Targeted carrier, targeted drug, method for preparing same, and use thereof

Abstract

Provided are a targeted carrier, a targeted drug, a method for preparing same, and use thereof. The targeted carrier comprises: (a) a metal-phospholipid complex particle, and (b) a targeted structure connected to the outer surface of the metal-phospholipid complex particle. The targeted carrier provided by the present application does not require the use of a cationic lipid or ionizable lipid, and is thus beneficial to carrying a negatively charged drug in organisms and applicable to drugs of different sizes.

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-phospholipid complex particles, comprising:

[0010] (i) a metal-phospholipid complex, wherein the metal-phospholipid complex is formed by reacting a phospholipid molecule portion, a linker molecule portion, and a metal ion portion, the phospholipid molecule portion is connected to the linker molecule portion, the linker molecule portion is connected to the metal ion portion via a coordination bond, and the metal-phospholipid complex is not a cationic lipid or an ionizable lipid;

[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) a non-cationic lipid or a non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation;

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

[0014] Furthermore, in the (i) metal-phospholipid complex, the phospholipid molecule portion is selected from phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphoceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysophosphatidylcholine LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, 1-phospho-sphingosine S1P, and a combination of one or more of their derivatives;

[0015] Preferably, the phospholipid molecule is selected from

[0016] Phosphatidylcholine (PC) (Formula 1)

[0017] Phosphatidylethanolamine (PE) (Formula 2)

[0018] Phosphatidylserine (PS) (Formula 3)

[0019] Phosphatidic acid (PA) (Formula 4)

[0020] Phosphatidylglycerol (PG) (Formula 5)

[0021] 1-phosphoceramide (SP) (Formula 6)

[0022] Phosphoinositide (PI) (Formula 7)

[0023] Phosphatidylthreonine (PT) (Formula 8)

[0024] Sphingomyelin (SM) (Formula 9)

[0025] Lysolecithin (LPC) (Formula 10)

[0026] Lysophosphoethanolamine (LPE) (Formula 11)

[0027] Lysophosphatidylserine (LPS) (Formula 12)

[0028] Lysophosphatidic acid (LPA) (Formula 13)

[0029] Lysophosphatidylglycerol (LPG) (Formula 14)

[0030] Lysophosphatidylinositol (LPI) (Formula 15)

[0031] Lysophosphatidylthreonine (LPT) (Formula 16)

[0032] Lysosphingomyelin (LSM) (Formula 17)

[0033] Sphingosine 1-phosphate (S1P) (Formula 18) and combinations of one or more of their derivatives;

[0034] Wherein, R1 and R2 are independently:

[0035] Capryloyl Lauroyl

[0036] Myristoyl Palmitoyl

[0037] Stearyl Oleoyl

[0038] Linoleyl erucyl

[0039] Arachidoyl or phytanoyl

[0040] Preferably, the phospholipid molecule is selected from the group consisting of phosphatidylcholine PC (Formula 1), phosphatidylethanolamine PE (Formula 2), phosphatidic acid PA (Formula 4), phosphatidylglycerol (PG) (Formula 5), ​​and a combination of one or more thereof.

[0041] Preferably, the phospholipid molecule is selected from the group consisting of DSPC, DSPE, DSPA, DSPG, and a combination of one or more of their derivatives;

[0042] Preferably, the phospholipid molecule is selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48), DSPG (Formula 49), and a combination of one or more of their derivatives;

[0043]

[0044]

[0045]

[0046]

[0047] Preferably, the linker molecule is selected from the group consisting of curcumin, chlorogenic acid, anthocyanidin, quercetin, dihydromyricetin, hesperetin, naringenin, apigenin, catechin, tea polyphenols, epigallocatechin gallate, ellagic acid, morin, epicatechin gallate, catechin gallate, epigallocatechin gallate or picoflavine C, and combinations of one or more thereof.

[0048] Preferably, the linker molecule is selected from

[0049] Curcumin (Formula 19)

[0050] Chlorogenic acid (Formula 20)

[0051] Anthocyanin (Formula 21) Wherein, R1 and R2 are H, OH or OCH3, R3 is H or glycosyl, R4, R5 and R6 are OH or glycosyl,

[0052] Quercetin (Formula 22) Dihydromyricetin (Formula 23)

[0053] Hesperetin (Formula 24) Naringenin (Formula 25)

[0054] Apigenin (Formula 26) Catechin (Formula 27)

[0055] Tea polyphenols (Formula 28)

[0056] Epigallocatechin gallate (Formula 29)

[0057] Ellagic acid (Formula 30) Morin (Formula 31)

[0058] Epicatechin gallate (Formula 32)

[0059] Catechin gallate (Formula 33)

[0060] Epigallocatechin gallate (Formula 34)

[0061] Pingbei alkaloid C (Formula 35) and combinations of one or more of their derivatives;

[0062] Preferably, the linker molecule is selected from one or more combinations of curcumin (Formula 19), dihydrocurcumin (Formula 36), hexahydrocurcumin (Formula 37), curcumin sulfate (Formula 38), and bisdemethoxycurcumin (Formula 39);

[0063]

[0064]

[0065]

[0066]

[0067] Preferably, the linker molecule is selected from the group consisting of curcumin (Formula 19), hesperetin (Formula 24), tea polyphenols (Formula 28), and a combination of one or more of their derivatives;

[0068] Preferably, the linker molecule is selected from curcumin (Formula 19), hesperetin (Formula 24) or tea polyphenols (Formula 28);

[0069] 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:

[0070] 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:

[0071] Preferably, the metal ion moiety is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ .

[0072] Further, the (ii) conjugated lipid that inhibits particle aggregation comprises PEG-lipid conjugate and / or PEG-DAA;

[0073] Preferably, the PEG-lipid conjugate is selected from

[0074] Phosphatidylethanolamine-polyethylene glycol 2000 (Formula 42)

[0075] Phosphatidylethanolamine-polyethylene glycol 700 (Formula 43)

[0076] Phosphatidylethanolamine-polyethylene glycol 1000 (Formula 44)

[0077] Phosphatidylethanolamine-polyethylene glycol 5000 (Formula 45)

[0078] and a combination of one or more of its derivatives, wherein R1 and R2 are independently: capryloyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytanoyl;

[0079] Preferably, the PEG-lipid conjugate is selected from a combination of one or more of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000 or DSPE-PEG5000;

[0080] 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);

[0081]

[0082]

[0083]

[0084]

[0085] Furthermore, the non-cationic lipid or non-ionizable lipid in (iii) is a combination of one or more of cholesterol and its derivatives;

[0086] Preferably, the non-cationic lipid or non-ionizable lipid in (iii) is cholesterol (Formula 40)

[0087] Preferably, the non-cationic lipid or non-ionizable lipid in (iii) further comprises a combination of one or more selected from phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphoceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysophosphatidylcholine LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, 1-phospho-sphingosine S1P, cholesterol sulfate and derivatives thereof;

[0088] Preferably, the non-cationic lipid or non-ionizable lipid described in (iii) further comprises at least one selected from phosphatidylcholine PC (Formula 1), phosphatidylethanolamine PE (Formula 2), phosphatidylserine PS (Formula 3), phosphatidic acid PA (Formula 4), phosphatidylglycerol PG (Formula 5), ​​1-phosphoceramide SP (Formula 6), phosphatidylinositol PI (Formula 7), phosphatidylthreonine PT (Formula 8), sphingomyelin SM (Formula 9), lysophosphatidylcholine LPC (Formula 10), lysophosphatidylethanolamine LPE (Formula 11), lysophosphatidylserine LPS (Formula 12), lysophosphatidic acid LPA (Formula 13), lysophosphatidylglycerol LPG (Formula 14), lysophosphatidylinositol LPI (Formula 15), lysophosphatidylthreonine LPT (Formula 16), lysophosphophospholipid LSM (Formula 17), 1-phospho-sphingosine S1P (Formula 18), cholesterol sulfate (Formula 41), and derivatives thereof;

[0089]

[0090] 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;

[0091] Preferably, the non-cationic lipid or non-ionizable lipid in (iii) comprises cholesterol (Formula 40) and DSPC (Formula 46).

[0092] Furthermore, the metal-phospholipid complex is made of a phospholipid molecule portion, a linker molecule portion and a metal ion portion, the phospholipid molecule portion is selected from DSPC, DSPE, DSPA or DSPG, the linker molecule portion is selected from curcumin, hesperidin or tea polyphenols, and the metal ion portion is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ ;

[0093] Preferably, the metal-phospholipid complex is made of a phospholipid molecule portion, a linker molecule portion and a metal ion portion, the phospholipid molecule portion is selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48) or DSPG (Formula 49), the linker molecule portion is selected from curcumin (Formula 19), hesperidin (Formula 24) or tea polyphenols (Formula 28), and the metal ion portion is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ ;

[0094] Preferably, the molar ratio of the phospholipid molecule portion, the linker molecule portion, and the metal ion portion is 1:1:(0.5-2);

[0095] Preferably, the phospholipid molecule portion is DSPC (Formula 46), the linker molecule portion is selected from curcumin (Formula 19), and the metal ion portion is selected from Fe 3+ Mg 2+ or Al 3+ The molar ratio of the phospholipid molecule part, the linker molecule part and the metal ion part is 1:1:1.

[0096] Furthermore, the metal-phospholipid complex particles are made of (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the metal-phospholipid complex accounts for 5% to 50% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 1% to 10% by mole in the raw material, the cholesterol accounts for 15% to 80% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 0% to 51% by mole in the raw material;

[0097] Preferably, the metal-phospholipid complex accounts for 5% to 40% by mole in the raw material, preferably 10% to 40%;

[0098] Preferably, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%;

[0099] Preferably, the molar proportion of cholesterol in the raw material is 25% to 75%, preferably 35% to 75%;

[0100] Preferably, the molar proportion of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 0% to 50%, preferably 0% to 40%.

[0101] 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-phospholipid complex particle based on hydrophilicity and hydrophobicity;

[0102] Preferably, the hydrophobic region comprises at least one or more of DSPE and its derivatives;

[0103] Preferably, the linker region comprises at least one or more of PEG-2000 and its derivatives;

[0104] 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;

[0105] Preferably, the target binding region comprises at least one of a nucleic acid, a polypeptide, a protein, and a small molecule;

[0106] Preferably, the target binding region comprises one of an aptamer, an antibody, an antigen binding portion, and galnac;

[0107] 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,

[0108] 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,

[0109] 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

[0110] 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,

[0111] 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;

[0112] 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,

[0113] Preferably, the targeting binding region is an aptamer, and the target cell of the targeting binding region is a liver cancer cell;

[0114] 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.

[0115] The method for preparing the above-mentioned targeting carrier comprises connecting the targeting structure to the outer surface of the metal-phospholipid complex particles to form the targeting carrier.

[0116] Further, (i) the metal-phospholipid complex, (ii) the conjugated lipid that inhibits particle aggregation, and (iii) the non-cationic lipid or the non-ionizable lipid are mixed to obtain the metal-phospholipid complex particles;

[0117] 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;

[0118] 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;

[0119] Preferably, the DSPE-PEG2000-aptamer forms micelles and then connects to the outer surface of the metal-phospholipid complex particles to form the targeting carrier;

[0120] Preferably, the preparation method of the micelles includes direct dissolution method, ethanol injection method, dialysis method or ultrasound method.

[0121] Furthermore, the preparation method comprises:

[0122] Step 1: reacting phospholipid molecules, linker molecules and metal ions to form a metal-phospholipid complex;

[0123] Step 2: mixing the metal-phospholipid complex prepared in step 1, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid to prepare metal-phospholipid complex particles;

[0124] Step 3: mixing the metal-phospholipid complex particles prepared in step 2 with the targeting structure to prepare the targeting carrier;

[0125] Preferably, in step 1, the phospholipid molecules, linker molecules and metal ions are dissolved in ethanol for reaction, the molar ratio of the phospholipid molecules, linker molecules and metal ions is preferably 1:1:1, and the reaction conditions are preferably 40-60° C. for 1-5 hours;

[0126] Preferably, in step three, the reaction conditions of the metal-phospholipid complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.

[0127] Furthermore, the preparation method comprises:

[0128] Step 1: Reaction and connection of phospholipid molecules with linker molecules to form phospholipid complexes;

[0129] Step 2: reacting the phospholipid complex prepared in step 1 with metal ions through coordination bonds to form a metal-phospholipid complex;

[0130] Step 3: mixing the metal-phospholipid complex prepared in step 2, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid to prepare metal-phospholipid complex particles;

[0131] Step 4: mixing the metal-phospholipid complex particles prepared in step 3 with the targeting structure to prepare the targeting carrier;

[0132] Preferably, in step 1, the phospholipid molecules and the linker molecules are dissolved in ethanol for reaction, and then n-hexane is added for precipitation to obtain the phospholipid complex. The molar ratio of the phospholipid molecules to the linker molecules is preferably 1:1. The reaction conditions are preferably 65° C. for 2 hours.

[0133] Preferably, in step 2, the phospholipid complex and the metal ion are dissolved in ethanol, and triethylamine is added to react to obtain the metal-phospholipid complex. The molar ratio of the phospholipid complex to the metal ion is preferably 1:(1-2), and the molar ratio of the phospholipid complex to the triethylamine is preferably 1:1. The reaction conditions are preferably 60° C. for 2 hours.

[0134] Preferably, in step 4, the reaction conditions of the metal-phospholipid complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.

[0135] The above-mentioned targeting carrier or preparation method is used for drug delivery, imaging agent or vaccine application.

[0136] A targeted drug comprises a drug and the above-mentioned targeting carrier, wherein the drug is encapsulated in the metal-phospholipid complex particles of the targeting carrier.

[0137] 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;

[0138] Preferably, the nucleic acid is selected from one or more combinations of mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, and artificial nucleic acid;

[0139] Preferably, the drug is mRNA, and the mRNA encodes a chimeric antigen receptor CAR or TCR;

[0140] 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;

[0141] 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,

[0142] 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,

[0143] 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,

[0144] 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,

[0145] 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;

[0146] Preferably, the drug is an mRNA having a nucleotide sequence as shown in SEQ ID No.43.

[0147] The preparation method of the above-mentioned targeted drug comprises: encapsulating the drug in a targeting carrier to obtain the targeted drug.

[0148] Furthermore, the targeting carrier includes metal-phospholipid complex particles and a targeting structure, and the drug is encapsulated in the metal-phospholipid complex particles to obtain drug-metal-phospholipid complex particles; the targeting structure is connected to the outer surface of the drug-metal-phospholipid complex particles to form the targeted drug.

[0149] Furthermore, the drug, (i) the metal-phospholipid complex, (ii) the conjugated lipid for inhibiting particle aggregation, and (iii) the non-cationic lipid or the non-ionizable lipid are mixed to obtain the drug-metal-phospholipid complex particles.

[0150] Furthermore, the preparation method comprises:

[0151] Step 1: reacting phospholipid molecules, linker molecules and metal ions to form a metal-phospholipid complex;

[0152] Step 2: mixing the metal-phospholipid complex prepared in step 1, (ii) a conjugated lipid that inhibits particle aggregation, (iii) a non-cationic lipid or a non-ionizable lipid, and a drug to prepare drug-metal-phospholipid complex particles;

[0153] Step 3: mixing the drug-metal-phospholipid complex particles prepared in step 2 with the targeting structure to prepare the targeted drug;

[0154] Preferably, in step 1, the phospholipid molecules, linker molecules and metal ions are dissolved in ethanol for reaction, the molar ratio of the phospholipid molecules, linker molecules and metal ions is preferably 1:1:1, and the reaction conditions are preferably 40-60° C. for 1-5 hours;

[0155] Preferably, in step 2, the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer to form an aqueous phase, and the organic phase and the aqueous phase are mixed to obtain drug-metal-phospholipid complex particles, wherein the organic compound is preferably ethanol; preferably, the mixing method of the organic phase and the aqueous phase includes a microfluidic chip or ultrasound;

[0156] Preferably, in step three, the reaction conditions of the drug-metal-phospholipid complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.

[0157] Furthermore, the preparation method comprises:

[0158] Step 1: Reaction and connection of phospholipid molecules with linker molecules to form phospholipid complexes;

[0159] Step 2: reacting the phospholipid complex prepared in step 1 with metal ions through coordination bonds to form a metal-phospholipid complex;

[0160] Step 3: mixing the metal-phospholipid complex prepared in step 2, (ii) a conjugated lipid that inhibits particle aggregation, (iii) a non-cationic lipid or a non-ionizable lipid, and a drug to prepare drug-metal-phospholipid complex particles;

[0161] Step 4: mixing the drug-metal-phospholipid complex particles prepared in step 3 with the targeting structure to prepare the targeted drug;

[0162] Preferably, in step 1, the phospholipid molecules and the linker molecules are dissolved in ethanol for reaction, and then n-hexane is added for precipitation to obtain the phospholipid complex. The molar ratio of the phospholipid molecules to the linker molecules is preferably 1:1. The reaction conditions are preferably 65° C. for 2 hours.

[0163] Preferably, in step 2, the phospholipid complex and the metal ion are dissolved in ethanol, and triethylamine is added to react to obtain the metal-phospholipid complex. The molar ratio of the phospholipid complex to the metal ion is preferably 1:(1-2), and the molar ratio of the phospholipid complex to the triethylamine is preferably 1:1. The reaction conditions are preferably 60° C. for 2 hours.

[0164] Preferably, in step 3, the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer to form an aqueous phase, and the organic phase and the aqueous phase are mixed to obtain drug-metal-phospholipid complex particles, wherein the organic compound is preferably ethanol; preferably, the mixing method of the organic phase and the aqueous phase includes a microfluidic chip or an ultrasonic

[0165] Preferably, in step 4, the reaction conditions of the drug-metal-phospholipid complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.

[0166] 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;

[0167] 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;

[0168] Preferably, the DSPE-PEG2000-aptamer forms micelles and then connects to the outer surface of the drug-metal-phospholipid complex particles to form the targeted drug;

[0169] Preferably, the preparation method of the micelles includes direct dissolution method, ethanol injection method, dialysis method or ultrasound method.

[0170] Application of the above-mentioned targeted drugs or preparation methods in drug delivery, imaging drugs, and vaccines.

[0171] Furthermore, the targeted drug is used for drug delivery, treatment and / or prevention;

[0172] 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;

[0173] 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;

[0174] Preferably, the mammal is a human;

[0175] Preferably, the treatment of the disease or condition is associated with the expression of a gene comprising a target sequence for the drug;

[0176] 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;

[0177] Preferably, the viral infection comprises hepatitis B virus, hepatitis C virus, SARS-Cov-2, human immunodeficiency virus, cytomegalovirus, invasive Aspergillus or conjugate virus;

[0178] Preferably, the cancer includes hematological tumors and solid tumors;

[0179] 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;

[0180] Preferably, the diffuse large B-cell lymphoma (DLBCL) includes Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL);

[0181] 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;

[0182] 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;

[0183] 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);

[0184] Preferably, the metabolic disease includes atherosclerosis, congenital hyperinsulinemia, non-alcoholic steatohepatitis or non-obese diabetes (NOD);

[0185] 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;

[0186] Preferably, the skin disease includes keloid or wound healing;

[0187] Preferably, the administration route of the targeted drug includes intrathecal injection, intramuscular administration, intracranial injection, intravenous injection or intratumoral injection;

[0188] Preferably, the targeted drug is used in combination therapy.

[0189] 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.

[0190] Compared with the prior art, the technical effects of this application are:

[0191] 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

[0192] 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:

[0193] Figure 1-1 is the eGFP-mRNA@MPP(Fe 3+ ) The percentage of eGFP-positive cells caused by 293T transfection;

[0194] Figure 1-2 is the RBD-mRNA@MPP(Fe 3+ ) Expression of RBD caused by 293T transfection;

[0195] Figure 1-3 is the RBD-mRNA@MPP(Fe 3+ ) ability to induce humoral immunity;

[0196] Figures 1-4 are NY-ESO-1-mRNA@MPP(Fe 3+ ) ability to induce humoral immunity;

[0197] Figure 1-5 is the RBD-mRNA@MPP(Fe 3+ ) ability to induce cellular immunity;

[0198] Figure 1-6 is NY-ESO-1-mRNA@MPP(Fe 3+ ) ability to induce cellular immunity;

[0199] Figure 1-1-2 is the eGFP-mRNA@MPP (Al 3+ ) The percentage of eGFP-positive cells caused by 293T transfection;

[0200] Figure 1-2-2 is the RBD-mRNA@MPP (Al 3+ ) Expression of RBD caused by 293T transfection;

[0201] Figure 1-3-2 is the RBD-mRNA@MPP (Al3+ ) ability to induce humoral immunity;

[0202] Figure 1-4-2 is NY-ESO-1-mRNA@MPP (Al 3+ ) ability to induce humoral immunity;

[0203] Figure 1-5-2 is the RBD-mRNA@MPP (Al 3+ ) ability to induce cellular immunity;

[0204] Figure 1-6-2 is NY-ESO-1-mRNA@MPP(Al 3+ ) ability to induce cellular immunity;

[0205] Figure 1-1-3 is the eGFP-mRNA@MPP(Mg 2+ ) The percentage of eGFP-positive cells caused by 293T transfection;

[0206] Figure 1-2-3 is the RBD-mRNA@MPP(Mg 2+ ) Expression of RBD caused by 293T transfection;

[0207] Figure 1-3-3 is the RBD-mRNA@MPP(Mg 2+ ) ability to induce humoral immunity;

[0208] Figure 1-4-3 is NY-ESO-1-mRNA@MPP(Mg 2+ ) ability to induce humoral immunity;

[0209] Figure 1-5-3 is the RBD-mRNA@MPP(Mg 2+ ) ability to induce cellular immunity;

[0210] Figure 1-6-3 is NY-ESO-1-mRNA@MPP(Mg 2+ ) ability to induce cellular immunity;

[0211] Figure 1-7 is a diagram of Bcl-2-siRNA@MPP(Fe 3+ ) ability to silence target genes;

[0212] Figure 1-8 is a PLK1-siRNA@MPP(Fe 3+ ) ability to silence target genes;

[0213] Figure 1-9 is a Gal-1-siRNA@MPP(Fe 3+ ) ability to silence target genes;

[0214] Figure 1-7-2 is Bcl-2-siRNA@MPP (Al 3+ ) ability to silence target genes;

[0215] Figure 1-8-2 is the PLK1-siRNA@MPP (Al 3+ ) ability to silence target genes;

[0216] Figure 1-9-2 is a Gal-1-siRNA@MPP (Al 3+ ) ability to silence target genes;

[0217] Figure 1-7-3 is Bcl-2-siRNA@MPP(Mg 2+ ) ability to silence target genes;

[0218] Figure 1-8-3 is the PLK1-siRNA@MPP(Mg 2+ ) ability to silence target genes;

[0219] Figure 1-9-3 is the Gal-1-siRNA@MPP(Mg 2+ ) ability to silence target genes;

[0220] Figure 1-10 is a STAT3-ASO@MPP (Fe 3+ ) the ability to silence target genes in cells;

[0221] Figure 1-11 is the α-syn-ASO@MPP(Fe 3+ ) the ability to silence target genes in cells;

[0222] Figure 1-12 is a diagram of Bcl-2-ASO@MPP(Fe 3+ ) the ability to silence target genes in cells;

[0223] Figure 1-10-2 is a STAT3-ASO@MPP (Al 3+ ) the ability to silence target genes in cells;

[0224] Figure 1-11-2 is the α-syn-ASO@MPP (Al 3+ ) the ability to silence target genes in cells;

[0225] Figure 1-12-2 is a diagram of Bcl-2-ASO@MPP (Al 3+ ) the ability to silence target genes in cells;

[0226] Figure 1-10-3 is a diagram of STAT3-ASO@MPP(Mg 2+ ) the ability to silence target genes in cells;

[0227] Figure 1-11-3 is the α-syn-ASO@MPP(Mg 2+ ) the ability to silence target genes in cells;

[0228] Figure 1-12-3 shows Bcl-2-ASO@MPP(Mg 2+ ) the ability to silence target genes in cells;

[0229] Figure 1-13 is a diagram of S-mRNA@MPP(Fe 3+ ) Expression of S protein caused by transfection of 293T cells;

[0230] Figure 1-14 shows the drug (dsDNA and ssDNA)@MPP(Fe 3+ ) function;

[0231] Figure 1-13-2 is the S-mRNA@MPP (Al 3+ ) Expression of S protein caused by transfection of 293T cells;

[0232] Figure 1-14-2 shows the drug (dsDNA and ssDNA)@MPP (Al 3+ ) function;

[0233] Figure 1-13-3 is the S-mRNA@MPP(Mg 2+ ) Expression of S protein caused by transfection of 293T cells;

[0234] Figure 1-14-3 shows the drug (dsDNA and ssDNA)@MPP(Mg 2+ ) function;

[0235] Figure 1-15 shows the drug (dsDNA and ssDNA)@MPP(Fe 3+ ) of transfected cells;

[0236] Figure 2-1 is a differential scanning calorimetry diagram of the phospholipid complex provided in Example 2.1.1 of the present application;

[0237] Figure 2-2 is a metal-phospholipid complex (Fe 3+ )’s UV absorption pattern;

[0238] Figure 2-2-2 is a metal-phospholipid complex (Al 3+ )’s UV absorption pattern;

[0239] Figure 2-2-3 is a metal-phospholipid complex (Mg 2+ )’s UV absorption pattern;

[0240] Figure 2-3 is provided in Example 2.2 of this application under low pH conditions (pH = 5.0) Fe 3+ characterization of shedding from metal-phospholipid complexes;

[0241] Figure 2-4 shows the drug-metal-phospholipid complex particles (Fe 3+ ) elemental analysis;

[0242] Figure 2-5 is a diagram of siRNA / mRNA@MPP(Fe 3+ 、Al 3+ or Mg 2+ ) and the efficiency of siRNA / mRNA@LNP encapsulation of nucleic acids (mRNA and siRNA);

[0243] Figure 2-6 is a diagram of siRNA / mRNA@MPP(Fe 3+ 、Al 3+ or Mg 2+ ) and statistical diagram of nucleic acid lysosomal escape ability of siRNA / mRNA@LNP;

[0244] Figure 2-7 is the MPP (Fe 3+ 、Al 3+ or Mg 2+) and the eGFP-positive cell rate of LNP;

[0245] Figure 2-8 is the MPP (Fe 3+ 、Al 3+ or Mg 2+ ) compared with the ability of LNP to promote RBD-mRNA expression;

[0246] Figure 2-9 is the MPP (Fe 3+ 、Al 3+ or Mg 2+ ) compared with the ability of LNP to promote humoral immunity;

[0247] Figure 2-10 is the MPP (Fe 3+ 、Al 3+ or Mg 2+ ) compared with the ability of LNP to promote cellular immunity;

[0248] Figure 2-11 is a diagram of siRNA / mRNA@MPP(Fe 3+ ) and the results of the nucleic acid lysosomal escape function test of siRNA / mRNA@LNP;

[0249] Figure 3-1 shows the drug-metal-phospholipid complex particles (Fe 3+ ) The effect of intratumoral injection in the treatment of liver cancer;

[0250] Figure 3-2 shows the drug-metal-phospholipid complex particles (Al 3+ ) The effect of intratumoral injection in the treatment of liver cancer;

[0251] Figure 3-3 shows the drug-metal-phospholipid complex particles (Mg 2+ ) The effect of intratumoral injection in the treatment of liver cancer;

[0252] Figure 4-1 is a diagram of the targeted drug (CD19 CAR mRNA@Apt-MPP) (Fe 3+ ), drug-metal-phospholipid particles (CD19 CAR mRNA@MPP)(Fe 3+ ) Statistical graph of the proportion of CD19-CAR positive cells;

[0253] Figure 4-1-2 is a diagram of the targeted drug (CD19 CAR mRNA@Apt-MPP) in Example 6.3 of this application (Al 3+ ), drug-metal-phospholipid particles (CD19 CAR mRNA@MPP) (Al 3+) Statistical graph of the proportion of CD19-CAR positive cells;

[0254] Figure 4-1-3 shows the targeted drug (CD19 CAR mRNA@Apt-MPP) (Mg 2+ ), drug-metal-phospholipid particles (CD19 CAR mRNA@MPP)(Mg 2+ ) Statistical graph of the proportion of CD19-CAR positive cells;

[0255] Figure 4-2 is a diagram of the targeted drug (CD19 CAR mRNA@Apt-MPP) (Fe 3+ ), drug-metal-phospholipid particles (CD19 CAR mRNA@MPP)(Fe 3+ ) Comparison of therapeutic effects on mice with acute B-lymphocytic leukemia;

[0256] Figure 4-2-2 is a diagram of the targeted drug (CD19 CAR mRNA@Apt-MPP) in Example 6.4 of this application (Al 3+ ), drug-metal-phospholipid particles (CD19 CAR mRNA@MPP) (Al 3+ ) Comparison of therapeutic effects on mice with acute B-lymphocytic leukemia;

[0257] Figure 4-2-3 shows the targeted drug (CD19 CAR mRNA@Apt-MPP) (Mg 2+ ), drug-metal-phospholipid particles (CD19 CAR mRNA@MPP)(Mg 2+ ) Comparison of therapeutic effects on mice with acute B-lymphocytic leukemia;

[0258] Figure 5-1 is a diagram of the targeted drug CD19 CAR mRNA@Apt(CD8)-MPP(Fe 3+ ) and CD19 CAR mRNA@MPP(Fe 3+ ) Comparison of the overall survival rate of mice treated with anti-acute B lymphocytic leukemia;

[0259] Figure 5-1-2 is a diagram of the targeted drug CD19 CAR mRNA@Apt(CD8)-MPP(Al 3+ ) and CD19 CAR mRNA@MPP(Al 3+ ) Comparison of the overall survival rate of mice treated with anti-acute B lymphocytic leukemia;

[0260] Figure 5-1-3 shows the targeted drug CD19 CAR mRNA@Apt(CD8)-MPP(Mg 2+ ) and CD19 CAR mRNA@MPP(Mg 2+ ) Comparison of the overall survival rate of mice treated with anti-acute B lymphocytic leukemia;

[0261] Figure 5-2 is a diagram of the targeted drug ASO@Apt-MPP (Fe 3+ ) and ASO@MPP(Fe 3+ ) Comparison of the overall survival rate of mice treated with lung cancer;

[0262] Figure 5-2-2 is a diagram of the targeted drug ASO@Apt-MPP (Al 3+ ) and ASO@MPP(Al 3+ ) Comparison of the overall survival rate of mice treated with lung cancer;

[0263] Figure 5-2-3 shows the targeted drug ASO@Apt-MPP (Mg 2+ ) and ASO@MPP(Mg 2+ ) Comparison of the overall survival rate of mice treated with lung cancer;

[0264] Figure 5-3 is a diagram of the targeted drug siRNA@Apt-MPP (Fe 3+ ) and siRNA@MPP(Fe 3+ ) Comparison of the overall survival rates of pancreatic cancer-resistant mice;

[0265] Figure 5-3-2 is the targeted drug siRNA@Apt-MPP (Al 3+ ) and siRNA@MPP(Al 3+ ) Comparison of the overall survival rates of pancreatic cancer-resistant mice;

[0266] Figure 5-3-3 is a diagram of the targeted drug siRNA@Apt-MPP (Mg 2+ ) and siRNA@MPP(Mg 2+ ) Comparison of the overall survival rates of pancreatic cancer-resistant mice;

[0267] Figure 6-1 shows the Fe 3+, a comparison of the survival rates of anti-acute B-lymphocytic leukemia mice treated with CD19 CAR mRNA@Apt(CD62L)-MPP and CD19 CAR mRNA@Apt(CD62L)-LNP when the drug is CD19 CAR and the aptamer is CD62L-targeted;

[0268] Figure 6-1-2 shows the Al 3+ , a comparison of the survival rates of anti-acute B-lymphocytic leukemia mice treated with CD19 CAR mRNA@Apt(CD62L)-MPP and CD19 CAR mRNA@Apt(CD62L)-LNP when the drug is CD19 CAR and the aptamer is CD62L-targeted;

[0269] Figure 6-1-3 shows the method of Mg in Example 8.2 of this application. 2+ , a comparison of the survival rates of anti-acute B-lymphocytic leukemia mice treated with CD19 CAR mRNA@Apt(CD62L)-MPP and CD19 CAR mRNA@Apt(CD62L)-LNP when the drug is CD19 CAR and the aptamer is CD62L-targeted;

[0270] Figure 6-2 shows the Fe 3+ , when the drug is ASO and the aptamer is AS1411, the treatment survival rate comparison of ASO@Apt(AS1411)-MPP and ASO@Apt(AS1411)-LNP in anti-lung cancer mice;

[0271] Figure 6-2-2 Al in Example 8.2 of this application 3+ , when the drug is ASO and the aptamer is AS1411, the treatment survival rate comparison of ASO@Apt(AS1411)-MPP and ASO@Apt(AS1411)-LNP in anti-lung cancer mice;

[0272] Figure 6-2-3 Mg in Example 8.2 of this application 2+ , when the drug is ASO and the aptamer is AS1411, the treatment survival rate comparison of ASO@Apt(AS1411)-MPP and ASO@Apt(AS1411)-LNP in anti-lung cancer mice;

[0273] Figure 6-3 shows the Fe 3+ , when the drug is siRNA and the aptamer is P19, the treatment survival rate comparison of siRNA@Apt(P19)-MPP and siRNA@Apt(P19)-LNP against pancreatic cancer mice;

[0274] Figure 6-3-2 shows the Al 3+ , when the drug is siRNA and the aptamer is P19, the treatment survival rate comparison of siRNA@Apt(P19)-MPP and siRNA@Apt(P19)-LNP against pancreatic cancer mice;

[0275] Figure 6-3-3 shows the Mg 2+ , when the drug is siRNA and the aptamer is P19, the treatment survival rate comparison of siRNA@Apt(P19)-MPP and siRNA@Apt(P19)-LNP against pancreatic cancer mice;

[0276] Figure 7-1 is a diagram of Fe in Examples 6.4, 9.1, and 9.2 of this application. 3+ Comparison of the overall survival rate of CD19 CAR mRNA@MPP, CD19 CAR mRNA@Apt(CD62L)-MPP, CAR mRNA@Apt(CD8)-MPP, and CD19 CAR mRNA@Apt(CD3)-MPP in mice infected with acute B-lymphocytic leukemia;

[0277] Figure 7-1-2 shows Al in Examples 6.4, 9.1 and 9.2 of this application. 3+ Comparison of the overall survival rate of CD19 CAR mRNA@MPP, CD19 CAR mRNA@Apt(CD62L)-MPP, CAR mRNA@Apt(CD8)-MPP, and CD19 CAR mRNA@Apt(CD3)-MPP in mice infected with acute B-lymphocytic leukemia;

[0278] Figure 7-1-3 shows the Mg content in Examples 6.4, 9.1 and 9.2 of this application. 2+ Comparison of the overall survival rate of CD19 CAR mRNA@MPP, CD19 CAR mRNA@Apt(CD62L)-MPP, CAR mRNA@Apt(CD8)-MPP, and CD19 CAR mRNA@Apt(CD3)-MPP in mice infected with acute B-lymphocytic leukemia;

[0279] Figure 8-1 is a CD19 CAR mRNA@MPP (Fe 3+ 、Al 3+ or Mg 2+ ) and the percentage of CAR-positive cells of CD19 CAR mRNA@LNP in myeloid cells;

[0280] Figure 8-2 is CD19 CAR mRNA@Apt-MPP (Fe in Example 6.5 of this application 3+ 、Al 3+ or Mg 2+ ) and the percentage of CAR-positive cells in peripheral blood T cells expressed by CD19 CAR mRNA@Apt-LNP;

[0281] Figure 8-3 shows the CD19 CAR mRNA@Apt-MPP (Ca) modified with different target head ratios in Example 6.6. 2+ ) and CD19 CAR mRNA@Apt-LNP(Ca 2+ ) CAR positivity rate;

[0282] Figure 8-4 shows the CD19 CAR mRNA@Apt-MPP (Mg 2+) modified with different target head ratios in Example 6.6. 2+ ) and CD19 CAR mRNA@Apt-LNP(Mg 2+ ) CAR positivity rate;

[0283] Figure 8-5 shows the CD19 CAR mRNA@Apt-MPP (Fe 3+ 、Al 3+ or Mg 2+ ) and the percentage of CAR-positive cells in peripheral blood myeloid cells by CD19 CAR mRNA@Apt-LNP. DETAILED DESCRIPTION

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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.

[0288] Although the numerical ranges and parameters used to define the broader scope of this application are approximate, the numerical values ​​of the specific examples have been 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.

[0289] 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.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] 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.

[0294] The phrase "phospholipid molecule portion" herein refers to the structure originally belonging to the phospholipid molecule after the phospholipid molecule reacts with other substances.

[0295] 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.

[0296] The phrase "part of a linker molecule" as used herein refers to a structure that originally belongs to the linker molecule after the linker molecule reacts with other substances.

[0297] The phrase "metal ion portion" herein refers to the structure originally belonging to the metal ion after the metal ion reacts with other substances.

[0298] The phrase "phospholipid complex" herein refers to a complex formed by reacting and linking the above-mentioned phospholipid molecule portion having a phosphate group with the above-mentioned linker molecule portion.

[0299] The phrase "metal-phospholipid complex" herein refers to a complex formed by the reaction of the above-mentioned phospholipid molecule portion having a phosphate group, the above-mentioned linker molecule portion, and the above-mentioned metal ion portion, wherein the above-mentioned phospholipid molecule portion is connected to the above-mentioned linker molecule portion, and the above-mentioned linker portion is connected to the above-mentioned metal ion portion through a coordination bond, and the metal-phospholipid complex is neither a cationic lipid nor an ionizable lipid.

[0300] The term "drug-metal-phospholipid complex particles" refers to drug-loaded metal-phospholipid complex particles, where the drug is connected to the metal-phospholipid complex by encapsulation, loading, chemical bonds or non-chemical bonds.

[0301] 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.

[0302] 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.

[0303] 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,

[0304] 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.

[0305] 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).

[0306] 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.

[0307] 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.

[0308] 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.

[0309] 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.

[0310] 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.

[0311] 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.

[0312] The term "P19" refers to a nucleic acid sequence that can specifically recognize pancreatic cancer cells.

[0313] The term "targeted drug" (also known as targeted preparation) refers to a drug or its preparation that is endowed with targeting ability.

[0314] 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.

[0315] 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.

[0316] 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.

[0317] 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.

[0318] 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.

[0319] 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.

[0320] 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.

[0321] 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 bispecific aptamers), and cell therapy drugs (such as CAR-T (Chimeric Antigen Receptor T-cell), TCR-T (T-cell Receptor Engineered T-cell), etc.). These drugs can accurately identify specific targets on the surface or inside immune cells, and intervene in the body's immune response by genetically modifying immune cells or regulating their functions, such as activating, inhibiting or changing their differentiation state, thereby treating diseases.

[0322] 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.

[0323] 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.

[0324] 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-phospholipid complex particles (MPPs) are "lipid vesicles," and drugs, such as nucleic acid mRNA, are encapsulated in MPPs as encapsulated components, and the "encapsulation" can be full encapsulation and / or partial encapsulation.

[0325] 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.

[0326] 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.

[0327] Among the components of the metal-phospholipid complex particles, the "non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation" in (iii) means that the non-cationic lipids or non-ionizable lipids in (iii) are the lipids remaining in the metal-phospholipid complex particles after excluding the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation.

[0328] 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.

[0329] In the metal-phospholipid complex particles, non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation are primarily present as vesicle-forming lipids, and the term "vesicle-forming lipid" is intended to include any amphiphilic lipid having a hydrophobic portion and a polar head group and which can spontaneously form bilayer vesicles in water, exemplified by most phospholipids.

[0330] In the metal-phospholipid 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).

[0331] 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.

[0332] The term "diacylglycerol" refers to a compound having a 2-fatty acyl chain, wherein R1 and R2 independently have 2-30 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:

[0333]

[0334] 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.

[0335] 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:

[0336]

[0337] 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:

[0338] 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.

[0339] 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).

[0340] 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.

[0341] 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:

[0342]

[0343] 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.

[0344] 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.

[0345] 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.

[0346] 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.

[0347] 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.

[0348] 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.

[0349] 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.

[0350] 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.

[0351] 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.

[0352] 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.

[0353] 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.

[0354] 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.

[0355] 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).

[0356] As used herein, "gene product" refers to the product of a gene such as, but not limited to, the transcript of DNA, mRNA.

[0357] 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.

[0358] 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.

[0359] As used herein, the term "aqueous solution" refers to a composition that comprises, in whole or in part, water.

[0360] As used herein, the term "organic lipid solution" refers to a composition comprising in whole or in part an organic solvent with lipids.

[0361] 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.

[0362] 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.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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)).

[0367] 1. Metal-phospholipid complex

[0368] In the present application, the metal-phospholipid complex is composed of a phospholipid molecule portion, a linker molecule portion, and a metal ion portion. The phospholipid molecule portion is connected to the linker molecule portion, and the linker molecule portion is connected to the metal ion portion via a coordination bond. The metal-phospholipid complex is not a cationic lipid or an ionizable lipid.

[0369] Regarding the phospholipid molecule, it should be noted that the cis-trans isomers of the phospholipid molecule of the present application will not affect the effect to be achieved by the protection content of the present application.

[0370] In some embodiments, the phospholipid molecule portion is selected from phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphoceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysophosphatidylcholine LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, 1-phosphate sphingosine S1P, and a combination of one or more of their derivatives. Here, the “their” in “and their derivatives” refers to “phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), 1-phosphoceramide (SP), phosphatidylinositol (PI), phosphatidylthreonine (PT), sphingomyelin (SM), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysosphingomyelin (LSM), and 1-sphingosine 1-phosphate (S1P)”. Specifically, the phospholipid molecule portion can be, for example, but not limited to, selected from phosphatidylcholine (PC), phosphatidylcholine (PC) derivatives, phosphatidylethanolamine (PE), phosphatidylethanolamine (PE) derivatives, phosphatidylglycerol (PG), phosphatidylglycerol (PG) derivatives, phosphatidylglycerol (PG) and phosphatidylcholine (PC), phosphatidylcholine (PC) and phosphatidylcholine (PC) derivatives, etc. In the present application, "and their derivatives" have similar meanings.

[0371] In some embodiments, the phospholipid molecule portion is selected from phosphatidylcholine (PC) (Formula 1), phosphatidylethanolamine (PE) (Formula 2), phosphatidylserine (PS) (Formula 3), phosphatidic acid (PA) (Formula 4), phosphatidylglycerol (PG) (Formula 5), ​​1-phosphoceramide (SP) (Formula 6), phosphatidylinositol (PI) (Formula 7), phosphatidylthreonine (PT) (Formula 8), sphingomyelin (SM) (Formula 9), hemolytic phosphatidylcholine (LPC) (Formula 10), hemolytic phosphatidylethanolamine (LPE) (Formula 11), hemolytic phosphatidylserine (LPS) (Formula 12), hemolytic phosphatidic acid (LPA) (Formula 13), hemolytic phosphatidylglycerol (LPG) (Formula 14), hemolytic phosphatidylinositol (LPI) (Formula 15), hemolytic phosphatidylthreonine (LPT) (Formula 16), hemolytic sphingomyelin (LSM) (Formula 17), 1-sphingosine phosphate (S1P) (Formula 18), and a combination of one or more of their derivatives. Preferably, the phospholipid molecule is selected from at least one of phosphatidylcholine (PC) (Formula 1), phosphatidylethanolamine (PE) (Formula 2), phosphatidic acid (PA) (Formula 4), phosphatidylglycerol (PG) (Formula 5), ​​and derivatives thereof. Preferably, the phospholipid molecule is selected from at least one of DSPC, DSPE, DSPA, DSPG, and derivatives thereof. Preferably, the phospholipid molecule is selected from at least one of DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48), DSPG (Formula 49), and derivatives thereof.

[0372] As for the linker molecule, it is mainly derived from natural plant extracts, such as curcumin, which has a wide range of biological effects, including antibacterial, antiviral, antifungal, antioxidant and anti-inflammatory activities. In addition, it is also an effective immunomodulator that can regulate the activity of various immune cells such as T cells, B cells, macrophages, neutrophils, natural killer cells and dendritic cells, promote the balance of immunity, and enhance the body's immunity. Based on the potential immune-enhancing, anti-inflammatory, antioxidant and anti-SARS-CoV-2 effects of curcumin molecules, it is expected to become a potential auxiliary treatment for COVID-19. In addition, the safety of curcumin molecules is extremely high and has been listed in the catalog of food additives and pharmaceutical excipients. Its safety is conducive to the overall clinical drug registration of targeted drugs and shortens the length of clinical drug registration.

[0373] In some embodiments, the linker molecule portion is selected from curcumin, chlorogenic acid, anthocyanidin, quercetin, dihydromyricetin, hesperetin, naringenin, apigenin, catechin, tea polyphenols, epigallocatechin gallate, ellagic acid, morin, epicatechin gallate, catechin gallate, epigallocatechin gallate or picarapine C, and a combination of one or more of their derivatives. Preferably, the linker molecule is selected from at least one of curcumin (Formula 19), chlorogenic acid (Formula 20), anthocyanin (Formula 21), quercetin (Formula 22), dihydromyricetin (Formula 23), hesperetin (Formula 24), naringenin (Formula 25), apigenin (Formula 26), catechin (Formula 27), tea polyphenols (Formula 28), epigallocatechin gallate (Formula 29), ellagic acid (Formula 30), morin (Formula 31), epicatechin gallate (Formula 32), catechin gallate (Formula 33), gallocatechin gallate (Formula 34), flavonoid C (Formula 35), and derivatives thereof. Preferably, the linker molecule is selected from at least one of curcumin (Formula 19), dihydrocurcumin (Formula 36), hexahydrocurcumin (Formula 37), curcumin sulfate (Formula 38), and bisdemethoxycurcumin (Formula 39). Preferably, the linker molecule is selected from at least one of curcumin (Formula 19), hesperetin (Formula 24), tea polyphenols (Formula 28), and derivatives thereof. Preferably, the linker molecule is selected from curcumin (Formula 19), hesperetin (Formula 24), or tea polyphenols (Formula 28).

[0374] Regarding the metal ion portion, the coordination bond between the linker molecule portion and the metal ion portion in the metal-phospholipid complex will break under low pH conditions such as lysosomes (pH = 5.0), and the metal ion will fall off from the metal-phospholipid complex.

[0375] 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+ 、V3+ 、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+ .

[0376] The ratio of each component in the metal-phospholipid complex can be adjusted based on the structure of the specific metal-phospholipid complex components. This adjustment is based on the following: Because phospholipid molecules and linker molecules are linked by hydrogen bonds, as long as the phospholipid molecule contains multiple phosphate groups, the ratio of phospholipid to linker molecules can be adjusted based on the number of phosphate groups contained in the phospholipid molecule during phospholipid complex synthesis. For example, when the phospholipid molecule contains two phosphate groups, the ratio of phospholipid to linker molecules can be adjusted to 1:2; when the phospholipid molecule contains three phosphate groups, the ratio of phospholipid to linker molecules can be adjusted to 1:3. Because the hydroxyl groups of the linker molecule are linked to the metal ion by coordination bonds, the ratio of linker to metal ion can be adjusted based on the number of binding sites contained in the linker, as long as the linker molecule contains multiple binding sites. When used to encapsulate drugs (such as nucleic acids), the metal ion functions to connect the phospholipid complex to the nucleic acid. Therefore, when the linker molecule is attached to as few complexing sites as possible for the metal ion, the metal-phospholipid complex can maximize nucleic acid encapsulation.

[0377] In some embodiments, the phospholipid molecule portion is selected from DSPC, DSPE, DSPA or DSPG, the linker molecule portion is selected from curcumin, hesperidin or tea polyphenols, and the metal ion portion is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ Preferably, the metal-phospholipid complex is made of a phospholipid molecule portion, a linker molecule portion and a metal ion portion, the phospholipid molecule portion is selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48) or DSPG (Formula 49), the linker molecule portion is selected from curcumin (Formula 19), hesperidin (Formula 24) or tea polyphenols (Formula 28), and the metal ion portion is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+Preferably, the molar ratio of the phospholipid molecule portion, the linker molecule portion, and the metal ion portion is 1:(1-3):(0.5-2). The molar ratio of the phospholipid molecule portion, the linker molecule portion, and the metal ion portion can be, but is not limited to, 1:1:0.5, 1:1:1, 1:1:1.5, 1:1:2, 1:2:1, 1:2:2, 1:2:0.5, 1:3:0.5, 1:3:1, or 1:3:2.

[0378] In one embodiment, the phospholipid molecule portion is DSPC (Formula 46), the linker molecule portion is selected from curcumin (Formula 19), and the metal ion portion is selected from Fe 3+ Mg 2+ or Al 3+ The molar ratio of the phospholipid molecule part, the linker molecule part and the metal ion part is 1:1:1.

[0379] In the present application, the metal-phospholipid complex can be prepared by a "one-step method" or by a "step-by-step method". The "one-step method" and the "step-by-step method" have equivalent capabilities for constructing metal-phospholipid complexes. In a preferred embodiment, the "one-step method" can significantly shorten the time required for the industrialization process, which is crucial in the scenario of industrial large-scale production. The "one-step method" reduces the problems of process connection and time intervals brought about by step-by-step operations, greatly improving the continuity and compactness of production, thereby effectively reducing production costs and improving production efficiency. From the perspective of industrial application, the "one-step method" is more in line with the needs of large-scale production, and has broad application prospects and extremely high practical value.

[0380] In a one-step process, phospholipid molecules, linker molecules, and metal ions react to form a metal-phospholipid complex.

[0381] In some embodiments, the phospholipid molecules, linker molecules, and metal ions are dissolved in ethanol for reaction. The molar ratio of the phospholipid molecules, linker molecules, and metal ions is preferably 1:1:(1-2). The reaction conditions are preferably 40-60° C. for 1-5 hours. The molar ratio of the phospholipid molecules, linker molecules, and metal ions can be, but is not limited to, 1:1:1, 1:1:1.5, or 1:1:2. The reaction temperature can be, but is not limited to, 40° C., 42° C., 46° C., 48° C., 50° C., 52° C., 54° C., 56° C., 58° C., or 60° C. The reaction time can be, but is not limited to, 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours.

[0382] In the step-by-step method, the phospholipid molecules are first reacted with the linker molecules to form a phospholipid complex; then the prepared phospholipid complex is reacted with the metal ion through a coordination bond to form a metal-phospholipid complex.

[0383] In some embodiments, the phospholipid molecules and the linker molecules are dissolved in ethanol for reaction, and then n-hexane is added for precipitation to obtain a phospholipid complex. The molar ratio of the phospholipid molecules to the linker molecules is preferably 1:1; the reaction conditions are preferably 65° C. for 2 hours.

[0384] In some embodiments, the phospholipid complex and the metal ion are dissolved in ethanol, and triethylamine is added to react to obtain a metal-phospholipid complex. The molar ratio of the phospholipid complex to the metal ion is preferably 1: (1-2), and the molar ratio of the phospholipid complex to triethylamine is preferably 1: 1. The reaction conditions are preferably 60 ° C for 2 hours.

[0385] 2. Metal-chelated phospholipid complex nanoparticles (MPP)

[0386] In the present application, the metal-phospholipid complex particles contain: (i) a metal-phospholipid complex; (ii) a conjugated lipid that inhibits particle aggregation, wherein the conjugated lipid that inhibits particle aggregation is not a cationic lipid or an ionizable lipid; and (iii) a non-cationic lipid or a non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation.

[0387] Conjugated lipids that inhibit particle aggregation refer to conjugated lipids that inhibit the aggregation of drug-metal-phospholipid complex particles or targeted drugs. Their primary function is to prevent the aggregation of drug-metal-phospholipid complex particles or targeted drugs. Examples include PEG conjugated to dialkoxypropyl groups, PEG conjugated to diacylglycerols, PEG conjugated to phosphatidylethanolamines, and PEG conjugated to ceramides, preferably PEG-lipid conjugates. Cis- and trans-isomers of the lipids do not affect the effects to be achieved in the present application.

[0388] 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 at least one of phosphatidylethanolamine-polyethylene glycol 2000 (Formula 42), phosphatidylethanolamine-polyethylene glycol 700 (Formula 43), phosphatidylethanolamine-polyethylene glycol 1000 (Formula 44), phosphatidylethanolamine-polyethylene glycol 5000 (Formula 45), and derivatives thereof. Preferably, the PEG-lipid conjugate is selected from at least one 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).

[0389] The non-cationic lipid or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation is at least one of cholesterol (preferably cholesterol formula 40) and its derivatives. In a preferred embodiment, in addition to cholesterol or its derivatives, it further comprises a combination of one or more selected from phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphoceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysophosphatidylcholine LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, 1-phospho-sphingosine S1P, cholesterol sulfate, and its derivatives. Preferably, in addition to cholesterol or its derivatives, it also includes at least one selected from phosphatidylcholine PC (Formula 1), phosphatidylethanolamine PE (Formula 2), phosphatidylserine PS (Formula 3), phosphatidic acid PA (Formula 4), phosphatidylglycerol PG (Formula 5), ​​1-phosphoceramide SP (Formula 6), phosphatidylinositol PI (Formula 7), phosphatidylthreonine PT (Formula 8), sphingomyelin SM (Formula 9), hemolytic phosphatidylcholine LPC (Formula 10), hemolytic phosphatidylethanolamine LPE (Formula 11), hemolytic phosphatidylserine LPS (Formula 12), hemolytic phosphatidic acid LPA (Formula 13), hemolytic phosphatidylglycerol LPG (Formula 14), hemolytic phosphatidylinositol LPI (Formula 15), hemolytic phosphatidylthreonine LPT (Formula 16), hemolytic sphingomyelin LSM (Formula 17), 1-phosphoinosine S1P (Formula 18), cholesterol sulfate (Formula 41), and its derivatives. Preferably, the non-cationic lipid or non-ionizable lipid in (iii) includes cholesterol, and a combination of one or more selected from DSPC, DSPE, DSPA or DSPG, for example, the non-cationic lipid or non-ionizable lipid in (iii) includes cholesterol (Formula 40) and DSPC (Formula 46).

[0390] In the present application, the metal-phospholipid complex as an integral component reacts with component (ii) and component (iii) to self-assemble to obtain metal-phospholipid complex particles. The metal-phospholipid complex particles can be used as drug carriers, wherein the drug is a negatively charged drug, such as a nucleic acid. The principle of loading the drug into the metal-phospholipid complex particles assembled by the metal-phospholipid complex is as follows: the linker molecule and the phospholipid molecule are bound together by hydrogen bonds, and the linker molecule is connected to the metal ion through a coordination bond to form a metal-phospholipid complex. The metal ion of the metal-phospholipid complex is connected to the negatively charged drug through a coordination bond, thereby ensuring that the metal-phospholipid complex and other components (conjugated lipids that inhibit particle aggregation, and non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation) self-assemble into MPPs, while the negatively charged drug is loaded into the nanoparticle MPPs to obtain drug-metal-phospholipid complex particles. Herein, the “non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation” refers to component (iii) in the metal-phospholipid complex particles, which can be simply referred to as “non-cationic lipids or non-ionizable lipids”.

[0391] The proportions of the components in the metal-phospholipid complex particles can be as follows: the metal-phospholipid complex comprises 5% to 50% by mole of the raw material, the conjugated lipid that inhibits particle aggregation comprises 1% to 10% by mole of the raw material, the cholesterol comprises 15% to 80% by mole of the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol comprises 0% to 51% by mole of the raw material. Here, "raw material" refers to the sum of the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, cholesterol, and the non-cationic lipid or non-ionizable lipid other than cholesterol.

[0392] The molar proportion of the metal-phospholipid complex in the raw material can be, but is not limited to, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%. In some embodiments, the molar proportion of the metal-phospholipid complex in the raw material is 5% to 40%, preferably 10% to 40%.

[0393] The molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material can be, but is not limited to, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In some embodiments, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%.

[0394] The non-cationic lipid or non-ionizable lipid is cholesterol, and the molar proportion of cholesterol in the raw material can be, but is not limited to, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125 %, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%. In some embodiments, the molar proportion of cholesterol in the raw material is 25% to 75%, preferably 35% to 75%, and preferably 35% to 55%.

[0395] The molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material can be, but is not limited to, 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or 51%. In some embodiments, the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 0% to 50%, preferably 0% to 40%, preferably 5% to 30%, and further 20% to 25%.

[0396] The metal-phospholipid complex particles are prepared by mixing the metal-phospholipid complex, a conjugated lipid that inhibits particle aggregation, and a non-cationic lipid or a non-ionizable lipid to obtain the metal-phospholipid complex particles. In some embodiments, the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or the non-ionizable lipid are mixed in a homogeneous phase, such as an organic phase (in some embodiments, the solvent is ethanol).

[0397] When the metal-phospholipid complex particles are loaded with a drug, the preparation method comprises mixing the metal-phospholipid complex, a conjugated lipid that inhibits particle aggregation, a non-cationic lipid or a non-ionizable lipid, and the drug to prepare the drug-metal-phospholipid complex particles. In some embodiments, the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer to form an aqueous phase, and the organic and aqueous phases are mixed to produce the drug-metal-phospholipid complex particles. In some embodiments, the buffer can be PBS or Tris-HCl. In some embodiments, the organic and aqueous phases can be mixed using a microfluidic chip or ultrasound.

[0398] 3. Targeting vector (containing MPP)

[0399] 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 a carrier protein. Preferably, the lipid delivery system is a metal-phospholipid 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.

[0400] In one embodiment, the carrier delivery system is a metal-phospholipid complex particle (MPP), and the targeting structure is attached to the outer surface of the metal-phospholipid complex particle (MPP).

[0401] 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-phospholipid complex particle based on hydrophilicity and hydrophobicity.

[0402] 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-phospholipid complex particle through this portion.

[0403] In some embodiments, the linking region comprises at least one or more of PEG-2000 and its derivatives.

[0404] 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 and metal-phospholipid 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 includes at least one of a nucleic acid, a polypeptide, a protein, or a small molecule. In some embodiments, the targeting binding region includes 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 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 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.

[0405] 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.

[0406] 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.

[0407] 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-phospholipid complex particles (MPP)) to form a targeted carrier. In some embodiments, the micelles are prepared by direct dissolution, ethanol infusion, dialysis, or ultrasound.

[0408] When the targeting carrier is used for drug encapsulation, the drug, metal-phospholipid complex, conjugated lipids that inhibit particle aggregation, non-cationic lipids or non-ionizable lipids are first prepared into drug-lipid particles (i.e., drug-metal-phospholipid complex particles), and then the targeting structure is reacted and connected with the drug-lipid particles to obtain the targeted drug.

[0409] 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.

[0410] 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.

[0411] 4. Targeted drugs

[0412] 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-phospholipid complex particles.

[0413] 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.

[0414] 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.

[0415] 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 an antigen binding domain-transmembrane domain connecting region. 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-transmembrane domain connecting region is selected from at least one of SEQ ID No. 40, SEQ ID No. 41, and SEQ ID No. 42.

[0416] In one embodiment, the drug is an mRNA having a nucleotide sequence as shown in SEQ ID No.43.

[0417] Furthermore, the nucleic acid encapsulated in the targeted drug of the present application is resistant to degradation by nucleases in aqueous solution.

[0418] In some embodiments, the drug is fully encapsulated inside the metal-phospholipid complex particles to avoid drug degradation, while the drug is delivered into specific cells through the action of the targeting binding region in the targeting structure.

[0419] In some embodiments, the targeted drugs provided herein have a small diameter suitable for systemic delivery.

[0420] 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.

[0421] The targeted drug or preparation method of the present application can be used for drug delivery, imaging drugs, and vaccines.

[0422] In some embodiments, targeted drugs are used for drug delivery, treatment and / or prevention.

[0423] 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.

[0424] 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.

[0425] 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.

[0426] 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.

[0427] 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.

[0428] 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.

[0429] 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).

[0430] In some embodiments, the metabolic disease comprises atherosclerosis, congenital hyperinsulinemia, nonalcoholic steatohepatitis, or non-obese diabetes mellitus (NOD).

[0431] 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.

[0432] In some embodiments, the administration route of the targeted drug includes intrathecal injection, intramuscular administration, intracranial injection, intravenous injection, or intratumoral injection.

[0433] In some embodiments, the targeted drug is used in combination therapy.

[0434] In some embodiments, the targeted drug is an immune cell targeted drug, and the immune cells are T cells and / or myeloid cells.

[0435] In some embodiments, the immune cell targeted drug is a chimeric antigen receptor (CAR) drug.

[0436] The present application provides a pharmaceutical agent containing a targeted drug, which is preferably a vaccine, and more preferably a new coronavirus vaccine.

[0437] 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.

[0438] 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.

[0439] 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.

[0440] 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.

[0441] 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.

[0442] 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.

[0443] 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)).

[0444] 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)).

[0445] 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)).

[0446] 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.

[0447] 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.

[0448] 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.

[0449] 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.

[0450] 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).

[0451] 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.

[0452] 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.

[0453] 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.

[0454] 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.

[0455] 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.

[0456] 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.

[0457] 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.

[0458] 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.

[0459] 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.

[0460] The drug-lipid particles of this application refer to drug-lipid particles other than those containing cationic / ionizable lipids, i.e., drug-loaded metal-chelated phospholipid complex nanoparticles (drug@MPP), which are linked to a targeting structure to form a targeted drug. After removing the drug from the targeted drug, the remaining components include the targeting carrier.

[0461] Metal (containing Fe 3+ 、Al 3+ or Mg 2+Preparation of Metal-chelated phospholipid complex nanoparticles (MPP)

[0462] Example 1: Preparation of drug-metal-phospholipid complex particles

[0463] Example 1.1. Preparation of phospholipid complexes

[0464] To connect a phospholipid molecule with a phosphate group to a linker molecule: Distearoylphosphatidylcholine (DSPC, Formula 46) and curcumin (Formula 19) were added to a reaction flask at a molar ratio of 1:1. An appropriate amount of ethanol was added to dissolve the mixture. After reacting at 65°C for 2 hours, the mixture was concentrated and n-hexane was added. The resulting phospholipid complex was precipitated, filtered, and vacuum dried to obtain the phospholipid complex. The structure of the phospholipid complex is shown below:

[0465] Result analysis: The yield of the target product obtained by reacting curcumin with DSPC at 65°C for 2 hours was 94%.

[0466] Example 1.2: Preparation of Metal-Phospholipid Complex

[0467] Example 1.2.1 Step-by-step preparation of metal ions: Fe 3+ Metal-phospholipid complexes

[0468] The phospholipid complex prepared in Example 1.1 was partially linked to the metal ion: the phospholipid complex and FeCl3 were added to a reaction flask at a molar ratio of 1:1, and ethanol was added to dissolve the mixture. Triethylamine was then added at a molar ratio of 1:1 to the phospholipid complex. The mixture was reacted at 60°C for 2 hours. The reaction solution was then suspended to dryness, washed with ultrapure water, and vacuum-dried to obtain a metal-phospholipid complex. The structure of the metal-phospholipid complex is shown below.

[0469] Result analysis: The yield of the target product obtained when the phospholipid complex and FeCl3 were reacted at 60℃ for 2 hours, the phospholipid complex feed concentration was 4.5 mg / mL and the feed ratio of phospholipid complex to FeCl3 was 1:1 was 95%.

[0470] Example 1.2.2 Step-by-step preparation of metal ions: Al 3+ Metal-phospholipid complexes

[0471] The difference between this example and example 1.2.1 is that FeCl3 is replaced by Al(NO3)3·9H2O. The structure of the prepared metal-phospholipid complex is shown below.

[0472] Result analysis: The yield of the target product obtained when the phospholipid complex and Al(NO3)3·9H2O were reacted at 60℃ for 2 hours, the phospholipid complex feed concentration was 4.5 mg / mL and the feed ratio of phospholipid complex to Al(NO3)3·9H2O was 1:1 was 95%.

[0473] Example 1.2.3 Step-by-step preparation of metal ions: Mg 2+ Metal-phospholipid complexes

[0474] The difference between this example and example 1.2.1 is that FeCl3 is replaced by MgCl2. The structure of the prepared metal-phospholipid complex is shown below.

[0475] Result analysis: The yield of the target product obtained when the phospholipid complex and MgCl2 were reacted at 60℃ for 2 hours, the phospholipid complex feed concentration was 4.5 mg / mL and the feed ratio of phospholipid complex to MgCl2 was 1:1 was 95.5%.

[0476] Example 1.2.4. Preparation of Metal-Phospholipid Complexes by One-Step Method

[0477] Phospholipid molecules with phosphate groups are connected with linker molecules and metal ions: Distearoylphosphatidylcholine (DSPC, Formula 46), curcumin (Formula 19), and FeCl3 are added to the reaction bottle at a molar ratio of 1:1:1, and an appropriate amount of ethanol is added to dissolve. After reacting at 50±10℃ for 2h, a metal-phospholipid complex is obtained. 3+ The yield of the target product of the metal-phospholipid complex is 95.6%. 3+ Replaced with Mg 2+ 、Al 3+ , Ca 2+ The reaction temperature is 50±10°C, and the reaction time is 1-5h, preferably 2h.

[0478] The metal ion is Fe 3+ The performance comparison analysis of the metal-phospholipid complex prepared by the "one-step method" and the step-by-step method is as follows:

[0479] Phospholipid molecules with phosphate groups are connected with linker molecules and metal ions: Distearoylphosphatidylcholine (DSPC, Formula 46), curcumin, and Al(NO3)3·9H2O are added to the reaction bottle at a molar ratio of 1:1:1, and an appropriate amount of ethanol is added to dissolve. After reacting at 50±10℃ for 2h, a metal-phospholipid complex is obtained. The Al obtained by this "one-step" reaction 3+ The yield of the target metal-phospholipid complex was 95.6%.

[0480] The metal ion is Al 3+ The performance comparison analysis of the metal-phospholipid complex prepared by the "one-step method" and the step-by-step method is as follows:

[0481] The phospholipid molecules with phosphate groups are connected with the linker molecules and metal ions: Distearoylphosphatidylcholine (DSPC, Formula 46), curcumin, and CaCl2 are added to the reaction bottle at a molar ratio of 1:1:1, and an appropriate amount of ethanol is added to dissolve. After reacting at 50±10℃ for 2h, a metal-phospholipid complex is obtained. The CaCl2 obtained by this "one-step" reaction is 2+ The yield of the target product of metal-phospholipid complex is 95%. 2+ Step-by-step preparation protocol of mRNA-metal-phospholipid complex particles with reference to the metal ion Fe 3+ Step-by-step preparation scheme of mRNA-metal-phospholipid complex particles, the metal ion is Ca 2+ mRNA-metal-phospholipid complex particles only convert metal ions from Fe 3+ Replaced by Ca 2+ , FeCl3 was replaced by CaCl2, and the rest remained unchanged. The target product metal ion obtained when the phospholipid complex and CaCl2 were reacted at 60℃ for 2 hours, the phospholipid complex feed concentration was 4.5mg / mL and the feed ratio of phospholipid complex to CaCl2 was 1:1 was Ca 2+ The yield of the metal-phospholipid complex was 95%.

[0482] The metal ion is Ca 2+ The performance comparison analysis of the metal-phospholipid complex prepared by the "one-step method" and the step-by-step method is as follows:

[0483] The phospholipid molecules with phosphate groups are connected with the linker molecules and metal ions: Distearoylphosphatidylcholine (DSPC, Formula 46), curcumin, and MgCl2 are added to the reaction bottle at a molar ratio of 1:1:1, and an appropriate amount of ethanol is added to dissolve. After reacting at 50±10℃ for 2h, a metal-phospholipid complex is obtained. The Mg obtained by this "one-step" reaction is 2+ The yield of the target metal-phospholipid complex was 95%.

[0484] The metal ion is Mg 2+ The performance comparison analysis of the metal-phospholipid complex prepared by the "one-step method" and the step-by-step method is as follows:

[0485] In the preparation process of metal-phospholipid complexes, the characteristics and effectiveness of metal-phospholipid complexes prepared by the "one-step method" and the step-by-step method were compared. The two methods had similar encapsulation efficiency values, and both showed stable performance in ensuring the effective encapsulation of metals in the complexes. In terms of eGFP positive expression, the data of the "one-step method" and the step-by-step method were also similar, proving that the "one-step method" and the step-by-step method have equivalent capabilities for constructing metal-phospholipid complexes. However, the "one-step method" can significantly shorten the time required for the industrialization process. In the scenario of industrial large-scale production, time efficiency is crucial. The "one-step method" reduces the problems of process connection and time intervals caused by step-by-step operations, greatly improving the continuity and compactness of production, thereby effectively reducing production costs and improving production efficiency. From the perspective of industrial application, compared with the step-by-step method, the "one-step method" is more in line with the needs of large-scale production, and has broad application prospects and extremely high practical value.

[0486] Example 1.3: Preparation of Metal Ions: Fe 3+ mRNA-loaded metal-chelated phospholipid complex nanoparticles (mRNA@MPP)

[0487] A metal-phospholipid complex was prepared according to the method described in Example 1.2.1, with DSPC, curcumin, and FeCl3 added at a 1:1:1 ratio. The metal-phospholipid complex, distearoylphosphatidylcholine (DSPC) as a non-cationic or non-ionizable lipid, cholesterol (CHOL) as a non-cationic or non-ionizable lipid, and DSPE-PEG2000 (a conjugated lipid to inhibit particle aggregation) were dissolved in ethanol at varying molar ratios as the organic phase. The metal-phospholipid complex, DSPC, CHOL, and DSPE-PEG2000 accounted for 15%, 35%, 46%, and 4%, respectively. mRNA was dissolved at a concentration of 20 μg / mL in PBS (PBS composed of 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.0018 M potassium dihydrogen phosphate) as the aqueous phase. The total mass of the metal phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) was mixed with the mRNA mass at a mass ratio of 40:1 in a microfluidic chip. The volume ratio of the aqueous phase to the organic phase was 3:1. The flow rate of the organic phase and the aqueous phase in the microfluidic chip was 12 ml / min. Among them, the drug mRNA was mRNA encoding the fluorescent protein eGFP, and its sequence was SEQ ID NO.1 (720 nt). The prepared eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPP without drug loading. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0488] Example 1.4: Preparation of Metal Ions as Al 3+ mRNA-loaded metal-chelated phospholipid complex nanoparticles (mRNA@MPP)

[0489] A metal-phospholipid complex was prepared according to the method described in Example 1.2.2, with DSPC, curcumin, and Al(NO₃)₃·9H₂O added at a 1:1:1 ratio. The metal-phospholipid complex, distearoylphosphatidylcholine (DSPC) as a non-cationic or non-ionizable lipid, cholesterol (CHOL) as a non-cationic or non-ionizable lipid, and DSPE-PEG2000 (a conjugated lipid to inhibit particle aggregation) were dissolved in ethanol at varying molar ratios as the organic phase. The metal-phospholipid complex, DSPC, CHOL, and DSPE-PEG2000 accounted for 7%, 34%, 56%, and 3%, respectively. mRNA was dissolved at a concentration of 20 μg / mL in PBS (PBS composed of 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.0018 M potassium dihydrogen phosphate) as the aqueous phase. The total mass of the metal phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) was mixed with the mRNA mass at a mass ratio of 40:1 in a microfluidic chip. The volume ratio of the aqueous phase to the organic phase was 3:1. The flow rate of the organic phase and the aqueous phase in the microfluidic chip was 12 ml / min. Among them, the drug mRNA was mRNA encoding the fluorescent protein eGFP, and its sequence was SEQ ID NO.1 (720 nt). The prepared eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPP without drug loading. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0490] Example 1.5: Preparation of Metal Ions as Mg 2+ mRNA-loaded metal-chelated phospholipid complex nanoparticles (mRNA@MPP)

[0491] A metal-phospholipid complex was prepared according to the one-step method described in Example 1.2.4, wherein DSPC, curcumin, and MgCl2 were added at a 1:1:1 ratio. The metal-phospholipid complex, distearoylphosphatidylcholine (DSPC) as a non-cationic or non-ionizable lipid, cholesterol (CHOL) as a non-cationic or non-ionizable lipid, and DSPE-PEG2000 (as a conjugated lipid to inhibit particle aggregation) were dissolved in ethanol at varying molar ratios as the organic phase. The metal-phospholipid complex, DSPC, CHOL, and DSPE-PEG2000 accounted for 15%, 40%, 43.2%, and 1.8%, respectively. mRNA was dissolved at a concentration of 20 μg / mL in PBS (PBS composed of 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.0018 M potassium dihydrogen phosphate) as the aqueous phase. The total mass of the metal phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) was mixed with the mRNA mass at a mass ratio of 40:1 in a microfluidic chip. The volume ratio of the aqueous phase to the organic phase was 3:1. The flow rate of the organic phase and the aqueous phase in the microfluidic chip was 12 ml / min. Among them, the drug mRNA was mRNA encoding the fluorescent protein eGFP, and its sequence was SEQ ID NO.1 (720 nt). The prepared eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPP without drug loading. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0492] The particle size, surface potential, and stability of the eGFP-mRNA@MPP prepared in Examples 1.3-1.5 were tested, and the efficiency of nucleic acid encapsulation in the eGFP-mRNA@MPP was calculated.

[0493] 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.

[0494] 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.

[0495] 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.

[0496] Method for calculating nucleic acid encapsulation efficiency: Specifically, agarose gel electrophoresis method is used. First, the nucleic acid feed amount of each group of lipid nanoparticles is set to 10 μg / mL, the mass ratio of lipid to nucleic acid is 40:1, and the same concentration of nucleic acid is 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 do not allow lipid nanoparticles to pass through. When the free nucleic acid bands are electrophoresed to a clearly distinguishable state, the electrophoresis is stopped. Image J software is used to count the grayscale values ​​of free nucleic acids in different groups. The positive control group is set to 100%. The ratio of free nucleic acid in each group to the positive control is the relative amount of free nucleic acid. The encapsulation rate of each group is (100-relative amount of free nucleic acid)%. A nucleic acid encapsulation rate of more than 50% is considered to be within the acceptable range.

[0497] 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.

[0498] Flow cytometry analysis of eGFP-positive cell percentage: 293T cells were seeded in 24-well plates at a seeding density of 5 × 10 5 Cells were plated at 1 mL of MPP or eGFP-mRNA@MPP 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.

[0499] The principle of loading nucleic acid into metal-chelated phospholipid complex nanoparticles (MPP) assembled by metal-phospholipid complex is that curcumin is bound to DSPC through hydrogen bonds, while curcumin is bound to Fe through coordination bonds. 3+ , Ca 2+ 、Al 3+ or Mg 2+ connected to form a metal-phospholipid complex, the Fe 3+ , Ca 2+ 、Al 3+ or Mg 2+The metal-phospholipid complex is connected to the nucleic acid through a coordination bond, thereby ensuring that the metal-phospholipid complex self-assembles with other lipid components into an MPP and simultaneously loads the nucleic acid into the nanoparticle. There are two possibilities for curcumin's contribution to MPP loading of nucleic acids: ① Curcumin interacts with nucleic acids and assists MPP loading of nucleic acids, for example, by inserting into the minor groove of nucleic acids to assist in loading nucleic acids; ② Curcumin may not directly interact with nucleic acids.

[0500] Example 1.3.1: Metal ion is Fe 3+ The proportion of the components of the metal-phospholipid complex

[0501] DSPC, curcumin, and FeCl3 in Example 1.3 were added at different molar ratios (1:1:1, 3:3:2, and 2:2:1). Other steps were the same as in Example 1.3 to prepare different eGFP-mRNA@MPPs, and their nucleic acid encapsulation efficiencies were tested.

[0502] Result analysis: As shown in Table 1-1, when the molar ratio of DSPC, curcumin, and FeCl3 was 1:1:1, the eGFP-mRNA encapsulation efficiency of the prepared metal-phospholipid complex particles was 87%; when the molar ratio of DSPC, curcumin, and FeCl3 was 3:3:2, the eGFP-mRNA encapsulation efficiency of the prepared metal-phospholipid complex particles was 70%; when the molar ratio of DSPC, curcumin, and FeCl3 was 2:2:1, the eGFP-mRNA encapsulation efficiency of the prepared metal-phospholipid complex particles was 60%. In the metal-phospholipid complex particles, Fe 3+ The function of Fe is to connect phospholipid complexes with nucleic acids. 3+ Since there are a maximum of three complexation sites, the molar ratio of DSPC, curcumin, and FeCl₃ in the drug-lipid particles should be 1:1:1 to maximize nucleic acid loading within the metal-phospholipid complex. Experimental results also confirmed that a 1:1:1 molar ratio of DSPC, curcumin, and FeCl₃ resulted in the highest eGFP-mRNA loading efficiency in the metal-phospholipid complex particles. Nucleic acid loading rates exceeded 60% when the molar ratios of DSPC, curcumin, and FeCl₃ ranged from 1:1:1 to 2:2:1.

[0503] Table 1-1 Metal ions are Fe 3+ Molar ratio of components of metal-phospholipid complex and function of metal-phospholipid complex particles prepared therefrom

[0504] Example 1.4.1: Metal ion is Al 3+The proportion of the components of the metal-phospholipid complex

[0505] DSPC, curcumin, and Al(NO3)3·9H2O in Example 1.4 were added at different molar ratios (1:1:1, 3:3:2, and 2:2:1). Other steps were the same as in Example 1.4 to prepare different eGFP-mRNA@MPPs, and their nucleic acid encapsulation efficiencies were tested.

[0506] Result analysis: As shown in Table 1-2, when the molar ratio of DSPC, curcumin, and Al(NO3)3·9H2O was 1:1:1, the eGFP-mRNA encapsulation efficiency of the prepared metal-phospholipid complex particles was 92%; when the molar ratio of DSPC, curcumin, and Al(NO3)3·9H2O was 3:3:2, the eGFP-mRNA encapsulation efficiency of the prepared metal-phospholipid complex particles was 72%; when the molar ratio of DSPC, curcumin, and Al(NO3)3·9H2O was 2:2:1, the eGFP-mRNA encapsulation efficiency of the prepared metal-phospholipid complex particles was 58%. In the metal-phospholipid complex particles, Al 3+ The function of Al is to connect the phospholipid complex with the nucleic acid. 3+ Since there are at most three complexation sites, the molar ratio of DSPC, curcumin, and Al(NO₃)₃·9H₂O in the drug-lipid particles should be 1:1:1 to maximize nucleic acid loading within the metal-phospholipid complex. Experimental results also confirmed that a 1:1:1 molar ratio of DSPC, curcumin, and Al(NO₃)₃·9H₂O resulted in the highest eGFP-mRNA loading efficiency within the metal-phospholipid complex. Nucleic acid loading rates exceeded 58% within the metal-phospholipid complex particles when the molar ratios of DSPC, curcumin, and Al(NO₃)₃·9H₂O ranged from 1:1:1 to 2:2:1.

[0507] Table 1-2 Metal ions are Al 3+ Molar ratio of components of metal-phospholipid complex and function of metal-phospholipid complex particles prepared therefrom

[0508] Example 1.5.1: Metal ion is Mg 2+ The proportion of the components of the metal-phospholipid complex

[0509] DSPC, curcumin, and MgCl2 in Example 1.5 were added at different molar ratios (1:1:1, 3:3:2, and 2:2:1). Other steps were the same as in Example 1.5 to prepare different eGFP-mRNA@MPPs, and their nucleic acid encapsulation efficiencies were tested.

[0510] Results: As shown in Tables 1-3, when the molar ratio of DSPC, curcumin, and MgCl2 was 1:1:1, the eGFP-mRNA encapsulation efficiency of the prepared metal-phospholipid complex particles reached 93.9%. When the molar ratio of DSPC, curcumin, and MgCl2 was 3:3:2, the eGFP-mRNA encapsulation efficiency of the prepared metal-phospholipid complex particles reached 72.6%. The function of MgCl2 in the metal-phospholipid complex particles is to connect the phospholipid complex with the nucleic acid. Each MgCl2 has a maximum of two complexation sites. Therefore, the molar ratio of DSPC, curcumin, and MgCl2 in the drug-lipid particles should be 1:1:1 to maximize nucleic acid encapsulation. Experimental results also confirmed that the metal-phospholipid complex particles prepared with a molar ratio of DSPC, curcumin, and MgCl2 of 1:1:1 achieved the highest eGFP-mRNA encapsulation efficiency. When the molar ratio of DSPC, curcumin, and MgCl2 ranges from 1:1:1 to 3:3:2, the nucleic acid encapsulation efficiency of the metal-phospholipid complex particles is above 72.6%.

[0511] Table 1-3 Metal ions are Mg 2+ Molar ratio of components of metal-phospholipid complex and function of metal-phospholipid complex particles prepared therefrom

[0512] Example 1.3.2: Metal ion is Fe 3+ The ratio of metal-phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000 and cholesterol (CHOL) in the preparation of metal-phospholipid complex particles

[0513] Compared with Example 1.3, the metal-phospholipid complex (metal ion is Fe 3+ ), distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) in proportions as shown in Tables 1-4, and the other conditions were the same.

[0514] Analysis of results: As shown in Tables 1-4, when the proportion of metal-phospholipid complexes is in the range of (10-40)%, the proportion of DSPC is in the range of (0-40)%, the proportion of CHOL is in the range of (35-75)%, and the proportion of DSPE-PEG2000 is in the range of (2-10)%, the particle size of the metal-phospholipid complex particles is in the range of 50-400 nm, the surface potential is in the range of -10-10 mV, the in vitro stability is >3 days, the mRNA encapsulation rate is >50%, and the positive expression rate of eGFP protein is above 70%. Among them, when the metal-phospholipid complex accounts for 15%, distearoylphosphatidylcholine (DSPC) accounts for 35%, cholesterol (CHOL) accounts for 46% and DSPE-PEG2000 accounts for 4%, the performance of the metal-phospholipid complex particles is optimal, that is, the particle size is in the range of 110nm, the surface potential is in the range of -2.04mV, the in vitro stability is >7 days, the mRNA encapsulation rate is 87%, and the positive expression rate of eGFP protein is 97%. Because mRNA@MPP mainly relies on metal-phospholipid complexes to adsorb nucleic acids, the proportion of metal-phospholipid complexes cannot be too low; when the DSPC content is in the range of 0-40%, the stability of its nanoparticles is within an acceptable range. When the DSPC content is 0%, the stability of its nanoparticles can be maintained because the metal-phospholipid complex contains DSPC; the role of DSPE-PEG2000 is to prevent nanoparticle aggregation and increase circulation time in the body. Its performance will be better when its content is in the range of 2-10%; the role of CHOL is to enhance the fluidity of nanoparticles, and maintaining a certain content is beneficial to the stability of nanoparticles.

[0515] The above results suggest that when the proportion of metal-phospholipid complexes is in the range of (10-40)%, when the proportion of DSPC is in the range of (0-40)%, when the proportion of CHOL is in the range of (35-75)%, and when the proportion of DSPE-PEG2000 is in the range of (2-10)%, mRNA@MPP has better drug loading performance.

[0516] Table 1-4 Metal ions are Fe 3+ Performance test of eGFP-mRNA@MPP with different component ratios

[0517] Example 1.4.2: Metal ion is Al 3+ The ratio of metal-phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000 and cholesterol (CHOL) in the preparation of metal-phospholipid complex particles

[0518] Compared with Example 1.4, the metal-phospholipid complex (metal ion is Al 3+), distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) in proportions as shown in Table 1-5, and the other conditions were the same.

[0519] Result analysis: As shown in Table 1-5, when the metal-phospholipid complex (metal ion is Al 3+ When the metal-phospholipid complex accounted for 7%, distearoylphosphatidylcholine (DSPC) accounted for 34%, cholesterol (CHOL) accounted for 56%, and DSPE-PEG2000 accounted for 3%, the drug-lipid particle performance was optimal, with a particle size of 100 nm, a surface potential of -1.57 mV, in vitro stability of more than 7 days, an mRNA entrapment efficiency of 92%, and an eGFP protein positive expression rate of 98%. Because mRNA@MPP mainly relies on metal-phospholipid complexes to adsorb nucleic acids, the proportion of metal-phospholipid complexes cannot be too low; when the DSPC content is in the range of 0-51%, the stability of its nanoparticles is within an acceptable range. When the DSPC content is 0%, the stability of its nanoparticles can be maintained because the metal-phospholipid complex contains DSPC; the role of DSPE-PEG2000 is to prevent nanoparticle aggregation and increase circulation time in the body. Its performance will be better when its content is in the range of 2-10%; the role of CHOL is to enhance the fluidity of nanoparticles, and maintaining a certain content is beneficial to the stability of nanoparticles.

[0520] The above results suggest that metal-phospholipid complexes (metal ions are Al 3+ ) accounts for (5-50)%, when DSPC accounts for (0-51)%, when CHOL accounts for (15-80)%, when DSPE-PEG2000 accounts for (2-10)%, mRNA@MPP has better drug loading performance.

[0521] Table 1-5 Metal ions are Al 3+ eGFP-mRNA@MPP(Al 3+ ) Performance testing

[0522] Example 1.5.2: Metal ion is Mg 2+The ratio of metal-phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000 and cholesterol (CHOL) in the preparation of metal-phospholipid complex particles

[0523] Compared with Example 1.5, the metal-phospholipid complex (metal ion is Mg 2+ ), distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) in proportions as shown in Table 1-6, and the other conditions were the same.

[0524] Analysis of results: As shown in Tables 1-6, when the proportion of metal-phospholipid complexes is in the range of (5-40)%, the proportion of DSPC is in the range of (0-50)%, the range of CHOL is in the range of (25-75)%, and the proportion of DSPE-PEG2000 is in the range of (1-10)%, the particle size of the metal-phospholipid complex particles is in the range of 50-400 nm, the surface potential is in the range of -10-10 mV, the in vitro stability is >3 days, the mRNA encapsulation rate is >50%, and the positive expression rate of eGFP protein is above 70%. Among them, when the metal-phospholipid complex accounted for 15%, distearoylphosphatidylcholine (DSPC) accounted for 40%, cholesterol (CHOL) accounted for 43.2% and DSPE-PEG2000 accounted for 1.8%, the performance of the metal-phospholipid complex particles was optimal, that is, the particle size was in the range of 115nm, the surface potential was in the range of -2.9mV, the in vitro stability was >7 days, the mRNA encapsulation rate was 93.9%, and the positive expression rate of eGFP protein was 98%. Because mRNA@MPP mainly relies on metal-phospholipid complexes to adsorb nucleic acids, the proportion of metal-phospholipid complexes cannot be too low; when the DSPC content is in the range of 0-50%, the stability of its nanoparticles is within an acceptable range. When the DSPC content is 0%, the stability of its nanoparticles can be maintained because the metal-phospholipid complex contains DSPC; the role of DSPE-PEG2000 is to prevent nanoparticle aggregation and increase circulation time in the body. Its performance will be better when its content is in the range of 1-10%; the role of CHOL is to enhance the fluidity of nanoparticles, and maintaining a certain content is beneficial to the stability of nanoparticles.

[0525] The above results suggest that when the proportion of metal-phospholipid complexes is in the range of (5-40)%, when the proportion of DSPC is in the range of (0-50)%, when the proportion of CHOL is in the range of (25-75)%, and when the proportion of DSPE-PEG2000 is in the range of (1-10)%, mRNA@MPP has better drug loading performance.

[0526] Table 1-6 Metal ions are Mg 2+ Performance test of eGFP-mRNA@MPP with different component ratios

[0527] Example 1.3.3 Preparation of non-cationic lipids or non-ionizable lipids in eGFP-mRNA@MPP

[0528] Example 1.3.3.1 Preparation of eGFP-mRNA@MPP(Fe 3+ ) in non-cationic lipids or non-ionizable lipid species

[0529] Compared with Example 1.3, the replacement of distearoylphosphatidylcholine (DSPC) is shown in Table 1-7, and the other conditions are the same.

[0530] Results analysis: To explore the eGFP-mRNA@MPP(Fe 3+ ) can be replaced by other non-cationic lipids or non-ionizable lipids. We selected three other non-cationic lipids or non-ionizable lipids, namely DSPE, DSPA and DSPG, to replace DSPC, and tested the particle size, surface potential, stability and mRNA encapsulation efficiency to prove that eGFP-mRNA@MPP(Fe 3+ ) can be replaced by other non-cationic lipids or non-ionizable lipids, and its function after replacement is equivalent to that of eGFP-mRNA@MPP(Fe 3+ ) (Table 1-7). Because non-cationic lipids or non-ionizable lipids DSPC in eGFP-mRNA@MPP(Fe 3+ ) is to make the liposome membrane fusion better, more stable and less toxic, while other non-cationic lipids or non-ionizable lipids also have the function of making the liposome membrane fusion better, more stable and less toxic. Therefore, DSPC in drug-lipid particles can be replaced by other non-cationic lipids or non-ionizable lipids except metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation, and its efficacy will not be affected.

[0531] The structural formulas of three non-cationic lipids or non-ionizable lipids (DSPE, DSPA, and DSPG) are shown below. DSPE (Formula 47) DSPA (Equation 48) DSPG (Equation 49)

[0532] Table 1-7 eGFP-mRNA@MPP(Fe) containing different types of non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation 3+ ) performance

[0533] Example 1.3.3.2, Preparation of eGFP-mRNA@MPP(Al 3+ ) in non-cationic lipids or non-ionizable lipid species

[0534] Compared with Example 1.4, the replacement of distearoylphosphatidylcholine (DSPC) is shown in Table 1-8, and the other conditions are the same.

[0535] Results analysis: To explore the eGFP-mRNA@MPP(Al 3+ ) can be replaced by other non-cationic lipids or non-ionizable lipids. We selected three other non-cationic lipids or non-ionizable lipids, namely DSPE (Formula 47), DSPA (Formula 48) and DSPG (Formula 49) to replace DSPC, and tested the particle size, surface potential, stability and mRNA encapsulation efficiency to prove that eGFP-mRNA@MPP(Al 3+ ) can be replaced by other non-cationic lipids or non-ionizable lipids, and its function after replacement is equivalent to that of eGFP-mRNA@MPP(Al 3+ ) Efficacy Table 1-8 (metal ions are Al 3+ Because non-cationic lipids or non-ionizable lipids DSPC in eGFP-mRNA@MPP (Al 3+ ) is to make the liposome membrane fusion better, more stable and less toxic, while other non-cationic lipids or non-ionizable lipids also have the function of making the liposome membrane fusion better, more stable and less toxic. Therefore, DSPC in drug-lipid particles can be replaced by other non-cationic lipids or non-ionizable lipids except metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation, and its efficacy will not be affected.

[0536] Table 1-8 eGFP-mRNA@MPP (Al) containing different types of non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation 3+ ) performance

[0537] Example 1.3.3.3 Preparation of eGFP-mRNA@MPP(Mg 2+ ) in non-cationic lipids or non-ionizable lipid species

[0538] Compared with Example 1.5, the replacement of distearoylphosphatidylcholine (DSPC) is shown in Table 1-9, and the other conditions are the same.

[0539] Results analysis: To explore the eGFP-mRNA@MPP(Mg 2+) can be replaced by other non-cationic lipids or non-ionizable lipids. We selected three other non-cationic lipids or non-ionizable lipids, namely DSPE (Formula 47), DSPA (Formula 48) and DSPG (Formula 49) to replace DSPC, and tested the particle size, surface potential, stability and mRNA encapsulation efficiency to prove that eGFP-mRNA@MPP(Mg 2+ ) can be replaced by other non-cationic lipids or non-ionizable lipids, and its function after replacement is equivalent to that of eGFP-mRNA@MPP(Mg 2+ ) (Table 1-9). Because non-cationic lipids or non-ionizable lipids DSPC in eGFP-mRNA@MPP(Mg 2+ ) is to make the liposome membrane fusion better, more stable and less toxic, while other non-cationic lipids or non-ionizable lipids also have the function of making the liposome membrane fusion better, more stable and less toxic. Therefore, DSPC in drug-lipid particles can be replaced by other non-cationic lipids or non-ionizable lipids except metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation, and its efficacy will not be affected.

[0540] Table 1-9 eGFP-mRNA@MPP(Mg) containing different types of non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation 2+ ) performance

[0541] Example 1.3.4. Preparation of conjugated lipid species for inhibiting particle aggregation in eGFP-mRNA@MPP

[0542] Example 1.3.4.1. Preparation of eGFP-mRNA@MPP(Fe 3+ ) Conjugated lipid species that inhibit particle aggregation

[0543] Compared with Example 1.3, the substitution of DSPE-PEG2000 (Formula 53) is shown in Table 1-10 (metal ion is Fe 3+ ), and the other conditions were the same. Three other conjugated lipids that inhibit particle aggregation are DSPE-PEG700 (Formula 50), DSPE-PEG5000 (Formula 52), and DSPE-PEG1000 (Formula 51).

[0544] Results analysis: To explore the eGFP-mRNA@MPP(Fe 3+) can be replaced by other conjugated lipids that inhibit particle aggregation. We selected three other conjugated lipids that inhibit particle aggregation, namely DSPE-PEG700, DSPE-PEG5000 and DSPE-PEG1000, to replace DSPE-PEG2000, and tested the particle size, surface potential, stability and mRNA packaging efficiency to prove that eGFP-mRNA@MPP(Fe 3+ ) can be replaced by other conjugated lipids that inhibit particle aggregation, and its function after replacement is equivalent to that of eGFP-mRNA@MPP(Fe 3+ ) (Table 1-10). 3+ ) is mainly used to inhibit aggregation, and other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation. Therefore, eGFP-mRNA@MPP(Fe 3+ ) can be replaced by other conjugated lipids that inhibit particle aggregation without affecting its efficacy.

[0545] Table 1-10 eGFP-mRNA@MPP(Fe) containing different types of conjugated lipids that inhibit particle aggregation 3+ ) performance

[0546] Example 1.3.4.2, Preparation of eGFP-mRNA@MPP(Al 3+ ) Conjugated lipid species that inhibit particle aggregation

[0547] Compared with Example 1.4, the substitution of DSPE-PEG2000 (Formula 53) is shown in Table 1-11 (metal ion is Al 3+ ), and the other conditions were the same. Three other conjugated lipids that inhibit particle aggregation are DSPE-PEG700 (Formula 50), DSPE-PEG5000 (Formula 52), and DSPE-PEG1000 (Formula 51).

[0548] Results analysis: To explore the eGFP-mRNA@MPP(Al 3+ ) can be replaced by other conjugated lipids that inhibit particle aggregation. We selected three other conjugated lipids that inhibit particle aggregation, namely DSPE-PEG700, DSPE-PEG5000 and DSPE-PEG1000, to replace DSPE-PEG2000, and tested the particle size, surface potential, stability and mRNA encapsulation efficiency to prove that eGFP-mRNA@MPP(Al 3+) can be replaced by other conjugated lipids that inhibit particle aggregation, and its function after replacement is equivalent to that of eGFP-mRNA@MPP (Al 3+ ) (Table 1-11). Because DSPE-PEG2000 is effective in eGFP-mRNA@MPP(Al 3+ ) is mainly used to inhibit aggregation, and other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation, so eGFP-mRNA@MPP(Al 3+ ) can be replaced by other conjugated lipids that inhibit particle aggregation without affecting its efficacy.

[0549] Table 1-11 eGFP-mRNA@MPP (Al) containing different types of conjugated lipids that inhibit particle aggregation 3+ ) performance

[0550] Example 1.3.4.3, Preparation of eGFP-mRNA@MPP(Mg 2+ ) Conjugated lipid species that inhibit particle aggregation

[0551] Compared with Example 1.5, the substitution of DSPE-PEG2000 (Formula 53) is shown in Table 1-12 (metal ion is Mg 2+ ), and the other conditions were the same. Three other conjugated lipids that inhibit particle aggregation are DSPE-PEG700 (Formula 50), DSPE-PEG5000 (Formula 52), and DSPE-PEG1000 (Formula 51).

[0552] Results: To explore whether DSPE-PEG2000 in eGFP-mRNA@MPP can be replaced by other conjugated lipids that inhibit particle aggregation, we selected three other conjugated lipids that inhibit particle aggregation, namely DSPE-PEG700, DSPE-PEG5000 and DSPE-PEG1000 to replace DSPE-PEG2000, and tested the particle size, surface potential, stability and mRNA encapsulation efficiency to prove that eGFP-mRNA@MPP(Mg 2+ ) can be replaced by other conjugated lipids that inhibit particle aggregation, and its function after replacement is equivalent to that of eGFP-mRNA@MPP(Mg 2+ ) (Table 1-12). Because DSPE-PEG2000 is effective in eGFP-mRNA@MPP(Mg 2+) is mainly used to inhibit aggregation, and other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation. Therefore, eGFP-mRNA@MPP(Mg 2+ ) can be replaced by other conjugated lipids that inhibit particle aggregation without affecting its efficacy.

[0553] Table 1-12 eGFP-mRNA@MPP(Mg) containing different types of conjugated lipids that inhibit particle aggregation 2+ ) performance

[0554] Example 1.3.5 Preparation and Effect Characterization of mRNA@MPP

[0555] Example 1.3.5.1, mRNA@MPP(Fe 3+ ) preparation and effect characterization

[0556] The mRNA in Example 1.3 was replaced with the other two mRNAs, and three mRNA@MPP (Fe 3+ ). The three different mRNA sequences are: ① the mRNA sequence encoding the fluorescent protein eGFP is SEQ ID NO.1 (720nt); ② the mRNA sequence encoding the receptor binding domain (RBD) of the novel 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)-metal-phospholipid complex particles is the same as that in Example 1.3, and eGFP-mRNA@MPP(Fe 3+ )、RBD-mRNA@MPP(Fe 3+ )、NY-ESO-1-mRNA@MPP(Fe 3+ ).

[0557] eGFP-mRNA@MPP(Fe 3+) 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 MPP. After 48 hours, the cell suspension was collected, and the percentage of eGFP-positive cells was detected by flow cytometry. The results are shown in Figure 1-1; RBD-mRNA@MPP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA), and the control group was incubated with MPP. After 24 hours, the supernatant was centrifuged and frozen at -20°C for later use. The expression level of the new coronavirus antigen RBD protein on the cells was detected using a commercially available new coronavirus antigen RBD ELISA detection kit. The results are shown in Figure 1-2.

[0558] ELISA method for detecting RBD expression level:

[0559] 1. Sample collection: Place whole blood samples at room temperature for 2 hours, centrifuge at 1000 × g for 20 minutes, and collect the supernatant;

[0560] 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.

[0561] 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.

[0562] 4. Discard the liquid in the wells and wash the plate 3 times, soaking for 1-2 minutes each time;

[0563] 5. Add 100 μL of prepared streptavidin HRP working solution to each well, mix well, and incubate at 37°C for 45 minutes;

[0564] 6. Discard the liquid in the wells and wash the plate 3 times, soaking for 1-2 minutes each time;

[0565] 7. Add 100 μL of TMB substrate solution (TMB) to each well and incubate at 37°C in the dark for 15 minutes;

[0566] 8. Add 100 μL of stop solution to each well to terminate the reaction;

[0567] 9. Measure the optical density (OD value) of each well at a wavelength of 450 nm.

[0568] Data analysis: Draw a standard curve with the concentration of the standard as the horizontal axis and the OD value as the vertical axis.

[0569] The experimental animals were randomly divided into two groups (experimental group and control group), with 5 animals in each group. 3+) The animal model was BALB / c mice. Each mouse received the first intramuscular administration on the first day and the second intramuscular administration on the 14th day. The experimental group was injected with RBD-mRNA@MPP(Fe 3+ ), and the control group was injected with metal-phospholipid complex particles (MPP) without mRNA. The dose of each administration was 100 μL, in which RBD-mRNA@MPP(Fe 3+ ) formulation contains 30 mg of mRNA. Blood was collected from mice 28 days after the first dose, and serum was separated and serially diluted. Total IgG antibodies against the RBD of the novel coronavirus S1 subunit produced in the mice were detected using a commercially available ELISA kit. The results are shown in Figures 1-3.

[0570] NY-ESO-1-mRNA@MPP(Fe 3+ The animal model of the experiment was C57BL / 6 mice. Each mouse was intramuscularly administered four times on days 1, 7, 14, and 21. The experimental group was injected with NY-ESO-1-mRNA@MPP(Fe 3+ ), and the control group was injected with metal-phospholipid complex particles (MPP) without mRNA. The dose of each administration was 100 μL, of which NY-ESO-1-mRNA@MPP(Fe 3+ ) formulation contained 30 mg of mRNA. Twenty-eight days after the first administration, mouse blood was collected, serum was separated and serially diluted, and total anti-NY-ESO-1 IgG antibodies produced in the mice were detected by ELISA. The results are shown in Figures 1-4.

[0571] Method for detecting total anti-NY-ESO-1 IgG antibodies in mice:

[0572] Preparation of reagents used in ELISA method:

[0573] 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.

[0574] 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.

[0575] 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.

[0576] 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.

[0577] 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.

[0578] 6. Stop solution: 2M H2SO4: concentrated sulfuric acid 55.5mL, add DDW to 500mL.

[0579] ELISA method was used to determine the titer of antibodies in mouse serum:

[0580] 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.

[0581] 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°C for 2 hours.

[0582] 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.

[0583] 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.

[0584] In the case of RBD-mRNA@MPP(Fe 3+) 28 days after the inoculation, the spleens of normal mice were collected and prepared into single cell suspensions under sterile conditions. 100,000 spleen cells / well were plated in a cell plate, and 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 1-5. 3+ ) 28 days after inoculation, spleens of normal mice were harvested and prepared into single-cell suspensions under sterile conditions. 100,000 spleen cells were plated in a cell plate at a density of 10 mg / mL. NY-ESO-1 protein was added at a final concentration of 10 mg / mL and cultured for 48 h. The supernatant was removed by centrifugation, and the expression levels of IFN-γ, IL-2, and TNF-α were determined using ELISA kits. The results are shown in Figures 1-6.

[0585] Result analysis: As shown in Figure 1-1, eGFP-mRNA@MPP(Fe 3+ ) experimental group eGFP positive cell rate was 97.7%, while MPP (Fe 3+ ) No eGFP signal was detected in the control group; As shown in Figure 1-2, the RBD protein encoded by the MPP-encapsulated RBD-mRNA was 193.3 ng / mL in the supernatant of 293T cells, while the empty vector MPP (Fe 3+ The RBD protein content in the supernatant of 293T cells was 0. The results suggest that mRNA-MPP (Fe 3+ ) can encapsulate and deliver any mRNA and directly encode polypeptides in cells. As shown in Figures 1-3 and 1-4, RBD-mRNA@MPP(Fe 3+ ) and NY-ESO-1-mRNA@MPP(Fe 3+ ) can effectively induce humoral immunity in mice and produce high levels of antigen-specific binding antibodies. 3+ ) treatment group mice IgG antibody titer reached 117268.8; NY-ESO-1-mRNA@MPP(Fe 3+ ) treatment group mice IgG antibody titer reached 5319.52. As shown in Figure 1-5 and Figure 1-6, RBD-mRNA@MPP(Fe 3+ ) and NY-ESO-1-mRNA@MPP(Fe 3+ ) can effectively induce cellular immunity in mice, namely activate immune cells and produce a large number of cytokines. 3+) made the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 252.8 pg / mL, 207.6 pg / mL, and 56.6 pg / mL, respectively; NY-ESO-1-mRNA@MPP(Fe 3+ ) made the expression levels of cytokines IFN-γ, IL-2, and TNF-α reach 70.79 pg / mL, 75.29 pg / mL, and 75.27 pg / mL, respectively. 3+ ) can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), and then effectively inducing the humoral immunity and cellular immunity of 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.

[0586] Example 1.3.5.2: Metal ion is Al 3+ Preparation and effect characterization of mRNA@MPP

[0587] The mRNA in Example 1.4 was replaced with the other two mRNAs, and three mRNA@MPP (Al 3+ ). For specific mRNA information and experimental procedures, refer to Example 1.3.5.1.

[0588] Result analysis: As shown in Figure 1-1-2, eGFP-mRNA@MPP(Al 3+ ) experimental group eGFP positive cell rate was 98.02%, while MPP (Al 3+ ) No eGFP signal was detected in the control group; As shown in Figure 1-2-2, MPP (Al 3+ The RBD protein encoded by the RBD-mRNA encapsulated by the mRNA was 212.6 ng / mL in the supernatant of 293T cells, while the RBD protein content in the supernatant of 293T cells transfected with the empty vector MPP was 0. 3+ ) can encapsulate and deliver any mRNA and directly encode polypeptides in cells. As shown in Figure 1-3-2 and Figure 1-4-2, RBD-mRNA@MPP(Al 3+ ) and NY-ESO-1-mRNA@MPP(Al 3+ ) can effectively induce humoral immunity in mice and produce high levels of antigen-specific binding antibodies. 3+ ) treatment group mice IgG antibody titer reached 129113; NY-ESO-1-mRNA@MPP(Al 3+) treatment group mice IgG antibody titer reached 6507.4. As shown in Figure 1-5-2 and Figure 1-6-2, RBD-mRNA@MPP(Al 3+ ) and NY-ESO-1-mRNA@MPP(Al 3+ ) can effectively induce cellular immunity in mice, namely activate immune cells and produce a large number of cytokines. 3+ ) made the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 271.8 pg / mL, 234.6 pg / mL, and 68.4 pg / mL, respectively; NY-ESO-1-mRNA@MPP(Al 3+ ) made the expression levels of cytokines IFN-γ, IL-2, and TNF-α reach 83.8 pg / mL, 98 pg / mL, and 97.8 pg / mL, respectively. 3+ ) can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), and then effectively inducing the humoral immunity and cellular immunity of 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.

[0589] Example 1.3.5.3: Metal ion is Mg 2+ Preparation and effect characterization of mRNA@MPP

[0590] The mRNA in Example 1.5 was replaced with the other two mRNAs, and three mRNA@MPP(Mg 2+ ). For specific mRNA information and experimental procedures, refer to Example 1.3.5.1.

[0591] Result analysis: As shown in Figure 1-1-3, eGFP-mRNA@MPP(Mg 2+ ) experimental group, the eGFP positive cell rate was 98.3%, while MPP (Mg 2+ ) No eGFP signal was detected in the control group; As shown in Figure 1-2-3, MPP (Mg 2+ )-encoded RBD protein in the supernatant of 293T cells was 218.50 ng / mL, while the RBD protein content in the supernatant of 293T cells transfected with empty vector MPP was 0. The results suggest that mRNA-MPP (Mg 2+ ) can encapsulate and deliver any mRNA and directly encode polypeptides in cells. As shown in Figure 1-3-3 and Figure 1-4-3, RBD-mRNA@MPP(Mg 2+ ) and NY-ESO-1-mRNA@MPP(Mg2+ ) can effectively induce humoral immunity in mice and produce high levels of antigen-specific binding antibodies. 2+ ) treatment group, the IgG antibody titer in mice reached 130614.40; NY-ESO-1-mRNA@MPP(Mg 2+ ) treatment group mice IgG antibody titer reached 6842.78. As shown in Figure 1-5-3 and Figure 1-6-3, RBD-mRNA@MPP(Mg 2+ ) and NY-ESO-1-mRNA@MPP(Mg 2+ ) can effectively induce cellular immunity in mice, namely activate immune cells and produce a large number of cytokines. 2+ ) made the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 296.40 pg / mL, 243.33 pg / mL, and 75.30 pg / mL, respectively; NY-ESO-1-mRNA@MPP(Mg 2+ ) made the expression levels of cytokines IFN-γ, IL-2, and TNF-α reach 85.35pg / mL, 98.58pg / mL, and 98.02pg / mL, respectively. The results suggest that mRNA@MPP(Mg 2+ ) can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), and then effectively inducing the humoral immunity and cellular immunity of 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.

[0592] Example 1.3.6. Preparation and Effect of siRNA-loaded Metal-chelated Phospholipid Complex Nanoparticles (siRNA@MPP)

[0593] Example 1.3.6.1: Metal ion is Fe 3+ Preparation and efficacy of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPP)

[0594] The mRNA in Example 1.3 was replaced with siRNA, and three siRNA@MPP(Fe 3+). The genes, sequences and corresponding random control sequences of the three different siRNA targeting genes are: 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 siRNA@MPP is the same as that in Example 1.3.

[0595] 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.

[0596] U251 cells were cultured at 1 × 10 6 After about 24 hours of cell seeding in 6-well plates, each well of cells was inoculated with siRNA@MPP(Fe 3+ ) (wherein the concentration of siRNA was 2 μg / mL) were incubated for 72 hours, the cells were collected, and the total RNA was extracted. The mRNA expression levels of the target genes (Bcl-2, PLK1, Gal-1) were detected by RT-PCR. The expression of siRNA@MPP(Fe 3+ ) ability to silence target genes in cells.

[0597] RT-PCR specific process:

[0598] 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.

[0599] 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.

[0600] 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

[0601] Calculation method of gene silencing efficiency: Calculation method of gene silencing efficiency: 100% - gene expression level of experimental group / gene expression level of control group.

[0602] Results analysis: As shown in Figures 1-7 (“scr siRNA” in the figure is “Scramble siRNA”, which serves as a negative control, the same below), Figures 1-8, and 1-9, three siRNA@MPP(Fe 3+ ) can significantly interfere with their corresponding target genes. 3+ ) achieved an inhibition rate of 76% on the target gene Bcl-2; PLK1-siRNA@MPP(Fe 3+ ) could inhibit the target gene PLK1 by 86%; Gal-1-siRNA@MPP(Fe 3+ ) can inhibit the target gene Gal-1 by 73%. 3+ ) can carry any siRNA for target gene intervention therapy, and play the role of siRNA-loaded drugs, vaccines or other products.

[0603] Example 1.3.6.2: Metal ion is Al 3+ Preparation and effect of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPP)

[0604] The mRNA in Example 1.4 was replaced with siRNA, and three siRNA@MPP (Al 3+ ). For specific siRNA information and experimental procedures, refer to Example 1.3.6.1.

[0605] Result analysis: As shown in Figure 1-7-2, Figure 1-8-2, and Figure 1-9-2, three siRNA@MPP(Al 3+ ) can significantly interfere with their corresponding target genes. 3+ ) achieved an inhibition rate of 81% on the target gene Bcl-2; PLK1-siRNA@MPP(Al 3+ ) can inhibit the target gene PLK1 by 90%; Gal-1-siRNA@MPP(Al 3+ ) can inhibit the target gene Gal-1 by 79%. 3+ ) can carry any siRNA for target gene intervention therapy, and play the role of siRNA-loaded drugs, vaccines or other products.

[0606] Example 1.3.6.3: Metal ion is Mg 2+ Preparation and effect of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPP)

[0607] The mRNA in Example 1.5 was replaced with siRNA, and three siRNA@MPP(Mg 2+) containing different siRNA sequences were prepared according to the method of Example 1.5. 2+ ). For specific siRNA information and experimental procedures, refer to Example 1.3.6.1.

[0608] Result analysis: As shown in Figure 1-7-3, Figure 1-8-3, and Figure 1-9-3, three siRNA@MPP(Mg 2+ ) can significantly interfere with their corresponding target genes. 2+ ) achieved an inhibition rate of 82% on the target gene Bcl-2; PLK1-siRNA@MPP(Mg 2+ ) could inhibit the target gene PLK1 by 92%; Gal-1-siRNA@MPP(Mg 2+ ) can inhibit the target gene Gal-1 by 86%. 2+ ) can carry any siRNA for target gene intervention therapy, and play the role of siRNA-loaded drugs, vaccines or other products.

[0609] Example 1.3.7. Preparation and Effects of ASO-loaded Metal-chelated Phospholipid Complex Nanoparticles (ASO@MPP)

[0610] Example 1.3.7.1: Metal ion is Fe 3+ Preparation and effect of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP)

[0611] The mRNA in Example 1.3 was replaced with ASO, and three ASO@MPP (Fe 3+). The genes, sequences and corresponding random control sequences of the three different ASO targets are: 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 ASO-metal-phospholipid complex particles is the same as that in Example 1.3. Different ASO@MPP (Fe 3+ ) were incubated with different cells: ASO@MPP targeting STAT3 gene was incubated with U251 human glioblastoma cells; ASO@MPP targeting α-syn gene (Fe 3+ ) were incubated with SH-SY5Y human neuroblastoma cells; ASO@MPP(Fe 3+ ) were incubated with Daudi human lymphoma cells. 1×10 6 After about 24 hours of cell seeding in 6-well plates, each well of cells was inoculated with ASO@MPP(Fe 3+ )(wherein the concentration of ASO was 2 μg / mL) were incubated for 48 hours, the cells were collected, and the total RNA of the cells was extracted. The mRNA expression levels of the target genes (STAT3, α-syn, Bcl-2) were detected by RT-PCR technology, and the expression of ASO@MPP(Fe 3+ ) ability to silence target genes in cells.

[0612] 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.

[0613] RT-PCR specific process:

[0614] 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.

[0615] 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.

[0616] 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

[0617] The gene silencing efficiency was calculated as follows: 100% - gene expression level in the experimental group / gene expression level in the control group.

[0618] Results analysis: As shown in Figures 1-10, 1-11, and 1-12, the three ASO@MPP(Fe 3+ ) can significantly interfere with their corresponding target genes, among which STAT3-ASO@MPP(Fe 3+ ) achieved an inhibition rate of 77% on the target gene STAT3; α-syn-ASO@MPP(Fe 3+ ) can inhibit the target gene α-syn by 75%; Bcl-2-ASO@MPP(Fe 3+ ) achieved an inhibition rate of 69% on the target gene Bcl-2. 3+ ) can carry any ASO for target gene intervention therapy, and play the role of ASO-carrying drugs, vaccines or other products.

[0619] Example 1.3.7.2: Metal ion is Al 3+ Preparation and effect of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP)

[0620] The mRNA in Example 1.4 was replaced with ASO, and three ASO@MPP (Al 3+ ). For specific ASO information and experimental procedures, refer to Example 1.3.7.1.

[0621] Result analysis: As shown in Figure 1-10-2, Figure 1-11-2, and Figure 1-12-2, three ASO@MPP (Al 3+ ) can significantly interfere with their corresponding target genes, among which STAT3-ASO@MPP(Al 3+ ) achieved an inhibition rate of 79% on the target gene STAT3; α-syn-ASO@MPP(Al 3+ ) can inhibit the target gene α-syn by 80%; Bcl-2-ASO@MPP(Al 3+ ) achieved an inhibition rate of 74% on the target gene Bcl-2. 3+ ) can carry any ASO for target gene intervention therapy, and play the role of ASO-carrying drugs, vaccines or other products.

[0622] Example 1.3.7.3: Metal ion is Mg 2+Preparation and effect of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP)

[0623] The mRNA in Example 1.5 was replaced with ASO, and three ASO@MPP (Mg2+) containing different ASO sequences were prepared according to the method of Example 1.5. 2+ ). For specific ASO information and experimental procedures, refer to Example 1.3.7.1.

[0624] Result analysis: As shown in Figure 1-10-3, Figure 1-11-3, and Figure 1-12-3, three ASO@MPP(Mg 2+ ) can significantly interfere with their corresponding target genes, among which STAT3-ASO@MPP(Mg 2+ ) achieved an inhibition rate of 82% on the target gene STAT3; α-syn-ASO@MPP(Mg 2+ ) can inhibit the target gene α-syn by 84%; Bcl-2-ASO@MPP(Mg 2+ ) can inhibit the target gene Bcl-2 by 78%. 2+ ) can carry any ASO for target gene intervention therapy, and play the role of ASO-carrying drugs, vaccines or other products.

[0625] Example 1.3.8 Preparation of drug (different types of nucleic acids)-metal-phospholipid complex particles and their effects

[0626] Example 1.3.8.1: Metal ion is Fe 3+ Preparation of drug (different types of nucleic acids)-metal-phospholipid complex particles and their effects

[0627] The mRNA in Example 1.3 was replaced by double-stranded RNA (siRNA), single-stranded RNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively. The different types of nucleic acid sequences are: ① The sequence of double-stranded RNA (Bcl-2-siRNA) is SEQ ID NO.4 (antisense chain) and SEQ ID No.21 (sense chain) (19 bp), and its random control sequence is SEQ ID NO.5 (antisense chain) and SEQ ID No.22 (sense chain) (19 bp); ② The sequence of single-stranded RNA (STAT3-ASO) is SEQ ID NO.10 (17 nt), and its random control sequence is SEQ ID NO.11 (18 nt); ③ The sequence of single-stranded RNA (mRNA encoding wild-type SARS-CoV-2 S protein) is SEQ ID No.16 (3822 nt); ④ 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). The drug-metal-phospholipid complex particles (Bcl-2-siRNA@MPP(Fe 3+ )、STAT3-ASO@MPP(Fe 3+ )、S-mRNA@MPP(Fe 3+ )、dsDNA@MPP(Fe 3+ )、ssDNA@MPP(Fe 3+ )), the preparation process of the remaining drug-lipid particles is the same as that in Example 1.3.

[0628] U251 cells were cultured at 1 × 10 5 After about 24 hours of cell seeding in 12-well plates, each well of cells was inoculated with siRNA@MPP(Fe 3+ )(wherein the concentration of siRNA was 2 μg / mL) or ASO@MPP(Fe 3+ ) (wherein the concentration of ASO was 2 μg / mL) were incubated for 72 hours, the cells were collected, and the total RNA of the cells was extracted. The mRNA expression levels of the target genes (Bcl-2, STAT3) were detected by RT-PCR technology, and the expression of siRNA@MPP(Fe 3+ ) or ASO@MPP(Fe 3+ ) ability to silence cellular target genes, as shown in Figures 1-7 of Example 1.3.6 and Figures 1-10 of Example 1.3.7.

[0629] S-mRNA@MPP(Fe 3+ ) was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of mRNA contained), and the control group was incubated with MPP (Fe 3+ ) incubated for 24 hours, centrifuged, and the supernatant was 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 levels of S protein in both the cell supernatant and the cell lysate were detected using a commercially available SARS-CoV-2 S protein ELISA kit. The results are shown in Figures 1-13.

[0630] ds-DNA@MPP(Fe 3+ ) A549 lung cancer cells were incubated with 100 nM of the drug-lipid particle (the concentration of the DNA contained) for 2 hours, and then the remaining drug-lipid particles were removed. The cells were washed twice with PBS, and the nuclei were stained with Hochest 33342 dye for 3 minutes, after which the dye was removed. The cells were washed twice with PBS and observed using a high-content imaging system. The efficiency of DNA transfection with the drug-lipid particle was calculated. The results are shown in Figures 1-14.

[0631] ss-DNA@MPP(Fe 3+ ) After incubating HT22 mouse hippocampal neurons with 200 nM of the drug-lipid particles (the concentration of the DNA contained) for 2 hours, the remaining drug-lipid particles were removed. After washing the cells twice with PBS, the cells were observed using a high-content imaging system, and the efficiency of drug-lipid particle transfection into DNA was calculated. The results are shown in Figures 1-14.

[0632] The culture method of human glioblastoma U251 cells is the same as that in Example 1.3.6.

[0633] The culture method of 293T cells is the same as that in Example 1.3.5.

[0634] Culture method of HT22 mouse hippocampal neurons: culture in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0635] The RT-PCR method is the same as in Example 1.3.6.

[0636] ELISA detection of S protein expression level: replace the "anti-RBD antibody working solution" in the ELISA method for detecting RBD in Example 1.3.5 with "anti-S protein antibody working solution", and the remaining steps are the same as Example 1.3.5.

[0637] The calculation method of gene silencing efficiency is the same as Example 1.3.6.

[0638] To calculate transfection efficiency: Use a high-content imaging system to randomly select 3-5 fields of view. Obtain cell morphology under a standard light source, fluorescence signals under excitation / emission light of 550nm / 570nm (excitation light of the fluorescent dye Cy3 that labels DNA), and fluorescence signals under excitation / emission light of 352nm / 461nm (excitation light of the fluorescent dye Hoechst33342 that labels the cell nucleus) in the same field of view (results are shown in Figure 1-15). Calculate the ratio of cells with Cy3 fluorescence signals in the randomly selected field of view to the number of cells with Hoechst33342 fluorescence signals in the same field of view, which is the transfection efficiency.

[0639] Results analysis: As shown in Figures 1-7 of Example 1.3.6, drug (double-stranded RNA)-metal-phospholipid complex particles (Bcl-2-siRNA@MPP(Fe 3+ )) The inhibition rate of the target gene Bcl-2 reached 76%; as shown in Figure 1-10 of Example 1.3.7, the drug (single-stranded RNA)-metal-phospholipid complex particles (STAT3-ASO@MPP(Fe 3+ )) achieved an inhibition rate of 77% on the target gene STAT3; as shown in Figure 1-13, drug (single-stranded RNA)-metal-phospholipid complex particles (S-mRNA@MPP(Fe 3+ The S protein expression level in the supernatant of 293T cells transfected with MPP was 161.3 ng / mL, while the S protein content in the supernatant of 293T cells transfected with empty vector MPP was 0; drug (double-stranded DNA)-metal-phospholipid complex particles (dsDNA@MPP(Fe 3+ )) The efficiency of transfection of double-stranded DNA into cells was 100% (Figure 1-14); drug (single-stranded DNA)-metal-phospholipid complex particles (ssDNA@MPP(Fe 3+ The efficiency of transfection of single-stranded DNA into cells was 100% (Figures 1-14). These results suggest that the drug-metal-phospholipid complex particles can encapsulate and function with any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA), with the length of the nucleic acids ranging from 16 to 3822 nt.

[0640] Example 1.3.8.2: Preparation of Metal Ions: Al 3+ Drug (different types of nucleic acids)-metal-phospholipid complex particles and their effects

[0641] The mRNA in Example 1.4 was replaced with double-stranded RNA (siRNA), single-stranded RNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively. The specific sequences and experimental procedures were as described in Example 1.3.8.1.

[0642] Results analysis: As shown in Figure 1-7-2 of Example 1.3.6.2, drug (double-stranded RNA)-metal-phospholipid complex particles (Bcl-2-siRNA@MPP(Al 3+ )) The inhibition rate of the target gene Bcl-2 reached 81%; as shown in Figure 1-10-2 of Example 1.3.7.2, drug (single-stranded DNA)-metal-phospholipid complex particles (STAT3-ASO@MPP (Al 3+ )) achieved an inhibition rate of 79% on the target gene STAT3; as shown in Figure 1-13-2, drug (single-stranded RNA)-metal-phospholipid complex particles (S-mRNA@MPP(Al 3+ The S protein expression level in the supernatant of 293T cells transfected with MPP was 178.7 ng / mL, while the S protein content in the supernatant of 293T cells transfected with empty vector MPP was 0; drug (double-stranded DNA)-metal-phospholipid complex particles (dsDNA@MPP(Al 3+ )) The efficiency of transfection of double-stranded DNA into cells was 100% (Figure 1-14-2); drug (single-stranded DNA)-metal-phospholipid complex particles (ssDNA@MPP(Al 3+ The efficiency of transfection of single-stranded DNA into cells was 100% (Figure 1-14-2). These results suggest that the drug-metal-phospholipid complex particles can encapsulate and function with any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA), ranging in length from 16 to 3822 nt.

[0643] Example 1.3.8.3: Preparation of Metal Ions as Mg 2+ Drug (different types of nucleic acids)-metal-phospholipid complex particles and their effects

[0644] The mRNA in Example 1.5 was replaced with double-stranded RNA (siRNA), single-stranded RNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively. The specific sequences and experimental procedures were as described in Example 1.3.8.1.

[0645] Results analysis: As shown in Figure 1-7-3 of Example 1.5.6, drug (double-stranded RNA)-metal-phospholipid complex particles (Bcl-2-siRNA@MPP(Mg 2+ )) achieved an inhibition rate of 82% on the target gene Bcl-2; as shown in Figure 1-11-3 of Example 1.5.7, drug (single-stranded RNA)-metal-phospholipid complex particles (STAT3-ASO@MPP(Mg 2+))) The inhibition rate of the target gene STAT3 reached 82%; As shown in Figure 1-13-3, the drug (single-stranded RNA)-metal-phospholipid complex particles (S-mRNA@MPP(Mg 2+ The S protein expression level in the supernatant of 293T cells transfected with the empty vector MPP (Mg 2+ The S protein content in the supernatant of 293T cells was 0; the drug (double-stranded DNA)-metal-phospholipid complex particles (dsDNA@MPP(Mg 2+ The efficiency of transfection of double-stranded DNA into cells was 100% (Figure 1-14-3); drug (single-stranded DNA)-metal-phospholipid complex particles (ssDNA@MPP(Mg 2+ The efficiency of transfection of single-stranded DNA into cells was 100% (Figure 1-14-3). These results suggest that the drug-metal-phospholipid complex particles can encapsulate and function with any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA), ranging in length from 16 to 3822 nt.

[0646] Example 2: Performance Characterization of Drug-Metal-Phospholipid Complex Particles

[0647] Example 2.1 Synthesis and characterization of metal-phospholipid complexes

[0648] Example 2.1.1: Metal ion is Fe 3+ Synthesis and characterization of metal-phospholipid complexes

[0649] The connection between DSPC and curcumin was characterized by differential scanning calorimetry. The following conditions were used: 3-5 mg of the test sample was weighed and the temperature was raised at a rate of 10°C / min over a range of 30°C to 300°C. Curcumin, DSPC, and the curcumin-DSPC complex were scanned separately. Curcumin-DSPC complex data were plotted based on the obtained data, as shown in Figure 2-1. The curves show that curcumin has a lattice structure, with a distinct melting peak at 185°C. DSPC is a mixture, with multiple depressions on the curve, likely due to thermal variations in the different components under different temperature conditions. The curcumin-DSPC complex exhibits no peaks near its melting peak, essentially forming a straight line. This indicates that curcumin and DSPC bind to each other in an amorphous form, confirming the successful preparation of the complex.

[0650] Phospholipid complex with Fe 3+ The connection is characterized by spectrophotometry: As shown in Figure 2-2, the phospholipid complex (CUR-DSPC) and Fe 3+After binding, the maximum absorption wavelength shifted from 420nm to 375nm, and the conjugated structure of the phospholipid complex changed, proving that Fe 3+ Successfully complexed with curcumin.

[0651] Example 2.1.2: Metal ion is Al 3+ Synthesis and characterization of metal-phospholipid complexes

[0652] The difference between this embodiment and embodiment 2.1.1 is that the phospholipid complex and Al 3+ The connection is characterized by spectrophotometry: As shown in Figure 2-2-2, the phospholipid complex (CUR-DSPC) and Al 3+ After binding, the maximum absorption wavelength shifted from 420nm to 433nm, and the conjugated structure of the phospholipid complex changed, proving that Al 3+ Successfully complexed with curcumin.

[0653] Example 2.1.3: Metal ion is Mg 2+ Synthesis and characterization of metal-phospholipid complexes

[0654] The difference between this embodiment and embodiment 2.1.1 is that the phospholipid complex and Mg 2+ The connection is characterized by spectrophotometry: As shown in Figure 2-2-3, the phospholipid complex (CUR-DSPC) and Mg 2+ After binding, the maximum absorption peak shifted from 426nm to 420nm, and the conjugated structure of the phospholipid complex changed, proving that Mg 2+ Successfully complexed with curcumin.

[0655] Example 2.2: Metal ion is Fe 3+ At low pH, Fe 3+ Characterization of shedding from metal-phospholipid complexes

[0656] The phospholipid complex in the metal-phospholipid complex binds Fe through coordination bonds 3+ Under the low pH conditions of lysosomes, phospholipid complexes and Fe 3+ The coordination bonds between them will be protonated (absorb hydrogen ions) and broken. 3+ Indeed, it is through the above mechanism that the metal-phospholipid complex is detached from the lipid complex. We designed the following experiment: Under physiological pH (pH = 7.4) and lysosomal low pH (pH = 5.0), the color of the metal-phospholipid complex was observed. As shown in Figure 2-3, the metal-phospholipid 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, Fe3+ Can be detached from the metal-phospholipid complex.

[0657] At low pH, Fe 3+ The principle of shedding from the metal-phospholipid 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-phospholipid complex (Figure 2-3).

[0658] Example 2.3: Metal ion is Fe 3+ Elemental Analysis of MPP in Drug-Metal-Phospholipid Complex Particles

[0659] The mRNA in Example 1.3 was replaced with thiol-modified siRNA, and the drug-metal-phospholipid complex particles siRNA@MPP (Fe 3+ ). Elemental analysis was performed using a transmission electron microscope. The results are shown in Figure 2-4: C, N, O, and P are common elements, and the Fe elemental analysis diagram shows that Fe 3+ Evenly distributed on the lipid nanoparticles, because the siRNA is modified with sulfhydryl groups, the S element analysis diagram can specifically indicate the location of the siRNA. It can be seen from the figure that the siRNA is well complexed with Fe 3+ , proving that the drug-lipid nanoparticles successfully encapsulated siRNA.

[0660] Example 2.4: Metal ion is Fe 3+ 、Al 3+ or Mg 2+ The efficiency of nucleic acid (siRNA and mRNA) encapsulation of metal-phospholipid complex particles (MPP) and its comparison with LNP

[0661] The mRNA in Example 1.3.5 was replaced with siRNA targeting the Bcl-2 gene (SEQ ID No. 4, 19 bp) and mRNA encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit (SEQ ID No. 2, 669 nt), respectively, to prepare nucleic acid-encapsulated drug-metal-phospholipid complex particles siRNA@MPP and mRNA@MPP, respectively. The preparation process of the remaining drug-metal-phospholipid complex particles was the same as that in Example 1.3.5.

[0662] siRNA@LNPs and mRNA@LNPs were prepared using the same drug loading as described in Example 1.3.6 for siRNA@MPP and Example 1.3.5 for mRNA@MPP. The organic phase solution was prepared according to the Onpattro lipid nanoparticle formulation, where the ionizable lipid ALC0315, DSPE-PEG2000, DSPC, and cholesterol were dissolved in ethanol at a molar ratio of 50%:1.5%:10%:38.5%. Bcl-siRNA or RBD-mRNA was added to the aqueous phase (0.1 M acetic acid-sodium acetate buffer, pH 4.0). The amino lipid to phosphate nucleotide ratio (N / P) was 6:1, ensuring the same nucleic acid loading as described for the siRNA@MPP and mRNA@MPP formulations. The aqueous and organic phases were rapidly mixed at a volume ratio of 3:1 at a flow rate of 14 mL / min. After mixing, the mixture was diluted tenfold with enzyme-free PBS buffer solution and concentrated to one-tenth using a 100 kDa ultrafiltration tube. After repeating the dilution and concentration operation three times, the ethanol concentration in the mixture was reduced to below 0.0005% and the pH value of the solution was increased to the normal pH value of PBS buffer solution (7.2-7.4), thus producing siRNA@LNP and mRNA@LNP, respectively.

[0663] Agarose gel electrophoresis was used to examine the nucleic acid (siRNA and mRNA) loading efficiency of siRNA@MPP, mRNA@MPP, 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)%.

[0664] Result analysis: As shown in Figure 2-5, MPP(Fe 3+ ) encapsulation efficiency of siRNA and mRNA was 90.11% and 89.78% respectively; MPP (Al 3+ ) encapsulation efficiency of siRNA and mRNA was 92.81% and 91.48% respectively; MPP (Mg 2+The efficiency of MPP and LNP in encapsulating siRNA and mRNA was 93.69% and 92.02%, respectively; the efficiency of LNP in encapsulating siRNA and mRNA was 89.02% and 89.36%, respectively. The results showed that there was no significant difference in the efficiency of nucleic acid encapsulation between MPP and LNP.

[0665] Example 2.5: Metal ion is Fe 3+ 、Al 3+ or Mg 2+ Nucleic acid lysosomal escape ability of metal-phospholipid complex particles (MPP) and its comparison with LNP

[0666] The Bcl-2-siRNA (SEQ ID No. 4) of Example 1.3.6 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@MPP (the concentration of the siRNA contained was 100 nM), and the Bcl-2-siRNA (SEQ ID No. 4) of Example 2.4 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@LNP (the concentration of the siRNA contained was 100 nM); the eGFP-mRNA (SEQ ID No. 1) of Example 1.3.5 was replaced with Cy5-labeled eGFP-mRNA to prepare Cy5-mRNA@MPP (the concentration of the mRNA contained was 2 μg / mL), and the RBD-mRNA of Example 2.4 was replaced with Cy5-labeled RBD-mRNA to prepare Cy5-mRNA@LNP (the concentration of the mRNA contained was 2 μg / mL), and they were respectively mixed with the cell lysosome probe Lysotracker After incubating A549 cells with Cy5 and Lysotracker Green for 3 hours, the high-content imaging system was used to observe the overlap of the Cy5 fluorescence signal (red) and Lysotracker Green fluorescence signal (green). 3+ The results are shown in Figures 2-11), and the ability of the drug-lipid particles to promote nucleic acid lysosomal escape was determined.

[0667] The ability of drug-metal-phospholipid complex particles to promote nucleic acid lysosomal escape was determined by incubating cells with the drug-lipid nanoparticles for 3 hours. High-content imaging was used to observe the overlap between the Cy5 fluorescence signal (red) and the Lysotracker Green fluorescence signal (green). ImageJ software was used to calculate the overlap ratio between the red and green fluorescence signals. A 3-hour overlap ratio of less than 50% between the red and green fluorescence signals indicated rapid nucleic acid escape from lysosomes, indicating that the lipid nanoparticles possessed robust lysosomal escape ability.

[0668] Result analysis: As shown in Figure 2-6, when Cy5-siRNA@MPP(Fe 3+ ) and Cy5-mRNA@MPP(Fe 3+ ) incubated A549 cells for 3 hours, the overlap rates of red fluorescence signal and green fluorescence signal were 39.11% and 43.44%, respectively, that is, the lysosomal escape rates were 60.89% and 56.56%, respectively; when Cy5-siRNA@MPP(Al 3+ ) and Cy5-mRNA@MPP(Al 3+ ) incubated A549 cells for 3 hours, the overlap rates of red fluorescence signal and green fluorescence signal were 33.48% and 40.15%, respectively, that is, the lysosomal escape rates were 66.52% and 59.85%, respectively; when Cy5-siRNA@MPP(Mg 2+ ) and Cy5-mRNA@MPP(Mg 2+ ) incubated A549 cells for 3 hours, the overlap rates of red fluorescence signal and green fluorescence signal were 54.86% and 57.36%, respectively, that is, the lysosomal escape rates were 45.14% and 42.64%, respectively; while after incubating A549 cells for 3 hours with Cy5-siRNA@LNP and Cy5-mRNA@LNP, the overlap rates of red fluorescence signal and green fluorescence signal were 77.69% and 84.69%, respectively, that is, the lysosomal escape abilities were 22.31% and 15.31%, respectively. This suggests that the drug-lipid nanoparticles (Fe 3+ 、Al 3+ or Mg 2+ )MPP has a good ability to promote nucleic acid lysosomal escape, and (Fe 3+ 、Al 3+ or Mg 2+ ) The ability of MPP to promote lysosomal escape is significantly stronger than that of LNP.

[0669] Example 2.6: Metal ion is Fe 3+ 、Al 3+ or Mg 2+ The ability of metal-phospholipid complex particles (MPP) to promote nucleic acid expression and its comparison with LNP

[0670] The RBD-mRNA in Example 2.4 was replaced with mRNA encoding the fluorescent protein eGFP, and the rest of the preparation method was the same as Example 2.4 to obtain eGFP-mRNA@LNP.

[0671] The eGFP-mRNA@MPP prepared in Example 1.3 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 MPP or LNP. After 48 h, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0672] The method for analyzing the eGFP-positive cell rate by flow cytometry is as described in Example 1.3.

[0673] Result analysis: As shown in Figure 2-7, MPP(Fe 3+ )、MPP(Al 3+ ) or MPP(Mg 2+ ) and LNP treated 293T cells, the percentages of eGFP-positive cells were 97.7%, 98.02%, 98.3% and 63.03% respectively. The results suggest that MPP is better than LNP in promoting nucleic acid expression. The possible reason is that, as described in Example 2.5, MPP has a stronger ability to promote lysosomal escape of nucleic acids than LNP, so more nucleic acids loaded by MPP can be effectively released into the cytoplasm and translated into proteins. The current method of using the percentage of eGFP-positive cells to analyze the expression of different metal ions (Fe 3+ 、Al 3+ or Mg 2+ ) composed of MPP (such as MPP (Fe 3+ )、MPP(Al 3+ ) or MPP(Mg 2+ ) to characterize the expression capacity of nucleic acids, because the performance of eGFP protein itself is insufficient to distinguish subtle differences, it is unable to distinguish metal ions (Fe 3+ 、Al 3+ or Mg 2+ ) composed of MPP (such as MPP (Fe 3+ )、MPP(Al 3+ ) or MPP(Mg 2+ )) have different abilities to promote nucleic acid expression.

[0674] Example 2.7: Metal ion is Fe 3+ 、Al 3+ or Mg 2+ The ability of drug-metal-phospholipid complex particles (MPP) to promote humoral and cellular immunity and its comparison with LNP

[0675] The RBD-mRNA@MPP of Example 1.3.5 and the RBD-mRNA@LNP of Example 2.4 were incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPP. After 24 hours, the supernatant was centrifuged and frozen at -20°C for later use; the cell pellet was resuspended in 100 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 2-8.

[0676] The ELISA method for detecting RBD expression levels is as described in Example 1.3.5.

[0677] The experimental animals were randomly divided into two groups (experimental group and control group), with 5 mice in each group. The animal model was BALB / c mice. Each mouse received the first intramuscular administration on day 1 and the second intramuscular administration on day 14. The experimental group was injected with RBD-mRNA@MPP and RBD-mRNA@LNP, respectively, while the control group was injected with MPP and LNP without mRNA. The dose of each administration was 100 μL, of which the RBD-mRNA@MPP and RBD-mRNA@LNP preparations in the experimental group each contained 30 mg of mRNA. On the 28th day after the first administration, the mouse blood was collected, the serum was separated and serially diluted, and the titer of the total RBD IgG antibody against the novel coronavirus S1 subunit produced in the mouse body was detected using a commercially available ELISA kit. The results are shown in Figure 2-9.

[0678] The ELISA method for detecting the titer of total RBD IgG antibodies against the novel coronavirus S1 subunit is as described in Example 1.3.5.

[0679] On the 28th day after administration of RBD-mRNA@MPP and RBD-mRNA@LNP, spleens of normal mice were collected and prepared into single-cell suspensions 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 measured using ELISA kits. The results are shown in Figure 2-10.

[0680] The ELISA method for detecting the expression levels of IFN-γ, IL-2, and IL-4 is as described in Example 1.3.5.

[0681] Result analysis: As shown in Figure 2-8, RBD-mRNA@MPP(Fe 3+ )、RBD-mRNA@MPP(Al 3+ )、RBD-mRNA@MPP(Mg 2+) and RBD-mRNA@LNP can induce 293T cells to express a certain amount of RBD, but RBD-mRNA@MPP(Mg 2+ ) was significantly more effective in inducing cell expression of RBD than RBD-mRNA@MPP(Al 3+ )、RBD-mRNA@MPP(Fe 3+ ), and the ability of the three to induce cell expression of RBD was significantly stronger than that of RBD-mRNA@LNP:RBD-mRNA@MPP(Fe 3+ ) treatment group, the expression level of RBD in the cell supernatant was 218.93 ng / mL, and RBD-mRNA@MPP(Al 3+ ) treatment group, the expression level of RBD in the cell supernatant was 239.93 ng / mL, and the expression level of RBD-mRNA@MPP(Mg 2+ ) treatment group in the supernatant of RBD was 303.63 ng / mL, and the RBD-mRNA@LNP treatment group in the supernatant of RBD was 126.67 ng / mL. As shown in Figure 2-9, RBD-mRNA@MPP effectively induced humoral immunity in mice and produced high levels of antigen-specific binding antibodies, and RBD-mRNA@MPP(Mg 2+ ) was significantly better than RBD-mRNA@MPP(Al 3+ )、RBD-mRNA@MPP(Fe 3+ ), and the ability of the three to induce humoral immunity in mice was significantly better than that of RBD-mRNA@LNP:RBD-mRNA@MPP(Mg 2+ ) treatment group mice IgG antibody titer reached 171782.00; RBD-mRNA@MPP(Fe 3+ ) treatment group mice IgG antibody titer reached 122666.67; RBD-mRNA@MPP(Al 3+ ) treatment group mice IgG antibody titer reached 134833.33; while the IgG antibody titer in the RBD-mRNA@LNP treatment group mice was only 73694.00. As shown in Figure 2-10, RBD-mRNA@MPP(Mg 2+ ) can effectively induce cellular immunity in mice, namely activate immune cells and produce a large number of cytokines, and RBD-mRNA@MPP(Mg 2+ ) was significantly better than RBD-mRNA@MPP(Fe 3+ )、RBD-mRNA@MPP(Al 3+ ), RBD-mRNA@MPP(Fe 3+) was significantly better than RBD-mRNA@LNP in inducing cellular immunity in mice: RBD-mRNA@MPP(Fe 3+ ) made the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 286.2 pg / mL, 209.67 pg / mL, and 58.02 pg / mL, respectively; RBD-mRNA@MPP(Al 3+ ) made the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 306 pg / mL, 239.67 pg / mL, and 71 pg / mL, respectively; RBD-mRNA@MPP(Mg 2+ ) made the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 333pg / mL, 270.33pg / mL, and 73pg / mL, respectively; while RBD-mRNA@LNP made the expression levels of cytokines IFN-γ, IL-2, and IL-4 only 95pg / mL, 75.67pg / mL, and 24pg / mL. The results suggest that mRNA@MPP(Mg 2+ ) is significantly better than RBD-mRNA@MPP(Fe 3+ )、mRNA@MPP(Al 3+ ), RBD-mRNA@MPP(Fe 3+ ) is significantly better than RBD-mRNA@LNP in inducing cellular immunity in mice: RBD-mRNA@MPP can more effectively promote cell expression of target proteins and can more effectively activate humoral immunity and cellular immunity in the body. Therefore, drug (mRNA)-lipid particles are significantly better than existing technology LNP in terms of the effects of mRNA-loaded drugs, vaccines or other products. The possible reasons are: 1) Compared with LNP, MPP has a stronger ability to promote nucleic acid lysosomal escape; 2) Compared with LNP, MPP has a stronger ability to promote nucleic acid expression into protein (antigen); 3) Compared with LNP, the curcumin in MPP is separated from DSPC in the body and acts as an immune adjuvant (also known as an immunomodulator), which can activate humoral immunity and cellular immunity to enhance the effect of MPP in delivering mRNA vaccines, and can also suppress the immune factor storm to suppress excessive and harmful immune responses to the body.

[0682] MPPs composed of different metal ions (e.g. MPP(Fe 3+ )、MPP(Al 3+ ))Compared with MPP(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.), 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.) and enhance immune response. 2+ It can regulate LFA-1 downstream signal transduction by inducing conformational changes in the co-stimulatory molecule LFA-1 on the surface of CD8+ T cells, thereby promoting T cell activation and cytotoxicity (Cell. 2022 Feb 17; 185(4): 585-602.e29.).

[0683] Example 2.8: Metal ion is Fe 3+ 、Al 3+ or Mg 2+ Comparison of the expression duration of drug-metal-phospholipid complex particles MPP and LNP

[0684] Preparation of CD19 CAR mRNA:

[0685] 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.

[0686] Signal transduction domain, signal peptide (SP): helps the CAR expressed in T cells to be directed to the T cell membrane.

[0687] 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.

[0688] The spacer connects the antigen-binding domain scFV and the transmembrane domain. Its flexibility ensures that scFV can better recognize antigens.

[0689] Transmembrane(TM): These combinations anchor the CAR expression to the T cell membrane.

[0690] 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.

[0691] The amino acid sequence expressed by CD19 CAR mRNA is shown in SEQ ID NO.60.

[0692] Referring to Example 2.4, mRNA was replaced with CD19 CAR mRNA to prepare CD19 CAR mRNA@MPP and CD19 CAR 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 Incubate the labeled CAR linker antibody and CD11b-PE antibody at 4°C for 30 minutes. Add red blood cell lysis buffer and lyse for 5-10 minutes in the dark. Wash twice with buffer and resuspend in 100-30,000 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.

[0693] The results are shown in Figure 8-1. CD19 CAR mRNA@MPP(Mg 2+ ) myeloid cells had the highest proportion of CAR-positive cells, and the duration of CAR-positive cells was significantly longer than that of CD19 CAR mRNA@MPP(Al 3+ ), CD19 CAR mRNA@MPP(Al 3+ ) is superior to CD19 CAR mRNA@MPP(Fe 3+ ), CD19 CAR mRNA@MPP(Fe 3+ ) is better than LNP, suggesting that MPP (Mg 2+ ) has stronger expression and better expression duration function.

[0694] Example 2.9: Metal ion is Fe 3+ 、Al 3+ or Mg 2+In vivo safety evaluation of metal-chelated phospholipid complex nanoparticles (MPP)

[0695] A 20-day subchronic toxicity study of MPP was conducted using SD rats, with a 20-day recovery period. The specific experimental methods are as follows:

[0696] Fifty-six SPF Sprague-Dawley rats (220 ± 20 g), half male and half female, were housed at 25°C, 45%-55% humidity, and 12 hours of light. After 3-5 days of acclimatization, they were randomly divided into the following groups: an experimental group (32 rats) and a recovery group (24 rats). The blank control group consisted of 14 rats (8 in the experimental group and 6 in the recovery group), half male and half female; the low-dose MPP group (25 mg / kg) consisted of 14 rats (8 in the experimental group and 6 in the recovery group), half male and half female; the medium-dose MPP group (50 mg / kg) consisted of 14 rats (8 in the experimental group and 6 in the recovery group), half male and half female; and the high-dose MPP group (100 mg / kg) consisted of 14 rats (8 in the experimental group and 6 in the recovery group), half male and half female. The experimental group (32 rats) was autopsied after the end of drug administration. The recovery group (24 rats) was autopsied after 20 days of continued normal feeding.

[0697] 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 MPP was dissolved in DPBS. The control group was injected with an equal volume of DPBS, while the low-dose MPP group, medium-dose MPP group, and high-dose MPP group were injected with 25 mg / kg, 50 mg / kg, and 100 mg / kg of MPP, respectively.

[0698] 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%.

[0699] 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.

[0700] 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.

[0701] 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.

[0702] 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.

[0703] 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.

[0704] 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 MPP 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 MPP groups; compared with the control group, there was no significant difference in the organ-to-body ratios of the low-, medium- and high-dose MPP groups.

[0705] 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 doses of MPP groups were normal; compared with the control group, the blood biochemical indicators in the low, medium and high doses of MPP groups, 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 MPP 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.

[0706] 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 MPP groups was intact, the tissue staining was normal, the cell morphology was intact, and there was no nuclear pyknosis or inflammatory cell infiltration; the myocardial tissue structure was intact, the myocardial cells were arranged neatly, continuously and tightly, the cell nuclei were clearly visible, and there was no obvious cell congestion, edema or necrosis; the liver cell morphology was normal, without inflammatory cell aggregation and necrosis; the spleen structure was normal, with clear boundaries between red and white pulp; the lung tissue structure was intact, the alveoli were of uniform size, and there was no obvious inflammatory cell aggregation or infiltration; and the kidney structure was normal.

[0707] The above results suggest that no obvious chronic toxicity was found in SD rats after long-term and large-scale injection of MPP, indicating that MPP is relatively safe.

[0708] Table 2-1 In vivo safety evaluation of MPP

[0709] 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.

[0710] Example 2.10: Metal ion is Fe 3+ 、Al 3+ or Mg 2+ Comparison of the in vivo safety of metal-phospholipid complex particles (MPP) and LNP

[0711] 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. Metal-chelated phospholipid complex nanoparticles (MPP) replace the cationic lipids / ionizable lipids in LNP with metal-phospholipid complexes. Therefore, we studied the median lethal dose (IC50) of metal-phospholipid complexes and cationic lipids / ionizable lipids on biological cells. 50 ), and compare the differences in toxicity between LNP and MPP.

[0712] 293T cells were incubated with different concentrations of the metal-phospholipid complex (0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM), cationic lipid (DOTAP, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM, structural formula as follows) and ionizable lipid (ALC0315, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM, structural formula as follows) for 48 hours, and then the cell viability was detected using a CCK8 activity detection kit. The median lethal dose (IC50) of the metal-phospholipid complex, cationic lipid (DOTAP) and ionizable lipid (ALC0315) on 293T cells was calculated. 50 .

[0713] DOTAP structure:

[0714] ALC0315 structural formula:

[0715] CCK8 detection method:

[0716] 1. Cell culture: Culture cells in DMEM containing 10% FBS and 1% double-antibody until the cell density reaches 80%-90% of the culture flask;

[0717] 2. Wash the remaining culture medium in the culture flask with PBS, add trypsin, and quickly transfer the culture flask to a 37°C incubator with 5% CO2. Observe carefully until the cells slightly round up, 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.

[0718] 3. 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.

[0719] 4. After drug incubation for 48 hours, add 10% CCK8 and incubate for 1-3 hours. Measure the absorbance at 450 nm with a microplate reader.

[0720] 5. Survival rate (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] x 100%.

[0721] 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 .

[0722] To compare the in vivo safety of MPP and LNP, MPP (8 mg / kg) and LNP (3.24 mg / kg) capable of carrying an equal amount of nucleic acid (200 μg / kg mRNA) were used to conduct an in vivo experiment according to the method of Example 11 to evaluate and compare the in vivo toxicity of MPP and LNP.

[0723] Results analysis: As shown in Table 2-2, metal-phospholipid complex (MPP (Fe 3+ )、MPP(Al 3+ )、MPP(Mg 2+ )) IC 50 The results showed that the toxicity of metal-phospholipid complexes was significantly lower than that of cationic lipids (DOTAP) and ionizable lipids (ALC0315).

[0724] As shown in Table 2-3, at the end of the treatment period and the end of the recovery period, MPP (Fe 3+ )、MPP(Al 3+ )、MPP(Mg 2+ There were no significant abnormalities in liver function (ALT, AST, ALP) and cytokines (IL-6, IL-1β) in the LNP group. However, compared with the control group, liver function (ALT, AST, ALP) and cytokines (IL-6, IL-1β) in the LNP group were significantly increased. The results suggest that MPP (Fe 3+ ) or MPP(Al 3+ ) is safer in vivo than LNP because 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 MPP (Fe 3+ ) or MPP(Al 3+ ) or MPP(Mg 2+The core component of MPP (Fe 2+) is a metal-phospholipid complex, which is composed of phospholipid molecules, a highly safe natural small molecule substance, 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+ ) or MPP(Al 3+ ) components do not contain cationic lipids / ionizable lipids and will not cause toxic side effects related to cationic lipids / ionizable lipids, so MPP(Fe 3+ )、MPP(Al 3+ )、MPP(Mg 2+ ) is safer than LNP.

[0725] Table 2-2 Metal ions are Fe 3+ 、Al 3+ or Mg 2+ IC of metal-phospholipid complexes with cationic lipids (DOTAP) and ionizable lipids (ALC0315) 50 Compared with

[0726] Table 2-3 Comparison of chronic toxicity test indicators of MPP and LNP

[0727] Table 2-4 Historical safety data of metals

[0728] References: "Dietary Reference Intakes of Nutrients for Chinese Residents" - 2023 Edition, "Risk Assessment of Dietary Aluminum Exposure of Chinese Residents", and ICH Harmonized Guidelines - Elemental Impurities Guidelines Q3D (R2).

[0729] The above historical data show that the safety of MPP or Apt-MPP prepared based on various metals is ranked as follows: Mg>Fe>Zn>Al>Mn>Cr. 2+ ) has the best efficacy and the best safety.

[0730] Example 3: Metal ion is Fe 3+ 、Al 3+ or Mg 2+ Clinical application and administration route of drug-metal-phospholipid complex particles

[0731] The mRNA in Example 1.3.5 was replaced with siRNA targeting the B7-H4 gene (B7-H4-siRNA) and its control (scr-siRNA), and mRNA encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit (RBD-mRNA).

[0732] The sequences of the different nucleic acids are as follows: ① The sequences of B7-H4-siRNA are SEQ ID No. 19 (sense strand) and SEQ ID No. 26 (antisense strand) (25 bp), and the randomized control sequences are SEQ ID No. 20 (sense strand) and SEQ ID No. 27 (antisense strand) (19 bp); ② The sequence of the mRNA encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit is SEQ ID No. 2 (669 nt). Drug-metal-phospholipid complex particles (B7-H4-siRNA@MPP and RBD-mRNA@MPP) encapsulating the different nucleic acids were prepared according to the method of Example 1.3.5. The remaining preparation processes of the drug-metal-phospholipid complex particles were the same as those of Example 1.3.5. The two different drug-metal-phospholipid complex particles (B7-H4-siRNA@MPP and RBD-mRNA@MPP) were used to treat glioma and as mRNA vaccines for the prevention of novel coronavirus, respectively.

[0733] To evaluate the effect of RBD-mRNA@MPP as an mRNA vaccine in preventing the new coronavirus, the experimental process and experimental methods are as shown in the previous Example 1.3.5.

[0734] The ELISA detection method is as described in Example 1.3.5.

[0735] To evaluate the B7-H4-siRNA@MPP(Fe 3+ ) and B7-H4-siRNA@MPP(Al 3+ )、B7-H4-siRNA@MPP(Mg 2+ ) to treat liver cancer. Animal models of liver cancer were established using HepG2 cells. When the tumor size increased to about 100 mm 3 The mice with liver cancer were randomly divided into 10 groups (5 mice in each group): PBS control group, blank vector MPP (Fe 3+ ) group, blank carrier MPP (Al 3+ ) group, blank carrier MPP (Mg 2+ ) group, Scr-siRNA@MPP(Fe 3+ ) control group, B7-H4-siRNA@MPP(Fe3+ ) treatment group, Scr-siRNA@MPP(Al 3+ ) control group, B7-H4-siRNA@MPP(Al 3+ ) treatment group, Scr-siRNA@MPP(Mg 2+ ) control group, B7-H4-siRNA@MPP(Mg 2+ ) treatment group. Each group of mice was injected intratumorally with PBS, MPP (Fe 3+ )、MPP(Al 3+ )、MPP(Mg 2+ )、Scr-siRNA@MPP(Fe 3+ )、B7-H4siRNA@MPP(Fe 3+ )、Scr-siRNA@MPP(Al 3+ )、B7-H4 siRNA@MPP(Al 3+ )、Scr-siRNA@MPP(Mg 2+ )、B7-H4-siRNA@MPP(Mg 2+ ) once, at a dose of 200 μg siRNA / kg, for eight injections. Tumor volume was measured and recorded every three days. The results are shown in Figures 3-1, 3-2, and 3-3.

[0736] 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 and kept on ice before inoculation. 100 μL of the cell suspension was then injected subcutaneously into the dorsal region near the hind legs of female Balb / c nude mice to establish a liver cancer mouse model.

[0737] Result analysis:

[0738] As shown in Figures 3-1, 3-2 and 3-3, Scr-siRNA@MPP(Fe 3+ )、Scr-siRNA@MPP(Al 3+ )、Scr-siRNA@MPP(Mg 2+ ) had almost no inhibitory effect on the growth of HepG2 cells, while B7-H4-siRNA@MPP(Fe 3+ )、B7-H4 siRNA@MPP(Al 3+ ) and B7-H4 siRNA@MPP(Mg 2+ ) showed a highly effective therapeutic effect, effectively inhibiting the growth of liver cancer tumors. The results suggest that drug-metal-phospholipid complex particles can encapsulate and deliver B7-H4 siRNA, inhibiting the expression of the target gene, thereby inhibiting the development of liver cancer.

[0739] As shown in Figures 1-3 and 1-5 of Example 1.3.5, RBD-mRNA@MPP(Fe 3+ ) increased the expression level of mouse IgG antibody to 117268.8 (Figure 1-3), and the expression levels of cytokines IFN-γ, IL-2, and IL-4 to 252.8 pg / mL, 207.6 pg / mL, and 56.6 pg / mL, respectively (Figure 1-5). RBD-mRNA@MPP(Al 3+ ) increased the expression level of mouse IgG antibody to 129113 (Figure 1-3-2), and the expression levels of cytokines IFN-γ, IL-2, and IL-4 to 271.8 pg / mL, 234.6 pg / mL, and 68.4 pg / mL, respectively (Figure 1-5-2). RBD-mRNA@MPP(Mg 2+ ) increased the expression level of mouse IgG antibodies to 130614.40 (Figure 1-3-3), and the expression levels of cytokines IFN-γ, IL-2, and IL-4 to 296.40 pg / mL, 243.33 pg / mL, and 75.30 pg / mL, respectively (Figure 1-5-3). The results suggest that RBD-mRNA@MPP 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, RBD-mRNA@MPP can effectively prevent infection with the new coronavirus.

[0740] B7-H4-siRNA@MPP is effective in treating liver cancer via intratumoral injection, while RBD-mRNA@MPP, via intramuscular injection, can activate humoral and cellular immunity, thereby preventing infection with the novel coronavirus. These results suggest that drug-metal-phospholipid complex particles can be administered via multiple routes.

[0741] MPPs composed of other metal ions (such as MPP (Fe 3+ )、MPP(Al 3+ ))Compared with MPP(Mg 2+ ) can more effectively inhibit the growth of liver cancer and more effectively prevent the infection of the new coronavirus. 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 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.) 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) 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.).

[0742] Example 4: DSPC, curcumin, Fe 3+ 、Al 3+ or Mg 2+ Function after being replaced by similar products

[0743] Example 4.1, DSPC, curcumin, Fe 3+ Function after being replaced by similar products

[0744] With reference to Example 1.1, Example 1.2 and Example 1.3, DSPC, curcumin, Fe 3+ The analogs of DSPC, curcumin, Fe 3+ 37 different eGFP-mRNA@MPPs were prepared by different combinations, and the concentration of mRNA in each eGFP-mRNA@MPP was 2 μg / mL. 3+ The names and structures of their analogs are shown in Table 4-1. Among the 37 mRNA@MPPs, DSPC, curcumin, Fe 3+ The combination of the same is shown in Table 4-2. In Example 1.1, the reaction temperature is 65°C and the reaction time is 2 hours, and in Example 1.2, the reaction temperature is 60°C and the reaction time is 2 hours, and other conditions remain unchanged.

[0745] To compare the effects of the 37 different eGFP-mRNA@MPPs and eGFP-mRNA@LNPs, we prepared LNPs encapsulating equal amounts of eGFP-mRNA with reference to Example 2.4 to obtain eGFP-mRNA@LNPs.

[0746] The above 37 different eGFP-mRNA@MPPs 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 MPP or LNP. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0747] The method for analyzing the eGFP-positive cell rate by flow cytometry is as described in Example 1.3.

[0748] 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. Metal-chelated phospholipid complex nanoparticles (MPP) replace the cationic lipids / ionizable lipids in LNP with metal-phospholipid complexes. Therefore, we studied the median lethal dose (IC50) of 37 metal-phospholipid complexes and cationic lipids (DOTAP) / ionizable lipids (ALC0315) on biological cells. 50 ), and compared the differences in toxicity between LNP and 37 MPPs.

[0749] IC 50 The calculation method of is as described in Example 2.10.

[0750] Results analysis: As shown in Table 4-2, the percentage of eGFP-positive cells in 293T cells treated with 37 different eGFP-mRNA@MPPs was significantly higher than that of eGFP-mRNA@LNPs, among which DSPC, curcumin, Fe 3+ The percentage of eGFP-positive cells of mRNA@MPP was the highest. 3+ The function of mRNA@MPP formed after being replaced by its congeners is not as good as that of DSPC, curcumin, Fe 3+ The mRNA@MPP composed of mRNA@MPP is superior to mRNA@LNP in function. The possible reason is that, as described in Example 8, MPP has a stronger ability to promote lysosomal escape of nucleic acids than LNP, so more nucleic acids loaded by MPP can be effectively released into the cytoplasm and translated into proteins.

[0751] The above results suggest that as long as the following conditions are met, DSPC, curcumin, Fe 3+The function of drug-metal-phospholipid complex particles formed after being replaced by its congeners is not affected: ① DSPC congeners are amphiphilic phospholipid molecules; ② Fe 3+ The congeners of curcumin are metal ions; ③ The congeners of curcumin and DSPC form phospholipid complexes and can also complex with metal ions; ④ Curcumin and Fe 3+ The coordination bonds between them can be broken in response to the low pH environment of the lysosome.

[0752] As shown in Table 4-3, the IC values ​​of 37 metal-phospholipid complexes 50 The toxicity of metal-phospholipid complexes is obviously lower than that of cationic lipids (DOTAP) and ionizable lipids (ALC0315). 3+ The safety of lipid nanoparticles (MPP) composed of DSPC 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 MPP is metal-phospholipid complex, which is composed of phospholipid molecules, natural small molecules with high safety (among 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, the drug composed of DSPC, curcumin, Fe 3+ The lipid particles (MPP) 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 MPP is higher than that of LNP.

[0753] Table 4-1 DSPC, curcumin, Fe 3+ The names and structures of its analogs

[0754] Table 4-2DSPC, curcumin, Fe 3+ List of composition combinations and functions of metal-phospholipid complexes in drug-lipid particles prepared from and their analogs

[0755] Table 4-3DSPC, curcumin, Fe 3+ IC of metal-phospholipid complexes prepared from its analogs 50

[0756] Example 4.2, DSPC, curcumin and its analogs, Fe 3+Component dosage ratio and function of drug-metal-phospholipid complex particles prepared therefrom

[0757] According to Example 1.3, mRNA-metal-phospholipid complex particles were prepared, and curcumin was replaced with its analog hesperetin (one hesperetin molecule contains four hydroxyl groups) and tea polyphenol (one tea polyphenol molecule contains eight hydroxyl groups), respectively, to prepare three types of mRNA-metal-phospholipid complex particles (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP38). The ratios of DSPC, curcumin or its analog, and FeCl3 used in the preparation of these three mRNA-metal-phospholipid complex particles were 1:1:1, 1:1:1, and 1:1:2, respectively. The mRNA encoding the eGFP fluorescent protein was SEQ ID No. 1 (720 nt). The mRNA loading efficiency of these four drug-lipid nanoparticles and their ability to promote eGFP fluorescent protein expression after treatment with 293T cells were tested according to the experimental procedures and methods described in Example 1.3.5.

[0758] Analysis of results: As shown in Table 4-4, the mRNA encapsulation efficiency and the ability to promote the expression of target proteins of drug-metal-phospholipid complex particles prepared using different dosage ratios based on the chemical structure of the metal-phospholipid complex components are comparable. The results suggest that the dosage ratio of the metal-phospholipid complex components can be adjusted according to the structure of the specific metal-phospholipid complex components. The basis for adjusting the dosage ratio is: because DSPC and its analogs are connected to curcumin and its analogs by hydrogen bonds, as long as DSPC and its analogs contain multiple phosphate groups, then when synthesizing phospholipid complexes, the dosage ratio of DSPC and its analogs and curcumin and its analogs can be adjusted according to the number of phosphate groups contained in DSPC and its analogs, that is, when DSPC and its analogs contain two phosphate groups, the dosage ratio of DSPC and its analogs to curcumin and its analogs can be adjusted to 1:2; when DSPC and its analogs contain three phosphate groups, the dosage ratio of DSPC and its analogs to curcumin and its analogs can be adjusted to 1:3; because the hydroxyl group of curcumin and its analogs reacts with Fe 3+ As long as curcumin and its analogs contain multiple binding sites, curcumin and its analogs and Fe 3+ The dosage ratio of curcumin and its analogs can be adjusted according to the number of binding sites contained in curcumin and its analogs.

[0759] Table 4-4 Component ratios of different metal-phospholipid complexes and functions of the drug-lipid particles prepared therefrom

[0760] Example 4.3, DSPC, curcumin and its analogs, Al3+ Component dosage ratio and function of drug-metal-phospholipid complex particles prepared therefrom

[0761] According to Example 1.4, mRNA metal-phospholipid complexes were prepared, and curcumin was replaced with its analog hesperetin (one hesperetin molecule contains four hydroxyl groups) and tea polyphenols (one tea polyphenol molecule contains eight hydroxyl groups), respectively, to prepare three metal-phospholipid complexes (mRNA@MPP2, mRNA@MPP5, and mRNA@MPP39). The ratios of DSPC, curcumin or its analog, and Al(NO3)3·9H2O used to prepare these three metal-phospholipid complexes were 1:1:1, 1:1:1, and 1:1:2, respectively. These three metal-phospholipid complexes (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP29) were then used to prepare corresponding drug-metal-phospholipid complex nanoparticles. The mRNA encoding the eGFP fluorescent protein is shown in SEQ ID No. 1 (720 nt). The mRNA encapsulation efficiency of these four drug-lipid nanoparticles and their ability to promote eGFP fluorescent protein expression after treating 293T cells were detected according to the experimental process and experimental methods described in Example 3.5.

[0762] Analysis of results: As shown in Tables 4-5, the mRNA encapsulation efficiency and the ability to promote the expression of target proteins of drug-metal-phospholipid complex particles prepared using different dosage ratios based on the chemical structure of the metal-phospholipid complex components are comparable. The results suggest that the dosage ratio of the metal-phospholipid complex components can be adjusted according to the structure of the specific metal-phospholipid complex components. The basis for adjusting the dosage ratio is: because the analogs of DSPC and the analogs of curcumin are connected by hydrogen bonds, as long as the analogs of DSPC contain multiple phosphate groups, then when synthesizing phospholipid complexes, the dosage ratio of the analogs of DSPC and the analogs of curcumin can be adjusted according to the number of phosphate groups contained in the analogs of DSPC, that is, when the analogs of DSPC contain two phosphate groups, the dosage ratio of the analogs of DSPC and the analogs of curcumin can be adjusted to 1:2; when the analogs of DSPC contain three phosphate groups, the dosage ratio of the analogs of DSPC and the analogs of curcumin can be adjusted to 1:3; because the hydroxyl groups of the analogs of curcumin react with Al 3+ As long as the congeners of curcumin contain multiple binding sites, the congeners of curcumin and Al 3+ The dosage ratio of the congeners can be adjusted according to the number of binding sites contained in the curcumin congeners.

[0763] Table 4-5 Component ratios of different metal-phospholipid complexes and functions of the drug-lipid particles prepared therefrom

[0764] Example 4.4, DSPC, curcumin and its analogs, Mg 2+ Component dosage ratio and function of drug-metal-phospholipid complex particles prepared therefrom

[0765] According to Example 1.5, mRNA-metal-phospholipid complex particles were prepared, and curcumin was replaced with its analogs hesperetin (one hesperetin molecule contains four hydroxyl groups) and tea polyphenols (one tea polyphenol molecule contains eight hydroxyl groups), respectively, to prepare three types of mRNA-metal-phospholipid ...

Claims

1. A targeting vector, characterized in that The targeting vector contains: (a) Metal-phospholipid complex particles, comprising: (i) a metal-phospholipid complex, wherein the metal-phospholipid complex is formed by reacting a phospholipid molecule portion, a linker molecule portion, and a metal ion portion, the phospholipid molecule portion is connected to the linker molecule portion, the linker molecule portion is connected to the metal ion portion via a coordination bond, and the metal-phospholipid complex is not a cationic lipid or an ionizable lipid; (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; as well as (iii) a non-cationic lipid or a non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation; (b) a targeting structure, wherein the targeting structure is connected to the outer surface of the metal-phospholipid complex particle.

2. The targeting vector according to claim 1, wherein In the (i) metal-phospholipid complex, the phospholipid molecule portion is selected from phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphoceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysophosphatidylcholine LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, 1-phospho-sphingosine S1P, and a combination of one or more of their derivatives; Preferably, the phospholipid molecule 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 phospholipid molecule is selected from the group consisting of phosphatidylcholine PC (Formula 1), phosphatidylethanolamine PE (Formula 2), phosphatidic acid PA (Formula 4), phosphatidylglycerol (PG) (Formula 5), and a combination of one or more thereof. Preferably, the phospholipid molecule is selected from the group consisting of DSPC, DSPE, DSPA, DSPG, and a combination of one or more of their derivatives; Preferably, the phospholipid molecule is selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48), DSPG (Formula 49), and a combination of one or more of their derivatives; Preferably, the linker molecule is selected from the group consisting of curcumin, chlorogenic acid, anthocyanidin, quercetin, dihydromyricetin, hesperetin, naringenin, apigenin, catechin, tea polyphenols, epigallocatechin gallate, ellagic acid, morin, epicatechin gallate, catechin gallate, epigallocatechin gallate or picoflavine C, and combinations of one or more thereof. Preferably, the linker molecule is selected from Curcumin (Formula 19) Chlorogenic acid (Formula 20) Anthocyanin (Formula 21) Wherein, R1 and R2 are H, OH or OCH3, R3 is H or a glycosyl group, R4, R5 and R6 are OH or a glycosyl group, quercetin (Formula 22) Dihydromyricetin (Formula 23) Hesperetin (Formula 24) Naringenin (Formula 25) Apigenin (Formula 26) Catechin (Formula 27) Tea polyphenols (Formula 28) Epigallocatechin gallate (Formula 29) Ellagic acid (Formula 30) Mulberry pigment (Formula 31) Epicatechin gallate (Formula 32) Catechin gallate (Formula 33) Epigallocatechin gallate (Formula 34) Pingbei alkaloid C (Formula 35) and combinations of one or more of their derivatives; Preferably, the linker molecule is selected from one or more combinations of curcumin (Formula 19), dihydrocurcumin (Formula 36), hexahydrocurcumin (Formula 37), curcumin sulfate (Formula 38), and bisdemethoxycurcumin (Formula 39); Preferably, the linker molecule is selected from the group consisting of curcumin (Formula 19), hesperetin (Formula 24), tea polyphenols (Formula 28), and a combination of one or more of their derivatives; Preferably, the linker molecule is selected from curcumin (Formula 19), hesperetin (Formula 24) or tea polyphenols (Formula 28); 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 a combination of one or more 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 a combination of one or more of cholesterol and its derivatives; Preferably, the non-cationic lipid or non-ionizable lipid in (iii) is cholesterol (Formula 40) Preferably, the non-cationic lipid or non-ionizable lipid in (iii) further comprises a combination of one or more selected from phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphoceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysophosphatidylcholine LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, 1-phospho-sphingosine S1P, cholesterol sulfate and derivatives thereof; Preferably, the non-cationic lipid or non-ionizable lipid described in (iii) further comprises at least one selected from phosphatidylcholine PC (Formula 1), phosphatidylethanolamine PE (Formula 2), phosphatidylserine PS (Formula 3), phosphatidic acid PA (Formula 4), phosphatidylglycerol PG (Formula 5), 1-phosphoceramide SP (Formula 6), phosphatidylinositol PI (Formula 7), phosphatidylthreonine PT (Formula 8), sphingomyelin SM (Formula 9), lysophosphatidylcholine LPC (Formula 10), lysophosphatidylethanolamine LPE (Formula 11), lysophosphatidylserine LPS (Formula 12), lysophosphatidic acid LPA (Formula 13), lysophosphatidylglycerol LPG (Formula 14), lysophosphatidylinositol LPI (Formula 15), lysophosphatidylthreonine LPT (Formula 16), lysophosphophospholipid LSM (Formula 17), 1-phospho-sphingosine S1P (Formula 18), cholesterol sulfate (Formula 41), and derivatives thereof; 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 DSPC (Formula 46).

5. The targeting vector according to claim 1, wherein The metal-phospholipid complex is made of a phospholipid molecule part, a linker molecule part and a metal ion part, the phospholipid molecule part is selected from DSPC, DSPE, DSPA or DSPG, the linker molecule part is selected from curcumin, hesperidin or tea polyphenols, and the metal ion part is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ ; Preferably, the metal-phospholipid complex is made of a phospholipid molecule portion, a linker molecule portion and a metal ion portion, the phospholipid molecule portion is selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48) or DSPG (Formula 49), the linker molecule portion is selected from curcumin (Formula 19), hesperidin (Formula 24) or tea polyphenols (Formula 28), and the metal ion portion is selected from Fe 3+ Mg 2+ , Ca 2+ or Al 3+ ; Preferably, the molar ratio of the phospholipid molecule portion, the linker molecule portion, and the metal ion portion is 1:1:(0.5-2); Preferably, the phospholipid molecule portion is DSPC (Formula 46), the linker molecule portion is selected from curcumin (Formula 19), and the metal ion portion is selected from Fe 3+ Mg 2+ or Al 3+ The molar ratio of the phospholipid molecule part, the linker molecule part and the metal ion part is 1:1:

1.

6. The targeting vector according to claim 4, characterized in that The metal-phospholipid complex particles are made of (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the metal-phospholipid complex accounts for 5% to 50% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 1% to 10% by mole in the raw material, the cholesterol accounts for 15% to 80% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 0% to 51% by mole in the raw material; Preferably, the molar proportion of the metal-phospholipid complex in the raw material is 5% to 40%, preferably 10% to 40%; 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 25% to 75%, preferably 35% to 75%; Preferably, the molar proportion of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 0% to 50%, preferably 0% to 40%.

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-phospholipid complex particle based on hydrophilicity and hydrophobicity; Preferably, the hydrophobic region comprises at least one or more of DSPE and its derivatives; Preferably, the linker 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 vector according to any one of claims 1 to 7, characterized in that: The targeting structure is connected to the outer surface of the metal-phospholipid complex particle to form the targeting carrier.

9. The preparation method according to claim 8, characterized in that mixing (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid to obtain the metal-phospholipid 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-phospholipid 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 phospholipid molecules, linker molecules and metal ions to form a metal-phospholipid complex; Step 2: mixing the metal-phospholipid complex prepared in step 1, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid to prepare metal-phospholipid complex particles; Step 3: mixing the metal-phospholipid complex particles prepared in step 2 with the targeting structure to prepare the targeting carrier; Preferably, in step 1, the phospholipid molecules, linker molecules and metal ions are dissolved in ethanol for reaction, the molar ratio of the phospholipid molecules, linker molecules and metal ions is preferably 1:1:1, and the reaction conditions are preferably 40-60° C. for 1-5 hours; Preferably, in step three, the reaction conditions of the metal-phospholipid complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.

11. The preparation method according to claim 8, characterized in that The preparation method comprises: Step 1: Reaction and connection of phospholipid molecules with linker molecules to form phospholipid complexes; Step 2: reacting the phospholipid complex prepared in step 1 with metal ions through coordination bonds to form a metal-phospholipid complex; Step 3: mixing the metal-phospholipid complex prepared in step 2, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid to prepare metal-phospholipid complex particles; Step 4: mixing the metal-phospholipid complex particles prepared in step 3 with the targeting structure to prepare the targeting carrier; Preferably, in step 1, the phospholipid molecules and the linker molecules are dissolved in ethanol for reaction, and then n-hexane is added for precipitation to obtain the phospholipid complex. The molar ratio of the phospholipid molecules to the linker molecules is preferably 1:

1. The reaction conditions are preferably 65° C. for 2 hours. Preferably, in step 2, the phospholipid complex and the metal ion are dissolved in ethanol, and triethylamine is added to react to obtain the metal-phospholipid complex. The molar ratio of the phospholipid complex to the metal ion is preferably 1:(1-2), and the molar ratio of the phospholipid complex to the triethylamine is preferably 1:

1. The reaction conditions are preferably 60° C. for 2 hours. Preferably, in step 4, the reaction conditions of the metal-phospholipid complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.

12. 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 11 for drug delivery, imaging agent or vaccine.

13. 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-phospholipid complex particles of the targeting carrier.

14. The targeted drug according to claim 13, 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.

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

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

17. The preparation method according to claim 16, characterized in that The drug, (i) a metal-phospholipid 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 drug-metal-phospholipid complex particles.

18. The preparation method according to claim 16, characterized in that The preparation method comprises: Step 1: reacting phospholipid molecules, linker molecules and metal ions to form a metal-phospholipid complex; Step 2: mixing the metal-phospholipid complex prepared in step 1, (ii) a conjugated lipid that inhibits particle aggregation, (iii) a non-cationic lipid or a non-ionizable lipid, and a drug to prepare drug-metal-phospholipid complex particles; Step 3: mixing the drug-metal-phospholipid complex particles prepared in step 2 with the targeting structure to prepare the targeted drug; Preferably, in step 1, the phospholipid molecules, linker molecules and metal ions are dissolved in ethanol for reaction, the molar ratio of the phospholipid molecules, linker molecules and metal ions is preferably 1:1:1, and the reaction conditions are preferably 40-60° C. for 1-5 hours; Preferably, in step 2, the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer to form an aqueous phase, and the organic phase and the aqueous phase are mixed to obtain drug-metal-phospholipid complex particles, wherein the organic compound is preferably ethanol; preferably, the mixing method of the organic phase and the aqueous phase includes a microfluidic chip or ultrasound; Preferably, in step three, the reaction conditions of the drug-metal-phospholipid complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.

19. The preparation method according to claim 16, characterized in that The preparation method comprises: Step 1: Reaction and connection of phospholipid molecules with linker molecules to form phospholipid complexes; Step 2: reacting the phospholipid complex prepared in step 1 with metal ions through coordination bonds to form a metal-phospholipid complex; Step 3: mixing the metal-phospholipid complex prepared in step 2, (ii) a conjugated lipid that inhibits particle aggregation, (iii) a non-cationic lipid or a non-ionizable lipid, and a drug to prepare drug-metal-phospholipid complex particles; Step 4: mixing the drug-metal-phospholipid complex particles prepared in step 3 with the targeting structure to prepare the targeted drug; Preferably, in step 1, the phospholipid molecules and the linker molecules are dissolved in ethanol for reaction, and then n-hexane is added for precipitation to obtain the phospholipid complex. The molar ratio of the phospholipid molecules to the linker molecules is preferably 1:

1. The reaction conditions are preferably 65° C. for 2 hours. Preferably, in step 2, the phospholipid complex and the metal ion are dissolved in ethanol, and triethylamine is added to react to obtain the metal-phospholipid complex. The molar ratio of the phospholipid complex to the metal ion is preferably 1:(1-2), and the molar ratio of the phospholipid complex to the triethylamine is preferably 1:

1. The reaction conditions are preferably 60° C. for 2 hours. Preferably, in step 3, the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer to form an aqueous phase, and the organic phase and the aqueous phase are mixed to obtain drug-metal-phospholipid complex particles, wherein the organic compound is preferably ethanol; preferably, the mixing method of the organic phase and the aqueous phase includes a microfluidic chip or an ultrasonic Preferably, in step 4, the reaction conditions of the drug-metal-phospholipid complex particles and the targeting structure are incubation at 2-10° C. for 0.2-12 h.

20. The preparation method according to claim 16, 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-metal-phospholipid complex particles to form the targeted drug; Preferably, the preparation method of the micelles includes direct dissolution method, ethanol injection method, dialysis method or ultrasound method.

21. Use of the targeted drug according to claim 13 or 14 or the preparation method according to any one of claims 15 to 20 in drug delivery, imaging drugs, and vaccines.

22. The use according to claim 21, 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 blood 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.

23. A pharmaceutical agent containing the targeted drug according to claim 13 or 14, wherein the pharmaceutical agent is preferably a vaccine, and the vaccine is preferably a new coronavirus vaccine.

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