Method for preparing metal-phospholipid complex and use thereof

Abstract

Provided are a "one-step" method for preparing a metal-phospholipid complex and use thereof. The method for preparing the metal-phospholipid complex consists of the following step: reacting a phospholipid molecule, a linker molecule, and a metal ion together to form a metal-phospholipid complex. The metal-phospholipid complex is formed by the reaction of a phospholipid molecule portion, a linker molecule portion, and a metal ion portion; the phospholipid molecule portion is linked to the linker molecule portion; the linker molecule portion is linked to the metal ion portion by means of a coordination bond; and the metal-phospholipid complex is not a cationic lipid or an ionizable lipid.

Description

Preparation methods and applications of metal-phospholipid complexes Cross-reference to related applications This application claims priority to Chinese Patent Application No. 2025101181750, filed on January 24, 2025, entitled "Preparation method of metal-phospholipid complex and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0001] This application relates to the field of biotechnology, specifically providing a method for preparing metal-phospholipid complexes and their applications. Background Technology

[0002] With the continuous development of molecular biology techniques, our understanding of the link between genes and diseases is deepening. Nucleic acid drugs, as artificially synthesized DNA or RNA fragments with disease-treating functions, regulate the expression of pathogenic genes at the gene level, possessing potential therapeutic value. Compared with traditional small molecule drugs and antibody drugs, nucleic acid drugs are not limited by the druggability of target proteins, can treat a wider range of diseases, and have significant advantages such as high efficiency, low toxicity, and high specificity, providing new possibilities for disease treatment and showing promise as the third major type of drug after small molecule drugs and antibody drugs.

[0003] However, nucleic acid drugs are easily degraded by nucleases in vivo, and their large molecular weight and negative charge make it difficult for them to cross cell membranes to exert their effects. Therefore, finding a safe and effective nucleic acid drug delivery system is a bottleneck problem that urgently needs to be solved in nucleic acid drug development. Currently, carriers capable of delivering nucleic acid drugs can be mainly divided into viral vectors and non-viral vectors. Viral vectors pose an immunogenicity risk; among non-viral vectors, nanoparticles and small molecule conjugates are commonly used. Compared with small molecule conjugates that are directly conjugated to nucleic acid drugs, nanoparticles can more effectively encapsulate nucleic acid drugs, preventing them from being rapidly degraded by nucleases in vivo, thereby increasing their circulation time in vivo. The mechanism by which nanoparticles encapsulate nucleic acids is through the adsorption of negatively charged nucleic acids by positively charged cationic lipids. However, cationic lipids have significant cytotoxicity, and their toxic mechanisms include: ① causing cell shrinkage, reducing the number of mitotic cells, and vacuolation of the cytoplasm; ② interacting with biological proteins such as protein kinase C, thereby destroying their activity; ③ triggering the secretion of various pro-inflammatory cytokines and chemokines by activating p38 mitogen-activated protein kinase and nuclear factor κB transcription factor. Furthermore, ionizable lipids are lipids containing positively charged ionizable amine groups. They are uncharged under physiological conditions (pH = 7.4), but become positively charged at lower pH values ​​through protonation. Therefore, ionizable lipids can be used to partially or completely replace cationic lipids as the main component of nanoparticles responsible for nucleic acid adsorption. When nanoparticles containing ionizable lipids enter lysosomes of biological cells, the ionizable lipids become positively charged in the low pH environment (pH = 4.0-6.5) of the lysosome. Although ionizable lipids reduce some of the cytotoxic and highly inflammatory effects of permanently positively charged cationic lipids, their cytotoxicity and immunogenicity remain high. Lipid nanoparticles (LNPs) based on cationic lipids and / or ionizable lipids are currently available as nanoparticle nucleic acid drug delivery systems for clinical use. Cationic lipids and / or ionizable lipids are the main components of LNPs and are responsible for adsorbing nucleic acids. However, the cytotoxicity and immunogenicity mediated by cationic lipids and / or ionizable lipids remain important reasons for the high toxicity of LNPs.

[0004] Therefore, when delivering negatively charged drugs (such as nucleic acid drugs, protein drugs, peptide drugs, small molecule drugs, etc.) using delivery systems, 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 safer liposome delivery system and its efficient preparation method.

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

[0006] One of the objectives of this application is to provide a novel and efficient method for preparing metal-phospholipid complexes.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] A method for preparing a metal-phospholipid complex, wherein the preparation method comprises the following steps: reacting phospholipid molecules, linker molecules, and metal ions together to form a metal-phospholipid complex; the metal-phospholipid complex is composed of a phospholipid molecule portion, a linker molecule portion, and a metal ion portion, wherein the phospholipid molecule portion is connected to the linker molecule portion, the linker molecule portion is connected to the metal ion portion through a coordinate bond, and the metal-phospholipid complex is not a cationic lipid or an ionizable lipid.

[0009] In one embodiment, the phospholipid molecules, the linker molecules, and the metal ions are dissolved in ethanol and reacted to obtain the metal-phospholipid complex.

[0010] In one embodiment, the molar ratio of the phospholipid molecules, the linker molecules, and the metal ions is 1:1:(0.5-2).

[0011] In one embodiment, the molar ratio of the phospholipid molecules, the linker molecules, and the metal ions is 1:1:1, 1:1:1.5, 1:1:2, 3:3:2, or 2:2:1.

[0012] In one embodiment, the reaction conditions include reacting at 40-60°C for 1 to 5 hours.

[0013] In one embodiment, the phospholipid molecule portion is selected from one or more combinations of lecithin (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), 1-phosphoceramide (SP), phosphatidylinositol (PI), phosphatidylthreonine (PT), sphingomyelin (SM), lysophosphatidylcosinate (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysophosphatidylsphingomyelin (LSM), 1-phosphosphoamine (S1P), and derivatives thereof.

[0014] Preferably, the phospholipid molecule is partially selected from lecithin (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) Lysophosphatidylcholine (LPA) Lysophosphatidylglycerol (LPG) Lysophosphatidylinositol (LPI) Lysophosphatidylthreonine (LPT) Lysosphingolipid (LSM) 1-Sphingosine monophosphate (S1P) One or more of its derivatives; wherein R1 and R2 are each independently: decanoyl group Lauroyl Myristoyl Palmitoyl Stearoyl oleoyl Linoleyl erucic acid Arachidyl or phytyl

[0015] Preferably, the phospholipid molecule is selected from one or more combinations of lecithin (PC, Formula 1), phosphatidylethanolamine (PE, Formula 2), phosphatidic acid (PA, Formula 4), phosphatidylglycerol (PG, Formula 5), ​​and their derivatives.

[0016] Preferably, the phospholipid molecule is selected from one or more combinations of DSPC, DSPE, DSPA, DSPG, and their derivatives.

[0017] Preferably, the phospholipid molecule is selected from DSPC (Formula 46). DSPE (Equation 47) DSPA (Formula 48) DSPG (Formula 49) One or more of their derivatives.

[0018] Preferably, the phospholipid molecule portion is selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48), or DSPG (Formula 49).

[0019] In one embodiment, the linker molecule is selected from one or more combinations of curcumin, chlorogenic acid, anthocyanin, quercetin, dihydromyricetin, hesperidin, naringenin, apigenin, catechin, tea polyphenols, epigallocatechin gallate, ellagic acid, morin, epigallocatechin gallate, catechin gallate, gallocatechin gallate, or piperine C, and their derivatives.

[0020] Preferably, the linker molecule is selected from curcumin. chlorogenic acid anthocyanin Among them, R7 and R8 are H, OH, or OCH3, R3 is H or a glycosyl group, and R4, R5, and R6 are OH or a glycosyl group, quercetin. Dihydromyricetin hesperidin Naringin Celery Catechins Tea polyphenols Epigallocatechin gallate Ellagic acid Morin Epicatechin gallate Catechin gallate Gallocatechin gallate Fritillaria C One or more of their derivatives.

[0021] In one embodiment, the linker molecule is partially selected from curcumin (Formula 19) and dihydrocurcumin (Formula 36). Hexahydrocurcumin (Formula 37) Curcumin sulfate (Formula 38) Didemethoxycurcumin (Formula 39) One or more combinations thereof.

[0022] Preferably, the linker molecule is selected from one or more combinations of curcumin (Formula 19), hesperidin (Formula 24), tea polyphenols (Formula 28), and their derivatives.

[0023] Preferably, the linker molecule is selected from curcumin (Formula 19), hesperidin (Formula 24), or tea polyphenols (Formula 28).

[0024] In one embodiment, the metal ion is selected from Fe. 3+ Ag + Ba 2+ Ca 2+ Cd 2+ Cu2+ Fe 2+ Mn 2+ Mg 2+ Mo 2+ Zn 2+ Pt 2+ Au 2+ Al 3+ Ce 3+ Co 3+ Cr 3+ Eu 3+ Gd 3+ Ni 3+ W 3+ V 3+ Zr 3+ One or more combinations thereof.

[0025] Preferably, the metal ion portion is selected from Fe. 3+ Ca 2+ Al 3+ Mg 2+ One or more combinations thereof.

[0026] Preferably, the metal ion portion is selected from Fe. 3+ Ca 2+ Mg 2+ Or Al 3+ .

[0027] In one embodiment, 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+ Ca 2+ Mg 2+ Or Al 3+ .

[0028] Preferably, 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+ Ca 2+ Mg 2+ Or Al 3+ .

[0029] The second objective of this application is to provide a method for preparing metal-phospholipid complex particles.

[0030] A method for preparing metal-phospholipid complex particles, wherein the method comprises mixing (i) the aforementioned metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, 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, to obtain the metal-phospholipid complex particles; wherein the conjugated lipid that inhibits particle aggregation is not a cationic lipid or an ionizable lipid.

[0031] In one embodiment, the conjugated lipids that inhibit particle aggregation include PEG-lipid conjugates and / or PEG-DAA.

[0032] 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 one or more of their derivatives.

[0033] R1 and R2 are each independently: decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, squalyl, arachidoyl, or phytanoyl.

[0034] Preferably, the PEG-lipid conjugate is selected from one or more combinations of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000 or DSPE-PEG5000.

[0035] Preferably, the PEG-lipid conjugate is selected from DSPE-PEG2000 (Formula 53).

[0036] In one embodiment, the non-cationic lipid or non-ionizable lipid described in (iii) is one or more of cholesterol and its derivatives.

[0037] Preferably, the non-cationic lipid or non-ionizable lipid described in (iii) is cholesterol (Formula 40).

[0038] In one embodiment, the non-cationic lipids or non-ionizable lipids described in (iii) further include one or more combinations selected from lecithin 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, lysophosphatidylglycerol LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysophosphatidylsphingomyelin LSM, 1-phosphosphingosine S1P, cholesterol sulfate, and their derivatives.

[0039] Preferably, the non-cationic lipids or non-ionizable lipids described in (iii) further include those selected from lecithin (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), lysophosphatidylsphingomyelin (LSM, Formula 17), 1-phosphosphoin (S1P, Formula 18), and cholesterol sulfate. One or more of their derivatives.

[0040] Preferably, the non-cationic lipids or non-ionizable lipids described in (iii) include cholesterol, and a combination of one or more selected from DSPC, DSPE, DSPA or DSPG.

[0041] Preferably, the non-cationic lipids or non-ionizable lipids described in (iii) include cholesterol (Formula 40) and DSPC (Formula 46).

[0042] In one embodiment, the metal-phospholipid complex particles are made of (i) a metal-phospholipid complex, (ii) conjugated lipids that inhibit particle aggregation, and (iii) non-cationic lipids or non-ionizable lipids, wherein the metal-phospholipid complex accounts for 5% to 50% of the raw material in molar proportion, the conjugated lipids that inhibit particle aggregation account for 1% to 10% of the raw material in molar proportion, the cholesterol accounts for 15% to 80% of the raw material in molar proportion, and the non-cationic lipids or non-ionizable lipids other than cholesterol account for 0% to 51% of the raw material in molar proportion.

[0043] Preferably, the metal-phospholipid complex accounts for 5% to 40% of the raw material, more preferably 10% to 40%, and even more preferably 15% to 25%.

[0044] Preferably, the conjugated lipids that inhibit particle aggregation have a molar percentage of 2% to 10% in the raw material, more preferably 4% to 10%.

[0045] Preferably, the cholesterol has a molar percentage of 25% to 75% in the raw material, more preferably 35% to 75%, and even more preferably 40% to 46%.

[0046] Preferably, the non-cationic lipids or non-ionizable lipids other than cholesterol account for 0% to 50% of the raw material, more preferably 0% to 40%, and even more preferably 25% to 35%.

[0047] In one embodiment, the metal-phospholipid complex comprises 15%–25% of the raw material in molar proportion, the conjugated lipid that inhibits particle aggregation comprises 4%–10% of the raw material in molar proportion, the cholesterol comprises 40%–46% of the raw material in molar proportion, and the DSPC comprises 25%–35% of the raw material in molar proportion, wherein the metal ion portion of the metal-phospholipid complex is selected from Fe. 3+ .

[0048] Preferably, the metal-phospholipid complex accounts for 15% of the raw material in molar proportion, the conjugated lipid that inhibits particle aggregation accounts for 4% of the raw material in molar proportion, the cholesterol accounts for 46% of the raw material in molar proportion, and the DSPC accounts for 35% of the raw material in molar proportion; or the metal-phospholipid complex accounts for 25% of the raw material in molar proportion, the conjugated lipid that inhibits particle aggregation accounts for 10% of the raw material in molar proportion, the cholesterol accounts for 40% of the raw material in molar proportion, and the DSPC accounts for 25% of the raw material in molar proportion; the metal ion portion of the metal-phospholipid complex is selected from Fe. 3+ .

[0049] In one embodiment, the metal-phospholipid complex accounts for 5% to 50% of the raw material in molar proportion, the conjugated lipid that inhibits particle aggregation accounts for 2% to 10% of the raw material in molar proportion, the cholesterol accounts for 15% to 80% of the raw material in molar proportion, and the DSPC accounts for 0% to 51% of the raw material in molar proportion; the metal ion portion of the metal-phospholipid complex is selected from Al. 3+ .

[0050] Preferably, the metal-phospholipid complex accounts for 7% of the raw material in molar proportion, the conjugated lipid that inhibits particle aggregation accounts for 3% of the raw material in molar proportion, the cholesterol accounts for 56% of the raw material in molar proportion, and the DSPC accounts for 34% of the raw material in molar proportion; the metal ion portion of the metal-phospholipid complex is selected from Al. 3+ .

[0051] In one embodiment, the metal-phospholipid complex accounts for 5% to 40% of the raw material in molar proportion, the conjugated lipid that inhibits particle aggregation accounts for 1% to 10% of the raw material in molar proportion, the cholesterol accounts for 25% to 75% of the raw material in molar proportion, the DSPC accounts for 0% to 50% of the raw material in molar proportion, and the metal ion portion of the metal-phospholipid complex is selected from Mg. 2+ .

[0052] Preferably, the metal-phospholipid complex accounts for 15% of the raw material in molar proportion, the conjugated lipid that inhibits particle aggregation accounts for 1.8% of the raw material in molar proportion, the cholesterol accounts for 43.2% of the raw material in molar proportion, the DSPC accounts for 40% of the raw material in molar proportion, and the metal ion portion of the metal-phospholipid complex is selected from Mg. 2+ .

[0053] The third objective of this application is to provide a method for preparing drug-lipid particles.

[0054] A method for preparing drug-lipid particles, wherein a drug is encapsulated in metal-phospholipid complex particles prepared by the aforementioned preparation method to obtain the drug-lipid particles.

[0055] In one embodiment, drug-lipid particles are prepared by mixing (i) the metal-phospholipid complex prepared by the aforementioned preparation method, (ii) the conjugated lipid that inhibits particle aggregation, (iii) the non-cationic lipid or non-ionizable lipid, and the drug.

[0056] In one embodiment, the metal-phospholipid complex, the conjugated lipids that inhibit particle aggregation, and the non-cationic lipids or non-ionizable lipids are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer solution to form an aqueous phase, and the organic phase and the aqueous phase are mixed to obtain drug-lipid particles, wherein the drug is a negatively charged molecule.

[0057] Preferably, the organic compound is ethanol.

[0058] Preferably, the mixing method between the organic phase and the aqueous phase includes microfluidic chips or ultrasound.

[0059] Preferably, the mass ratio of the total mass of the metal-phospholipid complex, the conjugated lipids that inhibit particle aggregation, and the non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation to the mass of the drug is (20-100):1.

[0060] In one embodiment, the drug is selected from one or more combinations of nucleic acids, proteins, peptides, small molecules, nucleic acid analogs, protein analogs, and peptide analogs.

[0061] In one embodiment, the nucleic acid is selected from one or more combinations of mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, and artificial nucleic acids.

[0062] In one embodiment, the nucleic acid is the mRNA sequence encoding eGFP shown in SEQ ID No. 1, the mRNA sequence encoding the receptor-binding domain RBD of the novel coronavirus S1 subunit shown in SEQ ID No. 2, the mRNA sequence encoding NY-ESO-1 shown in SEQ ID No. 3, the siRNA sequence of the Bcl-2 gene with antisense strand as SEQ ID No. 4 and sense strand as SEQ ID No. 21, the siRNA sequence of the PLK1 gene with antisense strand as SEQ ID No. 6 and sense strand as SEQ ID No. 23, the siRNA sequence of the Gal-1 gene shown in SEQ ID No. 8, the ASO sequence of the STAT-3 gene shown in SEQ ID No. 10, the ASO sequence of the α-syn gene shown in SEQ ID No. 12, the ASO sequence of the Bcl-2 gene shown in SEQ ID No. 14, the mRNA sequence encoding the wild-type novel coronavirus S protein shown in SEQ ID No. 16, the double-stranded DNA sequence with antisense strand as SEQ ID No. 17 and sense strand as SEQ ID No. 25, the single-stranded DNA sequence shown in SEQ ID No. 18, or the siRNA sequence encoding NY-ESO-1 shown in SEQ ID No. 16. No. 19 and the antisense strand are the siRNA sequences of the B7-H4 gene shown in SEQ ID No. 26.

[0063] Preferably, the drug is mRNA, and the mRNA encodes a chimeric antigen receptor (CAR) or a TCR.

[0064] In one embodiment, the drug is an mRNA encoding a chimeric antigen receptor (CAR), wherein the CAR includes a transmembrane domain, a signal transduction domain, an antigen-binding domain, a co-stimulatory signal transduction region, and a junction region between the antigen-binding domain and the transmembrane domain.

[0065] 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 signal transduction 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 co-stimulatory signal transduction region is selected from at least one of SEQ ID No. 37, SEQ ID No. 39, and SEQ ID No. 75; and / or, the junction region between the antigen-binding domain and the transmembrane domain is selected from at least one of SEQ ID No. 40, SEQ ID No. 41, and SEQ ID No. 42.

[0066] Preferably, the drug is an mRNA with a nucleotide sequence as shown in SEQ ID No. 43.

[0067] The drug-lipid particles are mixed with the targeting structure to prepare a targeted drug; wherein the targeting structure is connected to the outer surface of the metal-phospholipid complex particles.

[0068] Preferably, the reaction conditions between the drug-lipid particles and the target structure are incubation at 2-10°C for 0.2-12 hours.

[0069] Preferably, the targeting structure is a DSPE-PEG2000-aptamer, and the preparation process is as follows: DSPE-PEG2000 and the aptamer are linked by an intermediate pair reaction to obtain the DSPE-PEG2000-aptamer.

[0070] 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, and more preferably DSPE-PEG2000-MAL and aptamer-C6-SH.

[0071] In one embodiment, the DSPE-PEG2000-aptamer forms micelles and then attaches to the outer surface of the drug-lipid particles to form the targeted drug.

[0072] Preferably, the preparation method of the micelles includes direct dissolution, ethanol injection, dialysis, or ultrasound.

[0073] This application also provides the application of the aforementioned method for preparing metal-phospholipid complexes in the preparation of metal-phospholipid complex particles.

[0074] This application also provides the application of the aforementioned method for preparing metal-phospholipid complex particles in the preparation of drug-lipid particles.

[0075] Compared with the prior art, the technical effects of this application are as follows:

[0076] The one-step preparation method for metal-phospholipid complexes can significantly shorten the time required for industrialization. In large-scale industrial 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 increasing production efficiency. From the perspective of industrial application, the one-step method is more suitable for the needs of large-scale production and has broad application prospects and extremely high practical value.

[0077] The metal-phospholipid complex prepared by the one-step method to prepare metal-phospholipid complex particles has a loading rate and eGFP positivity rate that are comparable to or even higher than those of the stepwise method.

[0078] The metal-phospholipid complex particles provided in this application, while achieving an efficacy no less than that of LNPs based on cationic lipids and / or ionizable lipids, do not use cationic or ionizable lipids, resulting in significantly reduced toxicity and enhanced biocompatibility. This makes them more suitable for delivering negatively charged drugs in vivo, broadening their applications to include drugs of various sizes. Targeted therapies can achieve high expression levels of nucleic acid drugs, exhibiting good targeting specificity and enabling highly effective treatment of a variety of diseases. Attached Figure Description

[0079] The various technical features of this application and their relationships will be further explained below with reference to the accompanying drawings. The drawings are exemplary; some technical features are not shown to scale, and some drawings may omit technical features commonly used in the art to which this application pertains that are not essential for understanding and implementing this application, or additionally show technical features that are not essential for understanding and implementing this application. In other words, the combination of various technical features shown in the drawings is not intended to limit this application. Furthermore, throughout this application, the same reference numerals refer to the same things. Specific descriptions of the drawings are as follows:

[0080] Figure 1-1-1 shows the eGFP-mRNA@MPP(Fe) provided in Example 1.3.5.1 of this application. 3+ Percentage of eGFP-positive cells resulting from 293T transfection;

[0081] Figure 1-2-1 shows the RBD-mRNA@MPP(Fe) provided in Example 1.3.5.1 of this application. 3+ RBD expression induced by 293T transfection;

[0082] Figure 1-3-1 shows the RBD-mRNA@MPP(Fe) provided in Example 1.3.5.1 of this application. 3+ The ability to induce humoral immunity;

[0083] Figure 1-4-1 shows the NY-ESO-1-mRNA@MPP(Fe) provided in Example 1.3.5.1 of this application. 3+ The ability to induce humoral immunity;

[0084] Figure 1-5-1 shows the RBD-mRNA@MPP(Fe) provided in Example 1.3.5.1 of this application. 3+ The ability to induce cellular immunity;

[0085] Figure 1-6-1 shows the NY-ESO-1-mRNA@MPP(Fe) provided in Example 1.3.5.1 of this application. 3+ The ability to induce cellular immunity;

[0086] Figure 1-1-2 shows the eGFP-mRNA@MPP(Al) provided in Example 1.3.5.2 of this application. 3+ Percentage of eGFP-positive cells resulting from 293T transfection;

[0087] Figure 1-2-2 shows the RBD-mRNA@MPP(Al) provided in Example 1.3.5.2 of this application. 3+ RBD expression induced by 293T transfection;

[0088] Figure 1-3-2 shows the RBD-mRNA@MPP(Al) provided in Example 1.3.5.2 of this application. 3+ The ability to induce humoral immunity;

[0089] Figure 1-4-2 shows the NY-ESO-1-mRNA@MPP(Al) provided in Example 1.3.5.2 of this application. 3+ The ability to induce humoral immunity;

[0090] Figure 1-5-2 shows the RBD-mRNA@MPP(Al) provided in Example 1.3.5.2 of this application. 3+ The ability to induce cellular immunity;

[0091] Figure 1-6-2 shows the NY-ESO-1-mRNA@MPP(Al) provided in Example 1.3.5.2 of this application. 3+ The ability to induce cellular immunity;

[0092] Figure 1-1-3 shows the eGFP-mRNA@MPP(Mg) provided in Example 1.3.5.3 of this application. 2+ Percentage of eGFP-positive cells resulting from 293T transfection;

[0093] Figures 1-2-3 show the RBD-mRNA@MPP(Mg) provided in Example 1.3.5.3 of this application. 2+ RBD expression induced by 293T transfection;

[0094] Figure 1-3-3 shows the RBD-mRNA@MPP(Mg) provided in Example 1.3.5.3 of this application. 2+ The ability to induce humoral immunity;

[0095] Figure 1-4-3 shows the NY-ESO-1-mRNA@MPP(Mg) provided in Example 1.3.5.3 of this application. 2+ The ability to induce humoral immunity;

[0096] Figure 1-5-3 shows the RBD-mRNA@MPP(Mg) provided in Example 1.3.5.3 of this application. 2+ The ability to induce cellular immunity;

[0097] Figure 1-6-3 shows the NY-ESO-1-mRNA@MPP(Mg) provided in Example 1.3.5.3 of this application. 2+ The ability to induce cellular immunity;

[0098] Figure 1-7-1 shows the Bcl-2-siRNA@MPP(Fe) provided in Example 1.3.6.1 of this application. 3+ The ability to silence target genes;

[0099] Figure 1-8-1 shows the PLK1-siRNA@MPP(Fe) provided in Example 1.3.6.1 of this application. 3+ The ability to silence target genes;

[0100] Figure 1-9-1 shows the Gal-1-siRNA@MPP(Fe) provided in Example 1.3.6.1 of this application. 3+ The ability to silence target genes;

[0101] Figure 1-7-2 shows the Bcl-2-siRNA@MPP(Al) provided in Example 1.3.6.2 of this application. 3+ The ability to silence target genes;

[0102] Figure 1-8-2 shows the PLK1-siRNA@MPP(Al) provided in Example 1.3.6.2 of this application. 3+ The ability to silence target genes;

[0103] Figure 1-9-2 shows the Gal-1-siRNA@MPP(Al) provided in Example 1.3.6.2 of this application. 3+ The ability to silence target genes;

[0104] Figure 1-7-3 shows the Bcl-2-siRNA@MPP(Mg) provided in Example 1.3.6.3 of this application. 2+ The ability to silence target genes;

[0105] Figure 1-8-3 shows the PLK1-siRNA@MPP(Mg) provided in Example 1.3.6.3 of this application. 2+ The ability to silence target genes;

[0106] Figure 1-9-3 shows the Gal-1-siRNA@MPP(Mg) provided in Example 1.3.6.3 of this application. 2+ The ability to silence target genes;

[0107] Figure 1-10-1 shows the STAT3-ASO@MPP(Fe) provided in Embodiment 1.3.7.1 of this application. 3+ The ability to silence target genes in cells;

[0108] Figure 1-11-1 shows the α-syn-ASO@MPP(Fe) provided in Embodiment 1.3.7.1 of this application. 3+ The ability to silence target genes in cells;

[0109] Figure 1-12-1 shows the Bcl-2-ASO@MPP(Fe) provided in Embodiment 1.3.7.1 of this application. 3+ The ability to silence target genes in cells;

[0110] Figure 1-10-2 shows the STAT3-ASO@MPP(Al) provided in Embodiment 1.3.7.2 of this application. 3+ The ability to silence target genes in cells;

[0111] Figure 1-11-2 shows the α-syn-ASO@MPP(Al) provided in Embodiment 1.3.7.2 of this application. 3+ The ability to silence target genes in cells;

[0112] Figure 1-12-2 shows the Bcl-2-ASO@MPP(Al) provided in Embodiment 1.3.7.2 of this application. 3+ The ability to silence target genes in cells;

[0113] Figure 1-10-3 shows the STAT3-ASO@MPP(Mg) provided in Embodiment 1.3.7.3 of this application. 2+ The ability to silence target genes in cells;

[0114] Figure 1-11-3 shows the α-syn-ASO@MPP(Mg) provided in Embodiment 1.3.7.3 of this application. 2+ The ability to silence target genes in cells;

[0115] Figure 1-12-3 shows the Bcl-2-ASO@MPP(Mg) provided in Embodiment 1.3.7.3 of this application. 2+ The ability to silence target genes in cells;

[0116] Figure 1-13-1 shows the S-mRNA@MPP(Fe) provided in Example 1.3.8.1 of this application. 3+ Transfection of 293T cells induced S protein expression;

[0117] Figure 1-14-1 shows the drug (dsDNA and ssDNA)@MPP (Fe) provided in Example 1.3.8.1 of this application. 3+ The function of );

[0118] Figure 1-13-2 shows the S-mRNA@MPP(Al) provided in Example 1.3.8.2 of this application. 3+ Transfection of 293T cells induced S protein expression;

[0119] Figure 1-14-2 shows the drug (dsDNA and ssDNA)@MPP (Al) provided in Example 1.3.8.2 of this application. 3+ The function of );

[0120] Figure 1-13-3 shows the S-mRNA@MPP(Mg) provided in Example 1.3.8.3 of this application. 2+ Transfection of 293T cells induced S protein expression;

[0121] Figure 1-14-3 shows the drug (dsDNA and ssDNA)@MPP(Mg) provided in Example 1.3.8.4 of this application. 2+ The function of );

[0122] Figure 1-15 shows the drug (dsDNA and ssDNA)@MPP (Fe) provided in Example 1.3.8.1 of this application. 3+ Fluorescence image of transfected cells;

[0123] Figure 2-1 is a differential scanning calorimeter of the phospholipid complex provided in Example 2.1.1 of this application;

[0124] Figure 2-2-1 shows the metal-phospholipid complex (Fe) provided in Example 2.1.1 of this application. 3+ The ultraviolet absorption spectrum of )

[0125] Figure 2-2-2 shows the metal-phospholipid complex (Al) provided in Example 2.1.2 of this application. 3+ The ultraviolet absorption spectrum of )

[0126] Figure 2-2-3 shows the metal-phospholipid complex (Mg) provided in Example 2.1.3 of this application. 2+ The ultraviolet absorption spectrum of )

[0127] Figure 2-3 shows the Fe under low pH (pH = 5.0) conditions provided in Example 2.2 of this application. 3+ Characterization of detachment from metal-phospholipid complexes;

[0128] Figures 2-4 show the drug-metal-phospholipid complex particles (Fe) provided in Example 2.3 of this application. 3+ Elemental analysis of )

[0129] Figures 2-5 show the siRNA / mRNA@MPP(Fe) provided in Example 2.4 of this application. 3+ Al 3+ or Mg 2+ The efficiency of nucleic acid (mRNA and siRNA) packaged by siRNA / mRNA@LNP;

[0130] Figures 2-6 show the siRNA / mRNA@MPP(Fe) provided in Example 2.5 of this application. 3+ Al 3+ or Mg 2+ ) and statistical graph of the nucleolysosomal escape ability of siRNA / mRNA@LNP;

[0131] Figures 2-7 show the MPP (Fe) provided in Embodiment 2.6 of this application. 3+ Al 3+ or Mg 2+ The percentage of eGFP-positive cells compared to LNP;

[0132] Figure 2-8 shows the MPP (Fe) provided in Embodiment 2.7 of this application. 3+ Al 3+ or Mg 2+ Comparison of the ability of LNP to promote RBD-mRNA expression;

[0133] Figure 2-9 shows the MPP (Fe) provided in Embodiment 2.7 of this application. 3+ Al 3+ or Mg 2+ Comparison of the humoral immunity-promoting abilities of LNP with those of LNP;

[0134] Figure 2-10 shows the MPP (Fe) provided in Embodiment 2.7 of this application. 3+ Al 3+ or Mg2+ Comparison of the ability of LNP to promote cellular immunity;

[0135] Figure 2-11 shows the siRNA / mRNA@MPP(Fe) provided in Example 2.5 of this application. 3+ Figure 1 shows the results of detecting the nucleolysosomal escape function of siRNA / mRNA@LNP.

[0136] Figure 3-1 shows the drug-metal-phospholipid complex particles (Fe) provided in Example 3 of this application. 3+ The role of intratumoral injection in the treatment of liver cancer;

[0137] Figure 3-2 shows the drug-metal-phospholipid complex particles (Al) provided in Example 3 of this application. 3+ The role of intratumoral injection in the treatment of liver cancer;

[0138] Figure 3-3 shows the drug-metal-phospholipid complex particles (Mg) provided in Example 3 of this application. 2+ The role of intratumoral injection in the treatment of liver cancer;

[0139] Figure 4-1-1 shows the targeted drug (CD19 CAR mRNA@Apt-MPP) in Example 6.3 of this application (Fe 3+ ), drug-metal-phospholipid particles (CD19 CAR mRNA@MPP) (Fe 3+ A statistical chart showing the percentage of CD19-CAR-positive cells;

[0140] Figure 4-1-2 shows 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+ A statistical chart showing the percentage of CD19-CAR-positive cells;

[0141] Figure 4-1-3 shows the targeted drug (CD19 CAR mRNA@Apt-MPP) (Mg) in Example 6.3 of this application. 2+ ), drug-metal-phospholipid particles (CD19 CAR mRNA@MPP) (Mg 2+ A statistical chart showing the percentage of CD19-CAR-positive cells;

[0142] Figure 4-2-1 shows the targeted drug (CD19 CAR mRNA@Apt-MPP) in Example 6.4 of this application (Fe 3+ ), drug-metal-phospholipid particles (CD19 CAR mRNA@MPP) (Fe 3+ Comparison of treatment effects on mice with acute B-lymphoblastic leukemia;

[0143] Figure 4-2-2 shows 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 treatment effects on mice with acute B-lymphoblastic leukemia;

[0144] Figure 4-2-3 shows the targeted drug (CD19 CAR mRNA@Apt-MPP) (Mg) in Example 6.4 of this application. 2+ ), drug-metal-phospholipid particles (CD19 CAR mRNA@MPP) (Mg 2+ Comparison of treatment effects on mice with acute B-lymphoblastic leukemia;

[0145] Figure 5-1-1 shows the targeted drug CD19 CAR mRNA@Apt(CD8)-MPP(Fe) in Example 7.1 of this application. 3+ ) and CD19 CAR mRNA@MPP(Fe 3+ A comparison of overall survival rates in mice treated with anti-acute B-lymphoblastic leukemia therapy;

[0146] Figure 5-1-2 shows the targeted drug CD19 CAR mRNA@Apt(CD8)-MPP(Al) in Example 7.1 of this application. 3+ ) and CD19 CAR mRNA@MPP (Al 3+ A comparison chart of overall survival rates in mice treated with anti-acute B-lymphoblastic leukemia therapy.

[0147] Figure 5-1-3 shows the targeted drug CD19 CAR mRNA@Apt(CD8)-MPP(Mg) in Example 7.1 of this application. 2+ ) and CD19 CAR mRNA@MPP(Mg 2+ A comparison of overall survival rates in mice treated with anti-acute B-lymphoblastic leukemia therapy;

[0148] Figure 5-2-1 shows the targeted drug ASO@Apt-MPP(Fe) in Example 7.2 of this application. 3+ ) and ASO@MPP(Fe 3+ A comparison chart of the overall survival rates of mice treated with anti-lung cancer therapy;

[0149] Figure 5-2-2 shows the targeted drug ASO@Apt-MPP (Al) in Example 7.2 of this application. 3+ ) and ASO@MPP(Al 3+ A comparison chart of the overall survival rates of mice treated with anti-lung cancer therapy;

[0150] Figure 5-2-3 shows the targeted drug ASO@Apt-MPP(Mg) in Example 7.2 of this application. 2+ ) and ASO@MPP(Mg 2+ A comparison chart of the overall survival rates of mice treated with anti-lung cancer therapy;

[0151] Figure 5-3-1 shows the targeted drug siRNA@Apt-MPP(Fe) in Example 7.3 of this application. 3+ ) and siRNA@MPP(Fe 3+ A comparison chart of overall survival rates in mice treated with pancreatic cancer.

[0152] Figure 5-3-2 shows the targeted drug siRNA@Apt-MPP(Al) in Example 7.3 of this application. 3+ ) and siRNA@MPP(Al 3+ A comparison chart of overall survival rates in mice treated with pancreatic cancer.

[0153] Figure 5-3-3 shows the targeted drug siRNA@Apt-MPP(Mg) in Example 7.3 of this application. 2+ ) and siRNA@MPP(Mg 2+ A comparison chart of overall survival rates in mice treated with pancreatic cancer.

[0154] Figure 6-1-1 shows Fe in Embodiment 8.2 of this application. 3+ A comparison of the survival rates of mice with acute B-lymphocytic leukemia 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.

[0155] Figure 6-1-2 shows the Al in Embodiment 8.2 of this application. 3+ A comparison of the survival rates of mice with acute B-lymphocytic leukemia 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.

[0156] Figure 6-1-3 shows the use of Mg in Example 8.2 of this application. 2+ A comparison of the survival rates of mice with acute B-lymphocytic leukemia 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.

[0157] Figure 6-2-1 shows Fe in Embodiment 8.2 of this application. 3+ A comparison of the treatment survival rates of lung cancer mice treated with ASO@Apt(AS1411)-MPP and ASO@Apt(AS1411)-LNP when the drug is ASO and the aptamer is AS1411.

[0158] Figure 6-2-2 shows Al in Embodiment 8.2 of this application. 3+ A comparison of the treatment survival rates of lung cancer mice treated with ASO@Apt(AS1411)-MPP and ASO@Apt(AS1411)-LNP when the drug is ASO and the aptamer is AS1411.

[0159] Figure 6-2-3 shows Mg in Example 8.2 of this application. 2+ A comparison of the treatment survival rates of lung cancer mice treated with ASO@Apt(AS1411)-MPP and ASO@Apt(AS1411)-LNP when the drug is ASO and the aptamer is AS1411.

[0160] Figure 6-3-1 shows Fe in Embodiment 8.2 of this application. 3+ A comparison of the survival rates of mice with pancreatic cancer treated with siRNA@Apt(P19)-MPP and siRNA@Apt(P19)-LNP when the drug is siRNA and the aptamer is P19.

[0161] Figure 6-3-2 shows the Al in Embodiment 8.2 of this application. 3+ A comparison of the survival rates of mice with pancreatic cancer treated with siRNA@Apt(P19)-MPP and siRNA@Apt(P19)-LNP when the drug is siRNA and the aptamer is P19.

[0162] Figure 6-3-3 shows the Mg used in Example 8.2 of this application. 2+ A comparison of the survival rates of mice with pancreatic cancer treated with siRNA@Apt(P19)-MPP and siRNA@Apt(P19)-LNP when the drug is siRNA and the aptamer is P19.

[0163] Figure 7-1-1 shows Fe in Embodiments 6.4, 9.1, and 9.2 of this application. 3+ A comparison of the overall survival rates of CD19 CAR mRNA@MPP versus CD19 CAR mRNA@Apt(CD62L)-MPP, CAR mRNA@Apt(CD8)-MPP, and CD19 CAR mRNA@Apt(CD3)-MPP in mice with acute B-lymphocytic leukemia.

[0164] Figure 7-1-2 shows Al in Embodiments 6.4, 9.1, and 9.2 of this application. 3+ A comparison of the overall survival rates of CD19 CAR mRNA@MPP versus CD19 CAR mRNA@Apt(CD62L)-MPP, CAR mRNA@Apt(CD8)-MPP, and CD19 CAR mRNA@Apt(CD3)-MPP in mice with acute B-lymphocytic leukemia.

[0165] Figure 7-1-3 shows the Mg in Examples 6.4, 9.1, and 9.2 of this application. 2+ A comparison of the overall survival rates of CD19 CAR mRNA@MPP versus CD19 CAR mRNA@Apt(CD62L)-MPP, CAR mRNA@Apt(CD8)-MPP, and CD19 CAR mRNA@Apt(CD3)-MPP in mice with acute B-lymphocytic leukemia.

[0166] Figure 8-1 shows the CD19 CAR mRNA@MPP(Fe) in Example 2.8 of this application. 3+ Al 3+ or Mg 2+ The percentage of CAR-positive cells in myeloid cells and CD19 CAR mRNA@LNP.

[0167] Figure 8-2 shows the CD19 CAR mRNA@Apt-MPP(Fe) in Example 6.5 of this application. 3+ Al 3+ or Mg 2+ The percentage of CAR-positive cells in peripheral blood T cells is shown in the figure for CD19 CAR mRNA@Apt-LNP.

[0168] Figure 8-3 shows CD19 CAR mRNA@Apt-MPP(Ca) modified with different target ratios in Example 6.6. 2+ ) and CD19 CAR mRNA@Apt-LNP(Ca 2+ CAR positivity rate;

[0169] Figure 8-4 shows CD19 CAR mRNA@Apt-MPP(Mg) modified with different target ratios in Example 6.6. 2+ ) and CD19 CAR mRNA@Apt-LNP(Mg 2+ CAR positivity rate;

[0170] Figure 8-5 shows the CD19 CAR mRNA@Apt-MPP(Fe) in Example 6.5 of this application. 3+Al 3+ or Mg 2+ The percentage of CAR-positive cells in peripheral blood myeloid cells is shown in the figure. Detailed Implementation

[0171] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions described in this application will be further described in detail below with reference to specific embodiments.

[0172] For ease of explanation, specific terms described herein, in the embodiments, and in the appended claims are explained collectively. Unless otherwise defined in this specification, scientific and technical terms used herein have the same meaning as understood and commonly used by those skilled in the art. Furthermore, unless the context otherwise requires, it should be understood that singular terms should include the same plural form, and plural terms should include the singular. Specifically, unless the context clearly indicates otherwise, the terms “at least one” and “one or more” as used herein and in the appended claims include one, two, three, or more. In this application, terms such as “multiple,” “various,” “multiple times,” and “multi-faceted” refer to quantities greater than or equal to two unless otherwise specified. For example, “one or more” means one or more of two.

[0173] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0174] In this application, "preferred," "preferred," and "ideal" refer to options that are optional, meaning they are selected from either "with" or "without." If multiple "preferred" options appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "preferred" option is independent.

[0175] While the numerical ranges and parameters used to define the broader scope of this application are approximate values, the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Here, "approximately" 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 "approximately" may mean that the actual value falls within the acceptable standard error of the mean, as determined by those skilled in the art. Except for experimental examples, or unless explicitly stated otherwise, it is understood that all ranges, quantities, values, and percentages used herein (e.g., to describe material usage, duration, temperature, operating conditions, quantity ratios, and the like) are modified with "approximately". Therefore, unless otherwise stated, the numerical parameters disclosed in this specification and the accompanying claims are approximate values ​​and are subject to change as needed. At a minimum, these numerical parameters should be understood as the indicated significant digits and values ​​obtained by applying general rounding.

[0176] The term "lipid" refers to a group of organic compounds, including, but not limited to, lipids of fatty acids. They are generally classified into three categories: "simple lipids," "compound lipids," and "derived lipids." "Simple lipids" include fats and oils as well as waxes; "compound lipids" include phospholipids and glycolipids; and "derived lipids" include steroids.

[0177] The term "ionizable lipid" refers to a lipid containing a positively charged ionizable amine group that can be protonated and become positively charged at lower pH values, while remaining uncharged under physiological pH conditions.

[0178] The term "neutral lipid" refers to any of a variety of lipids that exist at a selected pH as either uncharged or in a neutral zwitterionic form. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, cerebrosides, cholesterol, cerebrosides, and diacylglycerols.

[0179] The term "anionic lipid" refers to any lipid that carries a negative charge at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoylphosphatidylglycerol (POPG), and other anionic groups linked to neutral lipids.

[0180] The term “cationic lipid” refers to any of many types of lipids that carry a net positive charge at a selected pH, such as physiological pH. These lipids include, but are not limited to, N,N-dioleoyl-N,N-dimethylammonium chloride (“DODAC”); N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (“DOTMA”); N,N-distearate-N,N-dimethylammonium bromide (“DDAB”); N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (“DOTAP”); 3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (“DC-Chol”); and N-(1,2-dimyristyloxypropyl-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.

[0181] In this article, the phrase "phospholipid molecule part" refers to the structural part of the phospholipid molecule that constitutes the "metal-phospholipid complex," that is, the structure that originally belonged to the phospholipid molecule after the phospholipid molecule reacts with other substances.

[0182] The term "phospholipid" refers to lipids containing phosphate groups, belonging to complex lipids, also known as phospholipids or phospholipid lipids. Phospholipids are the main components of biological membranes, divided into two main categories: glycerophospholipids and sphingomyelins, composed of glycerol and sphingosine, respectively. Phospholipids are amphoteric molecules, with one end being a hydrophilic nitrogen- or phosphorus-containing head and the other end being a hydrophobic (lipophilic) long hydrocarbon chain. For this reason, the hydrophilic ends of phospholipid molecules are close to each other, and the hydrophobic ends are close to each other, often forming a phospholipid bilayer, i.e., the structure of the cell membrane, together with other molecules such as proteins, glycolipids, and cholesterol.

[0183] In this article, the phrase "linker molecule portion" refers to the structural portion of the "metal-phospholipid complex" that originates from the linker molecule, that is, the structure that originally belonged to the linker molecule after the linker molecule reacts with other substances.

[0184] In this article, the phrase "metal ion part" refers to the structural part that constitutes the "metal-phospholipid complex" and originates from the metal ion, that is, the structure that originally belonged to the metal ion after the metal ion reacts with other substances.

[0185] In this article, the phrase "phospholipid complex" refers to a complex formed by the reaction and linkage of the aforementioned phospholipid molecule portion having a phosphate group with the aforementioned linker molecule portion.

[0186] In this article, the phrase "metal-phospholipid complex" refers to a complex composed of the aforementioned phospholipid molecule portion having a phosphate group, the aforementioned linker molecule portion, and the aforementioned metal ion portion, wherein the aforementioned phospholipid molecule portion is connected to the aforementioned linker molecule portion, the aforementioned linker portion is connected to the aforementioned metal ion portion through a coordinate bond, and the metal-phospholipid complex is neither a cationic lipid nor an ionizable lipid.

[0187] The term "drug-metal-phospholipid complex particle" (also known as drug-lipid particle) refers to a drug-loaded metal-phospholipid complex particle, in which the drug is embedded, loaded, or bound to the metal-phospholipid complex through chemical or non-chemical bonding.

[0188] The term "CD8" refers to a dimer coreceptor that enables T cells to recognize peptides presented by histocompatibility complex class I proteins; it is a surface marker protein of cytotoxic T lymphocytes (CTLs). One way CD8+ T cells initiate apoptosis is through the secretion of perforin and granzymes (two types of cytotoxic proteins). Perforin is a cytolytic protein that forms pores in the cell membrane of target cells. CTLs use these pores to guide the release of granzymes, a class of serine proteases that further induce apoptosis within the cytoplasm. These proteases shut down the target cell by cleaving viruses and cellular proteins involved in normal cell maintenance. The apoptotic target cell is then cleared by nearby phagocytes. In addition to directed apoptosis, CD8+ T cells can also indirectly kill target cells by releasing cytokines such as TNF-α.

[0189] The term "CD62L," also known as L-selectin, is a gene family of leukocyte-endothelial cell adhesion molecules. Its extracellular N-terminus contains one C-type lectin-like domain, one EGF-like domain, and two CCP domains. L-selectin is structurally expressed by leukocytes and mediates binding to oligosaccharide ligands expressed by vascular endothelial cells. "CD62L" is a family of vascular adhesion molecules with a close structural-functional relationship; its main function is to promote the rolling behavior of leukocytes along endothelial cells before firm adhesion and subsequent migration. They differ from other adhesion molecules, firstly because their adhesive properties involve only leukocyte-platelet-endothelial interactions within the vascular system, and secondly because, unlike other adhesion molecules, selectins form protein-protein bonds. Selectins possess protein-like lectin groups that bind to carbohydrate ligands, thereby forming protein-carbohydrate bonds. In 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.

[0190] The term "targeted delivery vehicle" refers to a system with specific targeting capabilities and drug delivery capacity. Targeted delivery vehicles can improve the pharmacokinetic properties of drugs in vivo, increase the targeted accumulation of drugs at specific therapeutic sites or even within specific cell types, enhance efficacy, and reduce toxic side effects. They can be nanoparticles or macromolecules in combination. In this application, the targeted delivery vehicle can be used to encapsulate CAR nucleic acid drugs.

[0191] The term "lipid delivery system" refers to a technology for delivering small or large molecules. It mainly 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 the bioavailability and therapeutic efficacy of the small or large molecules.

[0192] The term "exosome" refers to a nanoscale vesicle secreted by cells, which contains bioactive substances such as proteins and nucleic acids (e.g., mRNA, miRNA).

[0193] The term "pseudovirus" refers to an artificially constructed virus-like particle. It typically combines certain key structures of a virus (such as envelope proteins) with non-viral nucleic acids (such as reporter genes). It does not have the replication ability of a complete virus, but it can mimic some biological behaviors of viruses, such as entering cells. It can be used in many fields, including research on viral infection mechanisms, vaccine development, and drug screening.

[0194] 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 small hormone molecules (e.g., adrenaline) produced by metabolic processes in the body, or exogenous, such as small drug molecules (e.g., artemisinin) extracted from plants. They can exert their effects by interacting with biological macromolecules (e.g., proteins, nucleic acids), such as participating in processes like signal transduction, enzyme inhibition, or activation.

[0195] The term "phospholipid transport system" refers to a transport system constructed using phospholipid components, primarily used to transport various substances (such as drugs, nutrients, biomolecules, etc.) from one location to another.

[0196] The term "LNP" refers to a nanoscale colloidal delivery system formed by the self-assembly of components such as cationic lipids or ionizable lipids, auxiliary lipids, cholesterol, and polyethylene glycol-modified 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 interactions, thereby effectively protecting nucleic acids and promoting their delivery to target cells.

[0197] The term "aptamer," derived from the Latin word "aptus," refers to a single-stranded oligonucleotide (RNA) or single-stranded oligodeoxynucleotide (DNA) composed of 20-60 bases. "Ampamer" is used to refer to polymers 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 genomes, 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, including proteins, small molecules, ions, and cells. Nucleic acid aptamers are obtained through SELEX screening, a technique that uses random single-stranded nucleic acid sequence libraries to identify aptamers that specifically bind with high affinity to their targets.

[0198] The term "GalNac" in this specification refers to a partial structure of N-acetyl-D-galactosamine (GalNAc) capable of binding to the desialylate glycoprotein receptor (ASGPR) on hepatocytes of the liver. GalNAc contains a phosphate ester group or a thiophosphate ester group for binding a linear or branched linker structure to an oligonucleotide. Such a structure containing a phosphate ester group or a thiophosphate ester group for binding can be called "GalNAc". Unless otherwise stated, there is no limitation on the number of GalNacs contained in a GalNac, and those known and disclosed in this specification can be used. The structure of GalNAc can be modified as long as the ability to bind to ASGPR is maintained. This also includes GalNAc with protecting groups introduced during the preparation process. The term "Galnac" has its usual scientific meaning and here refers to N-acetylgalactosamine and its IUPAC name: 2-(acetylamino)-2-deoxy-D-galactose.

[0199] The term "P19" refers to a nucleic acid sequence that can specifically recognize pancreatic cancer cells.

[0200] The term "targeted drug" (also known as a targeted formulation) refers to a drug or formulation that has been endowed with the ability to target.

[0201] The term "transmembrane domain" refers to the transmembrane sequence of a CAR, which can be engineered to include a transmembrane domain that links the antigen-binding domain of the CAR to an intracellular domain. The transmembrane domain can be any protein structure that is thermodynamically stable in the membrane, typically an α-helix containing several hydrophobic residues.

[0202] The term "signal transduction domain" refers to the sequence encoding a signal peptide or a sequence encoding a substance that performs the same function as the signal peptide. A "signal transduction domain" is a functional portion of a protein that functions by transmitting information within the cell, thereby regulating cellular activity via a defined signal transduction pathway by generating a second messenger or by acting as an effector in response to such a messenger. The signal peptide causes the nascent protein to be directed to the endoplasmic reticulum and subsequently to the cell surface where it is expressed when CARs are expressed in cells such as T cells. The core of the signal peptide may contain long segments of hydrophobic amino acids that tend to form a single α-helix.

[0203] The term "antigen-binding domain" generally refers to a domain capable of specifically binding to an antigen, specifically a chimeric antigen receptor domain or fragment possessing the ability to bind specifically to a target antigen. The antigen-binding domain is the antigen-recognizing portion of a CAR. Many antigen-binding domains are known in the art, including those based on antibody, antibody mimicry, and T-cell receptor antigen-binding sites. For example, an antigen-binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single-domain antibody; an artificial single-chain binder such as Darpin (a designed ankyrin repeat protein); or a single chain derived from a T-cell receptor.

[0204] The term "co-stimulatory signal transduction domain" refers to an intracellular signal transduction domain derived from co-stimulatory protein receptors such as CD28, 41BB, and ICOS, which can enhance T cell activation through T cell receptors. Specifically, the "co-stimulatory signal transduction domain" refers to the domain within the CAR molecule responsible for providing a second activation signal to T cells, primarily 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, co-stimulating molecules and activating intracellular signals, enabling T cells to proliferate continuously and release cytokines, thereby enhancing the anti-tumor ability of T cells.

[0205] The term "transmembrane domain-to-transmembrane domain linker region" refers to the region responsible for linking the antigen-binding domain and the transmembrane domain. Spacer domains, such as oligopeptides or polypeptides, may be incorporated between the extracellular and transmembrane domains of a chimeric membrane protein, or between the cytoplasmic and transmembrane domains of a chimeric membrane protein, serving to link the transmembrane domain to the extracellular or cytoplasmic domain in the polypeptide chain. Spacer domains may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0206] The term "chimeric antigen receptor (CAR)" refers to an engineered receptor that is arbitrarily and specifically grafted onto immune effector cells. A CAR is modularly composed of four main domains: a single-chain variable domain (scFv) antibody at the extracellular terminal for target recognition, a spacer region linked to a transmembrane domain, and an intracellular signaling domain with or without a linked co-stimulatory domain. The involvement of homologous antigens on the surface of the target cell initiates the activation of the CAR-engineered immune cell, leading to a durable immune cell response. Each CAR domain has a unique function and influences the redirected immune cell activation; for each specific application, CAR design requires some adjustments to provide optimized targeting and immune cell activation. It cleverly integrates antigen recognition with intracellular activation elements, breaking the limitations of immune cell recognition and allowing immune cells to precisely target tumor cells. The CAR molecule comprises three main parts: an extracellular domain, a transmembrane domain, and an intracellular domain. The antigen-binding domain in the extracellular domain is typically an antibody-derived single-chain fragment (scFv) molecule, mainly composed of the antibody's variable light chain (VL) and variable heavy chain (VH) linked by an intermediate linker region, and then connected to the transmembrane domain via a hinge region, responsible for antigen recognition. The main function of the transmembrane domain is to anchor the CAR molecule to the cell membrane, playing a crucial role in the stability of CAR molecule expression. The intracellular domain includes a co-stimulatory domain and a signal transduction domain, which together are responsible for the complete activation of T cells.

[0207] The term "T cell receptor (TCR)" refers to a complex of membrane proteins involved in T cell activation in response to antigen presentation. It consists of variable (V) and constant (C) region domains, with the α and β chains forming the antigen recognition unit. Each variable region contains three complementarity-determining regions (CDR1, CDR2, and CDR3). During TCR ligand recognition, the less diverse CDR1 and CDR2 contact the α-helices flanking the pMHC, while CDR3 contacts the central peptide. CDR3 is the most critical sequence determining the TCR's antigen recognition specificity. TCR stimulation is triggered by the major histocompatibility complex (MHC) molecule on antigen-presenting cells, which present antigenic peptides to T cells and bind to the TCR complex, inducing a cascade of intracellular signaling.

[0208] 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 CRISPR-Cas9, CRISPR-Cas, etc.), antibody drugs (such as monoclonal antibodies, bispecific antibodies), immune-bridging therapies (such as TCE (T-cell Engager), ADC (Antibody-Drug Conjugate), APDC (Antibody-Peptide-Drug Conjugate), etc.), targeted therapies (including chemotherapy or radiotherapy) (such as ADC (Antibody-Drug Conjugate), APDC (Antibody-Peptide-Drug Conjugate), etc.), aptamer drugs (such as dual 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 precisely identify specific targets on or inside immune cells, and intervene in the body's immune response by genetically modifying immune cells or regulating their function, such as activating, inhibiting, or altering their differentiation state, thereby treating diseases.

[0209] The term "chimeric antigen receptor (CAR) drug" refers to a novel therapeutic agent that exerts its effect by genetically modifying target cells in vivo or in vitro to express a CAR. A CAR is a synthetically produced receptor whose structure includes a single-chain antibody variable region (scFv) capable of recognizing tumor antigens, a transmembrane region, and an intracellular signal transduction region. Typically, CAR drugs exert their therapeutic effect 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.).

[0210] The term "in situ CAR" refers to a CAR nucleic acid sequence encoded by mRNA technology. This sequence recodes a T-cell receptor using mRNA, enabling the production of CAR-expressing cells in vivo. The CAR contains an antigen domain, a transmembrane domain, a co-stimulatory signal transduction region, and a signal transduction domain. In situ CAR refers to the in-situ transformation 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 preparation process is significantly simplified, and costs are substantially reduced. Using messenger RNA as a carrier of the chimeric antigen receptor, the CAR is transferred into the patient's T cells, activating them and equipping them with the CAR (carrier antigen receptor) for targeting and navigation. This transforms ordinary T cells into "super soldiers," i.e., CAR-T cells. These cells utilize their CAR to specifically recognize tumor cells in the body and release a large number of effector factors through immune responses, which efficiently kill tumor cells, thereby achieving the goal of treating malignant tumors.

[0211] The term "lipid vesicle" refers to any lipid composition that can be used to deliver compounds, including, but not limited to, liposomes, in which an aqueous volume is encapsulated by an amphiphilic lipid bilayer; or in which lipids encapsulate the interior of a macromolecular component, such as mRNA, accompanied by a reduced aqueous interior; or lipid aggregates or micelles, in which the encapsulated component is contained within a relatively disordered lipid mixture. In this document, metal-phospholipid complex particles (MPPs) are referred to as "lipid vesicles," and drugs, such as nucleic acid mRNA, are encapsulated as components within MPPs; this "encapsulation" can be fully encapsulated and / or partially encapsulated.

[0212] The term "hydrophobic lipid" refers to a compound having nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and these groups are optionally substituted with one or more aromatic, alicyclic, or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerols, dialkylglycerols, N,N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0213] The term "non-cationic lipid or non-ionizable lipid" refers to lipids that are neither cationic nor non-ionizable, such as anionic lipids or neutral lipids.

[0214] In the components of metal-phospholipid complex particles, "non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation" in (iii) means that the non-cationic lipids or non-ionizable lipids in (iii) are the lipids remaining in the metal-phospholipid complex particles after excluding metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation.

[0215] The term "fusion" refers to the ability of liposomes, drug-lipid particles, targeted drugs, or other drug delivery systems to fuse with a cell membrane. The membrane can be the plasma membrane or the membrane surrounding organelles such as endosomes, the nucleus, etc.

[0216] In metal-phospholipid complex particles, non-cationic or non-ionizable lipids, other than the metal-phospholipid complex and conjugated lipids that inhibit particle aggregation, are mainly present as vesicle-forming lipids. The term "vesicle-forming lipids" tends to include any amphiphilic lipid with a hydrophobic portion and a polar head group that can spontaneously form bilayer vesicles in water, such as most phospholipids.

[0217] In metal-phospholipid complex particles, conjugated lipids that inhibit particle aggregation are primarily present as vesicle-embedded lipids. The term "vesicle-embedded lipids" tends to include any amphiphilic lipids that are stable and bound to the lipid bilayer, as well as other amphiphilic lipids whose hydrophobic portion contacts the hydrophobic region of the inner bilayer membrane and whose polar head group portion faces the polar surface of the membrane. Vesicle-embedded lipids include lipids that are independently adaptable to a non-layered phase and also capable of assuming 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 coupled with dialkoxypropyl, PEG coupled with diacylglycerol, PEG coupled with phosphatidylethanolamine, and PEG conjugated with ceramide (see U.S. Patent No. 5,885,613, which is incorporated herein by reference).

[0218] The term "amphiphilic lipid" refers to any suitable material in which the hydrophobic portion of the lipid material faces the hydrophobic phase, while the hydrophilic portion faces the aqueous phase. Amphiphilic lipids are often the main components of lipid vesicles. The hydrophilic nature arises from the presence of polar or charged groups such as carbohydrates, phosphates (esters), carboxyl, sulfate, amino, thiol, nitro, hydroxyl, and other similar groups. Hydrophobicity can be imparted by the inclusion of nonpolar 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, aminolipids, and sphingolipids. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, distearateoyl phosphatidylcholine, or dilinoleoyl phosphatidylcholine. Other phosphorus-deficient compounds, such as sphingomyelin, glycosphingolipids, diacylglycerols, and β-acyloxy acids, are also in the group referred to as amphiphilic lipids. Furthermore, the aforementioned amphiphilic lipids can be mixed with other lipids, including triglycerides and sterols.

[0219] The term "diacylglycerol" refers to a compound having a 2-fatty acyl chain, where R1 and R2 each have 2-30 carbon atoms independently bonded to the 1- and 2-positions of glycerol via ester bonds. The acyl group can be saturated or unsaturated to varying degrees. Diacylglycerol has the following formula 54:

[0220]

[0221] The term "diacylglycerol-coupled polyethylene glycol" refers to the conjugated lipids in this application that inhibit particle aggregation, which can be diacylglycerol-coupled polyethylene glycol, i.e., diacylglycerol-polyethylene glycol conjugates (DAG-PEG conjugates or PEG-DAG conjugates). 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 distearatelglycerol (C18)-PEG conjugate (DSG). Those skilled in the art will readily understand that other diacylglycerols can be used in the DAG-PEG conjugates of this application. Suitable DAG-PEG conjugates used in this application and methods for their preparation and use are disclosed in U.S. Application No. 10 / 136,707, published as USPA 2003 / 0077829, and PCT Patent Application No. CA 02 / 00669, the entire contents of which are incorporated herein by reference.

[0222] The term "dialkoxypropyl" refers to a compound having a 2-alkyl chain, where both R1 and R2 independently have 2-30 carbons. The alkyl group can be saturated or unsaturated to varying degrees. Dialkoxypropyl has the following formula 55:

[0223]

[0224] The term "dialkoxypropyl-coupled PEG" refers to the conjugated lipid that inhibits particle aggregation in this 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:

[0225] In Formula 56, R1 and R2 are independently selected and are long-chain alkyl groups having about 10 to about 22 carbon atoms. The 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 identical, i.e., both R1 and R2 are tetradecyl (i.e., bistetradecyl), both R1 and R2 are octadecyl (i.e., bisoctadecyl), etc. In Formula I, the PEG is polyethylene glycol having an average molecular weight of about 550 to about 10,000 Daltons and optionally substituted at the terminal hydroxyl position with an alkyl, alkoxy, acyl, or aryl group. In a preferred embodiment, the PEG has an average molecular weight of about 1,000 to about 5,000 Daltons, more preferably about 1,000 to about 3,000 Daltons, and even more preferably about 2,000 Daltons. The PEG may optionally be substituted with alkyl, alkoxy, acyl, or aryl groups. In Formula I, L is the linker moiety. Any linker moiety suitable for coupling the PEG to the dialkoxypropyl backbone may be used. Suitable linker moieties include, but are not limited to, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbonate (OC(O)O-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), succinyl (-(O)CCH2CH2C(O)-), ether, disulfide, and combinations thereof. Other suitable linkers are well known in the art.

[0226] Phosphatidylethanolamines can be conjugated with polyethylene glycol to serve as the conjugated lipids for inhibiting particle aggregation in this application, forming a bilayer stable component. The phosphatidylethanolamines contain 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, having carbon chain lengths in the C10-C20 range, are preferred. Phosphatidylethanolamines containing monounsaturated or diunsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, the following: bis(myristoyl)phosphatidylethanolamine (DMPE), dipalmitoyl)phosphatidylethanolamine (DPPE), dioleoyl)phosphatidylethanolamine (DOPE), and distearate (DSPE).

[0227] Ceramides, such as phosphatidylethanolamine, can be coupled with polyethylene glycol to form a conjugated lipid that inhibits particle aggregation as described in this application, creating a bilayer stable component. The ceramide has multiple acyl groups with different chain lengths and degrees of saturation. Those skilled in the art will understand that, compared to phosphatidylethanolamine, ceramide has only one acyl group, which can be readily varied according to its chain length and degree of saturation. Ceramides suitable for use according to this application are commercially available. Furthermore, ceramides can be isolated from eggs and brains, for example, 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 routes proposed in the preceding application, ceramides having saturated or unsaturated fatty acids with carbon chain lengths in the C2-C31 range can be prepared.

[0228] The terms “ATTA” or “polyamide” refer to, but are not limited to, the compounds disclosed in U.S. Patent Nos. 6,320,017 and 6,586,559, all of which are incorporated herein by reference. These compounds include those having the following formula 57:

[0229]

[0230] 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 bonded form an azide moiety; R2 is a member selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, and amino acid side chains; R3 is a member selected from the group consisting of hydrogen, halogen, hydroxyl, alkoxy, mercapto, 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. Those skilled in the art will appreciate that other polyamides may be used in the compounds of this application.

[0231] The term "similar" refers to an analogue that performs the same or similar function, or a derivative of the same parent nucleus that performs the same or similar function.

[0232] As used herein, the terms “mRNA” or “messenger RNA” or “messenger RNA” are used interchangeably and refer to a single-stranded polynucleotide transcribed from one strand of DNA as a template, carrying genetic information, and capable of directing protein synthesis.

[0233] As used herein, the terms "sgRNA," "small guide RNA," "guide RNA," or "gRNA" are used interchangeably. gRNA is a small non-coding RNA that guides the insertion or deletion of uridine residues into the kinetoplastid during RNA editing and can pair with pre-mRNA. gRNA edits RNA molecules that are approximately 60-80 nucleotides in length and are transcribed from a single gene.

[0234] As used herein, the terms “circRNA” or “circular RNA” or “circular polynucleotide” or “circular RNA” are used interchangeably and mean a polynucleotide molecule having a structure without free ends (i.e., without free 3' and / or 5' ends), such as polynucleotides that form a cyclic or ring structure by covalent or non-covalent bonds.

[0235] As used herein, the terms “microRNA” or “miRNA” or “microRNA” are used interchangeably and refer to a non-coding single-stranded polynucleotide of approximately 22 nucleotides in length with free 3' and 5' ends, which can regulate cellular biological functions by binding to the 3'-untranslated region (3'-UTR) of the mRNA of a target gene, thereby inhibiting the translation of the target gene protein.

[0236] 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 segment of a target gene or mRNA and can bind to the target gene / mRNA through the base complementarity principle, thereby blocking gene expression. These are single-stranded poly(deoxy)ribonucleotides, including antisense DNA and antisense RNA.

[0237] 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 that are 20 to 25 nucleotides in length and can induce the degradation of target gene mRNA.

[0238] As used herein, the terms “ecDNA” or “extrachromosomal circular DNA” are used interchangeably and refer to DNA that has detached from the chromosome and exists in a circular structure.

[0239] The term "nucleic acid derivative" refers to modifications or substitutions of nucleic acid sequences, including but not limited to chemical modifications of residues, substitutions of nucleotides or deoxynucleotides, modifications to improve sequence half-life or stability, and labeling modifications. For example, chemical modifications include, but are not limited to, phosphorylation, methylation, amination, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopization of any one or more bases. Substitutions of nucleotides or deoxynucleotides include, but are not limited to, nucleic acid analogs that replace the sugar-phosphate backbone with a polypeptide or other backbone (replacing DNA or RNA with PNA). Modifications to improve sequence half-life or stability include, but are not limited to, PEG-linked modifications and fluorine modifications. Labeling modifications include, but are not limited to, linking to fluorescent groups, amino groups, biotin, digoxigenin, small peptides, etc.

[0240] The term "artificial nucleic acid" refers to nucleic acid molecules that have been artificially modified, including but not limited to base modifications, ribose modifications, PNA, etc.

[0241] The term "nucleic acid" refers to a polymer consisting of at least two deoxynucleotides or nucleotides, existing in single or double strands. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, having similar binding properties to reference nucleic acids, and being metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by a mixture of 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” contains a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together by phosphate groups. "Bases" include purines and pyrimidines, further including 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 replace new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates (esters), and alkyl halides. DNA can exist as antisense, plasmid DNA, portions of plasmid DNA, pre-compressed DNA, polymerase chain reaction (PCR) products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. The terms nucleic acid, gene, cDNA, mRNA encoded by a gene, and interfering RNA molecules may be used interchangeably.

[0242] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that includes a partial or full-length coding sequence, which is necessary for the production of a polypeptide or polypeptide precursor (e.g., a polypeptide or polypeptide precursor from hepatitis A, B, C, D, E, or G viruses; or herpes simplex virus).

[0243] When used in this article, "gene product" refers to the product of a gene, such as, for example, transcripts of DNA, mRNA.

[0244] The phrase "silencing of target gene expression" refers to the ability of the siRNA of this application to silence a target gene. To determine the degree of gene silencing, a sample or assay of cells from a target organism or culture expressing a specific construct, and the control not expressing the construct, is compared to a control sample. The control sample (lacking construct expression) is set as 100% relative. Successful inhibition of target gene expression is achieved when the test value relative to the control is approximately 90%, preferably 50%, more preferably 25-0%. Suitable assays include, for example, detection of protein or mRNA levels using techniques known to those skilled in the art, such as spotting, RNA blotting, in situ hybridization, ELISA, immunoprecipitation, enzymatic action, and phenotypic assays known to those skilled in the art.

[0245] The "therapeutic effective amount" or "effective amount" of siRNA is an amount sufficient to produce a desired effect, which is, for example, a reduction in target sequence expression compared to the normal expression level detected in the absence of siRNA.

[0246] When used herein, the term "aqueous solution" refers to a composition which contains all or part of water.

[0247] When used herein, the term "organic lipid solution" refers to a composition which contains, in whole or in part, an organic solvent having lipids.

[0248] As used herein, "systemic delivery" refers to delivery that results in the widespread biodistribution of a compound in an organism. Some administration techniques may result in systemic delivery of certain compounds but not others. Systemic delivery means that an effective, preferably therapeutic, amount of the compound comes into contact with most of the body. To achieve widespread biodistribution, a blood survival period is typically required so that the compound is not rapidly degraded or cleared (e.g., by initial passage through an organ (liver, lung, etc.) or by rapid, nonspecific cell binding) before reaching the disease site distal to the administration site. Systemic delivery of a targeted drug can be carried out in any manner known in the art, including, for example, intravenous, subcutaneous, or intraperitoneal delivery. In a preferred embodiment, systemic delivery of the targeted drug is via intravenous delivery.

[0249] When used in this article, "local delivery" refers to the direct delivery of a compound to its target site within a living organism. For example, a compound can be locally delivered by direct injection into disease sites such as tumors or other target sites such as inflammatory sites or target organs such as the liver, heart, pancreas, and kidneys.

[0250] RNA ensembles can be used to provide long precursor RNAs, or long precursor RNAs that are substantially or completely identical to selected target sequences 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 RNAs can be a mixed ensemble (obtained from cells or tissues, transcribed from cDNA, etc.) or can represent a single target sequence. The RNAs can be naturally occurring, for example, isolated from tissue or cell samples, synthesized in vitro, for example, using T7 or SP6 polymerases and PCR products or cloned cDNA; or synthesized chemically.

[0251] To form long dsRNAs, for synthetic RNAs, complements can also be transcribed and hybridized in vitro to form dsRNAs. If naturally occurring RNA groups are used, for example by transcribing cDNAs corresponding to the RNA group, or by using RNA polymerases, RNA complements are also provided (e.g., forming dsRNAs, which are then digested by E. coli RNase III or digestion enzymes). The precursor RNAs then hybridize to form double-stranded RNAs for digestion. The dsRNAs can be directly encapsulated in SNALPs or can be digested in vitro before encapsulation.

[0252] Alternatively, one or more DNA plasmids encoding one or more siRNA templates can be encapsulated in nucleic acid-lipid particles. For example, based on naturally occurring transcription units of small nuclear RNA U6 or human RNase P RNA H1, siRNA can be transcribed from DNA templates in plasmids into sequences that automatically fold into double strands with hairpin loops, said plasmids having RNA polymerase III transcription units (see, Brummelkamp, ​​et al., Science 296:550 (2002)). 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. al., Nat. Biotech. 20: 497 (2002); Paul, 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 an H1-RNA or U6 promoter, and a termination sequence operatively linked to a template of the desired siRNA sequence for transcription. The termination sequence comprises 2-3 uridine residues and a polythymidine (T5) sequence (a polyadenylation signal) (Brummelkamp, ​​Science, ibid.). Selected promoters can provide constitutive or inducible transcription. A method for transcription of the composition and DNA-directed RNA interference molecule is 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 end (Elbashir, et al., Genes Dev. 15:188 (2001)). (et al., Cell 107:309 (2001)). Transcription units are incorporated into plasmids or DNA vectors, and interfering RNA is transcribed from said plasmid or DNA vector. 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 plasmids initially designed to express desired gene sequences can be modified to contain transcription unit cassettes for transing siRNA.

[0253] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, preparing and screening cDNA libraries, and performing PCR are well known in the art (see, for example, Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al., ibid.; Ausubel et al., ibid.), 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. Other fundamental books 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)).

[0254] 1. Metal-phospholipid complex

[0255] In this application, the metal-phospholipid complex is composed of a phospholipid molecular portion, a linker molecular portion, and a metal ion portion. The phospholipid molecular portion is linked to the linker molecular portion, and the linker molecular portion is linked to the metal ion portion through a coordinate bond. Furthermore, the metal-phospholipid complex is not a cationic lipid or an ionizable lipid.

[0256] Regarding the phospholipid molecule portion, it should be noted that the cis-trans isomers of the phospholipid molecules in this application will not affect the effect to be achieved by the content of protection in this application.

[0257] In some embodiments, the phospholipid molecule portion is selected from one or more combinations of lecithin 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, lysophosphatidylglycerol LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysophosphatidylsphingomyelin LSM, 1-phosphosphoamine S1P, and their derivatives. Here, "and its derivatives" refers to "lecithin (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), 1-phosphoceramide (SP), phosphatidylinositol (PI), phosphatidylthreonine (PT), sphingomyelin (SM), lysophosphatidylcosinate (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysophosphatidylsphingomyelin (LSM), and 1-phosphosphoamine (S1P)". Specifically, the phospholipid molecule moiety may, for example, but not limited to, be selected from lecithin (PC), lecithin (PC) derivatives, phosphatidylethanolamine (PE), phosphatidylethanolamine (PE) derivatives, phosphatidylglycerol (PG), phosphatidylglycerol (PG) derivatives, phosphatidylglycerol (PG) and lecithin (PC), lecithin (PC) and lecithin (PC) derivatives, etc. In this application, "and its derivatives" has a similar meaning.

[0258] In some embodiments, the phospholipid molecule portion is selected from one or more combinations of lecithin (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), lysophosphatidylsphingomyelin (LSM) (Formula 17), 1-phosphosphoin (S1P) (Formula 18), and derivatives thereof. Preferably, the phospholipid molecule portion is selected from at least one of lecithin PC (Formula 1), phosphatidylethanolamine PE (Formula 2), phosphatidic acid PA (Formula 4), phosphatidylglycerol (PG) (Formula 5), ​​and their derivatives. Preferably, the phospholipid molecule portion is selected from at least one of DSPC, DSPE, DSPA, DSPG, and their derivatives. Preferably, the phospholipid molecule portion is selected from at least one of DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48), DSPG (Formula 49), and their derivatives.

[0259] The linker molecule is primarily derived from natural plant extracts, such as curcumin, and possesses a wide range of biological activities, including antibacterial, antiviral, antifungal, antioxidant, and anti-inflammatory activities. Furthermore, it is an effective immunomodulator, capable of regulating the activity of various immune cells, including T cells, B cells, macrophages, neutrophils, natural killer cells, and dendritic cells, promoting immune balance and enhancing the body's immunity. Based on the potential immune-enhancing, anti-inflammatory, antioxidant, and anti-SARS-CoV-2 effects of curcumin molecules, it holds promise as a potential adjunctive treatment for COVID-19. Moreover, curcumin molecules have extremely high safety profiles and have been listed in the catalogues of food additives and pharmaceutical excipients. This safety profile facilitates the overall clinical drug registration of targeted therapies, shortening the clinical drug registration time.

[0260] In some embodiments, the linker molecule is selected from one or more combinations of curcumin, chlorogenic acid, anthocyanin, quercetin, dihydromyricetin, hesperidin, naringenin, apigenin, catechin, tea polyphenols, epigallocatechin gallate, ellagic acid, morin, epigallocatechin gallate, catechin gallate, gallocatechin gallate, or piperine C, and 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), hesperidin (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), epigallocatechin gallate (Formula 32), catechin gallate (Formula 33), gallocatechin gallate (Formula 34), piperine C (Formula 35), and their derivatives. 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), hesperidin (Formula 24), tea polyphenols (Formula 28), and their derivatives. Preferably, the linker molecule is selected from curcumin (Formula 19), hesperidin (Formula 24), or tea polyphenols (Formula 28).

[0261] For the metal ion portion, the coordination bond between the linker molecule portion and the metal ion portion in the metal-phospholipid complex breaks under low pH conditions, such as lysosomes (pH=5.0), and the metal ion is released from the metal-phospholipid complex.

[0262] In some embodiments, the metal ion is partially 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+ At least one of the following. Preferably, the metal ion portion is selected from Fe. 3+ Mg 2+ Ca 2+ Al 3+ At least one of the following. Preferably, the metal ion portion is selected from Fe. 3+ Mg 2+ Ca 2+ Or Al 3+ .

[0263] The proportions of each component in a metal-phospholipid complex can be adjusted according to the specific structure of the components. The basis for this adjustment is as follows: because phospholipid molecules are linked to linker molecules via hydrogen bonds, if a phospholipid molecule contains multiple phosphate groups, the ratio of phospholipid molecules to linker molecules during complex synthesis can be adjusted based on the number of phosphate groups in the phospholipid molecule. For example, if a phospholipid molecule contains two phosphate groups, the ratio can be 1:2; if it contains three phosphate groups, the ratio can be 1:3. Similarly, because the hydroxyl groups of the linker molecule are coordinated with metal ions, if the linker molecule contains multiple binding sites, the ratio of linker molecules to metal ions can be adjusted based on the number of binding sites. When used to encapsulate drugs (e.g., nucleic acids), the function of the metal ion is to connect the phospholipid complex to the nucleic acid. Therefore, minimizing the number of complexation sites between the linker molecule and the metal ion ensures that the metal-phospholipid complex can encapsulate as much nucleic acid as possible.

[0264] 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 composed 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). 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 may 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.

[0265] 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.

[0266] In this application, the metal-phospholipid complex can be prepared by either a one-step method or a stepwise method. Both methods offer comparable capabilities for constructing metal-phospholipid complexes. In a preferred embodiment, the one-step method significantly shortens the time required for industrialization. In large-scale industrial production, time efficiency is crucial. The one-step method reduces issues such as process connections and time intervals associated with stepwise operations, greatly improving the continuity and efficiency of production, thereby effectively reducing production costs and increasing production efficiency. From an industrial application perspective, the one-step method better meets the needs of large-scale production, possessing broad application prospects and high practical value.

[0267] In the one-step process, phospholipid molecules, linker molecules, and metal ions react together to obtain a metal-phospholipid complex.

[0268] In some embodiments of the one-step method, phospholipid molecules, linker molecules, and metal ions are dissolved in ethanol and reacted. The preferred molar ratio of phospholipid molecules, linker molecules, and metal ions is 1:1:(0.5-2), and the preferred reaction conditions are 40-60°C for 1-5 hours. The molar ratio of phospholipid molecules, linker molecules, and metal ions can be, but is not limited to, 1:1:1, 1:1:1.5, 1:1:2, 3:3:2, or 2:2:1. 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, and the reaction time can be, but is not limited to, 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours.

[0269] In the stepwise method, phospholipid molecules are first reacted with linker molecules to form phospholipid complexes; then the prepared phospholipid complexes are reacted with metal ions through coordination bonds to form metal-phospholipid complexes.

[0270] In some embodiments, phospholipid molecules and linker molecules are dissolved in ethanol and reacted, followed by the addition of n-hexane to precipitate and obtain a phospholipid complex. The molar ratio of phospholipid molecules to linker molecules is preferably 1:1. The reaction conditions are preferably 65°C for 2 hours.

[0271] In some embodiments, the phospholipid complex and metal ions are dissolved in ethanol, and then reacted with triethylamine to obtain a metal-phospholipid complex. The molar ratio of the phospholipid complex to the metal ions 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.

[0272] 2. Metal-chelated phospholipid complex nanoparticles (MPP)

[0273] In this application, the metal-phospholipid complex particles contain: (i) a metal-phospholipid complex; (ii) conjugated lipids that inhibit particle aggregation, wherein the conjugated lipids that inhibit particle aggregation are not cationic lipids or ionizable lipids; and (iii) non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation.

[0274] The lipid conjugation for inhibiting particle aggregation refers to lipid conjugation that inhibits the aggregation of drug-metal-phospholipid complex particles or targeted drugs. Its main function is to prevent the aggregation of drug-metal-phospholipid complex particles or targeted drugs. Examples include PEG conjugated with dialkoxypropyl, PEG conjugated with diacylglycerol, PEG conjugated with phosphatidylethanolamine, and PEG conjugated with ceramide, preferably PEG-lipid conjugations. The cis-trans isomers of the lipid do not affect the effects to be achieved by the present application.

[0275] In some embodiments, (ii) the conjugated lipids that inhibit particle aggregation include PEG-lipid conjugates 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).

[0276] For non-cationic or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation, the lipid is at least one of cholesterol (preferably cholesterol formula 40) and its derivatives. In a preferred embodiment, in addition to cholesterol or its derivatives, the lipid may also include one or more combinations of lecithin 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, lysophosphatidylcholine LSM, 1-phosphosphoamine S1P, cholesterol sulfate, and their derivatives. Preferably, in addition to cholesterol or its derivatives, it also includes at least one selected from lecithin 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), lysophosphatidylsphingomyelin LSM (Formula 17), 1-phosphosphoinositol S1P (Formula 18), cholesterol sulfate (Formula 41), and its derivatives. Preferably, the non-cationic lipids or non-ionizable lipids in (iii) include cholesterol, and a combination of one or more selected from DSPC, DSPE, DSPA or DSPG. For example, the non-cationic lipids or non-ionizable lipids in (iii) include cholesterol (Formula 40) and DSPC (Formula 46).

[0277] In this application, a metal-phospholipid complex, as a single component, reacts with components (ii) and (iii) to self-assemble into metal-phospholipid complex particles. These metal-phospholipid complex particles can serve as drug carriers, where the drug is negatively charged, such as nucleic acid. The principle of loading drugs onto metal-phospholipid complex particles assembled from the metal-phospholipid complex is as follows: the linker molecules and phospholipid molecules are bonded together by hydrogen bonds, while the linker molecules are simultaneously linked to metal ions through coordination bonds, forming a metal-phospholipid complex. The metal ions of this metal-phospholipid complex are then linked to the negatively charged drug through coordination bonds, thereby ensuring that the metal-phospholipid complex self-assembles with other components (conjugated lipids that inhibit particle aggregation, and non-cationic or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation) into an MPP, while simultaneously loading the negatively charged drug into the nanoparticle MPP, resulting in drug-metal-phospholipid complex particles. In this article, the “non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation” refers to component (iii) in metal-phospholipid complex particles, which can be simply referred to as “non-cationic lipids or non-ionizable lipids”.

[0278] The proportions of each component in the metal-phospholipid complex particles can be as follows: the metal-phospholipid complex accounts for 5% to 50% of the raw material in molar proportion; the conjugated lipids that inhibit particle aggregation account for 1% to 10% of the raw material in molar proportion; cholesterol accounts for 15% to 80% of the raw material in molar proportion; and non-cationic lipids or non-ionizable lipids other than cholesterol account for 0% to 51% of the raw material in molar proportion. Here, "raw material" refers to the sum of the metal-phospholipid complex, the conjugated lipids that inhibit particle aggregation, cholesterol, and non-cationic lipids or non-ionizable lipids other than cholesterol.

[0279] The molar percentage 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 percentage of the metal-phospholipid complex in the raw material is 5% to 40%, preferably 10% to 40%.

[0280] The molar percentage of the conjugated lipids that inhibit particle aggregation in the feedstock can be, but is not limited to, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some embodiments, the molar percentage of the conjugated lipids that inhibit particle aggregation in the feedstock is 2% to 10%.

[0281] The non-cationic lipid or non-ionizable lipid is cholesterol. The molar percentage of cholesterol in the raw material may 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%. The percentages are 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 percentage of cholesterol in the raw material is 25% to 75%, preferably 35% to 75%, and more preferably 35% to 55%.

[0282] The molar percentage of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw materials may 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 non-cationic lipids or non-ionizable lipids other than cholesterol account for 0% to 50% of the raw materials, preferably 0% to 40%, more preferably 5% to 30%, and further 20% to 25%.

[0283] The metal-phospholipid complex particles are prepared by mixing a metal-phospholipid complex, conjugated lipids that inhibit particle aggregation, and non-cationic lipids or non-ionizable lipids. In some embodiments, the metal-phospholipid complex, conjugated lipids that inhibit particle aggregation, and non-cationic lipids or non-ionizable lipids are mixed in a homogeneous phase, such as an organic phase (in some embodiments, the solvent is ethanol).

[0284] When a drug is encapsulated in metal-phospholipid complex particles, the preparation method involves mixing a metal-phospholipid complex, conjugated lipids that inhibit particle aggregation, non-cationic lipids or non-ionizable lipids, and the drug to obtain drug-metal-phospholipid complex particles. In some embodiments, the metal-phospholipid complex, the conjugated lipids that inhibit particle aggregation, and the non-cationic lipids or non-ionizable lipids are dissolved in an organic compound to form an organic phase, and the drug is dissolved in a buffer solution to form an aqueous phase. The organic phase and the aqueous phase are then mixed to obtain drug-metal-phospholipid complex particles. In some embodiments, the buffer solution can be PBS buffer or Tris-HCl buffer. In some embodiments, the mixing method for the organic phase and the aqueous phase can be a microfluidic chip or ultrasound.

[0285] 3. Targeted vector (containing MPP)

[0286] In this application, the targeting vector comprises a vector delivery system and a targeting structure. The vector delivery system includes at least one of a lipid delivery system, exosomes, pseudoviruses, small molecules, cells, or carrier proteins. Preferably, the lipid delivery system is a metal-phospholipid complex particle, a LNP, or a phospholipid delivery system. In one embodiment, the pseudovirus is an adenovirus. In one embodiment, the cell is a erythrocyte. In one embodiment, the carrier protein is a ferritin. In one embodiment, the LNP is a cationic lipid or an ionizable lipid.

[0287] In one embodiment, the carrier delivery system is a metal-phospholipid complex particle (MPP), and the targeting structure is connected to the outer surface of the metal-phospholipid complex particle (MPP).

[0288] In some embodiments, the targeting structure includes a hydrophobic region, a connecting region, and a targeting binding region connected in sequence, wherein the hydrophobic region is connected to the outer layer of the metal-phospholipid complex particle based on hydrophilicity and hydrophobicity.

[0289] In some embodiments, the hydrophobic region includes at least one or more of DSPE and its derivatives. This portion enables the targeting structure to connect to the outer layer of the metal-phospholipid complex particles.

[0290] In some embodiments, the linker region includes at least one or more of PEG-2000 and its derivatives.

[0291] In some embodiments, the targeting binding region can specifically recognize and guide the targeting carrier to a specific target (i.e., the target site or target cell of the targeting binding region), thereby enabling the drug-loaded LNP particles or metal-phospholipid complex particles to transport the drug and act on the target (i.e., the drug target site or target cell). In some embodiments, the targeting binding region can bind to at least one of CD62L, CD8, CD3, nucleolar 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 nucleic acid, peptide, protein, or small molecule. In some embodiments, the targeting binding region includes one of aptamer, antibody, antigen-binding part, or galnac. In some embodiments, the targeting binding region is an aptamer, the target site 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 site 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 pancreatic cancer cells, 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 liver cancer cells.

[0292] In one embodiment, the targeting structure is a DSPE-PEG2000-aptamer, preferably a DSPE-PEG2000-CD62L aptamer, the CD62L aptamer sequence of which is shown in SEQ ID NO.44.

[0293] In this application, the method for preparing the targeted structure involves first connecting the hydrophobic region and the linking region, and then connecting the hydrophobic region-linking region to the targeted binding region via an intermediate pair. Taking the DSPE-PEG2000-CD62L aptamer as an example, the preparation process is as follows: DSPE-PEG2000 and CD62L aptamer are reacted and linked through an intermediate pair 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.

[0294] 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 targeting carrier. In some embodiments, the micelles are prepared by methods including direct dissolution, ethanol injection, dialysis, or sonication.

[0295] When a targeted carrier is used for drug loading, 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 targeted structure is reacted and linked with the drug-lipid particles to obtain the targeted drug.

[0296] In some embodiments, the mass ratio of the target structure to the drug-lipid particles is 1:(14-84), and the mass ratio may 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.

[0297] In some embodiments, the reaction conditions between the drug-lipid particles and the target 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, or 10°C, and the reaction time may be, but is not limited to, 0.2 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0298] 4. Targeted drugs

[0299] In this application, the targeted drug includes a drug and a targeting carrier, wherein the drug is encapsulated in the targeting carrier, and more specifically, the drug is encapsulated in metal-phospholipid complex particles.

[0300] In this application, the drug is a negatively charged molecule, which may be selected from one or more combinations of nucleic acids, proteins, peptides, small molecules, nucleic acid analogs, protein analogs, and peptide analogs. In some embodiments, the nucleic acid is selected from one or more combinations of mRNA, siRNA, circular RNA, microRNA, ASO, sgRNA, DNA, ecDNA, and artificial nucleic acids.

[0301] In some implementations, the drug is mRNA encoding a chimeric antigen receptor (CAR) or a tumor receptor (TCR). In this case, the targeted drug achieves in situ CAR cell therapy or TCR cell therapy. Delivering this mRNA encoding the corresponding receptor to specific cells in vivo can induce these cells to express CAR or TCR in situ, thereby enabling them to target and kill specific tumor cells. In this case, in situ CAR cell therapy achieved through targeted drugs provides a new strategy for the treatment of diseases (especially cancer). This therapy has relatively stronger targeting and may reduce the complex preparation and reinfusion processes of traditional cell therapies, offering significant advantages.

[0302] In some embodiments, the drug is an mRNA encoding a chimeric antigen receptor (CAR), wherein the CAR includes a transmembrane domain, a signal transduction domain, an antigen-binding domain, a co-stimulatory signal transduction region, and a junction region between the antigen-binding domain and the transmembrane domain. In some embodiments, the transmembrane domain is selected from at least one of SEQ ID No. 28, SEQ ID No. 29, and SEQ ID No. 30; and / or, the signal transduction 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 co-stimulatory signal transduction region is selected from at least one of SEQ ID No. 37, SEQ ID No. 39, and SEQ ID No. 75; and / or, the junction region between the antigen-binding domain and the transmembrane domain is selected from at least one of SEQ ID No. 40, SEQ ID No. 41, and SEQ ID No. 42.

[0303] In one embodiment, the drug is mRNA with a nucleotide sequence as shown in SEQ ID No. 43.

[0304] Furthermore, the nucleic acids encapsulated in the targeted drug of this application are resistant to degradation by nucleases in aqueous solution.

[0305] In some implementations, the drug is fully encapsulated within the metal-phospholipid complex particles to prevent drug degradation, while the drug is delivered into specific cells through the targeting binding region in the targeting structure.

[0306] In some implementations, the targeted drug provided in this application has a small diameter suitable for systemic delivery.

[0307] In some embodiments, the medicament of this application is preferably a nucleic acid, the nucleic acid component typically including mRNA, interfering RNA (i.e., siRNA) which can be provided in several forms, said 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 transcript cassette in a DNA plasmid.

[0308] The targeted drug or preparation method of this application can be used for drug delivery, imaging drugs, and vaccines.

[0309] In some implementations, targeted drugs are used for drug delivery, treatment, and / or prevention.

[0310] In some embodiments, the targeted drug is used to deliver 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, cardiomyocytes, or stem cells.

[0311] In some embodiments, the targeted drug is used to express or silence a target sequence in a mammalian subject, to deliver the drug within a mammalian body, to deliver the drug from within a mammalian body to mammalian cells, to deliver the drug from within a mammalian body to mammalian cells by expressing or silencing 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 related to the expression of a gene containing the target sequence of the drug.

[0312] In some implementations, the disease or condition includes 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.

[0313] In some embodiments, the cancer includes hematologic malignancies and solid tumors. Preferably, the 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, with diffuse large B-cell lymphoma (DLBCL) preferably including Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). Preferably, the 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.

[0314] In some implementations, the viral infection includes hepatitis B virus, hepatitis C virus, SARS-CoV-2, human immunodeficiency virus, cytomegalovirus, invasive Aspergillus, or a conjugate virus.

[0315] In some implementations, autoimmune diseases include pemphigus vulgaris, systemic lupus erythematosus (SLE), hemophilia, myasthenia gravis, immune rejection of transplanted tissues or organs, type 1 diabetes mellitus (T1D), rheumatoid arthritis, systemic sclerosis, multiple sclerosis, idiopathic pulmonary fibrosis, Crohn's disease, or colitis.

[0316] In some implementations, diseases caused by overactivation of the immune system include cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), or graft-versus-host disease (GVHD).

[0317] In some implementations, metabolic diseases include atherosclerosis, congenital hyperinsulinemia, non-alcoholic steatohepatitis, or non-obese diabetes (NOD).

[0318] In some embodiments, 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, skin diseases include keloids or wound healing.

[0319] In some implementations, the routes of administration for the targeted drug include intrathecal injection, intramuscular administration, intracranial injection, intravenous injection, or intratumoral injection.

[0320] In some implementations, the targeted drug is used in combination therapy.

[0321] In some embodiments, the targeted drug is a drug that targets immune cells, and the immune cells are T cells and / or myeloid cells.

[0322] In some implementations, the targeted immune cell drug is a chimeric antigen receptor (CAR) drug.

[0323] This application provides a drug containing a targeted drug, preferably a vaccine, and more preferably a novel coronavirus vaccine.

[0324] When the drug is a nucleic acid, targeting vectors can be used to facilitate drug lysosomal escape and promote nucleic acid expression. Targeting vectors can also be used to deliver drugs, introducing them into cells to achieve the prevention and treatment of applicable diseases or symptoms.

[0325] In some embodiments, the pharmaceutical preparation provided in this application can achieve: silencing the expression of a target sequence in mammalian subjects, delivering a drug (e.g., a drug for treating tumors, a contrast agent, etc.) within a mammalian body, delivering a drug from within a mammalian body to mammalian cells, or treating diseases or conditions in mammals. The targeted drug is the main active ingredient in the pharmaceutical preparation, and it can be prepared into different dosage forms according to actual needs using different pharmaceutically acceptable excipients or preparation processes, such as solid dosage forms (powders, granules, pills, tablets, gels), semi-solid dosage forms (topical ointments, pastes), liquid dosage forms (decoctions, mixtures, syrups, tinctures, injections), and gaseous dosage forms (aerosols, smokes), etc.; for example, dosage forms administered via the gastrointestinal tract, dosage forms administered via the rectum, and dosage forms not administered via the gastrointestinal tract, etc. Products containing this pharmaceutical preparation can be, for example, but are not limited to, kits, pharmaceutical preparations, etc., and may optionally also contain other excipients.

[0326] For target genes of targeted drug action: Ideally, drug-lipid particles should be delivered to downregulate or silence the translation (i.e., expression) of the target gene product. Suitable classifications of gene products include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and conditions (e.g., diseases and conditions in which the liver is a target, and liver diseases and conditions), genes associated with tumorigenesis and cell transformation, angiogenesis genes, immunomodulatory genes such as those associated with inflammation and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative diseases.

[0327] Genes associated with viral infection and survival include those that bind, enter, and replicate in cells through viral expression, particularly viral sequences associated with chronic viral diseases. For example, viral sequences include those of hepatitis viruses (Hamasaki, et al., FEBS Lett. 543: 51 (2003); Yokota, et al., EMBO Rep. 4: 602 (2003); Schlomai, et al., Hepatology 37: 764 (2003); Wilson, et al., Proc. Natl. Acad. Sci. 100: 2783 (2003); Kapadia, et al., Proc. Natl. Acad. Sci. 100: 2014 (2003); and FIELDS VIROLOGY (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)), herpesvirus (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 hepatitis virus nucleic acid sequences that can be silenced include, but are not limited to: nucleic acid sequences involved in transcription and translation (e.g., En1, En2, X, P), nucleic acid sequences encoding structural proteins (e.g., core proteins including C and C-related proteins; capsid and envelope proteins including S, M, and / or L proteins, or fragments thereof) (see, for example, FIELDS VIROLOGY, 2001, ibid.). 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 mentioned, for example, in GenBank accession number NC_001489; hepatitis B nucleic acid sequences are mentioned, for example, in GenBank accession number NC_003977; hepatitis C nucleic acid sequences are mentioned, for example, in GenBank accession number NC_004102; hepatitis D nucleic acid sequences are mentioned, for example, in GenBank accession number NC_001653; hepatitis E nucleic acid sequences are mentioned, for example, in GenBank accession number NC_001434; and hepatitis G nucleic acid sequences are mentioned, for example, in GenBank accession number NC_001710. Silencing sequences encoding genes associated with viral infection and survival can be conveniently combined with the administration of conventional drugs used to treat viral diseases.

[0328] Genes associated with metabolic diseases and conditions (e.g., conditions in which the liver is targeted and liver diseases and conditions) include, for example, genes expressed in dyslipidemia (e.g., liver X receptors (e.g., LXRα and LXRβ, Genback registry NM_007121)), farnesoid X receptor (FXR) (Genbank registry NM_005123), sterol regulatory element-binding protein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), apolipoprotein (ApoB), and apolipoprotein (ApoE)) and diabetes (e.g., glucose-6-phosphate) (see, for example, 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 conditions (e.g., diseases and conditions in which the liver is targeted, and liver diseases and conditions) 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 conditions can be conveniently combined with the administration of conventional pharmaceutical agents for treating said diseases or conditions.

[0329] Examples of genes associated with tumorigenesis and cell transformation include translocation sequences such as MLL fusion genes, BCR-ABL (Wilda, et al., Oncogene, 21:5716 (2002); Scherr, et al., Blood 101:1566), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO, and AML1-MTG8 (Heidenreich, et al., Blood 101:3157 (2003)); overexpressed sequences such as multidrug resistance genes (Nieth, et al., FEBS Lett. 545:144 (2003); Wu, et al., Cancer Res. 63:1515 (2003)); and cyclins (Li, et al., Cancer Res. 63:3593 (2003); Zou ... et al., Genes Dev. 16: 2923 (2002)), β-linkin (Verma, et al., Clin Cancer Res. 9: 1291 (2003)), telomere terminal transferase 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 can be combined with the administration of chemotherapeutic agents (Collis, et al., Cancer Res. 63: 1550 (2003)). Genes encoding proteins associated with tumor migration are also target sequences, such as integrins, selectins, and metalloproteinases. Any complete or partial gene sequence that is favorable to or promotes tumorigenesis or cell transformation, tumor growth, or tumor migration can be included as a template sequence.

[0330] Angiogenesis genes can promote the formation of new blood vessels. Vascular endothelial growth factor (VEGF) is a key research area (Reich, et al., Mol. Vis. 9: 210 (2003)).

[0331] 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 target sequences of immunomodulators (Song, et al., Nat. Med. 9: 347 (2003)). Genes encoding secondary signaling molecules in hematopoietic and lymphoid cells are also included in this application, such as Tec family kinases, such as Bruton's tyrosine kinase (Btk) (Heinonen, et al., FEBS Lett. 527: 274 (2002)).

[0332] Cellular receptor ligands include ligands that bind to cell surface receptors (e.g., insulin receptor, EPO receptor, G-protein-coupled receptor, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) to regulate (e.g., inhibit, activate, etc.) physiological pathways involved in the receptors (e.g., glucose level regulation, blood cell development, mitosis, etc.). Examples of cellular receptor ligands include cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G-protein-coupled receptor ligands, etc.). Templates encoding the expansion of trinucleotide repeat sequences (e.g., CAG repeat sequences) have been found to silence pathogenic sequences in neurodegenerative diseases caused by the expansion of trinucleotide repeat sequences, such as spinal-medullobulal muscular atrophy and Huntington's disease (Caplen, et al., Hum. Mol. Genet. 11: 175 (2002)).

[0333] Injectable Delivery: In certain situations, as described in U.S. Patents 5,543,158, 5,641,515, and 5,399,363, delivery of the targeted drugs disclosed herein via parenteral, intravenous, intramuscular, subcutaneous, intradermal, or intraperitoneal administration is desirable. The targeted drugs can be injected locally into the target site (e.g., a disease site such as inflammation or tumor formation, or into a target organ or tissue) or systemically for broad distribution throughout the organism. Solutions of the targeted drugs can be prepared in water, suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and in oils. Optionally, these formulations contain preservatives to inhibit microbial growth. Typically, when administered intravenously, the targeted drug formulation is formulated with a suitable pharmaceutical carrier. Commonly, a plain buffered saline solution (135-150 mM NaCl) will typically be used as the pharmaceutical carrier, but other suitable carriers will be sufficient. Other 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, media, coatings, diluents, antimicrobial and antifungal agents, isotonic and absorption retardants, buffers, carrier solutions, suspensions, colloids, etc. The phrase "pharmaceutical" refers to molecular entities and compositions that, when administered to humans, do not produce allergic or similar adverse reactions. Formulations of aqueous compositions, containing proteins as active ingredients, are a conventional understanding in the art. Optionally, these compositions may be prepared as injections, liquid solutions, or suspensions; they may also be prepared in solid forms suitable for solutions or suspensions in liquids prior to injection. The formulations may also be emulsified.

[0334] Targeted drugs can be sterilized using conventional liposome sterilization techniques, such as filtration. The targeted drugs may contain pharmaceutical excipients, which are suitable physiological agents, such as pH adjusters and buffers, toxicity modifiers, wetting agents, etc. These compositions can be sterilized using the techniques described above, or alternatively, they can be produced under aseptic conditions. The resulting aqueous solutions can be packaged for use or filtered and freeze-dried under aseptic conditions, with the freeze-dried formulation combined with a sterile aqueous solution prior to administration.

[0335] Preventive and Therapeutic Treatment: In some embodiments, a targeted drug may be used for preventive or therapeutic treatment of a subject (e.g., a mammalian subject) suffering from a disease or condition associated with the expression or overexpression of a target sequence. The targeted drug is administered to the subject in an amount sufficient to elicit a therapeutic response in the patient. An amount sufficient to accomplish this is defined as a “therapeuticly 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 caused by the expression or overexpression of a target gene, the physician evaluates the circulating plasma level of the targeted drug, the toxicity of the targeted drug, and the progression of the disease associated with the expression or overexpression of the target gene. Administration may be performed by a single dose or in multiple doses.

[0336] For example, the targeted drug can be administered to a subject who is infected with or at risk of infection with a pathogenic microorganism. The drug should preferably correspond to a sequence that is unique to the microorganism (or at least absent in the natural genome of the patient undergoing treatment) and that plays a crucial role in the microorganism's life cycle. The targeted drug is delivered to target cells, tissues, or organs at a therapeutically effective dose via ex vivo or intravenous injection. Silencing sequences encoding genes associated with pathogen infection can be conveniently combined with the administration of conventional agents used to treat pathogenic diseases. The treatment can be administered prophylactically to individuals at risk of or already infected with the pathogenic microorganism.

[0337] In a preferred embodiment, the targeted drug of this 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 S, C, P, and X proteins, PRE, EnI, and EnII (see, for example, FIELDSVIROLOGY, 2001, ibid.). Those skilled in the art will understand that gene silencing associated with hepatitis infection can be associated with conventional treatments for hepatitis, such as immunoglobulins, interferon (e.g., PEGylated and unPEGylated interferon α) (see, for example, Medina et al., Antiviral Res. 60(2): 135-143 (2003); ribavirin (see, for example, Hugle and Cerny, Rev. Med. Virol. 13(6): 361-71 (2003); adefovir and lamivudine (see, for example, Kock et al., Hepatology 38(6): 1410-8 (2003); isoprenoidation inhibitors (see, for example, Bordier et al., J. Clin. Invest. 112(3): 407-414 (2003)); famciclovir (see, for example, Yurdaydin et al., J. Clin. Invest. 112(3): 407-414 (2003)); and famciclovir (see, for example, Yurdaydin et al., J. Clin. Invest. 112(3): 407-414 (2003)). Hepatol. 37(2): 266-71 (2002); and saikosaponins c and d (see, e.g., Chiang et al., Planta Med. 69(8): 705-9 (2003).

[0338] In another embodiment, the targeted drug of this application can be conveniently used to treat diseases and conditions characterized by the expression or overexpression of a gene or gene group. In some aspects, the targeted drug of this application can be used to treat metabolic diseases and conditions (e.g., diseases and conditions in which the liver is the target, and liver diseases and conditions) such as, for example, dyslipidemia and diabetes. Those skilled in the art will understand that the silencing of genes associated with metabolic diseases and conditions can be combined with conventional treatments for these diseases. For example, silencing genes involved in dyslipidemia can be achieved with the use of inhibin, bile acid chelating agents / resins and cholesterol absorption inhibitors such as ezetimibe, phytosterols / sterols, polyphenols, and nutritional products such as oat bran, flaxseed and soy protein, phytosterol analogs, squalene synthase inhibitors, bile acid transport inhibitors SREBP cleavage-activated protein (SCAP) activating ligands, niacin (nicotinic acid), acipimox, high-dose fish oil, antioxidants and sugarcane fatty alcohols, microsomal triglyceride transporter (MTP) inhibitors, acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors, gemcabene, ribofibrate, pantothenic acid analogs, niacin receptor agonists, anti-inflammatory agents (such as Lp-PLA(2) antagonists and AGI1067) functional oils, PPAR-α, γ, δ agonists, as well as dual PPAR-α, / γ and 'pan' PPAR-α / γ, / δ agonists, cholesterol 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 peptide, delayed-release niacin / lovastatin, atorvastatin / amlodipine, ezetimibe / simvastatin, atorvastatin / CETP inhibitors, statin / PPAR agonists, delayed-release niacin / simvastatin and pravastatin / aspirin under development, and therapeutic combinations of anti-obesity agents (see, for example, Bays and Stein, Expert Opin. Pharmacother. 4(11): 1901-38(2003)). Similarly, silencing genes involved in diabetes can be combined with insulin therapy, as well as dietary modifications and exercise.

[0339] In another embodiment, the targeted drug can be used to treat cancer, viral infections, autoimmune diseases, diseases caused by overactivation of the immune system, metabolic diseases, fibrotic diseases, tissue fibrosis, cellular senescence, atherosclerosis, diabetes, or osteoarthritis. The cancers include hematologic malignancies and solid tumors. The 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. The diffuse large B-cell lymphoma (DLBCL) includes Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). The solid tumors include liver cancer, glioma, gastric cancer, pancreatic cancer, lung cancer, non-small cell lung cancer, renal cell carcinoma, prostate cancer, osteosarcoma, breast cancer, colorectal cancer, ovarian cancer, melanoma, neuroblastoma, hemangioblastoma, anaplastic meningioma, recurrent glioblastoma, or differentiated thyroid cancer. The autoimmune diseases mentioned include pemphigus vulgaris, systemic lupus erythematosus (SLE), hemophilia, myasthenia gravis, immune rejection caused by transplanted tissues or organs, type 1 diabetes mellitus (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 mentioned include atherosclerosis, congenital hyperinsulinemia, non-alcoholic steatohepatitis, or non-obese diabetes mellitus (NOD). The fibrosis mentioned 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 mentioned include keloids or wound healing disorders.

[0340] Similar methods are used to inhibit the expression of endogenous receptor cellular genes associated with tumorigenesis and cell transformation, tumor growth, and tumor migration; inhibit the expression of angiogenesis genes; inhibit the expression of immunomodulatory 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 diseases; and inhibit the expression of other genes associated with viral infection and survival. The specific target gene sequences are as described above.

[0341] Detection of the particles: The targeted drug described herein is detected using any method known in the art. For example, a label is conjugated directly or indirectly to a component of the targeted drug or other lipid-based carrier system using methods well-known in the art. A wide variety of labels can be used, selected based on the required sensitivity, ease of conjugation to the targeted drug component, stability requirements, and the availability and readiness of the tools and processing. Suitable labeling includes, but is not limited to, spectral labeling, such as fluorescent dyes (e.g., fluorescein and its derivatives, such as fluorescein isothiocyanate (FITC) and Oregon Green™; rhodamine and its derivatives, such as Texas Red, tetrarhodimine isothiocynate (TRITC), etc., digitoxin, biotin, phycoerythrin, AMCA, CyDyes™, etc.); radioactive labeling, such as 3H, 125I, 35S, 14C, 32P, 33P, etc.; enzymes such as horseradish peroxidase, alkaline phosphatase, etc.; and spectral colorimetric labeling such as colloidal gold or colored glass or plastic beads, such as polystyrene, polypropylene, latex, etc.). The labeling shall be detected using any method known in the art.

[0342] Nucleic acid detection: The nucleic acids described herein are detected and quantified using any of a number of methods well known to those skilled in the art. Nucleic acid detection is performed using methods well known in the art, such as DNA blotting, RNA blotting, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Other analytical biochemical methods can also be applied, such as spectrophotometry, X-ray radiography, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and hyperdiffusion chromatography.

[0343] The sensitivity of hybridization assays can be improved by applying nucleic acid amplification systems that multiply the number of target nucleic acids being detected. In vitro amplification techniques suitable for amplifying sequences used as molecular probes or generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques that can guide technicians using these in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicaase amplification, and other RNA polymerase-mediated techniques (e.g., NASBA™), can be found in Sambrook, et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, 2000; and Ausubel et al., SHORT PROTOCOLSIN 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. Patent 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) 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).An improved method for cloning and amplifying nucleic acids in vitro is described in Wallace et al., U.S. Patent No. 5,426,039. Other methods described in the art are based on nucleic acid sequence amplification (NASBA™, Cangene, Mississauga, Ontario) and the Qβ replicase system.

[0344] As described in Needham VanDevanter et al., Nucleic Acids Res., 12:6159 (1984), oligonucleotides are typically chemically synthesized, for example, using an automated synthesizer, according to the solid-phase phosphoramidite method described in Beaucage and Caruthers, Tetrahedron Letts., 22(20):1859-1862 (1981). These oligonucleotides are used as probes in in vitro amplification methods, as gene probes, or as inhibitory components. If necessary, the oligonucleotides are typically purified by natural acrylamide gel electrophoresis or by anion-exchange HPLC, as described in Pearson and Renier, J. Chrom., 255:137-149 (1983). The sequence of the synthetic oligonucleotide can be confirmed using the chemical degradation method described by Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology, 65:499.

[0345] Compared to CD8 or CD3 as targets, CD62L as a target for immune cell drugs can result in higher drug expression levels, which is more conducive to the treatment of diseases.

[0346] The following embodiments are provided as examples, but are not intended to limit the claimed application. Those skilled in the art will readily identify various non-critical parameters that can produce substantially the same or similar results.

[0347] The drug-lipid particles in this application refer to drug-lipid particles other than those containing cationic / ionizable lipids, namely drug-loaded metal-chelated phospholipid complex nanoparticles (drug@MPP), which, when linked to a targeting structure, yield a targeted drug. After removing the drug portion, the remaining components of the targeted drug include a targeting carrier.

[0348] Metal (containing Fe) 3+ Al 3+ or Mg 2+Preparation of Metal-chelated Phospholipid Complex Nanoparticles (MPP)

[0349] Example 1: Preparation of drug-metal-phospholipid complex particles

[0350] Example 1.1: Preparation of phospholipid complex

[0351] Phospholipid molecules with phosphate groups were linked to a linker molecule: Distearate phosphatidylcholine (DSPC, formula 46) and curcumin (formula 19) were added to a reaction flask at a molar ratio of 1:1, dissolved in an appropriate amount of ethanol, reacted at 65°C for 2 hours, concentrated, and then hexane was added. The precipitated phospholipid complex was filtered and vacuum dried to obtain the phospholipid complex. The structure of the phospholipid complex is shown below:

[0352] Results analysis: The yield of the target product obtained by reacting curcumin with DSPC at 65℃ for 2 hours was 94%.

[0353] Example 1.2: Preparation of metal-phospholipid complexes

[0354] Example 1.2.1 Stepwise preparation of Fe metal ions 3+ Metal-phospholipid complexes at time

[0355] The phospholipid complex prepared in Example 1.1 was partially linked to a metal ion: The phospholipid complex and FeCl3 were added to a reaction flask at a molar ratio of 1:1, dissolved in ethanol, and then triethylamine was added at a molar ratio of 1:1 to the phospholipid complex. The mixture was reacted at 60°C for 2 hours, and the reaction solution was suspended to dryness. It was then washed with ultrapure water and dried under vacuum to obtain the metal-phospholipid complex. The structure of the metal-phospholipid complex is shown below.

[0356] Results analysis: When the phospholipid complex reacted with FeCl3 at 60℃ for 2 hours, the yield of the target product was 95% when the concentration of the phospholipid complex was 4.5 mg / mL and the ratio of the phospholipid complex to FeCl3 was 1:1.

[0357] Example 1.2.2 Stepwise preparation of metal ions as Al 3+ Metal-phospholipid complexes at time

[0358] The difference between this embodiment and Example 1.2.1 is that FeCl3 is replaced with Al(NO3)3·9H2O. The structure of the prepared metal-phospholipid complex is shown below.

[0359] Results analysis: When the phospholipid complex reacted with Al(NO3)3·9H2O at 60℃ for 2 hours, the yield of the target product was 95% when the concentration of the phospholipid complex was 4.5 mg / mL and the ratio of the phospholipid complex to Al(NO3)3·9H2O was 1:1.

[0360] Example 1.2.3 Stepwise preparation of Mg metal ions 2+ Metal-phospholipid complexes at time

[0361] The difference between this embodiment and Example 1.2.1 is that FeCl3 is replaced with MgCl2. The structure of the prepared metal-phospholipid complex is shown below.

[0362] Results analysis: When the phospholipid complex reacted with MgCl2 at 60℃ for 2 hours, the yield of the target product was 95.5% when the concentration of the phospholipid complex was 4.5 mg / mL and the ratio of the phospholipid complex to MgCl2 was 1:1.

[0363] Example 1.2.4: One-step preparation of metal-phospholipid complexes

[0364] Phospholipid molecules with phosphate groups are linked to linker molecules and metal ions: distearate phosphatidylcholine (DSPC, formula 46), curcumin (formula 19), and FeCl3 are added to a reaction flask in a molar ratio of 1:1:1, dissolved in an appropriate amount of ethanol, and reacted at 50±10℃ for 2 h to obtain a metal-phospholipid complex. The FeCl3 obtained by this one-step reaction... 3+ The yield of the target product, a metal-phospholipid complex, was 95.6%. Metal ions can also be converted from Fe... 3+ Replace with Mg 2+ Al 3+ Ca 2+ The reaction temperature was 50±10℃ and the reaction time was 1-5h, preferably 2h.

[0365] The metal ion is Fe 3+ The performance comparison analysis of the one-step method and the stepwise method for preparing metal-phospholipid complexes is as follows:

[0366] Phospholipid molecules with phosphate groups are linked to linker molecules and metal ions: distearate phosphatidylcholine (DSPC, formula 46), curcumin, and Al(NO3)3·9H2O are added to a reaction flask in a molar ratio of 1:1:1, dissolved in an appropriate amount of ethanol, and reacted at 50±10℃ for 2 h to obtain a metal-phospholipid complex. The Al obtained from this one-step reaction... 3+ The yield of the target product of the metal-phospholipid complex was 95.6%.

[0367] The metal ion is Al 3+ The performance comparison analysis of the one-step method and the stepwise method for preparing metal-phospholipid complexes is as follows:

[0368] Phospholipid molecules with phosphate groups are linked to linker molecules and metal ions: Distearate phosphatidylcholine (DSPC, formula 46), curcumin, and CaCl2 are added to a reaction flask in a molar ratio of 1:1:1, dissolved in an appropriate amount of ethanol, and reacted at 50±10℃ for 2 h to obtain a metal-phospholipid complex. The CaCl2 obtained by this one-step reaction... 2+ The yield of the target product, a metal-phospholipid complex, was 95%. Metal ions (Ca) 2+ The stepwise preparation method of mRNA-metal-phospholipid complex particles refers to the preparation method of metal ion Fe 3+ A stepwise method for preparing mRNA-metal-phospholipid complex particles, with Ca2+ as the metal ion. 2+ mRNA-metal-phospholipid complex particles, which only transfer metal ions from Fe 3+ Replace with Ca 2+ FeCl3 was replaced with CaCl2, while all other parameters remained unchanged. The phospholipid complex reacted with CaCl2 at 60℃ for 2 hours. The target product metal ion obtained when the phospholipid complex concentration was 4.5 mg / mL and the phospholipid complex to CaCl2 ratio was 1:1 was Ca. 2+ The yield of the metal-phospholipid complex was 95%.

[0369] The metal ion is Ca 2+ The performance comparison analysis of the one-step method and the stepwise method for preparing metal-phospholipid complexes is as follows:

[0370] Phospholipid molecules with phosphate groups are linked to linker molecules and metal ions: distearate phosphatidylcholine (DSPC, formula 46), curcumin, and MgCl2 are added to a reaction flask in a molar ratio of 1:1:1, dissolved in an appropriate amount of ethanol, and reacted at 50±10℃ for 2 h to obtain a metal-phospholipid complex. The MgCl2 obtained by this one-step reaction... 2+ The yield of the target product of the metal-phospholipid complex was 95%.

[0371] The metal ion is Mg 2+ The performance comparison analysis of the one-step method and the stepwise method for preparing metal-phospholipid complexes is as follows:

[0372] In the preparation process of metal-phospholipid complexes, the characteristics and effectiveness of the one-step and stepwise methods were compared. The two methods showed similar loading rates, demonstrating stable performance in ensuring effective metal loading within the complex. The eGFP positive expression levels were also similar for both methods, proving their comparable ability to construct metal-phospholipid complexes. However, the one-step method significantly shortens the time required for industrialization. In large-scale industrial production, time efficiency is crucial. The one-step method reduces the problems of process connections and time intervals associated with stepwise operations, greatly improving the continuity and compactness of production, thereby effectively reducing production costs and increasing efficiency. From an industrial application perspective, compared to the stepwise method, the one-step method is more suitable for the needs of large-scale production, possessing broad application prospects and high practical value.

[0373] Example 1.3: Preparation of Fe metal ions 3+ mRNA-loaded metal-chelated phospholipid complex nanoparticles (mRNA@MPP)

[0374] The metal-phospholipid complex was prepared according to the method in Example 1.2.1, wherein DSPC, curcumin, and FeCl3 were added in a 1:1:1 ratio. The metal-phospholipid complex, distearate phosphatidylcholine (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 that inhibits particle aggregation) were dissolved in ethanol at different molar proportions to form the organic phase. The proportions of the metal-phospholipid complex, DSPC, CHOL, and DSPE-PEG2000 were 15%, 35%, 46%, and 4%, respectively. mRNA was dissolved in PBS (composed of 0.137M sodium chloride, 0.0027M potassium chloride, 0.01M disodium hydrogen phosphate, and 0.0018M potassium dihydrogen phosphate) at a concentration of 20 μg / mL to form the aqueous phase. The total mass of metal-phospholipid complex, distearate phosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) was mixed with the mRNA 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 and aqueous phases in the microfluidic chip was 12 ml / min. The drug mRNA was the mRNA encoding the fluorescent protein eGFP, and its sequence is SEQ ID NO.1 (720 nt). The prepared eGFP-mRNA@MPP was then incubated with 293T cells at a concentration of 2 μg / mL (the concentration of mRNA contained). The control group was incubated with unloaded MPP. After 48 h, the cell suspension was collected, and the percentage of eGFP-positive cells was detected by flow cytometry.

[0375] Example 1.4: Preparation of metal ions as Al 3+ mRNA-loaded metal-chelated phospholipid complex nanoparticles (mRNA@MPP)

[0376] The metal-phospholipid complex was prepared according to the method in Example 1.2.2, wherein DSPC, curcumin, and Al(NO3)3·9H2O were added in a 1:1:1 ratio. The metal-phospholipid complex, distearate phosphatidylcholine (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 that inhibits particle aggregation) were dissolved in ethanol in different molar proportions as the organic phase. The proportions of the metal-phospholipid complex, DSPC, CHOL, and DSPE-PEG2000 were 7%, 34%, 56%, and 3%, respectively. mRNA was dissolved in PBS (composed of 0.137M sodium chloride, 0.0027M potassium chloride, 0.01M disodium hydrogen phosphate, and 0.0018M potassium dihydrogen phosphate) at a concentration of 20 μg / mL as the aqueous phase. The total mass of metal-phospholipid complex, distearate phosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) was mixed with the mRNA 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 and aqueous phases in the microfluidic chip was 12 ml / min. The drug mRNA was the mRNA encoding the fluorescent protein eGFP, and its sequence is SEQ ID NO.1 (720 nt). The prepared eGFP-mRNA@MPP was then incubated with 293T cells at a concentration of 2 μg / mL (the concentration of mRNA contained). The control group was incubated with unloaded MPP. After 48 h, the cell suspension was collected, and the percentage of eGFP-positive cells was detected by flow cytometry.

[0377] Example 1.5: Preparation of Mg metal ions 2+ mRNA-loaded metal-chelated phospholipid complex nanoparticles (mRNA@MPP)

[0378] The metal-phospholipid complex was prepared using the one-step method described in Example 1.2.4, wherein DSPC, curcumin, and MgCl2 were added in a 1:1:1 ratio. The metal-phospholipid complex, distearate phosphatidylcholine (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 that inhibits particle aggregation) were dissolved in ethanol at different molar proportions to form the organic phase. The proportions of the metal-phospholipid complex, DSPC, CHOL, and DSPE-PEG2000 were 15%, 40%, 43.2%, and 1.8%, respectively. mRNA was dissolved in PBS (composed of 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.0018 M potassium dihydrogen phosphate) at a concentration of 20 μg / mL to form the aqueous phase. The total mass of metal-phospholipid complex, distearate phosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) was mixed with the mRNA 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 and aqueous phases in the microfluidic chip was 12 ml / min. The drug mRNA was the mRNA encoding the fluorescent protein eGFP, and its sequence is SEQ ID NO.1 (720 nt). The prepared eGFP-mRNA@MPP was then incubated with 293T cells at a concentration of 2 μg / mL (the concentration of mRNA contained). The control group was incubated with unloaded MPP. After 48 h, the cell suspension was collected, and the percentage of eGFP-positive cells was detected by flow cytometry.

[0379] The particle size, surface potential, and stability of the eGFP-mRNA@MPP prepared in Examples 1.3-1.5 were detected, and the efficiency of the eGFP-mRNA@MPP in loading nucleic acids was calculated.

[0380] Except for the metal-phospholipid complex prepared by the "one-step method" in Example 1.2.4, the other metal ions were prepared according to the methods in Examples 1.3 and 1.4, respectively, with Fe as the metal ion. 3+ mRNA-loaded metal-chelated phospholipid complex nanoparticles (mRNA@MPP), and preparation of metal ions as Al 3+The prepared eGFP-mRNA@MPP was then analyzed for particle size, surface potential, and stability. The efficiency of the eGFP-mRNA@MPP in loading nucleic acids and the eGFP-positive cell rate were also calculated.

[0381] Methods for particle size detection and criteria for judging results: The particle size of nanoparticles was tested using a Malvern laser particle size analyzer (Zetasizer). Particle sizes within the range of 50–400 nm were considered acceptable.

[0382] Methods for detecting surface potential and criteria for judging results: The surface potential of nanoparticles is tested using a Malvern laser particle size analyzer (Zetasizer). A potential range of -10 to 10 mV is considered acceptable.

[0383] The method for testing stability and the criteria for judging the results are as follows: The nanoparticles are placed at 4°C for 7 days, and the particle size and surface potential of the nanoparticles are tested using a Malvern laser particle size analyzer (Zetasizer). When there is no significant change in the particle size and surface potential within 3-7 days, the stability is considered to be good.

[0384] The method for calculating nucleic acid loading efficiency was agarose gel electrophoresis. First, the nucleic acid loading amount for each group of lipid nanoparticles was set to 10 μg / mL, with a lipid-to-nucleic acid mass ratio of 40:1. Nucleic acid of equal concentration was dissolved in PBS buffer solution as a positive control, and PBS buffer solution was used as a negative control. The agarose gel concentration was 1.5%, at which point the gel pores only allowed free nucleic acid to pass through, not lipid nanoparticles. Electrophoresis was stopped when the free nucleic acid bands became clearly distinguishable. ImageJ software was used to analyze the grayscale values ​​of free nucleic acid in different groups, with the positive control group 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 rate for each group was then calculated as (100 - relative amount of free nucleic acid)%. A nucleic acid loading rate above 50% was considered acceptable.

[0385] Cell culture method: Human embryonic kidney cell line 293T was cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0386] Flow cytometry method for analyzing the percentage of eGFP-positive cells: 293T cells were seeded in 24-well plates at a seeding density of 5 × 10⁻⁶. 5Cells were incubated at 1 mL / well. When the cell density reached 80%, 1 mL of MPP or eGFP-mRNA@MPP was added for incubation, with the eGFP-mRNA@MPP concentration at 2 μg / mL. After 48 hours, the cell suspension was collected, and 20,000 cells were collected using the FITC channel of a flow cytometer. The percentage of eGFP-positive cells was analyzed using the formula: eGFP-positive cell rate = (number of cells expressing eGFP / total number of cells) × 100%. An eGFP-positive cell percentage of 40% or higher was considered acceptable.

[0387] The principle behind loading nucleic acids into metal-chelated phospholipid complex nanoparticles (MPPs) assembled from metal-phospholipid complexes is as follows: curcumin is bound to DSPC via hydrogen bonds, while curcumin also binds to Fe via coordination bonds. 3+ Ca 2+ Al 3+ or Mg 2+ They are linked together to form a metal-phospholipid complex, the Fe of which is... 3+ Ca 2+ Al 3+ or Mg 2+ By linking with nucleic acids through coordination bonds, the metal-phospholipid complex and other lipid components are ensured to self-assemble into MPPs while simultaneously loading nucleic acids into nanoparticles. The contribution of curcumin to nucleic acid loading in MPPs can be twofold: ① Curcumin interacts with nucleic acids, assisting in nucleic acid loading by intercalating into the minor grooves of nucleic acids; ② Curcumin may not directly interact with nucleic acids.

[0388] Example 1.3.1, The metal ion is Fe 3+ The dosage ratio of components in the metal-phospholipid complex

[0389] DSPC, curcumin, and FeCl3 from Example 1.3 were added in different molar ratios (1:1:1, 3:3:2, 2:2:1), and other steps were the same as in Example 1.3 to prepare different eGFP-mRNA@MPP. The nucleic acid loading rates of each were then detected.

[0390] Results Analysis: As shown in Table 1-1, when the molar ratio of DSPC, curcumin, and FeCl3 was 1:1:1, the eGFP-mRNA loading efficiency of the prepared metal-phospholipid complex particles was 87%; when the molar ratio was 3:3:2, the eGFP-mRNA loading efficiency was 70%; and when the molar ratio was 2:2:1, the eGFP-mRNA loading efficiency was 60%. In the metal-phospholipid complex particles, Fe... 3+ Its function is to connect phospholipid complexes to nucleic acids, each Fe 3+ There are a maximum of three complexation sites, so the molar ratio of DSPC, curcumin, and FeCl3 in the drug-lipid particles should be 1:1:1 to ensure that the metal-phospholipid complex particles can encapsulate as much nucleic acid as possible. Experimental results also confirmed that the eGFP-mRNA loading rate of the metal-phospholipid complex particles prepared using this 1:1:1 molar ratio was the highest. When the molar ratio of DSPC, curcumin, and FeCl3 ranged from 1:1:1 to 2:2:1, the nucleic acid loading rate of the metal-phospholipid complex particles was consistently above 60%.

[0391] Table 1-1 Metal ions are Fe 3+ The molar ratio of components in a metal-phospholipid complex and the function of the prepared metal-phospholipid complex particles

[0392] Example 1.4.1, the metal ion is Al 3+ The dosage ratio of components in the metal-phospholipid complex

[0393] DSPC, curcumin, and Al(NO3)3·9H2O from Example 1.4 were added in different molar ratios (1:1:1, 3:3:2, 2:2:1), and other steps were the same as in Example 1.4 to prepare different eGFP-mRNA@MPP, and their nucleic acid loading rates were detected.

[0394] Results Analysis: As shown in Tables 1-2, when the molar ratio of DSPC, curcumin, and Al(NO3)3·9H2O was 1:1:1, the eGFP-mRNA loading efficiency of the prepared metal-phospholipid complex particles was 92%; when the molar ratio was 3:3:2, the eGFP-mRNA loading efficiency was 72%; and when the molar ratio was 2:2:1, the eGFP-mRNA loading efficiency was 58%. In the metal-phospholipid complex particles, Al... 3+ Its function is to connect phospholipid complexes to nucleic acids, each Al 3+ There are a maximum of three complexation sites, so the molar ratio of DSPC, curcumin, and Al(NO3)3·9H2O in the drug-lipid particles should be 1:1:1 to ensure that the metal-phospholipid complex particles can encapsulate as much nucleic acid as possible. Experimental results also confirmed that the eGFP-mRNA loading rate of the metal-phospholipid complex particles prepared using this 1:1:1 molar ratio was the highest. When the molar ratio of DSPC, curcumin, and Al(NO3)3·9H2O ranged from 1:1:1 to 2:2:1, the nucleic acid loading rate of the metal-phospholipid complex particles was consistently above 58%.

[0395] Table 1-2 shows the metal ions as Al. 3+ The molar ratio of components in a metal-phospholipid complex and the function of the prepared metal-phospholipid complex particles

[0396] Example 1.5.1, Metal ion is Mg 2+ The dosage ratio of components in the metal-phospholipid complex

[0397] DSPC, curcumin, and MgCl2 from Example 1.5 were added in different molar ratios (1:1:1, 3:3:2, 2:2:1), and other steps were the same as in Example 1.5 to prepare different eGFP-mRNA@MPP, and their nucleic acid loading rates were detected.

[0398] Results Analysis: As shown in Tables 1-3, when the molar ratio of DSPC, curcumin, and MgCl2 was 1:1:1, the eGFP-mRNA loading efficiency of the prepared metal-phospholipid complex particles was 93.9%; when the molar ratio of DSPC, curcumin, and MgCl2 was 3:3:2, the eGFP-mRNA loading efficiency of the prepared metal-phospholipid complex particles was 72.6%. In the metal-phospholipid complex particles, the function of MgCl2 is to connect the phospholipid complex to the nucleic acid. Each MgCl2... 2+ Since there are at most two complexation sites, the molar ratio of DSPC, curcumin, and MgCl2 in the drug-lipid particles should be 1:1:1 to ensure that the metal-phospholipid complex particles can encapsulate as much nucleic acid as possible. Experimental results also confirmed that the eGFP-mRNA loading rate of the metal-phospholipid complex particles prepared using this 1:1:1 molar ratio was the highest. When the molar ratio of DSPC, curcumin, and MgCl2 ranged from 1:1:1 to 3:3:2, the nucleic acid loading rate of the metal-phospholipid complex particles was consistently above 72.6%.

[0399] Table 1-3 shows the metal ions as Mg. 2+ The molar ratio of components in a metal-phospholipid complex and the function of the prepared metal-phospholipid complex particles

[0400] Example 1.3.2, the metal ion is Fe 3+ The ratio of metal-phospholipid complex, distearate phosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) in the prepared metal-phospholipid complex particles was as follows:

[0401] Compared to Example 1.3, the metal-phospholipid complex (metal ion is Fe) 3+ The proportions of distearate phosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) are shown in Table 1-4, with the other conditions remaining the same.

[0402] Results analysis: As shown in Tables 1-4, when the proportion of metal-phospholipid complex 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)%, the proportion of DSPE-PEG2000 is in the range of (2-10)%, the particle size of 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 loading rate is >50%, and the positive expression rate of eGFP protein is over 70%. Among them, the metal-phospholipid complex particles exhibited the best performance when the proportions were 15% (metal-phospholipid complex), 35% (distearylphosphatidylcholine (DSPC), 46% (cholesterol), and 4% (DSPE-PEG2000), namely, a particle size in the range of 110 nm, a surface potential in the range of -2.04 mV, in vitro stability >7 days, mRNA loading rate of 87%, and positive expression rate of eGFP protein of 97%. Because mRNA@MPP mainly relies on the coordination between the metal-phospholipid complex and nucleic acid for encapsulation, the proportion of the metal-phospholipid complex cannot be too low. When the DSPC content is in the range of 0-40%, the stability of the nanoparticles is within an acceptable range. When the DSPC content is 0%, the stability of the nanoparticles is maintained because the metal-phospholipid complex contains DSPC. The role of DSPE-PEG2000 is to prevent nanoparticle aggregation and increase in vivo circulation time. Its performance is better when its content is in the range of 2-10%. The role of CHOL is to enhance the fluidity of nanoparticles. Maintaining a certain content is beneficial to the stability of nanoparticles.

[0403] The above results suggest that mRNA@MPP has good drug loading performance when the proportion of metal-phospholipid complex 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)%.

[0404] Table 1-4 shows the metal ions for Fe. 3+ Performance detection of eGFP-mRNA@MPP with different component ratios

[0405] Example 1.4.2, the metal ion is Al 3+ The ratio of metal-phospholipid complex, distearate phosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) in the prepared metal-phospholipid complex particles was as follows:

[0406] Compared to Example 1.4, the metal-phospholipid complex (metal ion is Al) was used. 3+The proportions of distearate phosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) are shown in Table 1-5, with the other conditions remaining the same.

[0407] Results Analysis: As shown in Tables 1-5, when the metal-phospholipid complex (metal ion is Al) 3+ When the proportions of metal-phospholipid complex (MSC) are in the range of 5-50%, DSPC is in the range of 0-51%, CHOL is in the range of 15-80%, and DSPE-PEG2000 is in the range of 2-10%, the drug-lipid particles have a particle size of 50-400 nm, a surface potential of -10-10 mV, in vitro stability of more than 3 days, mRNA loading rate of more than 50%, and positive expression rate of eGFP protein of more than 70%. Among these, the drug-lipid particles exhibit the best performance when the proportions of metal-phospholipid complex (MSC) are 7%, DSPC (distearate phosphatidylcholine) is 34%, CHOL (cholesterol) is 56%, and DSPE-PEG2000 (DSPE-PEG2000) is 3%, i.e., a particle size of 100 nm, a surface potential of -1.57 mV, in vitro stability of >7 days, mRNA loading rate of 92%, and positive expression rate of eGFP protein of 98%. Because mRNA@MPP mainly relies on the coordination between the metal-phospholipid complex and nucleic acid for encapsulation, the proportion of the metal-phospholipid complex cannot be too low. When the DSPC content is in the range of 0-51%, the stability of the nanoparticles is within an acceptable range. When the DSPC content is 0%, the stability of the nanoparticles is maintained because the metal-phospholipid complex contains DSPC. The role of DSPE-PEG2000 is to prevent nanoparticle aggregation and increase in vivo circulation time. Its performance is better when its content is in the range of 2-10%. The role of CHOL is to enhance the fluidity of nanoparticles. Maintaining a certain content is beneficial to the stability of nanoparticles.

[0408] The above results suggest that metal-phospholipid complexes (with Al as the metal ion) 3+ When the percentage of mRNA@MPP is in the range of (5-50)%, the percentage of DSPC is in the range of (0-51)%, the percentage of CHOL is in the range of (15-80)%, and the percentage of DSPE-PEG2000 is in the range of (2-10)%, mRNA@MPP exhibits good drug loading performance.

[0409] Table 1-5 Metal ions are Al 3+ eGFP-mRNA@MPP (Al) with different component ratios 3+ Performance testing

[0410] Example 1.5.2, Metal ion is Mg 2+The ratio of metal-phospholipid complex, distearate phosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) in the prepared metal-phospholipid complex particles was as follows:

[0411] Compared to Example 1.5, the metal-phospholipid complex (metal ion is Mg) 2+ The proportions of distearate phosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) are shown in Table 1-6, with the other conditions remaining the same.

[0412] Results analysis: As shown in Tables 1-6, when the proportion of metal-phospholipid complex is in the range of (5-40)%, the proportion of DSPC is in the range of (0-50)%, the proportion of CHOL is in the range of (25-75)%, the proportion of DSPE-PEG2000 is in the range of (1-10)%, the particle size of 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 loading rate is >50%, and the positive expression rate of eGFP protein is over 70%. Among them, the metal-phospholipid complex particles exhibited the best performance when the proportions were 15% (metal-phospholipid complex), 40% (distearylphosphatidylcholine (DSPC), 43.2% (cholesterol), and 1.8% (DSPE-PEG2000), namely, a particle size in the range of 115 nm, a surface potential in the range of -2.9 mV, in vitro stability >7 days, mRNA loading rate of 93.9%, and positive expression rate of eGFP protein of 98%. Because mRNA@MPP mainly relies on the coordination between the metal-phospholipid complex and nucleic acid for encapsulation, the proportion of the metal-phospholipid complex cannot be too low. When the content of DSPC is in the range of 0-50%, the stability of its nanoparticles is within an acceptable range. When the content of DSPC is 0%, the stability of its nanoparticles is maintained because the metal-phospholipid complex contains DSPC. The role of DSPE-PEG2000 is to prevent nanoparticle aggregation and increase in vivo circulation time. Its performance is better when its content is in the range of 1-10%. The role of CHOL is to enhance the fluidity of nanoparticles. Maintaining a certain content is beneficial to the stability of nanoparticles.

[0413] The above results suggest that mRNA@MPP has good drug loading performance when the proportion of metal-phospholipid complex is in the range of (5-40)%, DSPC is in the range of (0-50)%, CHOL is in the range of (25-75)%, and DSPE-PEG2000 is in the range of (1-10)%.

[0414] Table 1-6 shows the metal ions for Mg. 2+ Performance detection of eGFP-mRNA@MPP with different component ratios

[0415] Example 1.3.3: Preparation of non-cationic or non-ionizable lipid types in eGFP-mRNA@MPP

[0416] Example 1.3.3.1 Preparation of eGFP-mRNA@MPP(Fe 3+ Non-cationic lipids or non-ionizable lipids

[0417] Compared with Example 1.3, the substitution of distearylphosphatidylcholine (DSPC) is shown in Tables 1-7, with all other conditions remaining the same.

[0418] Results analysis: To explore the eGFP-mRNA@MPP(Fe 3+ In eGFP-mRNA@MPP, DSPC can be replaced by other non-cationic or non-ionizable lipids. We selected three other non-cationic or non-ionizable lipids, namely DSPE, DSPA, and DSPG, to replace DSPC, and demonstrated the effectiveness of eGFP-mRNA@MPP by detecting particle size, surface potential, stability, and mRNA loading rate. 3+ In this formula, DSPC can be replaced by other non-cationic or non-ionizable lipids, and its function after replacement is equivalent to that of eGFP-mRNA@MPP (Fe2+) containing DSPC. 3+ The efficacy of ) (Table 1-7). Because non-cationic lipids or non-ionizable lipids DSPC in eGFP-mRNA@MPP (Fe 3+ The main function of DSPC in liposomes is to improve liposome membrane fusion, stability, and toxicity. Other non-cationic or non-ionizable lipids also have the function of improving liposome membrane fusion, stability, and toxicity. Therefore, DSPC in drug-lipid particles can be replaced by other non-cationic or non-ionizable lipids, except for metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation, without affecting its efficacy.

[0419] The structural formulas of three non-cationic or non-ionizable lipids (DSPE, DSPA, and DSPG) are shown below. DSPE (Formula 47) DSPA (Formula 48) DSPG (Formula 49)

[0420] Table 1-7 eGFP-mRNA@MPP(Fe) containing different types of non-cationic or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation. 3+ ) performance

[0421] Example 1.3.3.2: Preparation of eGFP-mRNA@MPP(Al) 3+ Non-cationic lipids or non-ionizable lipids

[0422] Compared with Example 1.4, the substitution of distearylphosphatidylcholine (DSPC) is shown in Tables 1-8, with all other conditions remaining the same.

[0423] Results analysis: To explore the eGFP-mRNA@MPP(Al 3+ In this study, DSPC can be replaced by other non-cationic or non-ionizable lipids. We selected three other non-cationic or non-ionizable lipids, namely DSPE (Equation 47), DSPA (Equation 48), and DSPG (Equation 49), to replace DSPC. By detecting particle size, surface potential, stability, and mRNA loading rate, we demonstrated that eGFP-mRNA@MPP(Al) can replace DSPC. 3+ In this study, DSPC can be replaced by other non-cationic or non-ionizable lipids, and its function after replacement is equivalent to that of eGFP-mRNA@MPP (Al). 3+ Table 1-8 shows the efficacy of the metal ion Al. 3+ Because non-cationic lipids or non-ionizable lipids DSPC in eGFP-mRNA@MPP(Al) 3+ The main function of DSPC in liposomes is to improve liposome membrane fusion, stability, and toxicity. Other non-cationic or non-ionizable lipids also have the function of improving liposome membrane fusion, stability, and toxicity. Therefore, DSPC in drug-lipid particles can be replaced by other non-cationic or non-ionizable lipids, except for metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation, without affecting its efficacy.

[0424] Table 1-8 eGFP-mRNA@MPP (Al) containing different types of non-cationic or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation. 3+ ) performance

[0425] Example 1.3.3.3: Preparation of eGFP-mRNA@MPP(Mg 2+ Non-cationic lipids or non-ionizable lipids

[0426] Compared with Example 1.5, the substitution of distearylphosphatidylcholine (DSPC) is shown in Tables 1-9, with all other conditions remaining the same.

[0427] Results analysis: To explore eGFP-mRNA@MPP(Mg 2+In this study, DSPC can be replaced by other non-cationic or non-ionizable lipids. We selected three other non-cationic or non-ionizable lipids, namely DSPE (Equation 47), DSPA (Equation 48), and DSPG (Equation 49), to replace DSPC. By detecting particle size, surface potential, stability, and mRNA loading rate, we demonstrated that eGFP-mRNA@MPP(Mg 2+ In this formula, DSPC can be replaced by other non-cationic or non-ionizable lipids, and its function after replacement is equivalent to that of eGFP-mRNA@MPP(Mg) containing DSPC. 2+ The efficacy of ) (Table 1-9). Because non-cationic lipids or non-ionizable lipids DSPC in eGFP-mRNA@MPP(Mg 2+ The main function of DSPC in liposomes is to improve liposome membrane fusion, stability, and toxicity. Other non-cationic or non-ionizable lipids also have the function of improving liposome membrane fusion, stability, and toxicity. Therefore, DSPC in drug-lipid particles can be replaced by other non-cationic or non-ionizable lipids, except for metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation, without affecting its efficacy.

[0428] Table 1-9 eGFP-mRNA@MPP(Mg) containing different types of non-cationic or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation. 2+ ) performance

[0429] Example 1.3.4: Preparation of lipid conjugations for inhibiting particle aggregation in eGFP-mRNA@MPP

[0430] Example 1.3.4.1: Preparation of eGFP-mRNA@MPP(Fe 3+ Types of conjugated lipids that inhibit particle aggregation

[0431] Compared with Example 1.3, the substitutions for DSPE-PEG2000 (Formula 53) are shown in Tables 1-10 (metal ion is Fe). 3+ As shown in the figure, the other conditions are the same. Three other conjugated lipids that inhibit particle aggregation are DSPE-PEG700 (Equation 50), DSPE-PEG5000 (Equation 52), and DSPE-PEG1000 (Equation 51).

[0432] Results analysis: To explore the eGFP-mRNA@MPP(Fe 3+In this study, DSPE-PEG2000 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. By detecting particle size, surface potential, stability, and mRNA loading rate, we demonstrated that eGFP-mRNA@MPP(Fe 3+ In this formulation, DSPE-PEG2000 can be replaced by other conjugated lipids that inhibit particle aggregation. After replacement, its function is equivalent to that of eGFP-mRNA@MPP (Fe...). 3+ The efficacy of DSPE-PEG2000 (Table 1-10) is shown. Because DSPE-PEG2000 is effective in eGFP-mRNA@MPP (Fe... 3+ The main function of eGFP-mRNA@MPP is to inhibit aggregation, and other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation. Therefore, eGFP-mRNA@MPP(Fe 3+ DSPE-PEG2000 in the formula can be replaced by other conjugated lipids that inhibit particle aggregation without affecting its efficacy.

[0433] Table 1-10 eGFP-mRNA@MPP(Fe) containing different types of conjugated lipids that inhibit particle aggregation 3+ ) performance

[0434] Example 1.3.4.2: Preparation of eGFP-mRNA@MPP(Al) 3+ Types of conjugated lipids that inhibit particle aggregation

[0435] Compared with Example 1.4, the substitutions for DSPE-PEG2000 (Formula 53) are shown in Table 1-11 (metal ions are Al). 3+ As shown in the figure, the other conditions are the same. Three other conjugated lipids that inhibit particle aggregation are DSPE-PEG700 (Equation 50), DSPE-PEG5000 (Equation 52), and DSPE-PEG1000 (Equation 51).

[0436] Results analysis: To explore the eGFP-mRNA@MPP(Al 3+ In this study, DSPE-PEG2000 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. By detecting particle size, surface potential, stability, and mRNA loading rate, we demonstrated that eGFP-mRNA@MPP(Al 3+In this formulation, DSPE-PEG2000 can be replaced by other conjugated lipids that inhibit particle aggregation. After replacement, its function is equivalent to that of eGFP-mRNA@MPP (Al). 3+ The efficacy of DSPE-PEG2000 (Table 1-11). Because DSPE-PEG2000 in eGFP-mRNA@MPP (Al 3+ The main function of eGFP-mRNA@MPP is to inhibit aggregation, and other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation. Therefore, eGFP-mRNA@MPP(Al) 3+ DSPE-PEG2000 in the formula can be replaced by other conjugated lipids that inhibit particle aggregation without affecting its efficacy.

[0437] Table 1-11 eGFP-mRNA@MPP(Al) containing different types of conjugated lipids that inhibit particle aggregation 3+ ) performance

[0438] Example 1.3.4.3: Preparation of eGFP-mRNA@MPP(Mg 2+ Types of conjugated lipids that inhibit particle aggregation

[0439] Compared with Example 1.5, the substitutions for DSPE-PEG2000 (Formula 53) are shown in Tables 1-12 (metal ion is Mg). 2+ As shown in the figure, the other conditions are the same. Three other conjugated lipids that inhibit particle aggregation are DSPE-PEG700 (Equation 50), DSPE-PEG5000 (Equation 52), and DSPE-PEG1000 (Equation 51).

[0440] Results Analysis: To explore whether DSPE-PEG2000 in eGFP-mRNA@MPP could 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. By detecting particle size, surface potential, stability, and mRNA loading rate, we demonstrated that eGFP-mRNA@MPP(Mg 2+ In this formula, DSPE-PEG2000 can be replaced by other conjugated lipids that inhibit particle aggregation. After replacement, its function is equivalent to that of eGFP-mRNA@MPP(Mg) containing DSPE-PEG2000. 2+ The efficacy of DSPE-PEG2000 (Table 1-12) is shown. Because DSPE-PEG2000 is effective against eGFP-mRNA@MPP(Mg... 2+The main function of eGFP-mRNA@MPP(Mg) is to inhibit aggregation, and other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation. Therefore, eGFP-mRNA@MPP(Mg) 2+ DSPE-PEG2000 in the formula can be replaced by other conjugated lipids that inhibit particle aggregation without affecting its efficacy.

[0441] Table 1-12 eGFP-mRNA@MPP(Mg) containing different types of conjugated lipids that inhibit particle aggregation 2+ ) performance

[0442] Example 1.3.5: Preparation and Effect Characterization of mRNA@MPP

[0443] Example 1.3.5.1, mRNA@MPP(Fe 3+ Preparation and effect characterization of )

[0444] The mRNA in Example 1.3 was replaced with two other mRNAs, and three mRNA@MPP(Fe) samples containing different target protein mRNA sequences were prepared according to the method in Example 1.3. 3+ The three different mRNA sequences are: ① the mRNA sequence encoding the fluorescent protein eGFP is SEQ ID NO.1 (720 nt); ② the mRNA sequence encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit is SEQ ID NO.2 (669 nt); ③ the mRNA sequence encoding the tumor antigen NY-ESO-1 is SEQ ID NO.3 (543 nt). The preparation process of the remaining drug (mRNA)-metal-phospholipid complex particles is the same as in Example 1.3, yielding eGFP-mRNA@MPP(Fe 3+ ), RBD-mRNA@MPP(Fe 3+ ), NY-ESO-1-mRNA@MPP(Fe 3+ ).

[0445] eGFP-mRNA@MPP(Fe 3+293T cells were incubated with MPP at a concentration of 2 μg / mL (mRNA concentration), while the control group was incubated with MPP. After 48 h, the cell suspension was collected, and the percentage of eGFP-positive cells was detected by flow cytometry. The results are shown in Figure 1-1-1. RBD-mRNA@MPP was incubated with 293T cells at a concentration of 2 μg / mL (mRNA concentration), while the control group was incubated with MPP. After 24 h, the supernatant was centrifuged and stored at -20℃ for later use. The expression level of RBD protein on the cells was detected using a commercially available COVID-19 antigen RBD ELISA kit. The results are shown in Figure 1-2-1.

[0446] Methods for detecting RBD expression levels using ELISA:

[0447] 1. Sample collection: Whole blood samples were left at room temperature for 2 hours, then centrifuged at 1000×g for 20 min, and the supernatant was collected;

[0448] 2. Sample addition: The coated plate is equipped with blank wells, standard wells, and sample wells. Add 100 μL of sample diluent to the blank wells, add serially diluted standards to the standard wells, and add 100 μL of the sample to the sample wells. Incubate at 37°C for 60 min.

[0449] 3. Discard the liquid in the wells, wash the plate 3 times, soaking for 1-2 minutes each time. Add 100 μL of the prepared biotin-labeled anti-RBD antibody working solution to each well, mix well, and incubate at 37°C for 60 minutes;

[0450] 4. Discard the liquid in the well, wash the plate 3 times, soaking for 1-2 minutes each time;

[0451] 5. Add 100 μL of the prepared streptavidin HRP working solution to each well, mix well, and incubate at 37°C for 45 min;

[0452] 6. Discard the liquid in the well, wash the plate 3 times, soaking for 1-2 minutes each time;

[0453] 7. Add 100 μL of TMB substrate solution (TMB) to each well and incubate at 37°C in the dark for 15 min;

[0454] 8. Add 100 μL of stop solution to each well to terminate the reaction;

[0455] 9. Measure the optical density (OD value) of each aperture at a wavelength of 450 nm.

[0456] Data analysis: Plot a standard curve with the concentration of the standard on the x-axis and the OD value on the y-axis.

[0457] The experimental animals were randomly divided into two groups (experimental group and control group), with five animals in each group. Among them, RBD-mRNA@MPP(Fe 3+The animal model used was BALB / c mice. Each mouse received the first intramuscular injection on day 1 and the second intramuscular injection on day 14. The experimental group was injected with RBD-mRNA@MPP(Fe 3+ The control group was injected with metal-phospholipid complex particles (MPPs) without mRNA loading. Each administration dose was 100 μL, of which the experimental group contained RBD-mRNA@MPP (Fe... 3+ The formulation contained 30 μg of mRNA. Blood was collected from mice 28 days after the first administration, and the serum was separated and serially diluted. The total RBD IgG antibody against the S1 subunit of the novel coronavirus produced in mice was detected by a commercially available ELISA kit. The results are shown in Figure 1-3-1.

[0458] NY-ESO-1-mRNA@MPP(Fe 3+ The animal model used was the C57BL / 6 mouse. Each mouse received four intramuscular injections on days 1, 7, 14, and 21. The experimental group was injected with NY-ESO-1-mRNA@MPP(Fe 3+ The control group was injected with metal-phospholipid complex particles (MPPs) without mRNA loading. Each administration dose was 100 μL, of which the experimental group contained NY-ESO-1-mRNA@MPP (Fe... 3+ The formulation contained 30 mg of mRNA. Blood was collected from mice 28 days after the first administration, and the serum was separated and serially diluted. The total anti-NY-ESO-1 IgG antibody produced in the mice was detected by ELISA. The results are shown in Figure 1-4-1.

[0459] Methods for detecting total anti-NY-ESO-1 IgG antibodies in mice:

[0460] Preparation of reagents for ELISA:

[0461] 1. Coating solution: Accurately weigh 8.4g NaHCO3 and dissolve it in 1L distilled water (DDW). After the solid has completely dissolved, use 1M NaOH solution to adjust the pH of the entire solution to 9.6. Store the prepared coating solution at 4℃ for later use.

[0462] 2. Washing solution: Add 0.5 mL of Tween-20 to 1 L of 0.01 M PBS solution, mix well and let stand at room temperature.

[0463] 3. Blocking solution: Accurately weigh 20g of BSA and add it to 1L of 0.01M PBS solution. Sonicate the undissolved BSA powder in the solution until all the solid in the solution is dissolved and the solution turns pale yellow. Store in a refrigerator at 4℃ for later use.

[0464] 4. Antibody dilution solution: Accurately weigh 2.5g BSA and dissolve it in 250mL of 0.01M PBS solution. After the solid has completely dissolved, add 1.25mL of Tween-20, mix well, and store at 4℃ for later use.

[0465] 5. Colorimetric solutions: 0.1M citric acid: 19.2g citric acid added to DDW water to 1000mL (A) 0.2M disodium hydrogen phosphate: 28.4g anhydrous disodium hydrogen phosphate added to DDW water to 1000mL (B) 0.1M citric acid solution (A) 24.3mL, 0.2M phosphate buffer (B) 25.7mL, add DDW water 50mL. Add 50mg OPD (o-phenylenediamine) immediately before use, and add 0.15mL of 30% H2O2.

[0466] 6. Termination solution: 2M H2SO4: 55.5mL concentrated sulfuric acid, add DDW to 500mL.

[0467] ELISA method for determining antibody titers in mouse serum:

[0468] 1. Coating: Dilute NY-ESO-1 antigen to 1 μg / mL with coating buffer, add 50 μL / well to a 96-well plate, and coat overnight at 4°C.

[0469] 2. Sealing: Spin-dry the coating solution in the well plate, wash 3 times with sealing solution every 5 minutes and spin-dry, add 150 μL of sealing solution to each well, and incubate at 37°C for 2 hours.

[0470] 3. Drying: Shake off the sealing liquid and incubate at 37°C for 1-2 hours until the liquid at the bottom of the well plate is completely dry.

[0471] 4. Immunization: Initially dilute serum samples 1:1000 with antibody diluent, then serially dilute at a ratio of 1:2. Add 100 μL of the diluted serum sample to each well of a closed 96-well plate and incubate at 37°C for 2 hours. Shake off the liquid in the wells, add 300 μL of washing buffer per well, and gently shake for 40 seconds. Repeat this step three times. Add 100 μL of biotinylated goat anti-mouse IgG antibody diluted 1:1000 to each well and incubate at 37°C. 1 h; spin dry the liquid in the well plate, add washing buffer, and repeat the above washing steps; add freshly prepared streptavidin-labeled horseradish peroxidase (HRP) working solution, 100 μL / well, and incubate at 37°C for 1 h; spin dry the liquid in the well plate, add washing buffer, and repeat the above washing steps; add chromogenic solution, 100 μL / well, under light-protected conditions, react at room temperature for 5 min, then add stop solution, 50 μL / well; measure the absorbance at 450 nm using a microplate reader.

[0472] In giving RBD-mRNA@MPP(Fe 3+On day 28 post-treatment, spleens from normal mice were collected and prepared into single-cell suspensions under sterile conditions. Spinaching was performed at a rate of 100,000 spleen cells per well in cell culture plates. RBD protein at a final concentration of 10 mg / mL was added, and the cells were cultured for 48 h. After centrifugation and removal of the supernatant, the expression levels of IFN-γ, IL-2, and IL-4 were measured using an ELISA kit. The results are shown in Figure 1-5-1. [Further details about NY-ESO-1 mRNA@MPP (Fe...]] 3+ On day 28 after the event, spleens of normal mice were collected and prepared into single-cell suspensions under sterile conditions. The suspensions were seeded into cell plates at a ratio of 100,000 spleen cells / well. NY-ESO-1 protein at a final concentration of 10 mg / mL was added and the cells were cultured for 48 h. After centrifugation and removal of supernatant, the expression levels of IFN-γ, IL-2, and TNF-α were measured using an ELISA kit. The results are shown in Figure 1-6-1.

[0473] Results analysis: As shown in Figure 1-1-1, eGFP-mRNA@MPP(Fe 3+ The eGFP-positive cell rate in the experimental group was 97.7%, while the MPP (Fe) positive cell rate was 97.7%. 3+ No eGFP signal was detected in the control group; as shown in Figure 1-2-1, the RBD protein encoded by the RBD-mRNA loaded by MPP was 193.3 ng / mL in the supernatant of 293T cells, while the concentration of eGFP protein in cells transfected with 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+ RBD-mRNA@MPP (Fe) can encapsulate and deliver any mRNA, directly encoding polypeptides within the cell. As shown in Figures 1-3-1 and 1-4-1, the results demonstrate that RBD-mRNA@MPP (Fe) can encapsulate and deliver any mRNA, directly encoding polypeptides within the cell. 3+ ) and NY-ESO-1-mRNA@MPP(Fe 3+ Both can effectively induce humoral immunity in mice, producing high levels of antigen-specific binding antibodies. Among them, RBD-mRNA@MPP(Fe 3+ The IgG antibody titer in the treated mice reached 117268.8; NY-ESO-1-mRNA@MPP(Fe 3+ The IgG antibody titer in the treated group of mice reached 5319.52. As shown in Figures 1-5-1 and 1-6-1, RBD-mRNA@MPP(Fe 3+ ) and NY-ESO-1-mRNA@MPP(Fe 3+ Both RBD-mRNA@MPP and Fe2O3 can effectively induce cellular immunity in mice, that is, activate immune cells and produce a large number of cytokines. 3+This resulted in the expression levels of cytokines IFN-γ, IL-2, and IL-4 reaching 252.8 pg / mL, 207.6 pg / mL, and 56.6 pg / mL, respectively; NY-ESO-1-mRNA@MPP(Fe 3+ This resulted in the expression levels of cytokines IFN-γ, IL-2, and TNF-α reaching 70.79 pg / mL, 75.29 pg / mL, and 75.27 pg / mL, respectively. The results suggest that mRNA@MPP(Fe 3+ It can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), and effectively inducing humoral and cellular immunity in mice, producing high levels of antigen-specific binding antibodies and cytokines, thus playing the role of anti-COVID-19 mRNA vaccines and anti-tumor mRNA vaccines.

[0474] Example 1.3.5.2, the metal ion is Al 3+ Preparation and effect characterization of mRNA@MPP

[0475] The mRNA in Example 1.4 was replaced with two other mRNAs, and three mRNA@MPP(Al) samples containing different target protein mRNA sequences were prepared according to the method in Example 1.4. 3+ For specific mRNA information and experimental procedures, please refer to Example 1.3.5.1.

[0476] Results analysis: As shown in Figure 1-1-2, eGFP-mRNA@MPP(Al 3+ The eGFP-positive cell rate in the experimental group was 98.02%, while that of MPP (Al) was much lower. 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 transfected with the vector 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. These results suggest that mRNA-MPP (Al...) encodes 212.6 ng / mL of RBD protein. 3+ RBD-mRNA@MPP (Al) can encapsulate and deliver any mRNA, directly encoding polypeptides within the cell. As shown in Figures 1-3-2 and 1-4-2, the results demonstrate that RBD-mRNA@MPP (Al) can encapsulate and deliver any mRNA, directly encoding polypeptides within the cell. 3+ ) and NY-ESO-1-mRNA@MPP (Al 3+ Both can effectively induce humoral immunity in mice, producing high levels of antigen-specific binding antibodies. Among them, RBD-mRNA@MPP (Al) 3+ The IgG antibody titer in the treated mice reached 129113; NY-ESO-1-mRNA@MPP(Al 3+The IgG antibody titer in the treated group of mice reached 6507.4. As shown in Figures 1-5-2 and 1-6-2, RBD-mRNA@MPP(Al 3+ ) and NY-ESO-1-mRNA@MPP (Al 3+ Both can effectively induce cellular immunity in mice, that is, activate immune cells and produce a large number of cytokines. Among them, RBD-mRNA@MPP (Al) 3+ This resulted in the expression levels of cytokines IFN-γ, IL-2, and IL-4 reaching 271.8 pg / mL, 234.6 pg / mL, and 68.4 pg / mL, respectively; NY-ESO-1-mRNA@MPP(Al 3+ This resulted in the expression levels of cytokines IFN-γ, IL-2, and TNF-α reaching 83.8 pg / mL, 98 pg / mL, and 97.8 pg / mL, respectively. The results suggest that mRNA@MPP(Al)... 3+ It can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), and effectively inducing humoral and cellular immunity in mice, producing high levels of antigen-specific binding antibodies and cytokines, thus playing the role of anti-COVID-19 mRNA vaccines and anti-tumor mRNA vaccines.

[0477] Example 1.3.5.3, the metal ion is Mg 2+ Preparation and effect characterization of mRNA@MPP

[0478] The mRNA in Example 1.5 was replaced with two other mRNAs, and three mRNA@MPP(Mg) molecules containing different target protein mRNA sequences were prepared according to the method in Example 1.5. 2+ For specific mRNA information and experimental procedures, please refer to Example 1.3.5.1.

[0479] Results analysis: As shown in Figure 1-1-3, eGFP-mRNA@MPP(Mg 2+ The eGFP-positive cell rate in the experimental group was 98.3%, while the MPP(Mg)-positive cell rate was 98.3%. 2+ No eGFP signal was detected in the control group; as shown in Figure 1-2-3, MPP(Mg 2+ The RBD protein encoded by the RBD-mRNA-encoded mRNA was 218.50 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. These results suggest that mRNA-MPP (Mg...) 2+ RBD-mRNA@MPP(Mg) can encapsulate and deliver any mRNA, directly encoding polypeptides within the cell. As shown in Figures 1-3-3 and 1-4-3, the results demonstrate that RBD-mRNA@MPP(Mg) can encapsulate and deliver any mRNA, directly encoding polypeptides within the cell. 2+ ) and NY-ESO-1-mRNA@MPP(Mg2+ Both can effectively induce humoral immunity in mice, producing high levels of antigen-specific binding antibodies. Among them, RBD-mRNA@MPP(Mg 2+ The IgG antibody titer in the treated mice reached 130614.40; NY-ESO-1-mRNA@MPP(Mg 2+ The IgG antibody titer in the treated group of mice reached 6842.78. As shown in Figures 1-5-3 and 1-6-3, RBD-mRNA@MPP(Mg 2+ ) and NY-ESO-1-mRNA@MPP(Mg 2+ Both RBD-mRNA@MPP (Mg) can effectively induce cellular immunity in mice, that is, activate immune cells and produce a large number of cytokines. 2+ This resulted in the expression levels of cytokines IFN-γ, IL-2, and IL-4 reaching 296.40 pg / mL, 243.33 pg / mL, and 75.30 pg / mL, respectively; NY-ESO-1-mRNA@MPP(Mg 2+ This resulted in the expression levels of cytokines IFN-γ, IL-2, and TNF-α reaching 85.35 pg / mL, 98.58 pg / mL, and 98.02 pg / mL, respectively. The results suggest that mRNA@MPP(Mg 2+ It can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), and effectively inducing humoral and cellular immunity in mice, producing high levels of antigen-specific binding antibodies and cytokines, thus playing the role of anti-COVID-19 mRNA vaccines and anti-tumor mRNA vaccines.

[0480] Example 1.3.6: Preparation and Effects of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPP)

[0481] Example 1.3.6.1, Metal Ionization Fe 3+ Preparation and effects of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPP)

[0482] The mRNA in Example 1.3 was replaced with siRNA, and three siRNA@MPP(Fe) solutions containing different siRNAs were prepared according to the method in Example 1.3. 3+The three different siRNAs targeted genes, sequences, and their corresponding random control sequences were as follows: ① The siRNA targeting the Bcl-2 gene (Bcl-2-siRNA) had sequences SEQ ID No. 4 (antisense strand) and SEQ ID No. 21 (sense strand) (19bp), and its random control sequences were SEQ ID No. 5 (antisense strand) and SEQ ID No. 22 (sense strand) (19bp); ② The siRNA targeting the PLK1 gene (PLK1-siRNA) had sequences SEQ ID No. 6 (antisense strand) and SEQ ID No. 23 (sense strand) (21bp), and its random control sequences were SEQ ID No. 7 (antisense strand) and SEQ ID No. 24 (sense strand) (19bp); ③ The siRNA targeting the Gal-1 gene (Gal-1-siRNA) had a sequence SEQ ID No. 8 (19bp), and its random control sequence was SEQ ID No. 9 (19bp). The preparation process of the remaining siRNAs@MPP was the same as in Example 1.3.

[0483] Cell culture method: U251 human glioblastoma cells were grown in monolayer in high glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin and 2 ml glutamine (Bio Industries) medium, and cultured at 37°C and 5% CO2, and passaged twice a week.

[0484] U251 cells were used at a rate of 1 × 10⁻⁶ per well. 6 Approximately 24 hours after cell density seeding in 6-well plates, each well was inoculated with siRNA@MPP (Fe... 3+ (The concentration of siRNA was 2 μg / mL) After incubation for 72 hours, cells were collected, total RNA was extracted, and the mRNA expression levels of target genes (Bcl-2, PLK1, Gal-1) were detected by RT-PCR. The expression levels of siRNA@MPP (Fe) were statistically analyzed. 3+ The ability to silence target genes in cells.

[0485] Detailed RT-PCR procedure:

[0486] Total RNA extraction: Discard the culture medium from the six-well plate, wash three times with PBS buffer, and add 1 mL of Trizol to each well to lyse the cells. Add 200 μL of chloroform, shake well, incubate at room temperature for 10 min, and centrifuge at 13000 rpm at 4℃ for 15 min to obtain a three-phase liquid, in which RNA is dissolved in the upper aqueous phase. Transfer the upper aqueous phase to a new enzyme-free 1.5 mL centrifuge tube, add 500 μL of isopropanol, incubate at room temperature for 10 min, and centrifuge at 13000 rpm at 4℃ for 15 min to obtain RNA precipitate. Discard the supernatant, add 1 mL of freshly prepared 75% (v / v) ethanol with RNase-free water to each tube, carefully pipette to remove the white RNA precipitate from the bottom of the tube, centrifuge at 7500 rpm at 4℃ for 10 min, discard the supernatant, and aspirate as much liquid as possible from the bottom of the tube. After opening the tube and allowing it to air dry at room temperature, the RNA precipitate at the bottom was dissolved in 50 μL of enzyme-free water. The purity and concentration of the RNA were then determined using an ultra-micro UV-Vis spectrophotometer.

[0487] cDNA reverse transcription: using TaKaRa Prime Script TM The RT reagent kit with gDNA Eraser reverse transcribes RNA into cDNA. Removing genomic DNA (gDNA) before the reverse transcription step makes the results more accurate and reliable. The total RNA reverse transcription reaction mixture was prepared on ice: 1 μL Prime Script RT Enzyme Mix I, 1 μL RT Primer Mix, 4 μL 5×Prime Script Buffer II, and 4 μL RNase-Free dH2O. After preparing the reaction mixture, it was incubated at 37°C for 15 min, then incubated at 85°C for 5 sec to terminate the reaction, and subsequently stored at 4°C for later use.

[0488] RT-PCR Procedure: This detection method uses the SYBR Green dye and requires no probe. Specifically, Real-time PCR is performed using cDNA from different samples as templates. Prepare the reaction solution 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 cDNA template obtained in the previous step, and 3.2 μL dH2O. Add 10 μL to each well of a plate. After addition, centrifuge (1000 rpm, 5 min) to remove liquid residue and air bubbles from the reaction solution. Real-time PCR was performed using an ABI ViiA7 real-time quantitative PCR instrument. The reaction program was: 95℃, 30 sec (1 cycle) → 95℃, 5 sec; 55℃, 30 sec; 72℃, 30 sec (40 cycles) → 60℃-95℃, 2 min (1 cycle). The experiment was repeated three times, and the average value was used to obtain the Ct value for each group. The fold change in expression between the experimental group and the control group was calculated. The control gene was GAPDH. The RT-PCR primers are as follows: ① Bcl-2 primer: forward: 5'-AGGATTGTGGCCTTCTTTGAG-3' (SEQ ID NO.97), reverse: 5'-AGACAGCCAGGAGAAATC AAAC-3' (SEQ ID NO.98); ② PLK1 primer: forward: 5'-ACCAGCACGTCGTAGGATTC-3' (SEQ ID NO.99), reverse: 5'-CAAGCAATTTGCCGTAGG-3' (SEQ ID NO.100); ③ Gal-1 primer: forward: 5'-CAATCATGGCCTGTGGTCTG-3' (SEQ ID NO.101), reverse: 5'-GTGTAGGCACAGGTTGTTGCTG-3' (SEQ ID NO.102). ④ GAPDH primers: forward: 5'-TCAGGGGTTTCACATTTGGCA-3' (SEQ ID NO.103), reverse: 5'-GGAGCGGAAAACCA-3' (SEQ ID NO.104). The expression levels of each target gene are expressed using RQ values ​​(2). -ΔΔCT The formula is 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

[0489] The gene silencing efficiency (%) is calculated as follows: (1 - gene expression level in the experimental group / gene expression level in the control group) × 100%.

[0490] Results Analysis: As shown in Figure 1-7-1 (in the figure, "scr siRNA" refers to "Scramble siRNA", which serves as a negative control, the same applies below), Figures 1-8-1, and Figures 1-9-1, the three siRNAs@MPP (Fe 3+ All of these can significantly interfere with their corresponding target genes. Bcl-2-siRNA@MPP(Fe 3+ The inhibition rate of the target gene Bcl-2 reached 76%; PLK1-siRNA@MPP(Fe 3+ The inhibition rate of the target gene PLK1 reached 86%; Gal-1-siRNA@MPP(Fe 3+ The inhibition rate of the target gene Gal-1 reached 73%. These results suggest that siRNA@MPP(Fe) 3+ It can carry any siRNA to intervene in the treatment of target genes, and play the role of siRNA-loaded drugs, vaccines or other products.

[0491] Example 1.3.6.2, the metal ion is Al 3+ Preparation and effects of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPP)

[0492] The mRNA in Example 1.4 was replaced with siRNA, and three siRNA@MPP(Al) packages containing different siRNA sequences were prepared according to the method in Example 1.4. 3+ For specific siRNA information and experimental procedures, please refer to Example 1.3.6.1.

[0493] Results analysis: As shown in Figures 1-7-2, 1-8-2, and 1-9-2, the three siRNA@MPP(Al) 3+ All of these can significantly interfere with their corresponding target genes. Bcl-2-siRNA@MPP(Al 3+ The inhibition rate of the target gene Bcl-2 reached 81%; PLK1-siRNA@MPP (Al 3+ The inhibition rate of the target gene PLK1 reached 90%; Gal-1-siRNA@MPP (Al 3+The inhibition rate of the target gene Gal-1 reached 79%. These results suggest that siRNA@MPP(Al) has a significant inhibitory effect on Gal-1. 3+ It can carry any siRNA to intervene in the treatment of target genes, and play the role of siRNA-loaded drugs, vaccines or other products.

[0494] Example 1.3.6.3, the metal ion is Mg 2+ Preparation and effects of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPP)

[0495] The mRNA in Example 1.5 was replaced with siRNA, and three siRNA@MPP(Mg) molecules containing different siRNA sequences were prepared according to the method in Example 1.5. 2+ For specific siRNA information and experimental procedures, please refer to Example 1.3.6.1.

[0496] Results analysis: As shown in Figures 1-7-3, 1-8-3, and 1-9-3, the three siRNA@MPP(Mg 2+ All of these can significantly interfere with their corresponding target genes. Bcl-2-siRNA@MPP(Mg 2+ The inhibition rate of the target gene Bcl-2 reached 82%; PLK1-siRNA@MPP(Mg 2+ The inhibition rate of the target gene PLK1 reached 92%; Gal-1-siRNA@MPP(Mg 2+ The inhibition rate of the target gene Gal-1 reached 86%. These results suggest that siRNA@MPP(Mg 2+ It can carry any siRNA to intervene in the treatment of target genes, and play the role of siRNA-loaded drugs, vaccines or other products.

[0497] Example 1.3.7: Preparation and Effects of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP)

[0498] Example 1.3.7.1, The metal ion is Fe 3+ Preparation and effects of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP)

[0499] The mRNA in Example 1.3 was replaced with ASO, and three ASO@MPP(Fe3O4) solutions containing different ASOs were prepared according to the method in Example 1.3. 3+ The three different ASO-targeted genes, sequences, and corresponding random control sequences are as follows: ① ASO targeting the STAT3 gene (STAT3-ASO) sequence is SEQ ID No. 10 (17nt), and its random control sequence is SEQ ID No. 11 (18nt); ② ASO targeting the α-syn gene (α-syn-ASO) sequence is SEQ ID No. 12 (16nt), and its random control sequence is SEQ ID No. 13 (16nt); ③ ASO targeting the Bcl-2 gene (Bcl-2-ASO) sequence 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 in Example 1.3. Different ASO@MPP (Fe 3+ Incubation of different cell types: ASO@MPP targeting the STAT3 gene incubated U251 human glioblastoma cells; ASO@MPP targeting the α-syn gene (Fe... 3+ ) Incubation of SH-SY5Y human neuroblastoma cells; ASO@MPP (Fe) targeting the Bcl-2 gene 3+ Incubate Daudi human lymphoma cells. Use 1 × 10⁻⁶ cells per well. 6 Approximately 24 hours after seeding cells at the specified density in 6-well plates, each well was then inoculated with ASO@MPP (Fe3O4) containing the aforementioned ASO. 3+ After incubation for 48 hours (with ASO concentration of 2 μg / mL), cells were collected, total RNA was extracted, and the mRNA expression levels of target genes (STAT3, α-syn, Bcl-2) were detected by RT-PCR. The ASO@MPP (Fe 3+ The ability to silence target genes in cells.

[0500] Cell culture: ① U251 human glioblastoma cells were grown in monolayers in high glucose (4.5 g / L) DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mmol glutamine (Bio Industries), and cultured at 37°C and 5% CO2, passaged twice a week; ② SH-SY5Y human neuroblastoma cells were grown in monolayers in high glucose (4.5 g / L) DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mmol glutamine (Bio Industries), and cultured at 37°C and 5% CO2, passaged twice a week; ③ Daudi human lymphoma cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mmol glutamine (Bio Industries), and cultured at 37°C and 5% CO2, passaged twice a week.

[0501] Detailed RT-PCR procedure:

[0502] Total RNA extraction: Discard the culture medium from the six-well plate, wash three times with PBS buffer, and add 1 mL of Trizol to each well to lyse the cells. Add 200 μL of chloroform, shake well, incubate at room temperature for 10 min, and centrifuge at 13000 rpm at 4℃ for 15 min to obtain a three-phase liquid, in which RNA is dissolved in the upper aqueous phase. Transfer the upper aqueous phase to a new enzyme-free 1.5 mL centrifuge tube, add 500 μL of isopropanol, incubate at room temperature for 10 min, and centrifuge at 13000 rpm at 4℃ for 15 min to obtain RNA precipitate. Discard the supernatant, add 1 mL of freshly prepared 75% (v / v) ethanol with RNase-free water to each tube, carefully pipette to remove the white RNA precipitate from the bottom of the tube, centrifuge at 7500 rpm at 4℃ for 10 min, discard the supernatant, and aspirate as much liquid as possible from the bottom of the tube. After opening the tube and allowing it to air dry at room temperature, the RNA precipitate at the bottom was dissolved in 50 μL of enzyme-free water. The purity and concentration of the RNA were then determined using an ultra-micro UV-Vis spectrophotometer.

[0503] cDNA reverse transcription: using TaKaRa Prime Script TMThe RT reagent kit with gDNA Eraser reverse transcribes RNA into cDNA. Removing genomic DNA (gDNA) before the reverse transcription step makes the results more accurate and reliable. The total RNA reverse transcription reaction mixture was prepared 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, it was incubated at 37°C for 15 min, then incubated at 85°C for 5 sec to terminate the reaction, and subsequently stored at 4°C for later use.

[0504] RT-PCR Procedure: This detection method uses the SYBR Green dye and requires no probe. Specifically, Real-time PCR is performed using cDNA from different samples as templates. Prepare the reaction solution 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 cDNA template obtained in the previous step, and 3.2 μL dH2O. Add 10 μL to each well of a plate. After addition, centrifuge (1000 rpm, 5 min) to remove liquid residue and air bubbles from the reaction solution. Real-time PCR was performed using an ABI ViiA7 real-time quantitative PCR instrument. The reaction program was: 95℃, 30 sec (1 cycle) → 95℃, 5 sec; 55℃, 30 sec; 72℃, 30 sec (40 cycles) → 60℃-95℃, 2 min (1 cycle). The experiment was repeated three times, and the average value was used to obtain the Ct value for each group. The fold change in expression between the experimental group and the control group was calculated. The control gene was GAPDH. The RT-PCR primer sequences are as follows: ①STAT3 primer: forward: 5'-TGATCACCTTTGAGACCGAGG-3' (SEQ ID NO. 105), reverse: 5'-GATCACCACAACTGGCAAGG-3' (SEQ ID NO. 106); ②α-syn primer: forward: 5'-TGACGGGTGTGACAGCAGTAG-3' (SEQ ID NO. 107), reverse: 5'-CAGTGGCTGCTGCAATG-3' (SEQ ID NO. 108); ③Bcl-2 primer: forward: 5'-AGGATTGTG GCCTTCTTTGAG-3' (SEQ ID NO. 97), reverse: 5'-AGACAGCCAGGAGAAATCAAAC-3' (SEQ ID NO. 98); ④GAPDH primer: forward: 5'-TCAGGGG TTTCACATTTGGCA-3' (SEQ ID NO. 105). NO.103), reverse: 5'-GGAGCGGAAAACCA-3' (SEQ ID NO.104). The expression levels of each target gene are represented by RQ values ​​(2). -ΔΔCT The formula is 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

[0505] The calculation method for gene silencing efficiency is: 100% - gene expression level in the experimental group / gene expression level in the control group.

[0506] Results Analysis: As shown in Figures 1-10-1, 1-11-1, and 1-12-1, the three types of ASO@MPP(Fe 3+ All of them can significantly interfere with their corresponding target genes, among which STAT3-ASO@MPP(Fe 3+ The inhibition rate of the target gene STAT3 reached 77%; α-syn-ASO@MPP(Fe 3+ The inhibition rate of the target gene α-syn reached 75%; Bcl-2-ASO@MPP(Fe 3+ The inhibition rate of the target gene Bcl-2 reached 69%. These results suggest that ASO@MPP(Fe) 3+ It can carry any ASO for intervention and treatment of target genes, and play the role of ASO-carrying drugs, vaccines or other products.

[0507] Example 1.3.7.2, the metal ion is Al 3+ Preparation and effects of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP)

[0508] The mRNA in Example 1.4 was replaced with ASO, and three ASO@MPP (Al) molecules containing different ASO sequences were prepared according to the method in Example 1.4. 3+ For specific ASO information and experimental procedures, please refer to Example 1.3.7.1.

[0509] Results Analysis: As shown in Figures 1-10-2, 1-11-2, and 1-12-2, the three ASO@MPP (Al... 3+ All of them can significantly interfere with their corresponding target genes, among which STAT3-ASO@MPP(Al 3+ The inhibition rate of the target gene STAT3 reached 79%; α-syn-ASO@MPP (Al 3+ The inhibition rate of the target gene α-syn reached 80%; Bcl-2-ASO@MPP (Al 3+ The inhibition rate of the target gene Bcl-2 reached 74%. These results suggest that ASO@MPP (Al) has a significant inhibitory effect on this gene. 3+It can carry any ASO for intervention and treatment of target genes, and play the role of ASO-carrying drugs, vaccines or other products.

[0510] Example 1.3.7.3, the metal ion is Mg 2+ Preparation and effects of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP)

[0511] The mRNA in Example 1.5 was replaced with ASO, and three ASO@MPP(Mg) molecules containing different ASO sequences were prepared according to the method in Example 1.5. 2+ For specific ASO information and experimental procedures, please refer to Example 1.3.7.1.

[0512] Results Analysis: As shown in Figures 1-10-3, 1-11-3, and 1-12-3, the three types of ASO@MPP(Mg) 2+ All of them can significantly interfere with their corresponding target genes, among which STAT3-ASO@MPP(Mg 2+ The inhibition rate of the target gene STAT3 reached 82%; α-syn-ASO@MPP(Mg 2+ The inhibition rate of the target gene α-syn reached 84%; Bcl-2-ASO@MPP(Mg 2+ The inhibition rate of the target gene Bcl-2 reached 78%. The results suggest that ASO@MPP(Mg) 2+ It can carry any ASO for intervention and treatment of target genes, and play the role of ASO-carrying drugs, vaccines or other products.

[0513] Example 1.3.8: Preparation of drug (different types of nucleic acid)-metal-phospholipid complex particles and their effects

[0514] Example 1.3.8.1, The metal ion is Fe 3+ Preparation and Effects of Drug (Different Types of Nucleic Acid)-Metal-Phospholipid Complex Particles

[0515] The mRNA in Example 1.3 was replaced with 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 as follows: ① The sequences of double-stranded RNA (Bcl-2-siRNA) are SEQ ID NO.4 (antisense strand) and SEQ ID NO.21 (positive strand) (19bp), and the random control sequences are SEQ ID NO.5 (antisense strand) and SEQ ID NO.22 (positive strand) (19bp); ② The sequence of single-stranded DNA (STAT3-ASO) is SEQ ID NO.10 (17nt), and the random control sequence is SEQ ID NO.11 (18nt); ③ The sequence of single-stranded RNA (mRNA encoding the wild-type SARS-CoV-2 S protein) is SEQ ID NO.16 (3822nt); ④ The sequences of double-stranded DNA (dsDNA) are SEQ ID NO.17 (antisense strand) and SEQ ID NO.25 (positive strand) (22bp) (the 3' end of the sequence is labeled with the fluorescent probe Cy3); ⑤ The sequence of single-stranded DNA (ssDNA) is SEQ ID NO.18 (22nt) (the 3' end of the sequence is labeled with the fluorescent probe Cy3). Drug-metal-phospholipid complex particles (Bcl-2-siRNA@MPP(Fe)) loaded with the above-mentioned different types of nucleic acids were prepared according to the method in Example 1.3. 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 in Example 1.3.

[0516] U251 cells were used at a rate of 1 × 10⁻⁶ per well. 5 Approximately 24 hours after cell density seeding in 12-well plates, each well was inoculated with siRNA@MPP(Fe 3+ (where the concentration of siRNA is 2 μg / mL) or ASO@MPP (Fe 3+ After incubation for 72 hours (with ASO concentration of 2 μg / mL), cells were collected, total RNA was extracted, and the mRNA expression levels of target genes (Bcl-2 and STAT3) were detected by RT-PCR. The expression levels of siRNA@MPP (Fe) were calculated. 3+ ) or ASO@MPP(Fe 3+ The ability to silence cellular target genes was demonstrated, and the results are shown in Figure 1-7-1 of Example 1.3.6 and Figure 1-10-1 of Example 1.3.7.

[0517] S-mRNA@MPP(Fe 3+ ) was incubated with 293T cells at a concentration of 2 μg / mL (mRNA concentration), while the control group was incubated with MPP (Fe 3+ After incubation for 24 hours, the supernatant was centrifuged and stored at -20°C for later use. The cell pellet was resuspended in 100 μL of PBS buffer, frozen and thawed twice, and sonicated for 10 min before centrifugation to collect the supernatant. The expression level of S protein in both the cell supernatant and cell lysate was detected using a commercially available SARS-CoV-2 S protein ELISA kit. The results are shown in Figure 1-13-1.

[0518] ds-DNA@MPP(Fe 3+ A549 lung cancer cells were incubated with 100 nM (the concentration of DNA contained) for 2 hours. The remaining drug-lipid particles were removed, and the cells were washed twice with PBS. The cell nuclei were stained with Hochest 33342 dye for 3 minutes, and the dye was removed. The cells were washed twice with PBS. The cells were observed using a high-content imaging system, and the efficiency of drug-lipid particle transfection of DNA was calculated. The results are shown in Figure 1-14-1.

[0519] ss-DNA@MPP(Fe 3+ After incubating HT22 mouse hippocampal neurons for 2 hours with a concentration of 200 nM (the concentration of DNA contained), the remaining drug-lipid particles were removed. The cells were washed twice with PBS, and the cells were observed using a high-content imaging system. The efficiency of drug-lipid particle transfection of DNA was calculated. The results are shown in Figure 1-14-1.

[0520] The culture method for human glioblastoma U251 cells is the same as in Example 1.3.6.

[0521] The culture method for 293T cells is the same as in Example 1.3.5.

[0522] Culture method of hippocampal neurons in HT22 mice: cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0523] The RT-PCR method is the same as in Example 1.3.6.

[0524] ELISA detection of S protein expression level: Replace the "anti-RBD antibody working solution" with "anti-S protein antibody working solution" in the ELISA detection method for RBD in Example 1.3.5, and the remaining steps are the same as in Example 1.3.5.

[0525] The method for calculating gene silencing efficiency is the same as in Example 1.3.6.

[0526] Transfection efficiency was calculated as follows: 3-5 fields of view were randomly selected using a high-content imaging system to obtain cell morphology under normal light source, fluorescence signals at excitation / emission light of 550nm / 570nm (excitation light of the fluorescent dye Cy3 for labeled DNA), and fluorescence signals at excitation / emission light of 352nm / 461nm (excitation light of the fluorescent dye Hoechst33342 for labeled cell nuclei) in the same field of view (the results are shown in Figure 1-15). The proportion of cells with Cy3 fluorescence signals in the randomly selected field of view to the proportion of cells with Hoechst33342 fluorescence signals in the same field of view was calculated, which is the transfection efficiency.

[0527] Results Analysis: As shown in Figure 1-7-1 of Example 1.3.6, the 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 1-10-1 of Example 1.3.7, the drug (single-stranded DNA)-metal-phospholipid complex particles (STAT3-ASO@MPP(Fe)) achieved an inhibition rate of 76%; 3+ The inhibition rate of the target gene STAT3 reached 77%; as shown in Figure 1-13-1, the drug (single-stranded RNA)-metal-phospholipid complex particles (S-mRNA@MPP(Fe)) were transfected. 3+ The S protein expression level in the supernatant of 293T cells transfected with the empty vector MPP was 161.3 ng / mL, while the S protein content in the supernatant of 293T cells transfected with the empty vector MPP was 0; drug (double-stranded DNA)-metal-phospholipid complex particles (dsDNA@MPP(Fe 3+ The efficiency of transfecting double-stranded DNA into cells was 100% (Figure 1-14-1); drug (single-stranded DNA)-metal-phospholipid complex particles (ssDNA@MPP(Fe 3+ The efficiency of transfecting single-stranded DNA into cells was 100% (Figure 1-14-1). The results suggest that drug-metal-phospholipid complex particles can encapsulate any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and perform its function, with the length of the nucleic acid varying from 16 to 3822 nt.

[0528] Example 1.3.8.2: Preparation of metal ions as Al 3+ Drug (different types of nucleic acid)-metal-phospholipid complex particles and their effects

[0529] Replace the mRNA in Example 1.4 with double-stranded RNA (siRNA), single-stranded DNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively. Refer to Example 1.3.8.1 for specific sequences and experimental procedures.

[0530] Results Analysis: As shown in Figure 1-7-2 of Example 1.3.6.2, the 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, the drug (single-stranded DNA)-metal-phospholipid complex particles (STAT3-ASO@MPP(Al)) achieved an inhibition rate of 81%. 3+ The inhibition rate of the target gene STAT3 reached 79%; as shown in Figure 1-13-2, the drug (single-stranded RNA)-metal-phospholipid complex particles (S-mRNA@MPP(Al)) were transfected. 3+ The S protein expression level in the supernatant of 293T cells transfected with the empty vector MPP was 178.7 ng / mL, while the S protein content in the supernatant of 293T cells transfected with the empty vector MPP was 0; drug (double-stranded DNA)-metal-phospholipid complex particles (dsDNA@MPP(Al) 3+ The transfection efficiency 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 transfecting single-stranded DNA into cells was 100% (Figure 1-14-2). The results suggest that drug-metal-phospholipid complex particles can encapsulate any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and perform its function, with the length of the nucleic acid varying from 16 to 3822 nt.

[0531] Example 1.3.8.3: Preparation of Mg metal ions 2+ Drug (different types of nucleic acid)-metal-phospholipid complex particles and their effects

[0532] Replace the mRNA in Example 1.5 with double-stranded RNA (siRNA), single-stranded DNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively. Refer to Example 1.3.8.1 for specific sequences and experimental procedures.

[0533] Results Analysis: As shown in Figure 1-7-3 of Example 1.3.6.3, the drug (double-stranded RNA)-metal-phospholipid complex particles (Bcl-2-siRNA@MPP(Mg)) 2+ The inhibition rate of the target gene Bcl-2 reached 82%; as shown in Figure 1-10-3 of Example 1.3.7.3, the drug (single-stranded DNA)-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)) were transfected. 2+ The S protein expression level in the supernatant of 293T cells was 181.2 ng / mL, while the expression level in cells transfected with the empty vector MPP (Mg) was significantly higher. 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)) were also found to be negative. 2+ The efficiency of transfecting 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 transfecting single-stranded DNA into cells was 100% (Figure 1-14-3). The results suggest that drug-metal-phospholipid complex particles can encapsulate any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and perform its function, with the length of the nucleic acid varying from 16 to 3822 nt.

[0534] Example 2: Performance Characterization of Drug-Metal-Phospholipid Complex Particles

[0535] Example 2.1: Synthesis and Characterization of Metal-Phospholipid Complexes

[0536] Example 2.1.1, the metal ion is Fe 3+ Synthetic characterization of metal-phospholipid complexes

[0537] The characterization method for the connection between DSPC and curcumin was differential scanning calorimetry (DSC). The measurement conditions were as follows: 3–5 mg of the analyte was weighed, the heating rate was 10 °C / min, and the temperature range was 30–300 °C. Curcumin, DSPC, and the curcumin-DSPC complex were scanned separately, and curves were plotted based on the obtained data. The results are shown in Figure 2-1. The spectra show that curcumin has a crystalline structure and exhibits a distinct melting peak at 185 °C. DSPC is a mixture, and the curve shows several dips, possibly indicating thermal changes in different components under different temperature conditions. In the phospholipid complex, no peaks appeared near the curcumin melting peak, and the curve was essentially a straight line, indicating that curcumin and DSPC were bound together in an amorphous form, proving the successful preparation of the phospholipid complex.

[0538] Phospholipid complex and Fe 3+ The connection was characterized by spectrophotometry: As shown in Figure 2-2-1, the phospholipid complex (CUR-DSPC) and Fe... 3+After binding, its maximum absorption wavelength shifted from 420 nm to 375 nm, indicating a change in the conjugated structure of the phospholipid complex, proving that Fe... 3+ It successfully chelated with curcumin.

[0539] Example 2.1.2, the metal ion is Al 3+ Synthetic characterization of metal-phospholipid complexes

[0540] The difference between this embodiment and Embodiment 2.1.1 is that the phospholipid complex and Al 3+ The connection was characterized by spectrophotometry: As shown in Figure 2-2-2, the phospholipid complex (CUR-DSPC) and Al 3+ After binding, its maximum absorption wavelength shifted from 420 nm to 433 nm, and the conjugated structure of the phospholipid complex changed, proving that Al 3+ It successfully chelated with curcumin.

[0541] Example 2.1.3, the metal ion is Mg 2+ Synthetic characterization of metal-phospholipid complexes

[0542] The difference between this embodiment and Embodiment 2.1.1 is that the phospholipid complex and Mg 2+ The connection was 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, with its maximum absorption wavelength changing from 426 nm to 420 nm. This altered the conjugated structure of the phospholipid complex, indicating that Mg... 2+ It successfully chelated with curcumin.

[0543] Example 2.2, the metal ion is Fe 3+ Fe under low pH conditions 3+ Characterization of shedding from metal-phospholipid complexes

[0544] The phospholipid complex in the metal-phospholipid complex binds Fe through coordination bonds. 3+ Under the low pH conditions of lysosomes, phospholipid complexes react with Fe 3+ The coordination bonds between them will be protonated (absorbing hydrogen ions) and broken. This is to demonstrate the presence of Fe in the metal-phospholipid complex. 3+ Indeed, the metal-phospholipid complex detaches from the lipid complex through the aforementioned mechanism. We designed the following experiment: The color of the metal-phospholipid complex was observed under physiological pH (pH = 7.4) and lysosomal low pH (pH = 5.0) conditions. As shown in Figure 2-3, the metal-phospholipid complex changed from brownish-red to bright yellow under lysosomal low pH (pH = 5.0) conditions, suggesting that Fe... 3+ It has detached from the complex. The results suggest that under the low pH conditions of lysosomes, Fe...3+ It can be detached from the metal-phospholipid complex.

[0545] Fe under low pH conditions 3+ The principle behind the detachment of curcumin from metal-phospholipid complexes is that curcumin reacts with Fe... 3+ The coordination bonds between them are protonated under low pH conditions (pH = 5.0), meaning that curcumin will bind a large number of protons (H+) from the solution. + ), leading to Fe 3+ The coordination bond between curcumin and Fe breaks, thereby allowing Fe to... 3+ Separation from curcumin ultimately leads to Fe 3+ It was isolated from the metal-phospholipid complex (Figure 2-3).

[0546] Example 2.3, the metal ion is Fe 3+ Elemental analysis of MPP in drug-metal-phospholipid complex particles

[0547] The mRNA in Example 1.3 was replaced with thiol-modified siRNA, and drug-metal-phospholipid complex particles siRNA@MPP(Fe) were prepared according to the method in Example 1.3. 3+ Elemental analysis was performed using transmission electron microscopy. The results are shown in Figure 2-4: C, N, O, and P are common elements, and the elemental analysis diagram of Fe shows Fe... 3+ Uniformly distributed on lipid nanoparticles, the siRNA is modified with thiol groups, allowing the elemental analysis diagram to specifically represent its location. The diagram shows that the siRNA is well-complexed with Fe. 3+ The presence of the drug-lipid nanoparticles near the target area demonstrates that siRNA was successfully encapsulated.

[0548] Example 2.4, the metal ion is Fe 3+ Al 3+ or Mg 2+ The efficiency of metal-phospholipid complex particles (MPP) in encapsulating nucleic acids (siRNA and mRNA) and its comparison with LNP.

[0549] The mRNA in Example 1.3.5 was replaced with siRNA (SEQ ID No. 4, 19bp) targeting the Bcl-2 gene and mRNA (SEQ ID No. 2, 669nt) encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit, respectively, to prepare drug-metal-phospholipid complex particles siRNA@MPP and mRNA@MPP loaded with nucleic acids. The preparation process of the remaining drug-metal-phospholipid complex particles was the same as in Example 1.3.5.

[0550] siRNA@LNP and mRNA@LNP were prepared with the same drug loading as siRNA@MPP in Example 1.3.6 and mRNA@MPP in Example 1.3.5. Specifically, an organic phase solution was prepared according to the Onpattro lipid nanoparticle formulation, namely, ionizable lipids 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 (0.1M, pH=4.0 acetate-sodium acetate buffer). The ratio of amino esters to phosphate-containing nucleotides (N / P) was 6:1, ensuring that the nucleic acid drug loading was the same as that of siRNA@MPP and mRNA@MPP. 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 then concentrated to one-tenth using a 100kDa ultrafiltration tube. After repeating the dilution and concentration operations 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 obtaining siRNA@LNP and mRNA@LNP respectively.

[0551] The encapsulation rates of nucleic acids (siRNA and mRNA) by siRNA@MPP, mRNA@MPP, siRNA@LNP, and mRNA@LNP were determined using agarose gel electrophoresis. The encapsulation rate was measured as follows: the amount of nucleic acid (siRNA and mRNA) added to each lipid nanoparticle group was 10 μg / mL, with a lipid-to-nucleic acid mass ratio of 40:1. Nucleic acid was dissolved in PBS buffer solution as a positive control, and a PBS buffer solution without nucleic acid was used as a negative control. The concentration of the agarose gel was 1.5%, at which point the gel pores only allowed free nucleic acid to pass through, not lipid nanoparticles. Electrophoresis was stopped when the free nucleic acid bands became clearly distinguishable to prevent nucleic acid degradation due to prolonged electrophoresis. The gray values ​​of free nucleic acid in different groups were statistically analyzed using ImageJ software. The positive control group was set as 100%. The ratio of free nucleic acid in each group to the positive control was the relative amount of free nucleic acid. The encapsulation rate of each group was calculated as (100 - relative amount of free nucleic acid)%.

[0552] Results Analysis: As shown in Figure 2-5, MPP(Fe 3+ The efficiencies of siRNA and mRNA encapsulation were 90.11% and 89.78%, respectively; MPP(Al) 3+ The efficiencies of siRNA and mRNA encapsulation were 92.81% and 91.48%, respectively; MPP(Mg 2+The efficiencies of MPP and LNP in loading siRNA and mRNA were 93.69% and 92.02%, respectively; the efficiencies of LNP in loading siRNA and mRNA were 89.02% and 89.36%, respectively. These results suggest that there was no significant difference in the efficiency of nucleic acid loading between MPP and LNP.

[0553] Example 2.5, the metal ion is Fe 3+ Al 3+ or Mg 2+ Nucleotide-lysosomal escape ability of metal-phospholipid complex particles (MPPs) and their comparison with LNPs.

[0554] The Bcl-2-siRNA (SEQ ID No. 4) in Example 1.3.6 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@MPP (siRNA concentration of 100 nM); the Bcl-2-siRNA (SEQ ID No. 4) in Example 2.4 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@LNP (siRNA concentration of 100 nM); the eGFP-mRNA (SEQ ID No. 1) in Example 1.3.5 was replaced with Cy5-labeled eGFP-mRNA to prepare Cy5-mRNA@MPP (mRNA concentration of 2 μg / mL); the RBD-mRNA in Example 2.4 was replaced with Cy5-labeled RBD-mRNA to prepare Cy5-mRNA@LNP (mRNA concentration of 2 μg / mL). These were then respectively coupled with the lysosomal probe Lysotracker. After co-incubating A549 cells with Green for 3 hours, the overlap between the Cy5 fluorescence signal (red) and the Lysotracker Green fluorescence signal (green) was observed using a high-content imaging system (metal ion: Fe). 3+ The results are shown in Figure 2-11, which demonstrates the ability of the drug-lipid particles to promote the escape of nucleolysosomes.

[0555] Criteria for determining the ability of drug-metal-phospholipid complex particles to promote nucleic acid lysosome escape: After incubating cells with drug-lipid nanoparticles for 3 hours, the overlap between the Cy5 fluorescence signal (red) and the Lysotracker Green fluorescence signal (green) was observed using a high-content imaging system, and the overlap rate between the red and green fluorescence signals was calculated using ImageJ software. When the overlap rate between the red and green fluorescence signals was less than 50% after 3 hours of cell incubation with drug-metal-phospholipid complex particles, it indicated that nucleic acids could escape from cell lysosomes relatively quickly, and the lipid nanoparticles had a good ability to promote nucleic acid lysosome escape.

[0556] Results analysis: As shown in Figure 2-6, when Cy5-siRNA@MPP(Fe 3+ ) and Cy5-mRNA@MPP(Fe 3+ After incubating A549 cells for 3 hours, the overlap rates of red and green fluorescence signals were 39.11% and 43.44%, respectively, meaning the lysosomal escape rates were 60.89% and 56.56%, respectively; when Cy5-siRNA@MPP(Al 3+ ) and Cy5-mRNA@MPP (Al 3+ After incubating A549 cells for 3 hours, the overlap rates of red and green fluorescence signals were 33.48% and 40.15%, respectively, meaning the lysosomal escape rates were 66.52% and 59.85%, respectively; when Cy5-siRNA@MPP(Mg 2+ ) and Cy5-mRNA@MPP(Mg 2+ After incubating A549 cells for 3 hours, the overlap rates of red and green fluorescence signals were 54.86% and 57.36%, respectively, indicating lysosomal escape rates of 45.14% and 42.64%. However, after incubating A549 cells for 3 hours with Cy5-siRNA@LNP and Cy5-mRNA@LNP, the overlap rates of red and green fluorescence signals were 77.69% and 84.69%, respectively, indicating lysosomal escape rates of 22.31% and 15.31%. This suggests that the drug-lipid nanoparticles (Fe...)... 3+ Al 3+ or Mg 2+ The MPP (composed of Fe) has a good ability to promote the escape of nucleolysosomes, and (Fe 3+ Al 3+ or Mg 2+ MPP has a significantly stronger ability to promote lysosomal escape than LNP.

[0557] Example 2.6, the metal ion is Fe 3+ Al 3+ or Mg 2+ The ability of metal-phospholipid complex particles (MPPs) to promote nucleic acid expression and their comparison with LNPs.

[0558] 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 in Example 2.4 to obtain eGFP-mRNA@LNP.

[0559] 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.

[0560] The method for analyzing the eGFP-positive cell rate using flow cytometry is as described in Example 1.3.

[0561] Results Analysis: As shown in Figure 2-7, MPP(Fe 3+ ), MPP (Al) 3+ ) or MPP(Mg 2+ After treatment of 293T cells with MPP and LNP, the percentages of eGFP-positive cells were 97.7%, 98.02%, 98.3%, and 63.03%, respectively. The results suggest that MPP is superior to LNP in promoting nucleic acid expression. This may be because, as described in Example 2.5, MPP has a stronger ability to promote lysosomal escape from nucleic acids than LNP, so more nucleic acids loaded in MPP can be effectively released into the cytoplasm for translation into proteins. Currently, the percentage of eGFP-positive cells is used to determine the expression of different metal ions (Fe). 3+ Al 3+ or Mg 2+ MPP composed of (e.g., MPP(Fe)) 3+ ), MPP (Al) 3+ ) or MPP(Mg 2+ Characterization of nucleic acid expression capacity was performed, but because the eGFP protein itself lacked the ability to distinguish subtle differences, it failed to distinguish metal ions (Fe). 3+ Al 3+ or Mg 2+ MPP composed of (e.g., MPP(Fe)) 3+ ), MPP (Al) 3+ ) or MPP(Mg 2+ The difference in the ability to promote nucleic acid expression between ))

[0562] Example 2.7, the 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.

[0563] RBD-mRNA@MPP from Example 1.3.5 and RBD-mRNA@LNP from Example 2.4 were incubated with 293T cells at a concentration of 2 μg / mL (the concentration of mRNA contained). The control group was incubated with MPP. After 24 h, the cells were centrifuged and the supernatant was collected and stored at -20 °C for later use. The cell pellet was resuspended in 100 μL of PBS buffer, frozen and thawed twice, and sonicated for 10 min. The supernatant was then centrifuged and the expression level of RBD protein in the cell supernatant and cell lysate was detected using a commercially available SARS-CoV-2 antigen RBD ELISA kit. The results are shown in Figures 2-8.

[0564] The method for detecting RBD expression levels using ELISA is as described in Example 1.3.5.

[0565] 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 received RBD-mRNA@MPP and RBD-mRNA@LNP, respectively, while the control group received MPP and LNP without mRNA loading. Each administration dose was 100 μL, with each of the RBD-mRNA@MPP and RBD-mRNA@LNP formulations in the experimental group containing 30 μg of mRNA. Blood was collected from mice 28 days after the first administration, and the serum was serially diluted. The titer of total RBD IgG antibodies against the S1 subunit of the novel coronavirus produced in the mice was detected using a commercially available ELISA kit. The results are shown in Figures 2-9.

[0566] The method for detecting the titer of total IgG antibody against the S1 subunit of the novel coronavirus using ELISA is as described in Example 1.3.5.

[0567] On day 28 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. The suspensions were seeded into cell plates at a density of 100,000 spleen cells / well, and RBD protein at a final concentration of 10 mg / mL was added. The cells were cultured for 48 h, centrifuged, and the supernatant was removed. The expression levels of IFN-γ, IL-2, and IL-4 were measured using an ELISA kit. The results are shown in Figure 2-10.

[0568] The method for detecting the expression levels of IFN-γ, IL-2, and IL-4 using ELISA is as described in Example 1.3.5.

[0569] Results analysis: As shown in Figure 2-8, RBD-mRNA@MPP(Fe 3+ ), RBD-mRNA@MPP(Al 3+ ), RBD-mRNA@MPP(Mg 2+Both RBD-mRNA@LNP and RBD-mRNA@LNP can induce 293T cells to express a certain amount of RBD, but RBD-mRNA@MPP(Mg 2+ The ability to induce RBD expression in cells is significantly stronger than that of RBD-mRNA@MPP (Al). 3+ ), RBD-mRNA@MPP(Fe 3+ Of the three, the ability of RBD to induce cell expression of RBD is significantly stronger than that of RBD-mRNA@LNP: RBD-mRNA@MPP (Fe 3+ The expression level of RBD in the cell supernatant of the treatment group was 218.93 ng / mL, and the expression level of RBD-mRNA@MPP (Al) was [missing information]. 3+ The expression level of RBD in the cell supernatant of the treatment group was 239.93 ng / mL, and the expression level of RBD-mRNA@MPP(Mg) was [missing value]. 2+ The expression level of RBD in the cell supernatant of the RBD-mRNA@LNP treatment group was 303.63 ng / mL, while the expression level of RBD in the cell supernatant of the RBD-mRNA@LNP treatment group was 126.67 ng / mL. As shown in Figure 2-9, RBD-mRNA@MPP effectively induced humoral immunity in mice, producing high levels of antigen-specific binding antibodies, and RBD-mRNA@MPP(Mg 2+ The ability to induce humoral immunity in mice was significantly better than that of RBD-mRNA@MPP (Al). 3+ ), RBD-mRNA@MPP(Fe 3+ The ability of these three to induce humoral immunity in mice was significantly better than that of RBD-mRNA@LNP:RBD-mRNA@MPP(Mg 2+ The titer of IgG antibodies in mice treated with the RBD-mRNA@MPP(Fe) group reached 171782.00; 3+ The IgG antibody titer in the treated mice reached 122666.67; RBD-mRNA@MPP(Al 3+ The IgG antibody titer in mice treated with RBD-mRNA@LNP reached 134833.33, while the IgG antibody titer in mice treated with RBD-mRNA@LNP was only 73694.00. As shown in Figure 2-10, RBD-mRNA@MPP(Mg 2+ It can effectively induce cellular immunity in mice, that is, activate immune cells and produce a large number of cytokines, and RBD-mRNA@MPP(Mg 2+ The ability to induce cellular immunity in mice was significantly superior to that of RBD-mRNA@MPP (Fe 3+ ), RBD-mRNA@MPP(Al 3+ ), RBD-mRNA@MPP(Fe 3+The ability to induce cellular immunity in mice was significantly superior to that of RBD-mRNA@LNP: RBD-mRNA@MPP(Fe 3+ This resulted in the expression levels of cytokines IFN-γ, IL-2, and IL-4 reaching 286.2 pg / mL, 209.67 pg / mL, and 58.02 pg / mL, respectively; RBD-mRNA@MPP(Al 3+ This resulted in the expression levels of cytokines IFN-γ, IL-2, and IL-4 reaching 306 pg / mL, 239.67 pg / mL, and 71 pg / mL, respectively; RBD-mRNA@MPP(Mg 2+ The expression levels of cytokines IFN-γ, IL-2, and IL-4 were increased to 333 pg / mL, 270.33 pg / mL, and 73 pg / mL, respectively; while RBD-mRNA@LNP reduced the expression levels of IFN-γ, IL-2, and IL-4 to only 95 pg / mL, 75.67 pg / mL, and 24 pg / mL. These results suggest that mRNA@MPP (Mg 2+ The ability to deliver any mRNA and perform its function is significantly superior to RBD-mRNA@MPP (Fe 3+ ), mRNA@MPP(Al 3+ ), RBD-mRNA@MPP(Fe 3+ The ability of RBD-mRNA@MPP to induce cellular immunity in mice is significantly superior to that of RBD-mRNA@LNP: RBD-mRNA@MPP can more effectively promote the expression of target proteins in cells and more effectively activate humoral and cellular immunity in vivo. Therefore, drug (mRNA)-lipid particles are significantly superior to the existing technology LNP in terms of the efficacy of mRNA-loaded drugs, vaccines, or other products. The possible reasons are: 1) Compared with LNP, MPP has a stronger ability to promote the escape of nucleosomal proteins; 2) Compared with LNP, MPP has a stronger ability to promote the expression of nucleic acids into proteins (antigens); 3) Compared with LNP, curcumin in MPP, after being separated from DSPC in vivo, acts as an immune adjuvant (also known as an immunomodulator), which can both activate humoral and cellular immunity to enhance the effect of MPP in delivering mRNA vaccines and inhibit immune storms to suppress excessive and harmful immune responses.

[0570] MPPs composed of different metal ions (e.g., MPP(Fe)) 3+ ), MPP (Al) 3+ Compared to )), MPP(Mg 2+ ) can more effectively promote the expression of target proteins in cells because: Mg 2+By weakly chelating with RNA, Mg can enhance RNA function, including increasing its thermodynamic stability, chemical stability, and catalytic activity (Biochemistry. 2021 Aug 10; 60(31):2374-2386.). 2+ It can maintain the structural stability and translational activity of ribosomes, partially supplement the function of ribosomal proteins (Biosci Biotechnol Biochem. 2021 Jun 24; 85(7):1582-1593.), and enhance the immune response. Mg 2+ It can regulate downstream signal transduction of LFA-1 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.).

[0571] Example 2.8, the metal ion is Fe 3+ Al 3+ or Mg 2+ Comparison of the expression duration of drug-metal-phospholipid complex particles (MPP) with that of LNP

[0572] Preparation of CD19 CAR mRNA:

[0573] CAR mRNA includes a transmembrane domain, a signal transduction domain, an antigen-binding domain, a co-stimulatory signal transduction region, and a transmembrane domain junction region.

[0574] Signal transduction domain, signal peptide (SP): helps CAR expressed within T cells to be transported to the T cell membrane.

[0575] The antigen-binding domain, scFV, encodes the VH and VL portions of the FMC63 antibody. The VH and VL portions of the FMC63 antibody are linked by a linker and can recognize tumor cell antigens.

[0576] The spacer connects the antigen-binding domain scFV and the transmembrane domain, allowing for better antigen recognition by the scFV.

[0577] Transmembrane domain, transmembrane(TM): anchors these combinations of CAR expression to the T cell membrane.

[0578] The intracellular co-stimulatory signal transduction region is used to activate T cells. The activation signal is primarily generated by CD3-zeta, and other co-stimulatory domains such as CD28 and CD8, like 41BB, can be added to enhance the signal. The co-stimulatory signal transduction region can contain multiple co-stimulatory domains. Regardless of the number of co-stimulatory domains added, CD3-zeta should always be placed last. The CD28 co-stimulatory domain can effectively enhance T cell proliferation.

[0579] The amino acid sequence of CD19 CAR mRNA expression is shown in SEQ ID NO.60.

[0580] Referring to Example 2.4, the 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 percentage of CAR-positive cells in myeloid cells was detected by flow cytometry. The method for analyzing the percentage of CAR-positive cells by flow cytometry was as follows: peripheral blood was mixed with Alexa... CAR Linker antibody labeled with 647 and CD11b-PE antibody were co-incubated at 4°C for 30 minutes. After incubation, erythrocyte lysis buffer was added and lysed for 5–10 minutes in the dark. After washing twice with buffer, the cells were resuspended in 100–30,000 μL of buffer. The percentage of CAR-positive cells in myeloid cells was detected by flow cytometry. The calculation formula was: CAR-positive cell rate in myeloid cells = (Number of myeloid cells expressing CAR / Total number of myeloid cells) × 100%. CD19CAR-mRNA@LNP was detected using the same method.

[0581] The results are shown in Figure 8-1, CD19 CAR mRNA@MPP(Mg 2+ The proportion of CAR-positive cells was highest in myeloid cells, and its duration of CAR-positive cells was significantly longer than that of CD19 CAR mRNA@MPP (Al). 3+ ), CD19 CAR mRNA@MPP(Al 3+ Superior to CD19 CAR mRNA@MPP (Fe 3+ ), CD19 CAR mRNA@MPP(Fe 3+ MPP (Mg) is superior to LNP, suggesting that it is superior to LNP. 2+ It has stronger expression and better expression duration.

[0582] Example 2.9, the metal ion is Fe 3+ Al 3+ or Mg 2+In vivo safety evaluation of metal-chelated phospholipid complex nanoparticles (MPPs)

[0583] Using SD rats as the research subjects, a 20-day slow toxicity study of MPP was conducted, with a 20-day recovery period established. The specific experimental methods are as follows:

[0584] Fifty-six SPF-grade SD rats (220±20g), half male and half female, were housed in an environment with a temperature of 25℃, humidity of 45%-55%, and 12h light exposure. After 3-5 days of acclimatization, they were randomly divided into two groups according to sex: an experimental group (n=32) and a recovery group (n=24). A blank control group (n=14, including 8 from the experimental group and 6 from the recovery group) was also included; a low-dose MPP group (25mg / kg) included 14 rats (including 8 from the experimental group and 6 from the recovery group); a medium-dose MPP group (50mg / kg) included 14 rats (including 8 from the experimental group and 6 from the recovery group); and a high-dose group (100mg / kg) included 14 rats (including 8 from the experimental group and 6 from the recovery group) included 14 rats (including 8 from the experimental group and 6 from the recovery group) (half male and half female). The experimental group (n=32) underwent dissection and tissue collection after the drug administration was completed. The recovery group (n=24) continued normal housing for 20 days after the drug administration was completed, followed by dissection and tissue collection.

[0585] Administration method: Experimental animals were administered the drug via tail vein injection every 2 days for a total of 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.

[0586] General indicator detection methods: After each administration, observe the general condition of each group of animals, including survival, diet, appearance, behavior, weight, and whether there is a local reaction to the drug. Perform a gross necropsy, including timely weighing of major organs such as brain, heart, liver, spleen, lungs, and kidneys, calculating the organ-to-body weight ratio, and recording pathological changes in each organ. The organ-to-body weight ratio is calculated as: (Rat organ wet weight / Rat body weight) × 100%.

[0587] Obtaining and preserving whole blood and serum from SD rats: Rats were dissected 20 days after drug administration and during the 20-day recovery period. Blood was collected from the abdominal aorta. SD rats were anesthetized with isoflurane and fixed on a dissecting board. The abdomen was disinfected with 75% ethanol, and the abdomen was cut open with sterile ophthalmic scissors. The abdominal aorta was exposed by gently separating the internal organs with a cotton ball. Whole blood was collected using a 500 μL negative pressure EDTAK2 anticoagulant blood collection tube and stored at 4℃ for routine blood tests. Whole blood was also collected using a 5 mL negative pressure ordinary blood collection tube and allowed to stand at room temperature for 30 min. The blood was then centrifuged at 1500 rpm for 15 min at 4℃. The supernatant was collected in a 1.5 mL centrifuge tube and stored at -20℃ for the detection of blood biochemical and immunological indicators.

[0588] Methods for routine blood tests: Routine blood test indicators 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 (MCV), mean corpuscular hemoglobin (MCH) concentration, mean corpuscular distribution width (MCD) coefficient of variation, platelet count, mean platelet volume (MCV), platelet distribution width (CDW), and plateletcrit. The whole blood sample is gently inverted and mixed. A small amount of whole blood is taken and the results are automatically analyzed using a fully automated blood cell analyzer.

[0589] Methods for detecting blood biochemical indicators: Blood biochemical indicators include inorganic ions (Fe2+) 2+ Na + K + Cl - Ca 2+ The following parameters were measured: liver function indicators (ALT, AST, γ-GT, T-BIL, D-BIL, ALP, ALB), kidney function indicators (BUN, UA, CR), cardiac function indicators (LDH, CK), glucose metabolism indicators (GSP, GLU, INS), and lipid metabolism indicators (CHO, TG, LDL-C, HDL-C). Thawed serum samples were centrifuged at 3000 rpm for 15 min, and the supernatant was aliquoted and prepared for use. The appropriate parameters were set on the fully automated biochemical analyzer, the prepared working solution was added, and then the serum sample was added. The fully automated biochemical analyzer automatically measured the results.

[0590] Methods for detecting immunologically related indicators: Immunologically related indicators include 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). These indicators are detected using ELISA.

[0591] Methods for pathological examination of major organs in SD rats: At the end of the drug administration period and the end of the recovery period, after anesthetizing the rats in each group, the major organs of the rats, including the whole brain, heart, liver, spleen, lungs and kidneys, were removed by ophthalmology. They were gently rinsed with 0.9% physiological saline, fixed in 4% paraformaldehyde fixative, routinely embedded in paraffin, stained with H&E, and the histopathological changes of various organs of rats in the control group and experimental group were observed under an optical microscope.

[0592] Results analysis: As shown in Table 2-1, at the end of the administration 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, and normal appearance and behavior. No obvious adverse reactions were observed after administration. Compared with the control group, there was no significant difference in the weight gain of male 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 ratio in the low, medium and high dose MPP groups.

[0593] At the end of the treatment and recovery periods, compared with the control group, no abnormalities were observed in 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, and plateletcrit) in the low, medium, and high dose MPP groups. Compared with the control group, no abnormalities were found in the blood biochemical indicators, including inorganic ions (Fe2+, Fe ... 2+ Na + K + Cl - Ca 2+ Liver function indicators (ALT, AST, γ-GT, T-BIL, D-BIL, ALP, ALB), kidney function indicators (BUN, UA, CR), cardiac function indicators (LDH, CK), glucose metabolism indicators (GSP, GLU, INS), and lipid metabolism indicators (CHO, TG, LDL-C, HDL-C) were all normal. Compared with the control group, the immunological indicators of the low, medium, and high dose MPP groups, including 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), were all normal.

[0594] At the end of the administration period and the end of the recovery period, compared with the control group, the brain tissue of rats in the low, medium and high dose MPP groups was intact, with normal tissue staining, intact cell morphology and structure, and no nuclear pyknosis or inflammatory cell infiltration; the myocardial tissue was intact, with myocardial cells arranged neatly, continuously and tightly, and cell nuclei clearly visible, without obvious cell congestion, edema or necrosis; the hepatocytes were normal in morphology, without inflammatory cell aggregation or necrosis; the spleen was normal in structure, with clear boundaries between red and white pulp; the lung tissue was intact in structure, with alveoli of uniform size, and no obvious inflammatory cell aggregation or infiltration; and the kidneys were normal in structure.

[0595] The above results suggest that long-term, high-dose injection of MPP into SD rats did not reveal significant chronic toxicity, indicating that MPP has a high safety profile.

[0596] Table 2-1 In vivo safety evaluation of MPP

[0597] Note: ALT, alanine aminotransferase; AST, aspartate aminotransferase; γ-GT, gamma-glutamyl transferase; 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.

[0598] Example 2.10, the metal ion is Fe 3+ Al 3+ or Mg 2+ Comparison of in vivo safety of metal-phospholipid complex particles (MPP) and LNP

[0599] The main toxicity of LNP stems from its primary components—cationic lipids and / or ionizable lipids. During LNP metabolism in vivo, free cationic and / or ionizable lipids can exert significant toxicity on the body. The median lethal dose (IC50) of cationic and / or ionizable lipids for biological cells is... 50The median lethal dose (ICD) of metal-phospholipid complex nanoparticles (MPPs) is an important parameter for assessing the toxicity of LNPs to organisms. Metal-chelated phospholipid complex nanoparticles (MPPs) replace the cationic / ionizable lipids in LNPs with metal-phospholipid complexes. Therefore, we investigated the median lethal dose (ICD) of metal-phospholipid complexes versus cationic / ionizable lipids for biological cells. 50 To compare the differences in toxicity between LNP and MPP.

[0600] 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 below), and ionizable lipid (ALC0315, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM, structural formula below) were incubated with 293T cells for 48 hours. Cell viability was detected using a CCK8 activity assay kit, and the median lethal dose (IC50) of the metal-phospholipid complex, cationic lipid (DOTAP), and ionizable lipid (ALC0315) for 293T cells was calculated. 50 .

[0601] DOTAP structure:

[0602] ALC0315 structural formula:

[0603] CCK8 testing method:

[0604] 1. Cell culture: Culture cells in DMEM medium containing 10% FBS and 1% penicillin antibiotics until the cell density reaches 80%-90% of the culture flask.

[0605] 2. Wash the remaining culture medium in the culture flask with PBS, add trypsin, and quickly transfer the culture flask to an incubator containing 5% CO2 at 37°C. Observe carefully, and after the cells become slightly rounded, add culture medium to stop the digestion. Transfer to a centrifuge tube, centrifuge at 1500 RPM for 5 min, and resuspend the cells in fresh culture medium;

[0606] 3. Cell counting: Dilute the cell suspension to 100,000 cells per mL as intended, and insert 100 μL into each well of a 96-well plate, with at least 5 replicates per group. Incubate at 37°C and 5% CO2 for 24 h before adding the drug;

[0607] 4. After incubating the drug for 48 hours, add 10% CCK8 and incubate for 1-3 hours. Measure the absorbance at 450 nm using a microplate reader.

[0608] 5. Survival rate (%) = [A(medication administered) - A(blank)] / [A(0 medication administered) - A(blank)] × 100%.

[0609] IC 50 Calculation method: With survival rate as the ordinate and drug concentration as the abscissa, IC was calculated using GraphPad with the [Inhibitor] vs. normalized response - Variable slope analysis method. 50 .

[0610] To compare the in vivo safety of MPP and LNP, MPP (8 mg / kg) and LNP (3.24 mg / kg) capable of carrying equal amounts of nucleic acid (200 μg / kg mRNA) were taken and in vivo experiments were conducted according to the method in Example 2.9 to evaluate and compare the in vivo toxicity of MPP and LNP.

[0611] Results Analysis: As shown in Table 2-2, the metal-phospholipid complex (MPP(Fe)) 3+ ), MPP (Al) 3+ ), MPP(Mg 2+ IC 50 The toxicity was significantly greater than that of cationic lipids (DOTAP) and ionizable lipids (ALC0315). The results indicate that the metal-phospholipid complex is significantly less toxic than cationic lipids and ionizable lipids.

[0612] As shown in Table 2-3, at the end of the administration period and the end of the recovery period, compared with the control group, MPP(Fe) 3+ ), MPP (Al) 3+ ), MPP(Mg 2+ In the control group, liver function (ALT, AST, ALP) and cytokines (IL-6, IL-1β) were not significantly abnormal. However, compared with the control group, liver function (ALT, AST, ALP) and cytokines (IL-6, IL-1β) were significantly elevated in the LNP group. The results suggest that MPP (Fe 3+ ) or MPP (Al 3+ The in vivo safety profile of MPP (Fe2+) is higher than that of LNP because: the core components of LNP are artificially synthesized "cationic lipids / ionizable lipids," which have high cytotoxicity and immunogenicity, and their structure is relatively stable, making them difficult to break down and metabolize in vivo; while MPP (Fe2+) has a higher safety profile than LNP. 3+ ) or MPP (Al 3+ ) or MPP(Mg 2+The core component of MPP is a metal-phospholipid complex, which consists of phospholipid molecules, the highly safe natural small molecule curcumin (an FDA-approved food additive and pharmaceutical excipient), and safe metal ions. Furthermore, it is broken down into natural molecules in the body after drug delivery. In summary, because MPP (Fe... 3+ ) or MPP (Al 3+ The composition of MPP (Fe) contains no cationic lipids / ionizable lipids, and therefore will not cause toxic side effects related to cationic lipids / ionizable lipids. 3+ ), MPP (Al) 3+ ), MPP(Mg 2+ The security of ) is higher than that of LNP.

[0613] Table 2-2 shows the metal ions as Fe. 3+ Al 3+ or Mg 2+ IC50 of metal-phospholipid complexes with cationic lipids (DOTAP) and ionizable lipids (ALC0315) 50 Comparison

[0614] Table 2-3 Comparison of Chronic Toxicity Test Indicators for MPP and LNP

[0615] Table 2-4 Historical Safety Data for Metals

[0616] References: Chinese Dietary Reference Intakes - 2023 Edition, Chinese Dietary Aluminum Exposure Risk Assessment, ICH Harmonized Guidance - Elemental Impurities Guidance Q3D(R2).

[0617] The historical data above shows that the safety ranking of MPP or Apt-MPP based on various metals is: Mg > Fe > Zn > Al > Mn > Cr. This suggests that not only MPP (Mg...) 2+ The drug has the best efficacy and the best safety profile.

[0618] Example 3: The metal ion is Fe 3+ Al 3+ or Mg 2+ Clinical application and administration route of drug-metal-phospholipid complex particles

[0619] 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-mRNA) of the novel coronavirus S1 subunit.

[0620] The sequences of the different nucleic acids are as follows: ① The sequence of B7-H4-siRNA is SEQ ID No. 19 (positive strand) and SEQ ID No. 26 (antisense strand) (25bp), and its random control sequence is SEQ ID No. 20 (positive strand) and SEQ ID No. 27 (antisense strand) (19bp); ② The mRNA sequence encoding the receptor binding domain (RBD) of the S1 subunit of the novel coronavirus is SEQ ID No. 2 (669nt). Drug-metal-phospholipid complex particles (B7-H4-siRNA@MPP, RBD-mRNA@MPP) encapsulating the above different types of nucleic acids were prepared according to the method in Example 1.3.5. The preparation process of the remaining drug-metal-phospholipid complex particles was the same as in Example 1.3.5. The above two different drug-metal-phospholipid complex particles (B7-H4-siRNA@MPP, RBD-mRNA@MPP) were used to treat glioma and as an mRNA vaccine for the prevention of novel coronavirus, respectively.

[0621] To evaluate the effectiveness of RBD-mRNA@MPP as an mRNA vaccine against SARS-CoV-2, the experimental procedure and methods were as described in Example 1.3.5 above.

[0622] The ELISA detection method is as described in Example 1.3.5.

[0623] To evaluate B7-H4-siRNA@MPP(Fe 3+ ) and B7-H4-siRNA@MPP (Al 3+ ), B7-H4-siRNA@MPP(Mg 2+ The therapeutic effect on liver cancer was investigated using HepG2 cells to create an animal model of liver cancer. The tumor size was increased to approximately 100 mm. 3 Hepatocellular carcinoma mice were randomly divided into 10 groups (n=5 per group): PBS control group, blank vector MPP (Fe... 3+ Group ) and blank vector MPP (Al) 3+ Group ) and blank vector 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. Mice in each group were injected intratumorally with PBS or MPP (Fe2+) every 3 days. 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+ The drug was administered at a dose of 200 μg siRNA / kg, eight times in total. Tumor volume was measured and recorded every three days. The results are shown in Figures 3-1, 3-2, and 3-3.

[0624] Establishment of a mouse model of liver cancer: HepG2 cells were collected at a concentration of 1×10⁻⁶ cells / cells. 7 Cells at a density of 100 μL were resuspended in PBS and stored on ice before inoculation. A mouse model of liver cancer was then established by subcutaneous injection of 100 μL of the cell suspension into the back region near the hind legs of female Balb / c nude mice.

[0625] Results analysis:

[0626] 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 liver cancer cells, while B7-H4-siRNA@MPP(Fe 3+ ), B7-H4 siRNA@MPP (Al 3+ ) and B7-H4 siRNA@MPP(Mg 2+ This demonstrated a highly effective therapeutic effect, significantly inhibiting the growth of liver cancer tumors. The results suggest that drug-metal-phospholipid complex particles can encapsulate and deliver B7-H4 siRNA, thereby suppressing liver cancer development by inhibiting the expression of target genes.

[0627] As shown in Figures 1-3-1 and 1-5-1 of the previous examples 1.3.5, RBD-mRNA@MPP(Fe 3+ This resulted in the expression level of mouse IgG antibody reaching 117268.8 (Figure 1-3-1), and the expression levels of cytokines IFN-γ, IL-2, and IL-4 reaching 252.8 pg / mL, 207.6 pg / mL, and 56.6 pg / mL, respectively (Figure 1-5-1). RBD-mRNA@MPP(Al) 3+ This resulted in the expression level of mouse IgG antibody reaching 129113 (Figure 1-3-2), and the expression levels of cytokines IFN-γ, IL-2, and IL-4 reaching 271.8 pg / mL, 234.6 pg / mL, and 68.4 pg / mL, respectively (Figure 1-5-2). RBD-mRNA@MPP(Mg 2+ The study induced the expression level of IgG antibodies in mice to reach 130614.40 pg / mL (Figure 1-3-3), and the expression levels of cytokines IFN-γ, IL-2, and IL-4 to reach 296.40 pg / mL, 243.33 pg / mL, and 75.30 pg / mL, respectively (Figure 1-5-3). These results suggest that RBD-mRNA@MPP can effectively induce humoral immunity in mice, producing high levels of antigen-specific binding antibodies; simultaneously, it can effectively induce cellular immunity in mice, i.e., activate immune cells and produce a large number of cytokines. Therefore, RBD-mRNA@MPP can effectively prevent infection with the novel coronavirus.

[0628] B7-H4-siRNA@MPP, administered via intratumoral injection, is effective in treating liver cancer; RBD-mRNA@MPP, administered via intramuscular injection, activates humoral and cellular immunity, thereby preventing novel coronavirus infection. These results suggest that drug-metal-phospholipid complex particles can be administered via multiple routes.

[0629] MPP composed of other metal ions (e.g., MPP(Fe)) 3+ ), MPP (Al) 3+ Compared to )), MPP(Mg 2+ It can more effectively inhibit the growth of liver cancer and more effectively prevent infection with the novel coronavirus. The reason is: Mg 2+ Mg, which maintains a 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 structural stability and translational activity of ribosomes, partially supplement the function of ribosomal proteins (Biosci Biotechnol Biochem. 2021 Jun 24; 85(7):1582-1593.), and enhance the immune response. Mg 2+ The downstream signaling of LFA-1 can be regulated 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.) Mg 2+ As an enzymatic cofactor of the Ago protein in the 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.).

[0630] Example 4: DSPC, curcumin, Fe 3+ Al 3+ or Mg 2+ Function after being replaced by a similar object

[0631] Example 4.1, DSPC, curcumin, Fe 3+ Function after being replaced by a similar object

[0632] Referring to Examples 1.1, 1.2, and 1.3, DSPC, curcumin, and Fe were used. 3+ The same compounds were used to treat DSPC, curcumin, and Fe. 3+ By substitution, 37 different eGFP-mRNA@MPPs were prepared through different combinations, with each eGFP-mRNA@MPP containing an mRNA concentration of 2 μg / mL. DSPC, curcumin, Fe 3+ The names and structures of DSPC and its analogues are shown in Table 4-1. Among the 37 mRNA@MPPs, DSPC, curcumin, and Fe... 3+ The combinations of the substances and their analogues are shown in Table 4-2. In Example 1.1, the reaction temperature was 65°C and the reaction time was 2 hours, while in Example 1.2, the reaction temperature was 60°C and the reaction time was 2 hours, with other conditions remaining unchanged.

[0633] To compare the effects of the 37 different eGFP-mRNA@MPP and eGFP-mRNA@LNP, we prepared LNPs loaded with an equal amount of eGFP-mRNA according to Example 2.4, and obtained eGFP-mRNA@LNP.

[0634] The above 37 different eGFP-mRNA@MPP and the above eGFP-mRNA@LNP (the concentration of mRNA contained in each is 2 μg / mL) were incubated with 293T cells respectively, and 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.

[0635] The method for analyzing the eGFP-positive cell rate using flow cytometry is as described in Example 1.3.

[0636] The main toxicity of LNP comes from its main components—cationic lipids / ionizable lipids. During LNP metabolism in the body, free cationic / ionizable lipids can exert significant toxicity on the organism. The median lethal dose (IC50) of cationic / ionizable lipids for biological cells is... 50 Metal-phospholipid complex nanoparticles (MPPs) are an important parameter for assessing the toxicity of LNPs to the body. MPPs replace the cationic / ionizable lipids in LNPs with metal-phospholipid complexes. Therefore, we investigated the median lethal dose (IC50) of 37 metal-phospholipid complexes and cationic lipids (DOTAPs) / ionizable lipids (ALC0315) for biological cells as shown in Table 4-2. 50 The study compared the toxicity differences between LNP and 37 MPPs.

[0637] IC 50 The calculation method is as described in Example 2.10.

[0638] Results Analysis: As shown in Table 4-2, the percentage of eGFP-positive cells in 293T cells treated with 37 different eGFP-mRNA@MPP was significantly higher than that treated with eGFP-mRNA@LNP. Among them, the percentages of eGFP-positive cells treated with DSPC, curcumin, and Fe were significantly higher. 3+ The highest percentage of eGFP-positive cells were found in mRNA@MPP. Results suggest that DSPC, curcumin, and Fe... 3+ The mRNA@MPP formed after being replaced by its analogues is less functional than that formed by DSPC, curcumin, and Fe. 3+ The mRNA@MPP composition is superior to the mRNA@LNP in function, possibly because, as described in Example 2.5, MPP has a stronger ability to promote lysosomal escape from nucleic acid generation than LNP, so more nucleic acids loaded in MPP can be effectively released into the cytoplasm and translated into proteins.

[0639] The above results suggest that DSPC, curcumin, and Fe can be produced if the following conditions are met. 3+The function of the drug-metal-phospholipid complex particles formed after being replaced by its analogues remains unaffected: ① The analogue of DSPC is an amphiphilic phospholipid molecule; 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+ Coordination bonds between similar substances can break in response to the low pH environment of lysosomes.

[0640] As shown in Table 4-3, the IC50 values ​​of 37 metal-phospholipid complexes are... 50 All were significantly greater than cationic lipids (DOTAP) and ionizable lipids (ALC0315). This suggests that the toxicity of metal-phospholipid complexes is clearly less than that of cationic lipids and ionizable lipids, i.e., those composed of DSPC, curcumin, and Fe... 3+ Lipid nanoparticles (MPPs) composed of DSPC and its analogues have a higher safety profile than LNPs. This is because LNPs are primarily composed of synthetically produced cationic / ionizable lipids, which exhibit high cytotoxicity and immunogenicity, and their structure is relatively stable, making them difficult to metabolize in vivo. In contrast, MPPs are primarily composed of metal-phospholipid complexes, which consist of phospholipid molecules, highly safe natural small molecules (including curcumin, an FDA-approved food additive and pharmaceutical excipient), and safe metal ions. Furthermore, these metal-phospholipid complexes are broken down into natural molecules in vivo after drug delivery. In summary, MPPs composed of DSPC, curcumin, and Fe... 3+ Lipid particles (MPPs) composed of lipids and their analogues do not contain cationic lipids / ionizable lipids and will not cause toxic side effects associated with cationic lipids / ionizable lipids. Therefore, MPPs are safer than LNPs.

[0641] Table 4-1 DSPC, Curcumin, Fe 3+ The names and structures of its kind and similar products

[0642] Table 4-2 DSPC, Curcumin, Fe 3+ List of compositional combinations and functions of metal-phospholipid complexes in drug-lipid particles prepared from analogues and their counterparts.

[0643] Table 4-3 DSPC, Curcumin, Fe 3+ IC50 of metal-phospholipid complexes prepared from metals and their analogues 50

[0644] Example 4.2, DSPC of different metal-phospholipid complexes, curcumin and its analogues, Fe3+ The proportion of components added and the function of the prepared drug-metal-phospholipid complex particles

[0645] Following Example 1.3, mRNA-metal-phospholipid complex particles were prepared, and curcumin was replaced with its analogue hesperidin (1 molecule of hesperidin contains 4 hydroxyl groups) and tea polyphenols (1 molecule of tea polyphenols contains 8 hydroxyl groups) to prepare three types of mRNA-metal-phospholipid complex particles (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP38). The ratios of DSPC, curcumin or its analogues, and FeCl3 used in the preparation of these three types of mRNA-metal-phospholipid complex particles were 1:1:1, 1:1:1, and 1:1:2, respectively. The mRNA was the mRNA encoding eGFP fluorescent protein, and its sequence is SEQ ID No. 1 (720 nt). The mRNA loading rate of these four drug-lipid nanoparticles and their ability to promote eGFP fluorescent protein expression after treatment of 293T cells were detected according to the experimental procedures and methods described in Example 1.3.5.

[0646] Results Analysis: As shown in Table 4-4, the mRNA loading efficiency and the ability to promote the expression of target proteins in drug-metal-phospholipid complex particles prepared using different dosage ratios based on the chemical structure of the metal-phospholipid complex components were comparable. The results suggest that the dosage ratio of the metal-phospholipid complex components can be adjusted according to the specific structure of the metal-phospholipid complex components. The basis for adjusting the dosage ratio is as follows: because DSPC and its analogues are linked to curcumin and its analogues by hydrogen bonds, as long as DSPC and its analogues contain multiple phosphate groups, the dosage ratio of DSPC and its analogues to curcumin and its analogues during the synthesis of the phospholipid complex can be adjusted according to the number of phosphate groups contained in DSPC and its analogues. That is, when DSPC and its analogues contain two phosphate groups, the dosage ratio of DSPC and its analogues to curcumin and its analogues can be adjusted to 1:2; when DSPC and its analogues contain three phosphate groups, the dosage ratio of DSPC and its analogues to curcumin and its analogues can be adjusted to 1:3; because the hydroxyl groups of curcumin and its analogues are linked to Fe... 3+ Curcumin and its analogues are linked by coordination bonds. As long as curcumin and its analogues contain multiple binding sites, then curcumin and its analogues and Fe... 3+ The dosage ratio of curcumin and its analogues can be adjusted based on the number of binding sites contained in curcumin and its analogues.

[0647] Table 4-4 Component ratios of different metal-phospholipid complexes and the functions of the prepared drug-lipid particles

[0648] Example 4.3, DSPC of different metal-phospholipid complexes, curcumin and its analogues, Al 3+ The proportion of components added and the function of the prepared drug-metal-phospholipid complex particles

[0649] Following Example 1.4, mRNA-metal-phospholipid complexes were prepared, and curcumin was replaced with its analogue hesperidin (1 molecule of hesperidin contains 4 hydroxyl groups) and tea polyphenols (1 molecule of tea polyphenols contains 8 hydroxyl groups) to prepare three metal-phospholipid complexes (mRNA@MPP2, mRNA@MPP5, and mRNA@MPP39). The ratios of DSPC, curcumin or its analogues, and Al(NO3)3·9H2O used in the preparation of these three metal-phospholipid complexes were 1:1:1, 1:1:1, and 1:1:2, respectively. Corresponding drug-metal-phospholipid complex nanoparticles were prepared using these three metal-phospholipid complexes (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP29). The mRNA was the mRNA encoding the eGFP fluorescent protein, and its sequence is SEQ ID No. 1 (720 nt). The mRNA loading rate of these four drug-lipid nanoparticles and their ability to promote eGFP fluorescent protein expression after treatment of 293T cells were detected according to the experimental procedures and methods described in Example 3.5.

[0650] Results Analysis: As shown in Tables 4-5, the mRNA loading efficiency and the ability to promote the expression of target proteins in drug-metal-phospholipid complex particles prepared using different dosage ratios based on the chemical structure of the metal-phospholipid complex components were comparable. The results suggest that the dosage ratio of the metal-phospholipid complex components can be adjusted according to the specific structure of the metal-phospholipid complex components. The basis for adjusting the dosage ratio is as follows: because the analogues of DSPC and curcumin are linked by hydrogen bonds, as long as the analogue of DSPC contains multiple phosphate groups, the dosage ratio of the analogues of DSPC and curcumin during the synthesis of the phospholipid complex can be adjusted according to the number of phosphate groups contained in the analogue of DSPC. That is, when the analogue of DSPC contains two phosphate groups, the dosage ratio of the analogue of DSPC to curcumin can be adjusted to 1:2; when the analogue of DSPC contains three phosphate groups, the dosage ratio of the analogue of DSPC to curcumin can be adjusted to 1:3; because the hydroxyl groups of the analogue of curcumin are linked to Al... 3+ The conjugates of curcumin are linked by coordination bonds. As long as the conjugates of curcumin contain multiple binding sites, then the conjugates of curcumin and Al... 3+ The dosage ratio of similar substances can be adjusted based on the number of binding sites contained in the similar substances of curcumin.

[0651] Table 4-5 Component dosage ratios of different metal-phospholipid complexes and the functions of the prepared drug-lipid...

Claims

1. A method for preparing a metal-phospholipid complex, wherein, The preparation method consists of the following steps: Phospholipid molecules, linker molecules and metal ions react together to form metal-phospholipid complexes; 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 through a coordinate bond. The metal-phospholipid complex is not a cationic lipid or an ionizable lipid.

2. The production method according to claim 1, wherein The phospholipid molecules, the linker molecules, and the metal ions are dissolved in ethanol and reacted to obtain the metal-phospholipid complex. Preferably, the molar ratio of the phospholipid molecules, the linker molecules, and the metal ions is 1:1: (0.5～2)； Preferably, the molar ratio of the phospholipid molecules, the linker molecules, and the metal ions is 1:1:1, 1:1:1.5, 1:1:2, 3:3:2, or 2:2:

1. Preferably, the reaction conditions include reacting at 40-60°C for 1 to 5 hours.

3. The production method according to claim 1, wherein The phospholipid molecule is selected from one or more of the following: lecithin (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), 1-phosphoceramide (SP), phosphatidylinositol (PI), phosphatidylthreonine (PT), sphingomyelin (SM), lysophosphatidylcosinate (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysophosphatidylsphingomyelin (LSM), 1-phosphosphoamine (S1P), and their derivatives. Preferably, the phospholipid molecule portion is selected from... Lecithin (PC) phosphatidylethanolamine (PE) phosphatidylserine (PS) Phosphatidic acid (PA) phosphatidylglycerol (PG) 1 -phosphoacyl sphingosine (SP) phosphatidylinositol (PI) phosphotidylserine (PT) Sphingomyelin (SM) lysophosphatidylcholine (LPC) lysophosphatidylethanolamine (LPE) lysophosphatidylserine (LPS) lysophosphatidic acid (LPA) lysophosphatidylglycerol (LPG) lysophosphatidylinositol (LPI) lysophosphatidylthreonine (LPT) lysosphingomyelin (LSM) 1 -phospho sphingosine (S1P) One or more of their derivatives; Where R1 and R2 are both independent: decanoyl Lauryl myristoyl palmitoyl stearoyl oleoyl linoleoyl erucyl Arachidoyl or phytanoyl Preferably, the phospholipid molecule is selected from one or more combinations of lecithin (PC, formula 1), phosphatidylethanolamine (PE, formula 2), phosphatidic acid (PA, formula 4), phosphatidylglycerol (PG, formula 5), ​​and their derivatives; Preferably, the phospholipid molecule is selected from one or more combinations of DSPC, DSPE, DSPA, DSPG, and their derivatives; Preferably, the phospholipid molecule portion is selected from... DSPC (Formula 46) DSPE (Formula 47) DSPA (Formula 48) DSPG (Formula 49) One or more of their derivatives; Preferably, the phospholipid molecule portion is selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48), or DSPG (Formula 49); Preferably, the linker molecule is selected from one or more of the following: curcumin, chlorogenic acid, anthocyanin, quercetin, dihydromyricetin, hesperidin, naringin, apigenin, catechin, tea polyphenols, epigallocatechin gallate, ellagic acid, morin, epigallocatechin gallate, catechin gallate, gallocatechin gallate, or piperine C, and its derivatives. Preferably, the linker molecule portion is selected from... Curcumin chlorogenic acid Anthocyanins Among them, R7 and R8 are H, OH, or OCH3, R3 is H or a glycosyl group, and R4, R5, and R6 are OH or a glycosyl group. Quercetin Dihydromyricetin hesperidin Naringin Celery Catechins Tea polyphenols Epigallocatechin gallate ellagic acid morin Epicatechin gallate Catechin gallate Gallocatechin gallate Pinozolidine C One or more of their derivatives; Preferably, the linker molecule is selected from curcumin (Formula 19) and dihydrocurcumin (Formula 36). A combination of one or more of the following; Preferably, the linker molecule is selected from one or more combinations of curcumin (Formula 19), hesperidin (Formula 24), tea polyphenols (Formula 28), and their derivatives; Preferably, the linker molecule is selected from curcumin (Formula 19), hesperidin (Formula 24), or tea polyphenols (Formula 28); Preferably, the metal ion portion is selected from Fe. 3+ Ag + Ba 2+ Ca 2+ Cd 2+ Cu 2+ Fe 2+ Mn 2+ Mg 2+ Mo 2+ Zn 2+ Pt 2+ Au 2+ Al 3+ Ce 3+ Co 3+ Cr 3+ Eu 3+ Gd 3+ Ni 3+ W 3+ V 3+ Zr 3+ One or more combinations thereof; Preferably, the metal ion portion is selected from Fe. 3+ Ca 2+ Al 3+ Mg 2+ One or more combinations thereof; Preferably, the metal ion portion is selected from Fe. 3+ Ca 2+ Mg 2+ Or Al 3+ .

4. The preparation method according to any one of claims 1 to 3, wherein, 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+ Ca 2+ Mg 2+ Or Al 3+ ; Preferably, 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+ Ca 2+ Mg 2+ Or Al 3+ .

5. A method for preparing metal-phospholipid complex particles, wherein, The preparation method comprises mixing (i) a metal-phospholipid complex prepared by any one of claims 1 to 4, (ii) a conjugated lipid that inhibits particle aggregation, 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, to obtain the metal-phospholipid complex particles; wherein the conjugated lipid that inhibits particle aggregation is not a cationic lipid or an ionizable lipid.

6. The preparation method according to claim 5, wherein, The 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) One or more of the following, and their derivatives; R1 and R2 are both independently: decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucoyl, arachidoyl, or phytanoyl; Preferably, the PEG-lipid conjugate is selected from one or more combinations 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) 7. The preparation method according to claim 5 or 6, wherein, The non-cationic lipids or non-ionizable lipids mentioned in (iii) are one or more combinations of cholesterol and its derivatives; Preferably, the non-cationic lipid or non-ionizable lipid described in (iii) is cholesterol (Formula 40). Preferably, the non-cationic lipids or non-ionizable lipids described in (iii) further include one or more combinations of lecithin 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, lysophosphatidylglycerol LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysophosphatidylcholine LSM, 1-phosphosphoamine S1P, cholesterol sulfate, and their derivatives; Preferably, the non-cationic lipids or non-ionizable lipids described in (iii) further include those selected from lecithin (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), lysophosphatidylsphingomyelin (LSM, Formula 17), 1-phosphosphoin (S1P, Formula 18), and cholesterol sulfate. One or more of their derivatives; Preferably, the non-cationic lipids or non-ionizable lipids described in (iii) include cholesterol, and a combination of one or more selected from DSPC, DSPE, DSPA or DSPG; Preferably, the non-cationic lipids or non-ionizable lipids described in (iii) include cholesterol (Formula 40) and DSPC (Formula 46).

8. The production method according to claim 7, wherein The metal-phospholipid complex particles are made of (i) a metal-phospholipid complex, (ii) conjugated lipids that inhibit particle aggregation, and (iii) non-cationic lipids or non-ionizable lipids. The metal-phospholipid complex accounts for 5% to 50% of the raw material in molar proportion, the conjugated lipids that inhibit particle aggregation account for 1% to 10% of the raw material in molar proportion, the cholesterol accounts for 15% to 80% of the raw material in molar proportion, and the non-cationic lipids or non-ionizable lipids other than cholesterol account for 0% to 51% of the raw material in molar proportion. Preferably, the metal-phospholipid complex accounts for 5% to 40% of the raw material, more preferably 10% to 40%, and even more preferably 15% to 25%. Preferably, the conjugated lipids that inhibit particle aggregation have a molar percentage of 2% to 10% in the raw material, more preferably 4% to 10%; Preferably, the cholesterol in the raw material has a molar percentage of 25% to 75%, more preferably 35% to 75%, and even more preferably 40% to 46%; Preferably, the non-cationic lipids or non-ionizable lipids other than cholesterol account for 0% to 50% of the raw material, more preferably 0% to 40%, and even more preferably 25% to 35%.

9. A method of preparing drug-lipid particles, wherein, The drug is encapsulated in metal-phospholipid complex particles prepared by the preparation method according to any one of claims 5 to 8 to obtain the drug-lipid particles; Preferably, drug-lipid particles are prepared by mixing (i) the metal-phospholipid complex prepared by the preparation method according to any one of claims 1 to 4, (ii) the conjugated lipid that inhibits particle aggregation, (iii) the non-cationic lipid or non-ionizable lipid, and the drug. Preferably, the metal-phospholipid complex, the conjugated lipids that inhibit particle aggregation, and the non-cationic lipids or non-ionizable lipids are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer solution to form an aqueous phase, and the organic phase and the aqueous phase are mixed to obtain drug-lipid particles, wherein the drug is a negatively charged molecule.

10. The production method according to claim 9, wherein The organic compound is ethanol; Preferably, the mixing method between the organic phase and the aqueous phase includes microfluidic chip or ultrasound; Preferably, the mass ratio of the total mass of the metal-phospholipid complex, the conjugated lipids that inhibit particle aggregation, and the non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation to the mass of the drug is (20-100):

1.

11. The preparation method according to claim 9 or 10, wherein 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 acids; Preferably, the nucleic acid is the mRNA sequence encoding eGFP shown in SEQ ID No. 1, the mRNA sequence encoding the receptor-binding domain RBD of the novel coronavirus S1 subunit shown in SEQ ID No. 2, the mRNA sequence encoding NY-ESO-1 shown in SEQ ID No. 3, the siRNA sequence of the Bcl-2 gene with antisense strand as SEQ ID No. 4 and sense strand as SEQ ID No. 21, the siRNA sequence of the PLK1 gene with antisense strand as SEQ ID No. 6 and sense strand as SEQ ID No. 23, the siRNA sequence of the Gal-1 gene shown in SEQ ID No. 8, the ASO sequence of the STAT-3 gene shown in SEQ ID No. 10, the ASO sequence of the α-syn gene shown in SEQ ID No. 12, the ASO sequence of the Bcl-2 gene shown in SEQ ID No. 14, the mRNA sequence encoding the wild-type novel coronavirus S protein shown in SEQ ID No. 16, the double-stranded DNA sequence with antisense strand as SEQ ID No. 17 and sense strand as SEQ ID No. 25, the single-stranded DNA sequence shown in SEQ ID No. 18, or the siRNA sequence encoding NY-ESO-1 shown in SEQ ID No.

16. ID No. 19 and the antisense strand are the siRNA sequences of the B7-H4 gene shown in SEQ ID No. 26; Preferably, the drug is mRNA, and the mRNA encodes a chimeric antigen receptor (CAR) or a TCR. Preferably, the drug is an mRNA encoding a chimeric antigen receptor (CAR), wherein the CAR includes a transmembrane domain, a signal transduction domain, an antigen-binding domain, a co-stimulatory signal transduction region, and a transmembrane domain-transmembrane domain junction region. 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 signal transduction 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 co-stimulatory signal transduction region is selected from at least one of SEQ ID NO.37, SEQ ID No.39, and SEQ ID No.75; and / or, The antigen-binding domain and the transmembrane domain junction region are 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 with a nucleotide sequence as shown in SEQ ID No.

43.

12. The method of producing the drug-lipid particle according to claim 9, wherein, The drug-lipid particles are mixed with the targeting structure to prepare a targeted drug; wherein the targeting structure is connected to the outer surface of the metal-phospholipid complex particles. Preferably, the reaction conditions between the drug-lipid particles and the targeting structure are incubation at 2-10°C for 0.2-12 hours; Preferably, the targeting structure is a DSPE-PEG2000-aptamer, and the preparation process is as follows: DSPE-PEG2000 and the aptamer are linked by an intermediate pair to obtain the 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, and more 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-lipid particles to form the targeted drug; Preferably, the preparation method of the micelles includes direct dissolution, ethanol injection, dialysis, or ultrasound.

13. The application of the preparation method according to any one of claims 1 to 4 in the preparation of metal-phospholipid complex particles.

14. The use of the preparation method according to any one of claims 5 to 8 in the preparation of drug-lipid particles.

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