Targeted carrier, targeted drug, preparation method therefor, and use thereof

Abstract

Provided are a targeted carrier, a targeted drug, a preparation method therefor, and use thereof. The targeted carrier comprises: (a) metal-polyphenol complex particles, and (b) a targeted structure connected to an outer surface of the metal-polyphenol complex particles. The targeted carrier is free of a cationic lipid or an ionizable lipid. The targeted drug can realize a high expression level of a nucleic acid drug.

Description

Targeted carrier, targeted drug, preparation method and application CROSS-REFERENCE TO RELATED APPLICATIONS This application requires the application number 202410177519.0 submitted to the China Patent Office on February 8, 2024, and the name The present invention claims priority from a Chinese patent application entitled “Targeting vector, preparation method and application thereof, and application of CD62L as a target for immune cells”, the entire contents of which are incorporated herein by reference. Technical Field

[0001] 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. Background Art

[0002] With the continuous advancement of molecular biology techniques, our understanding of the relationship between genes and diseases is deepening. 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.

[0003] However, 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. Currently, 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. Compared with small molecule conjugates directly conjugated to nucleic acid drugs, 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. However, 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-κB transcription factors, triggering the secretion of various proinflammatory cytokines and chemokines. In addition, ionizable lipids are lipids containing positively charged ionizable amine groups. They are uncharged under physiological conditions (pH = 7.4), but are protonated and positively charged at lower pH values. Therefore, ionizable lipids can be used to partially or completely replace cationic lipids as the main component of nanoparticles, responsible for adsorbing nucleic acids. When nanoparticles containing ionizable lipids enter the lysosomes of biological cells, the ionizable lipids become positively charged lipids in the low pH environment (pH = 4.0-6.5) within the lysosomes. Although ionizable lipids reduce the cytotoxic and highly inflammatory effects of some permanently positively charged cationic lipids, their cytotoxicity and immunogenicity are still high. Lipid nanoparticles (LNPs) based on cationic lipids and / or ionizable lipids are currently available clinically as nanoparticle nucleic acid drug delivery systems. Cationic lipids and / or ionizable lipids are the main components of LNPs and are responsible for the adsorption of nucleic acids. At the same time, the cytotoxicity and immunogenicity mediated by cationic lipids and / or ionizable lipids remain one of the important reasons for the high toxicity of LNPs.

[0004] Therefore, when using a delivery system to deliver negatively charged drugs (such as nucleic acid drugs, protein drugs, polypeptide drugs, small molecule drugs, etc.), nanoparticle delivery systems developed based on cationic lipids and / or ionizable lipids cannot fundamentally solve the toxicity problem of nanoparticle delivery systems. There is an urgent need for a liposome delivery system with higher safety.

[0005] In view of this, this application is hereby filed. Summary of the Invention

[0006] 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.

[0007] In order to achieve the above objectives, this application adopts the following technical solutions:

[0008] A targeting vector, comprising:

[0009] (a) Metal-polyphenol composite particles, the metal-polyphenol composite particles comprising:

[0010] (i) a metal-polyphenol complex, which is composed of a polyphenol molecule portion and a metal ion portion reacting with each other, wherein the polyphenol molecule portion and the metal ion portion are connected by a coordination bond;

[0011] (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

[0012] (iii) non-cationic lipids or non-ionizable lipids other than conjugated lipids that inhibit particle aggregation;

[0013] (b) a targeting structure, wherein the targeting structure is connected to the outer surface of the metal-polyphenol composite particle.

[0014] Furthermore, in the (i) metal-polyphenol complex, the polyphenol molecule portion is selected from the group consisting of curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, paclitaxel, brown algae polyphenols, polyflavanol polyphenols, catechin, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, galloylglucose, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate, gallocatechin gallate, and one or more combinations thereof.

[0015] Preferably, the polyphenol molecule is selected from curcumin (Formula 19)

[0016] Quercetin (Formula 22) Kaempferol (Formula 59)

[0017] Rutin (Formula 60) Hesperetin (Formula 24)

[0018] Naringenin (Formula 25) Eriochoride (Formula 61)

[0019] Luteolin (Formula 62) Apigenin (Formula 26)

[0020] Taxol (Formula 63) Brown algae polyphenols (Formula 64)

[0021] Polyflavanol polyphenols (Formula 65) Catechin (Formula 27)

[0022] Ellagic acid (Formula 30) Gallic acid (Formula 66)

[0023] Digallic acid (Formula 67)

[0024] Propyl gallate (Formula 68)

[0025] Epigallocatechin gallate (Formula 29)

[0026] Galloylglucose (Formula 69)

[0027] Hydroxyhydroquinone (Formula 70) Morin (Formula 31)

[0028] Epicatechin gallate (Formula 32)

[0029] Catechin gallate (Formula 33)

[0030] Epigallocatechin gallate (Formula 34) and combinations of one or more of their derivatives;

[0031] Preferably, the polyphenol molecular moiety is selected from at least one of curcumin (Formula 19), dihydrocurcumin (Formula 36), hexahydrocurcumin (Formula 37), curcumin sulfate (Formula 38), and bisdemethoxycurcumin (Formula 39);

[0032]

[0033]

[0034]

[0035]

[0036] Preferably, the polyphenol molecular portion is selected from at least one of curcumin (Formula 19), hesperetin (Formula 24) or catechin (Formula 27), and derivatives thereof;

[0037] Preferably, the polyphenol molecule is selected from curcumin (Formula 19), hesperetin (Formula 24) or catechin (Formula 27);

[0038] Preferably, the metal ion moiety is selected from Fe 3+ 、Ag + 、Ba 2+ , Ca 2+ 、Cd 2+ 、Cu 2+ 、Fe 2+ 、Mn 2+ Mg 2+ 、Mo 2+ 、Zn 2+ , Pt 2+ 、Au 2+ 、Al 3+ 、Ce 3+ 、Co 3+ Cr 3+ 、Eu 3+ 、Gd 3+ 、Ni 3+ 、W 3+ 、V 3+ 、Zr 3+ A combination of one or more of the following:

[0039] Preferably, the metal ion moiety is selected from Fe 3+ Mg 2+ , Ca 2+ 、Al 3+ A combination of one or more of the following:

[0040] Preferably, the metal ion moiety is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ .

[0041] Further, the (ii) conjugated lipid that inhibits particle aggregation comprises PEG-lipid conjugate and / or PEG-DAA;

[0042] Preferably, the PEG-lipid conjugate is selected from

[0043] Phosphatidylethanolamine-polyethylene glycol 2000 (Formula 42)

[0044] Phosphatidylethanolamine-polyethylene glycol 700 (Formula 43)

[0045] Phosphatidylethanolamine-polyethylene glycol 1000 (Formula 44)

[0046] Phosphatidylethanolamine-polyethylene glycol 5000 (Formula 45) and at least one of its derivatives, wherein R1 and R2 are independently: capryloyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytanoyl;

[0047] Preferably, the PEG-lipid conjugate is selected from a combination of one or more of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000 or DSPE-PEG5000;

[0048] Preferably, the PEG-lipid conjugate is selected from DSPE-PEG2000 (Formula 53), DSPE-PEG700 (Formula 50), DSPE-PEG1000 (Formula 51) or DSPE-PEG5000 (Formula 52);

[0049]

[0050]

[0051]

[0052]

[0053] Furthermore, the non-cationic lipid or non-ionizable lipid in (iii) 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-phospho-sphingosine S1P, and a combination of one or more of their derivatives;

[0054] Preferably, the non-cationic lipid or non-ionizable lipid in (iii) is selected from

[0055] Phosphatidylcholine (PC) (Formula 1)

[0056] Phosphatidylethanolamine (PE) (Formula 2)

[0057] Phosphatidylserine (PS) (Formula 3)

[0058] Phosphatidic acid (PA) (Formula 4)

[0059] Phosphatidylglycerol (PG) (Formula 5)

[0060] 1-phosphoceramide (SP) (Formula 6)

[0061] Phosphoinositide (PI) (Formula 7)

[0062] Phosphatidylthreonine (PT) (Formula 8)

[0063] Sphingomyelin (SM) (Formula 9)

[0064] Lysolecithin (LPC) (Formula 10)

[0065] Lysophosphoethanolamine (LPE) (Formula 11)

[0066] Lysophosphatidylserine (LPS) (Formula 12)

[0067] Lysophosphatidic acid (LPA) (Formula 13)

[0068] Lysophosphatidylglycerol (LPG) (Formula 14)

[0069] Lysophosphatidylinositol (LPI) (Formula 15)

[0070] Lysophosphatidylthreonine (LPT) (Formula 16)

[0071] Lysosphingomyelin (LSM) (Formula 17)

[0072] Sphingosine 1-phosphate (S1P) (Formula 18) and combinations of one or more of their derivatives;

[0073] Wherein, R1 and R2 are independently:

[0074] Capryloyl Lauroyl

[0075] Myristoyl Palmitoyl

[0076] Stearyl Oleoyl

[0077] Linoleyl Erucyl

[0078] Arachidoyl or phytanoyl

[0079] Preferably, the non-cationic lipid or non-ionizable lipid in (iii) further comprises at least one of cholesterol and its derivatives;

[0080] Preferably, 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;

[0081] Preferably, the non-cationic lipid or non-ionizable lipid in (iii) comprises cholesterol (Formula 40), and a combination of one or more selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48) or DSPG (Formula 49);

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] Furthermore, the metal-polyphenol complex is composed of a polyphenol molecule portion and a metal ion portion, wherein the polyphenol molecule portion is selected from curcumin, hesperidin or catechin, and the metal ion portion is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ ;

[0088] Preferably, the metal-polyphenol complex is composed of a polyphenol molecule portion and a metal ion portion, wherein the polyphenol molecule portion is selected from curcumin (Formula 19), hesperidin (Formula 24) or catechin (Formula 27), and the metal ion portion is selected from Fe 3+ Mg 2+ , Ca 2+ or Al3+ ;

[0089] Preferably, the molar ratio of the polyphenol molecule portion to the metal ion portion is 1:(0.5-2);

[0090] Preferably, the polyphenol molecule portion is curcumin (Formula 19), and the metal ion portion is Fe 3+ Mg 2+ or Al 3+ , the molar ratio of the polyphenol molecule part to the metal ion part is 1:1.

[0091] Furthermore, the metal-polyphenol complex particles are made of (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the metal-polyphenol complex accounts for 1% to 30% 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 0% to 60% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 29% to 75% by mole in the raw material;

[0092] Preferably, the molar proportion of the metal-polyphenol complex in the raw material is 5% to 30%, preferably 10% to 30%, preferably 5% to 20%;

[0093] Preferably, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%;

[0094] Preferably, the molar proportion of cholesterol in the raw material is 0% to 48%;

[0095] Preferably, the molar proportion of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 30% to 75%, preferably 40% to 75%.

[0096] Furthermore, 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-polyphenol complex particle based on hydrophilicity and hydrophobicity;

[0097] Preferably, the hydrophobic region comprises at least one or more of DSPE and its derivatives;

[0098] Preferably, the linker region comprises at least one or more of PEG-2000 and its derivatives;

[0099] Preferably, 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;

[0100] Preferably, the target binding region comprises at least one of a nucleic acid, a polypeptide, a protein, and a small molecule;

[0101] Preferably, the target binding region comprises one of an aptamer, an antibody, an antigen binding portion, and galnac;

[0102] Preferably, the targeting binding region is an aptamer, the target of the targeting binding region is CD62L, and the aptamer is preferably represented by SEQ ID NO.44; or,

[0103] Preferably, 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,

[0104] Preferably, the targeting binding region is an aptamer, the target of the targeting binding region is CD3, and the aptamer is preferably represented by SEQ ID NO.95; or

[0105] Preferably, the targeting binding region is an aptamer, the target of the targeting binding region is nucleolin protein, and the aptamer is preferably represented by SEQ ID NO.86; or,

[0106] Preferably, the targeting binding region is an aptamer, the target cell of the targeting binding region is a T cell, and the aptamer is preferably represented by SEQ ID NO.44;

[0107] Preferably, 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 represented by SEQ ID NO.88; or,

[0108] Preferably, the targeting binding region is an aptamer, and the target cell of the targeting binding region is a liver cancer cell;

[0109] Preferably, the targeting structure is DSPE-PEG2000-aptamer, preferably DSPE-PEG2000-CD62L aptamer, and the CD62L aptamer sequence is shown in SEQ ID NO.44.

[0110] The method for preparing the above-mentioned targeting carrier comprises connecting the targeting structure with the outer surface of the metal-polyphenol complex particles to form the targeting carrier.

[0111] Further, (i) the metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid are mixed to obtain the metal-polyphenol complex particles;

[0112] Preferably, the targeting structure is DSPE-PEG2000-aptamer, and the preparation process is as follows: DSPE-PEG2000 and the aptamer are connected through an intermediate pair reaction to obtain DSPE-PEG2000-aptamer;

[0113] Preferably, the intermediate pair is selected from DSPE-PEG2000-MAL and aptamer-C6-SH, DSPE-PEG2000-NHS and aptamer-NH2, DSPE-PEG2000-COOH and aptamer-NH2, DSPE-PEG2000-NCO and aptamer-NH2, DSPE-PEG2000-N3 and aptamer-DBCO, preferably DSPE-PEG2000-MAL and aptamer-C6-SH;

[0114] Preferably, the DSPE-PEG2000-aptamer forms micelles and then connects to the outer surface of the metal-polyphenol complex particles to form the targeting carrier;

[0115] Preferably, the preparation method of the micelles includes direct dissolution method, ethanol injection method, dialysis method or ultrasound method.

[0116] Furthermore, the preparation method comprises:

[0117] Step 1: reacting the polyphenol molecule portion with the metal ion portion through a coordination bond to form a metal-polyphenol complex;

[0118] Step 2: mixing the metal-polyphenol complex prepared in step 1, a conjugated lipid that inhibits particle aggregation, a non-cationic lipid, or a non-ionizable lipid to prepare the metal-polyphenol complex particles;

[0119] Step 3: mixing the metal-polyphenol complex particles prepared in step 2 with the targeting structure to prepare the targeting carrier;

[0120] Preferably, in step 1, polyphenol molecules are dissolved in ethanol, and metal ions and triethylamine are added and reacted to obtain the metal-polyphenol complex, the molar ratio of polyphenol molecules to metal ions is preferably 1:(1-2), and the molar ratio of polyphenol molecules to triethylamine is preferably 1:1. The reaction conditions are preferably 60° C. for 2 hours;

[0121] Preferably, in step three, the reaction conditions of the metal-polyphenol complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.

[0122] The above-mentioned targeting carrier or preparation method is used for drug delivery, imaging agent or vaccine application.

[0123] A targeted medicine comprises a medicine and the above-mentioned targeting carrier, wherein the medicine is encapsulated in the metal-polyphenol complex particles of the targeting carrier.

[0124] Furthermore, 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;

[0125] Preferably, the nucleic acid is selected from one or more combinations of mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, and artificial nucleic acid;

[0126] Preferably, the drug is mRNA, and the mRNA encodes a chimeric antigen receptor CAR or TCR;

[0127] Preferably, 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;

[0128] Preferably, 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,

[0129] 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,

[0130] 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,

[0131] The costimulatory signaling region is selected from at least one of SEQ ID NO.37, SEQ ID No.39, and SEQ ID No.75; and / or,

[0132] 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;

[0133] Preferably, the drug is an mRNA having a nucleotide sequence as shown in SEQ ID No.43.

[0134] The preparation method of the above-mentioned targeted drug comprises: encapsulating the drug in a targeting carrier to obtain the targeted drug.

[0135] Furthermore, the targeting carrier includes metal-polyphenol complex particles and a targeting structure, and the drug is encapsulated in the metal-polyphenol complex particles to obtain drug-metal-polyphenol complex particles; the targeting structure is connected to the outer surface of the drug-metal-polyphenol complex particles to form the targeted drug.

[0136] Furthermore, the drug, (i) the metal-polyphenol 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-polyphenol complex particles.

[0137] Furthermore, the preparation method comprises:

[0138] Step 1: reacting the polyphenol molecule portion with the metal ion portion through a coordination bond to form a metal-polyphenol complex;

[0139] Step 2: mixing the metal-polyphenol complex prepared in step 1, a conjugated lipid for inhibiting particle aggregation, a non-cationic lipid or a non-ionizable lipid, and a drug to prepare the drug-metal-polyphenol complex particles;

[0140] Step 3: mixing the drug-metal-polyphenol complex particles prepared in step 2 with the targeting structure to prepare the targeted drug;

[0141] Preferably, in step 1, polyphenol molecules are dissolved in ethanol, and metal ions and triethylamine are added and reacted to obtain the metal-polyphenol complex, the molar ratio of polyphenol molecules to metal ions is preferably 1:(1-2), and the molar ratio of polyphenol molecules to triethylamine is preferably 1:1. The reaction conditions are preferably 60° C. for 2 hours;

[0142] Preferably, in step 2, the metal-polyphenol 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-polyphenol 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;

[0143] Preferably, in step three, the reaction conditions of the drug-metal-polyphenol complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.

[0144] Furthermore, the targeting structure is DSPE-PEG2000-aptamer, and the preparation process is as follows: DSPE-PEG2000 and the aptamer are connected through an intermediate pair reaction to obtain DSPE-PEG2000-aptamer;

[0145] Preferably, the intermediate pair is selected from DSPE-PEG2000-MAL and aptamer-C6-SH, DSPE-PEG2000-NHS and aptamer-NH2, DSPE-PEG2000-COOH and aptamer-NH2, DSPE-PEG2000-NCO and aptamer-NH2, DSPE-PEG2000-N3 and aptamer-DBCO, preferably DSPE-PEG2000-MAL and aptamer-C6-SH;

[0146] Preferably, the DSPE-PEG2000-aptamer forms micelles and then connects to the outer surface of the drug-carrier delivery system to form the targeted drug;

[0147] Preferably, the preparation method of the micelles includes direct dissolution method, ethanol injection method, dialysis method or ultrasound method.

[0148] Application of the above-mentioned targeted drugs or preparation methods in drug delivery, imaging drugs, and vaccines.

[0149] Furthermore, the targeted drug is used for drug delivery, treatment and / or prevention;

[0150] Preferably, 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;

[0151] Preferably, the targeted drug is used to express or silence a target sequence in a mammalian subject, to deliver a drug in a mammal, to deliver a drug from the body to a mammalian cell, to deliver a drug from 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;

[0152] Preferably, the mammal is a human;

[0153] Preferably, the treatment of the disease or condition is associated with the expression of a gene comprising a target sequence for the drug;

[0154] Preferably, 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;

[0155] Preferably, the viral infection comprises hepatitis B virus, hepatitis C virus, SARS-Cov-2, human immunodeficiency virus, cytomegalovirus, invasive Aspergillus or conjugate virus;

[0156] Preferably, the cancer includes hematological tumors and solid tumors;

[0157] Preferably, 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;

[0158] Preferably, the diffuse large B-cell lymphoma (DLBCL) includes Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL);

[0159] Preferably, the solid tumor comprises 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;

[0160] Preferably, 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;

[0161] Preferably, 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);

[0162] Preferably, the metabolic disease includes atherosclerosis, congenital hyperinsulinemia, non-alcoholic steatohepatitis or non-obese diabetes (NOD);

[0163] Preferably, 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;

[0164] Preferably, the skin disease includes keloid or wound healing;

[0165] Preferably, the administration route of the targeted drug includes intrathecal injection, intramuscular administration, intracranial injection, intravenous injection or intratumoral injection;

[0166] Preferably, the targeted drug is used in combination therapy.

[0167] A pharmaceutical agent containing the above-mentioned targeted drug, wherein the pharmaceutical agent is preferably a vaccine, and the vaccine is preferably a new coronavirus vaccine.

[0168] Compared with the prior art, the technical effects of this application are:

[0169] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0170] The following further illustrates the various technical features of the present application and the relationships between them with reference to the accompanying drawings. The accompanying drawings are exemplary, and some technical features are not shown in actual proportion. In addition, some drawings may omit technical features that are commonly used in the technical field to which the present application belongs and are not essential for understanding and implementing the present application, or additional technical features that are not essential for understanding and implementing the present application may be shown. In other words, the combination of the various technical features shown in the accompanying drawings is not intended to limit the present application. In addition, throughout the present application, the same figure numbers refer to the same content. The specific description of the drawings is as follows:

[0171] Figure 1-1 is an example of eGFP-mRNA@MPNP (Fe 3+ ) Statistical graph of the percentage of eGFP-positive cells caused by 293T transfection;

[0172] Figure 1-2 is a diagram of RBD-mRNA@MPNP (Fe 3+ ) Statistical graph of RBD expression caused by 293T transfection;

[0173] Figure 1-3 is a diagram of RBD-mRNA@MPNP (Fe 3+ ) Statistical graph of the ability to induce humoral immunity;

[0174] Figure 1-4 is a diagram of NY-ESO-1-mRNA@MPNP (Fe 3+ ) Statistical graph of the ability to induce humoral immunity;

[0175] Figure 1-5 is a diagram of RBD-mRNA@MPNP (Fe 3+ ) Statistical graph of the ability to induce cellular immunity;

[0176] Figure 1-6 is a diagram of NY-ESO-1-mRNA@MPNP (Fe3+ ) Statistical graph of the ability to induce cellular immunity;

[0177] Figure 1-1-2 is the eGFP-mRNA@MPNP (Al 3+ ) Statistical graph of the percentage of eGFP-positive cells caused by 293T transfection;

[0178] Figure 1-2-2 is RBD-mRNA@MPNP (Al 3+ ) Statistical graph of RBD expression caused by 293T transfection;

[0179] Figure 1-3-2 is RBD-mRNA@MPNP (Al 3+ ) Statistical graph of the ability to induce humoral immunity;

[0180] Figure 1-4-2 is NY-ESO-1-mRNA@MPNP (Al 3+ ) Statistical graph of the ability to induce humoral immunity;

[0181] Figure 1-5-2 is RBD-mRNA@MPNP (Al 3+ ) Statistical graph of the ability to induce cellular immunity;

[0182] Figure 1-6-2 is NY-ESO-1-mRNA@MPNP (Al 3+ ) Statistical graph of the ability to induce cellular immunity;

[0183] Figure 1-1-3 is the eGFP-mRNA@MPNP (Mg 2+ ) Statistical graph of the percentage of eGFP-positive cells caused by 293T transfection;

[0184] Figure 1-2-3 is the RBD-mRNA@MPNP(Mg 2+ ) Statistical graph of RBD expression caused by 293T transfection;

[0185] Figure 1-3-3 is RBD-mRNA@MPNP(Mg 2+ ) Statistical graph of the ability to induce humoral immunity;

[0186] Figure 1-4-3 is NY-ESO-1-mRNA@MPNP(Mg 2+ ) Statistical graph of the ability to induce humoral immunity;

[0187] Figure 1-5-3 is RBD-mRNA@MPNP(Mg 2+ ) Statistical graph of the ability to induce cellular immunity;

[0188] Figure 1-6-3 is NY-ESO-1-mRNA@MPNP(Mg 2+ ) Statistical graph of the ability to induce cellular immunity;

[0189] Figure 1-7 is a diagram of Bcl-2-siRNA@MPNP (Fe 3+ ) ability to silence target genes;

[0190] Figure 1-8 is a diagram of PLK1-siRNA@MPNP (Fe 3+ ) Statistical graph of the ability to silence target genes;

[0191] Figure 1-9 shows the Gal-1-siRNA@MPNP (Fe 3+ ) Statistical graph of the ability to silence target genes;

[0192] Figure 1-7-2 is Bcl-2-siRNA@MPNP (Al 3+ ) ability to silence target genes;

[0193] Figure 1-8-2 is the PLK1-siRNA@MPNP (Al 3+ ) Statistical graph of the ability to silence target genes;

[0194] Figure 1-9-2 is the Gal-1-siRNA@MPNP (Al 3+ ) Statistical graph of the ability to silence target genes;

[0195] Figure 1-7-3 shows Bcl-2-siRNA@MPNP(Mg 2+ ) ability to silence target genes;

[0196] Figure 1-8-3 is the PLK1-siRNA@MPNP (Mg 2+ ) Statistical graph of the ability to silence target genes;

[0197] Figure 1-9-3 shows the Gal-1-siRNA@MPNP (Mg 2+) Statistical graph of the ability to silence target genes;

[0198] Figure 1-10 is a diagram of STAT3-ASO@MPNP (Fe 3+ ) Statistical graph of the ability to silence cell target genes;

[0199] Figure 1-11 is a diagram of α-syn-ASO@MPNP (Fe 3+ ) Statistical graph of the ability to silence cell target genes;

[0200] Figure 1-12 shows Bcl-2-ASO@MPNP(Fe 3+ ) Statistical graph of the ability to silence cell target genes;

[0201] Figure 1-10-2 is a diagram of STAT3-ASO@MPNP (Al 3+ ) Statistical graph of the ability to silence cell target genes;

[0202] Figure 1-11-2 is a diagram of α-syn-ASO@MPNP (Al 3+ ) Statistical graph of the ability to silence cell target genes;

[0203] Figure 1-12-2 shows Bcl-2-ASO@MPNP (Al 3+ ) Statistical graph of the ability to silence cell target genes;

[0204] Figure 1-10-3 is a diagram of STAT3-ASO@MPNP (Mg 2+ ) Statistical graph of the ability to silence cell target genes;

[0205] Figure 1-11-3 shows the α-syn-ASO@MPNP (Mg 2+ ) Statistical graph of the ability to silence cell target genes;

[0206] Figure 1-12-3 shows Bcl-2-ASO@MPNP(Mg 2+ ) Statistical graph of the ability to silence cell target genes;

[0207] Figure 1-13 is a diagram of S-mRNA@MPNP (Fe 3+ ) Statistical graph of S protein expression caused by 293T transfection;

[0208] Figure 1-14 shows the drug (dsDNA and ssDNA)@MPNP (Fe 3+ ) of transfected cells;

[0209] Figure 1-14-1 shows the drug (dsDNA and ssDNA)@MPNP (Fe 3+ )’s functional test result diagram;

[0210] Figure 1-13-2 is S-mRNA@MPNP (Al 3+ ) Statistical graph of S protein expression caused by 293T transfection;

[0211] Figure 1-14-2 shows the drug (dsDNA and ssDNA)@MPNP (Al 3+ )’s functional test result diagram;

[0212] Figure 1-13-3 is a diagram of S-mRNA@MPNP (Mg 2+ ) Statistical graph of S protein expression caused by 293T transfection;

[0213] Figure 1-14-3 shows the drug (dsDNA and ssDNA)@MPNP (Mg 2+ )’s functional test result diagram;

[0214] Figure 2-1 is a metal-polyphenol complex (Fe 3+ )’s UV absorption pattern;

[0215] Figure 2-1-2 is a diagram of the metal-polyphenol complex (Al 3+ )’s UV absorption pattern;

[0216] Figure 2-1-3 shows the metal-polyphenol complex (Mg 2+ )’s UV absorption pattern;

[0217] Figure 2-2 shows the Fe 3+ Characterization images of shedding from metal-polyphenol complex;

[0218] Figure 2-3 shows the siRNA / mRNA@MPNP(Fe 3+ 、Al 3+ or Mg 2+ ) and siRNA / mRNA@LNP encapsulation efficiency statistics of nucleic acids (mRNA and siRNA);

[0219] Figure 2-4 is a diagram of siRNA / mRNA@MPNP (Fe 3+ 、Al 3+ or Mg 2+ ) and siRNA / mRNA@LNP nucleic acid lysosomal escape ability test results and statistical graphs;

[0220] Figure 2-5 shows the MPNP (Fe 3+ 、Al 3+ or Mg 2+ ) and the statistical graph of the eGFP-positive cell rate of LNP;

[0221] Figure 2-6 shows the MPNP (Fe 3+ 、Al 3+ or Mg 2+ ) and LNP's ability to promote RBD-mRNA expression statistical results;

[0222] Figure 2-7 is a diagram of MPNP (Fe 3+ 、Al 3+ or Mg 2+ ) and LNP's ability to promote humoral immunity;

[0223] Figure 2-8 is a diagram of MPNP (Fe 3+ 、Al 3+ or Mg 2+ ) and LNP's ability to promote cellular immunity;

[0224] FIG3-1 is a diagram of the drug-metal-polyphenol composite particles (Fe 3+ ) Statistical graph of the results of intratumoral injection treatment for liver cancer;

[0225] Figure 3-1-2 shows the drug-metal-polyphenol composite particles (Al 3+ ) Statistical graph of the results of intratumoral injection treatment for liver cancer;

[0226] Figure 3-1-3 shows the drug-metal-polyphenol composite particles (Mg 2+ ) Statistical graph of the results of intratumoral injection treatment for liver cancer;

[0227] Figure 4-1 is a diagram of the targeted drug (CD19 CAR-mRNA@Apt-MPNP) (Fe 3+ ), drug-metal-polyphenol particles (CD19 CAR mRNA@MPNP)(Fe 3+) Statistical graph of the proportion of CD19-CAR positive cells;

[0228] Figure 4-1-2 is a diagram of the targeted drug (CD19 CAR-mRNA@Apt-MPNP) in Example 5.2 of this application (Al 3+ ), drug-metal-polyphenol particles (CD19 CAR mRNA@MPNP) (Al 3+ ) Statistical graph of the proportion of CD19-CAR positive cells;

[0229] Figure 4-1-3 shows the targeted drug (CD19 CAR-mRNA@Apt-MPNP) (Mg 2+ ), drug-metal-polyphenol particles (CD19 CAR mRNA@MPNP)(Mg 2+ ) Statistical graph of the proportion of CD19-CAR positive cells;

[0230] Figure 5-1 is a diagram of Fe in Example 6.4 of this application. 3+ , a comparison of the survival rates of anti-acute B lymphoblastic leukemia mice treated with CD19 CAR mRNA@Apt(CD62L)-MPNP and CD19 CAR mRNA@Apt(CD62L)-LNP when the drug is CD19 CAR and the aptamer is CD62L-targeted;

[0231] Figure 5-1-2 shows the Al 3+ , a comparison of the survival rates of anti-acute B lymphoblastic leukemia mice treated with CD19 CAR mRNA@Apt(CD62L)-MPNP and CD19 CAR mRNA@Apt(CD62L)-LNP when the drug is CD19 CAR and the aptamer is CD62L-targeted;

[0232] Figure 5-1-3 shows the method of Mg in Example 6.4 of this application. 2+ , a comparison of the survival rates of anti-acute B lymphoblastic leukemia mice treated with CD19 CAR mRNA@Apt(CD62L)-MPNP and CD19 CAR mRNA@Apt(CD62L)-LNP when the drug is CD19 CAR and the aptamer is CD62L-targeted;

[0233] Figure 5-2 shows the Fe 3+ , when the drug is ASO and the aptamer is AS1411, the treatment survival rate comparison of ASO@Apt(AS1411)-MPNP and ASO@Apt(AS1411)-LNP in anti-lung cancer mice;

[0234] Figure 5-2-2 Al in Example 6.4 of this application 3+ , when the drug is ASO and the aptamer is AS1411, the treatment survival rate comparison of ASO@Apt(AS1411)-MPNP and ASO@Apt(AS1411)-LNP in anti-lung cancer mice;

[0235] Figure 5-2-3 Mg in Example 6.4 of this application 2+ , when the drug is ASO and the aptamer is AS1411, the treatment survival rate comparison of ASO@Apt(AS1411)-MPNP and ASO@Apt(AS1411)-LNP in anti-lung cancer mice;

[0236] Figure 5-3 shows the Fe 3+ , Comparison of the survival rates of pancreatic cancer mice treated with siRNA@Apt(P19)-MPNP and siRNA@Apt(P19)-LNP when the drug is siRNA and the aptamer is P19;

[0237] Figure 5-3-2 shows the Al 3+ , Comparison of the survival rates of pancreatic cancer mice treated with siRNA@Apt(P19)-MPNP and siRNA@Apt(P19)-LNP when the drug is siRNA and the aptamer is P19;

[0238] Figure 5-3-3 shows the method of Example 6.4 of this application using Mg 2+ , Comparison of the survival rates of pancreatic cancer mice treated with siRNA@Apt(P19)-MPNP and siRNA@Apt(P19)-LNP when the drug is siRNA and the aptamer is P19;

[0239] Figure 6-1 is the Fe content in Examples 7.1 and 7.2 of this application. 3+ Comparison of the overall survival rates of CD19 CAR mRNA@MPNP, CD19 CAR mRNA@Apt(CD62L)-MPNP, CAR mRNA@Apt(CD8)-MPNP, and CD19CAR mRNA@Apt(CD3)-MPNP anti-acute B lymphoblastic leukemia mice;

[0240] Figure 6-1-2 shows Al in Examples 7.1 and 7.2 of this application. 3+Comparison of the overall survival rates of CD19 CAR mRNA@MPNP, CD19 CAR mRNA@Apt(CD62L)-MPNP, CAR mRNA@Apt(CD8)-MPNP, and CD19 CAR mRNA@Apt(CD3)-MPNP anti-acute B lymphoblastic leukemia mice;

[0241] Figure 6-1-3 shows the Mg content in Examples 7.1 and 7.2 of this application. 2+ Comparison of the overall survival rates of CD19 CAR mRNA@MPNP, CD19 CAR mRNA@Apt(CD62L)-MPNP, CAR mRNA@Apt(CD8)-MPNP, and CD19 CAR mRNA@Apt(CD3)-MPNP anti-acute B lymphoblastic leukemia mice;

[0242] Figure 7-1 is a CD19 CAR mRNA@MPNP (Fe 3+ 、Al 3+ or Mg 2+ ) and the percentage of CAR-positive cells of CD19 CAR mRNA@LNP in myeloid cells;

[0243] Figure 7-2 shows the CD19 CAR mRNA@Apt-MPNP (Fe 3+ 、Al 3+ or Mg 2+ ) and the CAR positivity rate of CD19 CAR mRNA@Apt-LNP in myeloid cells. DETAILED DESCRIPTION

[0244] In order to enable those skilled in the art to better understand the technical solution of the present application, the technical solution of the present application is further described in detail below in conjunction with specific implementation methods.

[0245] For ease of explanation, specific terms described in this specification, the embodiments and the appended claims are collectively described here. Unless otherwise defined in this specification, the meanings of the scientific and technical terms used herein are the same as those understood and used by those skilled in the art. In addition, unless the context requires otherwise, it should be understood that singular terms should include the same plural forms, and plural terms should include the singular. Specifically, unless the context clearly indicates otherwise, the terms "at least one" and "one or more" used herein and in the appended claims include one, two, three or more. In this application, references to "multiple", "multiple", "multiple times", "multiple" and the like, unless otherwise specified, refer to quantities greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0246] In this application, “further”, “further”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present invention.

[0247] In this application, the terms "preferably," "preferred," and "preferred" are optional, meaning they refer to either option being selected from the two parallel options of "with" or "without." If multiple "preferably" items appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "preferably" item is independent of the others.

[0248] Although the numerical ranges and parameters used to define the broader scope of this application are approximate, the numerical values ​​of the specific examples are presented herein as precisely as possible. However, any numerical value inherently and inevitably contains standard deviations due to individual testing methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within an acceptable standard error of the mean, as determined by one skilled in the art. Except in the experimental examples, or unless otherwise expressly indicated, all ranges, amounts, values, and percentages used herein (e.g., to describe material amounts, time periods, temperatures, operating conditions, quantitative ratios, and the like) are to be understood as modified by the word "about." Therefore, unless otherwise indicated, the numerical parameters disclosed in this specification and the accompanying claims are approximate and are subject to change as needed. At a minimum, these numerical parameters should be understood to include the number of significant digits indicated and to apply normal rounding.

[0249] The term "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.

[0250] The term "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.

[0251] The term "neutral lipid" refers to any of a number of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0252] The term "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.

[0253] The term "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 bromide ("DMRIE"). The following lipids are cationic and have a positive charge below physiological pH: DODAP, DODMA, DMDMA, etc.

[0254] The term "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.

[0255] The phrase "metal ion portion" herein refers to the structure originally belonging to the metal ion after the metal ion reacts with other substances.

[0256] Herein, the "polyphenol molecule portion" refers to the structure originally belonging to the polyphenol molecule after the polyphenol molecule reacts with other substances.

[0257] The phrase "metal-polyphenol complex" herein is composed of the reaction of the polyphenol molecule portion and the metal ion portion, and the polyphenol molecule portion and the metal ion portion are connected via a coordination bond.

[0258] The term "drug-metal-polyphenol complex particles" refers to metal-polyphenol complex particles that have been drug-loaded, and the drug and the metal-polyphenol complex are connected by encapsulation, loading, chemical bonds or non-chemical bonds.

[0259] The term "CD8" refers to a dimeric co-receptor that enables T cells to recognize peptides presented by class I proteins of the histocompatibility complex and is a surface marker protein of cytotoxic T lymphocytes (CTLs). One way in which CD8+ T cells initiate apoptosis is through the secretion of perforins and granzymes, two types of cytotoxic proteins. 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. The targeted cells undergoing apoptosis are then cleared by nearby phagocytes. In addition to directed apoptosis, CD8+ T cells can also kill target cells indirectly by releasing cytokines such as TNF-α.

[0260] The term "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. They differ from other adhesion molecules primarily because their adhesive properties are limited to leukocyte-platelet-endothelial interactions within the vascular system and, secondly, because unlike other adhesion molecules, selectins form protein-protein bonds. Selectins possess a protein-like lectin group that binds to carbohydrate ligands, thereby forming protein-carbohydrate bonds. In the peripheral blood of healthy individuals, approximately 50% of peripheral blood lymphocytes, 40%-80% of monocytes, 95% of neutrophils, most B cells and primitive T cells express CD62L on their surface.

[0261] The term "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,

[0262] The term "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.

[0263] The term "exosomes" refers to a nanoscale vesicle secreted by cells, which contains bioactive substances such as proteins and nucleic acids (such as mRNA and miRNA).

[0264] The term "pseudovirus" 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.

[0265] The term "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.

[0266] The term "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.

[0267] The term "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.

[0268] The term "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. "Aptamer" is used to refer to a polymeric form of nucleotides of any length, ribonucleotides or deoxyribonucleotides. Therefore, 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.

[0269] The term "GalNAc" as used herein 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." Unless otherwise specified, the number of GalNAc groups contained in a GalNAc is not limited and may be those known and disclosed in this specification. 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.

[0270] The term "P19" refers to a nucleic acid sequence that can specifically recognize pancreatic cancer cells.

[0271] The term "targeted drug" (also known as targeted preparation) refers to a drug or its preparation that is endowed with targeting ability.

[0272] The term "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.

[0273] The term "signaling domain" refers to a sequence encoding a signal peptide or a sequence encoding a substance that plays the same role as a signal peptide. "Signaling 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 α-helix.

[0274] The term "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. For example, the antigen binding domain may include: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of a target antigen; a peptide with sufficient affinity for the target; a single domain antibody; an artificial single binder such as Darpin (designed ankyrin repeat protein); or a single chain derived from a T cell receptor.

[0275] The term "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.

[0276] The term "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.

[0277] The term "chimeric antigen receptor (CAR)" refers to an engineered receptor that is grafted onto immune effector cells with any specificity. CAR is modularly composed of four main domains: at the extracellular end is a single-chain variable region (scFv) antibody for target recognition, a spacer region connected to the transmembrane domain, and an intracellular signaling domain, with or without a connected co-stimulatory domain. Engagement of cognate antigens on the surface of target cells initiates the activation of CAR-engineered immune cells, leading to a lasting immune cell response. Each CAR domain has a unique function and affects redirected immune cell activation; for each specific application, CAR design requires some adjustments to provide optimized targeting and immune cell activation. It cleverly combines antigen recognition with intracellular activation elements, breaking the limitations of immune cell recognition and allowing immune cells to accurately target tumor cells. 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. 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.

[0278] The term "T Cell Receptor (T Cell Receptor, TCR)" is a complex of membrane proteins that participate 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, wherein the variable regions of the α chain and the β chain constitute the antigen recognition unit, and each variable region contains 3 complementary determining regions (CDR1, CDR2 and CDR3). When TCR recognizes a ligand, the less diverse CDR1 and CDR2 contact the α helix on both sides of the pMHC, while CDR3 contacts the central peptide. CDR3 is the most critical sequence that determines the specificity of TCR recognition of antigens. The stimulation of TCR is triggered by the major histocompatibility complex molecule (MHC) on the antigen presenting cell, which presents the antigen peptide to the T cell and binds to the TCR complex to induce a series of intracellular signal transduction cascades.

[0279] The term "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 biaptamers), 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 regulating their functions, such as activating, inhibiting or changing their differentiation state, thereby treating diseases.

[0280] The term "chimeric antigen receptor (CAR) drug" refers to a new type of therapeutic drug that works by genetically modifying target cells in vivo or in vitro to express CAR. 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. Generally, 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.

[0281] The term "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. The term "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. Using 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. 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.

[0282] The term "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. Herein, metal-polyphenol complex particles (MPNPs) are "lipid vesicles," and drugs, such as nucleic acid mRNA, are encapsulated in MPNPs as encapsulated components, and the "encapsulation" can be full encapsulation and / or partial encapsulation.

[0283] The term "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, NN-dialkylamino, 1,2-diacyloxy-3-aminopropane and 1,2-dialkyl-3-aminopropane.

[0284] The term "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.

[0285] Among the components of the metal-polyphenol complex particles, the "non-cationic lipids or non-ionizable lipids other than 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-polyphenol complex particles after excluding the conjugated lipids that inhibit particle aggregation.

[0286] 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.

[0287] In the metal-polyphenol complex particles, non-cationic lipids or non-ionizable lipids other than the conjugated lipids that inhibit particle aggregation are mainly present as vesicle-forming lipids. The term "vesicle-forming lipid" is intended to include any amphiphilic lipid having a hydrophobic part and a polar head group and which can spontaneously form bilayer vesicles in water, exemplified by most phospholipids.

[0288] In the metal-polyphenol complex particles, the conjugated lipids that inhibit particle aggregation are primarily present as vesicle-adopting lipids. The term "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).

[0289] The term "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. Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino lipids, and sphingolipids. The representative examples of phospholipid include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus are also in the group that is called as amphipathic lipid such as sphingomyelin, sphingosylglycolipid family, diacylglycerol and beta-acyloxy acid. In addition, above-mentioned amphipathic lipid can be mixed with other lipid, and this lipid comprises triglyceride and sterol.

[0290] The term "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:

[0291]

[0292] The term "diacylglycerol-conjugated polyethylene glycol" refers to the conjugated lipid that inhibits particle aggregation in the present application, which can be diacylglycerol-conjugated polyethylene glycol, i.e., a diacylglycerol-polyethylene glycol conjugate (DAG-PEG conjugate or PEG-DAG conjugate). In a preferred embodiment, 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). Those skilled in the art will readily understand that other diacylglycerols can be used in the DAG-PEG conjugates of the present application. Suitable DAG-PEG conjugates for use in the present application and methods of preparing and using them are disclosed in U.S. Application No. 10 / 136,707, published as USPA 2003 / 0077829, and PCT Patent Application No. CA 02 / 00669, each of which is incorporated by reference in its entirety.

[0293] The term "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:

[0294]

[0295] The term "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). In a preferred embodiment, the PEG-DAA conjugate has the following formula 56:

[0296] In 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). In a preferred embodiment, 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. In formula I, 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. In a preferred embodiment, 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. In Formula I, 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 (OC(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.

[0297] 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).

[0298] Like phosphatidylethanolamine, 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. In addition, 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. 5,820,873, which is incorporated herein by reference. Using the synthetic pathways proposed in the aforementioned applications, ceramides with saturated or unsaturated fatty acids can be prepared, wherein the fatty acids have carbon chain lengths ranging from C2 to C31.

[0299] The term "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:

[0300]

[0301] wherein: 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; and q is 0 or 1. It will be apparent to those skilled in the art that other polyamides can be used in the compounds of the present application.

[0302] The term "congener" refers to an analog that performs the same or similar function, or a derivative of the same parent core that performs the same or similar function.

[0303] As used herein, the terms "mRNA" or "messenger polyribonucleotide" or "messenger RNA" or "messenger RNA" are used interchangeably and refer to a single-stranded polyribonucleotide that is transcribed using one strand of DNA as a template, carries genetic information, and can direct protein synthesis.

[0304] As used herein, the terms "sgRNA," "small guide RNA," "guide RNA," or "gRNA" are used interchangeably and are small noncoding RNAs that pair with pre-mRNAs to guide the insertion or deletion of uridine residues into kinetoplastids during RNA editing. gRNAs edit RNA molecules, approximately 60-80 nucleotides in length, transcribed from separate genes.

[0305] As used herein, the terms "circRNA" or "circular RNA" or "cyclic polyribonucleotide" or "circular RNA" are used interchangeably and refer to a polyribonucleotide molecule having a structure without free ends (i.e., without free 3' and / or 5' ends), such as a polyribonucleotide that forms a ring or annular structure through covalent or non-covalent bonds.

[0306] As used herein, the terms "microRNA" or "miRNA" or "microRNA" are used interchangeably and refer to a non-coding single-stranded polyribonucleotide of approximately 22 nucleotides in length with 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 mRNA of the target gene, thereby regulating the biological function of the cell.

[0307] As used herein, the terms "ASO" or "antisense oligonucleotide" or "antisense oligonucleotide" are used interchangeably and refer to artificially synthesized nucleic acid fragments that are complementary to a certain segment of a 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, including antisense DNA and antisense RNA.

[0308] As used herein, the terms "siRNA" or "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.

[0309] As used herein, the terms "ecDNA" or "extrachromosomal circular DNA" are used interchangeably and refer to DNA that has fallen off chromosomes and exists in a circular structure.

[0310] The term "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. For example, 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.

[0311] The term "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.

[0312] The term "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. Specifically, degenerate codon substitutions can be achieved 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. The nucleotides are linked via the 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. The term nucleic acid is used interchangeably with gene, cDNA, mRNA encoded by a gene, and interfering RNA molecules.

[0313] The term "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).

[0314] As used herein, "gene product" refers to the product of a gene such as, but not limited to, the transcript of DNA, mRNA.

[0315] The phrase "expression silencing of target gene" refers to the ability of the siRNA of the present application to start silencing the target gene. In order to determine the degree of gene silencing, 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.

[0316] 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.

[0317] As used herein, the term "aqueous solution" refers to a composition that comprises, in whole or in part, water.

[0318] As used herein, the term "organic lipid solution" refers to a composition comprising in whole or in part an organic solvent with lipids.

[0319] As used herein, "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.

[0320] As used herein, "local delivery" refers to the delivery of a compound directly to a target site in the body. For example, 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.

[0321] 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.

[0322] To form long dsRNA, for synthetic RNAs, the complement can also be transcribed in vitro and hybridized to form dsRNA. If a naturally occurring RNA population is used, for example, by transcribing cDNAs corresponding to the RNA population, or by using RNA polymerase, RNA complements are also provided (e.g., to form dsRNA, which is digested by E. coli RNAse III or Dicer). The precursor RNA is then hybridized to form double-stranded RNAs and digested. The dsRNAs can be directly encapsulated in SNALPs or can be digested in vitro before encapsulation.

[0323] Alternatively, one or more DNA plasmids encoding one or more siRNA templates can be encapsulated within nucleic acid-lipid particles. For example, 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. Acad. Sci. 99:6047 (2002); Lee, et al., Nat. Biotech. 20:500 (2002); Miyagishi, et al., Nat. Biotech. 20:497 (2002); Paul, 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., Proc. Natl. Acad. Sci. 10:1017 (2002); et al., Nat. Biotech. 20: 505 (2002); and Sui, et al., Proc. Natl. Acad. Sci. 99: 5515 (2002). Typically, 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. Preferably, 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.

[0324] Methods for isolating 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. Additional basic texts disclosing the general methods used in this application include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

[0325] 1. Metal-polyphenol complex

[0326] In the present application, the metal-polyphenol complex is composed of a polyphenol molecule portion and a metal ion portion reacting with each other, and the polyphenol molecule portion and the metal ion portion are connected by a coordination bond.

[0327] Polyphenol molecules are 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.

[0328] In some embodiments, the polyphenol molecule portion is selected from curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, taxol, brown algae polyphenols, polyflavanol polyphenols, catechin, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, galloylglucose, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate, gallocatechin gallate, and one or more combinations thereof. Preferably, the polyphenol molecular portion is selected from curcumin (Formula 19), quercetin (Formula 22), kaempferol (Formula 59), rutin (Formula 60), hesperetin (Formula 24), naringenin (Formula 25), eriodictyol (Formula 61), luteolin (Formula 62), apigenin (Formula 26), paclitaxel (Formula 63), brown algae polyphenols (Formula 64), polyflavanol polyphenols (Formula 65), catechin (Formula 27), ellagic acid (Formula 30), gallic acid (Formula 66), digallic acid (Formula 67), propyl gallate (Formula 68), epigallocatechin gallate (Formula 29), galloylglucose (Formula 69), hydroxyhydroquinone (Formula 70), morin (Formula 31), epicatechin gallate (Formula 32), catechin gallate (Formula 33), gallocatechin gallate (Formula 34), and a combination of one or more thereof. Preferably, the polyphenol molecular portion is selected from at least one of curcumin (Formula 19), dihydrocurcumin (Formula 36), hexahydrocurcumin (Formula 37), curcumin sulfate (Formula 38), and bisdemethoxycurcumin (Formula 39). Preferably, the polyphenol molecular portion is selected from at least one of curcumin (Formula 19), hesperetin (Formula 24) or catechin (Formula 27), and derivatives thereof. Preferably, the polyphenol molecular portion is selected from curcumin (Formula 19), hesperetin (Formula 24) or catechin (Formula 27). Specifically, the polyphenol molecular portion, for example, can be but is not limited to curcumin, curcumin derivatives, quercetin, quercetin derivatives, naringenin, naringenin derivatives, eriodictyol and curcumin, apigenin and apigenin derivatives, etc. In this application, "and its derivatives" have similar meanings.

[0329] As for the metal ion part, the coordination bond between the polyphenol molecule part and the metal ion part in the metal-polyphenol complex will be broken under the low pH conditions of lysosomes (pH=5.0), and the metal ion will fall off from the metal-polyphenol complex.

[0330] In some embodiments, the metal ion moiety is selected from Fe 3+ 、Ag + 、Ba 2+ , Ca 2+ 、Cd 2+ 、Cu 2+ 、Fe 2+ 、Mn 2+Mg 2+ 、Mo 2+ 、Zn 2+ , Pt 2+ 、Au 2+ 、Al 3+ 、Ce 3+ 、Co 3+ Cr 3+ 、Eu 3+ 、Gd 3+ 、Ni 3+ 、W 3+ 、V 3+ 、Zr 3+ Preferably, the metal ion portion is selected from Fe 3+ Mg 2+ , Ca 2+ 、Al 3+ Preferably, the metal ion portion is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ .

[0331] In the present application, the dosage ratio of each component in the metal-polyphenol complex can be adjusted according to the structure of the specific metal-polyphenol complex components. The basis for adjusting the dosage ratio is that since the hydroxyl groups of the polyphenol molecules are connected to the metal ions by coordination bonds, as long as the polyphenol molecules contain multiple binding sites, the dosage ratio of the polyphenol molecules and the metal ions can be adjusted according to the number of binding sites contained in the polyphenol molecules. When used to encapsulate drugs (such as nucleic acids), the function of the metal ions is to connect the metal-polyphenol complex with the nucleic acids. Therefore, when the polyphenol molecules are connected to as few complexing sites of the metal ions as possible, it is possible to ensure that the metal-polyphenol complex encapsulates as much nucleic acid as possible.

[0332] In some embodiments, the polyphenol molecule portion is selected from curcumin, hesperetin or catechin, and the metal ion portion is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ Preferably, the polyphenol molecule is selected from curcumin (Formula 19), hesperidin (Formula 24) or catechin (Formula 27), and the metal ion is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ Preferably, the molar ratio of the polyphenol molecule portion to the metal ion portion is 1:(0.5-2). The molar ratio of the polyphenol molecule portion to the metal ion portion can be, but is not limited to, 1:0.5, 1:1, 1:1.5 or 1:2.

[0333] In one embodiment, the polyphenol molecule portion is curcumin (Formula 19), and the metal ion portion is Fe 3+ Mg 2+ or Al 3+ , the molar ratio of the polyphenol molecule part and the metal ion part is 1:1.

[0334] In the present application, the metal-polyphenol complex is prepared by reacting a polyphenol molecule with a metal ion via a coordination bond to form the metal-polyphenol complex. In one embodiment, the polyphenol molecules are dissolved in ethanol, and the metal ions and triethylamine are added for reaction to obtain the metal-polyphenol complex. The molar ratio of the polyphenol molecules to the metal ions is preferably 1:(1-2), and the molar ratio of the polyphenol molecules to the triethylamine is preferably 1:1. The reaction conditions are preferably 60°C for 2 hours.

[0335] 2. Metal-chelated polyphenol complex nanoparticles (MPNP)

[0336] In the present application, the metal-polyphenol complex particles contain: (i) a metal-polyphenol 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 conjugated lipid that inhibits particle aggregation.

[0337] Conjugated lipids that inhibit particle aggregation refer to conjugated lipids that inhibit the aggregation of drug-metal-polyphenol complex particles or targeted drugs. Their primary function is to prevent the aggregation of drug-metal-polyphenol complex particles or targeted drugs. Examples include PEG conjugated to dialkoxypropyl groups, PEG conjugated to diacylglycerols, PEG conjugated to phosphatidylethanolamine, and PEG conjugated to ceramides, preferably PEG-lipid conjugates. Cis- and trans-isomers of the lipids do not affect the effects to be achieved by the present disclosure.

[0338] In some embodiments, (ii) the conjugated lipid that inhibits particle aggregation includes a PEG-lipid conjugate and / or PEG-DAA. Preferably, the PEG-lipid conjugate is selected from 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 a combination of one or more of their derivatives. Preferably, the PEG-lipid conjugate is selected from a combination of one or more of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000, or DSPE-PEG5000. Preferably, the PEG-lipid conjugate is selected from DSPE-PEG2000 (Formula 53), DSPE-PEG700 (Formula 50), DSPE-PEG1000 (Formula 51), or DSPE-PEG5000 (Formula 52).

[0339] For non-cationic lipids or non-ionizable lipids other than the metal-polyphenol complex and the conjugated lipid that inhibits particle aggregation, they are 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 combinations of one or more thereof. Preferably, the non-cationic lipid or non-ionizable lipid in (iii) 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), lysophosphatidylcholine (LPC) (Formula 10), lysophosphatidylethanolamine (LPE) (Formula 11), lysophosphatidylserine (LPS) (Formula 12), lysophosphatidic acid (LPA) (Formula 13), lysophosphatidylglycerol (LPG) (Formula 14), lysophosphatidylinositol (LPI) (Formula 15), lysophosphatidylthreonine (LPT) (Formula 16), lysophosphocholine (LSM) (Formula 17), 1-sphingosine phosphate (S1P) (Formula 18), and a combination of one or more of their derivatives.

[0340] In some embodiments, the non-cationic lipid or non-ionizable lipid in (iii) further comprises at least one of cholesterol (preferably cholesterol of formula 40) and its derivatives in addition to the above components.

[0341] In some embodiments, 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. Preferably, the non-cationic lipid or non-ionizable lipid in (iii) comprises cholesterol (Formula 40), and a combination of one or more selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48) or DSPG (Formula 49).

[0342] In the present application, the metal-polyphenol complex as a whole reacts with component (ii) and component (iii) to self-assemble to obtain metal-polyphenol complex particles. The metal-polyphenol 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 nucleic acids on the metal-polyphenol complex particles assembled by the metal-polyphenol complex is that the polyphenol molecules are connected to the metal ions through coordination bonds to form a metal-polyphenol complex, and the metal ions of the metal-polyphenol complex are connected to the nucleic acids through coordination bonds, thereby ensuring that the metal-polyphenol complex and other components self-assemble into MPNPs while loading nucleic acids into nanoparticles to obtain drug-metal-polyphenol complex particles. In this article, the "non-cationic lipids or non-ionizable lipids other than the conjugated lipids that inhibit particle aggregation" refers to component (iii) in the metal-polyphenol complex particles, which can be simply referred to as "non-cationic lipids or non-ionizable lipids".

[0343] The proportions of the components in the metal-polyphenol complex particles can be as follows: the metal-polyphenol complex comprises 1% to 30% 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 0% to 60% by mole of the raw material; and the non-cationic lipid or non-ionizable lipid other than cholesterol comprises 29% to 75% by mole of the raw material. Here, "raw material" refers to the sum of the metal-polyphenol complex, the conjugated lipid that inhibits particle aggregation, cholesterol, and the non-cationic lipid or non-ionizable lipid other than cholesterol.

[0344] The molar proportion of the metal-polyphenol complex in the raw material can be, but is not limited to, 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% or 30%. In some embodiments, the molar proportion of the metal-polyphenol complex in the raw material is preferably 5% to 30%, preferably 10% to 30%, and preferably 5% to 20%.

[0345] 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%, preferably 3% to 5% or 5% to 10%, and more preferably 2.5%, 3%, 5% or 10%.

[0346] The non-cationic lipid or non-ionizable lipid optionally contains cholesterol, and the molar proportion of 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%, 51%, 52%, 53%, 54%, 55%, 56%, %, 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% or 60%. In some embodiments, the molar proportion of cholesterol in the raw material is 0% to 48%, preferably 10% to 30%, 30% to 47% or 10% to 20%, more preferably 10%, 30%, 37.5% or 47%.

[0347] In addition to cholesterol, the metal-polyphenol complex particles may also contain other non-cationic lipids or non-ionizable lipids, which may comprise, but are not limited to, 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% or 75% by mole in the raw material. In some embodiments, the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 30% to 75%, preferably 40% to 75%, preferably 45% to 55%, 60% to 65% or 50% to 65%, and further 45%, 50%, 55%, 60% or 65%.

[0348] The metal-polyphenol complex particles are prepared by mixing the metal-polyphenol complex, a conjugated lipid that inhibits particle aggregation, a non-cationic lipid, or a non-ionizable lipid to prepare drug-metal-polyphenol complex particles. In some embodiments, the metal-polyphenol complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or non-ionizable lipid are mixed in a homogeneous phase, such as an organic phase (in some embodiments, the solvent is ethanol).

[0349] When the metal-polyphenol complex particles are loaded with a drug, the preparation method is to mix the metal-polyphenol 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-polyphenol complex particles. In some embodiments, the metal-polyphenol 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 the drug-metal-polyphenol complex particles. In some embodiments, the buffer can be PBS buffer or Tris-HCl buffer. In some embodiments, the organic phase and the aqueous phase can be mixed using a microfluidic chip or ultrasound.

[0350] 3. Targeting carrier (containing MPNP)

[0351] In the present application, 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 carrier proteins. Preferably, the lipid delivery system is a metal-polyphenol complex particle, LNP, or a phospholipid delivery system. In one embodiment, the pseudovirus is an adenovirus. In one embodiment, the cell is a red blood cell. In one embodiment, the carrier protein is apoferritin. In one embodiment, the LNP is a cationic lipid or an ionizable lipid.

[0352] In one embodiment, the carrier delivery system is a metal-polyphenol complex particle (MPNP), and the targeting structure is attached to the outer surface of the metal-polyphenol complex particle (MPNP).

[0353] In some embodiments, 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-polyphenol complex particle based on hydrophilicity and hydrophobicity.

[0354] In some embodiments, 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-polyphenol complex particle through this portion.

[0355] In some embodiments, the linking region comprises at least one or more of PEG-2000 and its derivatives.

[0356] In some embodiments, 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) by specific recognition, thereby enabling the drug-loaded LNP particles or metal-polyphenol complex particles to transport the drug and act on the target (i.e., the target or target cell of the drug). In some embodiments, 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. In some embodiments, the targeting binding region comprises at least one of a nucleic acid, a polypeptide, a protein, or a small molecule. In some embodiments, the targeting binding region comprises one of an aptamer, an antibody, an antigen binding portion, or galnac. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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). In some embodiments, the targeting binding region is an aptamer, and the target cell of the targeting binding region is a liver cancer cell.

[0357] In one embodiment, the targeting structure is DSPE-PEG2000-aptamer, preferably DSPE-PEG2000-CD62L aptamer, and the CD62L aptamer sequence is shown in SEQ ID NO.44.

[0358] In this application, 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. Taking the DSPE-PEG2000-CD62L aptamer as an example, 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. In some embodiments, 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.

[0359] In one embodiment, the DSPE-PEG2000-aptamer is first prepared into micelles and then attached to the outer surface of a carrier delivery system (e.g., metal-polyphenol complex particles (MPNPs)) to form a targeted carrier. In some embodiments, the micelles are prepared by direct dissolution, ethanol infusion, dialysis, or ultrasound.

[0360] When the targeting carrier is used for drug encapsulation, the drug, metal-polyphenol 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-polyphenol complex particles), and then the targeting structure is reacted and connected with the drug-lipid particles to obtain the targeted drug.

[0361] In some embodiments, 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.

[0362] In some embodiments, 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., and 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.

[0363] 4. Targeted drugs

[0364] In the present application, 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-polyphenol composite particles.

[0365] In the present application, 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. In some embodiments, 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.

[0366] In some embodiments, the drug is mRNA, which is an mRNA encoding a chimeric antigen receptor CAR or TCR. At this time, 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. At this time, 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.

[0367] In some embodiments, 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 costimulatory signaling region, and a linker region between the antigen binding domain and the transmembrane domain. In some embodiments, 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 costimulatory 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 the transmembrane domain linker region is selected from at least one of SEQ ID No. 40, SEQ ID No. 41, and SEQ ID No. 42.

[0368] In one embodiment, the drug is an mRNA having a nucleotide sequence as shown in SEQ ID No.43.

[0369] Furthermore, the nucleic acid encapsulated in the targeted drug of the present application is resistant to degradation by nucleases in aqueous solution.

[0370] In some embodiments, the drug is fully encapsulated inside the metal-polyphenol 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.

[0371] In some embodiments, the targeted drugs provided herein have a small diameter suitable for systemic delivery.

[0372] In some embodiments, the drugs of the present application are preferably nucleic acids, and 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.

[0373] The targeted drug or preparation method of the present application can be used for drug delivery, imaging drugs, and vaccines.

[0374] In some embodiments, targeted drugs are used for drug delivery, treatment and / or prevention.

[0375] In some embodiments, 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.

[0376] In some embodiments, 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. Preferably, the mammal is a human. Preferably, the disease or condition being treated is associated with expression of a gene that contains the drug's target sequence.

[0377] In some embodiments, 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.

[0378] In some embodiments, the cancer includes hematologic malignancies and solid tumors. Preferably, 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). Preferably, 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.

[0379] In some embodiments, the viral infection comprises hepatitis B virus, hepatitis C virus, SARS-CoV-2, human immunodeficiency virus, cytomegalovirus, invasive Aspergillus, or a conjugate virus.

[0380] In some embodiments, 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.

[0381] In some embodiments, 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).

[0382] In some embodiments, the metabolic disease comprises atherosclerosis, congenital hyperinsulinemia, nonalcoholic steatohepatitis, or non-obese diabetes mellitus (NOD).

[0383] In some embodiments, 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. Preferably, skin diseases comprise keloids or wound healing.

[0384] In some embodiments, the administration route of the targeted drug includes intrathecal injection, intramuscular administration, intracranial injection, intravenous injection, or intratumoral injection.

[0385] In some embodiments, the targeted drug is used in combination therapy.

[0386] In some embodiments, the targeted drug is an immune cell targeted drug, and the immune cells are T cells and / or myeloid cells.

[0387] In some embodiments, the immune cell targeted drug is a chimeric antigen receptor (CAR) drug.

[0388] The present application provides a pharmaceutical agent containing a targeted drug, which is preferably a vaccine, and more preferably a new coronavirus vaccine.

[0389] When the drug is a nucleic acid, 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.

[0390] In some embodiments, 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. In the pharmaceutical agent, 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.

[0391] For 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.

[0392] Genes associated with viral infection and survival include those expressed by viruses to bind, enter, and replicate in cells, particularly viral sequences associated with chronic viral diseases. For example, viral sequences include sequences of hepatitis viruses (Hamasaki, et al., FEBS Lett. 543:51 (2003); Yokota, et al, EMBO Rep. 4:602 (2003); Schlomai, et al, Hepatology 37:764 (2003); Wilson, et al, Proc. Natl. Acad. Sci. 100:2783 (2003); Kapadia, et al, Proc. Natl. Acad. Sci. 100:2014 (2003); and FIELDSVIROLOGY (Knipe et al. eds. 2001)), human immunodeficiency virus (HIV) (Banerjea, et al, Mol Ther. 8:62 (2003); Song, et al, 2003). 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)), and human papillomavirus (HPV) (Hall, et al., J. Virol. 77:6066 (2003); Jiang, et al., Oncogene 21:6041 (2002)). 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). 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. NC_004102; hepatitis D nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_001653; hepatitis E nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_001434; and hepatitis G nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_001710. 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.

[0393] Genes associated with metabolic diseases and disorders (e.g., disorders in which the liver is targeted and liver diseases and disorders) include, for example, genes expressed in dyslipidemia (e.g., liver X receptors (e.g., LXRα and LXRβ Genbank Accession No. NM_007121)), farnesoid X receptor (FXR) (Genbank Accession No. NM_005123), sterol regulatory element binding protein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase (HMG-Coenzyme-A reductase), apolipoprotein (ApoB), and apolipoprotein (ApoE)) and 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, et al., Cell 85: 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. al., J. Biol. Chem. 272(6): 3137-3140 (1997); Janowski, et al., Nature 383:728-731 (199; Peet, et al., Cell 93:693-704 (1998)). Those skilled in the art will understand that genes associated with metabolic diseases and disorders (e.g., diseases and disorders in which the liver is targeted and liver 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 pharmaceutical agents for treating the disease or disorder.

[0394] Examples of genes associated with tumorigenesis and cell transformation include translocation sequences such as MLL fusion gene, BCR-ABL (Wilda, et al., Oncogene, 21:5716 (2002); Scherr, et al, Blood 101:1566), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO and AML1-MTG8 (Heidenreich, et al, Blood 101:3157 (2003)); overexpressed sequences such as multidrug resistance genes (Nieth, et al, FEBS Lett. 545:144 (2003); Wu, et al, Cancer Res. 63:1515 (2003)), cell cycle proteins (Li, et al, Cancer Res. 63:3593 (2003); Zou, et al, Cancer Res. 63:3607 (2003)). al., Genes Dev. 16:2923 (2002)), β-catenin (Verma, et al., Clin Cancer Res. 9:1291 (2003)), telomerase gene (Kosciolek, et al., Mol Cancer Ther. 2:209 (2003)), c-MYC, N-MYC, BCL-2, ERBB1 and ERBB2 (Nagy, et al. Exp. Cell Res. 285:39 (2003)); and mutant sequences such as RAS (reviewed in Tuschl and Borkhardt, Mol. Interventions, 2:158 (2002)). Silencing sequences encoding DNA repair enzymes is used in conjunction with administration of chemotherapeutic agents (Collis, et al., Cancer Res. 63:1550 (2003)). Genes encoding proteins associated with tumor migration, such as integrins, selectins, and metalloproteinases, are also target sequences of interest. Any complete or partial gene sequence that is beneficial to or promotes tumorigenesis or cell transformation, tumor growth, or tumor migration can be included as a template sequence.

[0395] Angiogenic genes can promote the formation of new blood vessels, and vascular endothelial growth factor (VEGF) is a key research direction (Reich, et al., Mol. Vis. 9: 210 (2003)).

[0396] Immunomodulator genes are genes that regulate one or more immune responses. Examples of immunomodulator genes include cytokines such as growth factors (e.g., TGF-α, TGF-β, 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-α, IFN-β, IFN-γ, etc.) and TNF. Fas and Fas ligand genes are also immunomodulator target sequences of interest (Song, et al., Nat. Med. 9: 347 (2003)). Also included are genes encoding secondary signaling molecules in hematopoietic and lymphoid cells, for example, Tec family kinases, such as Bruton's tyrosine kinase (Btk) (Heinonen, et al., FEBS Lett. 527:274 (2002)).

[0397] Cell receptor ligands include ligands that bind to cell surface receptors (e.g., insulin receptors, EPO receptors, G-protein coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) to modulate (e.g., inhibit, activate, etc.) physiological pathways involved in the receptors (e.g., glucose level regulation, blood cell development, mitogenesis, etc.). Examples of cell receptor ligands include cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G-protein coupled receptor ligands, etc.). Templates encoding trinucleotide repeat sequence (e.g., CAG repeat sequence) expansions have been found to be useful for silencing pathogenic sequences in neurodegenerative diseases caused by trinucleotide repeat sequence expansions, such as spinal bulbar muscular atrophy and Huntington's disease (Caplen, et al., Hum. Mol. Genet. 11: 175 (2002)).

[0398] 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. Patents 5,543,158; 5,641,515; and 5,399,363. The targeted drugs 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 throughout the organism. Solutions of the targeted drugs 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. Optionally, these formulations contain a preservative to prevent microbial growth. Typically, when administered intravenously, the targeted drug formulation is formulated with a suitable pharmaceutical carrier. Typically, a normal buffered saline solution (135-150 mM NaCl) is 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). As used herein, "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. The phrase "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 is conventional in the art, and the compositions contain the protein as the active ingredient. Optionally, these compositions are prepared as injectable solutions, liquid solutions, or suspensions; solid forms suitable for solution or suspension in a liquid prior to injection can also be prepared. The formulations can also be emulsified.

[0399] Can be sterilized by conventional liposome sterilization technology, such as filtration to targeted drug.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.

[0400] Prophylactic and therapeutic treatments: In some embodiments, 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." In determining the effective amount of a targeted drug to be administered for the treatment or prevention of a disease due to expression or overexpression of a target gene, 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.

[0401] For example, the targeted drug can be administered to a subject who is infected by pathogenic microorganisms or who is at risk of being infected by pathogenic microorganisms. The drug should preferably correspond to a sequence, and should also be unique (or at least lacking in the genome of the natural genome of the patient undergoing treatment) for microorganisms, and the sequence has a key role in the life history of microorganisms. By ex vivo or intravenous injection, the targeted drug is introduced into target cells, tissues or organs with a therapeutically effective dose. The sequence silencing of the gene encoding the gene relevant to pathogenic infection can be conveniently combined with the use of conventional reagents for the treatment of pathogenic diseases. The treatment can be prophylactically administered to a person who is at risk of being infected by pathogenic microorganisms or who has been infected by pathogenic microorganisms.

[0402] In a preferred embodiment, 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. For example, 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). Those skilled in the art will appreciate that 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 alpha) (see, e.g., Medina et al., Antiviral Res. 60(2):135-143 (2003); ribavirin (see, e.g., Hugle and Cerny, Rev. Med. Virol. 13(6):361-71 (2003); adefovir and lamivudine (see, e.g., Kock et al., Hepatology 38(6):1410-8 (2003); prenylation inhibitors (see, e.g., Bordier et al., J. Clin. Invest. 112(3):407-414 (2003)); famciclovir (see, e.g., Yurdaydin et al., J. Hepatol. 37(2):266-71 (2002); and saikosaponins c and d (see, e.g., Chiang et al., Planta Med. 69(8):705-9 (2003).

[0403] In another embodiment, 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.In some respects, 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.Those skilled in the art will appreciate that the silence of the gene relevant to metabolic diseases and illness can be combined with the conventional treatment of these diseases. For example, silencing of genes involved in dyslipidemia can be combined with the use of 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) antagonists and AGI1067) functional oils, PPAR-α, γ, Therapeutic combinations of delta agonists, as well as dual PPAR-α, / γ and 'pan' PPAR-α / γ, / δ agonists, cholesteryl ester transfer protein (CETP) inhibitors (such as torcetrapib), CETP vaccines, upregulators of ATP-binding cassette transporter (ABC) A1, lecithin cholesterol acyltransferase (LCAT) and scavenger receptor class B type 1 (SRB1), and synthetic apolipoprotein (Apo) E-related peptides, extended-release niacin / lovastatin, atorvastatin / amlodipine, ezetimibe / simvastatin, atorvastatin / CETP inhibitors, statin / PPAR agonists, extended-release niacin / simvastatin and pravastatin / aspirin in development, and anti-obesity agents (see, e.g., Bays and Stein, Expert Opin. Pharmacother. 4(11):1901-38 (2003)). Likewise, silencing of genes involved in diabetes can be combined with treatment with insulin, as well as dietary modifications and exercise.

[0404] In another embodiment, 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). 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.

[0405] 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.

[0406] Detecting the Particles: The targeted drug herein can be detected using any method known in the art. For example, 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., (e.g., fluorescein and derivatives, such as fluorescein isothiocyanate (FITC) and Oregon Green™; rhodamine and derivatives, such as Texas Red, tetrarhodimine isothiocynate (TRITC), etc., digoxigenin, biotin, phycoerythrin, AMCA, CyDyes™, 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 label is detected using any means known in the art.

[0407] Detection of Nucleic Acids: 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.

[0408] The sensitivity of hybridization assays can be increased by the use of nucleic acid amplification systems that multiply the amount of 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. Examples of techniques sufficient to guide one skilled in the art by these in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification, and other RNA polymerase-mediated techniques (e.g., NASBA™) are found in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2000, and Ausubel et al., SHORT PROTOCOLS IN MOLECULAR BIOLOGY, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (2002), and Mullis et al. (1987), U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October 2001). 1, 1990), C&EN 36; The Journal 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. al., J. Clin. Chem., 35: 1826 (1989); Landegren et al., Science, 241: 1077 (1988); Van Brunt, Biotechnology, 8: 291 (1990); Wu and Wallace, Gene, 4: 560 (1989); Barringer et al. al., Gene, 89: 117 (1990), and Sooknanan and Malek, Biotechnology, 13: 563 (1995).Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., US Patent No. 5,426,039. Other methods described in the art are nucleic acid sequence-based amplification (NASBA™, Cangene, Mississauga, Ontario) and the Qβ replicase system.

[0409] Oligonucleotides for use as probes in in vitro amplification methods, as gene probes, or as inhibitor components are chemically synthesized, for example, using an automated synthesizer, typically according to the solid-phase phosphoramidite triester method described by Beaucage and Caruthers, Tetrahedron Letts., 22(20): 1859-1862 (1981), as described by Needham VanDevanter et al., Nucleic Acids Res., 12: 6159 (1984). If necessary, oligonucleotides are typically purified by native acrylamide gel electrophoresis or by anion exchange HPLC, as described by Pearson and Regnier, J. Chrom., 255: 137-149 (1983). The sequence of synthetic oligonucleotides can be confirmed using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology, 65:499.

[0410] Compared with CD8 or CD3 as targets, CD62L as a target for immune cell drugs can increase the expression level of drugs and be more conducive to the treatment of diseases.

[0411] The examples that follow provide illustration, but do not limit the claimed application. Those skilled in the art will readily recognize a variety of noncritical parameters that can produce substantially the same or similar results.

[0412] The drug-lipid particles of this application refer to drug-lipid particles other than those containing cationic / ionizable lipids, namely drug-loaded metal-chelated polyphenol complex nanoparticles (drug@MPNPs), which are linked to a targeting structure to obtain a targeted drug. After removing the drug portion of the targeted drug, the remaining components include the targeting carrier.

[0413] Metal (containing Fe 3+ 、Al 3+ or Mg 2+Preparation of Metal-chelated polyphenol complex nanoparticles (MPNP)

[0414] The principle of loading nucleic acids into metal-polyphenol complex particles assembled by metal-polyphenol complexes is: polyphenol molecules are connected to metal ions through coordination bonds to form metal-polyphenol complexes, and the metal ions of the metal-polyphenol complex are connected to nucleic acids through coordination bonds, thereby ensuring that the metal-polyphenol complex and other components self-assemble into MPNPs while loading nucleic acids into nanoparticles.

[0415] Example 1: Preparation of drug-metal (Fe 3+ 、Al 3+ or Mg 2+ )-polyphenol complex particles

[0416] Example 1.1. Preparation of Metal-Polyphenol Complex

[0417] Example 1.1.1 Preparation of Metal Ions: Fe 3+ Metal-polyphenol complex

[0418] Curcumin (Formula 19) was dissolved in ethanol at 1.5 mg / ml, and anhydrous FeCl₃ was added at a molar ratio of 1:1. The reaction was refluxed at 60°C for 1 hour. After the reaction, the solution was dried, and the product was dissolved in ultrapure water and filtered. The resulting product, after lyophilization, was the metal-polyphenol complex. The structure of the metal-polyphenol complex is shown below.

[0419] Result analysis: Curcumin (Formula 19) and FeCl3 were reacted at 60°C for 1 hour, the curcumin (Formula 19) feed concentration was 1.5 mg / mL, and the feed ratio of curcumin (Formula 19) to FeCl3 was 1:1, and the yield of the target product obtained was 95%.

[0420] Example 1.1.2 Preparation of Metal Ions: Al 3+ Metal-polyphenol complex

[0421] The difference between this example and Example 1.1.1 is that FeCl3 is replaced by Al(NO3)3·9H2O. The structure of the prepared metal-polyphenol composite is shown below.

[0422] Result analysis: Curcumin (Formula 19) and Al(NO3)3·9H2O were reacted at 60°C for 1 hour, the curcumin (Formula 19) feed concentration was 1.5 mg / mL, and the feed ratio of curcumin (Formula 19) to Al(NO3)3·9H2O was 1:1, and the yield of the target product obtained was 98%.

[0423] Example 1.1.3 Preparation of Metal Ions as Mg 2+ Metal-polyphenol complex

[0424] The difference between this example and example 1.1.1 is that FeCl3 is replaced by MgCl2. The structure of the prepared metal-polyphenol complex is shown below.

[0425] Result analysis: Curcumin (Formula 19) and MgCl2 were reacted at 60°C for 1 hour, the curcumin (Formula 19) feed concentration was 1.5 mg / mL, and the feed ratio of curcumin (Formula 19) to MgCl2 was 1:1. The yield of the target product obtained was 98.5%.

[0426] Example 1.2, Preparation of mRNA-metal (Fe 3+ 、Al 3+ or Mg 2+ mRNA-loaded metal-chelated polyphenol complex nanoparticles (mRNA@MPNP)

[0427] Preparation of metal ions as Fe 3+ mRNA-metal-polyphenol complex particles

[0428] A metal-polyphenol complex was prepared according to the method described in Example 1.1.1, with curcumin and FeCl3 added at a 1:1 ratio. The metal-polyphenol 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-polyphenol complex, DSPC, CHOL, and DSPE-PEG2000 accounted for 5%, 60%, 30%, and 5%, respectively. mRNA was dissolved at a concentration of 20 μg / mL in enzyme-free PBS buffer (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. The total mass of the metal-polyphenol complex, the distearoylphosphatidylcholine (DSPC), the DSPE-PEG2000, and the cholesterol (CHOL) was mixed with the mRNA mass in a microfluidic chip at a mass ratio of 40:1. 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 an mRNA encoding the fluorescent protein eGFP, whose sequence was SEQ ID NO.1 (720 nt). Drug mRNA-metal-polyphenol complex particles (mRNA@MPNP) were prepared, which are drug-lipid particles. The eGFP-mRNA@MPNP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPNP. After 48 hours, the cell suspension was collected, and the percentage of eGFP-positive cells was detected by flow cytometry.

[0429] The particle size, surface potential and stability of the prepared mRNA-metal-polyphenol composite particles (mRNA@MPNP) were tested, and the efficiency of mRNA@MPNP encapsulation of nucleic acid was calculated.

[0430] 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.

[0431] Method for detecting surface potential and result judgment standard: The surface potential of nanoparticles was tested using a Malvern Zetasizer laser particle size analyzer, and a potential in the range of -10 to 10 mV was considered acceptable.

[0432] 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.

[0433] Method for calculating nucleic acid encapsulation efficiency: Specifically, agarose gel electrophoresis is used. First, the nucleic acid feed amount of each group of lipid nanoparticles is set at 10 μg / mL, and the mass ratio of metal-polyphenol complex particles to nucleic acid is 40:1. 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. Electrophoresis is stopped when the free nucleic acid bands are clearly distinguishable. Image J software is used to calculate the grayscale value of free nucleic acids in different groups. The positive control group is set as 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.

[0434] Cell culture method: Human embryonic kidney cell line 293T was cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0435] Flow cytometry analysis of eGFP-positive cell percentage: 293T cells were seeded in 12-well plates at a seeding density of 5 × 10 5 Cells were plated at 1 mL of MPNP or mRNA@MPNP at a concentration of 2 μg / mL at 80% cell density. After 48 hours, the cell suspension was harvested and 20,000 cells were collected using the FITC channel of a flow cytometer. The percentage of eGFP-positive cells was analyzed using the following formula: eGFP-positive cell percentage = number of eGFP-expressing cells / total number of cells × 100%. An eGFP-positive cell percentage of 40% or higher was considered acceptable.

[0436] The principle of loading nucleic acid on metal-chelated polyphenol complex nanoparticles (MPNPs) assembled by metal-polyphenol complexes is that curcumin binds to Fe through coordination bonds. 3+ connected to form a metal-polyphenol complex, the Fe 3+The metal-polyphenol complex is linked to the nucleic acid through a coordination bond, ensuring that the metal-polyphenol complex and other components self-assemble into MPNPs while simultaneously loading the nucleic acid into the nanoparticles. There are two possible possibilities for curcumin's contribution to MPNP loading: 1) Curcumin interacts with nucleic acids, assisting MPNP loading with nucleic acids, for example, by inserting into the minor groove of nucleic acids; 2) Curcumin may not directly interact with nucleic acids.

[0437] Preparation of metal ions as Al 3+ mRNA-metal-polyphenol complex particles

[0438] A metal-polyphenol complex was prepared according to the method of Example 1.1.2, wherein curcumin (Formula 19) and Al(NO₃)₃·9H₂O were added at a 1:1 feed ratio. The metal-polyphenol complex, distearoylphosphatidylcholine (DSPC, Formula 46, a non-cationic lipid or non-ionizable lipid), cholesterol (CHOL, Formula 40, a non-cationic lipid or non-ionizable lipid), and DSPE-PEG2000 (Formula 53, a conjugated lipid that inhibits particle aggregation) were dissolved in ethanol at varying molar ratios as the organic phase. The proportions of the metal-polyphenol complex, DSPC (Formula 46), CHOL (Formula 40), and DSPE-PEG2000 (Formula 53) were 5%, 45%, 47%, and 3%, respectively. mRNA was dissolved in PBS (PBS consisting of 0.137M sodium chloride, 0.0027M potassium chloride, 0.01M disodium hydrogen phosphate, and 0.0018M potassium dihydrogen phosphate) at a concentration of 20 μg / mL as the aqueous phase. The metal-polyphenol complex was mixed with the mRNA at a mass ratio of 18:1 in a microfluidic chip. The volume ratio of the aqueous phase to the organic phase was 3:1. The flow rate of the organic and aqueous phases in the microfluidic chip was 12 ml / min. The drug mRNA was mRNA encoding the fluorescent protein eGFP, whose sequence is SEQ ID NO.1 (720 nt). eGFP-mRNA@MPNP was prepared. eGFP-mRNA@MPNP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPNP. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0439] The particle size, surface potential and stability of the prepared eGFP-mRNA@MPNP were tested, and the efficiency of nucleic acid encapsulation of eGFP-mRNA@MPNP was calculated.

[0440] 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 30 to 400 nm was considered acceptable.

[0441] Method for detecting surface potential and result judgment standard: The surface potential of nanoparticles was tested using a Malvern Zetasizer laser particle size analyzer, and a potential in the range of -10 to 10 mV was considered acceptable.

[0442] 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.

[0443] Method for calculating nucleic acid encapsulation efficiency: Specifically, agarose gel electrophoresis was used. First, the nucleic acid loading for each lipid nanoparticle group was set at 10 μg / mL, and the mass ratio of the metal-polyphenol complex to nucleic acid was 18:1. Equal concentrations of nucleic acid were dissolved 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 a positive control, and 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 a negative control. The agarose gel concentration was 1.5%. At this point, the gaps in the gel only allow the passage of free nucleic acids, not lipid nanoparticles. Electrophoresis was stopped when the free nucleic acid bands were clearly distinguishable. Image J software was used to calculate the grayscale values ​​of free nucleic acids in different groups. The positive control group was set as 100%. The ratio of free nucleic acid in each group to the positive control was the relative amount of free nucleic acid. The entrapment efficiency of each group was calculated as (100 - relative amount of free nucleic acid)%. A nucleic acid entrapment efficiency of 50% or higher was considered acceptable.

[0444] Cell culture method: Human embryonic kidney cell line 293T was cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0445] Flow cytometry analysis of eGFP-positive cell percentage: 293T cells were seeded in 12-well plates at a seeding density of 5 × 10 5 Cells were plated at 1 mL of MPNP or eGFP-mRNA@MPNP at a concentration of 2 μg / mL at 80% cell density. After 48 hours, the cell suspension was harvested and 20,000 cells were collected using the FITC channel of a flow cytometer. The percentage of eGFP-positive cells was analyzed using the following formula: eGFP-positive cell percentage = number of eGFP-expressing cells / total number of cells × 100%. An eGFP-positive cell percentage of 40% or higher was considered acceptable.

[0446] The principle of loading nucleic acid on metal-chelated polyphenol complex nanoparticles (MPNPs) assembled by metal-polyphenol complexes is that curcumin binds to Fe through coordination bonds. 3+ or Al 3+ connected to form a metal-polyphenol complex, the Fe 3+ or Al 3+ The metal-polyphenol complex is linked to the nucleic acid through a coordination bond, ensuring that the metal-polyphenol complex and other components self-assemble into MPNPs while simultaneously loading the nucleic acid into the nanoparticles. There are two possible possibilities for curcumin's contribution to MPNP loading: 1) Curcumin interacts with nucleic acids, assisting MPNP loading with nucleic acids, for example, by inserting into the minor groove of nucleic acids; 2) Curcumin may not directly interact with nucleic acids.

[0447] Preparation of metal ions as Mg 2+ mRNA-metal-polyphenol complex particles

[0448] A metal-polyphenol complex was prepared according to the method of Example 1.1.3, wherein curcumin (Formula 19) and MgCl2 were added at a 1:1 feed ratio, and the metal-polyphenol complex, distearoylphosphatidylcholine (DSPC, Formula 46, a non-cationic lipid or non-ionizable lipid), cholesterol (CHOL, Formula 40, a non-cationic lipid or non-ionizable lipid), and DSPE-PEG2000 (Formula 53, a conjugated lipid that inhibits particle aggregation) were dissolved in ethanol at varying molar proportions as the organic phase. The proportions of the metal-polyphenol complex, DSPC (Formula 46), CHOL (Formula 40), and DSPE-PEG2000 (Formula 53) were 10%, 50%, 37.5%, and 2.5%, respectively. mRNA was dissolved in PBS (PBS consisting of 0.137M sodium chloride, 0.0027M potassium chloride, 0.01M disodium hydrogen phosphate, and 0.0018M potassium dihydrogen phosphate) at a concentration of 20 μg / mL as the aqueous phase. The metal-polyphenol complex was mixed with the mRNA at a mass ratio of 18:1 in a microfluidic chip. The volume ratio of the aqueous phase to the organic phase was 3:1. The flow rate of the organic and aqueous phases in the microfluidic chip was 12 ml / min. The drug mRNA was mRNA encoding the fluorescent protein eGFP, whose sequence is SEQ ID NO.1 (720 nt). eGFP-mRNA@MPNP was prepared. eGFP-mRNA@MPNP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPNP. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0449] The particle size, surface potential and stability of the prepared eGFP-mRNA@MPNP were tested, and the efficiency of nucleic acid encapsulation of eGFP-mRNA@MPNP was calculated.

[0450] 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 20 to 400 nm was considered acceptable.

[0451] Method for detecting surface potential and result judgment standard: The surface potential of nanoparticles was tested using a Malvern Zetasizer laser particle size analyzer, and a potential in the range of -10 to 10 mV was considered acceptable.

[0452] 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.

[0453] Method for calculating nucleic acid encapsulation efficiency: Specifically, agarose gel electrophoresis was used. First, the nucleic acid loading for each lipid nanoparticle group was set at 10 μg / mL, and the mass ratio of the metal-polyphenol complex to nucleic acid was 18:1. Equal concentrations of nucleic acid were dissolved 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 a positive control, and 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 a negative control. The agarose gel concentration was 1.5%. At this point, the gaps in the gel only allow the passage of free nucleic acids, not lipid nanoparticles. Electrophoresis was stopped when the free nucleic acid bands were clearly distinguishable. Image J software was used to calculate the grayscale values ​​of free nucleic acids in different groups. The positive control group was set as 100%. The ratio of free nucleic acid in each group to the positive control was the relative amount of free nucleic acid. The entrapment efficiency of each group was calculated as (100 - relative amount of free nucleic acid)%. A nucleic acid entrapment efficiency of 50% or higher was considered acceptable.

[0454] Cell culture method: Human embryonic kidney cell line 293T was cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0455] Flow cytometry analysis of eGFP-positive cell percentage: 293T cells were seeded in 12-well plates at a seeding density of 5 × 10 5Cells were plated at 1 mL of MPNP or eGFP-mRNA@MPNP at a concentration of 2 μg / mL at 80% cell density. After 48 hours, the cell suspension was harvested and 20,000 cells were collected using the FITC channel of a flow cytometer. The percentage of eGFP-positive cells was analyzed using the following formula: eGFP-positive cell percentage = number of eGFP-expressing cells / total number of cells × 100%. An eGFP-positive cell percentage of 40% or higher was considered acceptable.

[0456] The principle of loading nucleic acid on metal-chelated polyphenol complex nanoparticles (MPNPs) assembled by metal-polyphenol complexes is that curcumin binds to Fe through coordination bonds. 3+ or Al 3+ connected to form a metal-polyphenol complex, the Fe 3+ or Al 3+ Mg 2+ The metal-polyphenol complex is linked to the nucleic acid through a coordination bond, ensuring that the metal-polyphenol complex and other components self-assemble into MPNPs while simultaneously loading the nucleic acid into the nanoparticles. There are two possible possibilities for curcumin's contribution to MPNP loading: 1) Curcumin interacts with nucleic acids, assisting MPNP loading with nucleic acids, for example, by inserting into the minor groove of nucleic acids; 2) Curcumin may not directly interact with nucleic acids.

[0457] Example 1.2.1, (Fe 3+ 、Al 3+ or Mg 2+ ) The proportion of the components of the metal-polyphenol complex

[0458] The curcumin and FeCl3 in Example 1.2 were added in different ratios (1:1, 3:2, 2:1), and the other steps were the same as in Example 1.2 to prepare different mRNA@MPNP (Fe 3+ ), and their nucleic acid loading rates were detected respectively.

[0459] Result analysis: As shown in Table 1-1, when the ratio of curcumin and FeCl3 was 1:1, the mRNA encapsulation efficiency of the prepared drug-lipid particles was 85%; when the ratio of curcumin and FeCl3 was 3:2, the mRNA encapsulation efficiency of the prepared drug-lipid particles was 72%; when the ratio of curcumin and FeCl3 was 2:1, the mRNA encapsulation efficiency of the prepared drug-lipid particles was 63%. 3+ The function of curcumin is to connect with nucleic acid.3+ Since there are at most three complexation sites, the curcumin and FeCl₃ ratio in the drug-lipid particle should be 1:1 to ensure maximum nucleic acid loading. Our results also confirmed that a 1:1 curcumin:FeCl₃ ratio resulted in the highest mRNA loading efficiency. Nucleic acid loading efficiency in drug-lipid particles exceeded 60% when the curcumin:FeCl₃ ratio ranged from 1:1 to 2:1.

[0460] Table 1-1 Component ratios of metal-polyphenol complexes and functions of the drug-lipid particles prepared therefrom

[0461] Curcumin (Formula 19) and Al(NO3)3·9H2O in Example 1.2 were added in different ratios (1:1, 3:2, 2:1), and the other steps were the same as in Example 1.2 to prepare different eGFP-mRNA@MPNP (Al 3+ ), and their nucleic acid loading rates were detected respectively.

[0462] Result analysis: As shown in Table 1-1-2, when the ratio of curcumin (Formula 19) and Al(NO3)3·9H2O was 1:1, the eGFP-mRNA encapsulation efficiency of the prepared metal-polyphenol composite particles was 86%; when the ratio of curcumin (Formula 19) and Al(NO3)3·9H2O was 3:2, the eGFP-mRNA encapsulation efficiency of the prepared metal-polyphenol composite particles was 70%; when the ratio of curcumin (Formula 19) and Al(NO3)3·9H2O was 2:1, the eGFP-mRNA encapsulation efficiency of the prepared metal-polyphenol composite particles was 66%. In the metal-polyphenol composite particles, Al 3+ The function of Al is to connect the metal-polyphenol complex with nucleic acid. 3+ Since there are a maximum of three complexation sites, the ratio of curcumin and Al(NO3)3·9H2O in the drug-lipid particles should be 1:1 to ensure that the metal-polyphenol complex particles can encapsulate as many nucleic acids as possible. Experimental results also confirmed that when the curcumin and Al(NO3)3·9H2O ratio is 1:1, the metal-polyphenol complex particles prepared with it have the highest eGFP-mRNA encapsulation efficiency. When the curcumin and Al(NO3)3·9H2O ratio ranges from 1:1 to 2:1, the nucleic acid encapsulation efficiency of the metal-polyphenol complex particles exceeds 60%.

[0463] Table 1-1-2 Metal ions are Al 3+ The proportion of components of the metal-polyphenol complex and the function of the metal-polyphenol complex particles prepared by the method

[0464] The curcumin (Formula 19) and MgCl2 in Example 1.2 were added in different ratios (1:1, 3:2), and the other steps were the same as in Example 1.2 to prepare different eGFP-mRNA@MPNP (Mg 2+ ), and their nucleic acid loading rates were detected respectively.

[0465] Result analysis: As shown in Table 1-1-3, when the ratio of curcumin (Formula 19) and MgCl2 was 1:1, the eGFP-mRNA encapsulation efficiency of the prepared metal-polyphenol composite particles was 88%; when the ratio of curcumin (Formula 19) and MgCl2 was 3:2, the eGFP-mRNA encapsulation efficiency of the prepared metal-polyphenol composite particles was 75%. 2+ The function of Mg is to connect the metal-polyphenol complex with nucleic acid. 2+ Since there are at most two complexation sites, the curcumin and MgCl₂ ratio in the drug-lipid particles should be 1:1 to ensure maximum nucleic acid loading within the metal-polyphenol complex particles. Experimental results also confirmed that a 1:1 curcumin:MgCl₂ ratio resulted in the highest eGFP-mRNA loading efficiency. Nucleic acid loading rates of the metal-polyphenol complex particles exceeded 60% when the curcumin:MgCl₂ ratio ranged from 1:1 to 3:2.

[0466] Table 1-1-3 Metal ions are Mg 2+ The proportion of components of the metal-polyphenol complex and the function of the metal-polyphenol complex particles prepared by the method

[0467] Example 1.2.2 Preparation of Metals (Fe 3+ 、Al 3+ or Mg 2+ )-polyphenol complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000 and cholesterol (CHOL) ratio

[0468] Compared with Example 1.2, the ratio of the metal-polyphenol complex, the distearoylphosphatidylcholine (DSPC, Formula 46), the DSPE-PEG2000, and the cholesterol (CHOL) is as shown in Table 1-2 (the metal ion is Fe 3+ ), Table 1-2-2 (metal ions are Al 3+ ) and Table 1-2-3 (metal ion is Mg 2+ ), the other conditions are the same.

[0469] Result analysis: As shown in Table 1-2, the metal ion is Fe 3+ When the metal-polyphenol complex (metal ion is Fe 3+ When the metal-polyphenol complex accounted for 5% of the drug-lipid particle, the DSPC accounted for 40% of the drug-lipid particle, the CHOL accounted for 0% of the drug-lipid particle, and the DSPE-PEG2000 accounted for 2% of the drug-lipid particle, the particle size was in the range of 50-400 nm, the surface potential was in the range of -10-10 mV, the in vitro stability was greater than 3 days, the mRNA entrapment efficiency was greater than 50%, and the eGFP protein positive expression rate was greater than 40%. Among them, when the metal-polyphenol complex accounted for 5%, the distearoylphosphatidylcholine (DSPC) accounted for 60%, the cholesterol (CHOL) accounted for 30%, and the DSPE-PEG2000 accounted for 5%, the drug-lipid particle performance was optimal, that is, the particle size was in the range of 120 nm, the surface potential was in the range of -1.99 mV, the in vitro stability was greater than 7 days, the mRNA entrapment efficiency was 85%, and the eGFP protein positive expression rate was 97%. Because the metal-chelated polyphenol complex nanoparticles (MPNP) mainly rely on the metal-polyphenol complex to adsorb nucleic acids, the proportion of the metal-polyphenol complex cannot be too low; the function of the DSPC is to maintain the stability of the nanoparticle structure, and its performance will be better when its content is in the range of 40%-75%; the function of the DSPE-PEG2000 is to prevent the aggregation of nanoparticles and prolong the circulation time in the body, and its performance will be better when its content is in the range of 2%-10%; when the CHOL content is 0%, according to experimental results, the stability of its drug-lipid particles is within an acceptable range. When the CHOL content is greater than 0% and less than 48%, CHOL has the effect of enhancing the fluidity of the nanoparticles, which is beneficial to maintaining the stability of the nanoparticles.

[0470] The above results suggest that the metal ion is Fe 3+ When the proportion of metal-polyphenol complex is in the range of (5-20)%, the proportion of DSPC is in the range of (40-75)%, the proportion of CHOL is in the range of (0-48)%, and the proportion of DSPE-PEG2000 is in the range of (2-10)%, the drug-metal-polyphenol complex particles (mRNA@MPNP) have better drug loading performance.

[0471] Table 1-2 Metal ions are Fe 3+ The ratio of each component in the drug-metal-polyphenol composite particles (mRNA@MPNP)

[0472] Result analysis: As shown in Table 1-2-2, when the metal-polyphenol complex (metal ion is Al 3+ When the metal-polyphenol complexes accounted for 5% of the total metal-polyphenol complex, 45% of distearoylphosphatidylcholine (DSPC), 47% of cholesterol (CHOL), and 3% of DSPE-PEG2000, the particle size ranged from 30 to 400 nm, the surface potential ranged from -10 to 10 mV, the in vitro stability was ≥3 days, the mRNA loading efficiency was >50%, and the eGFP protein positive expression rate was above 40%. The metal-polyphenol complexes exhibited the best performance when the metal-polyphenol complexes accounted for 5%, distearoylphosphatidylcholine (DSPC) accounted for 45%, cholesterol (CHOL) accounted for 47%, and 3% of DSPE-PEG2000, with a particle size range of 100 nm, a surface potential range of -2.74 mV, in vitro stability >7 days, an mRNA loading efficiency of 86%, and an eGFP protein positive cell rate of 97%. Because mRNA@MPNP mainly relies on metal-polyphenol complexes to adsorb nucleic acids, the proportion of metal-polyphenol complexes cannot be too low; when the DSPC content is in the range of (30-75)%, the stability of its nanoparticles is within an acceptable range; the function of DSPE-PEG2000 is to prevent nanoparticle aggregation and increase the circulation time in the body, and its performance will be better when its content is in the range of (2-10)%; the function of CHOL is to enhance the fluidity of nanoparticles, and maintaining a certain content is beneficial to the stability of nanoparticles.

[0473] The above results suggest that the metal ion is Al 3+ When the metal-polyphenol complex (metal ion is Al 3+ ) accounts for (5-20)%, when DSPC accounts for (30-75)%, when CHOL accounts for (0-48)%, when DSPE-PEG2000 accounts for (2-10)%, mRNA@MPNP has better drug loading performance.

[0474] Table 1-2-2 Metal ions are Al 3+ The ratio of each component in the drug-metal-polyphenol composite particles (mRNA@MPNP)

[0475] Result analysis: As shown in Table 1-2-3, the metal ion is Mg 2+When the metal-polyphenol complex accounted for 1-30%, DSPC accounted for 29-75%, CHOL accounted for 0-60%, and DSPE-PEG2000 accounted for 1-10%, the drug-lipid particle size was in the range of 20-400 nm, the surface potential was in the range of -10-10 mV, the in vitro stability was ≥3 days, the mRNA entrapment efficiency was >50%, and the eGFP protein positive expression rate was above 40%. Among them, when the metal-polyphenol complex accounted for 10%, distearoylphosphatidylcholine (DSPC) accounted for 50%, cholesterol (CHOL) accounted for 37.5%, and DSPE-PEG2000 accounted for 2.5%, the drug-lipid particle performance was optimal, namely the particle size was 120 nm, the surface potential was -1.78 mV, the in vitro stability was >7 days, the mRNA entrapment efficiency was 88%, and the eGFP protein positive expression rate was 98%. Because mRNA@MPNP mainly relies on metal-polyphenol complexes to adsorb nucleic acids, the proportion of metal-polyphenol complexes cannot be too low; the function of the DSPC is to maintain the stability of the nanoparticle structure, and its performance will be better when its content is in the range of 29%-75%; the function of the DSPE-PEG2000 is to prevent nanoparticle aggregation and prolong the circulation time in the body, and its performance will be better when its content is in the range of 1%-10%; when the CHOL content is 0%, according to experimental results, the stability of its drug-lipid particles is within an acceptable range. When the CHOL content is greater than 0% and less than 60%, CHOL has the effect of enhancing the fluidity of the nanoparticles, which is beneficial to maintaining the stability of the nanoparticles.

[0476] The above results suggest that the metal ion is Mg 2+ When the proportion of metal-polyphenol complex is in the range of (1-30)%, the proportion of DSPC is in the range of (29-75)%, the proportion of CHOL is in the range of (0-60)%, and the proportion of DSPE-PEG2000 is in the range of (1-10)%, the drug-metal-polyphenol complex particles (mRNA@MPNP) have better drug loading performance.

[0477] Table 1-2-3 Metal ions are Mg 2+ The ratio of each component in the drug-metal-polyphenol composite particles (mRNA@MPNP)

[0478] Example 1.2.3, Preparation of mRNA-metal (Fe 3+ 、Al 3+ or Mg 2+ Types of non-cationic lipids or non-ionizable lipids in mRNA-polyphenol complex particles (mRNA@MPNP)

[0479] Compared with Example 1.2, the replacement of distearoylphosphatidylcholine (DSPC) is shown in Table 1-3 (metal ion is Fe 3+ ), Table 1-3-2 (metal ions are Al 3+ ) and Table 1-3-3 (metal ion is Mg 2+ ), the other conditions are the same.

[0480] Results: To explore whether DSPC in mRNA-metal-polyphenol complex particles (mRNA@MPNP) can be replaced by other non-cationic lipids or non-ionizable lipids in addition to the conjugated lipids that inhibit particle aggregation, DSPE, DSPA and DSPG were selected to replace DSPC, and the particle size, surface potential, stability and mRNA encapsulation efficiency were tested. It was proved that DSPC in mRNA-metal-polyphenol complex particles (mRNA@MPNP) can be replaced by other non-cationic lipids or non-ionizable lipids, and its function after replacement is equivalent to that of mRNA-metal-polyphenol complex particles (mRNA@MPNP) containing DSPC (Table 1-3 (metal ions are Fe 3+ ), Table 1-3-2 (metal ions are Al 3+ ) and Table 1-3-3 (metal ion is Mg 2+ Because the main function of the non-cationic lipid DSPC in the drug-metal-polyphenol complex particles (mRNA@MPNP) is to improve liposome membrane fusion, increase stability, and reduce toxicity, and other non-cationic lipids or non-ionizable lipids also have the function of improving liposome membrane fusion, increase stability, and reduce toxicity, DSPC in the drug-lipid particles can be replaced by other non-cationic lipids or non-ionizable lipids without affecting their efficacy.

[0481] Table 1-3 (metal ions are Fe 3+ The ratio of non-cationic lipids other than the conjugated lipids that inhibit particle aggregation in drug-metal-polyphenol complex particles (mRNA@MPNP)

[0482] Table 1-3-2 (metal ions are Al 3+ Performance of non-cationic lipids other than conjugated lipids in drug-metal-polyphenol composite particles (mRNA@MPNP) to inhibit particle aggregation

[0483] Table 1-3-3 Metal ions are Mg 2+ Performance of non-cationic lipids other than conjugated lipids in drug-metal-polyphenol composite particles (mRNA@MPNP) to inhibit particle aggregation

[0484] Example 1.2.4, Preparation of Drug mRNA-Metal (Fe 3+ 、Al 3+ or Mg 2+ Types of conjugated lipids that inhibit particle aggregation in mRNA-polyphenol complex particles (mRNA@MPNP)

[0485] Compared with Example 1.2, the substitution of DSPE-PEG2000 is shown in Table 1-4 (metal ion is Fe 3+ ), Table 1-4-2 (metal ions are Al 3+ ) and Table 1-4-3 (metal ion is Mg 2+ ), the other conditions are the same.

[0486] Analysis of results: In order to explore whether DSPE-PEG2000 in drug-metal-polyphenol complex particles (mRNA@MPNP) can be replaced by other conjugated lipids that inhibit particle aggregation, three other conjugated lipids that inhibit particle aggregation, namely DSPE-PEG700, DSPE-PEG5000 and DSPE-PEG1000, were selected to replace DSPE-PEG2000 respectively. By testing the particle size, surface potential, stability and mRNA loading rate, it was proved that DSPE-PEG2000 in the drug-metal-polyphenol complex particles (mRNA@MPNP) can be replaced by other conjugated lipids that inhibit particle aggregation, and its function after replacement is equivalent to the efficacy of drug-metal-polyphenol complex particles (mRNA@MPNP) containing DSPE-PEG2000 (Table 1-4, Table 1-4-2, Table 1-4-3). Because the main function of DSPE-PEG2000 in the drug-metal-polyphenol complex particles (mRNA@MPNP) is to inhibit aggregation, and other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation, DSPE-PEG2000 in the drug-metal-polyphenol complex particles (mRNA@MPNP) can be replaced by other conjugated lipids that inhibit particle aggregation, and its efficacy is not affected.

[0487] Table 1-4 Metal ions are Fe 3+ Conjugated lipid species for inhibiting particle aggregation in drug-metal-polyphenol complex particles (mRNA@MPNP)

[0488] Table 1-4-2 Metal ions are Al 3+ Conjugated lipid species for inhibiting particle aggregation in drug-metal-polyphenol complex particles (mRNA@MPNP)

[0489] Table 1-4-3 Metal ions are Mg 2+ Conjugated lipid species for inhibiting particle aggregation in drug-metal-polyphenol complex particles (mRNA@MPNP)

[0490] Example 1.2.5, Drug (mRNA)-Metal (Fe 3+ 、Al 3+ or Mg 2+ Preparation and effect characterization of mRNA-polyphenol complex particles (mRNA@MPNP)

[0491] Example 1.2.5.1 Metal ion is Fe 3+ Preparation and effect characterization of mRNA@MPNP

[0492] The mRNA in Example 1.2 was replaced with two other mRNAs, and three drug (mRNA)-lipid particles (mRNA@MPNP) containing different target protein mRNA sequences were prepared according to the method of Example 1.2. The three different mRNA sequences are: ① The mRNA sequence encoding the fluorescent protein eGFP is SEQ ID NO.1 (720nt); ② The mRNA sequence encoding the receptor binding domain (RBD) of the new coronavirus S1 subunit is SEQ ID NO.2 (669nt); ③ The mRNA sequence encoding the tumor antigen NY-ESO-1 is SEQ ID NO.3 (543nt). The preparation process of the remaining drug (mRNA)-lipid particles is the same as that in Example 1.2, and eGFP-mRNA@MPNP, RBD-mRNA@MPNP, and NY-ESO-1-mRNA@MPNP are obtained respectively.

[0493] eGFP-mRNA@MPNP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA), and the control group was incubated with MPNP. 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@MPNP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA), and the control group was incubated with MPNP. 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.

[0494] ELISA method for detecting RBD expression level:

[0495] 1. Sample collection: Place whole blood samples at room temperature for 2 hours, centrifuge at 1000 × g for 20 minutes, and collect the supernatant;

[0496] 2. Sample addition: Set up blank wells, standard wells, and test sample wells on the coated plate. Add 100 μL of sample diluent to the blank wells, add serially diluted standards to the standard wells, and add 100 μL of the test sample wells. Incubate at 37°C for 60 minutes.

[0497] 3. Discard the liquid in the wells and wash the plate 3 times, soaking for 1-2 minutes each time. Add 100 μL of the prepared biotinylated anti-RBD antibody working solution to each well, mix well, and incubate at 37°C for 60 minutes.

[0498] 4. Discard the liquid in the wells and wash the plate 3 times, soaking for 1-2 minutes each time;

[0499] 5. Add 100 μL of prepared streptavidin HRP working solution to each well, mix well, and incubate at 37°C for 45 minutes;

[0500] 6. Discard the liquid in the wells and wash the plate 3 times, soaking for 1-2 minutes each time;

[0501] 7. Add 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution to each well and incubate at 37°C in the dark for 15 minutes.

[0502] 8. Add 100 μL of stop solution to each well to terminate the reaction;

[0503] 9. Measure the optical density (OD) of each well at a wavelength of 450 nm.

[0504] Data analysis: Draw a standard curve with the concentration of the standard as the horizontal axis and the OD value as the vertical axis.

[0505] The experimental animals were randomly divided into two groups (experimental group and control group), with 5 animals in each group. Among them, the RBD-mRNA@MPNP 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@MPNP, and the control group was injected with metal-polyphenol complex particles (MPNP) without mRNA loading. The dose of each administration was 100μL, and the RBD-mRNA@MPNP preparation in the experimental group contained 30mg of mRNA. On the 28th day after the first administration, the blood of the mice was collected, the serum was separated and serially diluted, and the total RBD IgG antibodies against the new coronavirus S1 subunit produced in the mice were detected by commercially available ELISA kits. The results are shown in Figures 1-3.

[0506] The NY-ESO-1-mRNA@MPNP formulation was administered intramuscularly four times to each mouse on days 1, 7, 14, and 21. The experimental group received NY-ESO-1-mRNA@MPNP, while the control group received unloaded metal-polyphenol complex particles (MPNPs). Each dose was 100 μL, with the mRNA@MPNP formulation in the experimental group containing 30 mg of mRNA. Blood was collected from the 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.

[0507] Method for detecting total anti-NY-ESO-1 IgG antibodies in mice:

[0508] Preparation of reagents used in ELISA method:

[0509] 1. 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.

[0510] 2. Wash solution: Add 0.5 mL of Tween-20 to 1 L of 0.01 M PBS solution, mix well, and let it stand at room temperature.

[0511] 3. Blocking solution: Accurately weigh 20g of BSA and add it to 1L of 0.01M PBS solution. Ultrasonicate the undissolved BSA powder in the solution. When all the solids in the solution are dissolved and the solution turns light yellow, store it in a refrigerator at 4℃ until use.

[0512] 4. 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 ready to use.

[0513] 5. 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.

[0514] 6. Stop solution: 2M H2SO4: concentrated sulfuric acid 55.5mL, add DDW to 500mL.

[0515] ELISA method was used to determine the titer of antibodies in mouse serum:

[0516] 1. Coating: Dilute NY-ESO-1 antigen to 1 μg / mL with coating solution, add 50 μL / well to a 96-well plate, and coat overnight at 4°C.

[0517] 2. Blocking: Dry the coating solution in the well plate, wash with blocking solution 3 times, 5 minutes each time and dry, add 150 μL blocking solution to each well, and incubate at 37℃ for 2 hours.

[0518] 3. Drying: Shake off the blocking solution and incubate at 37°C for 1-2 hours until the liquid at the bottom of the plate is completely dry.

[0519] 4. Immunization: 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 μL / 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 μL / 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 μL / well and incubated at 37°C. 1h; shake dry the liquid in the well plate, add washing solution, and repeat the above washing steps; add freshly prepared streptavidin-labeled horseradish peroxidase HRP working solution, 100μL / well, incubate at 37℃ for 1h; shake dry the liquid in the well plate, add washing solution, and repeat the above washing steps; add color development solution, 100μL / well, react at room temperature for 5min, and then add stop solution, 50μL / well, to stop color development; use a microplate reader to measure the absorbance at 450nm.

[0520] On day 28 after administration of RBD-mRNA@MPNP, spleens from normal mice were harvested and aseptically prepared into single-cell suspensions. 100,000 spleen cells / well were plated in a cell plate. RBD protein was added at a final concentration of 10 mg / mL and cultured for 48 hours. The supernatant was removed by centrifugation, and the expression levels of IFN-γ, IL-2, and IL-4 were measured using ELISA kits. The results are shown in Figures 1-5. On day 28 after administration of NY-ESO-1-mRNA@MPBP, spleens from normal mice were harvested and aseptically prepared into single-cell suspensions. 100,000 spleen cells / well were plated in a cell plate. NY-ESO-1 protein was added at a final concentration of 10 mg / mL and cultured for 48 hours. The supernatant was removed by centrifugation, and the expression levels of IFN-γ, IL-2, and TNF-α were measured using ELISA kits. The results are shown in Figures 1-6.

[0521] Result analysis: As shown in Figure 1-1, the metal ion is Fe 3+The eGFP-mRNA@MPNP experimental group showed an eGFP-positive cell rate of 93.9%, while no eGFP signal was detected in the MPNP control group. As shown in Figures 1-2, the RBD protein encoded by the MPNP-encapsulated RBD-mRNA was present at 166 ng / mL in the supernatant of 293T cells, while the RBD protein content in the supernatant of 293T cells transfected with the empty MPNP vector was zero. These results suggest that the drug-metal-polyphenol complex particles (mRNA-MPNP) can encapsulate and deliver any mRNA and directly encode polypeptides within cells. As shown in Figures 1-3 and 1-4, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP effectively induced humoral immunity in mice, producing high levels of antigen-specific binding antibodies. The IgG antibody titer in mice treated with RBD-mRNA@MPNP reached 84363.4, while that in mice treated with NY-ESO-1-mRNA@MPNP reached 4283.56. As shown in Figures 1-5 and 1-6, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP effectively induced cellular immunity in mice, activating immune cells and producing large amounts of cytokines. RBD-mRNA@MPNP increased the expression of IFN-γ, IL-2, and IL-4 to 271.8 pg / mL, 269.6 pg / mL, and 75.8 pg / mL, respectively; while NY-ESO-1-mRNA@MPNP increased the expression of IFN-γ, IL-2, and TNF-α to 76.38 pg / mL, 74.56 pg / mL, and 69.31 pg / mL, respectively. The results suggest that the drug-metal-polyphenol complex particles (mRNA@MPNP) can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), and then effectively inducing humoral immunity and cellular immunity in mice, producing high levels of antigen-specific binding antibodies and cytokines, and playing the role of anti-new coronavirus mRNA vaccines and anti-tumor mRNA vaccines.

[0522] Example 1.2.5.2: Metal ion is Al 3+ Preparation and effect characterization of mRNA@MPNP

[0523] The difference between this embodiment and embodiment 1.2.5.1 is that the metal ion Fe in embodiment 1.2.5.1 is replaced by 3+ Replaced by Al 3+ .

[0524] Result analysis: As shown in Figure 1-1-2, the metal ion is Al 3+The eGFP-mRNA@MPNP experimental group showed an eGFP-positive cell rate of 97.03%, while no eGFP signal was detected in the MPNP control group. As shown in Figure 1-2-2, the RBD protein encoded by the MPNP-encapsulated RBD-mRNA was present at 207 ng / mL in the 293T cell supernatant, while the RBD protein content in the supernatant of 293T cells transfected with the empty MPNP vector was zero. These results suggest that mRNA-MPNPs can encapsulate and deliver any mRNA and directly encode polypeptides within cells. As shown in Figures 1-3-2 and 1-4-2, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP effectively induced humoral immunity in mice, producing high levels of antigen-specific antibodies. IgG antibody titers reached 94828.6 in mice treated with RBD-mRNA@MPNP, and 5848.02 in mice treated with NY-ESO-1-mRNA@MPNP. As shown in Figures 1-5-2 and 1-6-2, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP can effectively induce cellular immunity in mice, activating immune cells and producing large amounts of cytokines. RBD-mRNA@MPNP increased the expression of cytokines IFN-γ, IL-2, and IL-4 to 306.2 pg / mL, 289.6 pg / mL, and 88.2 pg / mL, respectively; while NY-ESO-1-mRNA@MPNP increased the expression of cytokines IFN-γ, IL-2, and TNF-α to 91.88 pg / mL, 85.32 pg / mL, and 80.22 pg / mL, respectively. The results suggest that mRNA@MPNP 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.

[0525] Example 1.2.5.3: Metal ion is Mg 2+ Preparation and effect characterization of mRNA@MPNP

[0526] The difference between this embodiment and embodiment 1.2.5.1 is that the metal ion Fe in embodiment 1.2.5.1 is replaced by 3+ Replaced by Mg 2+ .

[0527] Result analysis: As shown in Figure 1-1-3, the metal ion is Mg 2+The eGFP-mRNA@MPNP experimental group showed an eGFP-positive cell rate of 98.3%, while no eGFP signal was detected in the MPNP control group. As shown in Figure 1-2-3, the RBD protein encoded by the MPNP-encapsulated RBD-mRNA was present at 185.3 ng / mL in the supernatant of 293T cells, while the RBD protein content in the supernatant of 293T cells transfected with the empty MPNP vector was zero. These results suggest that mRNA-MPNPs can encapsulate and deliver any mRNA and directly encode polypeptides within cells. As shown in Figures 1-3-3 and 1-4-3, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP effectively induced humoral immunity in mice, producing high levels of antigen-specific antibodies. IgG antibody titers reached 113250.6 in mice treated with RBD-mRNA@MPNP, and 5967.5 in mice treated with NY-ESO-1-mRNA@MPNP. As shown in Figures 1-5-3 and 1-6-3, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP can effectively induce cellular immunity in mice, activating immune cells and producing large amounts of cytokines. RBD-mRNA@MPNP increased the expression of cytokines IFN-γ, IL-2, and IL-4 to 291.3 pg / mL, 231.1 pg / mL, and 81.7 pg / mL, respectively; while NY-ESO-1-mRNA@MPNP increased the expression of cytokines IFN-γ, IL-2, and TNF-α to 88.59 pg / mL, 89.73 pg / mL, and 90.40 pg / mL, respectively. The results suggest that mRNA@MPNP 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.

[0528] Example 1.2.6, Drug (siRNA)-Metal (Fe 3+ 、Al 3+ or Mg 2+ Preparation and Effect of siRNA-polyphenol Composite Particles (siRNA@MPNP)

[0529] Example 1.2.6.1: Metal ion is Fe 3+ Preparation and effect of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPNP)

[0530] The mRNA in Example 1.2 was replaced with siRNA, and three drug-metal-polyphenol composite particles (siRNA@MPNP) containing different siRNAs were prepared according to the method of Example 1.2. The genes, sequences and corresponding random control sequences of the three different siRNA targeting genes are as follows: ① The sequence of the siRNA targeting the Bcl-2 gene (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 the siRNA targeting the PLK1 gene (PLK1-siRNA) is SEQ ID NO.6 (antisense chain) and SEQ ID No.23 (sense chain) (21 bp), and its random control sequence is SEQ ID NO.7 (antisense chain) and SEQ ID No.24 (sense chain) (19 bp); ③ The sequence of the siRNA targeting the Gal-1 gene (Gal-1-siRNA) is SEQ ID NO.8 (19 bp); its random control sequence is SEQ ID NO.9 (19 bp). The preparation process of the remaining drug-metal-polyphenol composite particles (siRNA@MPNP) was the same as that in Example 1.2.

[0531] Cell culture methods: U251 human glioblastoma cells were grown as monolayers in high-glucose (4.5 g / L) DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (Bio Industries) at 37°C and 5% CO2, and passaged twice a week.

[0532] U251 cells were cultured at 1 × 10 6 After the cells were seeded in a 6-well plate at a high density for approximately 24 hours, the cells in each well were incubated with the drug-metal-polyphenol complex particles (siRNA@MPNP) containing the above-mentioned siRNA (wherein the concentration of siRNA was 2 μg / mL) for 72 hours, and the cells were collected and the total cellular RNA was extracted. The mRNA expression levels of the target genes (Bcl-2, PLK1, Gal-1) were detected by RT-PCR technology, and the ability of the drug-metal-polyphenol complex particles (siRNA@MPNP) to silence the target genes in the cells was calculated.

[0533] RT-PCR specific process:

[0534] 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 μL of chloroform, shake thoroughly, and let stand at room temperature for 10 minutes. Centrifuge at 13,000 rpm at 4°C for 15 minutes to obtain a three-phase liquid, with RNA dissolved in the upper aqueous phase. Pipette the upper aqueous phase into a new enzyme-free 1.5 mL centrifuge tube, add 500 μL of isopropanol, let stand at room temperature for 10 minutes, and centrifuge at 13,000 rpm at 4°C for 15 minutes to obtain an RNA precipitate. Remove the supernatant and add 1 mL of 75% (v / v) ethanol, freshly prepared with RNase-free water, to each tube. Carefully pipette to remove the white RNA precipitate at the bottom of the tube. Centrifuge at 7,500 rpm at 4°C for 10 minutes. Remove the supernatant and aspirate as much of the liquid at the bottom of the tube as possible. Open the lid and air dry the RNA precipitate at the bottom of the tube at room temperature, add 50 μL of enzyme-free water to dissolve it, and use an ultra-micro UV-visible spectrophotometer to detect the purity and concentration of the RNA.

[0535] cDNA reverse transcription: using Ta Ka Ra Prime Script TM The RT reagent kit with gDNA Eraser reverse transcribes RNA into cDNA, removing genomic DNA (gDNA) before the reverse transcription step for more accurate and reliable results. Prepare the total RNA reverse transcription reaction on ice: 1 μL Prime Script RT Enzyme Mix I, 1 μL RT Primer Mix, 4 μL 5× Prime Script Buffer 2, and 4 μL RNase-Free dH2O. After preparing the reaction mixture, incubate at 37°C for 15 minutes, then terminate the reaction by incubating at 85°C for 5 seconds. Store at 4°C until ready to use.

[0536] RT-PCR procedure: This detection method uses the SYBR Green dye method and does not require a probe. Specifically, real-time PCR reactions were performed using cDNA from different samples as templates. The reaction solution was prepared on ice: 5 μL SYBR Premix Dimer Eraser (2×), 0.3 μL PCR Forward primer (10 μM), 0.3 μL PCR Reverse primer (10 μM), 0.2 μL ROX Reference Dye II (50×), 1 μL of the cDNA template obtained in the previous step, and 3.2 μL dH2O. The sample was added to the well plate, with 10 μL per well. After addition, the sample was centrifuged (1000 rpm, 5 min) to eliminate liquid adhering to the wall and any bubbles in the reaction solution. 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: ①Bcl-2 primer: forward: 5'-AGGATTGTGGCCTTCTTTGAG-3', reverse: 5'-AGACAGCCAGGAGAAATC AAAC-3'; ②PLK1 primer: forward: 5'-ACCAGCACGTCGTAGGATTC-3', reverse: 5'-CAAGCAATTTGCCGTAGG-3'; ③Gal-1 primer: forward: 5'-CAATCAT GGCCTGTGGTCTG-3', reverse: 5'-GTG TAGGCACAGGTTGTTGCTG-3'. ④GAPDH primer: forward: 5'-TCAGGGGTTTCACATTTGGCA-3', reverse: 5'-GG AGCGGAA AACCA-3'. The expression level of each target gene was calculated using the RQ value (2 -ΔΔCT ) is expressed as follows: Fold Change = 2 -ΔΔCt Where ΔΔCt=ΔCt 实验组 -ΔCt 对照组 , ΔCt=Ct 目的基因 -Ct 内参基因

[0537] The calculation method of gene silencing efficiency is: 100% - gene expression level of experimental group / gene expression level of control group.

[0538] Result analysis: As shown in Figure 1-7, Figure 1-8, and Figure 1-9, the metal ion is Fe 3+ When the drug-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP, PLK1-siRNA@MPNP, and Gal-1-siRNA@MPNP) were added, they all significantly interfered with their corresponding target genes. Bcl-2-siRNA@MPNP achieved a 67% inhibition rate on the target gene Bcl-2; PLK1-siRNA@MPNP achieved an 87% inhibition rate on the target gene PLK1; and Gal-1-siRNA@MPNP achieved a 64% inhibition rate on the target gene Gal-1. These results suggest that the drug-metal-polyphenol complex particles (siRNA@MPNP) can carry any siRNA for target gene intervention therapy, acting as siRNA-loaded drugs, vaccines, or other products.

[0539] Example 1.2.6.2: Metal ion is Al 3+ Preparation and effect of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPNP)

[0540] In Example 1.2 (the metal ion is Al 3+ ) mRNA was replaced with siRNA, and three drug-metal-polyphenol composite particles (siRNA@MPNP) containing different siRNAs were prepared according to the method of Example 1.2. For siRNA information and experimental procedures, refer to Example 1.2.6.1.

[0541] Result analysis: The metal ion is Al 3+ As shown in Figures 1-7-2, 1-8-2, and 1-9-2, all three siRNA@MPNPs significantly interfered with their corresponding target genes. Bcl-2-siRNA@MPNP achieved a 72% inhibition rate against the target gene Bcl-2; PLK1-siRNA@MPNP achieved an 88.07% inhibition rate against the target gene PLK1; and Gal-1-siRNA@MPNP achieved a 70.11% inhibition rate against the target gene Gal-1. These results suggest that siRNA@MPNPs can carry any siRNA for target gene intervention therapy, potentially facilitating the development of siRNA-loaded drugs, vaccines, or other products.

[0542] Example 1.2.6.3: Metal ion is Mg 2+Preparation and effect of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPNP)

[0543] In Example 1.2 (the metal ion is Mg 2+ ) mRNA was replaced with siRNA, and three drug-metal-polyphenol composite particles (siRNA@MPNP) containing different siRNAs were prepared according to the method of Example 1.2. For siRNA information and experimental procedures, refer to Example 1.2.6.1.

[0544] Result analysis: The metal ion is Mg 2+ As shown in Figures 1-7-3, 1-8-3, and 1-9-3, all three siRNA@MPNPs significantly interfered with their corresponding target genes. Bcl-2-siRNA@MPNP achieved a 73.8% inhibition rate against the target gene Bcl-2; PLK1-siRNA@MPNP achieved a 76.6% inhibition rate against the target gene PLK1; and Gal-1-siRNA@MPNP achieved a 79.0% inhibition rate against the target gene Gal-1. These results suggest that siRNA@MPNPs can carry any siRNA for target gene intervention therapy, potentially facilitating the development of siRNA-loaded drugs, vaccines, or other products.

[0545] Example 1.2.7, Drug-Metal (Fe 3+ 、Al 3+ or Mg 2+ Preparation and effects of )-polyphenol composite particles (ASO@MPNP)

[0546] Example 1.2.7.1: Metal ion is Fe 3+ Preparation and effect of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPNP)

[0547] In Example 1.2 (the metal ion is Fe 3+) mRNA was replaced with ASO, and three drug-metal-polyphenol complex particles (ASO@MPNP) containing different ASOs were prepared respectively according to the method of Example 1.2. The genes, sequences and corresponding random control sequences of the three different ASOs are as follows: ① The sequence of the ASO targeting the STAT3 gene (STAT3-ASO) is SEQ ID NO.10 (17nt), and its random control sequence is SEQ ID NO.11 (18nt); ② The sequence of the ASO targeting the α-syn gene (α-syn-ASO) is SEQ ID NO.12 (16nt), and its random control sequence is SEQ ID NO.13 (16nt); ③ The sequence of the ASO targeting the Bcl-2 gene (Bcl-2-ASO) is SEQ ID NO.14 (18nt), and its random control sequence is SEQ ID NO.15 (20nt). The preparation process of the remaining drug (ASO)-metal-polyphenol complex particles is the same as that of Example 1.2. Different ASO@MPNPs were incubated with different cells: ASO@MPNPs targeting STAT3 gene were incubated with U251 human glioblastoma cells; ASO@MPNPs targeting α-syn gene were incubated with SH-SY5Y human neuroblastoma cells; ASO@MPNPs targeting Bcl-2 gene were incubated with Daudi human lymphoma cells. 6 After the cells were seeded in a 6-well plate at a high density for approximately 24 hours, the cells in each well were incubated with the drug-metal-polyphenol complex particles (ASO@MPNP) containing the above-mentioned ASO (wherein the concentration of ASO was 2 μg / mL) for 48 hours. The cells were then collected and total cellular RNA was extracted. The mRNA expression levels of the target genes (STAT3, α-syn, Bcl-2) were detected by RT-PCR, and the ability of the drug-metal-polyphenol complex particles (ASO@MPNP) to silence the target genes in the cells was calculated.

[0548] Cell culture: ① U251 human glioblastoma cells were grown as monolayers in high-glucose (4.5 g / L) DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (Bio Industries) at 37°C and 5% CO2, with passages twice weekly. ② SH-SY5Y human neuroblastoma cells were grown as monolayers in high-glucose (4.5 g / L) DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (Bio Industries) at 37°C and 5% CO2, with passages twice weekly. ③ Daudi human lymphoma cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (Bio Industries) at 37°C and 5% CO2, with passages twice weekly.

[0549] RT-PCR specific process:

[0550] 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 μL of chloroform, shake thoroughly, and let stand at room temperature for 10 minutes. Centrifuge at 13,000 rpm at 4°C for 15 minutes to obtain a three-phase liquid, with RNA dissolved in the upper aqueous phase. Pipette the upper aqueous phase into a new enzyme-free 1.5 mL centrifuge tube, add 500 μL of isopropanol, let stand at room temperature for 10 minutes, and centrifuge at 13,000 rpm at 4°C for 15 minutes to obtain an RNA precipitate. Remove the supernatant and add 1 mL of 75% (v / v) ethanol, freshly prepared with RNase-free water, to each tube. Carefully pipette to remove the white RNA precipitate at the bottom of the tube. Centrifuge at 7,500 rpm at 4°C for 10 minutes. Remove the supernatant and aspirate as much of the liquid at the bottom of the tube as possible. Open the lid and air dry the RNA precipitate at the bottom of the tube at room temperature, add 50 μL of enzyme-free water to dissolve it, and use an ultra-micro UV-visible spectrophotometer to detect the purity and concentration of the extracted RNA.

[0551] cDNA reverse transcription: using Ta Ka Ra Prime Script TMThe RT reagent kit with gDNA Eraser reverse transcribes RNA into cDNA, removing genomic DNA (gDNA) before the reverse transcription step for more accurate and reliable results. Prepare the total RNA reverse transcription reaction on ice: 1 μL Prime Script RT Enzyme Mix I, 1 μL RT Primer Mix, 4 μL 5× Prime Script Buffer 2, and 4 μL RNase-Free dH2O. After preparing the reaction mixture, incubate at 37°C for 15 minutes, then terminate the reaction by incubating at 85°C for 5 seconds. Store at 4°C until ready to use.

[0552] RT-PCR procedure: This detection method uses the SYBR Green dye method and does not require a probe. Specifically, real-time PCR reactions were performed using cDNA from different samples as templates. The reaction solution was prepared on ice: 5 μL SYBR Premix Dimer Eraser (2×), 0.3 μL PCR Forward primer (10 μM), 0.3 μL PCR Reverse primer (10 μM), 0.2 μL ROX Reference Dye II (50×), 1 μL of the cDNA template obtained in the previous step, and 3.2 μL dH2O. The sample was added to the well plate, with 10 μL per well. After addition, the sample was centrifuged (1000 rpm, 5 min) to eliminate liquid adhering to the wall and any bubbles in the reaction solution. 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. The RT-PCR primer sequences are as follows: ①STAT3 primer: forward: 5'-TGATCACCTTTGAGACCGAGG-3', reverse: 5'-GATCACCACAACTGG CAA GG-3'; ②α-syn primer: forward: 5'-TGACGGGTGTGACAGCAGTAG-3', reverse: 5'-CAGTGGCTGCTGCAATG-3'; ③Bcl-2 primer: forward: 5'-AGGATT GTG GCCTTCTTTGAG-3', reverse: 5'-AGACAGCCAGGAGAAATCAAAC-3'; ④GAPDH primer: forward: 5'-TCAGGGG TTTCACATTTGGCA-3', reverse: 5'-GGAGCGGAA AACCA-3'. The expression levels of each target gene were calculated using the RQ value (2 -ΔΔCT ) is expressed as follows: Fold Change = 2 -ΔΔCt Where ΔΔCt=ΔCt 实验组 -ΔCt 对照组 , ΔCt=Ct 目的基因 -Ct 内参基因

[0553] The calculation method of gene silencing efficiency is: 100% - gene expression level of experimental group / gene expression level of control group.

[0554] Result analysis: The metal ion is Fe 3+ As shown in Figures 1-10, 1-11, and 1-12, all three drug-metal-polyphenol complex particles (ASO@MPNP) significantly interfered with their corresponding target genes. The drug-metal-polyphenol complex particles (STAT3-ASO@MPNP) inhibited the target gene STAT3 by 72%, the drug-metal-polyphenol complex particles (α-syn-ASO@MPNP) inhibited the target gene α-syn by 78%, and the drug-metal-polyphenol complex particles (Bcl-2-ASO@MPNP) inhibited the target gene Bcl-2 by 62%. These results suggest that the drug-metal-polyphenol complex particles (ASO@MPNP) can carry any ASO for target gene intervention therapy, potentially acting as an ASO-carrying drug, vaccine, or other product.

[0555] Example 1.2.7.2: Metal ion is Al 3+ Preparation and effect of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPNP)

[0556] In Example 1.2 (the metal ion is Al 3+ ) mRNA was replaced with an ASO, and three drug-metal-polyphenol composite particles (ASO@MPNP) containing different ASOs were prepared according to the method of Example 1.2. ASO information and experimental procedures were similar to those of Example 1.2.7.1.

[0557] Result analysis: The metal ion is Al 3+ As shown in Figures 1-10-2, 1-11-2, and 1-12-2, all three ASO@MPNPs significantly interfered with their corresponding target genes. The STAT3-ASO@MPNP achieved a 75.4% inhibition rate on the target gene STAT3; the α-syn-ASO@MPNP achieved an 80.87% inhibition rate on the target gene α-syn; and the Bcl-2-ASO@MPNP achieved a 67.91% inhibition rate on the target gene Bcl-2. These results suggest that ASO@MPNPs can carry any ASO for target gene intervention therapy, potentially leveraging the potential of ASO-carrying drugs, vaccines, or other products.

[0558] Example 1.2.7.3: Metal ion is Mg 2+Preparation and effect of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPNP)

[0559] In Example 1.2 (the metal ion is Mg2 + ) mRNA was replaced with an ASO, and three drug-metal-polyphenol composite particles (ASO@MPNP) containing different ASOs were prepared according to the method of Example 1.2. ASO information and experimental procedures were similar to those of Example 1.2.7.1.

[0560] Result analysis: The metal ion is Mg2 + As shown in Figures 1-10-3, 1-11-3, and 1-12-3, all three ASO@MPNPs significantly interfered with their corresponding target genes. The STAT3-ASO@MPNP achieved a 76.2% inhibition rate on the target gene STAT3; the α-syn-ASO@MPNP achieved a 76.2% inhibition rate on the target gene α-syn; and the Bcl-2-ASO@MPNP achieved a 76.0% inhibition rate on the target gene Bcl-2. These results suggest that ASO@MPNPs can carry any ASO for target gene intervention therapy, potentially leveraging the potential of ASO-carrying drugs, vaccines, or other products.

[0561] Example 1.2.8, Preparation of Drugs (Different Types of Nucleic Acids)-Metal (Fe 3+ 、Al 3+ or Mg 2+ )-polyphenol complex particles and their effects

[0562] Example 1.2.8.1. Preparation of Metal Ions: Fe 3+ Drug (different types of nucleic acids)-metal-polyphenol complex particles and their effects

[0563] The mRNA in Example 1.2 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 The sequence of double-stranded DNA (dsDNA) is SEQ ID NO.17 (antisense chain) and SEQ ID NO.25 (sense chain) (22 bp) (the 3' end of the sequence is labeled with fluorescent probe Cy3); ⑤ The sequence of single-stranded DNA (ssDNA) is SEQ ID NO.18 (22 nt) (the 3' end of the sequence is labeled with fluorescent probe Cy3). Drug-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP, STAT3-ASO@MPNP, S-mRNA@MPNP, dsDNA@MPNP, ssDNA@MPNP) encapsulating the above-mentioned different types of nucleic acids were prepared respectively according to the method of Example 1.2. The preparation process of the remaining drug-lipid particles was the same as that of Example 1.2.

[0564] U251 cells were cultured at 1 × 10 6 After the cells were densely seeded in a 6-well plate for approximately 24 hours, the cells in each well were incubated with drug-metal-polyphenol complex particles (siRNA@MPNP) (wherein the concentration of siRNA was 2 μg / mL) or drug-metal-polyphenol complex particles (ASO@MPNP) (wherein the concentration of ASO was 2 μg / 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 technology, and the ability of the drug-metal-polyphenol complex particles (siRNA@MPNP) or drug-metal-polyphenol complex particles (ASO@MPNP) to silence the target genes in the cells was calculated. The results are shown in Figures 1-7 of Example 1.2.6 and Figures 1-10 of Example 1.2.7.

[0565] S-mRNA@MPNP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPNP. After 24 hours, the supernatant was centrifuged and stored at -20°C for later use. The cell pellet was resuspended in 100 μL 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.

[0566] A549 lung cancer cells were incubated with drug (double-stranded DNA)-metal-polyphenol complex particles (ds-DNA@MPNP) at a concentration of 100 nM (the concentration of the DNA contained) for 2 hours, and then the 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 DNA transfection was calculated. The results are shown in Figure 1-14-1.

[0567] HT22 mouse hippocampal neurons were incubated with drug (ss-DNA)-metal-polyphenol complex particles (ss-DNA@MPNP) at a concentration of 200 nM (the concentration of DNA contained). The drug-lipid particles were removed after 2 hours, 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 of DNA was calculated. The results are shown in Figure 1-14-1.

[0568] The culture method of human glioblastoma U251 cells is the same as that in Example 1.2.6.

[0569] The culture method of 293T cells is the same as that in Example 1.2.5.

[0570] Culture method of HT22 mouse hippocampal neurons: culture in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0571] The RT-PCR method is the same as in Example 1.2.6.

[0572] ELISA detection of S protein expression level: replace the "anti-RBD antibody working solution" in the ELISA method for detecting RBD in Example 1.2.5 with "anti-S protein antibody working solution", and the remaining steps are the same as Example 1.2.5.

[0573] The calculation method of gene silencing efficiency is the same as Example 1.2.6.

[0574] To calculate transfection efficiency, randomly select 3-5 fields of view using a high-content imaging system to 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 (Figure 1-14). Calculate the ratio of the number of cells with Cy3 fluorescent signals in the randomly selected field of view to the number of cells with Hoechst33342 fluorescent signals in the same field of view, which is the transfection efficiency.

[0575] Result analysis: The metal ion is Fe 3+ When, as shown in Figures 1-7 of Example 1.2.6, the inhibition rate of the drug (double-stranded RNA)-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP) on the target gene Bcl-2 reached 67%; as shown in Figures 1-10 of Example 1.2.7, the inhibition rate of the drug (single-stranded RNA)-metal-polyphenol complex particles (STAT3-ASO@MPNP) on the target gene STAT3 reached 72%; as shown in Figures 1-13, the drug (single-stranded RNA)-metal-polyphenol complex particles (S-mRNA) were transfected. The S protein expression level in the supernatant of 293T cells transfected with the drug (double-stranded DNA)-metal-polyphenol complex particles (dsDNA@MPNP) was 134 ng / mL, while the S protein content in the supernatant of 293T cells transfected with the empty vector MPNP was 0. The efficiency of double-stranded DNA transfection into cells by the drug (double-stranded DNA)-metal-polyphenol complex particles (dsDNA@MPNP) was 100% (Figure 1-14-1); the efficiency of single-stranded DNA transfection into cells by the drug (ssDNA)-metal-polyphenol complex particles (ssDNA@MPNP) was 100% (Figure 1-14-1). The results suggest that the drug-metal-polyphenol complex particles can encapsulate any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and achieve their function, where the nucleic acid length ranges from 16 to 3822 nt.

[0576] Example 1.2.8.2: Preparation of Metal Ions: Al 3+ Drug (different types of nucleic acids)-metal-polyphenol complex particles and their effects

[0577] In Example 1.2 (the metal ion is Al 3+ ) mRNA was replaced with double-stranded RNA (siRNA), single-stranded RNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively. For nucleic acid information and experimental procedures, refer to Example 1.2.8.1.

[0578] Result analysis: The metal ion is Al 3+As shown in Figure 1-7-2, the inhibition rate of drug (double-stranded RNA)-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP) on the target gene Bcl-2 reached 72%; as shown in Figure 1-10-2, the inhibition rate of drug (single-stranded DNA)-metal-polyphenol complex particles (STAT3-ASO@MPNP) on the target gene STAT3 reached 75.4%; as shown in Figure 1-13-2, drug (single-stranded RNA)-metal-polyphenol complex particles (S-mRNA@MPNP) were transfected with The S protein expression level in the supernatant of 293T cells transfected with MPNP (p) was 157 ng / mL, while the S protein content in the supernatant of 293T cells transfected with the empty vector MPNP was zero. The efficiency of double-stranded DNA transfection into cells by drug (double-stranded DNA)-metal-polyphenol complex particles (dsDNA@MPNP) was 100% (Figure 1-14-2); the efficiency of single-stranded DNA transfection into cells by drug (ssDNA)-metal-polyphenol complex particles (ssDNA@MPNP) was 100% (Figure 1-14-2). These results suggest that drug-metal-polyphenol complex particles can encapsulate and function with any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA), with nucleic acid lengths ranging from 16 to 3822 nt.

[0579] Example 1.2.8.3: Preparation of Metal Ions as Mg 2+ Drug (different types of nucleic acids)-metal-polyphenol complex particles and their effects

[0580] In Example 1.2 (the metal ion is Mg 2+ ) mRNA was replaced with double-stranded RNA (siRNA), single-stranded RNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively. For nucleic acid information and experimental procedures, refer to Example 1.2.8.1.

[0581] Result analysis: The metal ion is Mg 2+As shown in Figure 1-7-3, the inhibition rate of drug (double-stranded RNA)-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP) on the target gene Bcl-2 reached 73.8%; as shown in Figure 1-10-3, the inhibition rate of drug (single-stranded DNA)-metal-polyphenol complex particles (STAT3-ASO@MPNP) on the target gene STAT3 reached 76.2%; as shown in Figure 1-13-3, drug (single-stranded RNA)-metal-polyphenol complex particles (S-mRNA@MPNP) were transfected with The S protein expression level in the supernatant of 293T cells transfected with MPNP (p) was 168.7 ng / mL, while the S protein content in the supernatant of 293T cells transfected with the empty vector MPNP was zero. The efficiency of double-stranded DNA transfection into cells by drug (double-stranded DNA)-metal-polyphenol complex particles (dsDNA@MPNP) was 100% (Figure 1-14-3); the efficiency of single-stranded DNA transfection into cells by drug (ssDNA)-metal-polyphenol complex particles (ssDNA@MPNP) was 100% (Figure 1-14-3). These results suggest that drug-metal-polyphenol complex particles can encapsulate and function with any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA), with nucleic acid lengths ranging from 16 to 3822 nt.

[0582] Example 2: Drug-Metal (Fe 3+ 、Al 3+ or Mg 2+ )-polyphenol composite particles performance characterization

[0583] Example 2.1 Synthesis and characterization of metal-polyphenol complexes

[0584] Example 2.1.1: Metal ion is Fe 3+ Synthesis and characterization of metal-polyphenol complexes

[0585] Curcumin and Fe 3+ The connection is characterized by spectrophotometry: As shown in Figure 2-1, curcumin and Fe 3+ After binding, its maximum absorption wavelength shifted from 420nm to 372nm, and the conjugated structure of the metal-polyphenol complex changed, proving that curcumin successfully bound to Fe 3+ Complexation.

[0586] Example 2.1.2: Metal ion is Al 3+ Synthesis and characterization of metal-polyphenol complexes

[0587] Curcumin and Al 3+ The connection characterization is spectrophotometric: As shown in Figure 2-1-2, curcumin and Al 3+After binding, its maximum absorption wavelength shifted from 420nm to 433nm, and the conjugated structure of the metal-polyphenol complex changed, proving that curcumin successfully bound to Al 3+ Complexation.

[0588] Example 2.1.3: Metal ion is Mg 2+ Synthesis and characterization of metal-polyphenol complexes

[0589] Curcumin and Mg 2+ The connection characterization is spectrophotometric: As shown in Figure 2-1-3, curcumin and Mg 2+ After binding, its maximum absorption wavelength shifted from 425nm to 419nm, and the conjugated structure of the metal-polyphenol complex changed, proving that curcumin successfully bound to Mg. 2+ Complexation.

[0590] Example 2.2: Fe under low pH conditions 3+ Characterization of shedding from metal-polyphenol complexes

[0591] Curcumin in the metal-polyphenol complex binds Fe via coordination bonds 3+ Under the low pH conditions of lysosomes, curcumin and Fe 3+ The coordination bonds between the metal and polyphenols are protonated (absorb hydrogen ions) and broken. 3+ Indeed, it is through the above mechanism that the metal-polyphenol complex is detached from the lipid complex. We designed the following experiment: the color of the metal-polyphenol complex was observed under physiological pH (pH = 7.4) and lysosomal low pH (pH = 5.0). As shown in Figure 2-2, the metal-polyphenol complex changed from brown-red to bright yellow under lysosomal low pH (pH = 5.0), indicating that Fe 3+ The results suggest that under the low pH conditions of lysosomes, Fe 3+ Can be detached from the metal-polyphenol complex.

[0592] At low pH, Fe 3+ The principle of shedding from the metal-polyphenol complex is: curcumin and Fe 3+ The coordination bonds between them are protonated under low pH conditions (pH = 5.0), that is, curcumin binds a large number of protons (H + ), leading to Fe 3+ The coordination bond between Fe and curcumin is broken, which makes Fe 3+ Separation from curcumin, ultimately leading to Fe 3+ Separated from the metal-polyphenol complex (Figure 2-2).

[0593] Example 2.3, Drug-Metal (Fe3+ 、Al 3+ or Mg 2+ The efficiency of nucleic acid (siRNA and mRNA) encapsulation of )-polyphenol composite particles MPNP and its comparison with LNP

[0594] The mRNA in Example 1.2 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-polyphenol complex particles siRNA@MPNP and mRNA@MPNP, respectively. The preparation process of the remaining drug-metal-polyphenol complex particles was the same as that in Example 1.2.

[0595] siRNA@LNPs and mRNA@LNPs were prepared using the same drug loading as described in Example 1.2.6 for Bcl-2-siRNA@MPNPs and Example 1.2.5 for RBD-mRNA@MPNPs. The organic phase solution was prepared according to the Onpattro lipid nanoparticle formulation, dissolving the ionizable lipid ALC0315, DSPE-PEG2000, DSPC, and cholesterol 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@MPNPs and mRNA@MPNPs. 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.

[0596] Agarose gel electrophoresis was used to examine the nucleic acid (siRNA and mRNA) loading efficiency of siRNA@MPNP, mRNA@MPNP, siRNA@LNP, and mRNA@LNP. The loading efficiency was determined as follows: the nucleic acid (siRNA and mRNA) loading rate for each lipid nanoparticle group was set at 10 μg / mL, with a lipid-to-nucleic acid mass ratio of 40:1. The nucleic acids were dissolved in PBS buffer as a positive control, while a nucleic acid-free PBS buffer solution was used as a negative control. The agarose gel concentration was 1.5%, which allows only free nucleic acids to pass through the gel, not the lipid nanoparticles. Electrophoresis was stopped when the free nucleic acid bands were clearly distinguishable to prevent nucleic acid degradation due to prolonged electrophoresis. Image J software was used to calculate the grayscale value of free nucleic acid in each group. 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 loading efficiency for each group was calculated as (100 - relative amount of free nucleic acid)%.

[0597] Result analysis: As shown in Figure 2-3, MPNP(Fe 3+ ) encapsulated siRNA and mRNA with efficiencies of 89.11% and 87.4% respectively; MPNP (Al 3+ ) encapsulation efficiency of siRNA and mRNA was 90.7% and 86.1% respectively; MPNP (Mg 2+ The efficiency of MPNP encapsulation of siRNA and mRNA was 93.6% and 88.1%, respectively; the efficiency of LNP encapsulation of siRNA and mRNA was 86.3% and 87.1%, respectively. The results suggest that there is no significant difference in the efficiency of nucleic acid encapsulation between MPNP and LNP.

[0598] Example 2.4, Drug-Metal (Fe 3+ 、Al 3+ or Mg 2+ )-polyphenol complex particles MPNP nucleic acid lysosomal escape ability and its comparison with LNP

[0599] The Bcl-2-siRNA (SEQ ID NO.4) in Example 1.2.6 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@MPNP (the concentration of the siRNA contained was 100 nM); the Bcl-2-siRNA (SEQ ID NO.4) in Example 2.3 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.1) in Example 1.2.5 was replaced with Cy5-labeled mRNA (Cy5-mRNA) to prepare Cy5-mRNA@MPNP (the concentration of the mRNA contained was 2 μg / mL); the RBD-mRNA (SEQ ID NO.2) in Example 2.3 was replaced with Cy5-labeled mRNA (Cy5-mRNA), Cy5-mRNA@LNP (containing mRNA at a concentration of 2 μg / mL) was prepared and incubated with the lysosomal probe Lysotracker Green in A549 cells for 3 hours. The overlap of the Cy5 fluorescence signal (red) and the Lysotracker Green fluorescence signal (green) was observed using a high-content imaging system to determine the ability of the drug-lipid particles to promote nucleic acid lysosomal escape.

[0600] The ability of drug-metal-polyphenol complex particles to promote nucleic acid lysosomal escape was determined by incubating cells with the drug-metal-polyphenol complex 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 of the red and green fluorescence signals was calculated using imageJ software. When the overlap ratio of the red and green fluorescence signals was less than 50% after incubating cells with the drug-metal-polyphenol complex nanoparticles for 3 hours, it indicated that nucleic acids could escape from the cell lysosomes more quickly, indicating that the drug-metal-polyphenol complex nanoparticles had a strong ability to promote nucleic acid lysosomal escape.

[0601] Result analysis: As shown in Figure 2-4, when Cy5-siRNA@MPNP(Fe 3+ ) and Cy5-mRNA@MPNP(Fe 3+ ) incubated A549 cells for 3 hours, the overlap rates of red fluorescence signal and green fluorescence signal were 41.17% and 44.57%, respectively, that is, the lysosomal escape rates were 58.83% and 55.43%, respectively; when Cy5-siRNA@MPNP(Al 3+ ) and Cy5-mRNA@MPNP(Al 3+) incubated A549 cells for 3 hours, the overlap rates of red fluorescence signal and green fluorescence signal were 39.03% and 39.66%, respectively, that is, the lysosomal escape rates were 60.97% and 60.34%, respectively; when Cy5-siRNA@MPNP(Mg 2+ ) and Cy5-mRNA@MPNP(Mg 2+ After incubation of A549 cells with Cy5-siRNA@LNP and Cy5-mRNA@LNP for 3 hours, the overlap rates of the red and green fluorescence signals were 29.31% and 35.08%, respectively, indicating lysosomal escape rates of 70.69% and 64.92%, respectively. In contrast, after incubation of A549 cells with Cy5-siRNA@LNP and Cy5-mRNA@LNP for 3 hours, the overlap rates of the red and green fluorescence signals were 78.92% and 84.32%, respectively, indicating lysosomal escape abilities of 21.08% and 15.68%, respectively. This suggests that the drug-lipid nanoparticle MPNP has a good ability to promote nucleic acid lysosomal escape, and the ability of MPNP to promote lysosomal escape is significantly stronger than that of LNP.

[0602] Example 2.5, Drug-Metal (Fe 3+ 、Al 3+ or Mg 2+ The ability of )-polyphenol complex particles MPNP to promote nucleic acid expression and its comparison with LNP

[0603] The RBD-mRNA (SEQ ID NO. 2) in Example 2.3 was replaced with mRNA encoding the fluorescent protein eGFP, and the rest of the preparation method was the same as Example 2.3 to obtain eGFP-mRNA@LNP.

[0604] The eGFP-mRNA@MPNP prepared in Example 1.2.5 and the above-mentioned eGFP-mRNA@LNP (containing mRNA at a concentration of 2 μg / mL) were incubated with 293T cells, respectively. The control group was incubated with MPNP or LNP. After 48 h, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0605] The method for analyzing the eGFP-positive cell rate by flow cytometry is as described in Example 1.2.

[0606] Result analysis: As shown in Figure 2-5, MPNP (Fe 3+ )、MPNP(Al 3+ )、MPNP(Mg 2+) and LNP treated 293T cells, the percentage of eGFP-positive cells were 89.45%, 91.08%, 93.47% respectively, and LNP was 72.59%. The results suggest that MPNP is better than LNP in promoting nucleic acid expression. The possible reason is that as described in Example 2.4, MPNP has a stronger ability to promote nucleic acid lysosomal escape than LNP, so more nucleic acids loaded by MPNP can be effectively released into the cytoplasm and translated into proteins. However, because the method of using the percentage of eGFP-positive cells is not sensitive enough, it is not possible to distinguish MPNPs composed of various metal ions, such as MPNP (Fe 3+ )、MPNP(Al 3+ ) or MPNP(Mg 2+ ) between the two groups to determine the difference in their ability to promote nucleic acid expression.

[0607] Example 2.6, Drug-Metal (Fe 3+ 、Al 3+ or Mg 2+ The ability of )-polyphenol complex particles MPNP to promote humoral and cellular immunity and its comparison with LNP

[0608] The RBD-mRNA@MPNP in Example 1.2.5 and the RBD-mRNA@LNP in Example 2.3 were incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPNP. After 24 hours, the supernatant was centrifuged and frozen at -20°C for later use; the cell pellet was resuspended in 100 μL PBS buffer solution, frozen and thawed twice, and sonicated for 10 minutes before centrifugation to obtain the supernatant. The expression level of RBD protein in both the cell supernatant and the cell lysate was detected using a commercially available new coronavirus antigen RBD ELISA detection kit. The results are shown in Figures 2-6.

[0609] The ELISA method for detecting RBD expression levels is as described in Example 1.2.5.

[0610] 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@MPNP and RBD-mRNA@LNP, respectively, and the control group was injected with MPNP and LNP without mRNA. The dose of each administration was 100 μL, of which the RBD-mRNA@MPNP and RBD-mRNA@LNP preparations in the experimental group each contained 30 mg of mRNA. On the 28th day after the first administration, the blood of the mice was collected, the serum was separated and serially diluted, and the titer of the total RBD IgG antibody against the S1 subunit of the new coronavirus produced in the mice was detected by commercially available ELISA kits. The results are shown in Figures 2-7.

[0611] The ELISA method for detecting the titer of total RBD IgG antibodies against the novel coronavirus S1 subunit is as described in Example 1.2.5.

[0612] In the case of RBD-mRNA@MPNP(Fe 3+ )、RBD-mRNA@MPNP(Al 3+ )、RBD-mRNA@MPNP(Mg 2+ ) and RBD-mRNA@LNP, the spleens of normal mice were collected 28 days after injection, and single-cell suspensions were prepared under sterile conditions. 100,000 spleen cells were plated in a cell plate at a density of 10 mg / mL. RBD protein was added at a final concentration of 10 mg / mL and cultured for 48 hours. The supernatant was removed by centrifugation, and the expression levels of IFN-γ, IL-2, and IL-4 were determined by ELISA kits. The results are shown in Figures 2-8.

[0613] The ELISA method for detecting the expression levels of IFN-γ, IL-2, and IL-4 is as described in Example 1.2.5.

[0614] Result analysis: As shown in Figure 2-6, RBD-mRNA@MPNP(Fe 3+ )、RBD-mRNA@MPNP(Al 3+ )、RBD-mRNA@MPNP(Mg 2+ ) and RBD-mRNA@LNP can induce 293T cells to express a certain amount of RBD, but RBD-mRNA@MPNP(Mg 2+ ) was significantly more effective in inducing cell expression of RBD than RBD-mRNA@MPNP(Al 3+ )、RBD-mRNA@MPNP(Fe 3+ ), RBD-mRNA@MPNP(Al 3+ )、RBD-mRNA@MPNP(Fe 3+ ) was significantly stronger in inducing cell expression of RBD than RBD-mRNA@LNP: RBD-mRNA@MPNP(Fe 3+ ) treatment group, the expression level of RBD in the cell supernatant was 210.7 ng / mL, and the expression level of RBD-mRNA@MPNP(Al 3+ ) treatment group, the expression level of RBD in the cell supernatant was 255.3_ng / mL, and the expression level of RBD-mRNA@MPNP(Mg 2+) treatment group in the cell supernatant of RBD expression level was 296.5_ng / mL; RBD-mRNA@LNP treatment group in the cell supernatant of RBD expression level was 114.3ng / mL. As shown in Figure 2-7, RBD-mRNA@MPNP effectively induced humoral immunity in mice and produced high levels of antigen-specific binding antibodies, and RBD-mRNA@MPNP(Mg 2+ ) was significantly better than RBD-mRNA@MPNP(Al 3+ )、RBD-mRNA@MPNP(Fe 3+ ), RBD-mRNA@MPNP(Fe 3+ ) was significantly better than RBD-mRNA@LNP in inducing humoral immunity in mice: RBD-mRNA@MPNP(Fe 3+ ) treatment group mice IgG antibody titer reached 120940; RBD-mRNA@MPNP(Al 3+ ) treatment group mice IgG antibody titer reached 140096; RBD-mRNA@MPNP(Mg 2+ ) treatment group mice IgG antibody titer reached 171458; while the IgG antibody titer in the RBD-mRNA@LNP treatment group mice was only 68257. As shown in Figure 2-8, RBD-mRNA@MPNP can effectively induce cellular immunity in mice, i.e., activate immune cells and produce a large amount of cytokines, and RBD-mRNA@MPNP(Mg 2+ ) was significantly better than RBD-mRNA@MPNP(Al 3+ )、RBD-mRNA@MPNP(Fe 3+ ), RBD-mRNA@MPNP(Al 3+ )、RBD-mRNA@MPNP(Fe 3+ ) is clearly superior to RBD-mRNA@LNP in inducing cellular immunity in mice: RBD-mRNA@MPNP(Fe 3+ ) made the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 292 pg / mL, 211 pg / mL, and 62 pg / mL, respectively; RBD-mRNA@MPNP(Al 3+ ) made the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 306 pg / mL, 240 pg / mL, and 71 pg / mL, respectively; RBD-mRNA@MPNP(Mg 2+) made the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 335pg / mL, 273pg / mL, and 73pg / mL, respectively; while RBD-mRNA@LNP made the expression levels of cytokines IFN-γ, IL-2, and IL-4 only 98pg / mL, 81pg / mL, and 28pg / mL. The results suggest that RBD-mRNA@MPNP(Mg 2+ ) is significantly better than RBD-mRNA@MPNP(Al 3+ )、RBD-mRNA@MPNP(Fe 3+ ), RBD-mRNA@MPNP(Al 3+ )、RBD-mRNA@MPNP(Fe 3+ ) is significantly better than RBD-mRNA@LNP in inducing cellular immunity in mice: RBD-mRNA@MPNP can more effectively promote cells to express target proteins and can more effectively activate humoral immunity and cellular immunity in the body. Therefore, the drug (mRNA)-lipid particles are significantly better than the existing technology LNP in terms of the effects of mRNA-loaded drugs, vaccines or other products. The possible reasons are: 1) Compared with LNP, MPNP has a stronger ability to promote nucleic acid lysosomal escape; 2) Compared with LNP, MPNP has a stronger ability to promote nucleic acid expression into protein (antigen); 3) Compared with LNP, after the curcumin in MPNP is released, it acts as an immune adjuvant (also known as an immunomodulator), which can activate humoral immunity and cellular immunity to enhance the effect of MPNP in delivering mRNA vaccines, and can also suppress the immune factor storm to suppress excessive and harmful immune responses to the body.

[0615] MPNPs composed of other metal ions (e.g. MPNPs (Fe 3+ )、MPNP(Al 3+ )) Compared with MPNP(Mg 2+ ) can more effectively promote cell expression of target protein, the reason is: Mg 2+ Mg that maintains weak chelation with RNA 2+ It can enhance the function of RNA, including increasing its thermodynamic stability, chemical stability and catalytic activity (Biochemistry. 2021 Aug 10; 60(31): 2374-2386.) 2+ It can maintain the stability of ribosome structure and translation activity, partially supplement the function of ribosomal proteins (Biosci Biotechnol Biochem. 2021 Jun 24; 85(7): 1582-1593.) and enhance immune response. 2+It can regulate LFA-1 downstream signal transduction by inducing conformational changes of the costimulatory molecule LFA-1 on the surface of CD8+ T cells, thereby promoting T cell activation and cytotoxicity (Cell. 2022 Feb 17; 185(4): 585-602.e29.)

[0616] Example 2.7 Comparison of CAR editing rate and expression duration of CD19 CAR mRNA@MPNP and CD19 CAR mRNA@LNP

[0617] Preparation of CD19 CAR mRNA:

[0618] CAR mRNA 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.

[0619] Signal transduction domain, signal peptide (SP): helps the CAR expressed in T cells to be directed to the T cell membrane.

[0620] 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.

[0621] The spacer connects the antigen-binding domain scFV and the transmembrane domain. Its flexibility ensures that scFV can better recognize antigens.

[0622] Transmembrane(TM): These combinations of CARs anchor the CAR to the T cell membrane.

[0623] 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.

[0624] >The amino acid sequence expressed by CD19 CAR mRNA is shown in SEQ ID NO.60.

[0625] Referring to Example 1.2 and Example 2.3, mRNA was replaced with CD19 CAR mRNA to prepare CD19CAR mRNA@MPNP and CD19CAR mRNA@LNP. After intravenous administration according to the above method, 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 500 μg / mL. Incubate with 647-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 μL of buffer. Analyze the percentage of CAR-positive myeloid cells on a flow cytometer. Calculate the percentage using the formula: CAR-positive myeloid cell percentage = number of CAR-expressing myeloid cells / total number of myeloid cells × 100%. Detect CD19CAR-mRNA@LNP using the same method as above.

[0626] As shown in Figure 7-1, CD19 CAR mRNA@MPNP(Mg 2+ ) had the highest proportion of positive cells in myeloid cells, and the duration of positive cells was significantly longer than that of CD19 CAR mRNA@MPNP(Al 3+ ), CD19 CAR mRNA@MPNP(Al 3+ ) is superior to CD19 CAR mRNA@MPNP(Fe 3+ ), CD19 CAR mRNA@MPNP(Fe 3+ ) is better than LNP, suggesting that MPNP (Mg 2+ ) has stronger expression and better expression duration function.

[0627] Example 2.8, Metal (Fe 3+ 、Al 3+ or Mg 2+ In vivo safety evaluation of )-polyphenol complex particles (MPNP)

[0628] A 20-day subchronic toxicity study of MPNP was conducted on SD rats, with a 20-day recovery period. The specific experimental methods are as follows:

[0629] Fifty-six SPF Sprague-Dawley rats (220 ± 20 g), half male and half female, were housed at 25°C, 45%-55% humidity, and 12 h of light. After 3-5 days of acclimatization, they were randomly divided into the following sex groups: an experimental group (n=32) and a recovery group (n=24). 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 MPNP 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 MPNP 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 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.

[0630] Administration: Experimental animals were administered via tail vein injection every two days for 20 days, and the body weight of SD rats was recorded weekly. The prepared MPNPs were dissolved in DPBS. The control group was injected with an equal volume of DPBS, while the low-dose MPNP group, the medium-dose MPNP group, and the high-dose MPNP group were injected with 25 mg / kg, 50 mg / kg, and 100 mg / kg of MPNP, respectively.

[0631] General Index Testing Methods: After each administration, the general condition of each group of animals was observed, including survival, diet, appearance, behavior, body weight, and any local reactions to the administration. A gross autopsy was performed, including timely weighing of the wet weights of major organs, such as the brain, heart, liver, spleen, lungs, and kidneys. Organ-to-body ratios were calculated, and pathological changes in each organ were recorded. Organ-to-body ratio = organ wet weight / body weight × 100%.

[0632] Collection and storage of whole blood and serum from SD rats: 20 days after administration and a 20-day recovery period, rats were dissected and blood was collected from the abdominal aorta. The rats were anesthetized with isoflurane and fixed on a dissecting board. The abdomen was disinfected with 75% ethanol. Sterile ophthalmic scissors were used to cut the rat's abdomen open. The internal organs were gently parted with cotton balls to expose the abdominal aorta. Whole blood was collected using a 500μL negative pressure EDTAK2 anticoagulant blood collection tube and stored at 4°C for routine blood testing. Whole blood was collected using a 5mL negative pressure conventional blood collection tube and allowed to stand at room temperature for 30 minutes. The supernatant was collected in a 1.5mL centrifuge tube and stored at -20°C for the detection of blood biochemical and immunological indicators.

[0633] Routine blood test method: 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.

[0634] Blood biochemical index detection method: Blood biochemical index includes inorganic ions (Fe 2+ , Na + , K + , Cl - , Ca 2+ ), liver function indicators (ALT, AST, γ-GT, T-BIL, D-BIL, ALP, ALB), renal function indicators (BUN, UA, CR), cardiac function indicators (LDH, CK), glucose metabolism indicators (GSP, GLU, INS), lipid metabolism indicators (CHO, TG, LDL-C, HDL-C). Thaw serum samples and centrifuge at 3000 rpm for 15 minutes. Remove the supernatant and aliquot it for later use. Set the corresponding parameters on the automatic biochemical analyzer, add the prepared working solution, and then add the serum to be tested. The automatic biochemical analyzer will automatically determine the results.

[0635] Immunological indicators include thyroid function markers (TT3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN-γ, IFN-α, TNF-α), immunoglobulins (IgG, IgA, IgM), and serum complement (C3, CH50). These indicators are measured using ELISA.

[0636] 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.

[0637] Analysis of results: As shown in Table 2-1, at the end of the dosing period and the end of the recovery period, compared with the control group, the rats in the low-, medium- and high-dose MPNP groups survived well, had normal diet, normal appearance and behavioral activities, and no obvious adverse reactions were observed after administration; compared with the control group, there was no significant difference in the weight gain values ​​of male SD rats and female SD rats in the low-, medium- and high-dose MPNP groups; compared with the control group, there was no significant difference in the organ-to-body ratios of the low-, medium- and high-dose MPNP groups.

[0638] At the end of the administration period and the end of the recovery period, compared with the control group, 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) in the low, medium and high dose MPNP groups were normal. Compared with the control group, the blood biochemical indicators, including inorganic ions (Fe 2+ , Na + , K + , Cl - , Ca 2+ ), liver function indicators (ALT, AST, γ-GT, T-BIL, D-BIL, ALP, ALB), renal function indicators (BUN, UA, CR), cardiac function indicators (LDH, CK), glucose metabolism indicators (GSP, GLU, INS), lipid metabolism indicators (CHO, TG, LDL-C, HDL-C), all showed no abnormalities; compared with the control group, the immunological related indicators of the low, medium and high doses of MPNP groups included thyroid function indicators (TT3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN-γ, IFN-α, TNF-α), immunoglobulins (IgG, IgA, IgM), and serum complement (C3, CH50), all showed no abnormalities.

[0639] At the end of the administration period and the end of the recovery period, compared with the control group, the brain tissue structure of rats in the low-, medium-, and high-dose MPNP groups was intact, with normal tissue staining, intact cell morphology and structure, and no nuclear pyknosis or inflammatory cell infiltration; the myocardial tissue structure was intact, with myocardial cells arranged neatly, continuously, and tightly, and the cell nuclei were clearly visible, without obvious cell congestion, edema, or necrosis; the hepatocyte 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, with alveoli of uniform size, and no obvious inflammatory cell aggregation or infiltration; and the kidney structure was normal.

[0640] The above results suggest that long-term and large-scale injection of MPNP (metal ion Fe3+ 、Al 3+ or Mg 2+ ) No obvious chronic toxicity was found, indicating that MPNP is relatively safe.

[0641] Table 2-1MPNP (metal ion is Fe 3+ 、Al 3+ or Mg 2+ In vivo safety evaluation of

[0642] Note: ALT, alanine aminotransferase; AST, aspartate aminotransferase; γ-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; CHO, cholesterol; TG, triglycerides; 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; IL-4, interleukin-4; IFN-γ, interferon-γ; IFN-α, interferon-α; TNF-α, tumor necrosis factor-α; IgG, immunoglobulin G; IgA, immunoglobulin A; IgM, immunoglobulin M; C3, complement C3; CH50, total complement CH50

[0643] Example 2.9. Comparison of in vivo safety of metal-polyphenol composite particles (MPNP) and LNP

[0644] The main toxicity of LNP comes from its main components - cationic lipids and / or ionizable lipids. When LNP is metabolized in the body, the free cationic lipids and / or ionizable lipids will produce significant toxicity to the body. The median lethal dose (IC50) of cationic lipids and / or ionizable lipids to biological cells is 50 ) is an important parameter for evaluating the toxicity of LNP to the body. The drug-metal-polyphenol complex particles (MPNP) use metal-polyphenol complexes to replace the cationic lipids / ionizable lipids in LNPs. Therefore, we studied the median lethal dose (IC50) of metal-polyphenol complexes and cationic lipids / ionizable lipids on biological cells. 50 ), and compared the differences in toxicity between LNP and MPNP.

[0645] 293T cells were incubated with different concentrations of the metal-polyphenol complex (0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM), cationic lipid (DOTAP, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM) and ionizable lipid (ALC0315, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM) for 48 hours, and then the cell viability was detected using a CCK8 activity detection kit. The median lethal dose (IC50) of the metal-polyphenol complex, cationic lipid (DOTAP) and ionizable lipid (ALC0315) on 293T cells was calculated. 50 .

[0646] CCK8 detection method:

[0647] Cell culture: Culture cells in DMEM containing 10% FBS and 1% double-antibody until the cell density reaches 80%-90% of the culture flask.

[0648] Wash the remaining culture medium from the culture flask with PBS, add trypsin, and quickly transfer the flask to a 37°C incubator with 5% CO2. Observe carefully until the cells have slightly rounded, then add culture medium to terminate the digestion. Transfer the cells to a centrifuge tube, centrifuge at 1500 RPM for 5 minutes, and resuspend the cells in fresh culture medium.

[0649] Counting: Dilute the cell suspension to 10,000 cells per mL according to the intended cell count. Add 100 μL 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.

[0650] After 48 h of drug incubation, 10% CCK8 was added and incubated for 1-3 h. The absorbance was measured at 450 nm using a microplate reader.

[0651] Survival rate (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] x 100%.

[0652] IC 50 IC calculation method: With survival rate as the vertical axis and drug concentration as the horizontal axis, IC was calculated using Graphpad using the [Inhibitor] vs. normalized response--Variable slope analysis method. 50 .

[0653] To compare the in vivo safety of MPNP and LNP, MPNP (8 mg / kg) and LNP (3.24 mg / kg) capable of carrying an equal amount of nucleic acid (200 μg / kg mRNA) were used for in vivo experiments according to the method of Example 2.8 to evaluate and compare the in vivo toxicity of MPNP and LNP.

[0654] Result analysis: As shown in Table 2-2, the IC 50 The toxicity of the metal-polyphenol complex is significantly lower than that of cationic lipids (DOTAP) and ionizable lipids (ALC0315).

[0655] As shown in Table 2-3, at the end of the administration period and the end of the recovery period, MPNP (Fe 3+ )、MPNP(Al 3+ ) or MPNP(Mg 2+ ) group showed no significant abnormalities in liver function indicators ALT, AST, ALP and cytokine IL-6, IL-1β expression levels. However, compared with the control group, the liver function indicators ALT, AST, ALP and cytokine IL-6, IL-1β expression levels of the LNP group were significantly increased. The results suggest that MPNP is safer in vivo than LNP. The reason is that the core component of LNP is artificially synthesized "cationic lipid / ionizable lipid", which has high cytotoxicity and immunogenicity, and its structure is relatively stable and difficult to decompose and metabolize in the body; while MPNP (Fe 3+ )、MPNP(Al 3+ ) or MPNP(Mg 2+ The core component of MPNP (Fe) is a metal-polyphenol complex, which is composed of a highly safe natural small molecule curcumin (a food additive and pharmaceutical excipient approved by the FDA) and safe metal ions. After the drug is delivered, it is decomposed into natural molecules in the body. 3+ )、MPNP(Al 3+ ) or MPNP(Mg 2+ ) components do not contain cationic lipids / ionizable lipids and will not cause toxic side effects related to cationic lipids / ionizable lipids, so MPNP (Fe 3+ )、MPNP(Al 3+ ) or MPNP(Mg 2+ ) is safer than LNP.

[0656] The structural formula of DOTAP

[0657] The structural formula of ALC0315

[0658] Table 2-2 Metals (metal ions are Fe 3+ 、Al 3+ or Mg 2+ IC of )-polyphenol complex with cationic lipid (DOTAP) and ionizable lipid (ALC0315) 50 Compared with

[0659] Table 2-3MPNP (metal ion is Fe 3+ 、Al 3+ or Mg 2+ ) and LNP chronic toxicity test indicators comparison

[0660] Safety history data for various metals

[0661] References: "Dietary Reference Intakes for Chinese Residents" - 2023 Edition, "Risk Assessment of Dietary Aluminum Exposure for Chinese Residents", ICH Coordinated Guidelines - Elemental Impurities Guidelines Q3D (R2). The above historical data show that the safety ranking of MPNP or Apt-MPNP prepared based on various metals is: Mg>Fe>Zn>Al>Mn>Cr. This suggests that not only MPNP (Mg 2+ ) has the best efficacy and the best safety.

[0662] Example 3, Drug-Metal (Fe 3+ 、Al 3+ or Mg 2+ Clinical application and administration route of )-polyphenol complex particles MPNP

[0663] The mRNA in Example 1.2 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).

[0664] The sequences of the above-mentioned different nucleic acids are as follows: ① The sequence of B7-H4-siRNA is SEQ ID NO.19 (sense strand) and SEQ ID No.26 (antisense strand) (25bp), and its random control sequence is SEQ ID NO.20 (sense strand) and SEQ ID No.27 (antisense strand) (19bp); ② The mRNA sequence encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit is SEQ ID NO.2 (669nt). Referring to the method of Example 1.2, drug-metal-polyphenol complex particles (B7-H4-siRNA@MPNP and RBD-mRNA@MPNP) encapsulating the above-mentioned different types of nucleic acids were prepared respectively. The preparation process of the remaining drug-metal-polyphenol complex particles is the same as that of Example 1.2. The above two different drug-metal-polyphenol complex particles (B7-H4-siRNA@MPNP and RBD-mRNA@MPNP) are used to treat glioma and as mRNA vaccines to prevent novel coronavirus, respectively.

[0665] To evaluate the therapeutic effect of the drug-metal-polyphenol composite particles (B7-H4 siRNA@MPNP) on liver cancer, an animal model of liver cancer was established using HepG2 cells. 3 Mice bearing liver cancer were randomly divided into four groups (n=5 per group): a PBS control group, a blank MPNP vector group, a Scr siRNA@MPNP treatment group (control group), and a B7-H4 siRNA@MPNP treatment group. Each group received eight intratumoral injections of PBS, MPNP, Scr siRNA@MPNP, or B7-H4 siRNA@MPNP every three days at a dose of 200 μg siRNA / kg. Tumor volumes were measured and recorded every three days. The results are shown in Figure 3-1.

[0666] In order to evaluate the effect of the drug-metal-polyphenol complex particles (RBD-mRNA@MPNP) as an mRNA vaccine to prevent the new coronavirus, the experimental process and experimental methods are as shown in the previous Example 1.2.5.

[0667] The ELISA detection method is as described in Example 1.2.5.

[0668] Establishment of liver cancer mouse model: HepG2 cells were collected and cultured at 1×10 7 The cells were resuspended in PBS at a density of 100 μL / mL and kept on ice before inoculation. 100 μL of the cell suspension was then subcutaneously injected into the dorsal region near the hind legs of female Balb / c nude mice to establish a mouse model of liver cancer.

[0669] Result analysis:

[0670] As shown in Figure 3-1, Figure 3-1-2 and Figure 3-1-3, scr siRNA@MPNP(Fe 3+ )、Scr-siRNA@MPNP(Al 3+ )、Scr-siRNA@MPNP(Mg 2+ ) had almost no inhibitory effect on the growth of HepG2 cells, while B7-H4 siRNA@MPNP(Fe 3+ )、B7-H4 siRNA@MPNP(Al 3+ )、B7-H4siRNA@MPNP(Mg 2+ ) showed a highly effective therapeutic effect, effectively inhibiting the growth of liver cancer tumors. The results suggest that the drug-metal-polyphenol complex particles can encapsulate and deliver B7-H4 siRNA, inhibiting the expression of the target gene, thereby inhibiting the development of liver cancer.

[0671] As shown in Figures 1-3 and 1-5 of Example 1.2.5, drug-metal-polyphenol composite particles RBD-mRNA@MPNP (Fe 3+ ) increased the expression level of mouse IgG antibody to 84363.4 (Figure 1-3), and the expression levels of cytokines IFN-γ, IL-2, and IL-4 to 271.8 pg / mL, 269.6 pg / mL, and 75.8 pg / mL, respectively (Figure 1-5). RBD-mRNA@MPNP(Al 3+ ) increased the expression level of mouse IgG antibodies to 94828.6 (Figure 1-3-2), and the expression levels of cytokines IFN-γ, IL-2, and IL-4 to 306.2 pg / mL, 289.6 pg / mL, and 88.2 pg / mL, respectively (Figure 1-5-2); RBD-mRNA@MPNP(Mg2+) increased the expression level of mouse IgG antibodies to 113250.6 (Figure 1-3-3), and the expression levels of cytokines IFN-γ, IL-2, and IL-4 to 291.3 pg / mL, 231.1 pg / mL, and 81.7 pg / mL, respectively (Figure 1-5-3). The results suggest that the drug-metal-polyphenol complex particles (mRNA@MPNP) can effectively induce humoral immunity in mice and produce high levels of antigen-specific binding antibodies; at the same time, it can effectively induce cellular immunity in mice, that is, activate immune cells and produce a large amount of cytokines. Therefore, the drug-metal-polyphenol complex particles (mRNA@MPNP) can effectively prevent infection with the new coronavirus.

[0672] Intratumoral injection of scr siRNA@MPNP is effective in treating liver cancer, while intramuscular injection of RBD-mRNA@MPNP can activate humoral and cellular immunity, thereby preventing infection with the novel coronavirus. The results suggest that the drug-metal-polyphenol complex particles can be administered via multiple routes.

[0673] Example 4: Curcumin, Fe 3+ Function after being replaced by similar products

[0674] Example 4.1, Curcumin, Fe 3+ Function after being replaced by similar products

[0675] Refer to Example 1.1 with curcumin, Fe 3+ The analogs of curcumin, Fe 3+ Instead, 12 different drug-metal-polyphenol composite particles (eGFP-mRNA@MPNP) were prepared by different combinations according to Example 1.2, wherein the concentration of mRNA contained in each eGFP-mRNA@MPNP was 2 μg / mL. 3+ The names and structures of curcumin, Fe 3+ The combination of the same is shown in Table 3-2. In Example 1.1, the reaction temperature is 60°C and the reaction time is 2 hours, and other conditions remain unchanged.

[0676] To compare the effects of the 12 different eGFP-mRNA@MPNPs and eGFP-mRNA@LNPs, we prepared LNPs encapsulating equal amounts of eGFP mRNA according to Example 2.3 to obtain eGFP-mRNA@LNPs.

[0677] The above 12 different eGFP-mRNA@MPNPs and the above eGFP-mRNA@LNPs (all containing mRNA at a concentration of 2 μg / mL) were incubated with 293T cells, respectively. The control group was incubated with MPNP or LNP. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0678] The method for analyzing the eGFP-positive cell rate by flow cytometry is as described in Example 1.2.

[0679] The main toxicity of LNP comes from its main component - cationic lipids / ionizable lipids. When LNP is metabolized in the body, the free cationic lipids / ionizable lipids will produce significant toxicity to the body. The median lethal dose (IC50) of cationic lipids / ionizable lipids to biological cells is 50) is an important parameter for evaluating the toxicity of LNP to the body. The metal-polyphenol complex particles (MPNP) replace the cationic lipid / ionizable lipid in LNP with a metal-polyphenol complex. Therefore, we studied the median lethal dose (IC50) of the 12 metal-polyphenol complexes and cationic lipids (DOTAP) / ionizable lipids (ALC0315) in Table 3-2 to biological cells. 50 ), and compared the differences in toxicity between LNP and 12 MPNPs.

[0680] IC 50 The calculation method of is as described in Example 2.9.

[0681] Results analysis: As shown in Table 3-2, the percentage of eGFP-positive cells in 293T cells treated with 12 different eGFP-mRNA@MPNPs was significantly higher than that of eGFP-mRNA@LNP, among which mRNA@MPNP1 had the highest percentage of eGFP-positive cells. 3+ The function of mRNA@MPNP formed after being replaced by its analog is inferior to that of mRNA@MPNP1, but slightly better than that of mRNA@LNP. The possible reason is that, as described in Example 2.4, MPNP has a stronger ability to promote lysosomal escape of nucleic acids than LNP, so more nucleic acids loaded by MPNP can be effectively released into the cytoplasm and translated into proteins.

[0682] The above results suggest that as long as the following conditions are met, curcumin, Fe 3+ The function of the drug-metal-polyphenol composite particles formed after being replaced by its congeners is not affected: ① The congeners of curcumin are hydrophobic polyphenols that can complex with metals; ② Fe 3+ The congeners are metal ions; ③ Curcumin and Fe 3+ The coordination bonds between them can be broken in response to the low pH environment of the lysosome.

[0683] As shown in Table 3-3, the IC values ​​of 12 metal-polyphenol complexes 50 The toxicity of metal-polyphenol complex is significantly lower than that of cationic lipid (DOTAP) and ionizable lipid (ALC0315). 3+The safety of lipid particles (MPNP) composed of and its analogs is higher than that of LNP. The reasons are as follows: the core component of LNP is artificially synthesized "cationic lipid / ionizable lipid", which has high cytotoxicity and immunogenicity, and its structure is relatively stable and difficult to decompose and metabolize in the body; while the core component of MPNP is metal-polyphenol complex, which is composed of non-cationic lipid, natural small molecule with high safety (of which curcumin is a food additive and pharmaceutical excipient approved by the FDA) and safe metal ions, and it has been decomposed into natural molecules in the body after completing drug delivery. In summary, curcumin, Fe 3+ The components of MPNP composed of LNP and its 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 MPNP is higher than that of LNP.

[0684] Table 3-1 Curcumin, Fe 3+ The names and structures of its analogs

[0685] Table 3-2 Curcumin, Fe 3+ List of combinations and functions of metal-polyphenol complexes in drug-lipid nanoparticles prepared from their analogs

[0686] Table 3-3 Curcumin, Fe 3+ IC of metal-polyphenol complexes prepared from its analogs 50

[0687] Example 4.2, Curcumin and its analogs, Fe 3+ The dosage ratio of its similar components and the function of the drug-metal-polyphenol composite particles prepared therefrom

[0688] According to Example 1.2, mRNA-metal-polyphenol complex particles (metal ions are Fe 3+ ), and curcumin was replaced by its analogues hesperetin (1 molecule of hesperetin contains 4 hydroxyl groups) and catechin (1 molecule of catechin contains 5 hydroxyl groups), and three mRNA-metal-polyphenol complex particles (mRNA@MPNP1, mRNA@MPNP5, mRNA@MPNP13) were prepared. When preparing these three mRNA-metal-polyphenol complex particles, curcumin or its analogues were combined with Fe 3+The dosage ratios were 1:1, 1:1, and 1:2, respectively. The mRNA encoding the eGFP fluorescent protein was SEQ ID NO. 1 (720 nt). The mRNA encapsulation efficiency of the three drug-lipid particles and their ability to promote eGFP fluorescent protein expression after treatment in 293T cells were tested according to the experimental procedures and methods described in Example 1.2.5.

[0689] The metal ion mentioned above is Fe 3+ The steps of preparing metal ions are Al 3+ and Mg 2+ mRNA-metal-polyphenol complex particles: metal ion Al 3+ (mRNA@MPNP3, mRNA@MPNP7, mRNA@MPNP14), metal ions Mg 2+ (mRNA@MPNP4, mRNA@MPNP8, mRNA@MPNP15).

[0690] Results analysis: As shown in Table 3-4, Table 3-4-2 and Table 3-4-3, the mRNA encapsulation efficiency and the ability to promote the expression of target proteins of drug-metal-polyphenol complex particles prepared with different dosage ratios according to the chemical structure of the metal-polyphenol complex components are comparable. The results suggest that the dosage ratio of the metal-polyphenol complex components can be adjusted according to the structure of the specific metal-polyphenol complex components. The basis for adjusting the dosage ratio is: because the hydroxyl group of curcumin analogs reacts with Fe 3+ As long as the congeners of curcumin contain multiple binding sites, the congeners of curcumin and Fe 3+ The dosage ratio of the congeners can be adjusted according to the number of binding sites contained in the curcumin congeners.

[0691] Table 3-4 Different metals (Fe 3+ )-polyphenol complex component dosage ratio and the function of the drug-lipid particles prepared therefrom

[0692] Table 3-4-2 Different metals (Al 3+ )-polyphenol complex component dosage ratio and the function of the drug-lipid particles prepared therefrom

[0693] Table 3-4-3 Different metals (Mg 2+ )-polyphenol complex component dosage ratio and the function of the drug-lipid particles prepared therefrom

[0694] Example 5: Construction of targeted MPNP vector

[0695] Example 5.1 Preparation of Apt-MPNP

[0696] First, refer to Example 1.2 (the metal ions are Fe 3+ 、Al 3+ and Mg 2+ ) Metal-polyphenol complex particles (MPNPs) encapsulating mRNA were prepared. mRNA was dissolved 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) at a concentration of 60 μg / mL.

[0697] Preparation of targeted DSPE-PEG2000-Apt micelles

[0698] Apt-C6-SS-C6 is prepared by modifying the disulfide bond on Apt. Apt-C6-SS-C6 has six carbon chains modified on both sides of the disulfide bond, one end of which is connected to Apt. First, Apt-C6-SS-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-SS-C6 is incubated in 10mM phosphine hydrochloride (TCEP) at 25°C for 1 hour to break the disulfide bond and expose the sulfhydryl group. This reduction reaction of Apt-C6-SS-C6 produces Apt-C6-SH. In the third step, 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

[0699] 5'-TAGCCAAGGTAACCAGTACAAGGTGCTAAACGTAATGGCTTCGGCTT AC-3' (as shown in SEQ ID NO. 44), Apt is the target binding region, DSPE is the hydrophobic region, and PEG2000 is the linker region.

[0700] Next, 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.

[0701] In the preparation method of DSPE-PEG2000-Apt, the intermediates DSPE-PEG2000-MAL and Apt-C6-SH can also be changed to include but not limited to DSPE-PEG2000-NHS and Apt-NH2, DSPE-PEG2000-COOH and Apt-NH2, DSPE-PEG2000-NCO and Apt-NH2, and DSPE-PEG2000-N3 and Apt-DBCO, etc., with the ultimate goal of preparing DSPE-PEG2000-Apt.

[0702] 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.

[0703] The prepared DSPE-PEG2000-Apt micelles were mixed evenly with the MPNPs described above at a reaction mass ratio of 1:16.8 and incubated at 4°C (the acceptable reaction temperature is 0°C ± 10°C) for 2 hours (the acceptable incubation time is 0.2-12 hours). The DSPE-PEG2000-Apt micelles were inserted into the MPNPs to prepare Apt-MPNPs.

[0704] DSPE-PEG2000-Apt micelles were mixed uniformly with mRNA-encapsulated metal-polyphenol complex particles (MPNPs) at varying reaction mass ratios (1:84, 1:42, 1:21, 1:16.8, and 1:14). While varying the reaction mass ratio altered the distribution of Apt on the metal-polyphenol complex particles (MPNPs), the resulting targeting vectors (Apt-MPNPs) maintained their physicochemical properties despite varying modification rates.

[0705] MPNP allows DSPE-PEG2000-Apt micelles to hydrophobically insert into the outer surface of the metal-polyphenol complex particles MPNP, resulting in Apt-metal-polyphenol complex particles, known as the targeting carrier (Apt-MPNP). 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-polyphenol complex particles, Apt will be encapsulated within the metal-polyphenol complex particles, preventing Apt from exerting its targeting effect.

[0706] Example 5.2 Comparison of targeting between Apt-MPNP and MPNP

[0707] Referring to the preparation method of Example 5.1, the mRNA-metal-polyphenol complex particles (eGFP-mRNA@MPNP) prepared in Example 1.2 and DSPE-PEG2000-Apt can be prepared to obtain a targeting vector (eGFP-mRNA@Apt-MPNP).

[0708] Preparation of CD19 CAR mRNA:

[0709] CAR mRNA 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.

[0710] Signal transduction domain, signal peptide (SP): helps the CAR expressed in T cells to be directed to the T cell membrane.

[0711] 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.

[0712] The spacer connects the antigen-binding domain scFV and the transmembrane domain. Its flexibility ensures that scFV can better recognize antigens.

[0713] Transmembrane(TM): These combinations of CARs anchor the CAR to the T cell membrane.

[0714] 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.

[0715] The amino acid sequence expressed by CD19 CAR mRNA is shown in SEQ ID NO.60.

[0716] Referring to the preparation method of Example 5.1, mRNA was replaced with CD19 CAR mRNA to prepare mRNA-metal-polyphenol complex particles (CD19 CAR-mRNA@MPNP) and DSPE-PEG2000-Apt to prepare a targeting vector (CD19 CAR-mRNA@Apt-MPNP).

[0717] The targeting vector (eGFP-mRNA@Apt-MPNP), mRNA-metal-polyphenol complex particles (eGFP-mRNA@MPNP), mRNA-metal-polyphenol complex particles (CD19 CAR-mRNA@MPNP), and DSPE-PEG2000-Apt were respectively mixed with 4×10 4 Individual T cells were incubated and the cell suspension was collected after 48 hours and analyzed using Alexa The CAR Linker antibody labeled with 647 was used to detect the proportion of CD19-CAR positive cells.

[0718] The flow cytometry analysis method is as follows: The method for analyzing the percentage of CAR-positive cells by flow cytometry is as follows: T cells were seeded on a 12-well plate at a seeding density of 4×10 5 1 mL of MPNP, Apt-MPNP, CD19 CAR-mRNA@Apt-MPNP or CD19 CAR-mRNA@MPNP was added to the cells / well, where the concentration of CAR-mRNA@MPNP was 15 μg / mL. After 48 h, the cell suspension was collected and The cells were incubated with 647-labeled CAR Linker antibody at 4°C for 40 minutes. After washing with buffer, the supernatant was discarded and the percentage of CAR-positive cells was detected on a flow cytometer. The calculation formula was: CAR-positive cell rate = number of CAR-expressing cells / total number of cells × 100%; CAR-positive cell rate in T cells = number of CAR-expressing T cells / total number of T cells × 100%.

[0719] As shown in Figure 4-1, MPNP(Fe 3+ ) and Apt-MPNP(Fe 3+ ) were incubated with human T cells and MPNP (Fe 3+ ) and Apt-MPNP(Fe 3+ The percentages of CD19-CAR positive cells in the ) group were 1.83±0.97 and 8.40±0.75 respectively. This result shows that human T cells are sensitive to Apt-MPNP (Fe 3+ ) was better than MPNP(Fe 3+ This is because Apt-MPNP(Fe 3+ ) The surface-modified Apt can recognize receptor ligands with human T cells, effectively increasing cellular uptake and mRNA expression in cells, reducing the side effects of MPNP due to lack of targeting, and improving safety.

[0720] As shown in Figure 4-1-2, MPNP(Al 3+ ) and Apt-MPNP(Al 3+ ) were incubated with human T cells, and MPNP (Al 3+ ) and Apt-MPNP(Al 3+ The percentages of CD19-CAR positive cells in the ) group were 1.83±1.13 and 9.43±1.82 respectively. This result shows that human T cells are sensitive to Apt-MPNP (Al 3+ ) was better in uptake and expression than MPNP(Al 3+ This is because Apt-MPNP (Al 3+ ) The surface-modified Apt can recognize receptor ligands with human T cells, effectively increasing cellular uptake and mRNA expression in cells, reducing the side effects of MPNP due to lack of targeting, and improving safety.

[0721] As shown in Figure 4-1-3, MPNP (Mg 2+ ) and Apt-MPNP(Mg 2+ ) were incubated with human T cells, and MPNP (Mg 2+ ) and Apt-MPNP(Mg 2+ The percentages of CD19-CAR positive cells in the ) group were 3.05±0.96 and 14.27±1.02 respectively. This result showed that human T cells were sensitive to Apt-MPNP (Mg 2+ ) was better than MPNP (Mg 2+ This is because Apt-MPNP (Mg 2+ ) The surface-modified Apt can recognize receptor ligands with human T cells, effectively increasing cellular uptake and mRNA expression in cells, reducing the side effects of MPNP due to lack of targeting, and improving safety.

[0722] Example 6 Preparation and Detection of Apt-MPNP and Apt-LNP

[0723] Example 6.1. Preparation of CD19 CAR mRNA@Apt(CD62L)-LNP, ASO@Apt(AS1411)-LNP, and siRNA@Apt(P19)-LNP

[0724] The CD19 CAR mRNA in Example 2.7 was used to prepare the drug CD19 CAR mRNA@LNP according to Example 2.3. The targeted drug (CD19 CAR mRNA@Apt-LNP) was then prepared using the post-insertion method according to Example 5.1. The DSPE-PEG2000-Apt(CD62L) micelles prepared in Example 5.1 were mixed evenly with the CD19 CAR mRNA@LNP, reacted at 60°C for 10 minutes, and then incubated at 4°C overnight to allow the DSPE-PEG2000-Apt(CD62L) micelles to be inserted into the CD19 CAR mRNA@LNP to prepare the CD19 CAR mRNA@Apt(CD62L)-LNP.

[0725] Referring to Example 1.2.7, the drug was replaced with ASO to prepare ASO@LNP. The targeted drug (ASO@Apt-LNP) was then prepared using the post-insertion method, referring to Example 5.1. DSPE-PEG2000-Apt(AS1411) micelles were prepared by replacing Apt in Example 5.1 with Apt(AS1411). These micelles were then mixed with ASO@LNP and reacted at 60°C for 10 minutes. The mixture was then incubated overnight at 4°C to allow the DSPE-PEG2000-Apt(AS1411) micelles to intercalate into the ASO@LNP to prepare ASO@Apt(AS1411)-LNP.

[0726] Referring to Example 1.2.6, the drug was replaced with siRNA to prepare the drug siRNA@LNP. Then, referring to Example 5.1, the targeted drug (siRNA@Apt-LNP) was prepared using the post-insertion method. In Example 5.1, Apt was replaced with Apt(P19) to prepare DSPE-PEG2000-Apt(P19) micelles. These micelles were then mixed uniformly with the siRNA@LNP, reacted at 60°C for 10 minutes, and incubated overnight at 4°C to allow the DSPE-PEG2000-Apt(P19) micelles to intercalate into the siRNA@LNP to prepare siRNA@Apt(P19)-LNP.

[0727] Example 6.2. CD19 CAR mRNA@Apt(CD62L)-LNP, ASO@Apt(AS1411)-LNP, siRNA@Apt(P19)-LNP Animal Survival Rate Detection

[0728] Using the products in Example 6.1, the survival time of acute B-cell lymphoblastic leukemia model mice in the CD19 CAR mRNA@Apt(CD62L)-LNP treatment group, lung cancer mice in the ASO@Apt(AS1411)-LNP treatment group, and pancreatic cancer mice in the siRNA@Apt(P19)-LNP treatment group were recorded, and their respective survival rates were calculated.

[0729] Example 6.3. Preparation of CD19 CAR mRNA@Apt(CD62L)-MPNP, CD19 CAR mRNA@Apt(CD8)-MPNP, ASO@Apt(AS1411)-MPNP, and siRNA@Apt(P19)-MPNP

[0730] The drug CD19 CAR mRNA@MPNP (Fe 3+ Then, the targeted drug CD19 CAR mRNA@Apt(CD62L)-MPNP(Fe 3+ ). The DSPE-PEG2000-Apt(CD62L) micelles prepared in Example 5.1 were mixed with CD19 CAR mRNA@MPNP(Fe 3+ ) were mixed evenly and incubated at 4°C (the reaction temperature can be 0°C ± 10°C) for 2 h (the incubation time can be 0.2-12 h) to allow DSPE-PEG2000-Apt(CD62L) micelles to be inserted into CD19 CAR mRNA@MPNP(Fe 3+ ) was prepared from CD19 CAR mRNA@Apt(CD62L)-MPNP(Fe 3+ ).

[0731] The drug CD19 CAR mRNA@MPNP (Al) was prepared by using the CD19 CAR mRNA in Example 2.7 according to Example 1.2. 3+ Then, the targeted drug CD19 CAR mRNA@Apt(CD62L)-MPNP (Al 3+ ). The DSPE-PEG2000-Apt(CD62L) micelles prepared in Example 5.1 were mixed with CD19 CAR mRNA@MPNP(Al 3+ ) were mixed evenly and incubated at 4°C (the reaction temperature can be 0°C ± 10°C) for 2 h (the incubation time can be 0.2-12 h) to allow DSPE-PEG2000-Apt(CD62L) micelles to be inserted into CD19 CAR mRNA@MPNP(Al 3+) was prepared from CD19 CAR mRNA@Apt(CD62L)-MPNP(Al 3+ ).

[0732] The CD19 CAR mRNA in Example 2.7 was used to prepare the drug CD19 CAR mRNA@MPNP (Mg 2+ Then, the targeted drug CD19 CAR mRNA@Apt(CD62L)-MPNP(Mg 2+ ). The DSPE-PEG2000-Apt(CD62L) micelles prepared in Example 5.1 were mixed with CD19 CAR mRNA@MPNP(Mg 2+ ) were mixed evenly and incubated at 4°C (the reaction temperature can be 0°C ± 10°C) for 2 h (the incubation time can be 0.2-12 h) to allow DSPE-PEG2000-Apt(CD62L) micelles to be inserted into CD19 CAR mRNA@MPNP(Mg 2+ ) was prepared from CD19 CAR mRNA@Apt(CD62L)-MPNP(Mg 2+ ).

[0733] Referring to Example 1.2, the drug was replaced with ASO to prepare the drug ASO@MPNP(Fe 3+ ). Then, the targeted drug (ASO@Apt-MPNP) was prepared by referring to Example 5.1. Apt in Example 5.1 was replaced with Apt(AS1411) to prepare DSPE-PEG2000-Apt(AS1411) micelles, and ASO@MPNP(Fe 3+ ) were mixed evenly and incubated at 4 ° C (the reaction temperature can be used at 0 ° C ± 10 ° C) for 2 h (the incubation time can be used at 0.2-12 h) to allow DSPE-PEG2000-Apt (AS1411) micelles to be inserted into ASO@MPNP (Fe 3+ ) was prepared from ASO@Apt(AS1411)-MPNP(Fe 3+ ).

[0734] The drug was replaced with ASO to prepare drug ASO@MPNP (Al 3+ Then, the targeted drug (ASO@Apt-MPNP) was prepared by referring to Example 5.1. Apt in Example 5.1 was replaced with Apt (AS1411) to prepare DSPE-PEG2000-Apt (AS1411) micelles. 3+) were mixed evenly and incubated at 4 ° C (the reaction temperature can be used at 0 ° C ± 10 ° C) for 2 h (the incubation time can be used at 0.2-12 h) to allow DSPE-PEG2000-Apt (AS1411) micelles to be inserted into ASO@MPNP (Al 3+ ) was prepared from ASO@Apt(AS1411)-MPNP(Al 3+ ).

[0735] The drug was replaced with ASO to prepare drug ASO@MPNP(Mg 2+ ). Then, the targeted drug (ASO@Apt-MPNP) was prepared by referring to Example 5.1. Apt in Example 5.1 was replaced with Apt(AS1411) to prepare DSPE-PEG2000-Apt(AS1411) micelles, and ASO@MPNP(Mg 2+ ) were mixed evenly and incubated at 4 ° C (the reaction temperature can be used at 0 ° C ± 10 ° C) for 2 h (the incubation time can be used at 0.2-12 h) to allow DSPE-PEG2000-Apt (AS1411) micelles to be inserted into ASO@MPNP (Mg 2+ ) was prepared from ASO@Apt(AS1411)-MPNP(Mg 2+ ).

[0736] Referring to Example 1.2, the drug was replaced with siRNA to prepare the drug siRNA@MPNP(Fe 3+ ). Then, the targeted drug (siRNA@Apt-MPNP) was prepared by referring to Example 5.1. Apt in Example 5.1 was replaced with Apt(P19) to prepare DSPE-PEG2000-Apt(P19) micelles, and siRNA@MPNP(Fe 3+ ) were mixed evenly and incubated at 4°C (the reaction temperature can be 0°C ± 10°C) for 2 h (the incubation time can be 0.2-12 h) to allow DSPE-PEG2000-Apt(P19) micelles to be inserted into siRNA@MPNP(Fe 3+ ) was prepared from siRNA@Apt(P19)-MPNP(Fe 3+ ).

[0737] Referring to Example 1.2, the drug was replaced with siRNA to prepare the drug siRNA@MPNP (Al 3+ Then, the targeted drug (siRNA@Apt-MPNP) was prepared by referring to Example 5.1. Apt in Example 5.1 was replaced with Apt(P19) to prepare DSPE-PEG2000-Apt(P19) micelles. 3+) were mixed evenly and incubated at 4°C (the reaction temperature can be 0°C ± 10°C) for 2 h (the incubation time can be 0.2-12 h) to allow DSPE-PEG2000-Apt(P19) micelles to be inserted into siRNA@MPNP(Al 3+ ) was prepared from siRNA@Apt(P19)-MPNP(Al 3+ ).

[0738] Referring to Example 1.2, the drug was replaced with siRNA to prepare the drug siRNA@MPNP (Mg 2+ ). Then, the targeted drug (siRNA@Apt-MPNP) was prepared by referring to Example 5.1. Apt in Example 5.1 was replaced with Apt(P19) to prepare DSPE-PEG2000-Apt(P19) micelles, and siRNA@MPNP(Mg 2+ ) were mixed evenly and incubated at 4°C (the reaction temperature can be 0°C ± 10°C) for 2 h (the incubation time can be 0.2-12 h) to allow DSPE-PEG2000-Apt(P19) micelles to be inserted into siRNA@MPNP(Mg 2+ ) was prepared from siRNA@Apt(P19)-MPNP(Mg 2+ ).

[0739] Example 6.4. CD19 CAR mRNA@Apt(CD62L)-MPNP, CD19 CAR mRNA@Apt(CD8)-MPNP, ASO@Apt(AS1411)-MPNP, siRNA@Apt(P19)-MPNP Animal Survival Rate Detection

[0740] Using the product of Example 6.3, the survival time of acute B-lymphoid leukemia model mice in the CD19 CAR mRNA@Apt(CD62L)-MPNP treatment group, lung cancer mice in the ASO@Apt(AS1411)-MPNP treatment group, and pancreatic cancer mice in the siRNA@Apt(P19)-MPNP treatment group were recorded, and their respective survival rates were calculated.

[0741] Based on the results of Example 6.2 and Example 6.4, Figures 5-1, 5-1-2, 5-1-3, 5-2, 5-2-2, 5-2-3, 5-3, 5-3-2 and 5-3-3 are obtained. It can be seen from the results in Figures 5-1, 5-2 and 5-3 that under the same Apt, the same drug and the same drug dosage, the performance of Apt-MPNP is better than that of Apt-LNP; Apt-MPNP (Mg 2+ ) performance is better than Apt-MPNP(Al 3+ ), Apt-MPNP(Al3+ ) outperforms Apt-MPNP(Fe 3+ ); further proved that CD19CAR mRNA@Apt(CD62L)-MPNP(Mg 2+ ) has a better effect against acute B-lymphocytic leukemia; ASO@Apt(AS1411)-MPNP(Mg 2+ ) has a better anti-lung cancer effect; siRNA@Apt(P19)-MPNP(Mg 2+ ) has a better effect against pancreatic cancer.

[0742] Apt-MPNP(Mg 2+ ) performance and efficacy are better because: Mg maintains a weak chelation effect with RNA 2+ It 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+ It can maintain the stability of ribosome structure and translation activity, partially supplement the function of ribosomal proteins (Biosci Biotechnol Biochem. 2021 Jun 24; 85(7): 1582-1593.); enhance immune response. Mg 2+ It can regulate LFA-1 downstream signal transduction by inducing conformational changes of the co-stimulatory molecule LFA-1 on the surface of CD8+ T cells, thereby promoting T cell activation and cytotoxicity (Cell. 2022 Feb 17; 185(4): 585-602.e29.); Mg 2+ As an enzyme cofactor of Ago protein in RNA-induced silencing complex (RISC), it participates in catalysis and can enhance the RNAi effect of siRNA (Cell Rep. 2022 Oct 25; 41(4): 111533); Mg 2+ As an enzyme cofactor of RNase H1, it participates in catalysis and can enhance the gene silencing effect of ASO (DNA Repair (Amst). 2019 Dec; 84: 102672.).

[0743] Among them, according to the results in Figure 7-1, the duration of CAR expression positivity of Apt-MPNP was significantly stronger than that of Apt-LNP, among which Apt-MPNP (Mg 2+ ) is superior to Apt-MPNP(Al 3+ ), Apt-MPNP(Al 3+ ) is superior to Apt-MPNP(Fe 3+ ); Tip, Apt-MPNP (Mg 2+) has a long-lasting expression and strong ability, which may be an important reason for its optimal efficacy.

[0744] Example 7: Substituting different Apts in Apt-MPNP

[0745] Apt was replaced with an aptamer that recognizes CD8 protein, and the drug was CD19 CAR mRNA to prepare CD19 CAR mRNA@Apt(CD8)-MPNP.

[0746] The Apt in Example 5.1 was replaced with an aptamer that can specifically recognize the CD8 protein, and CD19 CAR mRNA@Apt(CD8)-MPNP was prepared according to the method of Example 5.1. The sequence of Apt(CD8) targeting CD8 is shown in SEQ ID NO.84 5'-CTACAGCTTGCTATGCTCCCCTTGGGGTA-3'. The preparation process of the remaining mRNA-metal-polyphenol complex particles is the same as that of Example 1.2. The targeted drugs CD19 CAR mRNA@Apt(CD62L)-MPNP and CD19 CAR mRNA@Apt(CD8)-MPNP (10 μg / mouse calculated based on the mass of mRNA) were respectively injected into acute B lymphoblastic leukemia model mice through the tail vein, once every five days, and human T cells equal to the number of cancer cells were injected two hours before the injection, for a total of three doses. There were five mice in each group. The survival time of the mice was recorded, and the survival rate was calculated.

[0747] As shown in Figure 6-1, CD19 CAR mRNA@Apt(CD62L)-MPNP(Fe 3+ ) group had a longer survival period than the CD19 CAR mRNA@Apt(CD8)-MPNP(Fe 3+ ). The results showed that the targeted drug CD19 CAR mRNA@Apt(CD62L)-MPNP(Fe 3+ ) can prolong the survival of mice with acute B-lymphocytic leukemia better than CD19 CAR mRNA@Apt(CD8)-MPNP(Fe 3+ ).

[0748] As shown in Figure 6-1-2, CD19 CAR mRNA@Apt(CD62L)-MPNP(Al 3+ ) group had a longer survival period than the CD19 CAR mRNA@Apt(CD8)-MPNP(Al 3+ ). The results showed that the targeted drug CD19 CAR mRNA@Apt(CD62L)-MPNP(Al 3+) can prolong the survival of mice with acute B-lymphocytic leukemia better than CD19 CAR mRNA@Apt(CD8)-MPNP(Al 3+ ).

[0749] As shown in Figure 6-1-3, CD19 CAR mRNA@Apt(CD62L)-MPNP(Mg 2+ ) group had a longer survival period than the CD19 CAR mRNA@Apt(CD8)-MPNP(Mg 2+ ). This result shows that the targeted drug CD19 CAR mRNA@Apt(CD62L)-MPNP(Mg 2+ ) was superior to CD19 CAR mRNA@Apt(CD8)-MPNP(Mg 2+ ).

[0750] Apt(CD62L)-MPNP is superior to Apt(CD8)-MPNP for the following reasons: (1) CD62L+T cells account for a larger proportion than CD8+T cells in the body, and targeting CD62L can mobilize a stronger immune system in the body; (2) Although CD8+T cells can secrete high levels of proteins such as granzymes and perforins, their survival time is very short and they cannot effectively kill tumor cells; (3) CD62L+T cells are in the initial stage of differentiation and have a longer lifespan. After being stimulated by antigens, they can quickly differentiate into effector T cells to exert anti-tumor effects and evolve immune memory functions; (4) Although B lymphocytes in acute B lymphoblastic leukemia also express CD62L, this application only uses CD19 mRNA to make cells express the corresponding protein, which will not affect the next generation of the cell, and will not affect the proliferation of the cell. Therefore, even if the material in this application recognizes and binds to B lymphocytes, it will theoretically not cause the patient's condition to worsen.

[0751] Example 7.2: Apt is replaced with an aptamer that recognizes CD3 protein

[0752] Apt was replaced with an aptamer that recognizes CD3 protein, and the drug was CD19 CAR mRNA to prepare CD19 CAR mRNA@apt(CD3)-MPNP.

[0753] The Apt in Example 5.1 was replaced with an aptamer that can specifically recognize the CD3 protein, and CD19 CAR mRNA@Apt(CD3)-MPNP was prepared according to the method of Example 5.1. The sequence of Apt(CD3) targeting CD3 is shown in SEQ ID NO.94. The preparation process of the remaining mRNA-metal-polyphenol complex particles is the same as that in Example 1.2. The targeted drugs CD19 CAR mRNA@Apt(CD62L)-MPNP and CD19 CAR mRNA@Apt(CD3)-MPNP (10 μg / mouse calculated based on the mass of mRNA) were respectively injected into acute B lymphoblastic leukemia model mice through the tail vein, once every five days and human T cells equal to the number of cancer cells were injected two hours before the injection, for a total of three doses. There were five mice in each group. The survival time of the mice was recorded, and the survival rate was calculated.

[0754] As shown in Figure 6-1, CD19 CAR mRNA@Apt(CD62L)-MPNP(Fe 3+ ) group had a longer survival period than the CD19 CAR mRNA@Apt(CD3)-MPNP(Fe 3+ ). The results showed that the targeted drug CD19 CAR mRNA@Apt(CD62L)-MPNP(Fe 3+ ) can prolong the survival of mice with acute B-lymphocytic leukemia better than CD19 CAR mRNA@Apt(CD3)-MPNP(Fe 3+ This is because CD62L+T cells account for a larger proportion than CD3+T cells in the body, and targeting CD62L can mobilize a more powerful immune system in the body.

[0755] As shown in Figure 6-1-2, CD19 CAR mRNA@Apt(CD62L)-MPNP(Al 3+ ) group had a longer survival period than the CD19 CAR mRNA@Apt(CD3)-MPNP(Al 3+ ). The results showed that the targeted drug CD19 CAR mRNA@Apt(CD62L)-MPNP(Al 3+ ) can prolong the survival of mice with acute B-lymphocytic leukemia better than CD19 CAR mRNA@Apt(CD3)-MPNP(Al 3+ This is because CD62L+T cells account for a larger proportion than CD3+T cells in the body, and targeting CD62L can mobilize a more powerful immune system in the body.

[0756] As shown in Figure 6-1-3, CD19 CAR mRNA@Apt(CD62L)-MPNP(Mg 2+) group had a longer survival period than the CD19 CAR mRNA@Apt(CD3)-MPNP(Mg 2+ ). This result shows that the targeted drug CD19 CAR mRNA@Apt(CD62L)-MPNP(Mg 2+ ) was superior to CD19 CAR mRNA@Apt(CD3)-MPNP(Mg 2+ This is because CD62L+T cells account for a larger proportion than CD3+T cells in the body, and targeting CD62L can mobilize a more powerful immune system in the body.

[0757] From the results of Example 7.1 and Example 7.2, as shown in Figure 6-1, under the same carrier, the same drug, and the same drug dose, Apt(CD62L)-MPNP performs better than Apt(CD8)-MPNP and Apt(CD3)-MPNP. This result further proves that CD19 CAR mRNA@Apt(CD62L)-MP...

Claims

1. A targeting vector, characterized in that The targeting vector comprises: (a) Metal-polyphenol composite particles, the metal-polyphenol composite particles comprising: (i) a metal-polyphenol complex, which is composed of a polyphenol molecule portion and a metal ion portion reacting with each other, wherein the polyphenol molecule portion and the metal ion portion are connected by a coordination bond; (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) non-cationic lipids or non-ionizable lipids other than conjugated lipids that inhibit particle aggregation; (b) a targeting structure, wherein the targeting structure is connected to the outer surface of the metal-polyphenol composite particle.

2. The targeting vector according to claim 1, wherein In the (i) metal-polyphenol complex, the polyphenol molecule portion is selected from the group consisting of curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, paclitaxel, fucoidan, polyflavanol polyphenols, catechin, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, galloylglucose, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate, epigallocatechin gallate, and combinations of one or more thereof; Preferably, the polyphenol molecule is selected from curcumin (Formula 19) Quercetin (Formula 22) Kaempferol (Formula 59) Rutin (Formula 60) Hesperetin (Formula 24) Naringenin (Formula 25) Eriochoride (Formula 61) Luteolin (Formula 62) Apigenin (Formula 26) Taxol (Formula 63) Brown algae polyphenols (Formula 64) Polyflavanol polyphenols (Formula 65) Catechin (Formula 27) Ellagic acid (Formula 30) Gallic acid (Formula 66) Digallic acid (Formula 67) Propyl gallate (Formula 68) Epigallocatechin gallate (Formula 29) Galloylglucose (Formula 69) Hydroxyhydroquinone (Formula 70) Mulberry pigment (Formula 31) Epicatechin gallate (Formula 32) Catechin gallate (Formula 33) Epigallocatechin gallate (Formula 34) and combinations of one or more of their derivatives; Preferably, the polyphenol molecular portion is selected from at least one of curcumin (Formula 19), dihydrocurcumin (Formula 36), hexahydrocurcumin (Formula 37), curcumin sulfate (Formula 38), and bisdemethoxycurcumin (Formula 39); Preferably, the polyphenol molecular moiety is selected from at least one of curcumin (Formula 19), hesperetin (Formula 24) or catechin (Formula 27), and derivatives thereof; Preferably, the polyphenol molecule is selected from curcumin (Formula 19), hesperetin (Formula 24) or catechin (Formula 27); Preferably, the metal ion moiety is selected from Fe 3+ 、Ag + 、Ba 2+ , Ca 2+ 、Cd 2+ 、Cu 2+ 、Fe 2+ 、Mn 2+ Mg 2+ 、Mo 2+ 、Zn 2+ , Pt 2+ 、Au 2+ 、Al 3+ 、Ce 3+ 、Co 3+ Cr 3+ 、Eu 3+ 、Gd 3+ 、Ni 3+ 、W 3+ 、V 3+ 、Zr 3+ A combination of one or more of the following: Preferably, the metal ion moiety is selected from Fe 3+ Mg 2+ , Ca 2+ 、Al 3+ A combination of one or more of the following: Preferably, the metal ion moiety is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ .

3. The targeting vector according to claim 1, wherein The (ii) conjugated lipids that inhibit particle aggregation include PEG-lipid conjugates and / or PEG-DAA; Preferably, the PEG-lipid conjugate is selected from 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 at least one of its derivatives, wherein R1 and R2 are independently: capryloyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytanoyl; Preferably, the PEG-lipid conjugate is selected from a combination of one or more of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000 or DSPE-PEG5000; Preferably, the PEG-lipid conjugate is selected from DSPE-PEG2000 (Formula 53), DSPE-PEG700 (Formula 50), DSPE-PEG1000 (Formula 51) or DSPE-PEG5000 (Formula 52); 4. The targeting vector according to claim 1, wherein The non-cationic lipid or non-ionizable lipid in (iii) 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-phospho-sphingosine S1P, and a combination of one or more of their derivatives; Preferably, the non-cationic lipid or non-ionizable lipid in (iii) 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) Phosphoinositide (PI) (Formula 7) Phosphatidylthreonine (PT) (Formula 8) Sphingomyelin (SM) (Formula 9) Lysolecithin (LPC) (Formula 10) Lysophosphoethanolamine (LPE) (Formula 11) Lysophosphatidylserine (LPS) (Formula 12) Lysophosphatidic acid (LPA) (Formula 13) Lysophosphatidylglycerol (LPG) (Formula 14) Lysophosphatidylinositol (LPI) (Formula 15) Lysophosphatidylthreonine (LPT) (Formula 16) Lysosphingomyelin (LSM) (Formula 17) Sphingosine 1-phosphate (S1P) (Formula 18) and combinations of one or more of their derivatives; Wherein, R1 and R2 are independently: Capryloyl Lauroyl Myristoyl Palmitoyl Stearyl Oleoyl Linoleyl Erucyl Arachidoyl or phytanoyl Preferably, the non-cationic lipid or non-ionizable lipid in (iii) further comprises at least one of cholesterol and its derivatives; Preferably, 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; Preferably, the non-cationic lipid or non-ionizable lipid in (iii) comprises cholesterol (Formula 40), and a combination of one or more selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48) or DSPG (Formula 49); 5. The targeting vector according to claim 1, wherein The metal-polyphenol complex is composed of a polyphenol molecule portion and a metal ion portion, wherein the polyphenol molecule portion is selected from curcumin, hesperidin or catechin, and the metal ion portion is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ ; Preferably, the metal-polyphenol complex is composed of a polyphenol molecule portion and a metal ion portion, wherein the polyphenol molecule portion is selected from curcumin (Formula 19), hesperidin (Formula 24) or catechin (Formula 27), and the metal ion portion is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ ; Preferably, the molar ratio of the polyphenol molecule portion to the metal ion portion is 1:(0.5-2); Preferably, the polyphenol molecule portion is curcumin (Formula 19), and the metal ion portion is Fe 3+ Mg 2+ or Al 3+ , the molar ratio of the polyphenol molecule part to the metal ion part is 1:

1.

6. The targeting vector according to claim 4, characterized in that The metal-polyphenol complex particles are made of (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the metal-polyphenol complex accounts for 1% to 30% 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 0% to 60% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 29% to 75% by mole in the raw material; Preferably, the molar proportion of the metal-polyphenol complex in the raw material is 5% to 30%, preferably 10% to 30%, preferably 5% to 20%; Preferably, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%; Preferably, the molar proportion of cholesterol in the raw material is 0% to 48%; Preferably, the molar proportion of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 30% to 75%, preferably 40% to 75%.

7. The targeting vector according to any one of claims 1 to 6, characterized in that 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-polyphenol complex particle based on hydrophilicity and hydrophobicity; Preferably, the hydrophobic region comprises at least one or more of DSPE and its derivatives; Preferably, the linking region comprises at least one or more of PEG-2000 and its derivatives; Preferably, 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; Preferably, the target binding region comprises at least one of a nucleic acid, a polypeptide, a protein, and a small molecule; Preferably, the target binding region comprises one of an aptamer, an antibody, an antigen binding portion, and galnac; Preferably, the targeting binding region is an aptamer, the target of the targeting binding region is CD62L, and the aptamer is preferably represented by SEQ ID NO.44; or, Preferably, 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, Preferably, the targeting binding region is an aptamer, the target of the targeting binding region is CD3, and the aptamer is preferably represented by SEQ ID NO.95; or Preferably, the targeting binding region is an aptamer, the target of the targeting binding region is nucleolin protein, and the aptamer is preferably represented by SEQ ID NO.86; or, Preferably, the targeting binding region is an aptamer, the target cell of the targeting binding region is a T cell, and the aptamer is preferably represented by SEQ ID NO.44; Preferably, 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 represented by SEQ ID NO.88; or, Preferably, the targeting binding region is an aptamer, and the target cell of the targeting binding region is a liver cancer cell; Preferably, the targeting structure is DSPE-PEG2000-aptamer, preferably DSPE-PEG2000-CD62L aptamer, and the CD62L aptamer sequence is shown in SEQ ID NO.

44.

8. The method for preparing the targeting carrier according to any one of claims 1 to 7, wherein the targeting structure is connected to the outer surface of the metal-polyphenol complex particles to form the targeting carrier.

9. The preparation method according to claim 8, characterized in that mixing (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid to obtain the metal-polyphenol complex particles; Preferably, the targeting structure is DSPE-PEG2000-aptamer, and the preparation process is as follows: DSPE-PEG2000 and the aptamer are connected through an intermediate pair reaction to obtain DSPE-PEG2000-aptamer; Preferably, the intermediate pair is selected from DSPE-PEG2000-MAL and aptamer-C6-SH, DSPE-PEG2000-NHS and aptamer-NH2, DSPE-PEG2000-COOH and aptamer-NH2, DSPE-PEG2000-NCO and aptamer-NH2, DSPE-PEG2000-N3 and aptamer-DBCO, preferably DSPE-PEG2000-MAL and aptamer-C6-SH; Preferably, the DSPE-PEG2000-aptamer forms micelles and then connects to the outer surface of the metal-polyphenol complex particles to form the targeting carrier; Preferably, the preparation method of the micelles includes direct dissolution method, ethanol injection method, dialysis method or ultrasound method.

10. The preparation method according to claim 8, characterized in that The preparation method comprises: Step 1: reacting the polyphenol molecule portion with the metal ion portion through a coordination bond to form a metal-polyphenol complex; Step 2: mixing the metal-polyphenol complex prepared in step 1, a conjugated lipid that inhibits particle aggregation, a non-cationic lipid, or a non-ionizable lipid to prepare the metal-polyphenol complex particles; Step 3: mixing the metal-polyphenol complex particles prepared in step 2 with the targeting structure to prepare the targeting carrier; Preferably, in step 1, polyphenol molecules are dissolved in ethanol, and metal ions and triethylamine are added and reacted to obtain the metal-polyphenol complex, the molar ratio of polyphenol molecules to metal ions is preferably 1:(1-2), and the molar ratio of polyphenol molecules to triethylamine is preferably 1:

1. The reaction conditions are preferably 60° C. for 2 hours; Preferably, in step three, the reaction conditions of the metal-polyphenol complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.

11. Use of the targeting vector according to any one of claims 1 to 7 or the preparation method according to any one of claims 8 to 10 for drug delivery, imaging agent or vaccine.

12. A targeted drug, characterized in that: The targeted drug comprises a drug and the targeting carrier according to any one of claims 1 to 7, wherein the drug is encapsulated in the metal-polyphenol complex particles of the targeting carrier.

13. The targeted drug according to claim 12, characterized in that The drug is selected from one or more combinations of nucleic acids, proteins, polypeptides, small molecules, nucleic acid analogs, protein analogs and polypeptide analogs; Preferably, the nucleic acid is selected from one or more combinations of mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, and artificial nucleic acid; Preferably, the drug is mRNA, and the mRNA encodes a chimeric antigen receptor CAR or TCR; Preferably, 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; Preferably, 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 costimulatory 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; Preferably, the drug is an mRNA having a nucleotide sequence as shown in SEQ ID No.

43.

14. The method for preparing the targeted drug according to claim 12 or 13, characterized in that: The preparation method comprises: encapsulating the drug in a targeting carrier to obtain the targeted drug.

15. The preparation method according to claim 14, characterized in that The targeting carrier includes metal-polyphenol complex particles and a targeting structure. The drug is encapsulated in the metal-polyphenol complex particles to obtain drug-metal-polyphenol complex particles; the targeting structure is connected to the outer surface of the drug-metal-polyphenol complex particles to form the targeted drug.

16. The preparation method according to claim 15, characterized in that The drug, (i) the metal-polyphenol 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 drug-metal-polyphenol complex particles.

17. The preparation method according to claim 15, characterized in that The preparation method comprises: Step 1: reacting the polyphenol molecule portion with the metal ion portion through a coordination bond to form a metal-polyphenol complex; Step 2: mixing the metal-polyphenol complex prepared in step 1, a conjugated lipid for inhibiting particle aggregation, a non-cationic lipid or a non-ionizable lipid, and a drug to prepare the drug-metal-polyphenol complex particles; Step 3: mixing the drug-metal-polyphenol complex particles prepared in step 2 with the targeting structure to prepare the targeted drug; Preferably, in step 1, polyphenol molecules are dissolved in ethanol, and metal ions and triethylamine are added and reacted to obtain the metal-polyphenol complex, the molar ratio of polyphenol molecules to metal ions is preferably 1:(1-2), and the molar ratio of polyphenol molecules to triethylamine is preferably 1:

1. The reaction conditions are preferably 60° C. for 2 hours; Preferably, in step 2, the metal-polyphenol 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-polyphenol 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; Preferably, in step three, the reaction conditions of the drug-metal-polyphenol complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.

18. The preparation method according to claim 15, characterized in that The targeting structure is DSPE-PEG2000-aptamer, and the preparation process is as follows: DSPE-PEG2000 and the aptamer are connected through an intermediate pair reaction to obtain DSPE-PEG2000-aptamer; Preferably, the intermediate pair is selected from DSPE-PEG2000-MAL and aptamer-C6-SH, DSPE-PEG2000-NHS and aptamer-NH2, DSPE-PEG2000-COOH and aptamer-NH2, DSPE-PEG2000-NCO and aptamer-NH2, DSPE-PEG2000-N3 and aptamer-DBCO, preferably DSPE-PEG2000-MAL and aptamer-C6-SH; Preferably, the DSPE-PEG2000-aptamer forms micelles and then connects to the outer surface of the drug-carrier delivery system to form the targeted drug; Preferably, the preparation method of the micelles includes direct dissolution method, ethanol injection method, dialysis method or ultrasound method.

19. Use of the targeted drug according to claim 12 or 13 or the preparation method according to any one of claims 14 to 18 in drug delivery, imaging drugs, and vaccines.

20. The use according to claim 19, characterized in that The targeted drug is used for drug delivery, treatment and / or prevention; Preferably, 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; Preferably, the targeted drug is used to express or silence a target sequence in a mammalian subject, to deliver a drug in a mammal, to deliver a drug from the body to a mammalian cell, to deliver a drug from 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; Preferably, the mammal is a human; Preferably, the treatment of the disease or condition is associated with the expression of a gene comprising a target sequence for the drug; Preferably, 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; Preferably, the viral infection comprises hepatitis B virus, hepatitis C virus, SARS-Cov-2, human immunodeficiency virus, cytomegalovirus, invasive Aspergillus or conjugate virus; Preferably, the cancer includes hematological tumors and solid tumors; Preferably, 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; Preferably, the diffuse large B-cell lymphoma (DLBCL) includes Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL); Preferably, the solid tumor comprises 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; Preferably, 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; Preferably, 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); Preferably, the metabolic disease includes atherosclerosis, congenital hyperinsulinemia, non-alcoholic steatohepatitis or non-obese diabetes (NOD); Preferably, 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; Preferably, the skin disease includes keloid or wound healing; Preferably, the administration route of the targeted drug includes intrathecal injection, intramuscular administration, intracranial injection, intravenous injection or intratumoral injection; Preferably, the targeted drug is used in combination therapy.

21. A pharmaceutical agent containing the targeted drug according to claim 12 or 13, wherein the pharmaceutical agent is preferably a vaccine, and the vaccine is preferably a new coronavirus vaccine.

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