co-stimulatory molecule LFA-1 on the surface of CD8+ T cells, thereby promoting T cell activation and cytotoxicity (Cell.2022Feb 17;185(4):585-602.e29.).
  • Example 2.8 Comparison of the Expression Duration of Drug-Metal-Phospholipid Complex Particles (MPP) with Fe 3+ , Al 3+ or Mg 2+ and LNP
  • CAR mRNA includes a transmembrane domain, a signal transduction domain, an antigen binding domain, a co-stimulatory signal transduction region, and a connecting region between the antigen binding domain and the transmembrane domain.
  • Antigen binding domain, scFV encodes the VH and VL parts of the FMC63 antibody.
  • the VH and VL parts of the FMC63 antibody are connected by a linker and can recognize tumor cell antigens.
  • the spacer connects the antigen-binding domain scFV and the transmembrane domain. Its flexibility ensures that scFV can better recognize antigens.
  • Transmembrane(TM) These combinations anchor the CAR expression to the T cell membrane.
  • the intracellular co-stimulatory signaling domain is used to activate T cells.
  • the activation signal is primarily provided by CD3-zeta, and other co-stimulatory domains, such as CD28 and CD8, can be added to enhance the signal.
  • the co-stimulatory signaling domain can contain multiple co-stimulatory domains. Regardless of the number of co-stimulatory domains, CD3-zeta should be placed last.
  • the CD28 co-stimulatory domain effectively enhances T cell proliferation.
  • the amino acid sequence expressed by CD19 CAR mRNA is shown in SEQ ID NO.60.
  • mRNA was replaced with CD19 CAR mRNA to prepare CD19 CAR mRNA@MPP and CD19 CAR mRNA@LNP.
  • the proportion of CAR-positive cells in myeloid cells was detected by flow cytometry.
  • Method for analyzing the percentage of CAR-positive cells by flow cytometry Peripheral blood was mixed with Alexa Fluor Incubate the labeled CAR linker antibody and CD11b-PE antibody at 4°C for 30 minutes. Add red blood cell lysis buffer and lyse for 5-10 minutes in the dark. Wash twice with buffer and resuspend in 100-30,000 ⁇ L of buffer.
  • CAR-positive myeloid cell percentage number of CAR-expressing myeloid cells / total number of myeloid cells ⁇ 100%.
  • Example 2.9 In vivo safety evaluation of metal-chelated phospholipid complex nanoparticles (MPP) containing Fe 3+ , Al 3+ or Mg 2+ metal ions
  • the blank control group consisted of 14 rats (8 in the experimental group and 6 in the recovery group), half male and half female; the low-dose MPP group (25 mg/kg) consisted of 14 rats (8 in the experimental group and 6 in the recovery group), half male and half female; the medium-dose MPP group (50 mg/kg) consisted of 14 rats (8 in the experimental group and 6 in the recovery group), half male and half female; and the high-dose MPP group (100 mg/kg) consisted of 14 rats (8 in the experimental group and 6 in the recovery group), half male and half female.
  • the experimental group 32 rats was autopsied after the end of drug administration.
  • the recovery group 24 rats was autopsied after 20 days of continued normal feeding.
  • mice were administered via tail vein injection every two days for 20 days, and the body weight of SD rats was recorded weekly.
  • the prepared MPP was dissolved in DPBS.
  • the control group was injected with an equal volume of DPBS, while the low-dose MPP group, medium-dose MPP group, and high-dose MPP group were injected with 25 mg/kg, 50 mg/kg, and 100 mg/kg of MPP, respectively.
  • Organ-to-body ratio organ wet weight / body weight ⁇ 100%.
  • Routine blood test parameters include: white blood cell count, lymphocyte count, monocyte count, neutrophil count, lymphocyte percentage, monocyte percentage, neutrophil percentage, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, coefficient of variation of red blood cell distribution width, platelet count, mean platelet volume, platelet distribution width, and platelet hematocrit. Gently invert the whole blood sample to mix thoroughly. A small amount of whole blood is then collected and analyzed using an automated hematology analyzer.
  • Blood biochemical marker testing method Blood biochemical markers include inorganic ions (Fe2 + , Na + , K + , Cl- , Ca2 + ), liver function markers (ALT, AST, ⁇ -GT, T-BIL, D-BIL, ALP, ALB), renal function markers (BUN, UA, CR), cardiac function markers (LDH, CK), glucose metabolism markers (GSP, GLU, INS), and lipid metabolism markers (CHO, TG, LDL-C, HDL-C).
  • inorganic ions Fe2 + , Na + , K + , Cl- , Ca2 +
  • liver function markers ALT, AST, ⁇ -GT, T-BIL, D-BIL, ALP, ALB
  • renal function markers BUN, UA, CR
  • cardiac function markers LH, CK
  • glucose metabolism markers GSP, GLU, INS
  • lipid metabolism markers CHO, TG, LDL-C, HDL-C.
  • Immunological indicators include thyroid function markers (TT3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN- ⁇ , IFN- ⁇ , TNF- ⁇ ), immunoglobulins (IgG, IgA, IgM), and serum complement (C3, CH50). These indicators are measured using ELISA.
  • Pathological examination method of the main organs of SD rats At the end of the administration period and the recovery period, the rats in each group were anesthetized, and the main organs of the rats, including the whole brain, heart, liver, spleen, lungs, and kidneys, were removed by ophthalmic scissors. The organs were gently rinsed with 0.9% saline, fixed in 4% paraformaldehyde fixative, routinely paraffin-embedded, 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 (white blood cell count, lymphocyte count, monocyte count, neutrophil count, lymphocyte percentage, monocyte percentage, neutrophil percentage, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, coefficient of variation of red blood cell distribution width, platelet count, mean platelet volume, platelet distribution width, platelet hematocrit) of the low, medium and high doses of MPP groups were normal; compared with the control group, the blood biochemical indicators of the low, medium and high doses of MPP groups, including 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
  • inorganic ions Fe 2+ , Na + , K + , Cl
  • the brain tissue structure of rats in the low-, medium- and high-dose MPP groups was intact, the tissue staining was normal, the cell morphology was intact, and there was no nuclear pyknosis or inflammatory cell infiltration;
  • the myocardial tissue structure was intact, the myocardial cells were arranged neatly, continuously and tightly, the cell nuclei were clearly visible, and there was no obvious cell congestion, edema or necrosis;
  • the liver cell morphology was normal, without inflammatory cell aggregation and necrosis;
  • the spleen structure was normal, with clear boundaries between red and white pulp;
  • the lung tissue structure was intact, the alveoli were of uniform size, and there was no obvious inflammatory cell aggregation or infiltration; and the kidney structure was normal.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ⁇ -GT glutamyl transpeptidase
  • T-BIL total bilirubin
  • D-BIL direct bilirubin
  • ALP alkaline phosphatase
  • ALB albumin
  • BUN blood 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
  • TT3 triiodothyronine
  • TT4 tetraiodothyronine
  • TSH thyroid-stimulating hormone
  • IL-1 interleukin 1
  • IL-2 interleukin 2
  • Example 2.10 Comparison of in vivo safety of metal-phospholipid complex particles (MPP) containing Fe 3+ , Al 3+ , or Mg 2+ and LNP
  • the primary toxicity of LNPs comes from their main components—cationic lipids and/or ionizable lipids. During LNP metabolism in the body, the free cationic lipids and/or ionizable lipids can produce significant toxicity.
  • the median lethal dose ( IC50 ) of cationic lipids and/or ionizable lipids to biological cells is an important parameter for assessing the toxicity of LNPs to the body.
  • Metal-chelated phospholipid complex nanoparticles (MPPs) replace the cationic lipids/ionizable lipids in LNPs with metal-phospholipid complexes. Therefore, we compared the toxicity of LNPs and MPPs by studying the median lethal dose ( IC50 ) of metal-chelated phospholipid complexes and cationic lipids/ionizable lipids to biological cells.
  • 293T cells were incubated with different concentrations of the metal-phospholipid complex (0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 ⁇ M), cationic lipid (DOTAP, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 ⁇ M, structural formula below), and ionizable lipid (ALC0315, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 ⁇ M, structural formula below) for 48 hours.
  • the metal-phospholipid complex 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 ⁇ M
  • DOTAP cationic lipid
  • ALC0315 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 ⁇ M, structural formula below
  • Cell culture Culture cells in DMEM containing 10% FBS and 1% double-antibody. Allow the cells to reach a density of 80%-90% of the total volume of the culture flask.
  • Counting Dilute the cell suspension to 10,000 cells per mL according to the intended cell count. Add 100 ⁇ L per well of a 96-well plate, with at least five replicates per group. Incubate at 37°C, 5% CO2 for 24 hours before adding the drug.
  • Survival rate (%) [A(drug added) - A(blank)]/[A(0 drug added) - A(blank)] x 100%.
  • IC 50 was calculated by plotting the survival rate on the ordinate and the drug concentration on the abscissa using Graphpad using the [Inhibitor] vs. normalized response--Variable slope analysis method.
  • MPP 8 mg/kg
  • LNP 3.24 mg/kg
  • nucleic acid 200 ⁇ g/kg mRNA
  • liver function ALT, AST, ALP
  • cytokines IL-6, IL-1 ⁇
  • MPP Fe 3+
  • MPP Al 3+
  • MPP Mg 2+
  • liver function ALT, AST, ALP
  • cytokines IL-6, IL-1 ⁇
  • the core component of LNP is the artificially synthesized "cationic lipid/ionizable lipid", which has high cytotoxicity and immunogenicity, and its structure is relatively stable and difficult to decompose and metabolize in the body; while the core component of MPP (Fe 3+ ) or MPP (Al 3+ ) or MPP (Mg 2+ ) is a metal-phospholipid complex, which is composed of phospholipid molecules, the highly safe natural small molecule curcumin (an FDA-approved food additive and pharmaceutical excipient) and safe metal ions, and the metal-phospholipid complex is decomposed into natural molecules in the body after drug delivery.
  • MPP (Fe 3+ ) or MPP (Al 3+ ) does not contain cationic lipids/ionizable lipids, it will not cause toxic side effects related to cationic lipids/ionizable lipids. Therefore, the safety of MPP (Fe 3+ ), MPP (Al 3+ ), and MPP (Mg 2+ ) is higher than LNP.
  • Table 2-2 IC 50 and comparison of metal-phospholipid complexes with cationic lipids (DOTAP) and ionizable lipids (ALC0315) when the metal ions are Fe 3+ , Al 3+ or Mg 2+
  • Example 1.3.5 The mRNA in Example 1.3.5 was replaced with siRNA targeting the B7-H4 gene (B7-H4-siRNA) and its control (scr-siRNA), and mRNA encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit (RBD-mRNA).
  • the sequences of the different nucleic acids are as follows: 1
  • the sequences of B7-H4-siRNA are SEQ ID No. 19 (sense strand) and SEQ ID No. 26 (antisense strand) (25 bp), and the random control sequences are SEQ ID No. 20 (sense strand) and SEQ ID No. 27 (antisense strand) (19 bp);
  • the mRNA sequence encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit is SEQ ID No. 2 (669 nt).
  • Drug-metal-phospholipid complex particles (B7-H4-siRNA@MPP and RBD-mRNA@MPP) encapsulating the different nucleic acids were prepared according to the method of Example 1.3.5. The preparation process of the remaining drug-metal-phospholipid complex particles was the same as that of Example 1.3.5.
  • the above two different drug-metal-phospholipid complex particles (B7-H4-siRNA@MPP and RBD-mRNA@MPP) are used to treat glioma and as mRNA vaccines to prevent the new coronavirus, respectively.
  • the ELISA detection method is as described in Example 1.3.5.
  • mice bearing liver cancer were randomly divided into 10 groups (5 mice in each group): PBS control group, blank vector MPP(Fe 3+ ) group, blank vector MPP(Al 3+ ) group, blank vector MPP(Mg 2+ ) 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, and Scr-siRNA@MPP(Mg 2+ ) group.
  • mice in each group were intratumorally injected with PBS, MPP(Fe 3+ ), MPP(Al 3+ ), MPP(Mg 2+ ), Scr-siRNA@MPP(Fe 3+ ), B7-H4siRNA@MPP(Fe 3+ ), Scr-siRNA@MPP(Al 3+ ), B7-H4 siRNA@MPP(Al 3+ ), Scr-siRNA@MPP(Mg 2+ ), and B7 - H4-siRNA@MPP(Mg 2+ ) once every three days, at a dose of 200 ⁇ g siRNA/kg, for eight injections. Tumor volume was measured and recorded every three days. The results are shown in Figures 3-1, 3-2, and 3-3.
  • HCC Mouse Model HepG2 cells were harvested and resuspended in PBS at a density of 1 ⁇ 10 7 /mL. The cells were kept on ice before inoculation. 100 ⁇ L of the cell suspension was then injected subcutaneously into the dorsal region near the hind legs of female Balb/c nude mice to establish a HCC mouse model.
  • RBD-mRNA@MPP(Fe 3+ ) increased the expression level of mouse IgG antibodies to 117,268.8 ( Figure 1-3), and the expression levels of cytokines IFN- ⁇ , IL-2, and IL-4 to 252.8 pg/mL, 207.6 pg/mL, and 56.6 pg/mL, respectively ( Figure 1-5).
  • RBD-mRNA@MPP(Al 3+ ) increased the expression level of mouse IgG antibodies to 129,113 ( Figure 1-3-2), and the expression levels of cytokines IFN- ⁇ , IL-2, and IL-4 to 271.8 pg/mL, 234.6 pg/mL, and 68.4 pg/mL, respectively ( Figure 1-5-2).
  • RBD-mRNA@MPP(Mg 2+ ) increased the expression level of mouse IgG antibodies to 130614.40 ( Figure 1-3-3), and the expression levels of cytokines IFN- ⁇ , IL-2, and IL-4 to 296.40 pg/mL, 243.33 pg/mL, and 75.30 pg/mL, respectively ( Figure 1-5-3).
  • B7-H4-siRNA@MPP is effective in treating liver cancer via intratumoral injection, while RBD-mRNA@MPP, via intramuscular injection, can activate humoral and cellular immunity, thereby preventing infection with the novel coronavirus.
  • MPP Compared with MPPs composed of other metal ions (such as MPP (Fe 3+ ) and MPP (Al 3+ )), MPP (Mg 2+ ) can more effectively inhibit the growth of liver cancer and more effectively prevent the infection of the new coronavirus.
  • Mg 2+ maintains a weak chelation effect with RNA, which can enhance the function of RNA, including increasing its thermodynamic stability, chemical stability and catalytic activity (Biochemistry. 2021 Aug 10; 60(31): 2374-2386.)
  • Mg 2+ can maintain the structural stability and translation activity of the ribosome , partially supplement the function of ribosomal proteins (Biosci Biotechnol Biochem.
  • Mg2 + can regulate LFA-1 downstream signal transduction by inducing conformational changes in the co-stimulatory molecule LFA-1 on the surface of CD8+T cells, thereby promoting T cell activation and cytotoxicity (Cell.2022Feb17;185(4):585-602.e29.)
  • Mg2 + participates in catalysis as an enzyme cofactor of Ago protein in the RNA-induced silencing complex (RISC), which can enhance the RNAi effect of siRNA (Cell Rep.2022Oct 25;41(4):111533)
  • RISC RNA-induced silencing complex
  • Rnase H1 which can enhance the gene silencing effect of ASO
  • Example 4 Functions of DSPC, curcumin, Fe 3+ , Al 3+ or Mg 2+ after replacement by similar substances
  • Example 4.1 Functions of DSPC, curcumin, and Fe 3+ after replacement by analogs
  • DSPC, curcumin, and Fe 3+ analogs were substituted for DSPC, curcumin, and Fe 3+ analogs, respectively.
  • Thirty-seven different eGFP-mRNA@MPPs were prepared using different combinations. Each eGFP-mRNA@MPP contained an mRNA concentration of 2 ⁇ g/mL.
  • the names and structures of DSPC, curcumin, Fe 3+ analogs, and their analogs are shown in Table 4-1.
  • the combinations of DSPC, curcumin, Fe 3+ analogs, and their analogs in the 37 mRNA@MPPs are shown in Table 4-2.
  • the reaction temperature in Example 1.1 was 65°C and the reaction time was 2 hours.
  • the reaction temperature in Example 1.2 was 60°C and the reaction time was 2 hours. All other conditions remained unchanged.
  • the above 37 different eGFP-mRNA@MPPs and the above eGFP-mRNA@LNPs were incubated with 293T cells, respectively.
  • the control group was incubated with MPP or LNP. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.
  • the method for analyzing the eGFP-positive cell rate by flow cytometry is as described in Example 1.3.
  • LNPs The primary toxicity of LNPs comes from their main components—cationic lipids and ionizable lipids. During LNP metabolism in the body, the free cationic lipids and ionizable lipids can produce significant toxicity.
  • the median lethal dose (IC50 ) of cationic lipids and ionizable lipids for biological cells is an important parameter for assessing the toxicity of LNPs.
  • Metal-chelated phospholipid complex nanoparticles (MPPs) replace the cationic lipids and ionizable lipids in LNPs with metal-chelated phospholipid complexes. Therefore, we compared the toxicity of LNPs with that of 37 MPPs (Table 4-2) by comparing their IC50s with those of cationic lipids (DOTAP) and ionizable lipids (ALC0315) for biological cells.
  • DOTAP cationic lipids
  • ALC0315 ionizable lipids
  • the IC50 was calculated as described in Example 2.10.
  • results As shown in Table 4-2, the percentage of eGFP-positive cells in 293T cells treated with 37 different eGFP-mRNA@MPPs was significantly higher than that of eGFP-mRNA@LNPs. Among them , the mRNA@MPP composed of DSPC, curcumin, and Fe 3+ had the highest percentage of eGFP-positive cells. The results suggest that the mRNA@MPP formed by replacing DSPC, curcumin, and Fe 3+ with their analogs is less functional than the mRNA@MPP composed of DSPC, curcumin, and Fe 3+, but is superior to the mRNA@LNP.
  • MPP has a stronger ability to promote lysosomal escape of nucleic acids than LNP, allowing more nucleic acids loaded with MPP to be effectively released into the cytoplasm and translated into proteins.
  • IC 50 values of all 37 metal-phospholipid complexes were significantly greater than those of cationic lipids (DOTAP) and ionizable lipids (ALC0315). This suggests that the toxicity of metal-phospholipid complexes is clearly less than that of cationic lipids and ionizable lipids, meaning that lipid nanoparticles (MPPs) composed of DSPC, curcumin, Fe 3+ , and their analogs are safer than LNPs.
  • MPPs lipid nanoparticles
  • the core component of LNPs is artificially synthesized "cationic lipids/ionizable lipids," which have high cytotoxicity and immunogenicity, and a relatively stable structure that is difficult to decompose and metabolize in the body.
  • the core component of MPPs is metal-phospholipid complexes, which are composed of phospholipid molecules, highly safe natural small molecules (of which curcumin is an FDA-approved food additive and pharmaceutical excipient), and safe metal ions. Furthermore, these metal-phospholipid complexes are decomposed into natural molecules in the body after drug delivery.
  • the lipid particles composed of DSPC, curcumin, Fe 3+ and their analogs do not contain cationic lipids/ionizable lipids and will not cause toxic side effects related to cationic lipids/ionizable lipids, so the safety of MPP is higher than that of LNP.
  • Table 4-2 List of composition combinations and functions of metal-phospholipid complexes in drug-lipid particles prepared from DSPC, curcumin, Fe 3+ and their analogs
  • Example 4.2 Functions of DSPC, curcumin and its analogs, and Fe 3+ components in different metal-phospholipid complexes and the resulting drug-metal-phospholipid complex particles
  • Example 1.3 mRNA-metal-phospholipid complex particles were prepared, and curcumin was replaced with its analog hesperetin (one hesperetin molecule contains four hydroxyl groups) and tea polyphenol (one tea polyphenol molecule contains eight hydroxyl groups), respectively, to prepare three types of mRNA-metal-phospholipid complex particles (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP38).
  • mRNA@MPP1, mRNA@MPP4, and mRNA@MPP38 three types of mRNA-metal-phospholipid complex particles
  • the ratios of DSPC, curcumin or its analog, and FeCl3 were 1:1:1, 1:1:1, and 1:1:2, respectively.
  • the mRNA encoding the eGFP fluorescent protein is SEQ ID No. 1 (720 nt).
  • the mRNA loading efficiency of these four drug-lipid nanoparticles and their ability to promote eGFP fluorescent protein expression after treatment with 293T cells were tested according to the experimental procedures
  • the basis for adjusting the dosage ratio is that: because DSPC and its congeners are connected to curcumin and its congeners by hydrogen bonds, as long as DSPC and its congeners contain multiple phosphate groups, when synthesizing phospholipid complexes, the dosage ratio of DSPC and its congeners to curcumin and its congeners can be adjusted according to the number of phosphate groups contained in DSPC and its congeners, that is, when DSPC and its congeners contain two phosphate groups, the dosage ratio of DSPC and its congeners to curcumin and its congeners can be adjusted to 1:2; when DSPC and its congeners contain three phosphate groups, the dosage ratio of DSPC and its congeners to curcumin and its congeners can be adjusted to 1:3; because the hydroxyl groups of curcumin and its congeners are connected to Fe 3+ and its congeners by coordination bonds, as long as curcumin and its congeners contain multiple binding sites, the dosage ratio of curcumin and
  • Example 4.3 Functions of drug-metal-phospholipid complex particles prepared by different ratios of DSPC, curcumin and its analogs, and Al 3+ components
  • Example 1.4 mRNA metal-phospholipid complexes were prepared, and curcumin was replaced with its analogs hesperetin (one hesperetin molecule contains four hydroxyl groups) and tea polyphenols (one tea polyphenol molecule contains eight hydroxyl groups), respectively, to prepare three metal-phospholipid complexes (mRNA@MPP2, mRNA@MPP5, and mRNA@MPP39).
  • the ratios of DSPC, curcumin or its analogs, and Al( NO3 ) 3 ⁇ 9H2O were 1:1:1, 1:1:1, and 1:1:2, respectively.
  • Corresponding drug-metal-phospholipid complex nanoparticles were prepared using these three metal-phospholipid complexes (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP29).
  • the mRNA encoding the eGFP fluorescent protein is shown in SEQ ID No. 1 (720 nt).
  • SEQ ID No. 1 720 nt.
  • the mRNA encapsulation efficiency of these four drug-lipid nanoparticles and their ability to promote eGFP fluorescent protein expression after treating 293T cells were detected according to the experimental process and experimental methods described in Example 3.5.
  • the basis for adjusting the dosage ratio is that because DSPC congeners and curcumin congeners are connected by hydrogen bonds, as long as DSPC congeners contain multiple phosphate groups, when synthesizing phospholipid complexes, the dosage ratio of DSPC congeners and curcumin congeners can be adjusted according to the number of phosphate groups contained in DSPC congeners, that is, when DSPC congeners contain two phosphate groups, the dosage ratio of DSPC congeners and curcumin congeners 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 groups of curcumin congeners are 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 adjusted according to the number of binding sites contained in curcumin congeners.
  • Example 4.4 Functions of drug-metal-phospholipid complex particles prepared by different ratios of DSPC, curcumin and its analogs, and Mg2 + components
  • Example 1.5 mRNA-metal-phospholipid complex particles were prepared, and curcumin was replaced with its analogs hesperetin (one hesperetin molecule contains four hydroxyl groups) and tea polyphenols (one tea polyphenol molecule contains eight hydroxyl groups), respectively, to produce three types of mRNA-metal-phospholipid complex particles (mRNA@MPP29, mRNA@MPP30, and mRNA@MPP40).
  • the ratios of DSPC, curcumin or its analogs, and MgCl2 used in the preparation of these three mRNA-metal-phospholipid complex particles were 1:1:1, 1:1:1, and 1:1:2, respectively.
  • the mRNA encoding the eGFP fluorescent protein is represented by SEQ ID No. 1 (720 nt).
  • SEQ ID No. 1 720 nt
  • the basis for adjusting the dosage ratio is that: because DSPC and its congeners are connected to curcumin and its congeners by hydrogen bonds, as long as DSPC and its congeners contain multiple phosphate groups, when synthesizing phospholipid complexes, the dosage ratio of DSPC and its congeners to curcumin and its congeners can be adjusted according to the number of phosphate groups contained in DSPC and its congeners, that is, when DSPC and its congeners contain two phosphate groups, the dosage ratio of DSPC and its congeners to curcumin and its congeners can be adjusted to 1:2; when DSPC and its congeners contain three phosphate groups, the dosage ratio of DSPC and its congeners to curcumin and its congeners can be adjusted to 1:3; because the hydroxyl groups of curcumin and its congeners are connected to Mg2 + and its congeners by coordination bonds, as long as curcumin and its congeners contain multiple binding sites, the dosage ratio of curcumin
  • Apt-C6-S-S-C6 is prepared by modifying the disulfide bond of Apt.
  • Apt-C6-S-S-C6 has six carbon chains modified on both sides of the disulfide bond, one end of which is connected to Apt.
  • Apt-C6-S-S-C6 is annealed on a gradient PCR instrument at 95°C for 5 minutes and then at 37°C for 15 minutes.
  • Second, Apt-C6-S-S-C6 is incubated in 10mM phosphine hydrochloride (TCEP) at 25°C for 1 hour to break the disulfide bond and expose the sulfhydryl group.
  • TCEP phosphine hydrochloride
  • Apt-C6-SH This reduction reaction of Apt-C6-S-S-C6 produces Apt-C6-SH.
  • Apt-C6-SH and DSPE-PEG2000-MAL were reacted at room temperature for 4 hours at a raw material molar ratio of 1:100 to allow the maleamide and thiol groups to fully react to form DSPE-PEG2000-Apt.
  • the unreacted DSPE-PEG2000-MAL was removed by ultrafiltration using a 10kD molecular weight cutoff ultrafiltration tube for 3-6 times.
  • the nucleotide sequence of Apt is
  • DSPE-PEG2000-Apt micelles were prepared using a thin-film hydration method. The steps are as follows: DSPE-PEG2000-Apt was dissolved in ethanol and rotary evaporated under vacuum for 30 minutes to form a uniform thin film. The film was then placed in a 4°C refrigerator overnight to allow the ethanol to evaporate completely. Finally, the film was hydrated with enzyme-free water at 60°C for 30 minutes to produce the DSPE-PEG2000-Apt micelles.
  • the intermediates DSPE-PEG2000-MAL and Apt-C6-SH can also be changed to include but not limited to DSPE-PEG2000-NHS and Apt-NH 2 , DSPE-PEG2000-COOH and Apt-NH 2 , DSPE-PEG2000-NCO and Apt-NH 2 , and DSPE-PEG2000-N 3 and Apt-DBCO, etc., with the ultimate goal of preparing DSPE-PEG2000-Apt.
  • the method for preparing DSPE-PEG2000-Apt into micelles can be varied, including but not limited to direct dissolution, ethanol injection, dialysis, and ultrasound, with the ultimate goal of obtaining DSPE-PEG2000-Apt micelles.
  • the DSPE-PEG2000-Apt micelles prepared in Example 5.1 were mixed uniformly with the mRNA-encapsulated metal-phospholipid complex particles (MPP) prepared in Examples 1.3 to 1.5 at different reaction mass ratios (1:84, 1:42, 1:21, 1:16.8, and 1:14) and incubated at 4°C (reaction temperature 0°C ⁇ 10°C) for 2 hours (incubation time 0.2-12 hours is acceptable). While varying the reaction mass ratios altered the distribution of Apt on the MPP, the resulting targeting vectors (Apt-MPP) with varying modification rates maintained their physicochemical properties.
  • MPP mRNA-encapsulated metal-phospholipid complex particles
  • the MPP allowed the DSPE-PEG2000-Apt micelles to hydrophobically insert into the outer surface of the MPP, resulting in Apt-metal-phospholipid complex particles, the targeting vectors (Apt-MPP).
  • Apt itself is hydrophilic and can adsorb metal ions. If DSPE-PEG2000-Apt is directly used to replace DSPE-PEG2000 in the preparation of metal-phospholipid complex particles, Apt will be encapsulated inside the metal-phospholipid complex particles and will not be able to exert the targeting effect of Apt.
  • CAR mRNA includes a transmembrane domain, a signal transduction domain, an antigen binding domain, a co-stimulatory signal transduction region, and a connecting region between the antigen binding domain and the transmembrane domain.
  • Antigen binding domain, scFV encodes the VH and VL parts of the FMC63 antibody.
  • the VH and VL parts of the FMC63 antibody are connected by a linker and can recognize tumor cell antigens.
  • the spacer connects the antigen-binding domain scFV and the transmembrane domain. Its flexibility ensures that scFV can better recognize antigens.
  • Transmembrane(TM) These combinations of CARs anchor the CAR to the T cell membrane.
  • the intracellular co-stimulatory signaling domain is used to activate T cells.
  • the activation signal is primarily provided by CD3-zeta, and other co-stimulatory domains, such as CD28 and CD8, can be added to enhance the signal.
  • the co-stimulatory signaling domain can contain multiple co-stimulatory domains. Regardless of the number of co-stimulatory domains, CD3-zeta should be placed last.
  • the CD28 co-stimulatory domain effectively enhances T cell proliferation.
  • signal-P The amino acid sequence of the CD8 leader sequence (signal peptide) is shown in SEQ ID NO.45.
  • the amino acid sequence of FMC63_VL is shown in SEQ ID NO.46.
  • amino acid sequence of the linker is shown in SEQ ID NO.47.
  • the amino acid sequence of FMC63_VH is shown in SEQ ID NO.48.
  • amino acid sequence of anti-CD19scFv is shown in SEQ ID NO.49.
  • the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO.50.
  • the amino acid sequence of the CD28 co-stimulatory domain is shown in SEQ ID NO.51.
  • the amino acid sequence of the CD3 ⁇ intracellular signaling domain is shown in SEQ NO. ID 52.
  • the DNA sequence of CD19 CAR is shown in SEQ ID NO.53.
  • CD8 leader sequence (signal peptide) DNA sequence is shown in SEQ ID NO.54.
  • the DNA sequence of the CD8 hinge region is shown in SEQ ID NO.56.
  • the DNA sequence of CD28 is shown as SEQ ID NO.58.
  • CD19 CAR-mRNA The sequence of CD19 CAR-mRNA is shown as SEQ ID NO.43.
  • the transmembrane domain can be selected from at least one or more of the CD8 transmembrane domain, the Fc ⁇ RI ⁇ (FCER1A) transmembrane domain, and the CD28 transmembrane domain.
  • amino acid sequence is shown as SEQ ID NO.61.
  • FCER1A Fc ⁇ RI ⁇ (FCER1A) transmembrane domain
  • amino acid sequence is shown in SEQ ID NO.63.
  • the mRNA sequence is shown as SEQ ID NO.29.
  • amino acid sequence is shown in SEQ ID NO.95.
  • the nucleotide sequence is shown as SEQ ID NO.96.
  • the mRNA sequence is shown as SEQ ID NO.30.
  • the signaling domain may be selected from at least one or more of a CD8 leader sequence, a mouse Kappa chain leader sequence, and an Fc ⁇ RI ⁇ leader sequence.
  • CD8 leader sequence
  • amino acid sequence is shown in SEQ ID NO.45.
  • the nucleotide sequence is shown as SEQ ID NO.54.
  • the mRNA sequence is shown as SEQ ID NO.31.
  • amino acid sequence is shown in SEQ ID NO.65.
  • the nucleotide sequence is shown as SEQ ID NO.66.
  • the mRNA sequence is shown as SEQ ID NO.32.
  • amino acid sequence is shown in SEQ ID NO.67.
  • the nucleotide sequence is shown as SEQ ID NO.68.
  • the mRNA sequence is shown as SEQ ID NO.33.
  • the antigen binding domain can be selected from one or more of anti-CD19 scFv, anti-EGFR scFv, and anti-HER2 scFv.
  • the DNA sequence is shown as SEQ ID NO.55.
  • the mRNA sequence is shown as SEQ ID NO.34.
  • Anti-EGFR scFv Anti-EGFR scFv
  • amino acid sequence is shown in SEQ ID NO.69.
  • the DNA sequence is shown as SEQ ID NO.70.
  • the mRNA sequence is shown as SEQ ID NO.71.
  • amino acid sequence is shown in SEQ ID NO.72.
  • the DNA sequence is shown as SEQ ID NO.73:.
  • the mRNA sequence is shown as SEQ ID NO.36.
  • the co-stimulatory signaling region can be selected from one or more of the CD28 co-stimulatory domain, the OX40 co-stimulatory domain, and the 41BB co-stimulatory domain.
  • amino acid sequence is shown as SEQ ID NO.51.
  • the DNA sequence is shown as SEQ ID NO.58.
  • the mRNA sequence is shown as SEQ ID NO.37.
  • amino acid sequence is shown in SEQ ID NO.38.
  • the DNA sequence is shown as SEQ ID NO.74.
  • the mRNA sequence is shown as SEQ ID NO.75.

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Abstract

一种靶向载体、靶向药物及制备方法与应用。靶向载体包括：（a）金属-磷脂复合物颗粒，（b）靶向结构，其中，所述靶向结构与所述金属-磷脂复合物颗粒的外表面连接。本申请提供的靶向载体未使用阳离子脂质和可电离脂质，有利于在生物体内运载负电荷药物，可以用于不同大小的药物。

Description

靶向载体、靶向药物及制备方法与应用
相关申请的交叉引用
本申请要求2024年02月08日提交中国专利局的申请号为202410177519.0，名
称为“靶向载体及制备方法和应用、CD62L作为靶向免疫细胞的应用”中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及生物技术领域，具体提供靶向载体、靶向药物及制备方法与应用。
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，急需一种安全性更高的脂质体递送系统。
有鉴于此，特提出本申请。
发明内容
本申请的目的之一在于提供靶向载体、靶向药物及制备方法与应用，以提供一种新的药物载体以及有效治疗药物。
为了实现上述目的，本申请采用如下技术方案：
一种靶向载体，所述靶向载体含有：
(a)金属-磷脂复合物颗粒，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；
(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；
(b)靶向结构，其中，所述靶向结构与所述金属-磷脂复合物颗粒的外表面连接。
进一步地，所述(i)金属-磷脂复合物中，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、磷酸酰肌醇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；
优选地，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)
磷酸酰肌醇(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)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
花生酰基或植烷酰基
优选地，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；
优选地，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；
优选地，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；
优选地，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；
优选地，所述连接物分子部分选自
姜黄素(式19)
绿原酸(式20)
花青素(式21)其中，R1和R2是H、OH或OCH3，R3是H或糖基，R4、R5和R6是OH或糖基、
槲皮素(式22)二氢杨梅素(式23)
橙皮素(式24)柚皮素(式25)
芹菜素(式26)儿茶素(式27)
茶多酚(式28)
表没食子儿茶素没食子酸酯(式29)
鞣花酸(式30)桑色素(式31)
表儿茶素没食子酸酯(式32)
儿茶素没食子酸酯(式33)
没食子儿茶素没食子酸酯(式34)
平贝碱C(式35)及其衍生物中一种或多种的组合；
优选地，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；
优选地，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；
优选地，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)；
优选地，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；
优选地，所述金属离子部分选自Fe3+、Mg2+、Ca2+、Al3+中一种或多种的组合；
优选地，所述金属离子部分选自Fe3+、Mg2+、Ca2+或Al3+。
进一步地，所述(ii)抑制颗粒聚集的缀合的脂质包括PEG-脂质缀合物和/或PEG-DAA；
优选地，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；
优选地，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；
优选地，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)；
进一步地，所述(iii)中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；
优选地，所述(iii)中的非阳离子脂质或非可电离脂质为胆固醇(式40)
优选地，所述(iii)中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、磷酸酰肌醇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；
优选地，(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)、磷酸酰肌醇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(式41)，及其衍生物中中的至少一种；
优选地，所述(iii)中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；
优选地，所述(iii)中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，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，磷脂分子部分选自DSPC、DSPE、DSPA或DSPG，连接物分子部分选自姜黄素、橙皮素或茶多酚，金属离子部分选自Fe3+、Mg2+、Ca2+或Al3+；
优选地，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，磷脂分子部分选自DSPC(式46)、DSPE(式47)、DSPA(式48)或DSPG(式49)，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)，金属离子部分选自Fe3+、Mg2+、Ca2+或Al3+；
优选地，磷脂分子部分、连接物分子部分和金属离子部分的摩尔比为1:1:(0.5～2)；
优选地，所述磷脂分子部分为DSPC(式46)，所述连接物分子部分选自姜黄素(式19)，所述金属离子部分选自Fe3+、Mg2+或Al3+，磷脂分子部分、连接物分子部分和金属离子部分的摩尔比为1:1:1。
进一步地，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％～50％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为1％～10％，所述胆固醇在原料中摩尔占比为15％～80％，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％～51％；
优选地，所述金属-磷脂复合物在原料中摩尔占比为5％～40％，优选为10％～40％；
优选地，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％；
优选地，所述胆固醇在原料中摩尔占比为25％～75％，优选为35％～75％；
优选地，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％～50％，优选为0％～40％。
进一步地，所述(b)靶向结构包括依次连接的疏水区、连接区和靶向结合区；所述疏水区与所述金属-磷脂复合物颗粒外层基于亲水性、疏水性进行连接；
优选地，所述疏水区包括DSPE及其衍生物中的至少一种或多种；
优选地，所述连接区包括PEG-2000及其衍生物中的至少一种或多种；
优选地，所述靶向结合区可结合CD62L、CD8、CD3、核仁素蛋白、T细胞、自然杀伤细胞、巨噬细胞、胰腺癌细胞或肝癌细胞中的至少一种；
优选地，所述靶向结合区包括核酸、多肽、蛋白、小分子中的至少一种；
优选地，所述靶向结合区包括适配体、抗体、抗原结合部、galnac中的一种；
优选地，所述靶向结合区为适配体，所述靶向结合区的靶点为CD62L，所述适配体优选为SEQ ID NO.44所示；或，
优选地，所述靶向结合区为适配体，所述靶向结合区的靶点为CD8，所述适配体优选为SEQ ID NO.84所示；或，
优选地，所述靶向结合区为适配体，所述靶向结合区的靶点为CD3，所述适配体优选为SEQ ID NO.95所示；或
优选地，所述靶向结合区为适配体，所述靶向结合区的靶点为核仁素蛋白，所述适配体优选为SEQ ID NO.86所示；或，
优选地，所述靶向结合区为适配体，所述靶向结合区的靶细胞为T细胞，所述适配体优选为SEQ ID NO.44所示；
优选地，所述靶向结合区为适配体，所述靶向结合区的靶细胞为胰腺癌细胞，所述适配体优选为SEQ ID NO.88所示；或，
优选地，所述靶向结合区为适配体，所述靶向结合区的靶细胞为肝癌细胞；
优选地，所述靶向结构为DSPE-PEG2000-适配体，优选为DSPE-PEG2000-CD62L适配体，所述CD62L适配体序列如SEQ ID NO.44所示。
上述靶向载体的制备方法，将所述靶向结构与所述金属-磷脂复合物颗粒的外表面连接形成所述靶向载体。
进一步地，将(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，得到所述金属-磷脂复合物颗粒；
优选地，所述靶向结构为DSPE-PEG2000-适配体，制备过程为：DSPE-PEG2000与适配体通过中间体对反应连接，得到DSPE-PEG2000-适配体；
优选地，所述中间体对选自DSPE-PEG2000-MAL和适配体-C6-SH、DSPE-PEG2000-NHS和适配体-NH2、DSPE-PEG2000-COOH和适配体-NH2、DSPE-PEG2000-NCO和适配体-NH2、DSPE-PEG2000-N3和适配体-DBCO，优选为DSPE-PEG2000-MAL和适配体-C6-SH；
优选地，所述DSPE-PEG2000-适配体形成胶束后与所述金属-磷脂复合物颗粒的外表面连接形成所述靶向载体；
优选地，所述胶束的制备方法包括直接溶解法、乙醇注入法、透析法或超声法。
进一步地，所述制备方法包括：
步骤一：将磷脂分子、连接物分子和金属离子反应形成金属-磷脂复合物；
步骤二：将步骤一中制备的所述金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质、(iii)非阳离子脂质或非可电离脂质混合制备得到金属-磷脂复合物颗粒；
步骤三：将步骤二中制备的所述金属-磷脂复合物颗粒与所述靶向结构混合制备得到所述靶向载体；
优选地，步骤一中，将磷脂分子、连接物分子和金属离子溶于乙醇中反应，磷脂分子、连接物分子和金属离子的摩尔比优选为1:1:1，所述的反应条件优选为40～60℃反应1～5h；
优选地，步骤三中，所述金属-磷脂复合物颗粒与所述靶向结构的反应条件为2-10℃孵育0.2-12h。
进一步地，所述制备方法包括：
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；
步骤三：将步骤二中制备的所述金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质、(iii)非阳离子脂质或非可电离脂质混合制备得到金属-磷脂复合物颗粒；
步骤四：将步骤三中制备的所述金属-磷脂复合物颗粒与所述靶向结构混合制备得到所述靶向载体；
优选地，步骤一中，将磷脂分子与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，所述磷脂分子与所述连接物分子的摩尔比优选为1:1；所述反应的条件优选为65℃反应2小时；
优选地，步骤二中，磷脂复合物与金属离子溶于乙醇，加入三乙胺后反应，得到所述金属-磷脂复合物，所述磷脂复合物与所述金属离子的摩尔比优选为1：(1～2)，所述磷脂复合物与所述三乙胺的摩尔比优选为1:1，所述反应的条件优选为60℃反应2小时；
优选地，步骤四中，所述金属-磷脂复合物颗粒与所述靶向结构的反应条件为2-10℃孵育0.2-12h。
上述靶向载体或制备方法用于药物运送、显影剂或疫苗的应用。
一种靶向药物，所述靶向药物包括药物和上述靶向载体，所述药物包封在所述靶向载体的金属-磷脂复合物颗粒中。
进一步地，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；
优选地，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；
优选地，所述药物为mRNA，所述mRNA为编码嵌合抗原受体CAR或TCR；
优选地，所述药物为编码嵌合抗原受体CAR的mRNA，所述CAR包括跨膜结构域、信号传导结构域、抗原结合结构域、共刺激信号传导区、抗原结合结构域与跨膜结构域连接区；
优选地，所述跨膜结构域选自SEQ ID No.28、SEQ ID No.29、SEQ ID No.30中的至少一种；和/或，
信号传导结构域选自SEQ ID No.31、SEQ ID No.32、SEQ ID No.33中的至少一种；和/或，
抗原结合结构域选自SEQ ID No.34、SEQ ID No.35、SEQ ID No.36中的至少一种；和/或，
共刺激信号传导区选自SEQ ID NO.37、SEQ ID No.39、SEQ ID No.75中的至少一种；和/或，
抗原结合结构域与跨膜结构域连接区选自SEQ ID No.40、SEQ ID No.41、SEQ ID No.42中的至少一种；
优选地，所述药物是核苷酸序列如SEQ ID No.43所示的mRNA。
上述靶向药物的制备方法，所述制备方法包括：将药物包载于靶向载体中，得到所述靶向药物。
进一步地，所述靶向载体包括金属-磷脂复合物颗粒和靶向结构，将药物包载于所述金属-磷脂复合物颗粒中，得到药物-金属-磷脂复合物颗粒；将所述靶向结构与所述药物-金属-磷脂复合物颗粒的外表面连接形成所述靶向药物。
进一步地，将药物、(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，得到所述药物-金属-磷脂复合物颗粒。
进一步地，所述制备方法包括：
步骤一：将磷脂分子、连接物分子和金属离子反应形成金属-磷脂复合物；
步骤二：将步骤一中制备的所述金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质、(iii)非阳离子脂质或非可电离脂质，以及药物混合制备得到药物-金属-磷脂复合物颗粒；
步骤三：将步骤二中制备的所述药物-金属-磷脂复合物颗粒与所述靶向结构混合制备得到所述靶向药物；
优选地，步骤一中，将磷脂分子、连接物分子和金属离子溶于乙醇中反应，磷脂分子、连接物分子和金属离子的摩尔比优选为1:1:1，所述的反应条件优选为40～60℃反应1～5h；
优选地，步骤二中，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-金属-磷脂复合物颗粒，所述有机化合物优选为乙醇；优选地，有机相与水相的混匀方式包含微流控芯片或超声；
优选地，步骤三中，所述药物-金属-磷脂复合物颗粒与所述靶向结构的反应条件为2-10℃孵育0.2-12h。
进一步地，所述制备方法包括：
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；
步骤三：将步骤二中制备的所述金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质、(iii)非阳离子脂质或非可电离脂质，以及药物混合制备得到药物-金属-磷脂复合物颗粒；
步骤四：将步骤三中制备的所述药物-金属-磷脂复合物颗粒与所述靶向结构混合制备得到所述靶向药物；
优选地，步骤一中，将磷脂分子与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，所述磷脂分子与所述连接物分子的摩尔比优选为1:1；所述反应的条件优选为65℃反应2小时；
优选地，步骤二中，磷脂复合物与金属离子溶于乙醇，加入三乙胺后反应，得到所述金属-磷脂复合物，所述磷脂复合物与所述金属离子的摩尔比优选为1：(1～2)，所述磷脂复合物与所述三乙胺的摩尔比优选为1:1，所述反应的条件优选为60℃反应2小时；
优选地，步骤三中，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-金属-磷脂复合物颗粒，所述有机化合物优选为乙醇；优选地，有机相与水相的混匀方式包含微流控芯片或超声
优选地，步骤四中，所述药物-金属-磷脂复合物颗粒与所述靶向结构的反应条件为2-10℃孵育0.2-12h。
进一步地，所述靶向结构为DSPE-PEG2000-适配体，制备过程为：DSPE-PEG2000与适配体通过中间体对反应连接，得到DSPE-PEG2000-适配体；
优选地，所述中间体对选自DSPE-PEG2000-MAL和适配体-C6-SH、DSPE-PEG2000-NHS和适配体-NH2、DSPE-PEG2000-COOH和适配体-NH2、DSPE-PEG2000-NCO和适配体-NH2、DSPE-PEG2000-N3和适配体-DBCO，优选为DSPE-PEG2000-MAL和适配体-C6-SH；
优选地，所述DSPE-PEG2000-适配体形成胶束后与所述药物-金属-磷脂复合物颗粒的外表面连接形成所述靶向药物；
优选地，所述胶束的制备方法包括直接溶解法、乙醇注入法、透析法或超声法。
上述靶向药物或制备方法在药物运送、显影药物、疫苗中的应用。
进一步地，所述靶向药物用于药物运送、治疗和/或预防；
优选地，所述靶向药物用于将药物引入细胞或血液；所述细胞优选包括免疫细胞、肿瘤细胞、生殖细胞、神经细胞、内分泌细胞、血细胞、吞噬细胞、白细胞、红细胞、上皮细胞、心肌细胞或干细胞；
优选地，所述靶向药物用于在哺乳动物受试者中使靶序列的表达或沉默、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、用于将药物从体内传递到哺乳动物细胞因进行靶序列表达或沉默、或用于治疗/或预防哺乳动物的疾病或病症；
优选地，所述哺乳动物为人；
优选地，所述疾病或病症的治疗与基因的表达相关，所述基因包含药物的靶序列；
优选地，所述疾病或病症包括癌症、病毒感染、自身免疫性疾病、免疫系统过度激活引发的疾病、代谢性疾病、纤维化疾病、组织纤维化、细胞衰老、动脉粥样硬化、糖尿病或骨关节炎；
优选地，所述病毒感染包括乙型肝炎病毒、丙肝炎病毒、SARS-Cov-2、人类免疫缺陷病毒、细胞巨细胞病毒、侵袭性曲霉菌或结合病毒；
优选地，所述癌症包括血液瘤和实体瘤；
优选地，所述血液瘤包括急性B细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、多发性骨髓瘤(MM)、急性髓系白血病(AML)或T细胞淋巴瘤；
优选地，所述弥漫性大B细胞淋巴瘤(DLBCL)包括霍奇金淋巴瘤(HL)和非霍奇金淋巴瘤(NHL)；
优选地，所述实体瘤包括肝癌、脑胶质瘤、胃癌、胰腺癌、肺癌、非小细胞肺癌、肾细胞癌、前列腺癌、骨肉瘤、乳腺癌、结直肠癌、卵巢癌、黑色素瘤、神经母细胞瘤、血管母细胞瘤、间变性脑膜瘤、复发性胶质母细胞瘤或分化型甲状腺癌；
优选地，所述自身免疫性疾病包括寻常型天疱疹、系统性红斑狼疮(SLE)、血友病、重症肌无力、移植组织器官引发的免疫排斥反应、I型糖尿病(T1D)、类风湿关节炎、系统性硬化症、多发性硬化症、特发性肺纤维化、克罗恩病或结肠炎；
优选地，所述免疫系统过度激活引发的疾病包括细胞因子释放综合征(CRS)、免疫效应细胞相关神经毒性综合征(ICANS)或移植物抗宿主病(GVHD)；
优选地，所述代谢性疾病包括动脉粥样硬化、先天性高胰岛素血症、非酒精性脂肪肝炎或非肥胖糖尿病(NOD)；
优选地，所述纤维化包括心肌纤维化、肥厚性心肌病、新冠肺炎、新冠心肌炎、缺血性心肌病、肝硬化、肝纤维化、原发性胆管炎、肺纤维化、骨骼肌纤维化、皮肤类疾病、骨髓纤维化、结节病或杜氏肌营养不良症；
优选地，所述皮肤类疾病包括瘢痕疙瘩或伤口愈合；
优选地，所述靶向药物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；
优选地，所述靶向药物用于联合用药。
一种含有上述靶向药物的药剂，所述药剂优选为疫苗，所述疫苗优选为新型冠状病毒疫苗。
与现有技术相比，本申请的技术效果为：
本申请提供的靶向载体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，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，应用范围广泛，可以用于不同大小的药物。靶向药物可以实现核酸药物的高表达量，生物安全性显著提升，靶向性好，能够实现多种疾病的高效治疗。
附图说明
下面参照附图来进一步说明本申请的各个技术特征和它们之间的关系。附图为示例性的，一些技术特征并不以实际比例示出，并且一些附图中可能省略了本申请所属技术领域中惯用的且对于理解和实现本申请并非必不可少的技术特征，或是额外示出了对于理解和实现本申请并非必不可少的技术特征，也就是说，附图所示的各个技术特征的组合并不用于限制本申请。另外，在本申请全文中，相同的附图标记所指代的内容也是相同的。具体的附图说明如下：
图1-1为本申请实施例1.3.5.1提供的eGFP-mRNA@MPP(Fe3+)转染293T所致eGFP阳性细胞百分比；
图1-2为本申请实施例1.3.5.1提供的RBD-mRNA@MPP(Fe3+)转染293T所致RBD的表达；
图1-3为本申请实施例1.3.5.1提供的RBD-mRNA@MPP(Fe3+)诱导体液免疫的能力；
图1-4为本申请实施例1.3.5.1提供的NY-ESO-1-mRNA@MPP(Fe3+)诱导体液免疫的能力；
图1-5为本申请实施例1.3.5.1提供的RBD-mRNA@MPP(Fe3+)诱导细胞免疫的能力；
图1-6为本申请实施例1.3.5.1提供的NY-ESO-1-mRNA@MPP(Fe3+)诱导细胞免疫的能力；
图1-1-2为本申请实施例1.3.5.2提供的eGFP-mRNA@MPP(Al3+)转染293T所致eGFP阳性细胞百分比；
图1-2-2为本申请实施例1.3.5.2提供的RBD-mRNA@MPP(Al3+)转染293T所致RBD的表达；
图1-3-2为本申请实施例1.3.5.2提供的RBD-mRNA@MPP(Al3+)诱导体液免疫的能力；
图1-4-2为本申请实施例1.3.5.2提供的NY-ESO-1-mRNA@MPP(Al3+)诱导体液免疫的能力；
图1-5-2为本申请实施例1.3.5.2提供的RBD-mRNA@MPP(Al3+)诱导细胞免疫的能力；
图1-6-2为本申请实施例1.3.5.2提供的NY-ESO-1-mRNA@MPP(Al3+)诱导细胞免疫的能力；
图1-1-3为本申请实施例1.3.5.3提供的eGFP-mRNA@MPP(Mg2+)转染293T所致eGFP阳性细胞百分比；
图1-2-3为本申请实施例1.3.5.3提供的RBD-mRNA@MPP(Mg2+)转染293T所致RBD的表达；
图1-3-3为本申请实施例1.3.5.3提供的RBD-mRNA@MPP(Mg2+)诱导体液免疫的能力；
图1-4-3为本申请实施例1.3.5.3提供的NY-ESO-1-mRNA@MPP(Mg2+)诱导体液免疫的能力；
图1-5-3为本申请实施例1.3.5.3提供的RBD-mRNA@MPP(Mg2+)诱导细胞免疫的能力；
图1-6-3为本申请实施例1.3.5.3提供的NY-ESO-1-mRNA@MPP(Mg2+)诱导细胞免疫的能力；
图1-7为本申请实施例1.3.6.1提供的Bcl-2-siRNA@MPP(Fe3+)沉默靶基因的能力；
图1-8为本申请实施例1.3.6.1提供的PLK1-siRNA@MPP(Fe3+)沉默靶基因的能力；
图1-9为本申请实施例1.3.6.1提供的Gal-1-siRNA@MPP(Fe3+)沉默靶基因的能力；
图1-7-2为本申请实施例1.3.6.2提供的Bcl-2-siRNA@MPP(Al3+)沉默靶基因的能力；
图1-8-2为本申请实施例1.3.6.2提供的PLK1-siRNA@MPP(Al3+)沉默靶基因的能力；
图1-9-2为本申请实施例1.3.6.2提供的Gal-1-siRNA@MPP(Al3+)沉默靶基因的能力；
图1-7-3为本申请实施例1.3.6.3提供的Bcl-2-siRNA@MPP(Mg2+)沉默靶基因的能力；
图1-8-3为本申请实施例1.3.6.3提供的PLK1-siRNA@MPP(Mg2+)沉默靶基因的能力；
图1-9-3为本申请实施例1.3.6.3提供的Gal-1-siRNA@MPP(Mg2+)沉默靶基因的能力；
图1-10为本申请实施例1.3.7.1提供的STAT3-ASO@MPP(Fe3+)沉默细胞靶基因的能力；
图1-11为本申请实施例1.3.7.1提供的α-syn-ASO@MPP(Fe3+)沉默细胞靶基因的能力；
图1-12为本申请实施例1.3.7.1提供的Bcl-2-ASO@MPP(Fe3+)沉默细胞靶基因的能力；
图1-10-2为本申请实施例1.3.7.2提供的STAT3-ASO@MPP(Al3+)沉默细胞靶基因的能力；
图1-11-2为本申请实施例1.3.7.2提供的α-syn-ASO@MPP(Al3+)沉默细胞靶基因的能力；
图1-12-2为本申请实施例1.3.7.2提供的Bcl-2-ASO@MPP(Al3+)沉默细胞靶基因的能力；
图1-10-3为本申请实施例1.3.7.3提供的STAT3-ASO@MPP(Mg2+)沉默细胞靶基因的能力；
图1-11-3为本申请实施例1.3.7.3提供的α-syn-ASO@MPP(Mg2+)沉默细胞靶基因的能力；
图1-12-3为本申请实施例1.3.7.3提供的Bcl-2-ASO@MPP(Mg2+)沉默细胞靶基因的能力；
图1-13为本申请实施例1.3.8.1提供的S-mRNA@MPP(Fe3+)转染293T所致S蛋白的表达；
图1-14为本申请实施例1.3.8.1提供的药物(dsDNA及ssDNA)@MPP(Fe3+)的功能；
图1-13-2为本申请实施例1.3.8.2提供的S-mRNA@MPP(Al3+)转染293T所致S蛋白的表达；
图1-14-2为本申请实施例1.3.8.2提供的药物(dsDNA及ssDNA)@MPP(Al3+)的功能；
图1-13-3为本申请实施例1.3.8.3提供的S-mRNA@MPP(Mg2+)转染293T所致S蛋白的表达；
图1-14-3为本申请实施例1.3.8.4提供的药物(dsDNA及ssDNA)@MPP(Mg2+)的功能；
图1-15为本申请实施例1.3.8.1提供的药物(dsDNA及ssDNA)@MPP(Fe3+)的转染细胞荧光图；
图2-1为本申请实施例2.1.1提供的磷脂复合物的差示扫描量热图；
图2-2为本申请实施例2.1.1提供的金属-磷脂复合物(Fe3+)的紫外吸收图；
图2-2-2为本申请实施例2.1.2提供的金属-磷脂复合物(Al3+)的紫外吸收图；
图2-2-3为本申请实施例2.1.3提供的金属-磷脂复合物(Mg2+)的紫外吸收图；
图2-3为本申请实施例2.2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2-4为本申请实施例2.3提供的药物-金属-磷脂复合物颗粒(Fe3+)的元素分析；
图2-5为本申请实施例2.4提供的siRNA/mRNA@MPP(Fe3+、Al3+或Mg2+)和siRNA/mRNA@LNP包载核酸(mRNA及siRNA)的效率；
图2-6为本申请实施例2.5提供的siRNA/mRNA@MPP(Fe3+、Al3+或Mg2+)和siRNA/mRNA@LNP的核酸溶酶体逃逸能力统计图；
图2-7为本申请实施例2.6提供的MPP(Fe3+、Al3+或Mg2+)与LNP的eGFP阳性细胞率；
图2-8为本申请实施例2.7提供的MPP(Fe3+、Al3+或Mg2+)与LNP的促RBD-mRNA表达的能力对比；
图2-9为本申请实施例2.7提供的MPP(Fe3+、Al3+或Mg2+)与LNP的促体液免疫的能力对比；
图2-10为本申请实施例2.7提供的MPP(Fe3+、Al3+或Mg2+)与LNP的促细胞免疫的能力对比；
图2-11为本申请实施例2.5提供的siRNA/mRNA@MPP(Fe3+)和siRNA/mRNA@LNP的促核酸溶酶体逃逸功能检测结果图；
图3-1为本申请实施例三提供的药物-金属-磷脂复合物颗粒(Fe3+)瘤内注射治疗肝癌的作用；
图3-2为本申请实施例三提供的药物-金属-磷脂复合物颗粒(Al3+)瘤内注射治疗肝癌的作用；
图3-3为本申请实施例三提供的药物-金属-磷脂复合物颗粒(Mg2+)瘤内注射治疗肝癌的作用；
图4-1为本申请实施例6.3中靶向药物(CD19 CAR mRNA@Apt-MPP)(Fe3+)、药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)(Fe3+)的CD19-CAR阳性细胞占比统计图；
图4-1-2为本申请实施例6.3中靶向药物(CD19 CAR mRNA@Apt-MPP)(Al3+)、药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)(Al3+)的CD19-CAR阳性细胞占比统计图；
图4-1-3为本申请实施例6.3中靶向药物(CD19 CAR mRNA@Apt-MPP)(Mg2+)、药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)(Mg2+)的CD19-CAR阳性细胞占比统计图；
图4-2为本申请实施例6.4中靶向药物(CD19 CAR mRNA@Apt-MPP)(Fe3+)、药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)(Fe3+)对急性B淋巴白血病小鼠的治疗效果对比图；
图4-2-2为本申请实施例6.4中靶向药物(CD19 CAR mRNA@Apt-MPP)(Al3+)、药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)(Al3+)对急性B淋巴白血病小鼠的治疗效果对比图；
图4-2-3为本申请实施例6.4中靶向药物(CD19 CAR mRNA@Apt-MPP)(Mg2+)、药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)(Mg2+)对急性B淋巴白血病小鼠的治疗效果对比图；
图5-1为本申请实施例7.1中靶向药物CD19 CAR mRNA@Apt(CD8)-MPP(Fe3+)与CD19 CAR mRNA@MPP(Fe3+)对比抗急性B淋巴细胞性白血病小鼠的治疗总生存率对比图；
图5-1-2为本申请实施例7.1中靶向药物CD19 CAR mRNA@Apt(CD8)-MPP(Al3+)与CD19 CAR mRNA@MPP(Al3+)对比抗急性B淋巴细胞性白血病小鼠的治疗总生存率对比图；
图5-1-3为本申请实施例7.1中靶向药物CD19 CAR mRNA@Apt(CD8)-MPP(Mg2+)与CD19 CAR mRNA@MPP(Mg2+)对比抗急性B淋巴细胞性白血病小鼠的治疗总生存率对比图；
图5-2为本申请实施例7.2中靶向药物ASO@Apt-MPP(Fe3+)与ASO@MPP(Fe3+)对比抗肺癌小鼠的治疗总生存率对比图；
图5-2-2为本申请实施例7.2中靶向药物ASO@Apt-MPP(Al3+)与ASO@MPP(Al3+)对比抗肺癌小鼠的治疗总生存率对比图；
图5-2-3为本申请实施例7.2中靶向药物ASO@Apt-MPP(Mg2+)与ASO@MPP(Mg2+)对比抗肺癌小鼠的治疗总生存率对比图；
图5-3为本申请实施例7.3中靶向药物siRNA@Apt-MPP(Fe3+)与siRNA@MPP(Fe3+)对比抗胰腺癌小鼠的治疗总生存率对比图；
图5-3-2为本申请实施例7.3中靶向药物siRNA@Apt-MPP(Al3+)与siRNA@MPP(Al3+)对比抗胰腺癌小鼠的治疗总生存率对比图；
图5-3-3为本申请实施例7.3中靶向药物siRNA@Apt-MPP(Mg2+)与siRNA@MPP(Mg2+)对比抗胰腺癌小鼠的治疗总生存率对比图；
图6-1为本申请实施例8.2中以Fe3+、药物为CD19 CAR、适配体为以CD62L为靶点时CD19 CAR mRNA@Apt(CD62L)-MPP、CD19 CAR mRNA@Apt(CD62L)-LNP的抗急性B淋巴细胞性白血病小鼠的治疗生存率对比图；
图6-1-2为本申请实施例8.2中以Al3+、药物为CD19 CAR、适配体为以CD62L为靶点时CD19 CAR mRNA@Apt(CD62L)-MPP、CD19 CAR mRNA@Apt(CD62L)-LNP的抗急性B淋巴细胞性白血病小鼠的治疗生存率对比图；
图6-1-3为本申请实施例8.2中以Mg 2+、药物为CD19 CAR、适配体为以CD62L为靶点时CD19 CAR mRNA@Apt(CD62L)-MPP、CD19 CAR mRNA@Apt(CD62L)-LNP的抗急性B淋巴细胞性白血病小鼠的治疗生存率对比图；
图6-2为本申请实施例8.2中以Fe3+、药物为ASO、适配体为AS1411时ASO@Apt(AS1411)-MPP、ASO@Apt(AS1411)-LNP的抗肺癌小鼠的治疗生存率对比图；
图6-2-2本申请实施例8.2中以Al3+、药物为ASO、适配体为AS1411时ASO@Apt(AS1411)-MPP、ASO@Apt(AS1411)-LNP的抗肺癌小鼠的治疗生存率对比图；
图6-2-3本申请实施例8.2中以Mg2+、药物为ASO、适配体为AS1411时ASO@Apt(AS1411)-MPP、ASO@Apt(AS1411)-LNP的抗肺癌小鼠的治疗生存率对比图；
图6-3为本申请实施例8.2中以Fe3+、药物为siRNA、适配体为P19时siRNA@Apt(P19)-MPP、siRNA@Apt(P19)-LNP的抗胰腺癌小鼠的治疗生存率对比图；
图6-3-2为本申请实施例8.2中以Al3+、药物为siRNA、适配体为P19时siRNA@Apt(P19)-MPP、siRNA@Apt(P19)-LNP的抗胰腺癌小鼠的治疗生存率对比图；
图6-3-3为本申请实施例8.2中以Mg2+、药物为siRNA、适配体为P19时siRNA@Apt(P19)-MPP、siRNA@Apt(P19)-LNP的抗胰腺癌小鼠的治疗生存率对比图；
图7-1为本申请实施例6.4、实施例9.1、实施例9.2中Fe3+的CD19 CAR mRNA@MPP与CD19 CAR mRNA@Apt(CD62L)-MPP、CAR mRNA@Apt(CD8)-MPP、CD19 CAR mRNA@Apt(CD3)-MPP的抗急性B淋巴细胞性白血病小鼠的治疗总生存率对比图；
图7-1-2为本申请实施例6.4、实施例9.1、实施例9.2中Al3+的CD19 CAR mRNA@MPP与CD19 CAR mRNA@Apt(CD62L)-MPP、CAR mRNA@Apt(CD8)-MPP、CD19 CAR mRNA@Apt(CD3)-MPP的抗急性B淋巴细胞性白血病小鼠的治疗总生存率对比图；
图7-1-3为本申请实施例6.4、实施例9.1、实施例9.2中Mg2+的CD19 CAR mRNA@MPP与CD19 CAR mRNA@Apt(CD62L)-MPP、CAR mRNA@Apt(CD8)-MPP、CD19 CAR mRNA@Apt(CD3)-MPP的抗急性B淋巴细胞性白血病小鼠的治疗总生存率对比图；
图8-1为本申请实施例2.8中CD19 CAR mRNA@MPP(Fe3+、Al3+或Mg2+)和CD19 CAR mRNA@LNP在髓系细胞中的CAR阳性细胞占比图；
图8-2为本申请实施例6.5中CD19 CAR mRNA@Apt-MPP(Fe3+、Al3+或Mg2+)和CD19 CAR mRNA@Apt-LNP在外周血T细胞中的CAR阳性细胞占比图；
图8-3为实施例6.6中不同靶头比例修饰的CD19 CAR mRNA@Apt-MPP(Ca2+)和CD19 CAR mRNA@Apt-LNP(Ca2+)的CAR阳性率；
图8-4为实施例6.6中不同靶头比例修饰的CD19 CAR mRNA@Apt-MPP(Mg2+)和CD19 CAR mRNA@Apt-LNP(Mg2+)的CAR阳性率；
图8-5为本申请实施例6.5中CD19 CAR mRNA@Apt-MPP(Fe3+、Al3+或Mg2+)和CD19 CAR mRNA@Apt-LNP在外周血髓系细胞中的CAR阳性细胞占比图。
具体实施方式
为了使本领域的技术人员更好的理解本申请的技术方案，下面结合具体实施方式对本申请所述技术方案作进一步的详细说明。
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领域技术人员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”和“一个或多个”包括一个，两个，三个或更多个。本申请中涉及“多个”、“多种”、“多次”、“多元”等，如无特别限定，指在数量上大于2或等于2。例如，“一种或多种”表示一种或大于等于两种。
本申请中，“进一步”、“更进一步”、“特别”等用于描述目的，表示内容上的差异，但并不应理解为对本发明保护范围的限制。
本申请中，“优选地”、“优选的”、“优选”，指可有可无，也即指选自“有”或“无”两种并列方案中的任一种。如果一个技术方案中出现多处“优选”，如无特别说明，且无矛盾之处或相互制约关系，则每项“优选”各自独立。
虽然用以界定本申请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，视本领域技术人员的考虑而定。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“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。
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。
术语“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“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的复合物，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，且该金属-磷脂复合物既不是阳离子脂质也不是可电离脂质。
术语“药物-金属-磷脂复合物颗粒”是指已完成载药的金属-磷脂复合物颗粒，药物与金属-磷脂复合物通过包埋、负载、化学键或非化学键等方式连接。
术语“CD8”是指一种二聚体共受体，可使T细胞识别组织相容性复合体I类蛋白呈递的肽，是细胞毒性T淋巴细胞(cytotoxic T lymphocyte，CTL)的表面标志蛋白。CD8+T细胞启动凋亡的一种方式是通过穿孔素和颗粒酶(两种类型的细胞毒性蛋白)的分泌。穿孔素是在靶细胞的细胞膜上形成孔的溶细胞蛋白。CTL使用这些孔来引导粒酶的释放，粒酶是一类丝氨酸蛋白酶，可继续在细胞质内凋亡。这些蛋白酶通过裂解参与正常细胞维持的病毒和细胞蛋白来关闭靶细胞。然后，经历凋亡的靶向细胞被附近的吞噬细胞清除。除定向凋亡外，CD8+T细胞还可以通过释放诸如TNF-α的细胞因子来间接杀死靶细胞。
术语“CD62L”又称L-选择素(L-Selectin)，是白细胞-内皮细胞黏附分子的基因家族之一。其胞膜外区N端有1个C型凝集素样结构域，1个EGF样结构域和2个CCP结构域。L-选择素由白细胞结构性表达，并介导与血管内皮细胞表达的寡糖配体的结合。“CD62L”是一类具有密切结构和功能关系的血管黏附分子家族，其主要作用是促进白细胞在牢固黏附和随后的迁移之前沿着内皮细胞的滚动行为。它们与其他黏附分子不同，首先是因为它们的黏附特性只涉及血管系统中白细胞-血小板-内皮的相互作用，其次是因为与其他黏附分子不同，选择素形成蛋白质-蛋白质键，选择素具有蛋白质类凝集素基团，与碳水化合物配体结合，从而形成蛋白质-碳水化合物键。健康人外周血中约50％的外周血淋巴细胞、40％-80％的单核细胞、95％的中性粒细胞、大多数B细胞和原始T细胞表面表达CD62L。
术语“靶向载体”是指具有特异性靶向作用和载药能力的系统。靶向载体具有改善药物在体内的代谢动力学特性、增加药物定向富集到特定治疗部位甚至特定细胞类型内部、提高疗效和降低毒副作用等功能。可以是纳米颗粒，也可以是组合物大分子。在本申请中，靶向载体可用于包载CAR核酸药物，
术语“脂质递送系统”是指一种小分子或大分子物质的递送技术。它主要利用脂质(如磷脂等)的特性来包裹、运输小分子或大分子物质，将小分子或大分子物质有效递送至靶细胞或靶组织，从而提高小分子或大分子物质的生物利用度和治疗效果。
术语“外泌体”是指指细胞分泌的一种纳米级别的囊泡，其内部包含蛋白质、核酸(如mRNA、miRNA)等生物活性物质。
术语“假病毒”是指一种人工构建的类似病毒的颗粒。它通常是将病毒的某些关键结构(如包膜蛋白)与非病毒的核酸(如报告基因)组合在一起，其不具有完整病毒的复制能力，但能够模拟病毒进入细胞等部分生物学行为，可用于病毒感染机制研究、疫苗研发和药物筛选等众多领域。
术语“小分子”是指能够调节生物体内生理过程的低分子量物质。这些小分子可以是内源性的，如体内代谢过程产生的激素小分子(如肾上腺素)，也可以是外源性的，比如从植物中提取的药物小分子(如青蒿素)，它们可以通过与生物大分子(如蛋白质、核酸)相互作用来发挥作用，如参与信号传导、酶抑制或激活等过程。
术语“磷脂运送系统”是指一种利用磷脂成分构建的运输体系，主要用于将各种物质(如药物、营养成分、生物分子等)从一个位置输送到另一个位置的机制。
术语“LNP”是指一种由阳离子脂质或可电离脂质，辅助脂质，胆固醇以及聚乙二醇化脂质等成分通过自组装形成的纳米级胶体递送系统；其中阳离子脂质在介导核酸的包封和细胞摄取过程中发挥关键作用，且阳离子脂质或可电离脂质电离后所携带的正电荷能够与带负电荷的核酸(如mRNA、siRNA、ASO等)通过静电作用结合，从而有效保护核酸并促进其递送至靶细胞。
术语“适配体”，Aptamer，即核酸适配体，源于拉丁语“aptus”，是由20-60个碱基组成的单链寡聚核苷酸(RNA)或单链寡聚脱氧核苷酸(DNA)。“适配体”被用来指任何长度的核苷酸的聚合形式，核糖核苷酸或脱氧核糖核苷酸。因此，该术语包括但不限于单链、双链或多链DNA或RNA，DNA基因组、cDNA，DNA-RNA杂交体、或包含嘌呤和嘧啶碱基或其他天然的、化学的或生物化学修饰的、非天然的或衍生的核苷酸碱基的聚合物。它能特异结合蛋白、小分子、离子和细胞等多种靶分子。核酸适配体是通过SELEX技术筛选得到的，利用该技术可以从随机单链核酸序列库中筛选出特异性与靶标高亲和力结合的核酸适配体(Aptamer)。
术语“GalNac”在本说明书中，是指含有能够与肝脏的肝实质细胞上的去唾液酸糖蛋白受体(ASGPR)结合的N-乙酰基-D-半乳糖胺(GalNAc)的部分结构。GalNAc含有磷酸酯基团或硫代磷酸酯基团，用于将直链或支链接头结构与寡核苷酸结合。这样的含有用于结合的一个磷酸酯基团或硫代磷酸酯基团的结构可称为“GalNAc”。除非另有说明，一个GalNac中包含的GalNac的数量没有限制，并且可以采用本说明书中己知和公开的那些。只要保持与ASGPR结合的能力，就可以修饰GalNAc的结构。此外，还包括在制备过程中导入的具有保护基团的GalNAc。术语“Galnac”具有其常规科学含义并且此处是指N-乙酰半乳糖胺及其IUPAC名称：2-(乙酰基氨基)-2-脱氧-D-半乳糖。
术语“P19”是指一段可特异性识别胰腺癌细胞的核酸序列。
术语“靶向药物”(也称作靶向制剂)是指被赋予了靶向(Targeting)能力的药物或其制剂。
术语“跨膜结构域”为CAR的跨越膜的序列，可以被设计以包括将CAR的抗原结合结构域连接至胞内结构域的跨膜结构域。跨膜结构域可以是在膜中热力学稳定的任何蛋白结构，通常为包含几个疏水残基的α螺旋。
术语“信号传导结构域”指编码信号肽的序列或编码与信号肽发挥相同作用物质的序列。“信号传导结构域”是指蛋白质的功能部分，该功能部分通过以下发挥作用：在细胞内传递信息，从而通过产生第二信使或通过响应这样的信使而作为效应子来经由确定的信号传导途径调节细胞活性。信号肽使得当CAR在细胞诸如T细胞内被表达时，新生的蛋白被引导至内质网并随后被引导至它被表达的细胞表面。信号肽的核心可以包含长段的疏水氨基酸，其具有形成单个α-螺旋的倾向。
术语“抗原结合结构域”通常是指能够与抗原特异性结合的结构域，具有能够与目标抗原特异性结合的能力的嵌合抗原受体结构域或片段。抗原结合结构域为CAR的识别抗原部分。许多抗原结合域是本领域已知的，包括基于抗体、抗体模拟物和T细胞受体的抗原结合位点的那些抗原结合结构域。例如，抗原结合结构域可以包含：来源于单抗隆抗体的单链可变片段(scFv)；靶抗原的天然配体；对靶具有足够亲和力的肽；单个结构域抗体；人工单个结合剂诸如Darpin(设计的锚蛋白重复蛋白)；或来源于T细胞受体的单链。
术语“共刺激信号传导区”是指能够通过T细胞受体增强T细胞激活的来自如CD28、41BB和ICOS等共刺激蛋白受体的细胞内信号传导结构域。“共刺激信号传导区”指在CAR分子中负责给T细胞提供第二个激活信号的结构域，主要包括CD28受体家族(CD28、ICOS)或肿瘤坏死因子受体家族(4-1BB、OX40、CD27)。共刺激结构域的主要功能为提供第二个激活信号，协同刺激分子和活化细胞内信号，使T细胞持续增殖并释放细胞因子，提高T细胞的抗肿瘤能力。
术语“抗原结合结构域与跨膜结构域连接区”是指负责将抗原结合结构域和跨膜结构域连接的区域。在嵌合膜蛋白的胞外结构域和跨膜结构域之间，或在嵌合膜蛋白的胞质结构域和跨膜结构域之间，可以并入间隔结构域，比如起将跨膜结构域连接至多肽链中的胞外结构域或胞质结构域作用的寡肽或多肽。间隔结构域可以包括多至300个氨基酸，优选地10至100个氨基酸和最优选地25至50个氨基酸。
术语“嵌合抗原受体(CAR)”指将任意特异性接枝到免疫效应细胞上的工程化受体，CAR由四个主要结构域模块化组成：在细胞外末端是用于目标识别的单链可变区(scFv)抗体，一个连接到跨膜结构域的间隔区，以及细胞内信号传导结构域，有或没有连接的共刺激结构域。目标细胞表面的同源抗原的参与启动了CAR工程化免疫细胞的激活，导致持久的免疫细胞反应。每个CAR结构域都有独特的功能，并对重定向的免疫细胞激活产生影响；对于每个特定应用，CAR设计需要进行一些调整，以提供优化的靶向和免疫细胞激活。它巧妙融合抗原识别与细胞内激活元件，打破免疫细胞识别局限，让免疫细胞精准狙击肿瘤细胞。CAR分子包括三个主要部分：胞外域、跨膜域和胞内域。胞外域中的抗原结合域通常是一个抗体来源的单链片段scFv分子，主要由抗体的可变轻链(VL)和可变重链(VH)及中间的Linker区连接组成，再通过铰链区(Hinge)连接到跨膜结构域，负责识别抗原。跨膜域的主要功能在于将CAR分子锚定在细胞膜上，其对CAR分子表达的稳定性具有重要作用。胞内域则包括共刺激结构域和信号转导结构域，二者合力负责T细胞的完全活化。
术语“T细胞受体(T Cell Receptor，TCR)”是响应于抗原的呈递参与T细胞的活化的膜蛋白的复合体。由可变区(V region)和恒定区(C region)结构域组成，其中α链和β链的可变区组成了抗原识别单位，每个可变区包含3个互补决定区(CDR1、CDR2和CDR3)。在TCR识别配体时，多样性程度较低的CDR1和CDR2与pMHC两侧的α螺旋接触，而CDR3则与中央的肽接触。CDR3是决定TCR识别抗原特异性的最关键序列。TCR的刺激由抗原呈递细胞上的主要组织相容性复合体分子(MHC)触发，所述抗原呈递细胞将抗原肽呈递至T细胞并且结合至TCR复合体以诱发一系列胞内信号传导级联。
术语“靶向免疫细胞药物”是指一类专门设计作用于免疫细胞的药物。这些药物包括：基因编辑类药物(例如Crisp cas9、CRISPR–Cas等)、抗体类药物(例如单抗、双抗)、免疫桥接类治疗药物(例如TCE(T-cell Engager)、ADC(Antibody-Drug Conjugate)、APDC(Antibody-Peptide-Drug Conjugate)等)、靶向治疗类药物(含化疗或放疗)(例如ADC(Antibody-Drug Conjugate)、APDC(Antibody-Peptide-Drug Conjugate)等)、适配体类药物(例如双适配体)、细胞治疗类药物(例如CAR-T(Chimeric Antigen Receptor T-cell)、TCR-T(T-cell Receptor engineered T-cell)等)。这些药物能够精准地识别免疫细胞表面或内部的特定靶点，通过对免疫细胞进行基因改造，或调节免疫细胞的功能，如激活、抑制或改变其分化状态等，来干预机体的免疫反应，从而治疗疾病。
术语“嵌合抗原受体(CAR)药物”是指通过在体内或体外将目标细胞进行基因改造，使其表达CAR，从而发挥作用的一种新型的治疗药物。CAR是一种人工合成的受体，其结构包括能够识别肿瘤抗原的单链抗体可变区(scFv)、跨膜区和细胞内信号转导区。通常，CAR药物通过将各种免疫细胞(例如T细胞、巨噬细胞、髓系细胞、NK细胞等)进行基因工程改造后，获得CAR-免疫细胞(例如CAR-T细胞、CAR-巨噬细胞、CAR-髓系细胞、CAR-NK细胞等)从而发挥治疗作用。
术语“原位CAR”基于mRNA技术编码的CAR核酸序列，通过mRNA重编码T细胞受体，用于体内产生CAR表达细胞，该CAR包含抗原结构域、跨膜结构域、共刺激信号传导区域和信号传导结构域。术语“原位CAR”是指以编码CAR基因的mRNA或组合mRNA为抗原，在体内原位将T细胞转化成为CAR-T细胞。相比传统体外CAR-T，流程制备大大简化，成本大幅下降。以信使RNA作为携带嵌合抗原受体的载体，转入患者T细胞后，将T细胞激活，并装上定位导航装置CAR(肿瘤嵌合抗原受体)，将T细胞这个普通“战士“改造成“超级战士“，即CAR-T细胞，他利用其“定位导航装置“CAR，专门识别体内肿瘤细胞，并通过免疫作用释放大量的多种效应因子，它们能高效地杀灭肿瘤细胞，从而达到治疗恶性肿瘤的目的。
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“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，(iii)中的非阳离子脂质或非可电离脂质为，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，靶向药物或其它药物传递系统与细胞膜融合的能力。所述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”，本申请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申请的DAG-PEG缀合物中。用在本申请中的合适的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”，本申请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I中，所述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，所述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。所述PEG可以任选地被烷基、烷氧基、酰基或芳基基团所取代。在式I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申请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申请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。适合于按照本申请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申请的化合物中。
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”指本申请的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)。靶向药物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，靶向药物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。
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。公开用在本申请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))。
1、金属-磷脂复合物
在本申请中，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。磷脂分子部分与连接物分子部分相连接，连接物分子部分与金属离子部分通过配位键连接，且金属-磷脂复合物不是阳离子脂质或可电离脂质。
对于磷脂分子部分，需要说明的是，本申请的磷脂分子的顺反异构体不会对本申请保护内容所要达到的效果产生影响。
在一些实施方式中，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、磷酸酰肌醇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。在此，“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)”。具体地，该磷脂分子部分，例如可以但不限于选自为卵磷脂(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。在本申请中，“及其衍生物”均为类似的含义。
在一些实施方式中，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)、磷酸酰肌醇(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)，及其衍生物中一种或多种的组合。优选地，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)，及其衍生物中的至少一种。优选地，磷脂分子部分选自DSPC、DSPE、DSPA、DSPG，及其衍生物中的至少一种。优选地，磷脂分子部分选自DSPC(式46)、DSPE(式47)、DSPA(式48)、DSPG(式49)，及其衍生物中的至少一种。
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，其安全性利于靶向药物整体的临床药物注册，缩短了临床药物注册的时间长度。
在一些实施方式中，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。优选地，连接物分子部分选自姜黄素(式19)、绿原酸(式20)、花青素(式21)、槲皮素(式22)、二氢杨梅素(式23)、橙皮素(式24)、柚皮素(式25)、芹菜素(式26)、儿茶素(式27)、茶多酚(式28)、表没食子儿茶素没食子酸酯(式29)、鞣花酸(式30)、桑色素(式31)、表儿茶素没食子酸酯(式32)、儿茶素没食子酸酯(式33)、没食子儿茶素没食子酸酯(式34)、平贝碱C(式35)，及其衍生物中的至少一种。优选地，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)中的至少一种。优选地，连接物分子部分选自姜黄素(式19)、橙皮素(式24)、茶多酚(式28)，及其衍生物中的至少一种。优选地，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)。
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。
在一些实施方式中，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+中的至少一种。优选地，金属离子部分选自Fe3+、Mg2+、Ca2+、Al3+中的至少一种。优选地，金属离子部分选自Fe3+、Mg2+、Ca2+或Al3+。
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。在用于包载药物(例如核酸)时，金属离子的功能是连接磷脂复合物与核酸，所以，当连接物分子与金属离子尽可能少的络合位点连接时，才能保证金属-磷脂复合物尽可能多的包载核酸。
在一些实施方式中，磷脂分子部分选自DSPC、DSPE、DSPA或DSPG，连接物分子部分选自姜黄素、橙皮素或茶多酚，金属离子部分选自Fe3+、Mg2+、Ca2+或Al3+。优选地，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，磷脂分子部分选自DSPC(式46)、DSPE(式47)、DSPA(式48)或DSPG(式49)，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)，金属离子部分选自Fe3+、Mg2+、Ca2+或Al3+。优选地，磷脂分子部分、连接物分子部分和金属离子部分的摩尔比为1:(1～3):(0.5～2)。磷脂分子部分、连接物分子部分和金属离子部分的摩尔比可以但不限于为1:1:0.5、1:1:1、1:1:1.5、1:1:2、1:2:1、1:2:2、1:2:0.5、1:3:0.5、1:3:1或1:3:2。
在一种实施方式中，磷脂分子部分为DSPC(式46)，连接物分子部分选自姜黄素(式19)，金属离子部分选自Fe3+、Mg2+或Al3+，磷脂分子部分、连接物分子部分和金属离子部分的摩尔比为1:1:1。
在本申请中，金属-磷脂复合物可通过“一步法”制备，也可通过“分步法”制备得到。“一步法”与“分步法”对于金属-磷脂复合物的构建能力相当。在优选地实施方式中，“一步法”能够显著缩短产业化进程所需的时间，这在工业化大规模生产的场景下，时间效率至关重要，“一步法”减少了分步操作所带来的流程衔接、时间间隔等问题，极大地提高了生产的连续性与紧凑性，从而有效降低生产成本、提高生产效率。从产业化应用的角度来看，“一步法”更契合大规模生产的需求，具有广阔的应用前景与极高的实用价值。
在一步法中，磷脂分子、连接物分子和金属离子反应得到金属-磷脂复合物。
在一些实施方式中，将磷脂分子、连接物分子和金属离子溶于乙醇中反应，磷脂分子、连接物分子和金属离子的摩尔比优选为1:1:(1～2)，反应条件优选为40～60℃反应1～5h。磷脂分子、连接物分子和金属离子的摩尔比可以但不限于为1:1:1、1:1:1.5或1:1:2，反应的温度可以但不限于为40℃、42℃、46℃、48℃、50℃、52℃、54℃、56℃、58℃或60℃，反应的时间可以但不限于为1h、2h、3h、4h或5h。
在分步法中，先将磷脂分子与连接物分子反应连接形成磷脂复合物；再将制备的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。
在一些实施方式中，将磷脂分子与连接物分子溶于乙醇中反应，之后加入正己烷，沉淀得到磷脂复合物，磷脂分子与连接物分子的摩尔比优选为1:1；反应的条件优选为65℃反应2小时。
在一些实施方式中，磷脂复合物与金属离子溶于乙醇，加入三乙胺后反应，得到金属-磷脂复合物，磷脂复合物与金属离子的摩尔比优选为1：(1～2)，磷脂复合物与三乙胺的摩尔比优选为1:1，反应的条件优选为60℃反应2小时。
2、金属-磷脂复合物颗粒(Metal-chelated phospholipid complex nanoparticles，MPP)
在本申请中，金属-磷脂复合物颗粒含有：(i)金属-磷脂复合物；(ii)抑制颗粒聚集的缀合的脂质，其中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。
对于抑制颗粒聚集的缀合的脂质，是指抑制药物-金属-磷脂复合物颗粒或靶向药物聚集的缀合的脂质，主要功能在于防止药物-金属-磷脂复合物颗粒或靶向药物的聚集，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申请保护内容所要达到得效果产生影响。
在一些实施方式中，(ii)抑制颗粒聚集的缀合的脂质包括PEG-脂质缀合物和/或PEG-DAA。优选地，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)，及其衍生物中的至少一种。优选地，PEG-脂质缀合物选自DSPE-PEG2000、DSPE-PEG700、DSPE-PEG1000或DSPE-PEG5000中的至少一种。优选地，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)。
对于除金属-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，其为胆固醇(优选为胆固醇式40)及其衍生物中的至少一种。在优选的实施方式中，除了胆固醇或其衍生物，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、磷酸酰肌醇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。优选地，除了胆固醇或其衍生物，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)、磷酸酰肌醇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(式41)，及其衍生物中中的至少一种。优选地，(iii)中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，例如，(iii)中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)。
在本申请中，金属-磷脂复合物作为一个整体组分与组分(ii)和组分(iii)进行反应自组装得到金属-磷脂复合物颗粒。该金属-磷脂复合物颗粒可以作为药物载体，其中，药物为带有负电荷的药物，例如核酸。由金属-磷脂复合物组装的金属-磷脂复合物颗粒装载药物的原理是：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，得到药物-金属-磷脂复合物颗粒。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)，可简称为“非阳离子脂质或非可电离脂质”。
金属-磷脂复合物颗粒中各组分的投放比例可以为：金属-磷脂复合物在原料中摩尔占比为5％～50％，抑制颗粒聚集的缀合的脂质在原料中摩尔占比为1％～10％，胆固醇在原料中摩尔占比为15％～80％，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％～51％。在此，“原料”指金属-磷脂复合物、抑制颗粒聚集的缀合的脂质、胆固醇、除胆固醇以外的非阳离子脂质或非可电离脂质的总和。
金属-磷脂复合物在原料中的摩尔占比可以但不限于为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％。在一些实施方式中，金属-磷脂复合物在原料中的摩尔占比为5％～40％，优选为10％～40％。
抑制颗粒聚集的缀合的脂质在原料中的摩尔占比可以但不限于为1％、2％、3％、4％、5％、6％、7％、8％、9％或10％。在一些实施方式中，抑制颗粒聚集的缀合的脂质在原料中摩尔占比为2％～10％。
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可以但不限于为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％。在一些实施方式中，胆固醇在原料中摩尔占比为25％～75％，优选为35％～75％，优选为35％～55％。
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可以但不限于为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％。在一些实施方式中，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0％～50％，优选为0％～40％，优选为5％～30％，进一步为20％～25％。
金属-磷脂复合物颗粒的制备方法为将金属-磷脂复合物、抑制颗粒聚集的缀合的脂质和非阳离子脂质或非可电离脂质混合得到金属-磷脂复合物颗粒。在一些实施方式中，金属-磷脂复合物、抑制颗粒聚集的缀合的脂质和非阳离子脂质或非可电离脂质在均一相中混合，例如有机相(在一些实施方式中，溶剂为乙醇)。
当金属-磷脂复合物颗粒包载药物时，制备方法为金属-磷脂复合物、抑制颗粒聚集的缀合的脂质、非阳离子脂质或非可电离脂质，以及药物混合制备得到药物-金属-磷脂复合物颗粒。在一些实施方式中，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-金属-磷脂复合物颗粒。在一些实施方式中，缓冲液可以为PBS缓冲液，也可以为Tris-HCl缓冲液。在一些实施方式中，有机相和水相的混匀方式可以为微流控芯片，也可以为超声。
3、靶向载体(含有MPP)
在本申请中，靶向载体含有载体运送系统和靶向结构。载体运送系统包括脂质递送系统、外泌体、假病毒、小分子、细胞或载运蛋白中的至少一种。优选地，脂质递送系统为金属-磷脂复合物颗粒、LNP或磷脂运送系统。在一种实施方式中，假病毒为腺病毒。在一种实施方式中，细胞为血红细胞。在一种实施方式中，载运蛋白为载铁蛋白。在一种实施方式中，LNP为阳离子脂质或可电离脂质。
在一种实施方式中，载体运送系统为金属-磷脂复合物颗粒(MPP)，靶向结构与金属-磷脂复合物颗粒(MPP)的外表面连接。
在一些实施方式中，靶向结构包括依次连接的疏水区、连接区和靶向结合区，疏水区与金属-磷脂复合物颗粒的外层基于亲水性、疏水性进行连接。
在一些实施方式中，疏水区包括DSPE及其衍生物中的至少一种或多种。通过该部分实现靶向结构与金属-磷脂复合物颗粒的外层的连接。
在一些实施方式中，连接区包括PEG-2000及其衍生物中的至少一种或多种。
在一些实施方式中，靶向结合区可通过特异性识别引导靶向载体至特定目标(即靶向结合区的靶点或靶细胞)，从而实现包载药物的LNP颗粒、金属-磷脂复合物颗粒将药物运输并作用于目标物(即药物的靶点或靶细胞)。在一些实施方式中，靶向结合区可结合CD62L、CD8、CD3、核仁素蛋白、T细胞、自然杀伤细胞、巨噬细胞、胰腺癌细胞或肝癌细胞中的至少一种。在一些实施方式中，靶向结合区包括核酸、多肽、蛋白、小分子中的至少一种。在一些实施方式中，靶向结合区包括适配体、抗体、抗原结合部、galnac中的一种。在一些实施方式中，靶向结合区为适配体，靶向结合区的靶点为CD62L，适配体优选为SEQ ID NO.44所示。在一些实施方式中，靶向结合区为适配体，靶向结合区的靶点为CD8，适配体优选为SEQ ID NO.84所示。在一些实施方式中，靶向结合区为适配体，靶向结合区的靶点为CD3，适配体优选为SEQ ID NO.95所示。在一些实施方式中，靶向结合区为适配体，靶向结合区的靶点为核仁素蛋白，适配体优选为SEQ ID NO.86所示。在一些实施方式中，靶向结合区为适配体，靶向结合区的靶细胞为T细胞，适配体优选为SEQ ID NO.44所示。在一些实施方式中，靶向结合区为适配体，靶向结合区的靶细胞为胰腺癌细胞，适配体优选为SEQ ID NO.88所示(例如P19)。在一些实施方式中，靶向结合区为适配体，靶向结合区的靶细胞为肝癌细胞。
在一种实施方式中，靶向结构为DSPE-PEG2000-适配体，优选为DSPE-PEG2000-CD62L适配体，CD62L适配体序列如SEQ ID NO.44所示。
本申请中，靶向结构的制备方法，可以先将疏水区和连接区连接，再通过中间体对将疏水区-连接区与靶向结合区连接。以DSPE-PEG2000-CD62L适配体为例，制备过程为：DSPE-PEG2000与CD62L适配体通过中间体对反应连接，得到DSPE-PEG2000-CD62L适配体。在一些实施方式中，中间体对选自DSPE-PEG2000-MAL和CD62L适配体-C6-SH、DSPE-PEG2000-NHS和CD62L适配体-NH2、DSPE-PEG2000-COOH和CD62L适配体-NH2、DSPE-PEG2000-NCO和CD62L适配体-NH2、DSPE-PEG2000-N3和CD62L适配体-DBCO，优选为DSPE-PEG2000-MAL和CD62L适配体-C6-SH。
在一种实施方式中，DSPE-PEG2000-适配体先制备成胶束再与载体运送系统(例如，金属-磷脂复合物颗粒(MPP))的外表面连接形成靶向载体。在一些实施方式中，胶束的制备方法包括直接溶解法、乙醇注入法、透析法或超声法。
当靶向载体用于药物包载时，先将药物、金属-磷脂复合物、抑制颗粒聚集的缀合的脂质、非阳离子脂质或非可电离脂质制备成药物-脂质颗粒(即药物-金属-磷脂复合物颗粒)，再将靶向结构与药物-脂质颗粒反应连接得到靶向药物。
在一些实施方式中，靶向结构与药物-脂质颗粒的反应投料质量比例为1：(14～84)，投料质量比可以但不限于为1:14、1:16、1:16.8、1:18、1:20、1:21、1:22、1:24、1:26、1:28、1:30、1:35、1:40、1:45、1:50、1:55、1:60、1:65、1:70、1:75、1:80、1:82或1:84。
在一些实施方式中，药物-脂质颗粒与靶向结构的反应条件为-10～10℃孵育0.2-12h。反应温度可以但不限于为-10℃、-9℃、-8℃、-7℃、-6℃、-5℃、-4℃、-3℃、-2℃、-1℃、0℃、1℃、2℃、3℃、4℃、5℃、6℃、7℃、8℃、9℃、10℃，反应时间可以但不限于为0.2h、0.5h、1h、2h、3h、4h、5h、6h、7h、8h、9h、10h、11h或12h。
4、靶向药物
在本申请中，靶向药物包括药物和靶向载体，该药物包封在靶向载体中，更具体地，该药物包封在金属-磷脂复合物颗粒中。
在本申请中，药物为带有负电荷的分子，该分子可以选自核酸、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。在一些实施方式中，核酸是选自mRNA、siRNA、环状RNA、microRNA、ASO、sgRNA、DNA、ecDNA、人工核酸中一种或多种组合的组中的成员。
在一些实施方式中，药物为mRNA，该mRNA为编码嵌合抗原受体CAR或TCR的mRNA。此时，靶向药物实现原位CAR细胞疗法或TCR细胞疗法。将这种编码相应受体的mRNA递送至体内特定细胞后，可促使这些细胞在体内原位表达CAR或TCR，进而使这些细胞具备靶向识别并杀伤特定肿瘤细胞等相关功能，此时，通过靶向药物实现的原位CAR细胞疗法，为疾病(尤其是肿瘤疾病)的治疗提供了一种新策略。这种疗法具有相对更强的靶向性，还可能减少传统细胞疗法中复杂的制备及回输流程，优势显著。
在一些实施方式中，药物为编码嵌合抗原受体CAR的mRNA，CAR包括跨膜结构域、信号传导结构域、抗原结合结构域、共刺激信号传导区、抗原结合结构域与跨膜结构域连接区。在一些实施方式中，跨膜结构域选自SEQ ID No.28、SEQ ID No.29、SEQ ID No.30中的至少一种；和/或，信号传导结构域选自SEQ ID No.31、SEQ ID No.32、SEQ ID No.33中的至少一种；和/或，抗原结合结构域选自SEQ ID No.34、SEQ ID No.35、SEQ ID No.36中的至少一种；和/或，共刺激信号传导区选自SEQ ID NO.37、SEQ ID No.39、SEQ ID No.75中的至少一种；和/或，抗原结合结构域与跨膜结构域连接区选自SEQ IDNo.40、SEQ ID No.41、SEQ ID No.42中的至少一种。
在一种实施方式中，药物是核苷酸序列如SEQ ID No.43所示的mRNA。
此外，包封在本申请的靶向药物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，同时通过靶向结构中靶向结合区的作用实现药物被递送进入特定细胞。
在一些实施方案中，本申请提供的靶向药物具有适合于全身传递的小直径。
在一些实施方案中，本申请的药物优选为核酸，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。
本申请的靶向药物或制备方法可用于药物运送、显影药物、疫苗。
在一些实施方式中，靶向药物用于药物运送、治疗和/或预防。
在一些实施方式中，靶向药物用于将药物引入细胞或血液；细胞优选包括免疫细胞、肿瘤细胞、生殖细胞、神经细胞、内分泌细胞、血细胞、吞噬细胞、白细胞、红细胞、上皮细胞、心肌细胞或干细胞。
在一些实施方式中，靶向药物用于在哺乳动物受试者中使靶序列的表达或沉默、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、用于将药物从体内传递到哺乳动物细胞因进行靶序列表达或沉默、或用于治疗/或预防哺乳动物的疾病或病症。优选地，哺乳动物为人。优选地，疾病或病症的治疗与基因的表达相关，基因包含药物的靶序列。
在一些实施方式中，疾病或病症包括癌症、病毒感染、自身免疫性疾病、免疫系统过度激活引发的疾病、代谢性疾病、纤维化疾病、组织纤维化、细胞衰老、动脉粥样硬化、糖尿病或骨关节炎。
在一些实施方式中，该癌症包括血液瘤和实体瘤。优选地，血液瘤包括急性B细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、多发性骨髓瘤(MM)、急性髓系白血病(AML)或T细胞淋巴瘤，弥漫性大B细胞淋巴瘤(DLBCL)优选包括霍奇金淋巴瘤(HL)和非霍奇金淋巴瘤(NHL)。优选地，实体瘤包括肝癌、脑胶质瘤、胃癌、胰腺癌、肺癌、非小细胞肺癌、肾细胞癌、前列腺癌、骨肉瘤、乳腺癌、结直肠癌、卵巢癌、黑色素瘤、神经母细胞瘤、血管母细胞瘤、间变性脑膜瘤、复发性胶质母细胞瘤或分化型甲状腺癌。
在一些实施方式中，该病毒感染包括乙型肝炎病毒、丙肝炎病毒、SARS-Cov-2、人类免疫缺陷病毒、细胞巨细胞病毒、侵袭性曲霉菌或结合病毒。
在一些实施方式中，自身免疫性疾病包括寻常型天疱疹、系统性红斑狼疮(SLE)、血友病、重症肌无力、移植组织器官引发的免疫排斥反应、I型糖尿病(T1D)、类风湿关节炎、系统性硬化症、多发性硬化症、特发性肺纤维化、克罗恩病或结肠炎。
在一些实施方式中，免疫系统过度激活引发的疾病包括细胞因子释放综合征(CRS)、免疫效应细胞相关神经毒性综合征(ICANS)或移植物抗宿主病(GVHD)。
在一些实施方式中，代谢性疾病包括动脉粥样硬化、先天性高胰岛素血症、非酒精性脂肪肝炎或非肥胖糖尿病(NOD)。
在一些实施方式中，纤维化包括心肌纤维化、肥厚性心肌病、新冠肺炎、新冠心肌炎、缺血性心肌病、肝硬化、肝纤维化、原发性胆管炎、肺纤维化、骨骼肌纤维化、皮肤类疾病、骨髓纤维化、结节病或杜氏肌营养不良症。优选地，皮肤类疾病包括瘢痕疙瘩或伤口愈合。
在一些实施方式中，靶向药物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。
在一些实施方式中，该靶向药物用于联合用药。
在一些实施方式中，该靶向药物为靶向免疫细胞药物，该免疫细胞为T细胞和/或髓系细胞。
在一些实施方式中，该靶向免疫细胞药物为嵌合抗原受体(CAR)药物。
本申请提供一种含有靶向药物的药剂，该药剂优选为疫苗，进一步优选为新型冠状病毒疫苗。
当药物为核酸时，靶向载体可用于促药物溶酶体逃逸中，以及促进核酸表达中。靶向载体也可用于递送药物，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，本申请提供药剂可实现：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，靶向药物为主要有效成分，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。含有该药剂的产品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。
对于靶向药物作用的靶基因：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申请中，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靶向药物是理想的。可以将所述靶向药物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靶向药物的溶液，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，将靶向药物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，诸如过滤来对靶向药物进行灭菌。所述靶向药物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，可以将靶向药物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。将所述靶向药物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靶向药物的有效量中，所述疾病由于靶基因的表达或过表达导致，所述内科医师评价靶向药物的循环血浆水平，靶向药物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，可以将所述靶向药物施用给受试者，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的基因组中是缺乏的)，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靶向药物引入靶细胞，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，本申请靶向药物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，本申请靶向药物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，本申请的靶向药物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。
在另一个实施方式中，所述靶向药物可用于治疗癌症、病毒感染、自身免疫性疾病、免疫系统过度激活引发的疾病、代谢性疾病、纤维化疾病、组织纤维化、细胞衰老、动脉粥样硬化、糖尿病或骨关节炎。所述癌症包括血液瘤和实体瘤。所述血液瘤包括急性B细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、多发性骨髓瘤(MM)、急性髓系白血病(AML)或T细胞淋巴瘤。所述弥漫性大B细胞淋巴瘤(DLBCL)包括霍奇金淋巴瘤(HL)和非霍奇金淋巴瘤(NHL)。所述实体瘤包括肝癌、脑胶质瘤、胃癌、胰腺癌、肺癌、非小细胞肺癌、肾细胞癌、前列腺癌、骨肉瘤、乳腺癌、结直肠癌、卵巢癌、黑色素瘤、神经母细胞瘤、血管母细胞瘤、间变性脑膜瘤、复发性胶质母细胞瘤或分化型甲状腺癌。所述自身免疫性疾病包括寻常型天疱疹、系统性红斑狼疮(SLE)、血友病、重症肌无力、移植组织器官引发的免疫排斥反应、I型糖尿病(T1D)、类风湿关节炎、系统性硬化症、特发性肺纤维化、克罗恩病或结肠炎。所述免疫系统过度激活引发的疾病包括细胞因子释放综合征(CRS)、免疫效应细胞相关神经毒性综合征(ICANS)或移植物抗宿主病(GVHD)。所述代谢性疾病包括动脉粥样硬化、先天性高胰岛素血症、非酒精性脂肪肝炎或非肥胖糖尿病(NOD)。所述纤维化包括心肌纤维化、肥厚性心肌病、新冠肺炎、新冠心肌炎、缺血性心肌病、肝硬化、肝纤维化、原发性胆管炎、肺纤维化、骨骼肌纤维化、皮肤类疾病、骨髓纤维化、结节病或杜氏肌营养不良症。所述皮肤类疾病包括瘢痕疙瘩或伤口愈合。
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靶向药物。例如，使用本领域众所周知的方法将标记直接或间接与靶向药物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，与靶向药物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，a joint 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。
与CD8或CD3作为靶点相比，CD62L作为靶向免疫细胞药物靶点可使药物的表达量更高，更利于疾病的治疗。
随后的实施例提供举例说明，但不限定被要求的本申请。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或相似的结果。
本申请的药物-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)，与靶向结构连接后得到靶向药物。靶向药物中除去药物后，剩下的成分中包括靶向载体。
金属(含有Fe3+、Al3+或Mg2+)-磷脂复合物颗粒制备(Metal-chelated phospholipid complex nanoparticles，MPP)
实施例一、制备药物-金属-磷脂复合物颗粒
实施例1.1、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1.2、制备金属-磷脂复合物
实施例1.2.1分步法制备金属离子为Fe3+时的金属-磷脂复合物
实施例1.1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，随后加入与磷脂复合物摩尔比1：1的三乙胺，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1.2.2分步法制备金属离子为Al3+时的金属-磷脂复合物
本实施例与实施例1.2.1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1.2.3分步法制备金属离子为Mg2+时的金属-磷脂复合物
本实施例与实施例1.2.1的区别在于，FeCl3替换为MgCl2。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MgCl2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MgCl2的投料比为1：1时获得的目标产物的产率为95.5％。
实施例1.2.4、“一步法”制备金属-磷脂复合物
将具有磷酸基团的磷脂分子与连接物分子、金属离子进行连接：将二硬脂酰基磷脂酰胆碱(DSPC，式46)和姜黄素(式19)、FeCl3按照摩尔比1：1：1加入反应瓶中，加入适量乙醇溶解，于50±10℃反应2h后，得到金属-磷脂复合物。该“一步法”反应所得Fe3+金属-磷脂复合物目标产物的产率为95.6％。也可将金属离子由Fe3+换为Mg2+、Al3+、Ca2+，反应温度均为50±10℃，反应时长均为1-5h，优选2h。
金属离子为Fe3+时“一步法”与分步法制备金属-磷脂复合物性能对比分析如下：
将具有磷酸基团的磷脂分子与连接物分子、金属离子进行连接：将二硬脂酰基磷脂酰胆碱(DSPC，式46)和姜黄素、Al(NO3)3·9H2O按照摩尔比1：1：1加入反应瓶中，加入适量乙醇溶解，于50±10℃反应2h后，得到金属-磷脂复合物。该“一步法”反应所得Al3+金属-磷脂复合物目标产物的产率为95.6％。
金属离子为Al3+时“一步法”与分步法制备金属-磷脂复合物性能对比分析如下：
将具有磷酸基团的磷脂分子与连接物分子、金属离子进行连接：将二硬脂酰基磷脂酰胆碱(DSPC，式46)和姜黄素、CaCl2按照摩尔比1：1：1加入反应瓶中，加入适量乙醇溶解，于50±10℃反应2h后，得到金属-磷脂复合物。该“一步法”反应所得Ca2+金属-磷脂复合物目标产物的产率为95％。其金属离子Ca2+的mRNA-金属-磷脂复合物颗粒分步法制备方案的参照金属离子Fe3+的mRNA-金属-磷脂复合物颗粒分步法制备方案，制备金属离子为Ca2+的mRNA-金属-磷脂复合物颗粒，仅将金属离子由Fe3+替换为Ca2+，使用CaCl2替换FeCl3，其余不变。磷脂复合物与CaCl2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CaCl2的投料比为1：1时获得的目标产物金属离子为Ca2+时的金属-磷脂复合物的产率为95％。
金属离子为Ca2+时“一步法”与分步法制备金属-磷脂复合物性能对比分析如下：
将具有磷酸基团的磷脂分子与连接物分子、金属离子进行连接：将二硬脂酰基磷脂酰胆碱(DSPC，式46)和姜黄素、MgCl2按照摩尔比1：1：1加入反应瓶中，加入适量乙醇溶解，于50±10℃反应2h后，得到金属-磷脂复合物。该“一步法”反应所得Mg2+金属-磷脂复合物目标产物的产率为95％。
金属离子为Mg2+时“一步法”与分步法制备金属-磷脂复合物性能对比分析如下：
在金属-磷脂复合物的制备工艺中，“一步法”与分步法制备出的金属-磷脂复合物的特性与成效。两种方法包载率数值相近，在保障复合物中金属有效包载方面都有着稳定的表现。在eGFP阳性表达量上，“一步法”与分步法二者的数据也相近，证明了“一步法”与分步法对于金属-磷脂复合物的构建能力相当。然而，“一步法”能够显著缩短产业化进程所需的时间。在工业化大规模生产的场景下，时间效率至关重要，“一步法”减少了分步操作所带来的流程衔接、时间间隔等问题，极大地提高了生产的连续性与紧凑性，从而有效降低生产成本、提高生产效率。从产业化应用的角度来看，相较于分步法，“一步法”更契合大规模生产的需求，具有广阔的应用前景与极高的实用价值。
实施例1.3、制备金属离子为Fe3+的mRNA-金属-磷脂复合物颗粒(mRNA-loaded metal-chelated phospholipid complex nanoparticles`，mRNA@MPP)
按照实施例1.2.1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磷脂酰胆碱(DSPC，作为非阳离子脂质或非可电离脂质)、胆固醇(CHOL，作为非阳离子脂质或非可电离脂质)、DSPE-PEG2000(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PBS(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)中作为水相。以金属磷脂复合物、二硬脂酰磷脂酰胆碱(DSPC)、DSPE-PEG2000、胆固醇(CHOL)的总质量与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。
实施例1.4、制备金属离子为Al3+的mRNA-金属-磷脂复合物颗粒(mRNA-loaded metal-chelated phospholipid complex nanoparticles`，mRNA@MPP)
按照实施例1.2.2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磷脂酰胆碱(DSPC，作为非阳离子脂质或非可电离脂质)、胆固醇(CHOL，作为非阳离子脂质或非可电离脂质)、DSPE-PEG2000(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PBS(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)中作为水相。以金属磷脂复合物、二硬脂酰磷脂酰胆碱(DSPC)、DSPE-PEG2000、胆固醇(CHOL)的总质量与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。
实施例1.5、制备金属离子为Mg2+的mRNA-金属-磷脂复合物颗粒(mRNA-loaded metal-chelated phospholipid complex nanoparticles`，mRNA@MPP)
按照实施例1.2.4中的一步法制备金属-磷脂复合物，其中DSPC、姜黄素、MgCl2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磷脂酰胆碱(DSPC，作为非阳离子脂质或非可电离脂质)、胆固醇(CHOL，作为非阳离子脂质或非可电离脂质)、DSPE-PEG2000(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％、40％、43.2％、1.8％。mRNA按20μg/mL的浓度溶于PBS(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)中作为水相。以金属磷脂复合物、二硬脂酰磷脂酰胆碱(DSPC)、DSPE-PEG2000、胆固醇(CHOL)的总质量与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1.3-1.5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50～400nm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，脂质与核酸的质量比为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。
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+、Ca2+、Al3+或Mg2+相连，形成了金属-磷脂复合物，该金属-磷脂复合物的Fe3+、Ca2+、Al3+或Mg2+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MPP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1.3.1、金属离子为Fe3+时金属-磷脂复合物的组分的投放比例
将实施例1.3中的DSPC、姜黄素、FeCl3按照不同的摩尔投放比例(1:1:1、3:3:2、2:2:1)投放，其他步骤同实施例1.3，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的投放摩尔比例为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87％；当DSPC、姜黄素、FeCl3的投放摩尔比例为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70％；当DSPC、姜黄素、FeCl3的投放摩尔比例为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60％。在金属-磷脂复合物颗粒中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-脂质颗粒中的投放摩尔比例应该为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、FeCl3的投放摩尔比例为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、FeCl3的投放摩尔比例从1:1:1到2:2:1范围内，其金属-磷脂复合物颗粒的核酸包载率均在60％以上。
表1-1金属离子为Fe3+时金属-磷脂复合物的组分投放摩尔比例及其制备的金属-磷脂复合物颗粒的功能
实施例1.4.1、金属离子为Al3+时金属-磷脂复合物的组分的投放比例
将实施例1.4中的DSPC、姜黄素、Al(NO3)3·9H2O按照不同的投放摩尔比例(1:1:1、3:3:2、2:2:1)投放，其他步骤同实施例1.4，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的投放摩尔比例为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92％；当DSPC、姜黄素、Al(NO3)3·9H2O的投放摩尔比例为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的投放摩尔比例为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-脂质颗粒中的投放摩尔比例应该为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的投放摩尔比例为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的投放摩尔比例从1:1:1到2:2:1范围内，其金属-磷脂复合物颗粒的核酸包载率均在58％以上。
表1-2金属离子为Al3+时金属-磷脂复合物的组分投放摩尔比例及其制备的金属-磷脂复合物颗粒的功能
实施例1.5.1、金属离子为Mg2+时金属-磷脂复合物的组分的投放比例
将实施例1.5中的DSPC、姜黄素、MgCl2按照不同的投放摩尔比例(1:1:1、3:3:2、2:2:1)投放，其他步骤同实施例1.5，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1-3所示，当DSPC、姜黄素、MgCl2的投放摩尔比例为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93.9％；当DSPC、姜黄素、MgCl2的投放摩尔比例为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72.6％。在金属-磷脂复合物颗粒中MgCl2的功能是连接磷脂复合物与核酸，每个MgCl2最多有2个络合位点，所以DSPC、姜黄素、MgCl2在药物-脂质颗粒中的投放摩尔比例应该为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、MgCl2的投放摩尔比例为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、MgCl2的投放摩尔比例从1:1:1到3:3:2范围内，其金属-磷脂复合物颗粒的核酸包载率均在72.6％以上。
表1-3金属离子为Mg2+时金属-磷脂复合物的组分投放摩尔比例及其制备的金属-磷脂复合物颗粒的功能
实施例1.3.2、金属离子为Fe3+时制备金属-磷脂复合物颗粒中金属-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1.3相比，以金属-磷脂复合物(金属离子为Fe3+)、二硬脂酰磷脂酰胆碱(DSPC)、DSPE-PEG2000、胆固醇(CHOL)的比例如表1-4所示，其余条件相同。
结果分析：如结果表1-4所示，当金属-磷脂复合物占比在(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，金属-磷脂复合物颗粒的粒径在50～400nm范围、表面电势在-10～10mV范围、体外稳定性>3天、mRNA包载率>50％，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，金属-磷脂复合物颗粒的性能最优，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1-4金属离子为Fe3+时不同组分比例的eGFP-mRNA@MPP性能检测
实施例1.4.2、金属离子为Al3+时制备金属-磷脂复合物颗粒中金属-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1.4相比，以金属-磷脂复合物(金属离子为Al3+)、二硬脂酰磷脂酰胆碱(DSPC)、DSPE-PEG2000、胆固醇(CHOL)的比例如表1-5所示，其余条件相同。
结果分析：如结果表1-5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1-5金属离子为Al3+时不同组分比例的eGFP-mRNA@MPP(Al3+)性能检测
实施例1.5.2、金属离子为Mg2+时制备金属-磷脂复合物颗粒中金属-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1.5相比，以金属-磷脂复合物(金属离子为Mg2+)、二硬脂酰磷脂酰胆碱(DSPC)、DSPE-PEG2000、胆固醇(CHOL)的比例如表1-6所示，其余条件相同。
结果分析：如结果表1-6所示，当金属-磷脂复合物占比在(5-40)％范围时、DSPC占比在(0-50)％范围时、CHOL在(25-75)％范围时，DSPE-PEG2000占比在(1-10)％范围时，金属-磷脂复合物颗粒的粒径在50～400nm范围、表面电势在-10～10mV范围、体外稳定性>3天、mRNA包载率>50％，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40％、胆固醇(CHOL)占比43.2％及DSPE-PEG2000占比1.8％时，金属-磷脂复合物颗粒的性能最优，即粒径在115nm范围、表面电势在-2.9mV范围、体外稳定性>7天、mRNA包载率为93.9％，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0-50％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1-10％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-磷脂复合物占比在(5-40)％范围时、DSPC占比在(0-50)％范围时、CHOL占比在(25-75)％范围时，DSPE-PEG2000占比在(1-10)％范围时，mRNA@MPP具备较好的载药性能。
表1-6金属离子为Mg2+时不同组分比例的eGFP-mRNA@MPP性能检测

实施例1.3.3、制备eGFP-mRNA@MPP中非阳离子脂质或非可电离脂质种类
实施例1.3.3.1制备eGFP-mRNA@MPP(Fe3+)中非阳离子脂质或非可电离脂质种类
与实施例1.3相比，二硬脂酰磷脂酰胆碱(DSPC)的替代如表1-7所示，其余条件相同。
结果分析：为探索eGFP-mRNA@MPP(Fe3+)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(Fe3+)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(Fe3+)的功效(表1-7)。因为非阳离子脂质或非可电离脂质DSPC在eGFP-mRNA@MPP(Fe3+)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。
DSPE(式47)
DSPA(式48)
DSPG(式49)
表1-7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

实施例1.3.3.2、制备eGFP-mRNA@MPP(Al3+)中非阳离子脂质或非可电离脂质种类
与实施例1.4相比，二硬脂酰磷脂酰胆碱(DSPC)的替代如表1-8所示，其余条件相同。
结果分析：为探索eGFP-mRNA@MPP(Al3+)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(式47)、DSPA(式48)及DSPG(式49)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(Al3+)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(Al3+)的功效表1-8(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(Al3+)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。
表1-8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1.3.3.3、制备eGFP-mRNA@MPP(Mg2+)中非阳离子脂质或非可电离脂质种类
与实施例1.5相比，二硬脂酰磷脂酰胆碱(DSPC)的替代如表1-9所示，其余条件相同。
结果分析：为探索eGFP-mRNA@MPP(Mg2+)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(式47)、DSPA(式48)及DSPG(式49)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(Mg2+)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(Mg2+)的功效(表1-9)。因为非阳离子脂质或非可电离脂质DSPC在eGFP-mRNA@MPP(Mg2+)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。
表1-9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(Mg2+)的性能
实施例1.3.4、制备eGFP-mRNA@MPP中抑制颗粒聚集的缀合的脂质种类
实施例1.3.4.1、制备eGFP-mRNA@MPP(Fe3+)中抑制颗粒聚集的缀合的脂质种类
与实施例1.3相比，DSPE-PEG2000(式53)的替代如表1-10(金属离子为Fe3+)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(Fe3+)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(Fe3+)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(Fe3+)的功效(表1-10)。因为DSPE-PEG2000在eGFP-mRNA@MPP(Fe3+)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(Fe3+)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1-10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
实施例1.3.4.2、制备eGFP-mRNA@MPP(Al3+)中抑制颗粒聚集的缀合的脂质种类
与实施例1.4相比，DSPE-PEG2000(式53)的替代如表1-11(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(Al3+)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(Al3+)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(Al3+)的功效(表1-11)。因为DSPE-PEG2000在eGFP-mRNA@MPP(Al3+)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(Al3+)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1-11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1.3.4.3、制备eGFP-mRNA@MPP(Mg2+)中抑制颗粒聚集的缀合的脂质种类
与实施例1.5相比，DSPE-PEG2000(式53)的替代如表1-12(金属离子为Mg2+)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(Mg2+)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(Mg2+)的功效(表1-12)。因为DSPE-PEG2000在eGFP-mRNA@MPP(Mg2+)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(Mg2+)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1-12含有不同种类抑制颗粒聚集的缀合的脂质的eGFP-mRNA@MPP(Mg2+)的性能
实施例1.3.5、mRNA@MPP的制备及效果表征
实施例1.3.5.1、mRNA@MPP(Fe3+)的制备及效果表征
将实施例1.3中的mRNA替换为其他两种mRNA，参照实施例1.3的方法分别制备三种含不同目的蛋白mRNA序列的mRNA@MPP(Fe3+)。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)-金属-磷脂复合物颗粒的制备过程与实施例1.3相同，分别得到eGFP-mRNA@MPP(Fe3+)、RBD-mRNA@MPP(Fe3+)、NY-ESO-1-mRNA@MPP(Fe3+)。
将eGFP-mRNA@MPP(Fe3+)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：全血样品于室温放置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(Fe3+)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(Fe3+)，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(Fe3+)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(Fe3+)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(Fe3+)，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(Fe3+)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％H2O2 0.15mL。
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(Fe3+)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(Fe3+)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(Fe3+)实验组eGFP阳性细胞率为97.7％，而MPP(Fe3+)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(Fe3+)的293T细胞上清RBD蛋白含量为0。结果提示，mRNA-MPP(Fe3+)可以包载、递送任意mRNA，并在细胞内直接编码多肽。如图1-3，图1-4结果所示，RBD-mRNA@MPP(Fe3+)和NY-ESO-1-mRNA@MPP(Fe3+)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(Fe3+)处理组小鼠体内IgG抗体滴度达117268.8；NY-ESO-1-mRNA@MPP(Fe3+)处理组小鼠IgG抗体滴度达5319.52。如图1-5，图1-6所示，RBD-mRNA@MPP(Fe3+)和NY-ESO-1-mRNA@MPP(Fe3+)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(Fe3+)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(Fe3+)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(Fe3+)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1.3.5.2、金属离子为Al3+的mRNA@MPP的制备及效果表征
将实施例1.4中的mRNA替换为其他两种mRNA，参照实施例1.4的方法分别制备三种含不同目的蛋白mRNA序列的mRNA@MPP(Al3+)。具体mRNA信息和实验操作参照实施例1.3.5.1。
结果分析：如图1-1-2所示，eGFP-mRNA@MPP(Al3+)实验组eGFP阳性细胞率为98.02％，而MPP(Al3+)对照组未检测到eGFP信号；如图1-2-2所示，MPP(Al3+)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(Al3+)可以包载、递送任意mRNA，并在细胞内直接编码多肽。如图1-3-2，图1-4-2结果所示，RBD-mRNA@MPP(Al3+)和NY-ESO-1-mRNA@MPP(Al3+)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(Al3+)处理组小鼠体内IgG抗体滴度达129113；NY-ESO-1-mRNA@MPP(Al3+)处理组小鼠IgG抗体滴度达6507.4。如图1-5-2，图1-6-2所示，RBD-mRNA@MPP(Al3+)和NY-ESO-1-mRNA@MPP(Al3+)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(Al3+)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(Al3+)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(Al3+)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1.3.5.3、金属离子为Mg2+的mRNA@MPP的制备及效果表征
将实施例1.5中的mRNA替换为其他两种mRNA，参照实施例1.5的方法分别制备三种含不同目的蛋白mRNA序列的mRNA@MPP(Mg2+)。具体mRNA信息和实验操作参照实施例1.3.5.1。
结果分析：如图1-1-3所示，eGFP-mRNA@MPP(Mg2+)实验组eGFP阳性细胞率为98.3％，而MPP(Mg2+)对照组未检测到eGFP信号；如图1-2-3所示，MPP(Mg2+)包载的RBD-mRNA所编码的RBD蛋白在293T细胞上清中为218.50ng/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(Mg2+)可以包载、递送任意mRNA，并在细胞内直接编码多肽。如图1-3-3，图1-4-3结果所示，RBD-mRNA@MPP(Mg2+)和NY-ESO-1-mRNA@MPP(Mg2+)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(Mg2+)处理组小鼠体内IgG抗体滴度达130614.40；NY-ESO-1-mRNA@MPP(Mg2+)处理组小鼠IgG抗体滴度达6842.78。如图1-5-3，图1-6-3所示，RBD-mRNA@MPP(Mg2+)和NY-ESO-1-mRNA@MPP(Mg2+)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(Mg2+)使细胞因子IFN-γ、IL-2、IL-4的表达量分别达296.40pg/mL、243.33pg/mL、75.30pg/mL；NY-ESO-1-mRNA@MPP(Mg2+)使细胞因子IFN-γ、IL-2、TNF-α的表达量分别达85.35pg/mL、98.58pg/mL、98.02pg/mL。结果提示，mRNA@MPP(Mg2+)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1.3.6、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1.3.6.1、金属离为Fe3+时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1.3中的mRNA替换为siRNA，参照实施例1.3的方法分别制备三种含不同siRNA的siRNA@MPP(Fe3+)。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1.3相同。
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(Fe3+)(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(Fe3+)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 ScriptTM RT 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1-7(图中“scr siRNA”为“Scramble siRNA”，作为阴性对照，下同)，图1-8，图1-9所示，三种siRNA@MPP(Fe3+)均能明显干扰其对应的靶基因。Bcl-2-siRNA@MPP(Fe3+)对靶基因Bcl-2的抑制率达到76％；PLK1-siRNA@MPP(Fe3+)对靶基因PLK1的抑制率达到86％；Gal-1-siRNA@MPP(Fe3+)对靶基因Gal-1的抑制率达到73％。结果提示，siRNA@MPP(Fe3+)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1.3.6.2、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
将实施例1.4中的mRNA替换为siRNA，参照实施例1.4的方法分别制备三种含不同siRNA序列的siRNA@MPP(Al3+)。具体siRNA信息和实验操作参照实施例1.3.6.1。
结果分析：如图1-7-2，图1-8-2，图1-9-2所示，三种siRNA@MPP(Al3+)均能明显干扰其对应的靶基因。Bcl-2-siRNA@MPP(Al3+)对靶基因Bcl-2的抑制率达到81％；PLK1-siRNA@MPP(Al3+)对靶基因PLK1的抑制率达到90％；Gal-1-siRNA@MPP(Al3+)对靶基因Gal-1的抑制率达到79％。结果提示，siRNA@MPP(Al3+)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1.3.6.3、金属离子为Mg2+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1.5中的mRNA替换为siRNA，参照实施例1.5的方法分别制备三种含不同siRNA序列的siRNA@MPP(Mg2+)。具体siRNA信息和实验操作参照实施例1.3.6.1。
结果分析：如图1-7-3，图1-8-3，图1-9-3所示，三种siRNA@MPP(Mg2+)均能明显干扰其对应的靶基因。Bcl-2-siRNA@MPP(Mg2+)对靶基因Bcl-2的抑制率达到82％；PLK1-siRNA@MPP(Mg2+)对靶基因PLK1的抑制率达到92％；Gal-1-siRNA@MPP(Mg2+)对靶基因Gal-1的抑制率达到86％。结果提示，siRNA@MPP(Mg2+)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1.3.7、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1.3.7.1、金属离子为Fe3+时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1.3中的mRNA替换为ASO，参照实施例1.3的方法分别制备三种含不同ASO的ASO@MPP(Fe3+)。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1.3相同。将不同的ASO@MPP(Fe3+)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(Fe3+)孵育SH-SY5Y人神经母细胞瘤细胞；靶向Bcl-2基因的ASO@MPP(Fe3+)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(Fe3+)(其中ASO的浓度为2μg/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(Fe3+)沉默细胞靶基因的能力。
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 ScriptTM RT 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1-10，图1-11，图1-12所示，三种ASO@MPP(Fe3+)均能明显干扰其对应的靶基因，其中STAT3-ASO@MPP(Fe3+)对靶基因STAT3的抑制率达到77％；α-syn-ASO@MPP(Fe3+)对靶基因α-syn的抑制率达到75％；Bcl-2-ASO@MPP(Fe3+)对靶基因Bcl-2的抑制率达到69％。结果提示，ASO@MPP(Fe3+)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1.3.7.2、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
将实施例1.4中的mRNA替换为ASO，参照实施例1.4的方法分别制备三种含不同ASO序列的ASO@MPP(Al3+)。具体ASO信息和实验操作参照实施例1.3.7.1。
结果分析：如图1-10-2，图1-11-2，图1-12-2所示，三种ASO@MPP(Al3+)均能明显干扰其对应的靶基因，其中STAT3-ASO@MPP(Al3+)对靶基因STAT3的抑制率达到79％；α-syn-ASO@MPP(Al3+)对靶基因α-syn的抑制率达到80％；Bcl-2-ASO@MPP(Al3+)对靶基因Bcl-2的抑制率达到74％。结果提示，ASO@MPP(Al3+)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1.3.7.3、金属离子为Mg2+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1.5中的mRNA替换为ASO，参照实施例1.5的方法分别制备三种含不同ASO序列的ASO@MPP(Mg2+)。具体ASO信息和实验操作参照实施例1.3.7.1。
结果分析：如图1-10-3，图1-11-3，图1-12-3所示，三种ASO@MPP(Mg2+)均能明显干扰其对应的靶基因，其中STAT3-ASO@MPP(Mg2+)对靶基因STAT3的抑制率达到82％；α-syn-ASO@MPP(Mg2+)对靶基因α-syn的抑制率达到84％；Bcl-2-ASO@MPP(Mg2+)对靶基因Bcl-2的抑制率达到78％。结果提示，ASO@MPP(Mg2+)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1.3.8、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1.3.8.1、金属离子为Fe3+时制备药物(不同种类核酸)-金属-磷脂复合物颗粒及其效果
将实施例1.3中的mRNA分别替换为双链的RNA(siRNA)、单链的RNA(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RNA(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1.3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(Fe3+)、STAT3-ASO@MPP(Fe3+)、S-mRNA@MPP(Fe3+)、dsDNA@MPP(Fe3+)、ssDNA@MPP(Fe3+))，其余药物-脂质颗粒的制备过程与实施例1.3相同。
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(Fe3+)(其中siRNA的浓度为2μg/mL)或ASO@MPP(Fe3+)(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(Fe3+)或ASO@MPP(Fe3+)沉默细胞靶基因的能力，结果如实施例1.3.6中图1-7及实施例1.3.7中图1-10所示。
将S-mRNA@MPP(Fe3+)按2μg/mL的浓度(所含mRNA的浓度)与293T细胞孵育，对照组用MPP(Fe3+)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1-13所示。
将ds-DNA@MPP(Fe3+)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1-14。
ss-DNA@MPP(Fe3+)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1-14。
人脑胶质母细胞瘤U251细胞的培养方法同实施例1.3.6。
293T细胞的培养方法同实施例1.3.5。
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1.3.6。
ELISA检测S蛋白的表达水平：将实施例1.3.5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1.3.5。
基因沉默效率的计算方法同实施例1.3.6。
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(结果如图1-15)，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1.3.6中图1-7所示，药物(双链RNA)-金属-磷脂复合物颗粒(Bcl-2-siRNA@MPP(Fe3+))对靶基因Bcl-2的抑制率达到76％；如实施例1.3.7中图1-10药物(单链RNA)-金属-磷脂复合物颗粒(STAT3-ASO@MPP(Fe3+))对靶基因STAT3的抑制率达到77％；如图1-13所示，转染了药物(单链RNA)-金属-磷脂复合物颗粒(S-mRNA@MPP(Fe3+))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(Fe3+))转染双链DNA入细胞的效率为100％(图1-14)；药物(单链DNA)-金属-磷脂复合物颗粒(ssDNA@MPP(Fe3+))转染单链DNA入细胞的效率为100％(图1-14)。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1.3.8.2、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
将实施例1.4中的mRNA分别替换为双链的RNA(siRNA)、单链的RNA(ASO)、单链的RNA(mRNA)、双链DNA、单链DNA。具体序列和实验操作参照实施例1.3.8.1。
结果分析：如实施例1.3.6.2中图1-7-2所示，药物(双链RNA)-金属-磷脂复合物颗粒(Bcl-2-siRNA@MPP(Al3+))对靶基因Bcl-2的抑制率达到81％；如实施例1.3.7.2中图1-10-2药物(单链DNA)-金属-磷脂复合物颗粒(STAT3-ASO@MPP(Al3+))对靶基因STAT3的抑制率达到79％；如图1-13-2所示，转染了药物(单链RNA)-金属-磷脂复合物颗粒(S-mRNA@MPP(Al3+))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(Al3+))转染双链DNA入细胞的效率为100％(图1-14-2)；药物(单链DNA)-金属-磷脂复合物颗粒(ssDNA@MPP(Al3+))转染单链DNA入细胞的效率为100％(图1-14-2)。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1.3.8.3、制备金属离子为Mg2+时药物(不同种类核酸)-金属-磷脂复合物颗粒及其效果
将实施例1.5中的mRNA分别替换为双链的RNA(siRNA)、单链的RNA(ASO)、单链的RNA(mRNA)、双链DNA、单链DNA。具体序列和实验操作参照实施例1.3.8.1。
结果分析：如实施例1.5.6中图1-7-3所示，药物(双链RNA)-金属-磷脂复合物颗粒(Bcl-2-siRNA@MPP(Mg2+))对靶基因Bcl-2的抑制率达到82％；如实施例1.5.7中图1-11-3药物(单链RNA)-金属-磷脂复合物颗粒(STAT3-ASO@MPP(Mg2+)))对靶基因STAT3的抑制率达到82％；如图1-13-3所示，转染了药物(单链RNA)-金属-磷脂复合物颗粒(S-mRNA@MPP(Mg2+)))的293T细胞的上清中S蛋白表达水平为181.2ng/mL，而转染了空载体MPP(Mg2+))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(Mg2+)))转染双链DNA入细胞的效率为100％(图1-14-3)；药物(单链DNA)-金属-磷脂复合物颗粒(ssDNA@MPP(Mg2+)))转染单链DNA入细胞的效率为100％(图1-14-3)。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。
实施例二、药物-金属-磷脂复合物颗粒的性能表征
实施例2.1、金属-磷脂复合物的合成表征
实施例2.1.1、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(curcumin)、DSPC、磷脂复合物(curcumin-DSPC complex)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-DSPC)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2.1.2、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2.1.1的区别在于，磷脂复合物与Al3+连接表征为分光光度法：如图2-2-2所示，磷脂复合物(CUR-DSPC)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2.1.3、金属离子为Mg2+时的金属-磷脂复合物的合成表征
本实施例与实施例2.1.1的区别在于，磷脂复合物与Mg2+连接表征为分光光度法：如图2-2-3所示，磷脂复合物(CUR-DSPC)与Mg2+结合后，最大吸收峰偏移，其最大吸收波长从426nm偏移至420nm，磷脂复合物的共轭结构发生改变，证明Mg2+成功与姜黄素络合。
实施例2.2、金属离子为Fe3+时在低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2-3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2-3)。
实施例2.3、金属离子为Fe3+时药物-金属-磷脂复合物颗粒中MPP的元素分析
将实施例1.3中的mRNA替换为巯基修饰的siRNA，参照实施例1.3的方法制备药物-金属-磷脂复合物颗粒siRNA@MPP(Fe3+)。用透射电镜仪器进行元素分析。结果如图2-4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2.4、金属离子为Fe3+、Al3+或Mg2+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1.3.5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1.3.5相同。
按实施例1.3.6中siRNA@MPP及实施例1.3.5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2-5所示，MPP(Fe3+)包载siRNA及mRNA的效率分别为90.11％及89.78％；MPP(Al3+)包载siRNA及mRNA的效率分别为92.81％及91.48％；MPP(Mg2+)包载siRNA及mRNA的效率分别为93.69％及92.02％；LNP包载siRNA及mRNA的效率分别为89.02％及89.36％。结果提示MPP与LNP包载核酸的效率无明显差异。
实施例2.5、金属离子为Fe3+、Al3+或Mg2+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1.3.6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2.4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1.3.5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2.4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(金属离子为Fe3+的结果如图2-11所示)，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2-6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39.11％和43.44％，即溶酶体逃逸率分别为60.89％和56.56％；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33.48％和40.15％，即溶酶体逃逸率分别为66.52％和59.85％；当Cy5-siRNA@MPP(Mg2+)和Cy5-mRNA@MPP(Mg2+)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54.86％和57.36％，即溶酶体逃逸率分别为45.14％和42.64％；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77.69％和84.69％，即溶酶体逃逸能力分别为22.31％和15.31％。提示所述药物-脂质纳米颗粒(Fe3+、Al3+或Mg2+)MPP具有较好的促核酸溶酶体逃逸能力，且(Fe3+、Al3+或Mg2+)MPP促溶酶体逃逸的能力明显强于LNP。
实施例2.6、金属离子为Fe3+、Al3+或Mg2+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2.4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2.4，获得eGFP-mRNA@LNP。
将实施例1.3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1.3所述。
结果分析：如图2-7所示，MPP(Fe3+)、MPP(Al3+)或MPP(Mg2+)与LNP处理293T细胞后其eGFP阳性细胞百分比分别为97.7％、98.02％、98.3％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2.5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。目前使用eGFP阳性细胞百分比的方法对不同金属离子(Fe3+、Al3+或Mg2+)组成的MPP(如MPP(Fe3+)、MPP(Al3+)或MPP(Mg2+)进行核酸表达能力的表征，因为eGFP蛋白本身的性能不足与区分细微的差别，因此未能区分出金属离子(Fe3+、Al3+或Mg2+)组成的MPP(如MPP(Fe3+)、MPP(Al3+)或MPP(Mg2+))之间促核酸表达能力的差异。
实施例2.7、金属离子为Fe3+、Al3+或Mg2+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1.3.5的RBD-mRNA@MPP和实施例2.4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2-8。
ELISA检测RBD表达水平的方法如实施例1.3.5所述。
将实验动物被随机分成2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和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2-9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1.3.5所述。
在给RBD-mRNA@MPP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2-10所示。
ELISA检测IFN-γ、IL-2、IL-4表达水平的方法如实施例1.3.5所述。
结果分析：如图2-8所示，RBD-mRNA@MPP(Fe3+)、RBD-mRNA@MPP(Al3+)、RBD-mRNA@MPP(Mg2+)和RBD-mRNA@LNP均能诱导293T细胞表达一定量的RBD，但RBD-mRNA@MPP(Mg2+)诱导细胞表达RBD的能力明显强于RBD-mRNA@MPP(Al3+)、RBD-mRNA@MPP(Fe3+)，三者的诱导细胞表达RBD的能力又明显强于RBD-mRNA@LNP：RBD-mRNA@MPP(Fe3+)处理组细胞上清中RBD的表达量为218.93ng/mL，RBD-mRNA@MPP(Al3+)处理组细胞上清中RBD的表达量为239.93ng/mL，RBD-mRNA@MPP(Mg2+)处理组细胞上清中RBD的表达量为303.63ng/mL，RBD-mRNA@LNP处理组细胞上清中RBD的表达量为126.67ng/mL。如图2-9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(Mg2+)诱导小鼠的体液免疫的能力明显优于RBD-mRNA@MPP(Al3+)、RBD-mRNA@MPP(Fe3+)，三者诱导小鼠的体液免疫的能力又明显优于RBD-mRNA@LNP：RBD-mRNA@MPP(Mg2+)处理组小鼠体内IgG抗体滴度达171782.00；RBD-mRNA@MPP(Fe3+)处理组小鼠体内IgG抗体滴度达122666.67；RBD-mRNA@MPP(Al3+)处理组小鼠体内IgG抗体滴度达134833.33；而RBD-mRNA@LNP处理组小鼠体内IgG抗体滴度达仅为73694.00。如图2-10所示，RBD-mRNA@MPP(Mg2+)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RBD-mRNA@MPP(Mg2+)诱导小鼠的细胞免疫的能力明显优于RBD-mRNA@MPP(Fe3+)、RBD-mRNA@MPP(Al3+)，RBD-mRNA@MPP(Fe3+)诱导小鼠的细胞免疫的能力明显优于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286.2pg/mL、209.67pg/mL、58.02pg/mL；RBD-mRNA@MPP(Al3+)使细胞因子IFN-γ、IL-2、IL-4的表达量分别达306pg/mL、239.67pg/mL、71pg/mL；RBD-mRNA@MPP(Mg2+)使细胞因子IFN-γ、IL-2、IL-4的表达量分别达333pg/mL、270.33pg/mL、73pg/mL；而RBD-mRNA@LNP使细胞因子IFN-γ、IL-2、IL-4的表达量仅为95pg/mL、75.67pg/mL、24pg/mL。结果提示，mRNA@MPP(Mg2+)递送任意mRNA并实现其功能的能力明显优于RBD-mRNA@MPP(Fe3+)、mRNA@MPP(Al3+)，RBD-mRNA@MPP(Fe3+)诱导小鼠的细胞免疫的能力明显优于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。
与不同金属离子组成的MPP(例如MPP(Fe3+)、MPP(Al3+))相比，MPP(Mg2+)能更有效的促进细胞表达目的蛋白，其原因是：Mg2+与RNA保持弱螯合作用的Mg2+可增强RNA的功能，包括增加其热力学稳定性、化学稳定性和催化性(Biochemistry.2021Aug10；60(31):2374-2386.)，Mg2+可以保持核糖体结构稳定性和翻译活性，部分补充核糖体蛋白的功能(Biosci Biotechnol Biochem.2021Jun 24；85(7):1582-1593.)增强免疫反应。Mg2+可以通过诱导CD8+T细胞表面共刺激分子LFA-1的构象变化调节LFA-1下游信号传导，进而促进T细胞的活化和细胞毒性作用(Cell.2022Feb 17；185(4):585-602.e29.)。
实施例2.8、金属离子为Fe3+、Al3+或Mg2+的药物-金属-磷脂复合物颗粒MPP的表达持续时间与LNP的对比
制备CD19 CAR mRNA：
CAR mRNA包括跨膜结构域、信号传导结构域、抗原结合结构域、共刺激信号传导区、抗原结合结构域与跨膜结构域连接区。
信号传导结构域，signal peptide(SP)：帮助T细胞内表达的CAR导到T细胞膜上。
抗原结合结构域，scFV：编码FMC63抗体的VH和VL部分，FMC63抗体的VH和VL两者用linker连接，可识别肿瘤细胞抗原。
抗原结合结构域与跨膜结构域连接区，spacer：将抗原结合结构域scFV和跨膜结构域transmembrane连接，其灵活性可以保证scFV更好的识别抗原。
跨膜结构域，transmembrane(TM)：将CAR表达的这些组合固定在T细胞膜上。
共刺激信号传导区，intracellular：用于激活T细胞。激活信号主要由CD3-zeta负责，可加带其它共刺激域CD28、CD8，比如41BB来增强信号。共刺激信号传导区可包含多个共刺激结构域，不管加多少共刺激域,CD3-zeta都要放在最后，CD28共刺激域，可有效增强T细胞增殖。
CD19 CAR mRNA表达的氨基酸序列，如SEQ ID NO.60所示。
参照实施例2.4，将mRNA替换为CD19 CAR mRNA，制备CD19 CAR mRNA@MPP、CD19 CAR mRNA@LNP。按照上述方法分别静脉给药后，用量流式细胞仪方法检测髓系细胞中的CAR阳性细胞占比。流式细胞术方法分析CAR阳性细胞百分比的方法：将外周血与Alexa标记的CAR Linker抗体和CD11b-PE抗体于4℃下共孵育30分钟，孵育结束后加入红细胞裂解液避光裂解5～10分钟。Buffer洗涤两次后加100～30000μL Buffer重悬。于流式仪上机检测髓系细胞中CAR阳性细胞百分比。计算公式为：髓系细胞中CAR阳性细胞率计算公式＝表达CAR的髓系细胞数/髓系细胞总数×100％。参照上述方法，对CD19CAR-mRNA@LNP进行检测。
结果如图8-1所示，CD19 CAR mRNA@MPP(Mg2+)的髓系细胞中CAR阳性细胞占比最高，且其持续的时间明显长于CD19 CAR mRNA@MPP(Al3+)，CD19 CAR mRNA@MPP(Al3+)优于CD19 CAR mRNA@MPP(Fe3+)，CD19 CAR mRNA@MPP(Fe3+)优于LNP，提示MPP(Mg2+)具有更强的表达及更优的表达持续时间长功能。
实施例2.9、金属离子为Fe3+、Al3+或Mg2+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(25mg/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(50mg/kg)14只(含实验组8只和恢复组6只)，雌雄各半；高剂量组(100mg/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25mg/kg、50mg/kg、100mg/kg的MPP。
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未发现明显的慢性毒性反应，提示MPP的安全性较高。
表2-1MPP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2.10、金属离子为Fe3+、Al3+或Mg2+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。
DOTAP结构式：
ALC0315结构式：
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(MPP(Fe3+)、MPP(Al3+)、MPP(Mg2+))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+)、MPP(Al3+)、MPP(Mg2+)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+)或MPP(Mg2+)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+)、MPP(Al3+)、MPP(Mg2+)的安全性高于LNP。
表2-2金属离子为Fe3+、Al3+或Mg2+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2-3MPP及LNP的慢性毒性实验指标对比
表2-4金属的安全性历史数据
参考：《中国居民膳食营养素参考摄入量》-2023版、《中国居民膳食铝暴露风险评估》、ICH协调指导原则-元素杂质指导原则Q3D(R2)。
以上历史数据表明，基于各种金属制备的MPP或Apt-MPP的安全性排序为：Mg>Fe>Zn>Al>Mn>Cr。提示不仅MPP(Mg2+)的药效最优，其安全性也最优。
实施例三、金属离子为Fe3+、Al3+或Mg2+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1.3.5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1.3.5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、RBD-mRNA@MPP)，其余药物-金属-磷脂复合物颗粒的制备过程与实施例1.3.5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)分别用于治疗胶质瘤及作为mRNA疫苗用于预防新型冠状病毒。
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1.3.5所示。
ELISA检测方法如实施例1.3.5所述。
为评价B7-H4-siRNA@MPP(Fe3+)和B7-H4-siRNA@MPP(Al3+)、B7-H4-siRNA@MPP(Mg2+)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10组(每组5只)：PBS对照组、空白载体MPP(Fe3+)组、空白载体MPP(Al3+)组、空白载体MPP(Mg2+)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、Scr-siRNA@MPP(Mg2+)对照组、B7-H4-siRNA@MPP(Mg2+)治疗组。每组小鼠每3天分别瘤内注射PBS、MPP(Fe3+)、MPP(Al3+)、MPP(Mg2+)、Scr-siRNA@MPP(Fe3+)、B7-H4siRNA@MPP(Fe3+)、Scr-siRNA@MPP(Al3+)、B7-H4 siRNA@MPP(Al3+)、Scr-siRNA@MPP(Mg2+)、B7-H4-siRNA@MPP(Mg2+)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、图3-2和图3-3所示。
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、图3-2和图3-3所示，Scr-siRNA@MPP(Fe3+)、Scr-siRNA@MPP(Al3+)、Scr-siRNA@MPP(Mg2+)对肝癌HepG2细胞的生长几乎没有抑制效果，而B7-H4-siRNA@MPP(Fe3+)、B7-H4 siRNA@MPP(Al3+)和B7-H4 siRNA@MPP(Mg2+)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1.3.5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1-3-2)，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1-5-2)。RBD-mRNA@MPP(Mg2+)使小鼠IgG抗体的表达水平达130614.40(图1-3-3)，使细胞因子IFN-γ、IL-2、IL-4的表达量分别达296.40pg/mL、243.33pg/mL、75.30pg/mL(图1-5-3)。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。
B7-H4-siRNA@MPP采用瘤内注射给药途径能有效治疗肝癌；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。
与其他金属离子组成的MPP(例如MPP(Fe3+)、MPP(Al3+))相比，MPP(Mg2+)能更有效抑制肝癌的生长和更有效的预防新型冠状病毒的感染，其原因是：Mg2+与RNA保持弱螯合作用的Mg2+可增强RNA的功能，包括增加其热力学稳定性、化学稳定性和催化性(Biochemistry.2021Aug 10；60(31):2374-2386.)Mg2+可以保持核糖体结构稳定性和翻译活性，部分补充核糖体蛋白的功能(Biosci Biotechnol Biochem.2021Jun 24；85(7):1582-1593.)增强免疫反应。Mg2+可以通过诱导CD8+T细胞表面共刺激分子LFA-1的构象变化调节LFA-1下游信号传导，进而促进T细胞的活化和细胞毒性作用(Cell.2022Feb17；185(4):585-602.e29.)Mg2+作为RNA诱导沉默复合体(RISC)中Ago蛋白的酶辅基参与催化，可增强siRNA的RNAi效应(Cell Rep.2022Oct 25；41(4):111533)Mg2+作为Rnase H1的酶辅基参与催化，可增强ASO的基因沉默效应(DNA Repair(Amst).2019Dec；84:102672.)。
实施例四：DSPC、姜黄素、Fe3+、Al3+或Mg2+被同类物替换后的功能
实施例4.1、DSPC、姜黄素、Fe3+被同类物替换后的功能
参照实施例1.1、实施例1.2及实施例1.3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37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，37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1.1中的反应温度为65℃、反应时间为2h，实施例1.2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37种不同的eGFP-mRNA@MPP与eGFP-mRNA@LNP的效果，我们参照实施例2.4制备了包载等量eGFP-mRNA的LNP，得到eGFP-mRNA@LNP。
将上述37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1.3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37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37种MPP的毒性的差异。
IC50的计算方法如实施例2.10所述。
结果分析：如表4-2所示，37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，37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4-1DSPC、姜黄素、Fe3+及其同类物的名称及结构
表4-2DSPC、姜黄素、Fe3+及其同类物制备的药物-脂质颗粒中金属-磷脂复合物的组成组合方式列表及功能

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

实施例4.2、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1.3，制备mRNA-金属-磷脂复合物颗粒，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mRNA-金属-磷脂复合物颗粒(mRNA@MPP1、mRNA@MPP4、mRNA@MPP38)。制备这三种mRNA-金属-磷脂复合物颗粒时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。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1.3.5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及其同类物与姜黄素及其同类物靠氢键相连，只要DSPC及其同类物含有多个磷酸基团，那么合成磷脂复合物时，DSPC及其同类物和姜黄素及其同类物的投放比例可以依据DSPC及其同类物所含磷酸基团的个数进行调整，即当DSPC及其同类物含两个磷酸基团时，DSPC及其同类物和姜黄素及其同类物的投放比例可调整为1：2；当DSPC及其同类物含三个磷酸基团时，DSPC及其同类物和姜黄素及其同类物的投放比例可调整为1：3；因为姜黄素及其同类物的羟基与Fe3+及其同类物靠配位键连接，只要姜黄素及其同类物含有多个结合位点，那么姜黄素及其同类物和Fe3+及其同类物的投放比例可以依据姜黄素及其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4.3、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1.4，制备mRNA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@MPP39)。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4.4、不同金属-磷脂复合物的DSPC、姜黄素及其同类物、Mg2+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1.5，制备mRNA-金属-磷脂复合物颗粒，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mRNA-金属-磷脂复合物颗粒(mRNA@MPP29、mRNA@MPP30、mRNA@MPP40)。制备这三种mRNA-金属-磷脂复合物颗粒时DSPC、姜黄素或其同类物、MgCl2的投放比例分别为：1:1:1，1:1:1，1:1:2。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1.3.5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4-6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及其同类物与姜黄素及其同类物靠氢键相连，只要DSPC及其同类物含有多个磷酸基团，那么合成磷脂复合物时，DSPC及其同类物和姜黄素及其同类物的投放比例可以依据DSPC及其同类物所含磷酸基团的个数进行调整，即当DSPC及其同类物含两个磷酸基团时，DSPC及其同类物和姜黄素及其同类物的投放比例可调整为1：2；当DSPC及其同类物含三个磷酸基团时，DSPC及其同类物和姜黄素及其同类物的投放比例可调整为1：3；因为姜黄素及其同类物的羟基与Mg2+及其同类物靠配位键连接，只要姜黄素及其同类物含有多个结合位点，那么姜黄素及其同类物和Mg2+及其同类物的投放比例可以依据姜黄素及其同类物所含结合位点的数量进行调整。
表4-6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
实施例五：靶向MPP载体构建
实施例5.1制备靶向结构DSPE-PEG2000-Apt胶束
在Apt上修饰二硫键制得Apt-C6-S-S-C6，其中Apt-C6-S-S-C6为二硫键两边各修饰六碳链，其中一端与Apt相连。首先将Apt-C6-S-S-C6在梯度PCR仪上进行变复性，变复性流程为95℃、5分钟，37℃、15分钟。第二步，将Apt-C6-S-S-C6在10mM膦盐酸盐(TCEP)中25℃孵育1小时，使二硫键断裂暴露出巯基，即Apt-C6-S-S-C6发生还原反应生成Apt-C6-SH。第三步，将Apt-C6-SH与DSPE-PEG2000-MAL以原料摩尔比1：100在室温下反应4小时，使马来酰胺与巯基充分反应生成DSPE-PEG2000-Apt，未反应的DSPE-PEG2000-MAL通过超滤法去除，超滤法为使用10kD截留分子量超滤管反复超滤3-6次。其中Apt的核苷酸序列为
5’-TAGCCAAGGTAACCAGTACAAGGTGCTAAACGTAATGGCTTCGGCTTAC-3’(如SEQ ID NO.44所示)，Apt为靶向结合区，DSPE为疏水区，PEG2000为连接区。
接下来，采用薄膜水化法将DSPE-PEG2000-Apt制备成胶束，具体步骤如下。将DSPE-PEG2000-Apt溶解在乙醇中，在一定的真空度下旋转蒸发30分钟形成均匀薄膜，再将其放置在4℃冰箱中过夜使乙醇完全挥发。最后使用无酶水在60℃条件下将薄膜水化30分钟，即制得DSPE-PEG2000-Apt胶束。
DSPE-PEG2000-Apt的制备方法中也可以将中间体DSPE-PEG2000-MAL和Apt-C6-SH更改，包括但不限于DSPE-PEG2000-NHS和Apt-NH2，DSPE-PEG2000-COOH和Apt-NH2，DSPE-PEG2000-NCO和Apt-NH2以及DSPE-PEG2000-N3和Apt-DBCO等，最终目的在于制备得到DSPE-PEG2000-Apt。
将DSPE-PEG2000-Apt制备成胶束的方法可以更改，包括但不限于直接溶解法，乙醇注入法，透析法以及超声法等，最终目的在于得到DSPE-PEG2000-Apt胶束。
实施例5.2.制备靶向载体(Apt-MPP)
将实施例5.1制备的DSPE-PEG2000-Apt胶束与实施例1.3至1.5中制备的包裹mRNA的金属-磷脂复合物颗粒MPP按照不同的的反应投料质量比(1:84、1:42、1:21、1:16.8、1:14)混合均匀，在4℃(可使用反应温度为0℃±10℃)孵育2h(可使用孵育时间为0.2-12h)。因不同的反应投料质量比改变Apt在金属-磷脂复合物颗粒MPP上的分布比例，其得到的不同修饰率的靶向载体(Apt-MPP)的理化性质不变。MPP使DSPE-PEG2000-Apt胶束通过疏水作用插入金属-磷脂复合物颗粒MPP外表面，得到Apt-金属-磷脂复合物颗粒，即为靶向载体(Apt-MPP)。Apt本身为亲水性，可与金属离子发生吸附，若直接使用DSPE-PEG2000-Apt替换DSPE-PEG2000参与金属-磷脂复合物颗粒制备，则Apt会被包裹在金属-磷脂复合物颗粒内部，无法发挥Apt的靶向性作用。
实施例六：靶向药物构建
实施例6.1.设计CD19 CAR mRNA
CAR mRNA包括跨膜结构域、信号传导结构域、抗原结合结构域、共刺激信号传导区、抗原结合结构域与跨膜结构域连接区。
信号传导结构域，signal peptide(SP)：帮助T细胞内表达的CAR导到T细胞膜上。
抗原结合结构域，scFV：编码FMC63抗体的VH和VL部分，FMC63抗体的VH和VL两者用linker连接，可识别肿瘤细胞抗原。
抗原结合结构域与跨膜结构域连接区，spacer：将抗原结合结构域scFV和跨膜结构域transmembrane连接，其灵活性可以保证scFV更好的识别抗原。
跨膜结构域，transmembrane(TM)：将CAR表达的这些组合固定在T细胞膜上。
共刺激信号传导区，intracellular：用于激活T细胞。激活信号主要由CD3-zeta负责，可加带其它共刺激域CD28、CD8，比如41BB来增强信号。共刺激信号传导区可包含多个共刺激结构域，不管加多少共刺激域,CD3-zeta都要放在最后，CD28共刺激域，可有效增强T细胞增殖。
>CD19 CAR mRNA表达的氨基酸序列，如SEQ ID NO.60所示。
(signal-P)：CD8前导序列(信号肽)的氨基酸序列如SEQ ID NO.45所示。
FMC63_VL的氨基酸序列如SEQ ID NO.46所示。
linker的氨基酸序列如SEQ ID NO.47所示。
FMC63_VH的氨基酸序列如SEQ ID NO.48所示。
抗CD19scFv的氨基酸序列如SEQ ID NO.49所示。
CD8铰链区的氨基酸序列如SEQ ID NO.50所示。
CD28共刺激域的氨基酸序列如SEQ ID NO.51所示。
CD3ζ胞内信号传导域的氨基酸序列如SEQ NO.ID 52所示。
>CD19 CAR的DNA序列如SEQ ID NO.53所示。
以上DNA序列分区：
1、CD8前导序列(信号肽)DNA序列如SEQ ID NO.54所示。
2、抗CD19scFv的DNA序列如SEQ ID NO.55所示。
3、CD8绞合区的DNA序列如SEQ ID NO.56所示。
4、CD8跨膜区的DNA序列如SEQ ID NO.57所示。
5、CD28的DNA序列如SEQ ID NO.58所示。
6、CD3ζ的DNA序列如SEQ ID NO.59所示。
CD19 CAR-mRNA的序列如SEQ ID NO.43所示。
其中，跨膜结构域可选自CD8跨膜域、FcεRIα(FCER1A)跨膜域、CD28跨膜域中的至少一种或多种。
CD8跨膜域：
氨基酸序列如SEQ ID NO.61所示。
核苷酸序列如SEQ ID NO.62所示。
mRNA序列如SEQ ID NO.28所示。
FcεRIα(FCER1A)跨膜域：
氨基酸序列如SEQ ID NO.63所示。
核苷酸序列如SEQ ID NO.64所示。
mRNA序列如SEQ ID NO.29所示。
CD28跨膜域：
氨基酸序列如SEQ ID NO.95所示。
核苷酸序列如SEQ ID NO.96所示。
mRNA序列如SEQ ID NO.30所示。
信号传导结构域可选自CD8前导序列、鼠Kappα链前导序列、FcεRIγ前导序列中的至少一种或多种。
CD8前导序列：
氨基酸序列如SEQ ID NO.45所示。
核苷酸序列如SEQ ID NO.54所示。
mRNA序列如SEQ ID NO.31所示。
鼠Kappα链前导序列：
氨基酸序列如SEQ ID NO.65所示。
核苷酸序列如SEQ ID NO.66所示。
mRNA序列如SEQ ID NO.32所示。
FcεRIγ前导序列：
氨基酸序列如SEQ ID NO.67所示。
核苷酸序列如SEQ ID NO.68所示。
mRNA序列如SEQ ID NO.33所示。
抗原结合结构域可选自抗CD19scFv、抗EGFR scFv、抗HER2scFv中的一种或多种。
抗CD19scFv：
DNA序列如SEQ ID NO.55所示。
mRNA序列如SEQ ID NO.34所示。
抗EGFR scFv：
氨基酸序列如SEQ ID NO.69所示。
DNA序列如SEQ ID NO.70所示。
mRNA序列如SEQ ID NO.71所示。
抗HER2scFv：
氨基酸序列如SEQ ID NO.72所示。
DNA序列如SEQ ID NO.73所示：。
mRNA序列如SEQ ID NO.36所示。
共刺激信号传导区可选自CD28共刺激域、OX40共刺激域、41BB共刺激域中的一种或多种。
CD28共刺激域：
氨基酸序列如SEQ ID NO.51所示。
DNA序列如SEQ ID NO.58所示。
mRNA序列如SEQ ID NO.37所示。
OX40共刺激域：
氨基酸序列如SEQ ID NO.38所示。
DNA序列如SEQ ID NO.74所示。
mRNA序列如SEQ ID NO.75所示。
41BB共刺激域：
氨基酸序列如SEQ ID NO.76所示。
DNA序列如SEQ ID NO.77所示。
mRNA序列如SEQ ID NO.39所示。
抗原结合结构域与跨膜结构域连接区可选自IgG4铰链、CD8α铰链、FcγRIIIα(FCGR3A)铰链中的一种或多种。
IgG4铰链：
氨基酸序列如SEQ ID NO.78所示。
DNA序列如SEQ ID NO.79所示。
mRNA序列如SEQ ID NO.42所示。
CD8α铰链
氨基酸序列如SEQ ID NO.80所示。
DNA序列如SEQ ID NO.81所示。
mRNA序列如SEQ ID NO.40所示。
FcγRIIIα(FCGR3A)铰链：
氨基酸序列如SEQ ID NO.82所示。
DNA序列如SEQ ID NO.83所示。
mRNA序列如SEQ ID NO.41所示。
实施例6.2靶向药物(CD19 CAR mRNA@Apt-MPP)、药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)制备
实施例6.2.1基于金属离子Fe3+的靶向药物(CD19 CAR mRNA@Apt-MPP)、药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)制备
使用实施例6.1中的CD19 CAR mRNA参照实施例1.3制备药物(CD19 CAR mRNA)-基于金属离子Fe3+的金属-磷脂复合物颗粒(CD19 CAR mRNA@MPP)，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60μg/mL的浓度溶于0.1M的PBS(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、二硬脂酰磷脂酰胆碱(DSPC)、DSPE-PEG2000、胆固醇(CHOL)的总质量与mRNA质量按100:1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。
然后参照实施例5.2制备基于金属离子Fe3+的靶向药物(CD19 CAR mRNA@Apt-MPP)。将实施例5.1中制备得到的DSPE-PEG2000-Apt胶束与上述CD19CAR mRNA@MPP按照1：16.8的反应投料质量比混合均匀，在4℃(可使用的反应温度为0℃±10℃)孵育2h(可使用的孵育时间为0.2-12h)，使DSPE-PEG2000-Apt胶束通过疏水作用插入CD19 CAR mRNA@MPP中制得CD19 CAR mRNA@Apt-MPP。
实施例6.2.2基于金属离子Al3+的靶向药物(CD19 CAR mRNA@Apt-MPP)、药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)制备
使用实施例6.1中的CD19 CAR mRNA参照实施例1.4制备药物(CD19 CAR mRNA)-基于金属离子Al3+的金属-磷脂复合物颗粒(CD19 CAR mRNA@MPP)，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60μg/mL的浓度溶于0.1M的PBS(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、二硬脂酰磷脂酰胆碱(DSPC)、DSPE-PEG2000、胆固醇(CHOL)的总质量与mRNA质量按100:1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。
然后参照实施例5.2制备基于金属离子Al3+的靶向药物(CD19 CAR mRNA@Apt-MPP)。将实施例5.1中制备得到的DSPE-PEG2000-Apt胶束与上述CD19CAR mRNA@MPP按照1：16.8的反应投料质量比混合均匀，在4℃(0℃±10℃)孵育2h(0.2-12h)，使DSPE-PEG2000-Apt胶束通过疏水作用插入CD19 CAR mRNA@MPP中制得CD19 CAR mRNA@Apt-MPP。
实施例6.2.3基于金属离子Mg2+的靶向药物(CD19 CAR mRNA@Apt-MPP)、药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)制备
使用实施例6.1中的CD19 CAR mRNA参照实施例1.5制备药物(CD19 CAR mRNA)-金属离子Mg2+的金属-磷脂复合物颗粒(CD19 CAR mRNA@MPP)，其中金属-磷脂复合物、DSPC、CHOL及DSPE-PEG2000的占比分别为15％、40％、43.2％、1.8％。mRNA按60μg/mL的浓度溶于0.1M的PBS(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、二硬脂酰磷脂酰胆碱(DSPC)、DSPE-PEG2000、胆固醇(CHOL)的总质量与mRNA质量按100:1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。
然后参照实施例5.2制备金属离子Mg2+的靶向药物(CD19 CAR mRNA@Apt-MPP)。将实施例5.1中制备得到的DSPE-PEG2000-Apt胶束与上述CD19 CAR mRNA@MPP按照不同的反应投料质量比(1:84(表示靶头比例1)、1:42(表示靶头比例2)、1:21(表示靶头比例3)、1:16.8(表示靶头比例4)、1:14(表示靶头比例5)，靶头在本实施例中指Apt，也可替换为其他具有靶向性的抗体、小分子探针、多肽等。)混合均匀，在4℃(0℃±10℃)孵育2h(0.2-12h)，使DSPE-PEG2000-Apt胶束通过疏水作用插入CD19 CAR mRNA@MPP中制得CD19 CAR mRNA@Apt-MPP。本实施例中，靶头代指具体的靶向结合区。
实施例6.3靶向载体(Apt-MPP)、金属-磷脂颗粒(MPP)、靶向药物(CD19CAR mRNA@Apt-MPP)、药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)的靶向性对比
将实施例6.2中制备的靶向载体(Apt-MPP)、金属-磷脂颗粒(MPP)以15μg/mL的浓度与1×106个人T细胞(购自远泰生物)孵育，48小时后收细胞悬液，使用Alexa标记的CAR Linker抗体检测细胞CD19-CAR阳性细胞占比。流式细胞术方法分析如下所述：流式细胞术方法分析CAR阳性细胞百分比的方法：将T细胞接种于12孔板上，接种密度为4×105个细胞/孔，加入1mL MPP或CAR-mRNA@MPP孵育细胞，其中CAR-mRNA@MPP浓度为15μg/mL。48h后收集细胞悬液，与CAR Linker抗体于4℃共孵育40分钟，Buffer洗涤后去上清，于流式仪上机检测CAR阳性细胞百分比。计算公式为：CAR阳性细胞率计算公式＝表达CAR细胞数/细胞总数×100％；T细胞中CAR阳性细胞率计算公式＝表达CAR的T细胞数/T细胞总数×100％。未进行Apt修饰的药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)作为对照。
如图4-1所示，经流式细胞术检测得到MPP(Fe3+)和Apt-MPP(Fe3+)组的CD19-CAR阳性细胞占比分别为2.12±1.32和9.40±2.00。该结果表明人T细胞对Apt-MPP的摄取优于MPP。Apt-MPP表面修饰的Apt能与人T细胞进行受体配体识别从而增加细胞摄取，降低了MPP因缺乏靶向性而产生的副作用，安全性得到了提升。
如图4-1-2所示，经流式细胞术检测得到MPP(Al3+)和Apt-MPP(Al3+)组的CD19-CAR阳性细胞占比分别为2.16±1.58和10.77±3.60。该结果表明人T细胞对Apt-MPP的摄取优于MPP。Apt-MPP表面修饰的Apt能与人T细胞进行受体配体识别从而增加细胞摄取，降低了MPP因缺乏靶向性而产生的副作用，安全性得到了提升。
如图4-1-3所示，经流式细胞术检测得到MPP(Mg2+)和Apt-MPP(Mg2+)组的CD19-CAR阳性细胞占比分别为3.38±1.13和15.6±4.42。该结果表明人T细胞对Apt-MPP的摄取优于MPP。Apt-MPP表面修饰的Apt能与人T细胞进行受体配体识别从而增加细胞摄取，降低了MPP因缺乏靶向性而产生的副作用，安全性得到了提升。
实施例6.4靶向药物(CD19 CAR mRNA@Apt-MPP)、药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)对急性B淋巴白血病小鼠的治疗效果
根据实施例6.2制备获得药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)以及靶向药物(CD19 CAR mRNA@Apt-MPP)。在人源化的急性B淋巴白血病模型小鼠，将靶向药物(以mRNA的质量计算30μg/只)通过尾静脉注射，每5天注射一次，共给药三次。
人源化急性B淋巴白血病模型小鼠的建立方法如下：将PBMC重悬后，以1×106个细胞/只剂量尾静脉注射至小鼠体内。待PBMC接种约15～24天，PBMC人源化程度均值为10-20％时，将PBS重悬后Raji-Luc肿瘤细胞以浓度为5×105个细胞/mL，200μL/只，尾静脉注射接种于小鼠体内(记为D0)。待Raji-Luc肿瘤细胞接种5～7天后，确定人源化急性B淋巴白血病小鼠模型造模成功后开始给药(记为D5～7)。
造模成功后，每五天对实验动物进行一次注射底物以及活体成像，以其荧光强度表示白血病的进展情况。
如图4-2(Fe3+)、图4-2-2(Al3+)和图4-2-3(Mg2+)所示，在药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)以及靶向药物(CD19 CAR mRNA@Apt-MPP)分别进行三次给药后，靶向药物(CD19 CAR mRNA@Apt-MPP)组小鼠肿瘤的平均荧光强度低于药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)。该结果提示：靶向药物(CD19 CAR mRNA@Apt-MPP)对急性B淋巴白血病的治疗效果优于药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)。其可能的原因是：靶向药物(CD19 CAR mRNA@Apt-MPP)表面修饰的Apt能与人T细胞进行受体配体识别并增加摄取，从而具有更多的CD19-CAR表达，具有更好的治疗效果。
之后对实验组以及对照组小鼠的生存时间进行了统计，评估了药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)以及靶向药物(CD19 CAR mRNA@Apt-MPP)对急性B淋巴白血病小鼠生存期的影响。
如图7-1(Fe3+，CD62L)、图7-1-2(Al3+，CD62L)和图7-1-3(Mg2+，CD62L)所示，第0天、第5天、第10天为给药时间点，在药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)以及靶向药物(CD19 CAR mRNA@Apt-MPP)分别进行三次给药后，靶向药物(CD19 CAR mRNA@Apt-MPP)组小鼠肿瘤的生存期长于药物-金属-磷脂颗粒(CD19CAR mRNA@MPP)。该结果表明靶向药物(CD19 CAR mRNA@Apt-MPP)对急性B淋巴白血病小鼠生存期的延长优于药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)。靶向药物(CD19 CAR mRNA@Apt-MPP)表面修饰的Apt能与人T细胞进行受体配体识别并增加摄取，从而具有更多的CD19-CAR表达，具有更好的治疗效果。
实施例6.5CD19 CAR mRNA@Apt-MPP和CD19 CAR mRNA@Apt-LNP的CAR阳性率及表达持续时间长能力对比
根据实施例6.2制备获得靶向药物(CD19 CAR mRNA@Apt-MPP)，并参照实施例6.2将MPP替换为LNP制备CD19 CAR mRNA@Apt-LNP。在人源化的急性B淋巴白血病模型小鼠，将靶向药物(CD19 CAR mRNA@Apt-MPP)(以mRNA的质量计算30μg/只)通过尾静脉注射，每5天注射一次，共给药三次，在最后一次给药后的第6h、24h、48h及72h取外周血，用流式细胞仪方法检测外周血T细胞或髓系细胞中的CAR阳性细胞占比。外周血T细胞中的CAR阳性细胞占比如图8-2所示；外周血髓系细胞中的CAR阳性细胞占比如图8-5所示；结果提示：CD19 CAR mRNA@Apt-MPP(Mg2+)组的T细胞CAR阳性细胞占比及髓系细胞CAR阳性细胞占均最高，且其持续的时间明显长于CD19CAR mRNA@Apt-MPP(Al3+)，CD19 CAR mRNA@Apt-MPP(Al3+)优于CD19 CAR mRNA@Apt-MPP(Fe3+)，CD19 CAR mRNA@Apt-MPP(Fe3+)优于Apt-LNP，提示Apt-MPP(Mg2+)具有更强的表达及更长的表达时间。
因CD62L在T细胞和髓系细胞均有较高的表达，所以靶向递送系统Apt-MPP能选择性编辑T细胞和髓系细胞。
实施例6.6基于金属离子Ca2+的不同靶头比例修饰的CD19 CAR mRNA@Apt-MPP和CD19 CAR mRNA@Apt-LNP的CAR阳性率及表达持续时间长能力对比：
参照实施例6.2制备获得靶向不同靶头修饰比例的药物(CD19 CAR mRNA@Apt-MPP，Ca2+)，并参照实施例6.2将MPP替换为LNP制备CD19 CAR mRNA@Apt-LNP。将实施例5.1中制备得到的DSPE-PEG2000-Apt胶束与上述CD19 CAR mRNA@MPP按照不同的反应投料质量比(1:84(表示靶头比例1)、1:42(表示靶头比例2)、1:21(表示靶头比例3)、1:16.8(表示靶头比例4)、1:14(表示靶头比例5)，靶头在本实施例中指Apt，也可替换为其他具有靶向性的抗体、小分子探针、多肽等。)混合均匀，在4℃(可使用的反应温度为0℃±10℃)，孵育2h(可使用的孵育时间为0.2-12h)，使DSPE-PEG2000-Apt胶束通过疏水作用插入CD19 CAR mRNA@MPP中制得CD19 CAR mRNA@Apt-MPP靶向比例1、CD19 CAR mRNA@Apt-MPP靶向比例2、CD19CAR mRNA@Apt-MPP靶向比例3、CD19 CAR mRNA@Apt-MPP靶向比例4、CD19 CAR mRNA@Apt-MPP靶向比例5；在人源化的急性B淋巴白血病模型小鼠，将不同靶向比例的靶向药物(以mRNA的质量计算30μg/只)通过尾静脉注射，每5天注射一次，共给药三次，在最后一次给药后的第6h、24h、48h及72h取外周血，用流式细胞仪方法检测外周血T细胞中的CAR阳性细胞占比。如图8-3所示，靶头比例4的靶向药物的CAR阳性T细胞占比最高，且其持续的时间最长，提示靶头比例4在体内实验中具有更强的表达及更优的表达持续时间长功能，因此具有最好的药效。
基于金属离子Mg2+的不同靶头比例修饰的CD19 CAR mRNA@Apt-MPP和CD19 CAR mRNA@Apt-LNP的CAR阳性率及表达持续时间长能力对比：
参照实施例6.2制备获得靶向不同靶头修饰比例的药物(CD19 CAR mRNA@Apt-MPP)，并参照实施例6.2将MPP替换为LNP制备CD19 CAR mRNA@Apt-LNP。将实施例5.1中制备得到的DSPE-PEG2000-Apt胶束与上述CD19 CAR mRNA@MPP按照不同的反应投料质量比(1:84(表示靶头比例1)、1:42(表示靶头比例2)、1:21(表示靶头比例3)、1:16.8(表示靶头比例4)、1:14(表示靶头比例5)，靶头在本实施例中指Apt，也可替换为其他具有靶向性的抗体、小分子探针、多肽等。)混合均匀，在4℃(可使用的反应温度为0℃±10℃)，孵育2h(可使用的孵育时间为0.2-12h)，使DSPE-PEG2000-Apt胶束通过疏水作用插入CD19 CAR mRNA@MPP中制得CD19 CAR mRNA@Apt-MPP靶向比例1、CD19 CAR mRNA@Apt-MPP靶向比例2、CD19 CAR mRNA@Apt-MPP靶向比例3、CD19 CAR mRNA@Apt-MPP靶向比例4、CD19 CAR mRNA@Apt-MPP靶向比例5；在人源化的急性B淋巴白血病模型小鼠，将不同靶向比例的靶向药物(以mRNA的质量计算30μg/只)通过尾静脉注射，每5天注射一次，共给药三次，在最后一次给药后的第6h、24h、48h及72h取外周血，用流式细胞仪方法检测外周血T细胞中的CAR阳性细胞占比。如图8-4所示，靶头比例4的靶向药物的CAR阳性细胞占比最高，且其持续的时间最长，提示靶头比例4在体内实验中具有更强的表达及更优的表达持续时间长功能，因此具有最好的药效。
实施例6.7靶向药物(CD19 CAR mRNA@Apt-MPP)、药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)、CD19 CAR mRNA@LNP、CD19 CAR mRNA@Apt-LNP、对系统性红斑狼疮的治疗效果
根据实施例6.2制备获得药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)以及靶向药物(CD19 CAR mRNA@Apt-MPP)，并参照实施例6.2将MPP替换为LNP制备CD19 CAR mRNA@LNP、CD19 CAR mRNA@Apt-LNP，将10μg(以mRNA的质量计算)的CD19 CAR mRNA@LNP、CD19 CAR mRNA@Apt-LNP、CD19 CAR mRNA@MPP和CD19 CAR mRNA@Apt-MPP分别与多种B细胞驱动的自身免疫性疾病(系统性红斑狼疮(SLE)/狼疮肾炎(LN)、类风湿性关节炎(RA)、干燥综合征(SS)、ANCA相关性血管炎(AAV)、重症肌无力(MG)、多发性硬化症(MS)、视神经脊髓炎谱系疾病(NMOSD))病人来源的外周血单个核细胞(PBMC)孵育72h后流式细胞术检测B细胞活率，减少的B细胞活率即为杀伤率。
B细胞杀伤率的检测方法为：将PBMC接种于24孔板上，接种密度为4×105个细胞/孔，加入受试物10μg与PBMC共孵育72h后，用7-氨基放线菌素D(7-AAD)、APC标记的CD3抗体和PE标记的CD19抗体在4℃黑暗环境下与PBMC孵育10分钟进行染色，流式细胞术检测B细胞活率。未加入受试物的PBMC组别作对照，检测B细胞活率。B细胞杀伤率的计算公式＝100％×(1-实验组B细胞活率)/对照组B细胞活率。
B细胞驱动的自身免疫性疾病是一类由自身抗体产生的疾病，这些抗体错误地攻击身体的正常组织，主要分三类：1.风湿病类，包括类风湿性关节炎(RA)、系统性硬化症/硬皮病(SSc)、干燥综合征(SS)、系统性红斑狼疮(SLE)和狼疮肾炎(LN)、特发性炎性肌病(IIM)、ANCA相关性血管炎(AAV)、系统性红斑狼疮相关免疫性血小板减少症(SLE-ITP)、系统性硬化症相关性间质性肺病(SSc-ILD)、特发性炎性肌病(IIM)包括抗合成酶抗体综合征(ASS)、皮肌炎(DM)、免疫介导坏死性肌病(IMNM)、多发性肌炎(PM)；2.神经病类，包括重症肌无力(MG)、多发性硬化症(MS)、视神经脊髓炎谱系疾病(NMOSD)、僵人综合征(SPS)、自身免疫性脑炎(例如抗NMDAR脑炎等)；3.皮肤病类，包括皮肤粘膜寻常型天疱疮(mcPV)。
目前尚无合适的动物模型来评估上述B细胞驱动的自身免疫性疾病的药效，原因是人源化动物建立自身免疫性疾病模型存在模型不稳定、病理指标不充分、不显著。因此，鉴于相关动物模型的缺陷和不足，多家公司均采用体外培养各种B细胞驱动的自身免疫性疾病病人的外周血单个核细胞(PBMC)，在体外检测受试物对其细胞B杀伤率，来评估药效。
表8-1不同药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)、靶向药物(CD19CAR mRNA@Apt-MPP)、CD19 CAR mRNA@LNP、CD19 CAR mRNA@Apt-LNP组B细胞杀伤率表
如表8-1所示，在药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)以及靶向药物(CD19 CAR mRNA@Apt-MPP)分别孵育病人PBMC后，靶向药物(CD19 CAR mRNA@Apt-MPP)组B细胞杀伤率明显高于药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)。该结果提示：靶向药物(CD19 CAR mRNA@Apt-MPP)对B细胞驱动性自身免疫性疾病的治疗效果优于药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)。其可能的原因是：靶向药物(CD19 CAR mRNA@Apt-MPP)表面修饰的Apt能与人T细胞进行受体配体识别并增加摄取，从而具有更多的CD19-CAR表达，具有更好的治疗效果。
而CD19 CAR mRNA@Apt-MPP(Mg2+)的效果优于CD19 CAR mRNA@Apt-MPP(Al3+)，CD19 CAR mRNA@Apt-MPP(Al3+)优于CD19 CAR mRNA@Apt-MPP(Fe3+)，CD19 CAR mRNA@Apt-MPP(Fe3+)优于LNP，其原因是Apt-MPP(Mg2+)具有更优的促核酸表达能力，及更优的表达持续时间长功能。
实施例七.靶向结构区替换
实施例7.1将特异性识别CD62L的Apt替换为特异性识别CD8的Apt,制备CD19 CAR mRNA@Apt(CD8)-MPP，CD19 CAR mRNA@Apt(CD8)-MPP与CD19 CAR mRNA@MPP对比抗急性B淋巴细胞性白血病的作用
将实施例6.2中的Apt替换为可特异性识别CD8的适配体，参照实施例6.2的方法制备CD19 CAR mRNA@Apt(CD8)-MPP。靶向CD8的Apt(CD8)序列如SEQ ID NO.84所示，其随机对照序列如SEQ ID NO.85所示。将靶向药物(CD19 CAR mRNA@Apt(CD8)-MPP)(以mRNA的质量计算10μg/只)通过尾静脉注射至急性B淋巴白血病模型小鼠，每五天注射一次并在打药前两小时注射与癌细胞数量相等的人T细胞，共给药三次。对照组则注射药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)，其他条件均与实验组相同。实验组与对照组小鼠数量均为五只。记录小鼠的生存时间，并统计生存率。
如图5-1(Fe3+)、图5-1-2(Al3+)和图5-1-3(Mg2+)所示，第0日、第5日、第10日为给药时间点，在药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)以及靶向药物(CD19 CAR mRNA@Apt(CD8)-MPP)分别进行三次给药后，靶向药物(CD19 CAR mRNA@Apt(CD8)-MPP)组小鼠肿瘤的生存期长于药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)。该结果表明靶向药物(CD19 CAR mRNA@Apt(CD8)-MPP)对急性B淋巴白血病小鼠生存期的延长优于药物-金属-磷脂颗粒(CD19 CAR mRNA@MPP)。靶向药物(CD19 CAR mRNA@Apt(CD8)-MPP)表面修饰的Apt能与人T细胞进行受体配体识别并增加摄取，从而具有更多的CD19-CAR表达，具有更好的治疗效果。
实施例7.2.靶向药物中核酸药物替换，Apt替换为AS1411、核酸药物为ASO制备ASO@Apt-MPP与ASO@MPP对比抗肺癌作用
将实施例6.2中的Apt替换为能识别癌细胞中高表达核仁素蛋白的AS1411，并将mRNA替换为ASO(序列如SEQ ID NO.86所示)，参照实施例6.2的方法制备ASO@Apt(AS1411)-MPP。靶向核仁素基因的Apt(AS1411)序列为ASO(序列如SEQ ID NO.86所示)，其随机对照序列为序列如SEQ ID NO.87所示，其余ASO-金属-磷脂复合物颗粒的制备过程与实施例1.3相同。
为评价ASO@Apt(AS1411)-MPP和ASO@MPP治疗肺癌的作用，用A549细胞制作肺癌动物模型，待肿瘤大小增加到约100mm3，将肺癌小鼠随机分为2组(每组5只)：ASO@Apt(AS1411)-MPP靶向治疗组和ASO@MPP对照组。每组小鼠每5天注射一次，共给药三次。记录小鼠的生存时间，并统计生存率。
肺癌小鼠模型的建立：收集将A549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，建立肺癌小鼠模型。
如图5-2(Fe3+)、图5-2-2(Al3+)和图5-2-3(Mg2+)所示，第0天、第5天、第10天为给药时间点，在药物-金属-磷脂颗粒(ASO@MPP)以及靶向药物(ASO@Apt-MPP)分别进行三次给药后，靶向药物(ASO@Apt-MPP)组小鼠肿瘤的生存期长于药物-金属-磷脂颗粒(ASO@MPP)。该结果表明靶向药物(ASO@Apt-MPP)对肺癌小鼠生存期的延长优于药物-金属-磷脂颗粒(ASO@MPP)。
实施例7.3Apt替换为可以特异性识别胰腺癌细胞的适配体P19,Kras siRNA制备siRNA@Apt-MPP、siRNA@MPP，siRNA@Apt-MPP与siRNA@MPP对比抗胰腺癌的作用。
将实施例6.2中的Apt替换为可特异性识别胰腺癌细胞的P19，并将实施例6.2中的mRNA替换为KRAS siRNA药物，参照实施例6.2的方法制备siRNA@Apt(P19)-MPP。靶向胰腺癌细胞的Apt(P19)序列如SEQ ID NO.88所示，其随机对照序列如SEQ ID NO.89所示。KRAS siRNA序列如SEQ ID NO.90所示、如SEQ ID NO.91所示。其随机对照序列为如SEQ ID NO.92所示'、如SEQ ID NO.93所示，其余siRNA-金属-磷脂复合物颗粒的制备过程与实施例1.3相同。
为评价siRNA@Apt(P19)-MPP和siRNA@MPP治疗肺癌的作用，用Panc-1细胞制作胰腺癌动物模型，待肿瘤大小增加到约100mm3，将肺癌小鼠随机分为2组(每组5只)：siRNA@Apt(P19)-MPP靶向治疗组和siRNA@MPP对照组。每组小鼠每5天注射一次，共给药三次。记录小鼠的生存时间，并统计生存率。
胰腺癌小鼠模型的建立：收集将Panc-1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，建立胰腺癌小鼠模型。
如图5-3(Fe3+)、图5-3-2(Al3+)和图5-3-3(Mg2+)所示，第0天、第5天、第10天为给药时间点，在药物-金属-磷脂颗粒(siRNA@MPP)以及靶向药物(siRNA@Apt-MPP)分别进行三次给药后，靶向药物(siRNA@Apt-MPP)组小鼠肿瘤的生存期长于药物-金属-磷脂颗粒(siRNA@MPP)。该结果表明靶向药物(siRNA@Apt-MPP)对胰腺癌小鼠生存期的延长优于药物-金属-磷脂颗粒(siRNA@MPP)。
实施例八Apt-MPP、Apt-LNP制备与检测
实施例8.1.CD19 CAR mRNA@Apt(CD62L)-LNP、ASO@Apt(AS1411)-LNP、siRNA@Apt(P19)-LNP制备
制备CD19 CAR mRNA：
CAR mRNA包括跨膜结构域、信号传导结构域、抗原结合结构域、共刺激信号传导区、抗原结合结构域与跨膜结构域连接区。
信号传导结构域，signal peptide(SP):帮助T细胞内表达的CAR导到T细胞膜上。
抗原结合结构域，scFV：编码FMC63抗体的VH和VL部分，FMC63抗体的VH和VL两者用linker连接，可识别肿瘤细胞抗原。
抗原结合结构域与跨膜结构域连接区，spacer：将抗原结合结构域scFV和跨膜结构域transmembrane连接，其灵活性可以保证scFV更好的识别抗原。
跨膜结构域，transmembrane(TM)：将CAR表达的这些组合固定在T细胞膜上。
共刺激信号传导区，intracellular：用于激活T细胞。激活信号主要由CD3-zeta负责，可加带其它共刺激域CD28、CD8，比如41BB来增强信号。共刺激信号传导区可包含多个共刺激结构域，不管加多少共刺激域，CD3-zeta都要放在最后，CD28共刺激域，可有效增强T细胞增殖。
>CD19 CAR mRNA表达的氨基酸序列，如SEQ ID NO.60所示。
制备CD19 CAR mRNA@LNP：
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CD19 CAR 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)，即制得CD19 CAR mRNA@LNP。
然后参照实施例5.2采用后插入法制备靶向药物(CD19 CAR mRNA@Apt-LNP)。将实施例5.1中制备得到得DSPE-PEG2000-Apt(CD62L)胶束，与CD19 CAR mRNA@LNP混合均匀，在60℃下反应10分钟，再在4℃下孵育过夜，使DSPE-PEG2000-Apt(CD62L)胶束插入CD19 CAR mRNA@LNP中制得CD19 CAR mRNA@Apt(CD62L)-LNP。
参照实施例1.3.7，将药物换为ASO制备药物ASO@LNP。然后参照实施例5.2采用后插入法制备靶向药物(ASO@Apt-LNP)。将实施例5.1中Apt换成Apt(AS1411)制备得到得DSPE-PEG2000-Apt(AS1411)胶束，与ASO@LNP混合均匀，在60℃下反应10分钟，再在4℃下孵育过夜，使DSPE-PEG2000-Apt(AS1411)胶束插入ASO@LNP中制得ASO@Apt(AS1411)-LNP。
参照实施例1.3.6，将药物换为siRNA制备药物siRNA@LNP。然后参照实施例5.2采用后插入法制备靶向药物(siRNA@Apt-LNP)。将实施例5.1中Apt换成Apt(P19)制备得到得DSPE-PEG2000-Apt(P19)胶束，与siRNA@LNP混合均匀，在60℃下反应10分钟，再在4℃下孵育过夜，使DSPE-PEG2000-Apt(P19)胶束插入siRNA@LNP中制得siRNA@Apt(P19)-LNP。
实施例8.2.CD19 CAR mRNA@Apt(CD62L)-LNP、ASO@Apt(AS1411)-LNP、siRNA@Apt(P19)-LNP动物存活率检测
参考实施例七(实施例7.1、实施例7.2和实施例7.3)的实验方法，将载体中的MPP替换为LNP，将药物分别换为CD19 CAR mRNA@Apt(CD62L)-LNP、ASO@Apt(AS1411)-LNP、siRNA@Apt(P19)-LNP，分别记录CD19 CAR mRNA@Apt(CD62L)-LNP治疗组的急性B淋巴白血病模型小鼠、ASO@Apt(AS1411)-LNP治疗组的肺癌小鼠和siRNA@Apt(P19)-LNP治疗组的胰腺癌小鼠的生存时间，并统计各自的生存率。
由实施例6.5、实施例6.6、实施例8.2的结果得到图6-1、图6-1-2、图6-1-3、图6-2、图6-2-2、图6-2-3、图6-3、图6-3-2、图6-3-3，由图6-1、图6-2、图6-3中结果可知，在相同Apt、相同药物、相同药量情况下，Apt-MPP性能优于Apt-LNP；Apt-MPP(Mg2+)性能优于Apt-MPP(Al3+)，Apt-MPP(Al3+)性能优于Apt-MPP(Fe3+)；进一步证明CD19 CAR mRNA@Apt(CD62L)-MPP(Mg2+)抗急性B淋巴细胞性白血病的效果最佳；ASO@Apt(AS1411)-MPP(Mg2+)抗肺癌的效果最佳；siRNA@Apt(P19)-MPP(Mg2+)抗胰腺癌的效果最佳。
Apt-MPP(Mg2+)性能和药效最佳的原因是：与RNA保持弱螯合作用的Mg2+可增强RNA的功能，包括增加其热力学稳定性、化学稳定性和催化性(Biochemistry.2021Aug 10；60(31):2374-2386.)；Mg2+可以保持核糖体结构稳定性和翻译活性，部分补充核糖体蛋白的功能(Biosci Biotechnol Biochem.2021 Jun 24；85(7):1582-1593.)；增强免疫反应。Mg2+可以通过诱导CD8+T细胞表面共刺激分子LFA-1的构象变化调节LFA-1下游信号传导，进而促进T细胞的活化和细胞毒性作用(Cell.2022 Feb 17；185(4):585-602.e29.)；Mg2+作为RNA诱导沉默复合体(RISC)中Ago蛋白的酶辅基参与催化，可增强siRNA的RNAi效应(Cell Rep.2022 Oct 25；41(4):111533)；Mg2+作为Rnase H1的酶辅基参与催化，可增强ASO的基因沉默效应(DNA Repair(Amst).2019 Dec；84:102672.)
其中，根据图8-1中结果显示，Apt-MPP的CAR表达阳性率的持续时间明显强于Apt-LNP，其中Apt-MPP(Mg2+)优于Apt-MPP(Al3+)，Apt-MPP(Al3+)优于Apt-MPP(Fe3+)；提示，Apt-MPP(Mg2+)的表达持续时间长能力较强，可能是其药效最优的重要原因。
实施例九Apt-MPP中替换不同Apt
实施例9.1、Apt替换为识别CD8蛋白的适配体
将实施例5.1中的Apt替换为可特异性识别CD8蛋白的适配体，参照实施例6.2的方法制备CD19 CAR mRNA@Apt(CD8)-MPP。靶向CD8的Apt(CD8)序列如SEQ ID NO.84所示：5’-CTACAGCTTGCTATGCTCCCCTTGGGGTA-3’。其余mRNA-金属-磷脂复合物颗粒的制备过程与实施例1.3至1.5相同。分别将靶向药物CD19 CAR mRNA@Apt(CD62L)-MPP和CD19 CAR mRNA@Apt(CD8)-MPP(以mRNA的质量计算10μg/只)通过尾静脉注射至急性B淋巴白血病模型小鼠，每五天注射一次并在打药前两小时注射与癌细胞数量相等的人T细胞，共给药三次。每组小鼠数量均为五只。记录小鼠的生存时间，并统计生存率。
如图7-1所示，CD19 CAR mRNA@Apt(CD62L)-MPP(Fe3+)组小鼠肿瘤的生存期长于CD19 CAR mRNA@Apt(CD8)-MPP(Fe3+)。该结果表明靶向药物CD19 CAR mRNA@Apt(CD62L)-MPP(Fe3+)对急性B淋巴白血病小鼠生存期的延长优于CD19 CAR mRNA@Apt(CD8)-MPP(Fe3+)。
如图7-1-2所示，CD19 CAR mRNA@Apt(CD62L)-MPP(Al3+)组小鼠肿瘤的生存期长于CD19 CAR mRNA@Apt(CD8)-MPP(Al3+)。该结果表明靶向药物CD19 CAR mRNA@Apt(CD62L)-MPP(Al3+)对急性B淋巴白血病小鼠生存期的延长优于CD19 CAR mRNA@Apt(CD8)-MPP。
如图7-1-3所示，CD19 CAR mRNA@Apt(CD62L)-MPP(Mg2+)组小鼠肿瘤的生存期长于CD19 CAR mRNA@Apt(CD8)-MPP(Mg2+)。该结果表明靶向药物CD19 CAR mRNA@Apt(CD62L)-MPP(Mg2+)对急性B淋巴白血病小鼠生存期的延长优于CD19 CAR mRNA@Apt(CD8)-MPP(Mg2+)。
Apt(CD62L)-MPP优于Apt(CD8)-MPP有以下原因：(1)生物体内CD62L+T细胞比CD8+T占比更多，靶向CD62L可以调动体内更强大的免疫系统；(2)CD8+T细胞虽然可以分泌高水平的颗粒酶和穿孔素等蛋白，但其存活时间很短，不能有效杀死肿瘤细胞；(3)CD62L+T细胞处于分化的初始阶段，有更长的寿命，在受到抗原刺激后可迅速分化为效应T细胞发挥抗肿瘤作用，且进化出免疫记忆功能；(4)虽然急性B淋巴白血病中的B淋巴细胞也表达CD62L，但本申请只是利用CD19 mRNA使细胞表达相应蛋白，并不会影响该细胞的下一代，也就不会对该细胞的增殖产生影响，所以就算本申请中的材料识别并结合B淋巴细胞了，理论上也不会造成患者病情恶化。
实施例9.2、Apt替换为识别CD3蛋白的适配体
将实施例5.1中的Apt替换为可特异性识别CD3蛋白的适配体，参照实施例6.2的方法制备CD19 CAR mRNA@Apt(CD3)-MPP。
靶向CD3的Apt(CD3)序列如SEQ ID NO.94所示：5’-TCTCGGACGCGTGTGGTCGGCCGAGTGGCCCACGGTAGAAGGGTTAGAACTGCTGG TTGGTGAATCTCGCTGCCTGGCCCTACAGTG-3’。其余mRNA-金属-磷脂复合物颗粒的制备过程与实施例1.3至1.5相同。分别将靶向药物CD19 CAR mRNA@Apt(CD62L)-MPP和CD19 CAR mRNA@Apt(CD3)-MPP(以mRNA的质量计算10μg/只)通过尾静脉注射至急性B淋巴白血病模型小鼠，每五天注射一次并在打药前两小时注射与癌细胞数量相等的人T细胞，共给药三次。每组小鼠数量均为五只。记录小鼠的生存时间，并统计生存率。
如图7-1所示，CD19 CAR mRNA@Apt(CD62L)-MPP(Fe3+)组小鼠肿瘤的生存期长于CD19 CAR mRNA@Apt(CD3)-MPP(Fe3+)。该结果表明靶向药物CD19 CAR mRNA@Apt(CD62L)-MPP(Fe3+)对急性B淋巴白血病小鼠生存期的延长优于CD19 CAR mRNA@Apt(CD3)-MPP(Fe3+)。这是因为生物体内CD62L+T细胞比CD3+T占比更多，靶向CD62L可以调动体内更强大的免疫系统。
如图7-1-2所示，CD19 CAR mRNA@Apt(CD62L)-MPP(Al3+)组小鼠肿瘤的生存期长于CD19 CAR mRNA@Apt(CD3)-MPP(Al3+)。该结果表明靶向药物CD19 CAR mRNA@Apt(CD62L)-MPP(Al3+)对急性B淋巴白血病小鼠生存期的延长优于CD19 CAR mRNA@Apt(CD3)-MPP(Al3+)。这是因为生物体内CD62L+T细胞比CD3+T占比更多，靶向CD62L可以调动体内更强大的免疫系统。
如图7-1-3所示，CD19 CAR mRNA@Apt(CD62L)-MPP(Mg2+)组小鼠肿瘤的生存期长于CD19 CAR mRNA@Apt(CD3)-MPP(Mg2+)。该结果表明靶向药物CD19 CAR mRNA@Apt(CD62L)-MPP(Mg2+)对急性B淋巴白血病小鼠生存期的延长优于CD19 CAR mRNA@Apt(CD3)-MPP(Mg2+)。这是因为生物体内CD62L+T细胞比CD3+T占比更多，靶向CD62L可以调动体内更强大的免疫系统。
由实施例9.1、实施例9.2的结果可知，如图7-1所示，在相同载体、相同药物、相同药量情况下，Apt(CD62L)-MPP性能优于Apt(CD8)-MPP和Apt(CD3)-MPP。该结果进一步证明CD19 CAR mRNA@Apt(CD62L)-MPP抗急性B淋巴细胞性白血病的效果最佳，Apt(CD62L)与人T细胞进行受体配体识别的能力最强、亲和力最高，使用Apt(CD62L)作为靶向结构制得的CD19 CAR mRNA@Apt(CD62L)-MPP抗急性B淋巴细胞性白血病的效果最佳。
除非另有定义，本申请全文所使用的所有技术和科学术语与本申请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，以本申请全文中所说明的含义或者根据本申请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申请实施例的目的，不是旨在限制本申请。
注意，上述仅为本申请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，本申请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申请的保护范围。因此，虽然通过以上实施例对本申请进行了较为详细的说明，但是本申请不仅仅限于以上实施例，在不脱离本申请的技术构思的情况下，还可以包括更多其他等效实施例，均属于本申请的保护范畴。

Claims (23)

  1. 一种靶向载体，其特征在于，所述靶向载体含有：
    (a)金属-磷脂复合物颗粒，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；
    (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；
    (b)靶向结构，其中，所述靶向结构与所述金属-磷脂复合物颗粒的外表面连接。
  2. 根据权利要求1所述的靶向载体，其特征在于，所述(i)金属-磷脂复合物中，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、磷酸酰肌醇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；
    优选地，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)磷酸酰肌醇(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)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或植烷酰基
    优选地，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；
    优选地，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；
    优选地，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；

    优选地，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；
    优选地，所述连接物分子部分选自
    姜黄素(式19)
    绿原酸(式20)花青素(式21)其中，R1和R2是H、OH或OCH3，R3是H或糖基，R4、R5和R6是OH或糖基、槲皮素(式22)二氢杨梅素(式23)橙皮素(式24)柚皮素(式25)芹菜素(式26)儿茶素(式27)茶多酚(式28)表没食子儿茶素没食子酸酯(式29)鞣花酸(式30)桑色素(式31)表儿茶素没食子酸酯(式32)儿茶素没食子酸酯(式33)没食子儿茶素没食子酸酯(式34)平贝碱C(式35)及其衍生物中一种或多种的组合；
    优选地，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；

    优选地，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；
    优选地，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)；
    优选地，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；
    优选地，所述金属离子部分选自Fe3+、Mg2+、Ca2+、Al3+中一种或多种的组合；
    优选地，所述金属离子部分选自Fe3+、Mg2+、Ca2+或Al3+。
  3. 根据权利要求1所述的靶向载体，其特征在于，所述(ii)抑制颗粒聚集的缀合的脂质包括PEG-脂质缀合物和/或PEG-DAA；
    优选地，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；
    优选地，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；
    优选地，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)；

  4. 根据权利要求1所述的靶向载体，其特征在于，所述(iii)中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；
    优选地，所述(iii)中的非阳离子脂质或非可电离脂质为胆固醇(式40)
    优选地，所述(iii)中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、磷酸酰肌醇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；
    优选地，(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)、磷酸酰肌醇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(式41)，及其衍生物中中的至少一种；
    优选地，所述(iii)中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；
    优选地，所述(iii)中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)。
  5. 根据权利要求1所述的靶向载体，其特征在于，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，磷脂分子部分选自DSPC、DSPE、DSPA或DSPG，连接物分子部分选自姜黄素、橙皮素或茶多酚，金属离子部分选自Fe3+、Mg2+、Ca2+或Al3+；
    优选地，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，磷脂分子部分选自DSPC(式46)、DSPE(式47)、DSPA(式48)或DSPG(式49)，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)，金属离子部分选自Fe3+、Mg2+、Ca2+或Al3+；
    优选地，磷脂分子部分、连接物分子部分和金属离子部分的摩尔比为1:1:(0.5～2)；
    优选地，所述磷脂分子部分为DSPC(式46)，所述连接物分子部分选自姜黄素(式19)，所述金属离子部分选自Fe3+、Mg2+或Al3+，磷脂分子部分、连接物分子部分和金属离子部分的摩尔比为1:1:1。
  6. 根据权利要求4所述的靶向载体，其特征在于，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％～50％，所述抑制颗粒聚集的缀合的脂质在原料中摩尔占比为1％～10％，所述胆固醇在原料中摩尔占比为15％～80％，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％～51％；
    优选地，所述金属-磷脂复合物在原料中摩尔占比为5％～40％，优选为10％～40％；
    优选地，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％；
    优选地，所述胆固醇在原料中摩尔占比为25％～75％，优选为35％～75％；
    优选地，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％～50％，优选为0％～40％。
  7. 根据权利要求1至6任一项所述的靶向载体，其特征在于，所述(b)靶向结构包括依次连接的疏水区、连接区和靶向结合区；所述疏水区与所述金属-磷脂复合物颗粒外层基于亲水性、疏水性进行连接；
    优选地，所述疏水区包括DSPE及其衍生物中的至少一种或多种；
    优选地，所述连接区包括PEG-2000及其衍生物中的至少一种或多种；
    优选地，所述靶向结合区可结合CD62L、CD8、CD3、核仁素蛋白、T细胞、自然杀伤细胞、巨噬细胞、胰腺癌细胞或肝癌细胞中的至少一种；
    优选地，所述靶向结合区包括核酸、多肽、蛋白、小分子中的至少一种；
    优选地，所述靶向结合区包括适配体、抗体、抗原结合部、galnac中的一种；
    优选地，所述靶向结合区为适配体，所述靶向结合区的靶点为CD62L，所述适配体优选为SEQ ID NO.44所示；或，
    优选地，所述靶向结合区为适配体，所述靶向结合区的靶点为CD8，所述适配体优选为SEQ ID NO.84所示；或，
    优选地，所述靶向结合区为适配体，所述靶向结合区的靶点为CD3，所述适配体优选为SEQ ID NO.95所示；或
    优选地，所述靶向结合区为适配体，所述靶向结合区的靶点为核仁素蛋白，所述适配体优选为SEQ ID NO.86所示；或，
    优选地，所述靶向结合区为适配体，所述靶向结合区的靶细胞为T细胞，所述适配体优选为SEQ ID NO.44所示；
    优选地，所述靶向结合区为适配体，所述靶向结合区的靶细胞为胰腺癌细胞，所述适配体优选为SEQ ID NO.88所示；或，
    优选地，所述靶向结合区为适配体，所述靶向结合区的靶细胞为肝癌细胞；
    优选地，所述靶向结构为DSPE-PEG2000-适配体，优选为DSPE-PEG2000-CD62L适配体，所述CD62L适配体序列如SEQ ID NO.44所示。
  8. 权利要求1至7任一项所述的靶向载体的制备方法，其特征在于，将所述靶向结构与所述金属-磷脂复合物颗粒的外表面连接形成所述靶向载体。
  9. 根据权利要求8所述的制备方法，其特征在于，将(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，得到所述金属-磷脂复合物颗粒；
    优选地，所述靶向结构为DSPE-PEG2000-适配体，制备过程为：DSPE-PEG2000与适配体通过中间体对反应连接，得到DSPE-PEG2000-适配体；
    优选地，所述中间体对选自DSPE-PEG2000-MAL和适配体-C6-SH、DSPE-PEG2000-NHS和适配体-NH2、DSPE-PEG2000-COOH和适配体-NH2、DSPE-PEG2000-NCO和适配体-NH2、DSPE-PEG2000-N3和适配体-DBCO，优选为DSPE-PEG2000-MAL和适配体-C6-SH；
    优选地，所述DSPE-PEG2000-适配体形成胶束后与所述金属-磷脂复合物颗粒的外表面连接形成所述靶向载体；
    优选地，所述胶束的制备方法包括直接溶解法、乙醇注入法、透析法或超声法。
  10. 根据权利要求8所述的制备方法，其特征在于，所述制备方法包括：
    步骤一：将磷脂分子、连接物分子和金属离子反应形成金属-磷脂复合物；
    步骤二：将步骤一中制备的所述金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质、(iii)非阳离子脂质或非可电离脂质混合制备得到金属-磷脂复合物颗粒；
    步骤三：将步骤二中制备的所述金属-磷脂复合物颗粒与所述靶向结构混合制备得到所述靶向载体；
    优选地，步骤一中，将磷脂分子、连接物分子和金属离子溶于乙醇中反应，磷脂分子、连接物分子和金属离子的摩尔比优选为1:1:1，所述的反应条件优选为40～60℃反应1～5h；
    优选地，步骤三中，所述金属-磷脂复合物颗粒与所述靶向结构的反应条件为2-10℃孵育0.2-12h。
  11. 根据权利要求8所述的制备方法，其特征在于，所述制备方法包括：
    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；
    步骤三：将步骤二中制备的所述金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质、(iii)非阳离子脂质或非可电离脂质混合制备得到金属-磷脂复合物颗粒；
    步骤四：将步骤三中制备的所述金属-磷脂复合物颗粒与所述靶向结构混合制备得到所述靶向载体；
    优选地，步骤一中，将磷脂分子与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，所述磷脂分子与所述连接物分子的摩尔比优选为1:1；所述反应的条件优选为65℃反应2小时；
    优选地，步骤二中，磷脂复合物与金属离子溶于乙醇，加入三乙胺后反应，得到所述金属-磷脂复合物，所述磷脂复合物与所述金属离子的摩尔比优选为1：(1～2)，所述磷脂复合物与所述三乙胺的摩尔比优选为1:1，所述反应的条件优选为60℃反应2小时；
    优选地，步骤四中，所述金属-磷脂复合物颗粒与所述靶向结构的反应条件为2-10℃孵育0.2-12h。
  12. 权利要求1至7任一项所述的靶向载体或权利要求8至11任一项所述制备方法用于药物运送、显影剂或疫苗的应用。
  13. 一种靶向药物，其特征在于，所述靶向药物包括药物和权利要求1至7任一项所述的靶向载体，所述药物包封在所述靶向载体的金属-磷脂复合物颗粒中。
  14. 根据权利要求13所述的靶向药物，其特征在于，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；
    优选地，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；
    优选地，所述药物为mRNA，所述mRNA为编码嵌合抗原受体CAR或TCR；
    优选地，所述药物为编码嵌合抗原受体CAR的mRNA，所述CAR包括跨膜结构域、信号传导结构域、抗原结合结构域、共刺激信号传导区、抗原结合结构域与跨膜结构域连接区；
    优选地，所述跨膜结构域选自SEQ ID No.28、SEQ ID No.29、SEQ ID No.30中的至少一种；和/或，
    信号传导结构域选自SEQ ID No.31、SEQ ID No.32、SEQ ID No.33中的至少一种；和/或，
    抗原结合结构域选自SEQ ID No.34、SEQ ID No.35、SEQ ID No.36中的至少一种；和/或，
    共刺激信号传导区选自SEQ ID NO.37、SEQ ID No.39、SEQ ID No.75中的至少一种；和/或，
    抗原结合结构域与跨膜结构域连接区选自SEQ ID No.40、SEQ ID No.41、SEQ ID No.42中的至少一种；
    优选地，所述药物是核苷酸序列如SEQ ID No.43所示的mRNA。
  15. 权利要求13或14所述靶向药物的制备方法，其特征在于，所述制备方法包括：将药物包载于靶向载体中，得到所述靶向药物。
  16. 根据权利要求15所述的制备方法，其特征在于，所述靶向载体包括金属-磷脂复合物颗粒和靶向结构，将药物包载于所述金属-磷脂复合物颗粒中，得到药物-金属-磷脂复合物颗粒；将所述靶向结构与所述药物-金属-磷脂复合物颗粒的外表面连接形成所述靶向药物。
  17. 根据权利要求16所述的制备方法，其特征在于，将药物、(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，得到所述药物-金属-磷脂复合物颗粒。
  18. 根据权利要求16所述的制备方法，其特征在于，所述制备方法包括：
    步骤一：将磷脂分子、连接物分子和金属离子反应形成金属-磷脂复合物；
    步骤二：将步骤一中制备的所述金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质、(iii)非阳离子脂质或非可电离脂质，以及药物混合制备得到药物-金属-磷脂复合物颗粒；
    步骤三：将步骤二中制备的所述药物-金属-磷脂复合物颗粒与所述靶向结构混合制备得到所述靶向药物；
    优选地，步骤一中，将磷脂分子、连接物分子和金属离子溶于乙醇中反应，磷脂分子、连接物分子和金属离子的摩尔比优选为1:1:1，所述的反应条件优选为40～60℃反应1～5h；
    优选地，步骤二中，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-金属-磷脂复合物颗粒，所述有机化合物优选为乙醇；优选地，有机相与水相的混匀方式包含微流控芯片或超声；
    优选地，步骤三中，所述药物-金属-磷脂复合物颗粒与所述靶向结构的反应条件为2-10℃孵育0.2-12h。
  19. 根据权利要求16所述的制备方法，其特征在于，所述制备方法包括：
    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；
    步骤三：将步骤二中制备的所述金属-磷脂复合物、(ii)抑制颗粒聚集的缀合的脂质、(iii)非阳离子脂质或非可电离脂质，以及药物混合制备得到药物-金属-磷脂复合物颗粒；
    步骤四：将步骤三中制备的所述药物-金属-磷脂复合物颗粒与所述靶向结构混合制备得到所述靶向药物；
    优选地，步骤一中，将磷脂分子与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，所述磷脂分子与所述连接物分子的摩尔比优选为1:1；所述反应的条件优选为65℃反应2小时；
    优选地，步骤二中，磷脂复合物与金属离子溶于乙醇，加入三乙胺后反应，得到所述金属-磷脂复合物，所述磷脂复合物与所述金属离子的摩尔比优选为1：(1～2)，所述磷脂复合物与所述三乙胺的摩尔比优选为1:1，所述反应的条件优选为60℃反应2小时；
    优选地，步骤三中，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-金属-磷脂复合物颗粒，所述有机化合物优选为乙醇；优选地，有机相与水相的混匀方式包含微流控芯片或超声
    优选地，步骤四中，所述药物-金属-磷脂复合物颗粒与所述靶向结构的反应条件为2-10℃孵育0.2-12h。
  20. 根据权利要求16所述的制备方法，其特征在于，所述靶向结构为DSPE-PEG2000-适配体，制备过程为：DSPE-PEG2000与适配体通过中间体对反应连接，得到DSPE-PEG2000-适配体；
    优选地，所述中间体对选自DSPE-PEG2000-MAL和适配体-C6-SH、DSPE-PEG2000-NHS和适配体-NH2、DSPE-PEG2000-COOH和适配体-NH2、DSPE-PEG2000-NCO和适配体-NH2、DSPE-PEG2000-N3和适配体-DBCO，优选为DSPE-PEG2000-MAL和适配体-C6-SH；
    优选地，所述DSPE-PEG2000-适配体形成胶束后与所述药物-金属-磷脂复合物颗粒的外表面连接形成所述靶向药物；
    优选地，所述胶束的制备方法包括直接溶解法、乙醇注入法、透析法或超声法。
  21. 权利要求13或14所述的靶向药物或权利要求15至20任一项所述的制备方法在药物运送、显影药物、疫苗中的应用。
  22. 根据权利要求21所述的应用，其特征在于，所述靶向药物用于药物运送、治疗和/或预防；
    优选地，所述靶向药物用于将药物引入细胞或血液；所述细胞优选包括免疫细胞、肿瘤细胞、生殖细胞、神经细胞、内分泌细胞、血细胞、吞噬细胞、白细胞、红细胞、上皮细胞、心肌细胞或干细胞；
    优选地，所述靶向药物用于在哺乳动物受试者中使靶序列的表达或沉默、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、用于将药物从体内传递到哺乳动物细胞因进行靶序列表达或沉默、或用于治疗/或预防哺乳动物的疾病或病症；
    优选地，所述哺乳动物为人；
    优选地，所述疾病或病症的治疗与基因的表达相关，所述基因包含药物的靶序列；
    优选地，所述疾病或病症包括癌症、病毒感染、自身免疫性疾病、免疫系统过度激活引发的疾病、代谢性疾病、纤维化疾病、组织纤维化、细胞衰老、动脉粥样硬化、糖尿病或骨关节炎；
    优选地，所述病毒感染包括乙型肝炎病毒、丙肝炎病毒、SARS-Cov-2、人类免疫缺陷病毒、细胞巨细胞病毒、侵袭性曲霉菌或结合病毒；
    优选地，所述癌症包括血液瘤和实体瘤；
    优选地，所述血液瘤包括急性B细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、多发性骨髓瘤(MM)、急性髓系白血病(AML)或T细胞淋巴瘤；
    优选地，所述弥漫性大B细胞淋巴瘤(DLBCL)包括霍奇金淋巴瘤(HL)和非霍奇金淋巴瘤(NHL)；
    优选地，所述实体瘤包括肝癌、脑胶质瘤、胃癌、胰腺癌、肺癌、非小细胞肺癌、肾细胞癌、前列腺癌、骨肉瘤、乳腺癌、结直肠癌、卵巢癌、黑色素瘤、神经母细胞瘤、血管母细胞瘤、间变性脑膜瘤、复发性胶质母细胞瘤或分化型甲状腺癌；
    优选地，所述自身免疫性疾病包括寻常型天疱疹、系统性红斑狼疮(SLE)、血友病、重症肌无力、移植组织器官引发的免疫排斥反应、I型糖尿病(T1D)、类风湿关节炎、系统性硬化症、多发性硬化症、特发性肺纤维化、克罗恩病或结肠炎；
    优选地，所述免疫系统过度激活引发的疾病包括细胞因子释放综合征(CRS)、免疫效应细胞相关神经毒性综合征(ICANS)或移植物抗宿主病(GVHD)；
    优选地，所述代谢性疾病包括动脉粥样硬化、先天性高胰岛素血症、非酒精性脂肪肝炎或非肥胖糖尿病(NOD)；
    优选地，所述纤维化包括心肌纤维化、肥厚性心肌病、新冠肺炎、新冠心肌炎、缺血性心肌病、肝硬化、肝纤维化、原发性胆管炎、肺纤维化、骨骼肌纤维化、皮肤类疾病、骨髓纤维化、结节病或杜氏肌营养不良症；
    优选地，所述皮肤类疾病包括瘢痕疙瘩或伤口愈合；
    优选地，所述靶向药物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；
    优选地，所述靶向药物用于联合用药。
  23. 一种含有权利要求13或14所述的靶向药物的药剂，所述药剂优选为疫苗，所述疫苗优选为新型冠状病毒疫苗。
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