Metal-polyphenol complex particles, drug-lipid particles, and their manufacturing methods and applications

Abstract

The present disclosure relates to the field of biopharmaceutical technology, specifically to metal-polyphenol complex particles, drug-lipid particles, and their manufacturing methods and applications. The present disclosure provides a metal-polyphenol complex as a drug carrier for drug stability, delivery, and transport, etc., and forms metal-polyphenol complex particles with other carriers, which act in concert to achieve effective administration of negatively charged drugs. While achieving efficient systemic drug delivery, toxicity is significantly reduced compared to LNPs containing cationic or ionizable lipids, enabling safe and effective treatment of diseases or conditions.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This disclosure claims priority to three Chinese patent applications: application number 202210950179.1, filed on August 9, 2022, with the Patent Office of China, entitled "Drug-Lipid Particles, and Their Preparation Methods and Applications"; application number 202210950180.4, filed on August 9, 2022, with the Patent Office of China, entitled "Metal-Polyphenol Complex Particles, and Their Preparation Methods and Applications"; and application number 202210958544.3, filed on August 9, 2022, with the Patent Office of China, entitled "Application of Metal-Polyphenol Complexes in Nucleic Acid Delivery Systems," the contents of which are incorporated herein by reference in their entireties. [Technical Field]

[0002] The present disclosure relates to the field of biomedical technology, and in particular to metal-polyphenol complex particles, drug-lipid particles, and their manufacturing methods and applications. [Background technology]

[0003] Nucleic acid drugs refer to functional DNA or RNA that specifically edit pathogenic genes or proteins through methods such as binding, fragmentation, knockout, and insertion. The discovery of this kind of nucleic acid not only breaks the conventional idea that nucleic acids only carry genetic information, but also provides powerful molecular tools for biomedicine and biosensing.

[0004] Nucleic acid drugs have drawbacks, such as rapid degradation by nucleases in the body, weak membrane penetration ability, and short half-life in the bloodstream, which severely limit their clinical application. Therefore, the development of safe and effective nucleic acid drug delivery systems has become a hotspot in the development of nucleic acid drugs. Currently, nucleic acid drug delivery carriers can be mainly divided into viral and non-viral carriers. Viral carriers (including adenoviral, retroviral, and lentiviral carriers) induce immune responses upon entry into the human body and are therefore not widely used. Among non-viral carriers, nanoparticles and small molecule conjugates are the most commonly used. Compared with small molecule conjugates directly conjugated to nucleic acid drugs, nanoparticles can more effectively encapsulate nucleic acid drugs, prevent rapid degradation by nucleases in the body, and extend their circulation time in the body. The mechanism of nucleic acid encapsulation in nanoparticles relies on the adsorption of negatively charged nucleic acids by positively charged cationic lipids.

[0005] Cationic liposomes are typically prepared by mixing cationic lipids with auxiliary lipids, such as dioleoylphosphatidylethanolamine (DOPE) and cholesterol, in a certain ratio. Cationic liposomes can be used to deliver genes or drug molecules to target cells. However, during transfection and other processes, cationic liposomes still contain a certain degree of cytotoxicity, which can cause toxic effects on normal cells while delivering drugs to treat cancer cells. This limits their clinical application. The cascade reactions induced by cationic liposomes include the generation of reactive oxygen species, enzyme activation, changes in mitochondrial membrane potential, and cell apoptosis caused by the release of cytochrome C and caspases.

[0006] Ionizable lipids are lipids containing positively charged, ionizable amine groups that are uncharged under physiological conditions (pH = 7.4) but become protonated and positively charged at relatively low pH values. Therefore, ionizable lipids can be used to partially or completely replace cationic lipids as the main component of nanoparticles responsible for nucleic acid adsorption. After nanoparticles containing ionizable lipids enter the lysosomes of living cells, the ionizable lipids become positively charged in the low pH (pH = 4.0-6.5) environment within the lysosome. Although ionizable lipids mitigate the cytotoxic and highly proinflammatory effects of some permanently positively charged cationic lipids, their cytotoxicity and immunogenicity remain relatively high. Lipid nanoparticles (LNPs) based on cationic and / or ionizable lipids are currently available as nanoparticle nucleic acid drug delivery systems for clinical use. Cationic and / or ionizable lipids are the main components of LNPs and are responsible for nucleic acid adsorption. At the same time, cytotoxicity and immunogenicity mediated by cationic and / or ionizable lipids remain an important reason for the relatively high toxicity of LNPs.

[0007] Therefore, when using a delivery system to deliver negatively charged drugs (e.g., nucleic acid drugs, protein drugs, polypeptide drugs, small molecule drugs, etc.), none of the nanoparticle delivery systems developed relying on cationic lipids and / or ionizable lipids can fundamentally solve the toxicity problem of nanoparticle delivery systems. There is an urgent need for a liposome delivery system with low toxicity that does not use cationic lipids and / or ionizable lipids.

[0008] The present disclosure aims to provide an application of metal-polyphenol complexes in nucleic acid delivery systems so as to solve at least one of the technical problems existing in the prior art.

[0009] In order to achieve the above object, the present disclosure employs the following inventions.

[0010] The present disclosure provides the application of a metal-polyphenol complex in a nucleic acid delivery system, which is formed by reacting a polyphenol molecular moiety with a metal ion moiety, and the polyphenol molecular moiety and the metal ion moiety are linked by a coordinate bond.

[0011] In some embodiments, the polyphenol moiety is one or a combination of two or more selected from curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, taxifolin, brown algae polyphenols, polyflavanol polyphenols, catechin, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, glucogallin, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate, gallocatechin gallate, and derivatives thereof.

[0012] The term "these" in "and derivatives thereof" refers to "curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, taxifolin, brown algae polyphenols, polyflavanol polyphenols, catechin, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, glucogallin, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate, or gallocatechin gallate." The polyphenol molecule moiety may be, for example, but is not limited to, curcumin, curcumin derivatives, hesperetin, hesperetin derivatives, catechin, catechin derivatives, curcumin and catechin, catechin and catechin derivatives, etc. In the present disclosure, "and derivatives thereof" all have the same meaning.

[0013] Furthermore, the polyphenol molecule portion may be selected from the group consisting of curcumin (formula 1), quercetin (formula 2), kaempferol (formula 3), rutin (formula 4), hesperetin (formula 5), naringenin (formula 6), eriodictyol (formula 7), luteolin (formula 8), apigenin (formula 9), taxifolin (formula 10), brown algae polyphenols (formula 11), polyflavanol polyphenols (formula 12), catechin (formula 13), ellagamine (formula 14), and the like. and combinations of one or more selected from the group consisting of catechin gallate (Formula 14), gallic acid (Formula 15), digallic acid (Formula 16), propyl gallate (Formula 17), epigallocatechin gallate (Formula 18), glucogallin (Formula 19), hydroxyhydroquinone (Formula 20), morin (Formula 21), epicatechin gallate (Formula 22), catechin gallate (Formula 23), gallocatechin gallate (Formula 24), and derivatives thereof. [ka] TIFF2025527207000002.tif245155TIFF2025527207000003.tif250153TIFF2025527207000004.tif77155

[0014] Furthermore, the polyphenol molecule portion is one or a combination of two or more selected from curcumin (Formula 1), dihydrocurcumin (Formula 25), hexahydrocurcumin (Formula 26), curcumin sulfate (Formula 27), and bisdemethoxycurcumin (Formula 28). [ka]

[0015] Furthermore, the polyphenol molecule portion is one or a combination of two or more selected from curcumin (formula 1), hesperetin (formula 5), or catechin (formula 13), and derivatives thereof.

[0016] Furthermore, the polyphenol moiety is selected from curcumin (Formula 1), hesperetin (Formula 5), or catechin (Formula 13).

[0017] In some embodiments, the metal ion moiety is 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+ It is one or a combination of several selected from the above.

[0018] Furthermore, the metal ion moiety is Fe 3+ , Ca 2+ , Al 3+ It is one or a combination of several selected from the above.

[0019] Furthermore, the metal ion moiety is Fe 3+ , Ca 2+ or Al 3+ is selected from.

[0020] In some embodiments, the disclosed metal-polyphenol complexes comprise a polyphenol moiety reacted with a metal ion moiety, wherein the polyphenol moiety is selected from curcumin, hesperetin, or catechin, and the metal ion moiety is Fe. 3+ , Ca 2+ or Al 3+ is selected from.

[0021] Furthermore, the metal-polyphenol complex is formed by reacting a polyphenol molecular portion with a metal ion portion, the polyphenol molecular portion being selected from curcumin (formula 1), hesperetin (formula 5), or catechin (formula 13), and the metal ion portion being Fe3+ , Ca 2+ or Al 3+ is selected from.

[0022] Furthermore, the molar ratio of the polyphenol molecule portion to the metal ion portion is 1:(0.5 to 2).

[0023] Furthermore, the polyphenol molecule portion is curcumin (Formula 1), and the metal ion portion is Fe 3+ is.

[0024] Furthermore, curcumin (Eq. 1) and Fe 3+ The molar ratio is 1:1.

[0025] Furthermore, the polyphenol molecule portion is curcumin (Formula 1), and the metal ion portion is Al 3+ is.

[0026] Furthermore, curcumin (formula 1) and Al 3+ The molar ratio is 1:1.

[0027] In some embodiments, the nucleic acid delivered by the nucleic acid delivery system is one or a combination of more selected from mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, and artificial nucleic acids.

[0028] Further, the nucleic acid may be an mRNA sequence encoding eGFP (Enhanced Green Fluorescent Protein) as shown in SEQ ID No. 1, an mRNA sequence encoding the receptor-binding domain RBD of the S1 subunit of the novel coronavirus as shown in SEQ ID No. 2, an mRNA sequence encoding NY-ESO-1 (New York esophageal squamous cell carcinoma-1) as shown in SEQ ID No. 3, an siRNA sequence of the Bcl-2 gene (B-cell lymphoma / Leukemia-2 gene) as shown in SEQ ID No. 4 as an antisense strand and SEQ ID No. 21 as a sense strand, an siRNA sequence of the PLK1 gene (Polo-like Kinase 1) as shown in SEQ ID No. 6 as an antisense strand and SEQ ID No. 23 as a sense strand, an siRNA sequence of the Gal-1 gene as shown in SEQ ID No. 8, an ASO sequence of the STAT-3 gene as shown in SEQ ID No. 10, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 11, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 12, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 13, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 14, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 15, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 16, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 17, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 18, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 19, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 20, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 21, an siRNA sequence of the The ASO sequence of the α-syn gene (α-synuclein) shown in 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 whose antisense strand is SEQ ID No. 17 and whose sense strand is SEQ ID No. 25, the single-stranded DNA shown in SEQ ID No. 18, or the siRNA sequence of the B7-H4 gene whose sense strand is SEQ ID No. 19 and whose antisense strand is SEQ ID No. 26.

[0029] Furthermore, the nucleic acid delivery system is used to introduce nucleic acids into cells.

[0030] Additionally, the nucleic acids are used to silence expression of a target sequence in a mammalian subject or to treat a disease or condition in a mammal.

[0031] Additionally, the mammal is a human.

[0032] Furthermore, the disease or condition is associated with the expression of a gene, the gene containing a target sequence for the drug.

[0033] Further, the disease or condition includes cancer, viral infection, autoimmune disease, diabetes or Alzheimer's disease.

[0034] Further, the viral infection includes hepatitis A, hepatitis B, hepatitis C, SARS-Cov-2, HIV, HPV, influenza, smallpox or syphilis.

[0035] Furthermore, the cancer includes liver cancer, glioma, melanoma, lung cancer, pancreatic cancer, or breast cancer.

[0036] Furthermore, the nucleic acid delivery system is used in the production of vaccines.

[0037] Furthermore, the vaccine is a novel coronavirus vaccine.

[0038] The present disclosure further provides metal-polyphenol complex particles, the metal-polyphenol complex particles comprising:

[0039] (i) a metal-polyphenol complex, which is formed by reacting a polyphenol molecular portion with a metal ion portion, and the polyphenol molecular portion and the metal ion portion are linked by a coordinate bond;

[0040] (ii) a conjugated lipid that inhibits particle aggregation, wherein the conjugated lipid that inhibits particle aggregation is not a cationic lipid or an ionizable lipid;

[0041] (iii) a non-cationic or non-ionizable lipid other than the conjugated lipid that inhibits particle aggregation.

[0042] In some embodiments, the polyphenol molecule portion and the metal ion portion of the metal-polyphenol complex in (i) of the metal-polyphenol complex particle are independently the same as the polyphenol molecule portion and the metal ion portion described in the above applications of the metal-polyphenol complex.

[0043] In some embodiments, the lipid conjugated to inhibit particle aggregation in (ii) of the metal-polyphenol complex particles comprises a polyethylene glycol (PEG)-lipid conjugate and / or a PEG-dialkyloxypropyl (DAA).

[0044] Furthermore, the PEG-lipid conjugate is one or more combinations selected from phosphatidylethanolamine-polyethylene glycol 2000 (formula 47), phosphatidylethanolamine-polyethylene glycol 700 (formula 48), phosphatidylethanolamine-polyethylene glycol 1000 (formula 49), phosphatidylethanolamine-polyethylene glycol 5000 (formula 50), and derivatives thereof. R1 and R2 are each independently as follows. [ka] TIFF2025527207000007.tif192142

[0045] Furthermore, the PEG-lipid conjugate is one or a combination of more selected from DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000, and DSPE-PEG5000.

[0046] Further, the PEG-lipid conjugate is selected from DSPE-PEG2000 (Formula 58), DSPE-PEG700 (Formula 55), DSPE-PEG1000 (Formula 56), or DSPE-PEG5000 (Formula 57). [ka] TIFF2025527207000009.tif195166

[0047] In some embodiments, the non-cationic or non-ionizable lipid in (iii) of the metal-polyphenol complex particles is selected from the group consisting of lecithin (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), ceramide-1-phosphate (SP), phosphatidylinositol (Pi), phosphatidylthreonine (PT), sphingomyelin (SM), The lysolecithin may be one or a combination of two or more selected from lysolecithin (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysosphingomyelin (LSM), sphingosine-1-phosphate (S1P), and derivatives thereof.

[0048] Further, the non-cationic lipid or non-ionizable lipid in (iii) may be lecithin (PC) (Formula 29), phosphatidylethanolamine (PE) (Formula 30), phosphatidylserine (PS) (Formula 31), phosphatidic acid (PA) (Formula 32), phosphatidylglycerol (PG) (Formula 33), ceramide-1-phosphate (SP) (Formula 34), phosphatidylinositol (Pi) (Formula 35), phosphatidylthreonine (PT) (Formula 36), sphingomyelin (SM) (Formula 37), lysolecithin (LPC) (Formula 38), lysophosphatidylethanolamine (LPE) (Formula 39), lysophosphatidylserine (LP) (Formula 40), lysophosphatidylethanolamine (LPE) (Formula 41), lysophosphatidylserine (LP) (Formula 42), lysophosphatidylglycerol (PG) (Formula 43), ceramide-1-phosphate (SP) (Formula 44), phosphatidylinositol (Pi) (Formula 45), phosphatidylthreonine (PT) (Formula 46), sphingomyelin (SM) (Formula 47), lysolecithin (LPC) (Formula 48), lysophosphatidylethanolamine (LPE) (Formula 49), lysophosphatidylserine (LP) (Formula 50), lysophosphatidylglycerol (LPC) (Formula 51), lysophosphatidylglycerol (LPC) (Formula 52), lysophosphatidylglycerol (LPC) (Form and a combination of one or more selected from the group consisting of lysophosphatidic acid (LPA) (formula 41), lysophosphatidylglycerol (LPG) (formula 42), lysophosphatidylinositol (LPI) (formula 43), lysophosphatidylthreonine (LPT) (formula 44), lysosphingomyelin (LSM) (formula 45), sphingosine-1-phosphate (S1P) (formula 46), and derivatives thereof, wherein R1 and R2 are each independently a capryl group, a lauroyl group, a tetradecanol group, a palmitoyl group, a stearoyl group, an oleoyl group, a linoleoyl group, an erucoyl group, an arachidonoyl group, or a diphytanoyl group. [ka] TIFF2025527207000011.tif227143TIFF2025527207000012.tif113126

[0049] It should be noted that in the present disclosure, any cis-trans isomers of the components used do not affect the technical effects achieved by the protected content of the present disclosure.

[0050] Furthermore, the non-cationic or non-ionizable lipid in (iii) comprises at least one of cholesterol and derivatives thereof.

[0051] In some embodiments, the non-cationic or non-ionizable lipid in (iii) comprises a combination of cholesterol and one or more selected from DSPC, DSPE, DSPA, or DSPG.

[0052] Preferably, the cholesterol structural formula is: [ka]

[0053] In one embodiment, the non-cationic or non-ionizable lipid in (iii) comprises a combination of cholesterol (Formula 59) and one or more selected from DSPC (Formula 51), DSPE (Formula 52), DSPA (Formula 53), or DSPG (Formula 54). [ka]

[0054] In some embodiments, the non-cationic or non-ionizable lipid in (iii) comprises cholesterol (Formula 59) and DSPC (Formula 51).

[0055] In some embodiments, the metal-polyphenol complex particles comprise (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or non-ionizable lipid (including cholesterol and non-cationic lipids or non-ionizable lipids other than cholesterol), wherein the molar fraction of the metal-polyphenol complex in the feedstock is 10% to 20%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the feedstock is 2% to 10%, the molar fraction of the cholesterol in the feedstock is 0% to 48%, and the molar fraction of the non-cationic lipid or non-ionizable lipid other than cholesterol in the feedstock is 40% to 75%.

[0056] In some embodiments, the metal-polyphenol complex particles comprise (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or non-ionizable lipid, wherein the molar fraction of the metal-polyphenol complex in the feedstock is 5% or more and less than 10%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the feedstock is 2% to 10%, the molar fraction of the cholesterol in the feedstock is 0% to 48%, and the molar fraction of the non-cationic lipid or non-ionizable lipid other than cholesterol in the feedstock is 30% or more and less than 40%, or 40% to 75%. Or,

[0057] In some embodiments, the metal-polyphenol complex particles comprise (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or non-ionizable lipid, wherein the molar fraction of the metal-polyphenol complex in the raw material is 10% to 20%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, the molar fraction of the cholesterol in the raw material is 0% to 48%, and the molar fraction of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 30% or more but less than 40%.

[0058] Furthermore, the molar fraction of the metal-polyphenol complex in the raw material is 5% or more and less than 10%, 10% to 15%, or 15% to 20%, and preferably 5%, 10%, or 15%.

[0059] Furthermore, the molar fraction of conjugated lipids that inhibit particle aggregation in the raw material is 3% to 5% or 5% to 10%, preferably 3%, 5%, or 10%.

[0060] Furthermore, the molar fraction of cholesterol in the raw material is 10% to 30%, 30% to 47%, or 10% to 20%, and preferably 10%, 30%, or 47%.

[0061] Furthermore, the molar fraction of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 45% to 55%, 60% to 65%, or 50% to 65%, preferably 45%, 55%, 60%, or 65%.

[0062] In some embodiments, the metal-polyphenol complex (metal ion moiety) in the feedstock is Fe 3+ The molar fraction of the polyphenol molecule moiety (selected from the group consisting of DSPE-PEG2000 and DSPC) is 5% or more and less than 10%, or 10% to 15%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 5% to 10%, the molar fraction of the cholesterol in the raw material is 10% to 30%, and the molar fraction of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 60% to 65%. Preferably, the polyphenol molecule moiety is selected from curcumin (Formula 1), the conjugated lipid that inhibits particle aggregation is DSPE-PEG2000, and the non-cationic lipid or non-ionizable lipid is cholesterol and DSPC.

[0063] In one embodiment, the metal-polyphenol complex (metal ion portion) in the raw material is Fe 3+ The molar fraction of the lipid conjugated to inhibit particle aggregation in the feedstock is 15%, the molar fraction of the cholesterol in the feedstock is 10%, and the molar fraction of non-cationic or non-ionizable lipids other than cholesterol (e.g., DSPC, DSPA, DSPE, or DSPG) in the feedstock is 65%.

[0064] In one embodiment, the metal-polyphenol complex (metal ion portion) in the raw material is Fe 3+ the molar fraction of the lipid conjugated to inhibit particle aggregation in the feedstock is 5%, the molar fraction of the cholesterol in the feedstock is 30%, and the molar fraction of non-cationic lipids or non-ionizable lipids other than cholesterol in the feedstock is 60%.

[0065] In some embodiments, the metal-polyphenol complex (metal ion portion) in the feedstock is Al 3+ The molar fraction of the polyphenol molecule moiety (selected from the group consisting of DSPE-PEG2000 and DSPC) is 5% or more but less than 10%, or 10%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 5%, the molar fraction of cholesterol in the raw material is 30% to 47%, and the molar fraction of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 45% to 55%. Preferably, the polyphenol molecule moiety is selected from curcumin (Formula 1), the conjugated lipid that inhibits particle aggregation is DSPE-PEG2000, and the non-cationic lipids or non-ionizable lipids are cholesterol and DSPC.

[0066] In one embodiment, the metal-polyphenol complex (metal ion portion) in the feedstock is Al 3+ the molar fraction of the particle aggregation-inhibiting conjugated lipid in the feedstock is 3%, the molar fraction of the cholesterol in the feedstock is 47%, and the molar fraction of non-cationic lipids or non-ionizable lipids other than cholesterol in the feedstock is 45%.

[0067] In one embodiment, the metal-polyphenol complex (metal ion portion) in the feedstock is Al 3+ the molar fraction of the lipid conjugated to inhibit particle aggregation in the feedstock is 5%, the molar fraction of the cholesterol in the feedstock is 30%, and the molar fraction of non-cationic lipids or non-ionizable lipids other than cholesterol in the feedstock is 55%.

[0068] The present disclosure provides a method for producing the metal-polyphenol complex particles by combining (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic or non-ionizable lipid.

[0069] Furthermore, the manufacturing method includes the following steps:

[0070] Step 1: reacting a polyphenol molecule portion with a metal ion portion through a coordinate bond to form a metal-polyphenol complex;

[0071] Step 2 includes mixing the metal-polyphenol complex produced in step 1 with a conjugation lipid that inhibits particle aggregation, a non-cationic lipid, or a non-ionizable lipid to produce the metal-polyphenol complex particles.

[0072] Furthermore, polyphenol molecules are dissolved in ethanol and then reacted with metal ions. The molar ratio of polyphenol molecules to metal ions is 1:(1-2), and the reaction conditions include reacting at 60°C for 1 hour.

[0073] The present disclosure provides:

[0074] (a) a drug, which is a negatively charged molecule;

[0075] (b) a metal-polyphenol complex particle, the metal-polyphenol complex particle comprising:

[0076] (i) a metal-polyphenol complex, which is formed by reacting a polyphenol molecular portion with a metal ion portion, and the polyphenol molecular portion and the metal ion portion are linked by a coordinate bond;

[0077] (ii) a conjugated lipid that inhibits particle aggregation, wherein the conjugated lipid that inhibits particle aggregation is not a cationic lipid or an ionizable lipid;

[0078] (iii) a non-cationic or non-ionizable lipid other than the conjugated lipid that inhibits particle aggregation.

[0079] Furthermore, the drug is encapsulated in the metal-polyphenol complex particles.

[0080] Furthermore, the drug is one or more combinations selected from nucleic acids, proteins, polypeptides, small molecules, nucleic acid analogs, protein analogs, and polypeptide analogs.Preferably, the nucleic acid is the same as the nucleic acid described in the above application of metal-polyphenol complexes.

[0081] The present disclosure provides a method for producing the drug-lipid particles, which are obtained by encapsulating a drug in a metal-polyphenol complex particle.

[0082] In one embodiment, the metal-polyphenol complex particles are obtained by combining (i) a metal-polyphenol complex, (ii) a conjugation lipid that inhibits particle aggregation, and (iii) a non-cationic or non-ionizable lipid.

[0083] In one embodiment, the drug-lipid particles are obtained by combining (a) a drug, (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic or non-ionizable lipid.

[0084] In one embodiment, a metal-polyphenol complex, a conjugated lipid that inhibits particle aggregation, and a non-cationic or non-ionizable lipid are dissolved in an organic compound to form an organic phase, a drug is dissolved in a buffer solution to form an aqueous phase, and the organic and aqueous phases are uniformly mixed to obtain drug-lipid particles. Preferably, the organic compound is ethanol. The buffer solution is an enzyme-free Tris-HCl buffer. The organic and aqueous phases can be mixed using a microfluidic chip or ultrasound.

[0085] The present disclosure provides an application of the metal-polyphenol complex particles in drug-lipid particles.

[0086] The present disclosure provides applications of the metal-polyphenol complex particles or the drug-lipid particles in compositions used for drug delivery.

[0087] Additionally, the compositions are used to introduce drugs into cells.

[0088] Additionally, the composition is a pharmaceutical agent.

[0089] Additionally, the agents are used to silence the expression of target sequences in mammalian subjects.

[0090] Additionally, the medicament is used to deliver drugs within a mammalian body.

[0091] Additionally, the agents are used to deliver drugs to mammalian cells from within the body.

[0092] Additionally, the medicament is used to treat a disease or condition in a mammal.

[0093] Further, the mammal is a human.

[0094] Furthermore, the disease or condition is associated with the expression of a gene, the gene containing a target sequence for the drug.

[0095] Further, the disease or condition includes cancer, viral infection, autoimmune disease, diabetes or Alzheimer's disease.

[0096] Furthermore, the viral infection includes hepatitis A, hepatitis B, hepatitis C, SARS-Cov-2 (2019 novel coronavirus), HIV (AIDS virus), HPV (human papillomavirus), influenza, smallpox, or syphilis.

[0097] Furthermore, the cancer includes liver cancer, glioma, melanoma, lung cancer, pancreatic cancer, or breast cancer.

[0098] Additionally, the agent is a vaccine.

[0099] Furthermore, the route of administration of the agent includes intrathecal injection, intramuscular injection, intracranial injection, intravenous injection, or intratumoral injection.

[0100] The present disclosure provides a medicament containing the metal-polyphenol complex particles or the drug-lipid particles.

[0101] Additionally, the agent is a vaccine.

[0102] Furthermore, the vaccine is a novel coronavirus vaccine.

[0103] The present disclosure provides applications of the metal-polyphenol complex particles or the drug-lipid particles in the prevention and / or treatment of mammalian diseases or conditions.

[0104] In some embodiments, the mammal is a human.

[0105] In some embodiments, the disease or condition is associated with expression of a gene, and the gene comprises a target sequence for the drug.

[0106] In some embodiments, the disease or condition comprises cancer, a viral infection, an autoimmune disease, diabetes, or Alzheimer's disease.

[0107] In some embodiments, the viral infection comprises hepatitis A, hepatitis B, hepatitis C, SARS-Cov-2, HIV, HPV, influenza, smallpox, or syphilis.

[0108] In some embodiments, the cancer comprises liver cancer, glioma, melanoma, lung cancer, pancreatic cancer, or breast cancer.

[0109] Compared with the prior art, the technical advantages of the present disclosure are as follows:

[0110] In the prior art, complexes of metal ions and polyphenol molecules (e.g., curcumin, etc.) are mainly used as active ingredients in the fields of antioxidants, anti-inflammatory agents, antivirals, etc. However, the present inventors have discovered through experimental research that the present disclosure provides applications of metal-polyphenol complexes in compositions or drugs that are mainly used as drug carriers for drug stability, delivery, transport, etc., and that work in conjunction with other carriers to achieve effective administration of negatively charged drugs.

[0111] The metal-polyphenol complex particles provided by the present disclosure have a small diameter suitable for systemic delivery and ensure efficacy not lower than that of LNPs. However, because they do not use cationic or ionizable lipids, the toxicity of the drug-lipid particles is significantly lower than that of LNPs, their biological safety is significantly improved, and they are more advantageous for transporting negatively charged drugs in vivo. [Brief explanation of the drawings]

[0112] In order to more clearly describe the invention according to the specific embodiments of the present disclosure or the prior art, the drawings necessary for describing the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present disclosure. Those skilled in the art can obtain other drawings based on these drawings without creative work.

[0113] [Figure 1-1] FIG. 10 is a statistical diagram of the results of the percentage of eGFP-positive cells caused by transfection of 293T with eGFP-mRNA@MPNP(Fe 3+ ) provided in Example 2.5.1 of the present disclosure.

[0114] [Figure 1-2] FIG. 11 is a statistical diagram of the expression level of RBD induced by transfection of 293T with RBD-mRNA@MPNP(Fe3+) in Example 2.5.1 of the present disclosure.

[0115] [Figure 1-3]FIG. 11 is a statistical diagram showing the results of the ability of RBD-mRNA@MPNP(Fe3+) to induce humoral immunity in Example 2.5.1 of the present disclosure.

[0116] [Figure 1-4] FIG. 10 is a statistical diagram showing the results of the ability of NY-ESO-1-mRNA@MPNP(Fe3+) to induce humoral immunity in Example 2.5.1 of the present disclosure.

[0117] [Figure 1-5] FIG. 11 is a statistical diagram showing the results of the ability of RBD-mRNA@MPNP(Fe3+) to induce cellular immunity in Example 2.5.1 of the present disclosure.

[0118] [Figure 1-6] FIG. 10 is a statistical diagram showing the results of the ability of NY-ESO-1-mRNA@MPNP(Fe3+) to induce cellular immunity in Example 2.5.1 of the present disclosure.

[0119] [Figure 1-7] FIG. 10 is a statistical diagram of the results of the percentage of eGFP-positive cells caused by transfection of 293T with eGFP-mRNA@MPNP(Al 3+ ) provided in Example 2.5.2 of the present disclosure.

[0120] [Figure 1-8] FIG. 10 is a statistical diagram of the expression level of RBD induced by transfection of 293T with RBD-mRNA@MPNP(Al3+) in Example 2.5.2 of the present disclosure.

[0121] [Figure 1-9] FIG. 10 is a statistical diagram showing the results of the ability of RBD-mRNA@MPNP(Al3+) to induce humoral immunity in Example 2.5.2 of the present disclosure.

[0122] [Figure 1-10] FIG. 10 is a statistical diagram showing the results of the ability of NY-ESO-1-mRNA @MPNP(Al3+) to induce humoral immunity in Example 2.5.2 of the present disclosure.

[0123] [Figure 1-11] FIG. 10 is a statistical diagram showing the results of the ability of RBD-mRNA@MPNP(Al3+) to induce cellular immunity in Example 2.5.2 of the present disclosure.

[0124] [Figure 1-12] FIG. 10 is a statistical diagram showing the results of the ability of NY-ESO-1-mRNA@MPNP(Al3+) to induce cellular immunity in Example 2.5.2 of the present disclosure.

[0125] [Figure 1-13] In Example 2.6.1 of the present disclosure, the ability of Bcl-2-siRNA@MPNP(Fe3+) to silence target genes is shown.

[0126] [Figure 1-14] FIG. 11 is a statistical diagram of the results of the ability of PLK1-siRNA@MPNP(Fe3+) to silence target genes in Example 2.6.1 of the present disclosure.

[0127] [Figure 1-15] FIG. 11 is a statistical diagram of the results of the ability of Gal-1-siRNA@MPNP(Fe3+) to silence target genes in Example 2.6.1 of the present disclosure.

[0128] [Figure 1-16] In Example 2.6.2 of the present disclosure, the ability of Bcl-2-siRNA@MPNP(Al3+) to silence target genes is shown.

[0129] [Figure 1-17] FIG. 10 is a statistical diagram of the results of the ability of PLK1-siRNA@MPNP(Al 3+ ) to silence target genes in Example 2.6.2 of the present disclosure.

[0130] [Figure 1-18]FIG. 10 is a statistical diagram of the results of the ability of Gal-1-siRNA@MPNP(Al 3+ ) to silence target genes in Example 2.6.2 of the present disclosure.

[0131] [Figure 1-19] Statistical diagram of the results of the ability of STAT3-ASO@MPNP(Fe3+) to silence cellular target genes in Example 2.7.1 of the present disclosure.

[0132] [Figure 1-20] Statistical diagram of the results of the ability of α-syn-ASO@MPNP(Fe3+) to silence cellular target genes in Example 2.7.1 of the present disclosure.

[0133] [Figure 1-21] FIG. 11 is a statistical diagram showing the results of the ability of Bcl-2-ASO@MPNP(Fe3+) to silence cellular target genes in Example 2.7.1 of the present disclosure.

[0134] [Figure 1-22] Statistical diagram of the results of the ability of STAT3-ASO@MPNP(Al3+) to silence cellular target genes in Example 2.7.2 of the present disclosure.

[0135] [Figure 1-23] This is a statistical diagram of the results of the ability of α-syn-ASO@MPNP(Al3+) to silence cellular target genes in Example 2.7.2 of the present disclosure.

[0136] [Figure 1-24] FIG. 11 is a statistical diagram showing the results of the ability of Bcl-2-ASO@MPNP(Al3+) to silence cellular target genes in Example 2.7.2 of the present disclosure.

[0137] [Figure 1-25]FIG. 11 is a statistical diagram of the expression level of S protein induced by transfection of 293T with S-mRNA@MPNP(Fe3+) in Example 2.8.1 of the present disclosure.

[0138] [Figure 1-26] FIG. 1 shows the results of a functional test of drug (dsDNA and ssDNA)-metal-polyphenol complex particles (Fe3+) in Example 2.8.1 of the present disclosure.

[0139] [Figure 1-27] FIG. 10 is a statistical diagram of the expression level of S protein induced by transfection of 293T with S-mRNA@MPNP(Al3+) in Example 2.8.2 of the present disclosure.

[0140] [Figure 1-28] FIG. 10 shows the results of a functional test of drug (dsDNA and ssDNA)-metal-polyphenol complex particles (Al 3+ ) in Example 2.8.2 of the present disclosure.

[0141] [Figure 2-1] FIG. 1 is an ultraviolet absorption diagram of a metal-polyphenol complex (Fe3+) in Example 3.1 of the present disclosure.

[0142] [Figure 2-2] FIG. 1 is an ultraviolet absorption diagram of a metal-polyphenol complex (Al 3+ ) in Example 3.2 of the present disclosure.

[0143] [Figure 2-3] This is a photograph evaluating the characteristics of Fe3+ released from metal-polyphenol complexes under low pH conditions (pH = 5.0) in Example 4 of the present disclosure.

[0144] [Figure 2-4] FIG. 10 is a statistical diagram of the efficiency of drug-lipid particles encapsulating nucleic acids (mRNA and siRNA) in Example 5 of the present disclosure.

[0145] [Figure 2-5] FIG. 10 shows the results and statistical charts of the nucleic acid lysosomal escape ability of siRNA / mRNA@MPNP and siRNA / mRNA@LNP in Example 6 of the present disclosure.

[0146] [Figure 2-6] FIG. 10 is a statistical diagram showing the results of the eGFP-positive cell rates of MPNPs and LNPs in Example 7 of the present disclosure.

[0147] [Figure 2-7] FIG. 10 is a statistical diagram showing the results of the ability of MPNPs and LNPs to promote mRNA expression in Example 8 of the present disclosure.

[0148] [Figure 2-8] FIG. 11 is a statistical diagram showing the results of the ability of MPNPs and LNPs to promote humoral immunity in Example 8 of the present disclosure.

[0149] [Figure 2-9] FIG. 10 is a statistical diagram showing the results of the ability of MPNPs and LNPs to promote cellular immunity in Example 8 of the present disclosure.

[0150] [Figure 3-1] FIG. 11 is a statistical diagram showing the results of intratumoral injection of drug-metal-polyphenol complex particles in the treatment of liver cancer in Example 11 of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0151] definition

[0152] For the sake of convenience, certain terms used in the specification, examples, and appended claims will be summarized here. Unless otherwise defined herein, scientific and technical terms used herein have the same meaning as commonly understood and commonly used by those skilled in the art. Furthermore, unless the context requires otherwise, singular terms should be understood to include the plural form of the same, and plural terms should be understood to include the singular form. Specifically, unless the context clearly dictates otherwise, the terms "at least one" and "one or more" as used in the specification and appended claims include one, two, three, or more species.

[0153] Although all numerical ranges and parameters used to define the broad scope of the present disclosure are approximations, the relevant numerical values in the specific examples herein are presented as precisely as possible. However, any numerical value inherently contains standard deviations resulting from the particular testing method employed. Herein, the term "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of the particular numerical value or range. Alternatively, the term "about" may mean that the actual numerical value falls within an acceptable standard error of the mean, as determined by a person of ordinary skill in the art to which the present disclosure pertains. Except in experimental examples, or unless otherwise expressly stated, all ranges, quantities, values, and percentages used herein (e.g., those used to describe amounts of materials, lengths of time, temperatures, operating conditions, proportions of quantities, and other similar terms) are understood to be modified by the term "about." Therefore, unless specifically stated to the contrary, all numerical parameters disclosed in this specification and the appended claims are approximations and can be modified as necessary. At the very least, these numerical parameters should be understood to mean the number of significant digits represented and the resulting values obtained by applying ordinary rounding techniques.

[0154] All ranges provided herein are intended to include each specific range within the stated range and any combination of subranges between the stated ranges. Unless otherwise specified, all ranges provided herein include the endpoints of the range. Thus, the range 1-5 specifically includes 1, 2, 3, 4, and 5, as well as subranges such as 2-5, 3-5, 2-3, 2-4, and 1-4.

[0155] All publications and patent applications cited in this specification are hereby incorporated by reference, and for any and all purposes, each individual publication or patent application is specifically and individually indicated to be incorporated by reference herein. In the event of a conflict between this specification and any publication or patent application incorporated by reference herein, the present specification shall control.

[0156] The term "lipid" refers to a group of organic compounds, including but not limited to lipids of fatty acids. These are typically divided into three categories: "simple lipids," "complex lipids," and "derived lipids." "Simple lipids" include glyceryl esters, glyceryl esters of fatty acids, waxes, long-chain fatty acids, and long-chain alcohols or sterols. "Complex lipids" refer to lipids that contain, in addition to fatty acids and alcohols, non-lipid components in the molecule, including phospholipids and glycolipids. "Derived lipids" are derived from simple or complex lipids.

[0157] The term "lipid vesicle" refers to any lipid composition that can be used to deliver a compound, including, but not limited to, liposomes. An aqueous volume is encapsulated by an amphiphilic lipid bilayer. Alternatively, the lipids are encapsulated in an aqueous interior containing a large molecular component such as mRNA, or in a reduced interior, or in lipid aggregates or micelles. The encapsulated component is contained in a relatively chaotic lipid mixture. As used herein, metal-polyphenol complex particles (MPNPs) are "lipid vesicles," and drugs, such as nucleic acids, such as mRNA, are encapsulated in the MPNPs as encapsulated components. The "encapsulation" can be complete and / or partial.

[0158] As used herein, the term "polyphenol molecule portion" refers to the structure that originally belonged to the polyphenol molecule after the polyphenol molecule has reacted with another substance.

[0159] As used herein, the phrase "metal ion moiety" refers to the structure originally belonging to the metal ion after the metal ion moiety has reacted with another substance.

[0160] As used herein, the term "metal-polyphenol complex" refers to a complex formed by a reaction between the polyphenol molecule portion and the metal ion portion, and the polyphenol portion and the metal ion portion are linked by a coordinate bond.

[0161] The term "ionizable lipid" refers to a lipid containing a positively charged, ionizable amine group that can be protonated and positively charged at relatively low pH values, but is uncharged at physiological pH values.

[0162] The term "neutral lipid" refers to any one of a number of lipid species that are uncharged or exist in a neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0163] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH, including, but not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic groups linked to neutral lipids.

[0164] The term "cationic lipid" refers to any one of a number of lipid species that carry a positive charge at a selected pH, such as physiological pH. These lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). The following lipids are cationic, carrying a positive charge at sub-physiological pH, for example, DODAP, DODMA, DMDMA.

[0165] The term "hydrophobic lipid" refers to a compound having a non-polar group, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, which are optionally substituted with one or more aromatic, alicyclic or heterocyclic groups.Suitable examples include but are not limited to diacylglycerol, dialkylglycerol, N-N-dialkylamino group, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0166] The term "non-cationic or non-ionizable lipid" refers to a lipid that is neither a cationic lipid nor a non-ionizable lipid, and can be, for example, an anionic lipid, a neutral lipid.

[0167] Among the components of the metal-polyphenol complex particles, the "non-cationic lipid or non-ionizable lipid other than the conjugated lipid that inhibits particle aggregation" in (iii) refers to the lipid that remains in the metal-polyphenol complex particles after the conjugated lipid that inhibits particle aggregation has been removed.

[0168] The term "fusogenic" refers to the ability of a liposome, drug-lipid particle, or other drug delivery system to fuse with a cell membrane, which may be the plasma membrane or a membrane surrounding an organelle such as an endosome, nucleus, etc.

[0169] In metal-polyphenol complex particles, non-cationic or non-ionizable lipids, other than the conjugated lipid that inhibits particle aggregation, are present primarily as vesicle-forming lipids, and the term "vesicle-forming lipid" tends to include any amphipathic lipid that has a hydrophobic portion and a polar head group and that can itself spontaneously form bilayer vesicles in water, examples of which are most phospholipids.

[0170] In metal-polyphenol complex particles, conjugated lipids that inhibit particle aggregation are primarily present as vesicle-adopting lipids. The term "vesicle-adopting lipids" tends to include any amphipathic lipid that stabilizes lipid bilayer bonds, as well as other amphipathic lipids, whose hydrophobic portion is in contact with the interior, hydrophobic region of the bilayer membrane, and whose polar head group is oriented toward the exterior, polar surface of the membrane. Vesicle-adopting lipids include lipids that are independently suitable for adopting a nonlamellar phase and can also adopt a bilayer structure in the presence of a bilayer-stabilizing component. Conjugated lipids that inhibit drug-lipid particle aggregation include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid derivatives), peptides, proteins, decontaminants, lipid derivatives, and PEG-lipid derivatives such as dialkyloxypropyl-conjugated PEG, diacylglycerol-conjugated PEG, phosphatidylethanolamine-conjugated PEG, and ceramide-conjugated PEG (see U.S. Pat. No. 5,885,613, incorporated herein by reference).

[0171] The term "amphipathic lipid" refers to any suitable lipid material in which the hydrophobic portion of the lipid material faces the hydrophobic phase and the hydrophilic portion faces the hydrophilic phase. Amphipathic lipids are typically the main component of lipid vesicles. Hydrophilic properties arise from the presence of polar or charged groups such as carbohydrates, phosphate (ester), carboxyl, sulfato, amino, sulfhydryl, 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 with one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphipathic 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, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine.Other compounds lacking phosphorus, such as sphingomyelin, glycosphingolipid family, diacylglycerol, and β-acyloxyacid, are also included in the group known as amphipathic lipids.In addition, the above amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.

[0172] The term "diacylglycerol" refers to a compound in which R1 and R2 each independently have a 2-fatty acyl chain of 2 to 30 carbon atoms attached to positions 1 and 2 of glycerol via an ester bond. The acyl group may be saturated or may have varying degrees of unsaturation. Diacylglycerol has the following formula: [ka]

[0173] In the term "diacylglycerol-conjugated polyethylene glycol," the lipid conjugated to inhibit particle aggregation in the present disclosure can be diacylglycerol-conjugated polyethylene glycol, i.e., diacylglycerol-polyethylene glycol conjugate (DAG-PEG conjugate or PEG-DAG conjugate). In a preferred embodiment, the DAG-PEG conjugate is a dilaurylglycerol (C12)-PEG conjugate, a dimyristylglycerol (C14)-PEG conjugate (DMG), a dipalmitoylglycerol (C16)-PEG conjugate, or a disterylglycerol (C18)-PEG conjugate (DSG). Those skilled in the art will readily understand that other diacylglycerols can be used in the DAG-PEG conjugates of the present disclosure. DAG-PEG conjugates suitable for use in the present disclosure, as well as methods for making and using them, are disclosed in U.S. Application No. 10 / 136,707, published as USPA 2003 / 0077829, and PCT Patent Application No. CA 02 / 00669, the entire contents of each of which are incorporated by reference.

[0174] The term "dialkyloxypropyl" refers to a compound in which R1 and R2 each independently have a 2-alkyl chain having 2 to 30 carbons. The alkyl groups may be saturated or have varying degrees of unsaturation. Dialkyloxypropyl has the following formula: [ka]

[0175] In the term "dialkyloxypropyl-conjugated PEG," the lipid of the conjugation that inhibits particle aggregation in the present disclosure can be a dialkyloxypropyl-conjugated PEG, i.e., a dialkyloxypropyl conjugate (PEG-DAA conjugate). In a preferred embodiment, the PEG-DAA conjugate has the following formula 62: [ka]

[0176] In Formula 62, R1 and R2 are independently selected long-chain alkyl groups having from about 10 to about 22 carbon atoms. The long-chain alkyl groups may be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, dodecyl (C12), tetradecyl (C14), hexadecyl (C16), octadecyl (C18), and icosyl (C20). In preferred embodiments, R1 and R2 are the same, i.e., both R1 and R2 are tetradecyl (i.e., ditetradecyl), both R1 and R2 are octadecyl (i.e., dioctadecyl), etc. In Formula 62, PEG is polyethylene glycol having an average molecular weight of from about 550 to about 10,000 daltons, 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 be optionally substituted with an alkyl, alkoxy, acyl, or aryl group. In Formula 62, L is a linker moiety. Any linker moiety suitable for conjugating PEG to a dialkyloxypropyl 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-), urethane (-NHC(O)O-), urea (-NHC(O)NH-), succinyl (-(O)CCHCHC(O)-), ether, disulfide, and combinations thereof. Other suitable linkers are well known in the art.

[0177] Phosphatidylethanolamines can be conjugated to polyethylene glycol to form bilayer-stabilizing components as conjugated lipids that inhibit particle aggregation in the present disclosure. The phosphatidylethanolamines have a variety of 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 with carbon chain lengths ranging from C10 to C20 are preferred. Also, such phosphatidylethanolamines containing monounsaturated or diunsaturated fatty acids, as well as mixtures of saturated and unsaturated fatty acids, can be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoylphosphatidylethanolamine (DSPE).

[0178] Similar to phosphatidylethanolamine, ceramide can be conjugated with polyethylene glycol to form a bilayer-stabilizing component as a conjugated lipid to inhibit particle aggregation in the present disclosure. The ceramide has multiple acyl chain groups of varying chain length and saturation. It should be apparent to those skilled in the art that, compared to phosphatidylethanolamine, ceramide has only one acyl group. The acyl group can be easily varied depending on its chain length and saturation. Ceramides suitable for application in accordance with the present disclosure are commercially available. Ceramides can be isolated from, for example, eggs and brains using well-known isolation 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 route proposed in the aforementioned application, ceramides with saturated or unsaturated fatty acids with carbon chain lengths ranging from C2 to C31 can be prepared.

[0179] The term "ATTA" or "polyamide" refers to, but is not limited to, compounds disclosed in U.S. Patent Nos. 6,320,017 and 6,586,559, both of which are incorporated herein by reference. These compounds include compounds having the following formula 63: [ka]

[0180] R is a member selected from the group consisting of hydrogen, an alkyl group, and an acyl group. R1 is a member selected from the group consisting of hydrogen and an alkyl group. Alternatively, optionally, R and R1 and the nitrogen atom to which they are attached form an azide group moiety. R2 is a member selected from the group consisting of hydrogen, an optionally substituted alkyl group, an optionally substituted aryl group, and an amino acid side chain. R3 is a member selected from the group consisting of hydrogen, halogen, hydroxyl, an alkoxy group, a mercapto group, a hydrazine group, an amino group, and NRR5, where R4 and R5 are independently hydrogen or an alkyl group, n is 4 to 80, m is 2 to 6, p is 1 to 4, and q is 0 or 1. It will be apparent to one skilled in the art that other polyamides may be used in the compounds of the present disclosure.

[0181] The term "congener" refers to an analog that fulfills the same or a similar function, or a derivative of the same parent nucleus that fulfills the same or a similar function.

[0182] As used herein, the terms "mRNA" or "messenger polyribonucleotide" or "messenger RNA" or "messenger RNA" may be used interchangeably and refer to a single-stranded polyribonucleotide that is transcribed using a single strand of DNA as a template, carries genetic information, and can direct protein synthesis.

[0183] As used herein, the terms "sgRNA" or "small guide RNA" or "guide RNA" or "gRNA" may be used interchangeably and refer to small non-coding RNAs that can pair with pre-mRNA and guide the insertion or deletion of uridine residues in kinetoplastids during RNA editing. gRNAs edit RNA molecules approximately 60-80 nucleotides in length that are transcribed by individual genes.

[0184] As used herein, the terms "circRNA" or "circular RNA" or "circular polyribonucleotide" or "circular RNA" may be used interchangeably and refer to a structure that has no free ends (i.e., no free 3' and / or 5' ends), e.g., a polyribonucleotide that forms a circular or ring structure by covalent or non-covalent bonds.

[0185] As used herein, the terms "microRNA" or "miRNA" or "microRNA" may be used interchangeably and refer to a non-coding, single-stranded polyribonucleotide that is approximately 22 nucleotides in length and has free 3' and 5' ends, which can inhibit the translation of target gene proteins by binding to the mRNA 3'-untranslated region (3'-UTR) of a target gene, thereby regulating the biological functions of cells.

[0186] As used herein, the terms "ASO" or "antisense oligonucleotide" or "antisense oligonucleotide" may be used interchangeably and refer to an artificially synthesized nucleic acid fragment complementary to a segment of a target gene or mRNA, and is a single-stranded poly(deoxy)ribonucleotide that can bind to a target gene / mRNA through the principle of base complementarity, thereby blocking gene expression, and includes antisense DNA and antisense RNA.

[0187] 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" may be used interchangeably and refer to a type of double-stranded RNA molecule that is 20 to 25 nucleotides in length and can induce degradation of target gene mRNA.

[0188] As used herein, the terms "ecDNA" or "extrachromosomal circular DNA" may be used interchangeably and refer to DNA that has been shed from a chromosome and exists in a circular structure away from the chromosome.

[0189] The term "nucleic acid derivative" refers to modifications or substitutions of nucleic acid sequences, including, but not limited to, chemical modifications of residues, nucleotide or deoxynucleotide substitutions, sequence modifications to enhance half-life or stability, and marking modifications. For example, chemical modifications include, but are not limited to, phosphorylation, methylation, amination, thiolation, sulfur substitution for oxygen, selenium substitution for oxygen, or isotope substitution of any one or more bases. Nucleotide or deoxynucleotide substitutions include, but are not limited to, nucleic acid analogs in which the sugar-phosphate backbone is replaced with a polypeptide or other backbone (e.g., replacing DNA or RNA with PNA (peptide nucleic acids)). Sequence modifications to enhance half-life or stability include, but are not limited to, PEG conjugation and fluorine modification. Marking modifications include, but are not limited to, the attachment of fluorescent groups, amino groups, biotin, digoxin, small peptides, etc.

[0190] The term "artificial nucleic acid" refers to an artificially modified nucleic acid molecule, including but not limited to base modifications, ribose modifications, PNA, etc.

[0191] The term "nucleic acid" refers to a polymer containing at least two deoxynucleotides or nucleotides, present in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. 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 with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). A "nucleotide" includes a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are attached via the phosphate group. "Bases" include purines and pyrimidines, as well as the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that substitute new reactive groups. Such reactive groups include, but are not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. DNA can exist as antisense, plasmid DNA, portions of plasmid DNA, pre-packaged DNA, polymerase chain reaction (PCR) products, carriers (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. The term nucleic acid can be used interchangeably to refer to genes, cDNAs, mRNAs encoded by genes, and interfering RNA molecules.

[0192] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence comprising a partial or full-length coding sequence necessary for the production of a polypeptide or polypeptide precursor (e.g., a polypeptide or polypeptide precursor from hepatitis A, B, C, D, E, G virus, or herpes simplex virus).

[0193] As used herein, "gene product" refers to a gene product, including, for example, a DNA transcript, mRNA, and the like.

[0194] The phrase "silencing expression of a target gene" refers to the ability of the disclosed siRNA to initiate silencing of a target gene. To determine the degree of gene silencing, a sample or measurement of cells in a target organism or culture expressing a particular construct is compared to a control sample not expressing the construct. The control sample (lacking expression of the construct) is set to a relative value of 100%. Successful inhibition of target gene expression is achieved when the test value relative to the control is approximately 90%, preferably 50%, and more preferably 25-0%. Suitable measurements include, for example, detection of protein or mRNA levels using techniques known to those skilled in the art, such as dotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzymatic action, and phenotypic measurements known to those skilled in the art.

[0195] A "therapeutically effective amount" or "effective amount" of an siRNA is an amount sufficient to produce a desired effect, such as a decrease in target sequence expression compared to normal expression levels detected in the absence of the siRNA.

[0196] As used herein, the term "aqueous solution" refers to a composition that comprises, in whole or in part, water.

[0197] As used herein, the term "organic lipid solution" refers to a composition comprising an organic solvent, in whole or in part, with a lipid.

[0198] As used herein, "systemic delivery" refers to delivery that results in widespread biodistribution of a compound within an organism. Some administration techniques can result in systemic delivery of some compounds but not others. Systemic delivery means that an effective, preferably therapeutic, amount of a compound comes into contact with most of the body. Achieving widespread biodistribution typically requires a blood survival period to prevent the compound from being rapidly degraded or eliminated (e.g., by first-pass organ (e.g., liver, lung) or rapid nonspecific cellular binding) before reaching disease sites distal to the administration site. Systemic delivery of drug-lipid particles can be achieved by any method known in the art, including, for example, intravenous, subcutaneous, or intraperitoneal delivery; in a preferred embodiment, the systemic delivery of drug-lipid particles is intravenous.

[0199] As used herein, "local delivery" refers to the direct delivery of a compound to a target site within an organism. For example, a compound can be delivered locally by direct injection into a disease site such as a tumor, or other target site such as a site of inflammation, or into a target organ such as the liver, heart, pancreas, or kidney.

[0200] The term "phospholipid" refers to a lipid containing a phosphate group. It belongs to the complex lipid family and is also called phospholipids or phospholipids. Phospholipids are the main components of biological membranes and are broadly classified into two categories: lysophosphatidylglycerol and sphingomyelin, each composed of glycerol and sphingosine. Phospholipids are amphipathic molecules with a hydrophilic nitrogen- or phosphorus-containing head group at one end and a hydrophobic (lipophilic) long alkyl chain at the other end. Therefore, the hydrophilic and hydrophobic ends of phospholipid molecules are close to each other, forming a phospholipid bilayer with other molecules such as proteins, glycolipids, and cholesterol, which forms the structure of cell membranes.

[0201] In this disclosure, the polyphenol moiety in the metal-polyphenol complex 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 that can regulate the activity of various immune cells, such as T cells, B cells, macrophages, neutrophil granulocytes, natural killer cells, and dendritic cells, promoting immune balance and enhancing the immune system. Based on the potential immune-enhancing, anti-inflammatory, antioxidant, and anti-SARS-CoV-2 effects of curcumin, it is expected to be a potential adjunct treatment for COVID-19. Furthermore, curcumin has an extremely high level of safety and is listed in the catalogs of food additives and pharmaceutical excipients. Its safety contributes to the clinical drug registration of the entire drug-lipid complex, shortening the clinical drug registration period.

[0202] In the present disclosure, the coordinate bond between the polyphenol molecule portion and the metal ion portion in the metal-polyphenol complex is broken under the low pH condition (pH=5.0) of the lysosome, for example, and the metal ion is released from the metal-polyphenol complex.

[0203] In the present disclosure, the ratio of each component in the metal-polyphenol complex can be adjusted depending on the structure of the specific metal-polyphenol complex component. The criteria for adjusting the ratio are as follows: Because the hydroxyl groups of polyphenol molecules are bound to metal ions by coordinate bonds, as long as there are multiple binding sites on a polyphenol molecule, the ratio of polyphenol molecules to metal ions can be adjusted based on the number of binding sites contained in the polyphenol molecule.

[0204] Metal-chelated polyphenol complex nanoparticles (MPNPs)

[0205] The principle of loading nucleic acids into metal-polyphenol complex particles assembled by metal-polyphenol complexes is that polyphenol molecules are linked to metal ions through coordination bonds to form metal-polyphenol complexes, and the metal ions of the metal-polyphenol complexes are linked to nucleic acids through coordination bonds, thereby ensuring that the metal-polyphenol complexes self-assemble with other components into MPNPs and the nucleic acids are loaded onto the nanoparticles.

[0206] As used herein, the term "non-cationic or non-ionizable lipid other than the lipid of the conjugation that inhibits particle aggregation" refers to component (iii) in the metal-polyphenol complex particles.

[0207] In some embodiments, the conjugated lipid that inhibits particle aggregation refers to the conjugated lipid that inhibits drug-lipid particle aggregation. Its main function is to prevent the aggregation of drug-lipid particles, such as PEG conjugated with dialkyloxypropyl, PEG conjugated with diacylglycerol, PEG conjugated with phosphatidylethanolamine, and PEG linked with ceramide, preferably PEG-lipid conjugates. The cis-trans isomers of lipids do not affect the effect achieved by the protected subject matter of the present disclosure.

[0208] In some embodiments, the molar fraction of the metal-polyphenol complex in the raw material is 5% to 30%, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, preferably 5% or more but less than 10%, 10% to 15%, or 15% to 20%, and more preferably 5%, 10%, or 15%.

[0209] In some embodiments, the molar fraction of conjugated lipids that inhibit particle aggregation in the feedstock is 2% to 10%, e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably 3% to 5% or 5% to 10%, more preferably 3%, 5%, or 10%.

[0210] In some embodiments, the non-cationic or non-ionizable lipid optionally contains cholesterol, and the mole fraction of cholesterol in the feedstock is between 0% and 48%, e.g., 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 1 3%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47% or 48%, preferably 10% to 30%, 30% to 47% or 10% to 20%, and more preferably 10%, 30% or 47%.

[0211] In some embodiments, the metal-polyphenol complex particles further contain, in addition to cholesterol, other non-cationic or non-ionizable lipids, whose molar fraction in the feedstock is 30% to 75%, for example, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%. The ratio is preferably 45% to 55%, 60% to 65%, or 50% to 65%, and more preferably 45%, 55%, 60%, or 65%.

[0212] Drug-lipid particles

[0213] The drug-lipid particles described herein typically contain a drug (a negatively charged molecule that may be selected from the group consisting of one or more combinations of nucleic acids, proteins, polypeptides, small molecules, nucleic acid analogs, protein analogs, and polypeptide analogs; the nucleic acid may be selected from the group consisting of one or more combinations of mRNA, siRNA, circular RNA, microRNA, sgRNA, DNA, ecDNA, and artificial nucleic acids), a metal-polyphenol complex, a non-cationic or non-ionizable lipid, and a bilayer-stabilizing component, such as a conjugated lipid that inhibits particle aggregation. The nucleic acids encapsulated in the drug-lipid particles of the present disclosure are resistant to degradation by nucleases in aqueous solution.

[0214] In some embodiments, the drug is sufficiently encapsulated within the metal-polyphenol complex particles, thereby avoiding drug degradation and achieving drug delivery to cells.

[0215] In some embodiments, the drug-lipid particles provided by the present disclosure have a small diameter suitable for systemic delivery, with a particle size of 30 to 400 nm, a surface potential of -10 to 10 mV, a stability of at least 3 days, preferably 7 days or more, and a cellular delivery efficiency of at least 40%, e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0216] In some embodiments, the drug of the drug-lipid particle is preferably a nucleic acid, and the nucleic acid component typically includes mRNA, interfering RNA (i.e., siRNA), which can be provided in several forms, including, for example, one or more isolated small interfering RNA (siRNA) duplexes, longer double-stranded RNA (dsRNA), or siRNA or dsRNA translated from a transcription cassette in a DNA plasmid.

[0217] A population of RNA can be used to provide long precursor RNAs, or long precursor RNAs with basic or complete identity to a selected target sequence, which can be used to produce siRNA. The RNAs can be isolated from cells or tissues, synthesized, and / or cloned according to methods well known to those of skill in the art. The RNA can be a mixed population (obtained from cells or tissues, transcribed from cDNA, etc.) or can represent a single target sequence. The RNA can be naturally occurring, e.g., isolated from a tissue or cell sample, synthesized in vitro using, e.g., T7 or SP6 polymerase and PCR products or cloned cDNA, or synthesized by chemical methods.

[0218] To form long dsRNA, in the case of synthetic RNAs, complements can be transcribed in vitro and hybridized to form dsRNA.When a naturally occurring RNA population is used, for example, by transcribing the cDNA corresponding to the RNA population or by using RNA polymerase, RNA complements (for example, by E. coli RNAse III or enzyme cleavage to form digested dsRNA) can be further provided.Precursor RNAs are then hybridized to form double-stranded RNAs, which are then digested.The dsRNAs can be directly encapsulated in SNALPs or can be digested in vitro before being encapsulated.

[0219] Alternatively, one or more DNA plasmids encoding one or more siRNA templates can be encapsulated in the nucleic acid-lipid particles. For example, based on the naturally occurring transcription units of the small nuclear RNA U6 or human RNase P RNA H1, siRNA can be transcribed from a DNA template in a plasmid into a sequence that automatically folds into a double-stranded form with a hairpin loop, and the plasmid contains an RNA polymerase III transcription unit (Brummelkamp et al., Science 296:550 (2002); Donze et al., Nucleic Acids Res. 30:e46 (2002); Paddison et al., Genes 2002). Dev. (Genetics Dev.) 16:948 (2002); Yu et al., Proc. Natl. Acad. Sci. (Proceedings of the National Academy of Sciences) 99:6047 (2002); Lee et al., Nat. Biotech. (National Biotechnology) 20:500 (2002); Miyagishi et al., Nat. Biotech. (National Biotechnology) 20:497 (2002); Paul et al., Nat. Biotech. (National Biotechnology) 20:505 (2002); and Sui et al., Proc. Natl. Acad. Sci. (Proceedings of the National Academy of Sciences) 99:5515 (2002). Typically, a transcription unit or cassette includes an RNA transcription promoter sequence, such as an H1-RNA or U6 promoter, and a termination sequence. The promoter sequence is operably linked to a template for transcribing the desired siRNA sequence, and the termination sequence includes 2-3 uridine residues and a polythymidine (T5) sequence (polyadenylation signal) (Brummelkamp, Science, supra). The selected promoter can provide for constitutive or inducible transcription. Compositions and methods for transcribing DNA-guided RNA interference molecules are described in detail in U.S. Pat. No. 6,573,099, incorporated herein by reference. Preferably, the synthetic or transcribed siRNA has a 3' overhang of about 1-4 nucleotides, preferably about 2-3 nucleotides, and a 5' phosphate terminus (Elbashir et al., Genes Dev. 15:188 (2001)). TIFF2025527207000019.tif929 et al., Cell 107:309 (2001). The transcription unit is attached to a plasmid or DNA carrier, and the interfering RNA is transcribed from the plasmid or DNA vector. Plasmids suitable for delivering genetic material into the body 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. The selected plasmid can provide instantaneous or stable delivery to target cells. It will be apparent to those skilled in the art that a plasmid originally designed to express a desired gene sequence can be modified to contain a transcription unit cassette that transcribes siRNA.

[0220] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, preparing and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see U.S. Pat. Nos. 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 of skill in the art. Additional basic books that disclose the general methods used in this disclosure 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).

[0221] Applications of metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles

[0222] In some embodiments, when the drug is a nucleic acid, the metal-polyphenol complex and metal-polyphenol complex particles can both be used to promote lysosomal escape of the drug and promote nucleic acid expression. The metal-polyphenol complex and metal-polyphenol complex particles can also be used to deliver the drug and introduce the drug into cells, thereby achieving the prevention and treatment of the corresponding disease or condition.

[0223] In some embodiments, the present disclosure provides applications of metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles in compositions capable of, for example, drug delivery or drug introduction into cells. The compositions are, for example, pharmaceuticals capable of silencing the expression of a target sequence in a mammalian subject, delivering drugs (e.g., drugs for treating tumors, imaging agents, etc.) into the mammalian body, delivering drugs from the body to mammalian cells, or treating mammalian diseases or conditions. In pharmaceuticals, the drug-lipid particles are the main active ingredient. Depending on actual needs, different dosage forms, such as solid dosage forms (powders, granules, pills, tablets, gels), semisolid dosage forms (external ointments, pastes), liquid dosage forms (decoctions, mixtures, syrups, medicinal liquors, injections), and gaseous dosage forms (aerosols, smoke) can be prepared using different pharmaceutically acceptable excipients or manufacturing processes. For example, examples include dosage forms for gastrointestinal administration, dosage forms for rectal administration, and dosage forms for parenteral administration.

[0224] In some embodiments, the present disclosure provides products made from the above-described metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles. The products have the above-described functions and uses of the metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles. Specific examples include, but are not limited to, kits, pharmaceuticals, etc. The products may optionally further contain other excipients.

[0225] Regarding target genes for drug-lipid particle action, it is generally desirable to deliver the drug-lipid particles so that the translation (i.e., expression) of the target gene product is downregulated or silenced. Suitable classes 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 targeting the liver, and liver diseases and conditions), genes associated with tumorigenesis and cell transformation, angiogenesis genes, immunomodulator genes such as those associated with inflammation and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative diseases.

[0226] Genes associated with viral infection and survival include those expressed by viruses to bind to, enter, and replicate within cells, 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); FIELDS VIROLOGY (Knipe et al., eds., 2001)), human immunodeficiency virus (HIV) (Banerjea et al., Mol Ther. (Journal of Molecular Therapy) 8:62 (2003); Song et al., J. Virol. (Journal of Virology) 77:7174 (2003); Stephenson JAMA (Stephenson JAMA) 289:1494 (2003); Qin et al., Proc. Natl. Acad. Sci. (Proceedings of the National Academy of Sciences of the United States of America) 100:183 (2003)), herpesviruses (Jia et al., J. Virol. (Journal of Virology) 77:3301 (2003)), and human papillomavirus (HPV) (Hall et al., J. Virol. (Journal of Virology) 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, e.g., FIELDS VIROLOGY, 2001, supra).Hepatitis C nucleic acid sequences that can be silenced include, but are not limited to, serine proteases (e.g., NS3 / NS), helicases (e.g., NS3), polymerases (e.g., NS5B), and envelope proteins (e.g., E1, E2, and p7). Hepatitis A nucleic acid sequences are, for example, referenced in Genbank accession number NC_001489, hepatitis B nucleic acid sequences are, for example, referenced in Genbank accession number NC_003977, hepatitis C nucleic acid sequences are, for example, referenced in Genbank accession number NC_004102, hepatitis D nucleic acid sequences are, for example, referenced in Genbank accession number NC_001653, hepatitis E nucleic acid sequences are, for example, referenced in Genbank accession number NC_001434, and hepatitis G nucleic acid sequences are, for example, referenced in Genbank accession number NC_001710. Silencing of sequences encoding genes associated with viral infection and survival can be advantageously used in combination with the administration of conventional drugs used to treat viral diseases.

[0227] Genes associated with metabolic diseases and conditions (e.g., diseases targeting the liver and diseases and conditions of the liver) include, for example, genes expressed in dyslipidemia (e.g., liver X receptors (e.g., LXRα and LXRβ Genbank Accession No. NM_007121)), farnesoid X receptor (FXR) (Genbank Accession No. NM_005123), sterol regulatory element-binding protein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG coenzyme A reductase), apolipoprotein (ApoB), apolipoprotein (ApoE), and diabetes (e.g., glucose-6-phosphate) (e.g., Forman et al., Cell 81:687 (1995); Seol et al., Mol. Endocrinol. 9:72 (1995); Zavacki et al., PNAS USA 94:7909(1997), Sakai et al., Cell 85:1037-1046(1996), Duncan et al., J. Biol. Chem. 272:12778-12785(1997), Willy et al., Genes Dev. 9(9):1033-45(1995), Lehmann et al., J. Biol. Chem. 272(6):3137-3140(1997), Janowski et al., Nature 383:728-731 (1999; see 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 targeted at the liver, and liver diseases and conditions) include genes expressed in the liver itself as well as genes expressed in other organs and tissues. Silencing of sequences encoding genes associated with metabolic diseases and conditions can be advantageously used in combination with the administration of conventional drugs used to treat the disease or condition.

[0228] Examples of genes involved in tumorigenesis and cell transformation include MLL fusion genes, translocation sequences such as 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)), cyclins (Li et al., Cancer Res. 63:3593 (2003); Zou et al., Genes Dev. (Genetics Development) 16:2923 (2002)), beta-catenin (Verma et al., Clin Cancer Res. (Clinical Cancer Research) 9:1291 (2003)), the telomerase gene Kosciolek et al., Mol Cancer Ther. (Molecular Cancer Ther.) 2:209 (2003)), c-MYC, N-MYC, BCL-2, ERBB1, and ERBB2 (Nagy et al., Exp. Cell Res. (Experimental Cell Research) 285:39 (2003)), and mutated sequences such as RAS (reviewed in Tuschl and Borkhardt, Mol. Interventions (Molecular Interventions) 2:158 (2002)). Silencing of sequences encoding DNA repair enzymes is used in combination with the administration of chemotherapeutic agents (Collis et al., Cancer Res. 63:1550 (2003)). Genes encoding proteins associated with tumor migration, such as integrins, selectins, metalloproteases, etc., are also suitable target sequences. Any complete or partial gene sequence that favors or promotes tumorigenesis or cell transformation, tumor growth, or tumor migration may be included as a template sequence.

[0229] Angiogenic genes can promote the formation of new blood vessels. Vascular endothelial growth factor (VEGF) is an important research direction (Reich et al., Mol. Vis. 9:210 (2003)).

[0230] Immunomodulatory genes are genes that regulate one or more immune responses. Examples of immunomodulatory 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 immunomodulatory target sequences (Song et al., Nat. Med. 9:347 (2003)). Genes encoding secondary signaling molecules in hematopoietic and lymphoid cells, for example, Tec family kinases such as Bruton's tyrosine kinase (Btk), are also included in the present disclosure (Heinonen et al., FEBS Lett. 527:274 (2002)).

[0231] Cell receptor ligands include ligands that bind to cell surface receptors (e.g., insulin receptors, EPO receptors, G protein-coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) and regulate (e.g., inhibit, activate, etc.) physiological pathways in which the receptor is involved (e.g., glucose level regulation, blood cell development, mitogenesis, etc.). Examples of cell receptor ligands include cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G protein-coupled receptor ligands, etc. Templates encoding trinucleotide repeat (e.g., CAG repeat) expansions have been found to be useful for silencing pathogenic sequences in neurodegenerative diseases, such as spinal-bulbar muscular atrophy and Huntington's disease (Caplen et al., Hum. Mol. Genet. (Human Molecular Genetics) 11:175 (2002)).

[0232] Injectable delivery, as described in U.S. Patent Nos. 5,543,158, 5,641,515, and 5,399,363, is desirable in some cases, including parenteral, intravenous, intramuscular, subcutaneous, intradermal, or intraperitoneal delivery of the drug-lipid particles disclosed herein. The drug-lipid particles can be injected locally at a target site (e.g., a disease site, such as inflammation or tumor formation, or a target organ or tissue) or systemically for widespread distribution within the organism. Solutions of the drug-lipid particles can be prepared in water, appropriately mixed with a surfactant. Dispersions can be prepared in glycerin, liquid polyethylene glycol, and mixtures thereof, and oils. Optionally, these formulations contain a preservative to prevent microbial growth. Typically, for intravenous administration, the drug-lipid particle formulation is formulated with a suitable pharmaceutical carrier. Buffered saline (135-150 mM NaCl) is commonly used as a pharmaceutical carrier, although other suitable carriers will suffice. Additional suitable carriers are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The term "medicinal" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans. The formulation of aqueous compositions containing proteins as active ingredients is conventionally understood in the art. Alternatively, the compositions may be prepared as injectables, liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection. Additionally, the preparations may be emulsified.

[0233] The drug-lipid particles can be sterilized by conventional liposome sterilization techniques such as filtration. The drug-lipid particles can contain pharmaceutical auxiliary substances that create appropriate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and wetting agents. These compositions can be sterilized using the techniques described above, or alternatively, can be produced under aseptic conditions. The resulting aqueous solution can be packaged for use, or filtered and lyophilized under sterile conditions, and the lyophilized preparation can be combined with a sterile aqueous solution before administration.

[0234] Prophylactic and Therapeutic Treatment: In some embodiments, drug-lipid particles can be used for the prophylactic 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 drug-lipid particles are administered to the subject in an amount sufficient to elicit a therapeutic response in the patient. An amount sufficient to achieve this is defined as a "therapeutically effective dose or amount" or "effective dose or amount." When determining the effective amount of drug-lipid particles to be administered to treat or prevent a disease caused by the expression or overexpression of a target gene, a physician will evaluate the circulating plasma levels of the drug-lipid particles, the toxicity of the drug-lipid particles, and the progression of the disease associated with the expression or overexpression of the target gene. Administration can be accomplished by single or divided dose administration.

[0235] For example, the drug-lipid particles can be administered to subjects infected with or at risk of infection by pathogenic microorganisms. The drug preferably corresponds to a sequence that plays an important role in the life cycle of the microorganism and should be unique to the microorganism (or at least absent from the native genome of the patient being treated). The drug-lipid particles are introduced into target cells, tissues, or organs in a therapeutically effective dose by ex vivo or intravenous injection. Silencing of sequences encoding genes associated with pathogenic infections can be advantageously used in combination with the administration of conventional reagents used to treat pathogenic diseases. The treatment can be administered prophylactically to individuals at risk of infection with pathogenic microorganisms or those already infected with pathogenic microorganisms.

[0236] In a preferred embodiment, the drug-lipid particles of the present disclosure can be advantageously used to treat cancer, viral infections, autoimmune diseases, diabetes, and Alzheimer's disease. Viral infections include hepatitis A, hepatitis B, hepatitis C, SARS-CoV-2, HIV, HPV, influenza, smallpox, and syphilis. For example, suitable sites for inhibiting hepatitis B virus include nucleic acid sequences encoding the S, C, P, and X proteins, PRE, EnI, and EnII (see, e.g., FIELDSVIROLOGY, 2001, supra). Those skilled in the art will appreciate that gene silencing associated with hepatitis infection may be achieved by conventional treatments for hepatitis, such as immunoglobulins, interferons (e.g., PEGylated and non-PEGylated interferon alpha) (see, e.g., Medina et al., Antiviral Res. 60(2):135-143 (2003)), ribavirin (see, e.g., Hugle and Cerny, Rev. Med. Virol. 13(6):361-71 (2003)), adefovir and lamivudine (see, e.g., Kock et al., Hepatology 38(6):1410-8 (2003)), prenylation inhibitors (see, e.g., Bordier et al., J. Clin. Invest. 112(3):407-414 (2003)), famciclovir (see, e.g., Yurdaydin et al., J. Hepatol. 37(2):266-71 (2002)), and saikosaponins c and d (see, e.g., Chiang et al., Planta Med. 69(8):705-9 (2003)).

[0237] In another embodiment, the drug-lipid particles of the present disclosure can be advantageously used to treat diseases and conditions characterized by the expression or overexpression of a gene or a group of genes. In some aspects, the drug-lipid particles of the present disclosure can be used to treat metabolic diseases and conditions (e.g., liver-targeted diseases and conditions, and liver diseases and conditions), such as dyslipidemia and diabetes. Those skilled in the art will understand that silencing genes associated with metabolic diseases and conditions can be combined with conventional treatments for these diseases.For example, silencing of genes involved in dyslipidemia has been demonstrated with statins, bile acid sequestrants / resins, cholesterol absorption inhibitors such as ezetimibe, phytostanols / sterols, polyphenols, dietary supplements such as oat bran, flaxseed, and soy protein, phytostanol analogs, squalene synthase inhibitors, bile acid transport inhibitors, SREBP cleavage activating protein (SCAP) activating ligands, niacin (nicotinic acid), acipimox, high-dose fish oil, antioxidants, and sugarcane fatty alcohols, microsomal triacylglycerol transporter (MTP) inhibitors, fatty acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors, gemcabene, lifevero, pantothenic acid analogs, niacin receptor agonists, anti-inflammatory agents (such as Lp-PLA(2) antagonists and AGI1067), and functional oils. , PPAR-α, gamma, delta agonists, dual PPAR-α, gamma, and "pan" PPAR-α / γ / δ agonists, cholesteryl ester transfer protein (CETP) inhibitors (such as torcetrapib), CETP vaccines, upregulators of ATP-binding cassette transport protein (ABC) A1, lecithin cholesterol acyltransferase (LCAT) and scavenger receptor class B type 1 (SRB1), and synthetic apolipoprotein (Apo) E-related peptides, sustained-release niacin / lovastatin, atorvastatin / amlodipine, ezetimibe / simvastatin, atorvastatin / CETP inhibitors, statins / PPAR agonists, developmental delayed-release niacin / simvastatin and pravastatin / aspirin, and anti-obesity agents (e.g., Bays and Stein, Expert See Opin. Pharmacother. 4(11):1901-38 (2003). Similarly, silencing of genes involved in diabetes can be combined with insulin treatment and dietary modification and exercise.

[0238] Similar methods are used to inhibit the expression of endogenous receptor cell genes associated with tumorigenesis and cell transformation, tumor growth and tumor migration, to inhibit the expression of angiogenic genes, to inhibit the expression of genes associated with inflammation and autoimmune responses, to inhibit the expression of ligand receptor genes, to inhibit the expression of genes associated with neurodegenerative diseases, and to inhibit the expression of additional genes associated with viral infection and survival. Specific targeted gene sequences are described above.

[0239] The drug-lipid particles of the present invention can be detected by any method known in the art. For example, a label can be directly or indirectly conjugated to a component of the drug-lipid particle or other lipid-based carrier system using a method well known in the art. A wide variety of labels can be used, selected based on the required sensitivity, ease of conjugation to the drug-lipid particle component, stability requirements, tool availability, processing readiness, etc. Suitable labels include, but are not limited to, spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives such as Texas Red, tetramethylrhodamine isothiocyanate (TRITC), digitoxin, biotin, phycoerythrin, AMCA, CyDyes™, radioactive labels such as 3H, 125I, 35S, 14C, 32P, 33P, enzymes such as horseradish peroxidase, alkaline phosphatase, and spectrocolorimetric labels such as colloidal gold, colored glass, plastic beads such as polystyrene, polypropylene, latex, etc.). Labels are detected using any means known in the art.

[0240] Nucleic acids herein can be detected and quantified by any of a number of means well known to those skilled in the art. Nucleic acids can be detected by methods well known in the art, such as Southern blot analysis, Northern blot analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Additional analytical biochemical techniques 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.

[0241] The sensitivity of hybridization assays can be improved by applying a nucleic acid width system that doubles the amount of target nucleic acid detected. In vitro amplification techniques are known that are suitable for amplifying sequences to be used as molecular probes or for generating nucleic acid fragments for subsequent subcloning. Examples of in vitro amplification methods sufficient to guide one of skill in the art, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ replicase amplification, and other RNA polymerase-mediated techniques (e.g., NASBA™), include Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2000; Ausubel et al., SHORT PROTOCOLS IN MOLECULAR BIOLOGY (eds.), Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (2002); Mullis et al., SHORT PROTOCOLS IN MOLECULAR BIOLOGY (eds.), Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (2002); et al., (1987), U.S. Patent No. 4,683,202, A Guide to PCR Protocol 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 (Proceedings of the National Academy of Sciences), 87:1874 (1990), Lomell et al., J. Clin. Chem.(Journal of the Japanese Society of Clinical Chemistry, 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., Gene, 89:117 (1990); Sooknanan and Malek, Biotechnology, 13:563 (1995). Improved methods for in vitro amplification and cloning of nucleic acids are described in U.S. Patent No. 5,426,039 to Wallace et al. Other methods described in the art are nucleic acid sequence-based amplification (NASBA™, Cangene, Mississauga, Ontario) and the Qβ replicase system.

[0242] As described in Needham VanDevanter et al., Nucleic Acids Res., 12:6159 (1984), this typically follows the solid-phase phosphoramidite method described by Beaucage and Caruthers, Tetrahedron Letts., 22(20):1859-1862 (1981). For example, automated synthesizers are used to chemically synthesize oligonucleotides for use as probes in in vitro amplification procedures, gene probes, or inhibitor components. If necessary, oligonucleotides are typically purified by native acrylamide gel electrophoresis or anion-exchange HPLC, as described in Pearson and Regnier, J. Chrom., 255:137-149 (1983). The sequence of synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.), Academic Press, New York, Methods in Enzymology, 65:499.

[0243] The following examples are provided to illustrate, but not to limit, the claimed disclosure. Those of skill in the art will readily identify a variety of non-critical parameters that could yield substantially similar results.

[0244] The drug-lipid particles sought to be protected in this disclosure refer to drug-lipid particles other than those containing cationic / ionizable lipids, i.e., drug-loaded metal-chelated polyphenol complex nanoparticles (drug@MPNP).

[0245] Experimental Example 1: Preparation of drug-metal-polyphenol complex particles (mRNA-loaded metal-chelated polyphenol complex nanoparticles, mRNA@MPN)

[0246] Example 1 Preparation of Metal-chelated Polyphenol Complex Nanoparticles (MPNPs)

[0247] Example 1.1 Metal ion is Fe 3+ Preparation of metal-polyphenol complexes when

[0248] Curcumin (formula 1) is dissolved in ethanol at 1.5 mg / ml, and anhydrous FeCl3 is added. The molar ratio of curcumin (formula 1) to anhydrous FeCl3 is 1:1, and the reaction is refluxed at 60°C for 1 hour. After the reaction is complete, the solution is suspended and dried, and the product is dissolved in ultrapure water and lyophilized. The resulting product is a metal-polyphenol complex. The structure of the metal-polyphenol complex is shown below. [ka]

[0249] The results showed that curcumin (Formula 1) and FeCl3 were reacted at 60°C for 1 hour, the input concentration of curcumin (Formula 1) was 1.5 mg / mL, and the input ratio of curcumin (Formula 1) to FeCl3 was 1:1. The yield of the target product was 95%.

[0250] Example 1.2 Metal ion is Al 3+ Preparation of metal-polyphenol complexes when

[0251] The difference between this example and Example 1.1 is that FeCl3 is replaced with Al(NO3)3·9H2O. The structure of the resulting metal-polyphenol complex is as follows: [ka]

[0252] Results analysis: Curcumin (formula 1) and Al(NO3)3·9H2O were reacted at 60°C for 1 hour. The input concentration of curcumin (formula 1) was 1.5 mg / mL, and the input ratio of curcumin (formula 1) to Al(NO3)3·9H2O was 1:1. The yield of the target product was 98%.

[0253] Example 2: Preparation of mRNA-loaded metal-chelated polyphenol complex nanoparticles (mRNA@MPNP)

[0254] Metal ion is Fe 3+ Preparation of mRNA-metal-polyphenol complex particles when

[0255] Metal-polyphenol complexes were prepared according to the method described in Example 1.1. Curcumin (Formula 1) and FeCl3 were added in a 1:1 ratio. The metal-polyphenol complexes, distearoylphosphatidylcholine (DSPC, Formula 51, as a non-cationic or non-ionizable lipid), cholesterol (CHOL, Formula 59, as a non-cationic or non-ionizable lipid), and DSPE-PEG2000 (Formula 58, as a conjugation lipid to inhibit particle aggregation) were dissolved in ethanol at different molar fractions to form an organic phase. The proportions of the metal-polyphenol complex, DSPC (Formula 51), CHOL (Formula 59), and DSPE-PEG2000 (Formula 58) were 5%, 60%, 30%, and 5%, respectively. mRNA was dissolved in enzyme-free Tris-HCl buffer (0.1 M molarity) at pH 5.0 at a concentration of 20 μg / mL to form an aqueous phase. The metal-polyphenol complex and mRNA were mixed in a microfluidic chip at a mass ratio of 20:1. The volume ratio of the aqueous phase to the organic phase was 3:1. The flow rate of the organic and aqueous phases in the microfluidic chip was 12 ml / min. The drug mRNA was mRNA encoding the fluorescent protein eGFP, and its sequence is SEQ ID NO. 1 (720 nt). eGFP-mRNA@MPNPs were produced and obtained. 293T cells were incubated with eGFP-mRNA@MPNPs at a concentration of 2 μg / mL (mRNA concentration). A control group was incubated with MPNPs without drug loading. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0256] The particle size, surface potential, and stability of the prepared eGFP-mRNA@MPNPs were detected, and the efficiency of eGFP-mRNA@MPNPs encapsulating nucleic acids was calculated.

[0257] The particle size detection method and criteria for the results are as follows: the particle size of nanoparticles is tested using a Malvern laser particle size analyzer, Zetasizer, and if the particle size is in the range of 30-400 nm, it is considered acceptable.

[0258] The method for detecting surface potential and the criteria for judging the results are as follows: the surface potential of nanoparticles is measured using a Malvern Zetasizer laser particle size analyzer; if the potential is in the range of -10 to 10 mV, it is considered acceptable.

[0259] The method for detecting stability and the criteria for judging the results are as follows: leave the nanoparticles at 4°C for 7 days, and measure the particle size and surface potential of the nanoparticles using a Malvern Zetasizer laser particle size analyzer; if the particle size and surface potential do not change significantly within 3 to 7 days, the stability is considered to be relatively good.

[0260] The nucleic acid encapsulation efficiency was calculated using agarose gel electrophoresis. The nucleic acid loading for each group of lipid nanoparticles was 10 μg / mL. The mass ratio of the metal-polyphenol complex to nucleic acid was 20:1. An equal concentration of nucleic acid was dissolved in enzyme-free Tris-HCl buffer. This served as the positive control, while enzyme-free Tris-HCl buffer served as the negative control. The agarose gel concentration was 1.5%, and the gaps in the gel allowed only free nucleic acid to pass through, while the lipid nanoparticles were blocked. Electrophoresis was stopped when the free nucleic acid bands became clearly distinguishable. Image J software was used to calculate the gray values of free nucleic acid for each group. The positive control group was set to 100%, and the ratio of free nucleic acid in each group to the positive control was the relative amount of free nucleic acid. The encapsulation rate for each group was calculated as (100 - relative amount of free nucleic acid)%. A nucleic acid encapsulation rate of 50% or higher was considered acceptable.

[0261] The cell culture method involved culturing the human embryonic kidney cell line 293T in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0262] Analysis of the percentage of eGFP-positive cells by flow cytometry was performed by seeding 5 x 10 293T cells in a 12-well plate. 5Cells were seeded at a density of 100 cells / well. When the cell density reached 80%, 1 mL of MPNPs or eGFP-mRNA@MPNPs was added and incubated. The concentration of eGFP-mRNA@MPNPs was 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 percentage = number of eGFP-expressing cells / total number of cells × 100%. A percentage of eGFP-positive cells of 40% or higher was considered acceptable.

[0263] Metal ion is Al 3+ Preparation of mRNA-metal-polyphenol complex particles when

[0264] Metal-polyphenol complexes were prepared according to the method described in Example 1.2. Curcumin (Formula 1) and Al(NO3)3·9H2O were added at a 1:1 ratio. The metal-polyphenol complex was dissolved in ethanol at different molar fractions with distearoylphosphatidylcholine (DSPC, Formula 51, a non-cationic or non-ionizable lipid), cholesterol (CHOL, Formula 59, a non-cationic or non-ionizable lipid), and DSPE-PEG2000 (Formula 58, a conjugated lipid that inhibits particle aggregation) to form the organic phase. The proportions of metal-polyphenol complex, DSPC (Formula 51), CHOL (Formula 59), and DSPE-PEG2000 (Formula 58) were 5%, 45%, 47%, and 3%, respectively. mRNA was dissolved at a concentration of 20 μg / mL in enzyme-free Tris-HCl buffer (0.1 M molarity) at pH 5.0 to form the aqueous phase. The metal-polyphenol complex and mRNA were mixed in a microfluidic chip at a mass ratio of 18:1. The volume ratio of the aqueous phase to the organic phase was 3:1. The flow rate of the organic and aqueous phases in the microfluidic chip was 12 ml / min. The drug mRNA was mRNA encoding the fluorescent protein eGFP, whose sequence is SEQ ID NO. 1 (720 nt). eGFP-mRNA@MPNPs were prepared and incubated with 293T cells at a concentration of 2 μg / mL (mRNA concentration). A control group was incubated with MPNPs alone. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0265] The particle size, surface potential, and stability of the produced eGFP-mRNA@MPNPs are detected, and the efficiency of eGFP-mRNA@MPNPs encapsulating nucleic acids is calculated.

[0266] The particle size detection method and criteria for judging the results are as follows: the particle size of nanoparticles is tested using a Malvern Zetasizer laser particle size analyzer; if the particle size is in the range of 30-400 nm, it is considered acceptable.

[0267] The method for detecting surface potential and the criteria for judging the results are as follows: the surface potential of nanoparticles is tested using a Malvern Zetasizer laser particle size analyzer; if the potential is in the range of -10 to 10 mV, it is considered acceptable.

[0268] The method for detecting stability and the criteria for judging the results are as follows: 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. If the particle size and surface potential do not change significantly within 3 to 7 days, the stability is considered to be relatively good.

[0269] The nucleic acid encapsulation efficiency was calculated using agarose gel electrophoresis. The nucleic acid loading for each group of lipid nanoparticles was 10 μg / mL. The mass ratio of the metal-polyphenol complex to nucleic acid was 18:1. An equal concentration of nucleic acid was dissolved in enzyme-free Tris-HCl buffer. This served as the positive control, while enzyme-free Tris-HCl buffer served as the negative control. The agarose gel concentration was 1.5%, and the gaps in the gel allowed only free nucleic acid to pass through, while the lipid nanoparticles were blocked. Electrophoresis was stopped when the free nucleic acid bands became clearly distinguishable. Image J software was used to calculate the gray values of free nucleic acid for each group. The positive control group was set to 100%, and the ratio of free nucleic acid in each group to the positive control was calculated as the relative amount of free nucleic acid. The encapsulation rate for each group was calculated as (100 - relative amount of free nucleic acid)%. A nucleic acid encapsulation rate of 50% or higher was considered acceptable.

[0270] For cell culture, human embryonic kidney cell line 293T was cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0271] Analysis of the percentage of eGFP-positive cells by flow cytometry was performed by seeding 5 x 10 293T cells in a 12-well plate. 5Cells were seeded at a density of 100 cells / well. When the cell density reached 80%, 1 mL of MPNPs or eGFP-mRNA@MPNPs was added and incubated. The concentration of eGFP-mRNA@MPNPs was 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 percentage = number of eGFP-expressing cells / total number of cells × 100%. A percentage of eGFP-positive cells of 40% or higher was considered acceptable.

[0272] The principle of loading nucleic acids into metal-chelated polyphenol complex nanoparticles (MPNPs) organized by metal-polyphenol complexes is that curcumin binds to Fe via coordination bonds. 3+ or Al 3+ The metal-polyphenol complex is linked to the Fe 3+ or Al 3+ is linked to nucleic acids by a coordinate bond, which ensures that nucleic acids are loaded onto the nanoparticles while the metal-polyphenol complex self-assembles with other components into MPNPs. There are two possibilities for the contribution of curcumin to the nucleic acid loading of MPNPs: (1) curcumin interacts with nucleic acids and assists in the loading of MPNPs with nucleic acids, for example, by inserting into the minor groove of nucleic acids. (2) It is also possible that curcumin does not directly interact with nucleic acids.

[0273] Example 2.1 Metal-Polyphenol Complex Component Dosage Ratio

[0274] In Example 2, curcumin (Formula 1) and FeCl3 were added in different ratios (1:1, 3:2, 2:1), and the other steps were the same as in Example 2 to produce different eGFP-mRNA@MPNPs and detect their nucleic acid encapsulation rates.

[0275] As shown in Table 1-1, the analysis of the results showed that when the administration ratio of curcumin (Formula 1) and FeCl3 was 1:1, the mRNA encapsulation efficiency of the manufactured metal-polyphenol complex particles was 85%. When the administration ratio of curcumin (Formula 1) and FeCl3 was 3:2, the mRNA encapsulation efficiency of the manufactured metal-polyphenol complex particles was 72%. When the administration ratio of curcumin (Formula 1) and FeCl3 was 2:1, the mRNA encapsulation efficiency of the manufactured metal-polyphenol complex particles was 63%. Fe in the drug-lipid particles 3+ The function of each Fe is to link curcumin to nucleic acids. 3+ Because curcumin has up to three conjugation sites, the metal-polyphenol complex particles can encapsulate as much nucleic acid as possible only when the curcumin to FeCl3 ratio in the drug-lipid particles is 1:1. The results also demonstrated that the mRNA encapsulation rate of the metal-polyphenol complex particles produced at a curcumin to FeCl3 ratio of 1:1 was the highest. When the curcumin to FeCl3 ratio was between 1:1 and 2:1, the nucleic acid encapsulation rate of the metal-polyphenol complex particles was always over 60%. [Table 1] Table 1-1 Metal ions are Fe 3+ The component input ratio of the metal-polyphenol complex when the metal-polyphenol complex is

[0276] In Example 2, curcumin (Formula 1) and Al(NO3)3·9H2O were added in different ratios (1:1, 3:2, 2:1), and the other steps were the same as in Example 2 to prepare different eGFP-mRNA@MPNPs and detect their nucleic acid encapsulation rates.

[0277] As shown in Table 1-2, when the dosage ratio of curcumin (Formula 1) and Al(NO3)3·9H2O is 1:1, the eGFP-mRNA entrapment efficiency of the produced metal-polyphenol complex particles is 86%. When the dosage ratio of curcumin (Formula 1) and Al(NO3)3·9H2O is 3:2, the eGFP-mRNA entrapment efficiency of the produced metal-polyphenol complex particles is 70%. When the dosage ratio of curcumin (Formula 1) and Al(NO3)3·9H2O is 2:1, the eGFP-mRNA entrapment efficiency of the produced metal-polyphenol complex particles is 66%. Al in the metal-polyphenol complex particles 3+ The function of each Al is to link the phospholipid complex with the nucleic acid. 3+ Since curcumin has up to three conjugation sites, the ratio of curcumin to Al(NO3)3·9H2O in the drug-lipid particles should be 1:1 to ensure that the metal-polyphenol complex particles can encapsulate as much nucleic acid as possible. Experimental results also demonstrated that the highest eGFP-mRNA encapsulation rate was achieved in metal-polyphenol complex particles prepared with a curcumin to Al(NO3)3·9H2O ratio of 1:1. When the curcumin to Al(NO3)3·9H2O encapsulation rate ranged from 1:1 to 2:1, the nucleic acid encapsulation rate of the metal-polyphenol complex particles was always above 60%. [Table 2] Table 1-2 Metal ions are Al 3+ The component input ratio of the metal-polyphenol complex when the metal-polyphenol complex is

[0278] Example 2.2 Proportions of metal-polyphenol complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) in the drug-lipid particles produced

[0279] Compared with Example 2, the proportions of metal-polyphenol complex, distearoylphosphatidylcholine (DSPC, Formula 51), DSPE-PEG2000 (Formula 58), and cholesterol (CHOL, Formula 59) are shown in Tables 1 to 3 (metal ions are Fe 3+ ) and Tables 1 to 4 (metal ions are Al 3+ ) and other conditions are the same.

[0280] The results of the analysis showed that the metal-polyphenol complex (metal ion is Fe) 3+When the ratio of DSPC to DSPE-PEG2000 is in the range of (5-20)%, the ratio of DSPC is in the range of (40-75)%, the ratio of CHOL is in the range of (0-48)%, and the ratio of DSPE-PEG2000 is in the range of (2-10)%, the particle size of the drug-lipid particles is in the range of 30-400 nm, the surface potential is in the range of -10-10 mV, the in vitro stability is ≥ 3 days, the mRNA encapsulation rate is > 50%, and the eGFP protein positive cell rate is ≥ 65%. Drug-lipid particles with a 5% metal-polyphenol complex, 60% distearoylphosphatidylcholine (DSPC), 30% cholesterol (CHOL), and 5% DSPE-PEG2000 exhibited the best performance, with particle diameters in the 120 nm range, surface potentials in the -1.99 mV range, in vitro stability of >7 days, mRNA encapsulation rates of 85%, and eGFP protein-positive cell rates of 97%. Because the metal-chelated polyphenol complex nanoparticles (MPNPs) primarily rely on the metal-polyphenol complex for nucleic acid uptake, the metal-polyphenol complex content should not be too low. The role of DSPC is to maintain the stability of the nanoparticle structure, and relatively good performance is achieved at a content of 40% to 75%. The role of DSPE-PEG2000 is to prevent nanoparticle aggregation and extend circulation time in the body, and relatively good performance is achieved at a content of 2% to 10%. When the CHOL content was 0%, the experimental results showed that the stability of the drug-lipid particles was within an acceptable range. When the CHOL content was greater than 0% and less than 48%, CHOL increased the fluidity of the nanoparticles, which was beneficial for maintaining the stability of the nanoparticles.

[0281] The above results indicate that the metal-polyphenol complex (metal ion is Fe 3+These results suggest that the drug-loading performance of the drug-metal-polyphenol complex particles (mRNA@MPNPs) is relatively excellent when the proportion of DSPC is in the range of (40-75)%, the proportion of CHOL is in the range of (0-48)%, and the proportion of DSPE-PEG2000 is in the range of (2-10)%. [Table 3] Table 1-3 Metal ions are Fe 3+ The ratio of each component in the drug-metal-polyphenol complex particles (mRNA@MPNP)

[0282] The results of the analysis are shown in Tables 1 to 4. The metal-polyphenol complex (metal ion is Al 3+When the ratio of DSPC to DSPE-PEG2000 is in the range of (5-20)%, (30-75)%, (0-48)%, or (2-10)%, the particle diameter of the metal-polyphenol complex particles is in the range of 30-400 nm, the surface potential is in the range of -10-10 mV, the in vitro stability is ≥ 3 days, the mRNA encapsulation rate is > 50%, and the positive expression rate of eGFP protein is ≥ 65%. The optimal performance of metal-polyphenol complex particles was achieved when the metal-polyphenol complex content was 5%, distearoylphosphatidylcholine (DSPC) content was 45%, cholesterol (CHOL) content was 47%, and DSPE-PEG2000 content was 3%. This resulted in particle diameters in the 100 nm range, surface potentials in the -2.74 mV range, in vitro stability of >7 days, mRNA entrapment rate of 86%, and eGFP protein-positive cell rate of 97%. Because mRNA@MPNPs primarily rely on the metal-polyphenol complex for nucleic acid adsorption, the metal-polyphenol complex content should not be too low. The stability of the nanoparticles was within an acceptable range when the DSPC content was in the range of 30-75%. The role of DSPE-PEG2000 was to prevent nanoparticle aggregation and extend circulation time in the body, and its content ranged from 2-10%. The role of CHOL was to enhance nanoparticle fluidity, and maintaining a constant content was beneficial to nanoparticle stability.

[0283] The above results indicate that the metal-polyphenol complex (metal ion is Al 3+ This suggests that the drug-loading performance of mRNA@MPNPs is relatively better when the proportion of DSPC is in the range of (30–75)%, the proportion of CHOL is in the range of (0–48)%, and the proportion of DSPE-PEG2000 is in the range of (2–10)%. [Table 4] Table 1-4 Metal ions are Al 3+The ratio of each component in the drug-metal-polyphenol complex particles (mRNA@MPNP)

[0284] Example 2.3: Types of non-cationic or non-ionizable lipids in the prepared eGFP-mRNA@MPNPs

[0285] Compared with Example 2, the substitution of distearoylphosphatidylcholine (DSPC) is as shown in Tables 1-5 and 1-6, and the other conditions are the same.

[0286] To explore whether the DSPC in eGFP-mRNA@MPNPs could be replaced with other non-cationic or non-ionizable lipids in addition to the conjugated lipids that inhibit particle aggregation, DSPE, DSPA, and DSPG were selected instead of DSPC. Particle size, surface potential, stability, and mRNA encapsulation rate were measured, demonstrating that the DSPC in eGFP-mRNA@MPNPs could be replaced with other non-cationic or non-ionizable lipids. The functionality after replacement was equivalent to that of eGFP-mRNA@MPNPs containing DSPC (Tables 1-5). The metal ion was Fe. 3+ ) and Table 1-6 (metal ions are Al 3+ The main role of the non-cationic lipid DSPC in eGFP-mRNA@MPNPs is to improve liposome membrane fusion, increase stability, and reduce toxicity. However, other cationic or non-ionizable lipids also have the functions of improving liposome membrane fusion, increasing stability, and reducing toxicity. Therefore, DSPC in the drug-lipid particles can be replaced by other non-cationic or non-ionizable lipids without affecting their efficacy. [Table 5] Table 1-5 Metal ions are Fe 3+ Ability of non-cationic lipids other than conjugated lipids to inhibit particle aggregation in drug-metal-polyphenol complex particles (mRNA@MPNP) [Table 6] Table 1-6 Metal ions are Al 3+ Ability of non-cationic lipids other than conjugated lipids to inhibit particle aggregation in drug-metal-polyphenol complex particles (mRNA@MPNP)

[0287] Example 2.4: Types of conjugated lipids that inhibit particle aggregation in the prepared eGFP-mRNA@MPNPs

[0288] Compared with Example 2, the substitution of DSPE-PEG2000 is shown in Table 1-7 (metal ion is Fe 3+ ), Table 1-8 (metal ions are Al 3+ ) and other conditions are the same.

[0289] To investigate whether the DSPE-PEG2000 in eGFP-mRNA@MPNPs could be replaced with other lipids that inhibit particle aggregation, three other lipids that inhibit particle aggregation, namely DSPE-PEG700, DSPE-PEG5000, and DSPE-PEG1000, were selected instead of DSPE-PEG2000. Particle size, surface potential, stability, and mRNA encapsulation efficiency were measured, demonstrating that the DSPE-PEG2000 in eGFP-mRNA@MPNPs could be replaced with other lipids that inhibit particle aggregation. The performance of the replacement lipids was comparable to that of eGFP-mRNA@MPNPs containing DSPE-PEG2000 (Tables 1-7 and 1-8). Although the main role of DSPE-PEG2000 in eGFP-mRNA@MPNPs is to inhibit aggregation, other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation, so DSPE-PEG2000 in eGFP-mRNA@MPNPs can be replaced by other conjugated lipids that inhibit particle aggregation without affecting its efficacy. [Table 7] Table 1-7 Metal ions are Fe 3+ Conjugation of lipids to inhibit particle aggregation in drug-metal-polyphenol complex particles (mRNA@MPNP) [Table 8] Table 1-8 Metal ions are Al 3+ Conjugation of lipids to inhibit particle aggregation in drug-metal-polyphenol complex particles (mRNA@MPNP)

[0290] Example 2.5 Preparation and Efficacy Characterization of mRNA@MPNPs

[0291] Example 2.5.1 Metal ion is Fe 3+ Preparation and efficacy characterization of mRNA@MPNPs

[0292] Using the method of Example 2 as a reference, three types of mRNA@MPNPs with different target protein mRNA sequences were prepared by replacing the mRNA in Example 2 with two other types of mRNA. The three different mRNA sequences were: (1) the mRNA sequence encoding the fluorescent protein eGFP (SEQ ID NO. 1, 720 nt); (2) the mRNA sequence encoding the receptor binding domain (RBD) of the S1 subunit of the novel coronavirus (COVID-19) (SEQ ID NO. 2, 669 nt); and (3) the mRNA sequence encoding the tumor antigen NY-ESO-1 (SEQ ID NO. 3, 543 nt). The remaining drug (mRNA)-lipid particle preparation process was the same as in Example 2, resulting in eGFP-mRNA@MPNPs, RBD-mRNA@MPNPs, and NY-ESO-1-mRNA@MPNPs, respectively.

[0293] 293T cells were incubated with eGFP-mRNA@MPNPs at a concentration of 2μg / mL (mRNA concentration), while the control group was incubated with MPNPs. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry. The results are shown in Figure 1-1. 293T cells were incubated with RBD-mRNA@MPNPs at a concentration of 2μg / mL (mRNA concentration), while the control group was incubated with MPNPs. After 24 hours, the supernatant was centrifuged and frozen at -20°C for later use. The expression level of the novel coronavirus antigen RBD protein in the cell supernatant was detected using a commercially available novel coronavirus antigen RBD ELISA detection kit. The results are shown in Figure 1-2.

[0294] The method for detecting RBD expression levels by ELISA is as follows:

[0295] 1. Sample collection involves leaving the cell supernatant at room temperature for 2 hours, centrifuging at 1000 xg for 20 minutes, and collecting the supernatant.

[0296] 2. To add samples, prepare blank wells, standard wells, and sample wells to be tested on the coated plate. Add 100 μL of sample dilution to the blank wells, add serially diluted standard solutions to the standard wells, and add 100 μL of the sample to be tested to the sample wells. Incubate at 37°C for 60 minutes.

[0297] 3. Discard the liquid in the wells and wash the plate three times, soaking for 1-2 minutes each time. Add 100 μL of the formulated biotin-labeled anti-RBD antibody working solution to each well, mix evenly, and incubate at 37°C for 60 minutes.

[0298] 4. Discard the liquid in the wells and wash the plate three times, soaking for 1-2 minutes each time.

[0299] 5. Add 100 μL of the prepared streptavidin-HRP working solution to each well, mix evenly, and incubate at 37°C for 45 minutes.

[0300] 6. Discard the liquid in the wells and wash the plate three times, soaking for 1-2 minutes each time.

[0301] 7. Add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution to each well and incubate at 37°C for 15 minutes away from light.

[0302] 8. Add 100 μL of stop solution to each well to stop the reaction.

[0303] 9. Measure the optical density (OD) value of each well at a wavelength of 450 nm.

[0304] For data analysis, a standard curve is drawn with the concentration of the standard on the horizontal axis and the OD value on the vertical axis.

[0305] Experimental animals were randomly divided into two groups (experimental and control groups) with five animals per group. The RBD-mRNA@MPNP animal model was BALB / c mice. Each mouse received a first intramuscular injection on day 1 and a second intramuscular injection on day 14. The experimental group received RBD-mRNA@MPNPs, while the control group received metal-polyphenol complex particles (MPNPs) without mRNA particles. Each injection was 100 μL, and the RBD-mRNA@MPNP formulation in the experimental group contained 30 mg of mRNA. Blood was collected from the mice 28 days after the first injection, and serum was separated and gradient diluted. RBD total IgG antibodies against the S1 subunit of the novel coronavirus produced in the mice were detected using a commercially available ELISA kit. The results are shown in Figures 1–3.

[0306] The animal model for NY-ESO-1-mRNA@MPNPs was C57BL / 6 mice. Each mouse received four intramuscular injections on days 1, 7, 14, and 21. The experimental group received NY-ESO-1-mRNA@MPNPs, while the control group received metal-polyphenol complex particles (MPNPs) without mRNA. Each injection was 100 μL, and the experimental group received 30 mg of mRNA. On day 28 after the first injection, blood was collected from the mice, and serum was separated and gradient diluted. ELISA was used to detect anti-NY-ESO-1 total IgG antibodies produced in the mice. The results are shown in Figures 1-4.

[0307] The method for detecting anti-NY-ESO-1 total IgG antibodies in mice is as follows:

[0308] The preparation of reagents used in ELISA is as follows:

[0309] 1. To prepare the coating solution, precisely weigh 8.4 g of NaHCO3 and dissolve it in 1 L of distilled water (DDW). After the solid is completely dissolved, adjust the pH of the entire solution to 9.6 using 1 M NaOH solution. Store the prepared coating solution at 4°C for later use.

[0310] 2. For the washing solution, add 0.5 mL of Tween-20 to 1 L of 0.01 M PBS solution, mix evenly, and leave at room temperature.

[0311] 3. For the blocking solution, precisely weigh 20 g of BSA and add it to 1 L of 0.01 M PBS solution. Sonicate to remove any undissolved BSA powder. Once all solids have dissolved and the solution has turned pale yellow, store it in a 4°C refrigerator for later use.

[0312] 4. To prepare the antibody dilution solution, precisely weigh 2.5 g of BSA and dissolve it in 250 mL of 0.01 M PBS solution. Once the solid is completely dissolved, add 1.25 mL of Tween-20, mix thoroughly, and store at 4°C for later use.

[0313] 5. For the color developing solution, add 19.2 g of citric acid to DDW water to make 1000 mL. (A) For 0.2 M disodium hydrogen phosphate, add 28.4 g of anhydrous disodium hydrogen phosphate to DDW water to make 1000 mL. (B) Add 24.3 mL of 0.1 M citric acid solution (A), 25.7 mL of 0.2 M phosphate buffer (B), and 50 mL of DDW water. At the time of use, add 50 mg of OPD (o-phenylenediamine) and 20.15 mL of 30% HO.

[0314] 6. To prepare the stop solution, add 55.5 mL of 2M H2SO4: concentrated sulfuric acid and DDW to make a 500 mL solution.

[0315] The antibody titer in the mouse serum is measured by ELISA.

[0316] 1. For coating, dilute the NY-ESO-1 antigen in coating solution to 1 μg / mL, add 50 μL / well to a 96-well plate, and coat overnight at 4°C.

[0317] 2. For blocking, dehydrate the coating solution in the well plate, wash with blocking solution three times for 5 minutes each time, and then add 150 μL of blocking solution to each well and incubate at 37°C for 2 hours.

[0318] 3. Drying: Dehydrate the blocking solution and incubate at 37°C for 1-2 hours until all the liquid at the bottom of the well plate has dried.

[0319] 4. For immunization, serum samples were first diluted 1:1000 in antibody diluent, then serially diluted 1:2. The diluted serum samples were added to a sealed 96-well plate at 100 μL per well and incubated at 37°C for 2 hours. The wells were drained, and 300 μL of washing solution was added per well. The plate was gently shaken for 40 seconds. This step was repeated three times. Biotinylated goat anti-mouse IgG antibody diluted 1:1000 was added to the wells at 100 μL per well and incubated at 37°C for 1 hour. The wells were drained, washing solution was added, and the plate washing step was repeated. Freshly prepared streptavidin-labeled horseradish peroxidase HRP working solution was added at 100 μL per well and incubated at 37°C for 1 hour. The wells were drained, washing solution was added, and the plate washing step was repeated. Add 100 μL of color-developing solution to each well under dark conditions, and incubate at room temperature for 5 minutes. Add 50 μL of stop solution to each well to stop the color development. Measure the absorbance at 450 nm using a microplate reader.

[0320] On the 28th day after administration of RBD-mRNA@MPNPs, the spleens of normal mice were collected and prepared into single cell suspensions under sterile conditions. 100,000 splenocytes were plated onto cell well plates at a final concentration of 10 mg / mL of RBD protein and cultured for 48 hours. The supernatant was then centrifuged and the expression levels of IFN-γ, IL-2, and IL-4 were measured using ELISA kits. The results are shown in Figures 1-5.

[0321] On the 28th day after administration of NY-ESO-1-mRNA@MPBP, normal mouse spleens were collected and prepared into single cell suspensions under sterile conditions. 100,000 splenocytes were plated onto cell well plates at a final concentration of 10 mg / mL NY-ESO-1 protein, and the cells were cultured for 48 hours. The supernatant was then centrifuged and removed. The expression levels of IFN-γ, IL-2, and TNF-α were measured using ELISA. The results are shown in Figures 1-6.

[0322] As shown in Figure 1-1, the eGFP-positive cell rate in the eGFP-mRNA@MPNP experimental group was 93.7%, while no eGFP signal was detected in the MPNP control group. As shown in Figure 1-2, the RBD protein encoded by the RBD-mRNA encapsulated in MPNPs was 166 ng / mL in the supernatant of 293T cells, whereas the supernatant RBD protein content in 293T cells transfected with empty carrier MPNPs was 0. These results suggest that the drug-metal-polyphenol complex particles (mRNA-MPNPs) can encapsulate and deliver any mRNA to directly encode polypeptides within cells. As shown in Figures 1-3 and 1-4, both RBD-mRNA@MPNPs and NY-ESO-1-mRNA@MPNPs effectively induced humoral immunity in mice, generating high levels of antigen-specific binding antibodies. The IgG antibody titer in mice treated with RBD-mRNA@MPNPs reached 84,363.4, while the IgG antibody titer in mice treated with NY-ESO-1-mRNA@MPNPs reached 4,283.56. As shown in Figures 1-5 and 1-6, both RBD-mRNA@MPNPs and NY-ESO-1-mRNA@MPNPs effectively induced cellular immunity in mice, activating immune cells and producing large amounts of cytokines. With RBD-mRNA@MPNPs, the expression levels of cytokines IFN-γ, IL-2, and IL-4 reached 271.8 pg / mL, 269.6 pg / mL, and 75.8 pg / mL, respectively. With NY-ESO-1-mRNA@MPNPs, the expression levels of cytokines IFN-γ, IL-2, and TNF-α reached 76.38 pg / mL, 74.56 pg / mL, and 69.31 pg / mL, respectively. These results suggest that the drug-metal-polyphenol complex particles (mRNA@MPNPs) can encapsulate and deliver any mRNA, promoting the expression of the target protein (antigen), thereby effectively inducing humoral and cellular immunity in mice and producing high levels of antigen-specific binding antibodies and cytokines, thereby serving as an anti-coronavirus mRNA vaccine and an anti-tumor mRNA vaccine.

[0323] Example 2.5.2 Metal ion is Al 3+ Preparation and efficacy characterization of mRNA@MPNPs

[0324] The difference between this example and Example 2.5.1 is that the metal ion Fe in Example 2.5.1 3+ Al 3+ is to be replaced by

[0325] As shown in Figures 1-7, the eGFP-positive cell rate in the eGFP-mRNA@MPNP experimental group was 97.03%, while no eGFP signal was detected in the MPNP control group. As shown in Figures 1-8, the RBD protein encoded by the RBD-mRNA encapsulated in MPNPs was 207 ng / mL in the supernatant of 293T cells, while the RBD protein content in the supernatant of 293T cells transfected with empty carrier MPNPs was 0. These results suggest that mRNA-MPNPs can encapsulate and deliver any mRNA and directly encode polypeptides in cells. As shown in Figures 1-9 and 1-10, both RBD-mRNA@MPNPs and NY-ESO-1-mRNA@MPNPs effectively induced humoral immunity in mice, generating high levels of antigen-specific binding antibodies. The IgG antibody titer in mice treated with RBD-mRNA@MPNP reached 94828.6, while the IgG antibody titer in mice treated with NY-ESO-1-mRNA@MPNP reached 5848.02. As shown in Figures 1-11 and 1-12, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP effectively induced cellular immunity in mice, activating immune cells and producing large amounts of cytokines. The expression levels of cytokines IFN-γ, IL-2, and IL-4 reached 306.2 pg / mL, 289.6 pg / mL, and 88.2 pg / mL, respectively, with RBD-mRNA@MPNP. The expression levels of cytokines IFN-γ, IL-2, and TNF-α reached 91.88 pg / mL, 85.32 pg / mL, and 80.22 pg / mL, respectively, with NY-ESO-1-mRNA@MPNP. These results suggest that mRNA@MPNPs can encapsulate and deliver any mRNA, promoting the expression of target proteins (antigens), thereby effectively inducing humoral and cellular immunity in mice and producing high levels of antigen-specific binding antibodies and cytokines, thereby serving as an anti-coronavirus mRNA vaccine and an anti-tumor mRNA vaccine.

[0326] Example 2.6 Preparation and Effects of siRNA-Metal-Polyphenol Complex Particles (siRNA@MPNP)

[0327] Example 2.6.1 Metal ion is Fe 3+ Preparation and effect of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPNP)

[0328] Three siRNA@MPNPs containing different siRNAs were prepared using the same method as in Example 2, replacing the mRNA in Example 2 with siRNA. The genes and sequences targeted by the three different siRNAs, as well as their corresponding random control sequences, are as follows: (1) The sequences of the siRNA targeting the Bcl-2 gene (Bcl-2-siRNA) are SEQ ID NO. 4 (antisense strand) and SEQ ID NO. 21 (sense strand) (19 bp), and its random control sequences are SEQ ID NO. 5 (antisense strand) and SEQ ID NO. 22 (sense strand) (19 bp). (2) The sequences of the siRNA targeting the PLK1 gene (PLK1-siRNA) are SEQ ID NO. 6 (antisense strand) and SEQ ID NO. 23 (sense strand) (21 bp), and its random control sequences are SEQ ID NO. 7 (antisense strand) and SEQ ID NO. 24 (sense strand) (19 bp). (3) The sequence of siRNA targeting the Gal-1 gene (Gal-1-siRNA) is SEQ ID NO. 8 (19 bp), and its random control sequence is SEQ ID NO. 9 (19 bp). The remaining preparation process of siRNA@MPNPs was the same as in Example 2.

[0329] The sequence of Bcl-2-siRNA is as follows:

[0330] The antisense was 5′-CAGCUUAUAAUGGAUGUAC-3′ (SEQ ID No. 4).

[0331] The sense sequence is 5'-GUACAUCCAUUAUAAGCUG-3' (SEQ ID No. 21) (19 bp).

[0332] The random control sequence of Bcl-2-siRNA is as follows:

[0333] The antisense was 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID No. 5).

[0334] The sense sequence is 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID No. 22) (19 bp).

[0335] The sequence of PLK1-siRNA is as follows:

[0336] The antisense was 5'-UAAGGAGGGUGAUCUUCUUCA-3' (SEQ ID No. 6).

[0337] The sense sequence is 5'-UGAAGAAGAUCACCCUCCUUA-3' (SEQ ID No. 23) (21 bp).

[0338] The random control sequence of PLK1-siRNA is as follows:

[0339] The antisense was 5'-CUUACGCUGAGUACUUCGA-3' (SEQ ID No. 7).

[0340] The sense sequence is 5'-UCGAAGUACUCAGCGUAAG-3' (SEQ ID No. 24) (19 bp).

[0341] The sequence of Gal-1-siRNA is as follows:

[0342] 5'-GCUGCCAGAUGGAUACGAA-3' (SEQ ID No. 8) (19bp).

[0343] The random control sequence of Gal-1-siRNA is as follows:

[0344] 5'-GGAAAUCCCCAACAGUGA-3' (SEQ ID No. 9) (19bp).

[0345] Cell culture methods included culturing U251 human brain glioblastoma cells in monolayer in high-glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 m m l -glutamine (Bio Industries) medium at 37°C and 5% CO and passaged twice weekly.

[0346] U251 cells were plated in a 6-well plate at 1 × 10 cells per well. 6 The cells were seeded at a cell density of 100x for approximately 24 hours, and the cells in each well were incubated with siRNA@MPNPs containing the above siRNA (the concentration of siRNA was 2 μg / mL) for 72 hours, after which the cells were collected, total cellular RNA was extracted, and the mRNA expression levels of the target genes (Bcl-2, PLK1, Gal-1) were detected using RT-PCR technology to statistic the ability of the siRNA@MPNPs to silence the cellular target genes.

[0347] The specific process of RT-PCR is as follows:

[0348] To extract total RNA, discard the medium from the 6-well plate, rinse three times with PBS buffer, and add 1 mL of Trizol to each well to lyse the cells. Add 200 μL of chloroform, shake thoroughly, and leave at room temperature for 10 minutes. Centrifuge at 13,000 rpm and 4°C for 15 minutes to obtain a three-phase liquid with the RNA dissolved in the upper aqueous phase. Aspirate the upper aqueous phase and place it in a new 1.5 mL centrifuge tube without enzymes. Add 500 μL of isopropyl alcohol, leave at room temperature for 10 minutes, and centrifuge at 13,000 rpm and 4°C for 15 minutes to obtain the RNA precipitate. Remove the supernatant, add 1 mL of 75% (v / v) ethanol freshly prepared in RNase-free water to each tube, carefully blow off the white RNA precipitate at the bottom of the tube, and centrifuge at 7,500 rpm and 4°C for 10 minutes. Remove the supernatant and aspirate as much liquid as possible from the bottom of the tube. Open the lid, let the RNA precipitate at the bottom of the tube dry at room temperature, add 50 µL of enzyme-free water to dissolve it, and then use an ultra-microvolume UV-visible spectrophotometer to detect the purity and concentration of the RNA.

[0349] cDNA reverse transcription was performed using Ta Ka Ra Prime Script TM The RT Reagent Kit and the gDNA Eraser Kit are used to reverse transcribe RNA to cDNA, respectively. Removing genomic DNA (gDNA) before the reverse transcription step ensures more accurate and reliable results. Prepare the total RNA reverse transcription reaction on ice: 1 μL Prime Script RT Enzyme Mix, 1 μL RT Primer Mix, 4 μL 5x Prime Script Buffer 2, and 4 μL RNase-free dH2O. After preparing the reaction mixture, incubate at 37°C for 15 minutes, then heat at 85°C for 5 seconds to terminate the reaction. Store at 4°C for further use.

[0350] The RT-PCR procedure uses the SYBR Green dye method for detection, eliminating the need for a probe. Specifically, real-time PCR reactions are performed using cDNA from various samples as templates. Prepare the reaction mixture on ice: 5 μL SYBR Premix Dimer Eraser (2x), 0.3 μL PCR Forward Primer (10 μM), 0.3 μL PCR Reverse Primer (10 μM), 0.2 μL ROX Reference Dye (50x), 1 μL of the cDNA template obtained in the previous step, and 3.2 μL dH2O. Load 10 μL of sample per well into a well plate. After sample loading is complete, centrifuge (1000 rpm, 5 minutes) to remove any liquid and air bubbles in the reaction mixture. Perform real-time PCR reaction detection using an ABI ViiA7 real-time fluorescent quantitative PCR instrument. The reaction procedure is as follows: 95°C, 30 seconds (1 cycle) → 95°C, 5 seconds, 55°C, 30 seconds, and 72°C, 30 seconds (40 cycles) → 60°C to 95°C, 2 minutes (1 cycle). The experiment was repeated three times, and the average value was calculated to determine the Ct value for each group, and the fold difference in expression between the experimental and control groups was calculated. The control gene was GAPDH. The RT-PCR primers were as follows: (1) Bcl-2 primer, forward 5'-AGGATTGTGGCCTTCTTTGAG-3', reverse 5'-AGACAGCCAGGAGAAATC AAAC-3'; (2) PLK1 primer, forward 5'-ACCAGCACGTCGTAGGATTC-3', reverse 5'-CAAGCAATTTGCCGTAGG-3'; (3) Gal-1 primer, forward 5'-CAATCATGGCCTGTGGTCTG-3', reverse 5'-GTG TAGGCACAGGTTGTTGCTG-3'; (4) GAPDH primer, forward 5'-TCAGGGGTTTCACATTTGGCA-3', reverse 5'-GGAGCGGAAAACCA-3'. The expression level of each target gene was measured using the RQ value (2 -ΔΔCT ) The formula is as follows: Fold Change = 2 - ΔΔCt In the formula, ΔΔCt = ΔCt 実験群 - ΔCt 対照群 , ΔCt = Ct 標的遺伝子 -Ct 内部参照遺伝子

[0351] The gene silencing efficiency was calculated as follows: 100% - gene expression level in the experimental group / gene expression level in the control group.

[0352] As shown in Figures 1-13, 1-14, and 1-15 (scr siRNA was a random control sequence), all three drug-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP, PLK1-siRNA@MPNP, and Gal-1-siRNA@MPNP) significantly inhibited their corresponding target genes. The Bcl-2-siRNA@MPNPs inhibited the target gene Bcl-2 by 67%, the PLK1-siRNA@MPNPs inhibited the target gene PLK1 by 87%, and the Gal-1-siRNA@MPNPs inhibited the target gene Gal-1 by 64%. These results suggest that siRNA@MPNPs can be used to deliver any siRNA for targeted gene intervention and serve as siRNA-carrying drugs, vaccines, or other products.

[0353] Example 2.6.2 Metal ion is Al 3+ Preparation and effect of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPNP)

[0354] The difference between this example and Example 2.6.1 is that the metal ion Fe in Example 3.6.1 3+ Al 3+ is to be replaced by

[0355] Analysis of the results, as shown in Figures 1-16, 1-17, and 1-18, showed that all three types of siRNA@MPNPs significantly inhibited their corresponding target genes. The Bcl-2-siRNA@MPNPs inhibited the target gene Bcl-2 by 72%, the PLK1-siRNA@MPNPs inhibited the target gene PLK1 by 88.07%, and the Gal-1-siRNA@MPNPs inhibited the target gene Gal-1 by 70.11%. These results suggest that siRNA@MPNPs can deliver any siRNA for targeted gene intervention therapy and can serve as siRNA-carrying drugs, vaccines, or other products.

[0356] Example 2.7 Preparation and effects of ASO-metal-polyphenol composite particles (ASO@MPNP)

[0357] Example 2.7.1 Metal ion is Fe 3+ Preparation and effects of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPNP)

[0358] Using the same method as in Example 2, three types of drug-metal-polyphenol complex particles (ASO@MPNPs) containing different ASOs were prepared, replacing the mRNA in Example 2 with ASO. The target genes, sequences, and corresponding random control sequences of the three different ASOs were as follows: (1) ASO targeting the STAT3 gene (STAT3-ASO) sequence was SEQ ID NO. 10 (17 nt), and its random control sequence was SEQ ID NO. 11 (18 nt); (2) ASO targeting the α-syn gene (α-syn-ASO) sequence was SEQ ID NO. 12 (16 nt), and its random control sequence was SEQ ID NO. 13 (16 nt); and (3) ASO targeting the Bcl-2 gene (Bcl-2-ASO) sequence was SEQ ID NO. 14 (18 nt), and its random control sequence was SEQ ID NO. 15 (20 nt). The remaining drug (ASO)-metal-polyphenol complex particle manufacturing process was the same as in Example 2. Different cells were incubated with different ASO@MPNPs. U251 human brain glioblastoma cells were incubated with ASO@MPNPs targeting the STAT3 gene, SH-SY5Y human neuroblastoma cells were incubated with ASO@MPNPs targeting the α-syn gene, and Daudi human lymphoma cells were incubated with ASO@MPNPs targeting the Bcl-2 gene. 1 × 10 cells were cultured per well in a 6-well plate. 6 Approximately 24 hours after seeding at a density of cells, the cells in each well were incubated with drug-metal-polyphenol complex particles (ASO@MPNPs) containing the above ASO (the concentration of ASO was 1 μg / mL) for 48 hours, the cells were collected, total cellular RNA was extracted, and the mRNA expression levels of the target genes (STAT3, α-syn, Bcl-2) were detected using RT-PCR technology, respectively, to calculate the ability of ASO@MPNPs to silence the cellular target genes.

[0359] The sequence of SEQ ID No. 10 (the sequence of STAT3-ASO) is as follows:

[0360] 5'-GCTCCAGCATCTGCTTC-3' (17nt).

[0361] The sequence of SEQ ID No. 11 (random control sequence of STAT3-ASO) is as follows:

[0362] 5'-GAAGCAGCAGATGCTGGA-3'(18nt).

[0363] The sequence of SEQ ID No. 12 (the sequence of α-syn-ASO) is as follows:

[0364] 5'-GCTCCCTCCACTGTCT-3'(16nt).

[0365] The sequence of SEQ ID No. 13 (random control sequence of α-syn-ASO) is as follows:

[0366] 5'-ACTCCCGAACCTGTCT-3'(16nt).

[0367] The sequence of SEQ ID No. 14 (the sequence of Bcl-2-ASO) is as follows:

[0368] 5'-TCTCCCAGCGTGCGCCAT-3' (18nt).

[0369] The sequence of SEQ ID No. 15 (random control sequence of Bcl-2-ASO) is as follows:

[0370] 5'-CAGCGTGCGCCATCCTTCCC-3' (20nt).

[0371] Cell culture was as follows: (1) U251 human brain glioblastoma cells were grown in monolayers in high-glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (Bio Industries) at 37°C and 5% CO2 with two passages per week; (2) SH-SY5Y human neuroblastoma cells were grown in monolayers in high-glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (Bio Industries) at 37°C and 5% CO2 with two passages per week. (3) Daudi human lymphoma cells were grown in RPMI 1640 + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (Bio Industries) medium at 37°C and 5% CO2, and passaged twice a week.

[0372] The specific process of RT-PCR is as follows:

[0373] To extract total RNA, discard the medium from the 6-well plate, rinse three times with PBS buffer, and add 1 mL of Trizol to each well to lyse the cells. Add 200 μL of chloroform, shake thoroughly, and leave at room temperature for 10 minutes. Centrifuge at 13,000 rpm and 4°C for 15 minutes to obtain a three-phase liquid with the RNA dissolved in the upper aqueous phase. Aspirate the upper aqueous phase and place it in a new 1.5 mL centrifuge tube without enzymes. Add 500 μL of isopropyl alcohol, leave at room temperature for 10 minutes, and centrifuge at 13,000 rpm and 4°C for 15 minutes to obtain the RNA precipitate. Remove the supernatant, add 1 mL of 75% (v / v) ethanol freshly prepared in RNase-free water to each tube, carefully blow off the white RNA precipitate at the bottom of the tube, and centrifuge at 7,500 rpm and 4°C for 10 minutes. Remove the supernatant and aspirate as much liquid as possible from the bottom of the tube. Open the lid, let the RNA precipitate at the bottom of the tube dry at room temperature, add 50 µL of enzyme-free water to dissolve it, and then use an ultra-microvolume UV-visible spectrophotometer to detect the purity and concentration of the extracted RNA.

[0374] cDNA reverse transcription was performed using Ta Ka Ra Prime Script TM The RT Reagent Kit and the gDNA Eraser Kit are used to reverse transcribe RNA to cDNA, respectively. Removing genomic DNA (gDNA) before the reverse transcription step ensures more accurate and reliable results. Prepare the total RNA reverse transcription reaction on ice: 1 μL Prime Script RT Enzyme Mix, 1 μL RT Primer Mix, 4 μL 5x Prime Script Buffer 2, and 4 μL RNase-free dH2O. After preparing the reaction mixture, place it at 37°C for 15 minutes, then heat it at 85°C for 5 seconds to terminate the reaction, and store it at 4°C for further use.

[0375] The RT-PCR procedure uses the SYBR Green dye method for detection, eliminating the need for a probe. Specifically, real-time PCR reactions are performed using cDNA from various samples as templates. The reaction mixture is prepared on ice: 5 μL SYBR Premix Dimer Eraser (2x), 0.3 μL PCR Forward Primer (10 μM), 0.3 μL PCR Reverse Primer (10 μM), 0.2 μL ROX Reference Dye (50x), 1 μL of the cDNA template obtained in the previous step, and 3.2 μL dH2O. Add 10 μL of sample per well to a well plate. After sample loading is complete, centrifuge (1000 rpm, 5 minutes) to remove any liquid and air bubbles in the reaction mixture. The real-time PCR reaction is detected using an ABI ViiA7 real-time fluorescent quantitative PCR instrument. The reaction procedure is as follows: 95°C, 30 seconds (1 cycle) → 95°C, 5 seconds, 55°C, 30 seconds, and 72°C, 30 seconds (40 cycles) → 60°C to 95°C, 2 minutes (1 cycle). The experiment was repeated three times, and the average value was calculated to determine the Ct value for each group, and the fold difference 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: (1) STAT3 primer, forward 5'-TGATCACCTTTGAGACCGAGG-3', reverse 5'-GATCACCACAACTGG CAA GG-3'; (2) α-syn primer, forward 5'-TGACGGTGTGACAGCAGTAG-3', reverse 5'-CAGTGGCTGCTGCAATG-3'; (3) Bcl-2 primer, forward 5'-AGGATT GTG GCCTTCTTTGAG-3', reverse 5'-AGACAGCCAGGAGAAATCAAAC-3'; (4) GAPDH primer, forward 5'-TCAGGGG TTTCACATTTGGCA-3', reverse 5'-GGAGCGGAA AACCA-3'. The expression level of each target gene was measured using the RQ value (2 -ΔΔCT ) The formula is as follows: Fold Change = 2 - ΔΔCt In the formula, ΔΔCt = ΔCt 実験群 - ΔCt 対照群 , ΔCt = Ct 標的遺伝子 -Ct 内部参照遺伝子

[0376] The gene silencing efficiency was calculated as follows: 100% - gene expression level in the experimental group / gene expression level in the control group.

[0377] Analysis of the results, as shown in Figures 1-19, 1-20, and 1-21 (scr ASO was a random control sequence), showed that all three ASO@MPNPs significantly inhibited their corresponding target genes. The STAT3-ASO@MPNPs inhibited the target gene STAT3 by 72%, the α-syn-ASO@MPNPs inhibited the target gene α-syn by 78%, and the Bcl-2-ASO@MPNPs inhibited the target gene Bcl-2 by 62%. These results suggest that the drug-metal-polyphenol complex particles (ASO@MPNPs) can deliver any ASO for targeted gene intervention therapy and serve as ASO-loaded drugs, vaccines, or other products.

[0378] Example 2.7.2 Metal ion is Al 3+ Preparation and effects of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPNP)

[0379] The difference between this example and Example 2.7.1 is that the metal ion Fe in Example 2.7.2 3+ Al 3+ It has been replaced by.

[0380] Analysis of the results, as shown in Figures 1-22, 1-23, and 1-24, showed that all three ASO@MPNPs significantly inhibited their corresponding target genes. The STAT3-ASO@MPNPs inhibited the target gene STAT3 by 75.4%, the α-syn-ASO@MPNPs inhibited the target gene α-syn by 80.87%, and the Bcl-2-ASO@MPNPs inhibited the target gene Bcl-2 by 67.91%. These results suggest that ASO@MPNPs can carry any ASO for targeted gene intervention therapy and can serve as ASO-carrying drugs, vaccines, or other products.

[0381] Example 2.8 Preparation of drug (different types of nucleic acid)-metal-polyphenol complex particles and their effects

[0382] Example 2.8.1 Metal ion is Fe 3+ Preparation of drug (different kinds of nucleic acid)-metal-polyphenol complex particles and their effects when

[0383] In Example 2, mRNA was replaced with double-stranded RNA (siRNA), single-stranded DNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively. The different nucleic acid sequences were as follows: (1) The sequences of the double-stranded RNA (Bcl-2-siRNA) were SEQ ID NO. 4 (antisense strand) and SEQ ID NO. 21 (sense strand) (19 bp), and their random sequences were SEQ ID NO. 5 (antisense strand) and SEQ ID NO. 22 (sense strand) (19 bp). (2) The sequence of the single-stranded DNA (STAT3-ASO) was SEQ ID NO. 10 (17 nt), and its random control sequence was SEQ ID NO. 11 (18 nt). (3) The sequence of the single-stranded RNA (mRNA encoding the wild-type novel coronavirus S protein) was SEQ ID NO. 16 (3822 nt). (4) The double-stranded DNA (dsDNA) sequences were SEQ ID NO. 17 (antisense strand) and SEQ ID NO. 25 (sense strand) (22 bp), with the 3' end labeled with the fluorescent probe Cy3. (5) The single-stranded DNA (ssDNA) sequence was SEQ ID NO. 18 (22 nt), with the 3' end labeled with the fluorescent probe Cy3. Using the method of Example 2, the drug-metal-polyphenol complex particles encapsulating the above-mentioned different types of nucleic acids (Bcl-2-siRNA@MPNP, STAT3-ASO@MPNP, S-mRNA@MPNP, dsDNA@MPNP, ssDNA@MPNP) were prepared, respectively. The remaining drug-lipid particle preparation processes were the same as those of Example 2.

[0384] U251 cells were cultured at 1 × 10 per well. 6The cells were seeded into a 6-well plate at a density of 1000 x g / mL. After about 24 hours, the cells in each well were incubated with siRNA@MPNP (the concentration of siRNA@MPNP was 2 μg / mL) or ASO@MPNP (the concentration of ASO was 2 μg / mL) for 72 hours, respectively. Then, the cells were collected, and the total cellular RNA was extracted. The mRNA expression levels of the target genes (Bcl-2, STAT3) were detected using RT-PCR technology, respectively, and the ability of siRNA@MPNP or ASO@MPNP to silence the cellular target genes was calculated. The results are shown in Figures 1 to 13 in Example 2.6 and Figures 1 to 19 in Example 2.7.

[0385] 293T cells were incubated with S-mRNA @MPNPs at a concentration of 2 μg / mL (mRNA concentration), while the control group was incubated with MPNPs. After 24 hours, the cells were centrifuged and the supernatant was frozen at -20°C for later use. The cell pellet was resuspended in 100 μL of PBS buffer, frozen and thawed twice, sonicated for 10 minutes, and then centrifuged to collect the supernatant. The expression levels of S protein in both the cell supernatant and lysate were detected using a commercially available novel coronavirus S protein ELISA detection kit. The results are shown in Figures 1-25.

[0386] After incubating A549 lung cancer cells with ds-DNA@MPNPs at a concentration of 100 nM (concentration of DNA contained) for 2 hours, the drug-lipid particles were removed and washed twice with PBS. After staining the cell nuclei with Hochest33342 dye for 3 minutes, the dye was removed and the cells were washed twice with PBS. The cells were observed using a high-content imaging system, and the transfection efficiency of the drug-lipid particles into DNA was calculated. The results are shown in Figures 1-26.

[0387] After incubating ss-DNA@MPNPs with HT22 mouse hippocampal neurons at a concentration of 200 nM (concentration of DNA) for 2 hours, the drug-lipid particles were removed and the cells were washed twice with PBS. The cells were then observed using a high-content imaging system to calculate the transfection efficiency of the drug-lipid particles into DNA. The results are shown in Figures 1-26.

[0388] The method for culturing human glioblastoma U251 cells was the same as in Example 2.6.

[0389] The method for culturing 293T cells was the same as in Example 2.5.

[0390] HT22 mouse hippocampal neurons are cultured in DMEM medium containing 10% FBS and penicillin-streptomycin under conditions of 37°C and 5% CO2.

[0391] The RT-PCR method was the same as in Example 2.6.

[0392] To detect the expression level of S protein by ELISA, replace the "anti-RBD antibody working solution" in the method for detecting RBD by ELISA in Example 2.5 with the "anti-S protein antibody working solution," and the remaining steps are the same as in Example 2.5.

[0393] The method for calculating gene silencing efficiency is the same as in Example 2.6.

[0394] The transfection efficiency was calculated by randomly selecting 3 to 5 fields of view using a high-content imaging system, and obtaining the cell morphology under a normal light source, the fluorescent signal when the excitation light / emission light in the same field of view was 550 nm / 570 nm (excitation light for the fluorescent dye Cy3 that labels DNA), and the fluorescent signal when the excitation light / emission light in the same field of view was 352 nm / 461 nm (excitation light for the fluorescent dye Hoechst33342 that labels cell nuclei).Then, the transfection efficiency was calculated as the ratio of the number of cells with Cy3 fluorescent signals among the cells in the randomly selected field of view to the number of cells with Hoechst33342 fluorescent signals among the cells in the same field of view.

[0395] As shown in Figures 1-13 of Example 2.6, the drug (double-stranded RNA)-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP) inhibited the target gene Bcl-2 by 67%. As shown in Figure 1-19 of Example 2.7, the drug (single-stranded DNA)-metal-polyphenol complex particles (STAT3-ASO@MPNP) inhibited the target gene STAT3 by 72%. As shown in Figure 1-25, the S protein expression level in the supernatant of 293T cells transfected with drug (single-stranded RNA)-metal-polyphenol complex particles (S-mRNA@MPNP) was 134 ng / mL, while the S protein content in the supernatant of 293T cells transfected with empty carrier MPNP was 0. The drug (double-stranded DNA)-metal-polyphenol complex particles (dsDNA@MPNP) transfected double-stranded DNA into cells with 100% efficiency (Figure 1-26). The transfection efficiency of single-stranded DNA into cells using drug (ssDNA)-metal-polyphenol complex particles (ssDNA@MPNP) was 100% (Figure 1-26). This result suggests that the drug-metal-polyphenol complex particles can encapsulate any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and realize its function. The length of the nucleic acid can vary from 16 to 3822 nt.

[0396] Example 2.8.2 Metal ion is Al 3+ Preparation of drug (different kinds of nucleic acid)-metal-polyphenol complex particles and their effects when

[0397] The difference between this example and Example 2.8.1 is that the metal ion Fe in Example 2.8.1 3+ Al 3+ is to be replaced by

[0398] As shown in Figure 1-16 of Example 3.6.2, the drug (double-stranded RNA)-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP) inhibited the target gene Bcl-2 by 72%. As shown in Figure 1-22 of Example 3.7.2, the drug (single-stranded DNA)-metal-polyphenol complex particles (STAT3-ASO@MPNP) inhibited the target gene STAT3 by 75.4%. As shown in Figure 1-27, the S protein expression level in the supernatant of 293T cells transfected with drug (single-stranded RNA)-metal-polyphenol complex particles (S-mRNA@MPNP) was 157 ng / mL, while the S protein content in the supernatant of 293T cells transfected with empty carrier MPNP was 0. The drug (double-stranded DNA)-metal-polyphenol complex particles (dsDNA@MPNP) transfected double-stranded DNA into cells with 100% efficiency (Figure 1-28). The transfection efficiency of single-stranded DNA into cells using drug (ssDNA)-metal-polyphenol complex particles (ssDNA@MPNP) was 100% (Figure 1-28). This result suggests that drug-metal-polyphenol complex particles can encapsulate any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and achieve its function. The length of the nucleic acid can vary from 16 to 3822 nt.

[0399] Experimental Example 2: Performance Characterization of Drug-Metal-Polyphenol Complex Particles

[0400] Example 3 Synthesis and Characterization of Metal-Polyphenol Complexes

[0401] Example 3.1 Metal ion is Fe 3+ Synthesis and characterization of metal-polyphenol complexes in the case of

[0402] Curcumin and Fe 3+ The binding of Fe was characterized spectrophotometrically. As shown in Figure 2-1, curcumin binds Fe. 3+After binding with Fe, its maximum absorption wavelength shifted from 420 nm to 372 nm, and the conjugated structure of the metal-polyphenol complex changed. This is because curcumin binds to Fe. 3+ and has demonstrated successful conjugation.

[0403] Example 3.2 Metal ion is Al 3+ Synthesis and characterization of metal-polyphenol complexes in the case of

[0404] Curcumin and Al 3+ The binding of Al was characterized by spectrophotometry. As shown in Figure 2-2, curcumin binds Al. 3+ After binding with Al, its maximum absorption wavelength shifted from 420 nm to 433 nm, and the conjugated structure of the metal-polyphenol complex changed. 3+ and has demonstrated successful conjugation.

[0405] Example 4: Fe released from metal-polyphenol complexes under low pH conditions 3+ Characterization

[0406] Curcumin in the metal-polyphenol complex is coordinated with Fe 3+ Under the low pH conditions of lysosomes, curcumin and Fe bind to 3+ The coordination bond between the Fe and the polyphenol is protonated (absorbs a hydrogen ion) and broken. 3+ To prove that Fe is indeed released from the lipid complex through the above mechanism, the following experiment was designed. The color of the metal-polyphenol complex was observed under physiological pH (pH = 7.4) and lysosomal low pH (pH = 5.0). As shown in Figures 2-3, the color of the metal-polyphenol complex changed from reddish brown to bright yellow under lysosomal low pH (pH = 5.0). 3+ This result indicates that Fe is already released from the complex under the low pH conditions of the lysosome. 3+ This suggests that metal-polyphenol complexes may be shed.

[0407] Fe under low pH conditions 3+ The mechanism by which Fe is released from the metal-polyphenol complex is 3+ The coordinate bond between these two molecules is protonated at low pH (pH = 5.0). In other words, curcumin has many protons (H + ) and Fe 3+ The coordination bond between Fe and curcumin is broken, which results in the formation of Fe 3+ was isolated from curcumin and ultimately Fe 3+ is separated from the metal-polyphenol complex (Figure 2-3).

[0408] Example 5 Metal ion is Fe 3+ or Al 3+ Nucleic acid (siRNA and mRNA) encapsulation efficiency of drug-metal-polyphenol complex particles MPNP in the presence of HCl and its comparison with LNP

[0409] The mRNA in Example 2 was replaced with siRNA (SEQ ID NO. 4, 21 bp) targeting the Bcl-2 gene and mRNA (SEQ ID NO. 2, 669 nt) encoding the receptor binding domain (RBD) of the S1 subunit of the novel coronavirus, respectively, to produce drug-metal-polyphenol complex particles encapsulating nucleic acids, siRNA@MPNP and mRNA@MPNP, respectively. The remaining manufacturing process of the drug-metal-polyphenol complex particles was the same as in Example 2.

[0410] siRNA@LNPs and mRNA@LNPs were prepared using the same drug loading as the Bcl-2-siRNA@MPNPs in Example 2.6 and the RBD-mRNA@MPNPs in Example 2.5. The specific method is as follows: An organic phase solution was prepared according to the Onpattro lipid nanoparticle formulation: the ionizable lipid ALC0315, DSPE-PEG2000, DSPC, and cholesterol were dissolved in ethanol at a molar ratio of 50%:1.5%:10%:38.5%. Bcl-siRNA or RBD-mRNA was added to the aqueous phase (0.1 M acetic acid-sodium acetate buffer, pH 4.0). The ratio of amino lipids to phosphate-containing nucleotides (N / P) was 6:1, ensuring that the nucleic acid drug loading was the same as that of the siRNA@MPNPs and mRNA@MPNs described above. The aqueous and organic phases were rapidly mixed at a volume ratio of 3:1 and a flow rate of 14 ml / min. After mixing, the mixture was diluted 10-fold with pH 7.4 Tris-HCl buffer and concentrated to 1 / 10 using a 100 kDa ultrafiltration tube. After repeating the dilution and concentration procedure three times, the ethanol concentration in the mixture was adjusted to 0.0005% or less, and the pH value of the solution was raised to the normal pH value of Tris-HCl buffer (7.2-7.4), to obtain siRNA@LNP and mRNA@LNP, respectively.

[0411] The encapsulation rates of nucleic acids (siRNA and mRNA) in siRNA@MPNP, mRNA@MPNP, 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 the lipid nanoparticles in each group was set to 10 μg / mL. The mass ratio of lipid to nucleic acid was adjusted so that the metal in the metal-polyphenol complex was Fe. 3+ In the case of the metal-polyphenol complex, the mass ratio is 20:1, and the metal in the metal-polyphenol complex is Al 3+The mass ratio for the positive control was 18:1. The positive control was a nucleic acid solution in pH 5.0 Tris-HCl buffer, while the negative control was a nucleic acid-free PBS buffer solution. The agarose gel concentration was 1.5%, and the voids in the gel allowed only the free nucleic acid to pass through, but not the lipid nanoparticles. Electrophoresis was stopped when the free nucleic acid bands were clearly distinguishable to prevent degradation of the nucleic acid due to excessive electrophoresis time. Image J software was used to calculate the gray values of the free nucleic acids in the different groups. The positive control group was set to 100%, and the ratio of the free nucleic acid in each group to the positive control was calculated as the relative amount of free nucleic acid. The encapsulation rate for each group was calculated as (100 - relative amount of free nucleic acid)%.

[0412] The results were analyzed as shown in Figures 2-4. 3+ ) were 87.78% and 83.17%, respectively, and the efficiency of MPNPs (Al 3+ The efficiencies of LNPs encapsulating siRNA and mRNA were 87.32% and 79.94%, respectively. These results suggest that there is no significant difference in the encapsulation efficiency of nucleic acids between MPNPs and LNPs.

[0413] Example 6 Metal ions are Fe 3+ or Al 3+ Nucleic acid lysosomal escape ability of drug-metal-polyphenol complex particles MPNP in comparison with LNP

[0414] The Bcl-2-siRNA (SEQ ID NO. 4) in Example 2.6 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@MPNPs (the siRNA concentration was 100 nM). The Bcl-2-siRNA (SEQ ID NO. 4) in Example 5 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@LNPs (the siRNA concentration was 100 nM). The eGFP-mRNA (SEQ ID NO. 1) in Example 2.5 was replaced with Cy5-labeled mRNA (Cy5-mRNA) to prepare Cy5-mRNA@MPNPs (the mRNA concentration was 2 μg / mL). The RBD-mRNA (SEQ ID NO. 2) in Example 5 was replaced with Cy5-labeled mRNA (Cy5-mRNA) to prepare Cy5-mRNA@LNPs (the mRNA concentration was 2 μg / mL). After incubating A549 cells with the lysosomal probe Lysotracker Green for 3 hours, a high-content imaging system was used to observe the overlap of the Cy5 fluorescent signal (red) and the Lysotracker Green fluorescent signal (green) to determine and study the ability of the drug-lipid particles to promote nucleic acid lysosomal escape.

[0415] The ability of drug-metal-polyphenol conjugate particles to promote lysosomal escape of nucleic acids was assessed by incubating cells with drug-metal-polyphenol conjugate nanoparticles for 3 hours, then using a high-content imaging system to observe the overlap of the Cy5 fluorescent signal (red) and the Lysotracker Green fluorescent signal (green). ImageJ software was then used to calculate the overlap rate between the red and green fluorescent signals. After incubating cells with drug-metal-polyphenol conjugate particles for 3 hours, the overlap rate between the red and green fluorescent signals was less than 50%, suggesting that nucleic acids were able to escape from cellular lysosomes relatively quickly and that the drug-metal-polyphenol conjugate particles had a relatively good ability to promote lysosomal escape of nucleic acids.

[0416] The results were analyzed using Cy5-siRNA@MPNP(Fe 3+ ) and Cy5-mRNA@MPNP(Fe 3+ When A549 cells were incubated with Cy5-siRNA@MPNPs (Al) for 3 hours, the overlap rates of red and green fluorescent signals were 39.20% ± 8.89% and 44.96% ± 3.85%, respectively, i.e., the lysosomal escape rates were 60.80% ± 8.89% and 55.04% ± 3.85%, respectively. 3+ ) and Cy5-mRNA@MPNP(Al 3+ When A549 cells were incubated with Cy5-siRNA@LNPs and Cy5-mRNA@LNPs for 3 hours, the overlap rates of red and green fluorescent signals were 34.70% ± 4.98% and 39.10% ± 4.43%, respectively, i.e., the lysosomal escape rates were 65.30% ± 4.98% and 60.90% ± 4.43%, respectively. When A549 cells were incubated with Cy5-siRNA@LNPs and Cy5-mRNA@LNPs for 3 hours, the overlap rates of red and green fluorescent signals were 76.02% ± 7.90% and 85.33% ± 4.87%, respectively, i.e., the lysosomal escape capacities were 23.98% ± 7.90% and 14.67% ± 4.87%, respectively. This suggests that the drug-lipid nanoparticles MPNPs have a relatively superior ability to promote nucleic acid lysosomal escape, and that the ability of MPNPs to promote lysosomal escape is significantly stronger than that of LNPs.

[0417] Example 7 Metal ion is Fe 3+ or Al 3+ Nucleic acid expression promoting ability of drug-metal-polyphenol complex particles MPNP in the presence of HCl and its comparison with LNP

[0418] The RBD-mRNA (SEQ ID NO. 2) in Example 5 was replaced with mRNA encoding the fluorescent protein eGFP, and the remaining production methods were the same as in Example 5 to obtain eGFP-mRNA@LNP.

[0419] The eGFP-mRNA@MPNPs prepared in Example 2.5 and the above-mentioned eGFP-mRNA@LNPs (containing 2 μg / mL of mRNA) were incubated with 293T cells, respectively. The control group was incubated with MPNPs or LNPs. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0420] The method for analyzing the rate of eGFP-positive cells by flow cytometry is as described in Example 2. The results were analyzed as shown in Figure 2-6. 3+ ), MPNP(Al 3+ When 293T cells were treated with MPNPs and LNPs, the percentage of eGFP-positive cells was 93.47%, 97.06%, and 63.09%, respectively. This result suggests that the function of MPNPs in promoting lysosomal expression is superior to that of LNPs. A possible reason for this is that, as described in Example 6, MPNPs have a stronger ability to promote the escape of nucleic acids from lysosomes than LNPs, so more nucleic acids loaded into MPNPs are effectively released into the cytoplasm and can be translated into proteins.

[0421] Example 8 Ability of drug-metal-polyphenol complex particles MPNP to promote humoral and cellular immunity, and its comparison with LNP

[0422] 293T cells were incubated with the RBD-mRNA@MPNPs in Example 2.5 and the RBD-mRNA@LNPs in Example 5 at a concentration of 2 μg / mL (mRNA concentration), while the control group was incubated with only MPNPs. After 24 hours, the cells were centrifuged to collect the supernatant and frozen at -20°C for later use. The cell pellet was resuspended in 100 μL of PBS buffer, frozen and thawed twice, sonicated for 10 minutes, and then centrifuged to collect the supernatant. The expression levels of RBD protein in both the cell supernatant and cell lysate were detected using a commercially available novel coronavirus antigen RBD ELISA detection kit. The results are shown in Figures 2-7.

[0423] The method for detecting RBD expression levels by ELISA is as described in Example 2.5.

[0424] The experimental animals were randomly divided into three groups (experimental group and control group), with five animals assigned to each group. The animal model was BALB / c mice, and each mouse received the first intramuscular injection on day 1 and the second intramuscular injection on day 14. The experimental group received RBD-mRNA@MPNP (Fe 3+ ), RBD-mRNA@MPNP(Al 3+ ) or RBD-mRNA@LNP, respectively, and the control group was injected with MPNPs and LNPs not loaded with mRNA. The volume per injection was 100 μL, and the experimental group was injected with RBD-mRNA@MPNP (Fe 3+ ), RBD-mRNA@MPNP(Al 3+ The RBD-mRNA@LNP formulations each contained 30 mg of mRNA. Blood samples were collected from the mice 28 days after the first administration, and serum samples were separated and gradient diluted. The titers of RBD total IgG antibodies against the S1 subunit of the novel coronavirus produced in the mice were detected using a commercially available ELISA kit. The results are shown in Figures 2-8.

[0425] The method for detecting the titer of RBD total IgG antibodies against the S1 subunit of the novel coronavirus by ELISA is as described in Example 2.5.

[0426] RBD-mRNA@MPNP (Fe 3+ ), RBD-mRNA@MPNP(Al 3+ On the 28th day after administration of RBD-mRNA@LNP, spleens from normal mice were harvested and prepared into single cell suspensions under sterile conditions. 100,000 spleen cells were plated in well plates at a final concentration of 10 mg / mL of RBD protein. The cells were then cultured for 48 hours, centrifuged, and the supernatant was removed. The expression levels of IFN-γ, IL-2, and IL-4 were measured using ELISA kits. The results are shown in Figures 2-9.

[0427] The method for detecting the expression levels of IFN-γ, IL-2, and IL-4 by ELISA is as described in Example 2.5.

[0428] The results were analyzed using RBD-mRNA@MPNP(Fe 3+ ), RBD-mRNA@MPNP(Al 3+ Both RBD-mRNA@MPNP (Al) and RBD-mRNA@LNP can induce a certain level of RBD expression in 293T cells. 3+ ) was found to be able to induce RBD expression in cells. 3+ ) and RBD-mRNA@MPNP(Fe 3+ The ability of RBD-mRNA@MPNP(Fe) to induce RBD expression in cells was significantly stronger than that of RBD-mRNA@LNP. 3+ The RBD expression level in the cell supernatant of the RBD-mRNA@MPNP(Al 3+ The expression level of RBD in the cell supernatant of the RBD-mRNA@MPNP (Al)-treated group was 215 ng / mL, and the expression level of RBD in the cell supernatant of the RBD-mRNA@LNP-treated group was 115.67 ng / mL. As shown in Figure 2-8, RBD-mRNA@MPNP effectively induced humoral immunity in mice, producing high levels of antigen-specific binding antibodies, and RBD-mRNA@MPNP (Al 3+ The ability of RBD-mRNA@MPNP(Fe) to induce humoral immunity in mice was 3+ ) and RBD-mRNA@MPNP(Fe 3+ ) is clearly superior to RBD-mRNA@LNP in its ability to induce humoral immunity in mice. 3+ The IgG antibody titer in mice treated with RBD-mRNA@MPNP(Al 3+The IgG antibody titer in mice treated with RBD-mRNA@MPNP reached 96418, while that in mice treated with RBD-mRNA@LNP was only 67476. As shown in Figure 2-9, RBD-mRNA@MPNP could effectively induce cellular immunity in mice, activating immune cells and producing a large amount of cytokines, and the mRNA@MPNP(Al 3+ The ability of RBD-mRNA@MPNP(Fe) to induce cellular immunity in mice was 3+ ) and RBD-mRNA@MPNP(Fe 3+ ) is clearly superior to RBD-mRNA@LNP in its ability to induce cellular immunity in mice. 3+ ) resulted in the expression levels of cytokines IFN-γ, IL-2, and IL-4 reaching 274.4 pg / mL, 254.2 pg / mL, and 77.4 pg / mL, respectively. 3+ The expression levels of cytokines IFN-γ, IL-2, and IL-4 by RBD-mRNA@LNP reached 309 pg / mL, 299 pg / mL, and 91.2 pg / mL, respectively. However, the expression levels of cytokines IFN-γ, IL-2, and IL-4 by RBD-mRNA@LNP were only 104.2 pg / mL, 79.2 pg / mL, and 27 pg / mL. These results suggest the following: 3+ ) delivers any mRNA and its function is demonstrated by RBD-mRNA@MPNP(Fe 3+ ) and RBD-mRNA@MPNP(Fe 3+) is significantly superior to RBD-mRNA@LNPs in inducing cellular immunity in mice. RBD-mRNA@MPNPs can more effectively promote cellular expression of target proteins and activate humoral and cellular immunity in the body, making these drug (mRNA)-lipid particles significantly superior to conventional LNPs in the role of mRNA-carrying drugs, vaccines, and other products. Possible reasons for this are as follows: 1) Compared to LNPs, MPNPs have a stronger ability to promote lysosomal escape of nucleic acids. 2) Compared to LNPs, MPNPs have a stronger ability to promote nucleic acid expression into proteins (antigens). 3) Compared to LNPs, the released curcumin in MPNPs acts as an immune adjuvant (also known as an immunomodulator) that not only activates humoral and cellular immunity to enhance the delivery effect of MPNP-mediated mRNA vaccines, but also suppresses the storm of immune factors, suppressing excessive and harmful immune responses against organisms.

[0429] Example 9 In vivo safety evaluation of metal-polyphenol complex particles (MPNPs)

[0430] SD rats were used as study subjects. 3+ or Al 3+ A 20-day subchronic toxicity test was conducted on the rats, followed by a 20-day recovery period. The specific experimental method is as follows:

[0431] Fifty-six SPF-grade SD rats (220 ± 20 g), half male and half female, were housed at 25°C, 45%–55% humidity, and 12-hour light exposure. After 3–5 days of adaptation, they were randomly assigned to groups according to sex: 32 rats in the experimental group and 24 rats in the recovery group. Fourteen rats (half male and half female) were assigned to the blank control group (control), including 8 rats in the experimental group and 6 rats in the recovery group. Fourteen rats (half male and half female) were assigned to the low-dose MPNP group (25 mg / kg), including 8 rats in the experimental group and 6 rats in the recovery group. Fourteen rats (half male and half female) were assigned to the medium-dose MPNP group (50 mg / kg), including 8 rats in the experimental group and 6 rats in the recovery group. The high-dose group (100 mg / kg) consisted of 14 animals (8 experimental and 6 recovery), half male and half female. The experimental group (32 animals in total) was dissected and sampled after administration was completed, while the recovery group (24 animals in total) was dissected and sampled after 20 days of normal care following administration.

[0432] The experimental animals were administered the compound via tail vein injection every other day for a total of 20 days, and the body weights of the SD rats were recorded once a week. The prepared MPNPs were dissolved in Tris-HCl buffer (pH 7.4). The control group was injected with the same amount of Tris-HCl buffer, while the low-dose, medium-dose, and high-dose MPNP groups were injected with 8 mg / kg, 16 mg / kg, and 32 mg / kg of MPNPs, respectively.

[0433] The basis for the above MPNP dosage setting is that when 200 μg / kg of mRNA (the amount actually required in mRNA animal experiments) is contained, the required amount of empty carrier MPNP is 8 mg / kg. To fully demonstrate the safety of MPNP, 1, 2, and 4 times the dose of MPNP actually required in animal experiments, i.e., 8 mg / kg, 16 mg / kg, and 32 mg / kg, were selected and administered.

[0434] The general indicator detection method involves observing the general condition of each group of animals after each administration, including their survival status, feeding status, external characteristics, behavioral activity, body weight, and whether or not there is any local reaction to the administration. During autopsy, a gross dissection is performed, including timely weighing of the wet weights of major organs such as the brain, heart, liver, spleen, lungs, and kidneys, calculating the organ-to-body ratio, and recording pathological changes in each organ. Organ-to-body ratio = rat organ wet weight / rat body weight × 100%.

[0435] After 20 days of treatment and a 20-day recovery period, SD rats were anesthetized with isoflurane, fixed to a dissection board, and their abdomens disinfected with 75% ethanol. The rat's abdomen was incised with sterile ophthalmic scissors, and the internal organs gently pried open using a cotton ball to expose the abdominal aorta. Whole blood was collected using a 500 μL negative-pressure EDTAK2 anticoagulant blood collection tube and stored at 4°C for routine blood testing. Whole blood was collected using a 5 mL negative-pressure standard blood collection tube, allowed to stand at room temperature for 30 minutes, and centrifuged at 1500 rpm for 15 minutes at 4°C. The supernatant was transferred to a 1.5 mL centrifuge tube and stored at -20°C for blood biochemistry and immunological indices.

[0436] Routine blood testing methods include white blood cell count, lymphocyte count, monocyte count, neutrophil count, lymphocyte percentage, monocyte percentage, neutrophil percentage, red blood cell count, hemoglobin, hematocrit, mean red blood cell volume, mean red blood cell hemoglobin content, mean red blood cell hemoglobin concentration, red blood cell distribution width coefficient of variation, platelet count, mean platelet volume, platelet distribution width, and plateletcrit. A small amount of whole blood is collected by gently inverting the whole blood sample and the results are automatically analyzed using a fully automated blood cell analyzer.

[0437] The blood biochemical index detection method is as follows: 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). Serum samples are thawed and centrifuged at 3000 rpm for 15 minutes, and the supernatant is collected and aliquoted for later use. The corresponding parameters are set on the automated biochemistry instrument, and the formulated working fluid is added, followed by the serum to be tested. The automated biochemistry instrument then automatically measures the results.

[0438] Immunological 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 complements (C3, CH50). ELISA is used to detect these indicators.

[0439] The pathological examination method for the major organs of SD rats was as follows: at the end of the administration period and the end of the recovery period, rats in each group were anesthetized, and then the major organs of the rats, including the whole brain, heart, liver, spleen, lungs, and kidneys, were removed with ophthalmic scissors, rinsed lightly with 0.9% saline, fixed in 4% paraformaldehyde fixative, embedded in standard paraffin, and stained with H&E. Histopathological changes in each organ of the rats in the control and experimental groups were observed under an optical microscope.

[0440] As shown in Table 2-1, the results showed that at the end of the administration period and the end of the recovery period, rats in the low-, medium-, and high-dose MPNP groups survived well, had normal diet, normal appearance, and normal behavioral activity, and had no obvious adverse reactions after administration, compared with the control group. There was no significant difference in body weight gain in male and female SD rats in the low-, medium-, and high-dose MPNP groups compared with the control group. There was no significant difference in organ-to-body ratio in the low-, medium-, and high-dose MPNP groups compared with the control group.

[0441] At the end of the administration period and the end of the recovery period, no abnormalities were found in routine blood indices (white blood cell count, lymphocyte count, monocyte count, neutrophil count, lymphocyte percentage, monocyte percentage, neutrophil percentage, red blood cell count, hemoglobin, hematocrit, mean red blood cell volume, mean red blood cell hemoglobin content, mean red blood cell hemoglobin concentration, red blood cell distribution width coefficient of variation, platelet count, mean platelet volume, platelet distribution width, plateletcrit) in the low, medium, and high dose MPNP groups compared to the control group. 2+ , Na + , K. + , Cl - , Ca 2+ No abnormalities were found in blood biochemistry indices, including liver function indices (ALT, AST, γ-GT, T-BIL, D-BIL, ALP, ALB), kidney function indices (BUN, UA, CR), cardiac function indices (LDH, CK), glucose metabolism indices (GSP, GLU, INS), and lipid metabolism indices (CHO, TG, LDL-C, HDL-C).Compared to the control group, no abnormalities were found in immunology-related indices, including thyroid function indices (TT3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN-γ, IFN-α, TNF-α), immunoglobulins (IgG, IgA, IgM), and serum complements (C3, CH50), in the low, medium, and high-dose MPNP groups.

[0442] At the end of the administration period and the end of the recovery period, compared with the control group, the brain tissue structure of rats in the low, medium, and high dose MPNP groups was completely organized, the tissue staining was normal, the cell morphology and structure were completely organized, and there was no nuclear pyknosis or inflammatory cell infiltration; the myocardial tissue structure was completely organized, the myocardial cells were neatly, continuously, and firmly arranged, the cell nuclei were clearly visible, and there was no obvious cell congestion, edema, or necrosis; the hepatic cell morphology was normal, and there was no inflammatory cell aggregation or necrosis; the spleen structure was normal, and there was a clear boundary between the red pulp and the white pulp; the lung tissue structure was completely organized, the alveoli were consistent in size, and there was no obvious inflammatory cell aggregation or infiltration; and the kidney structure was normal.

[0443] The above results show that the effect of MPNP (Fe3+ or Al 3+ This suggests that no obvious chronic toxic reactions were found in long-term, large-dose injections of MPNPs, suggesting that the safety of MPNPs is relatively high. [Table 9] TIFF2025527207000031.tif56162Table 2-1 In vivo safety evaluation of MPNPs NOTE: ALT alanine aminotransferase, AST glutamic oxaloacetic transaminase, γ-GT glutamyl transpeptidase, T-BIL total bilirubin, D-BIL direct bilirubin, ALP alkaline phosphatase, ALB albumin, BUN urea nitrogen, UA uric acid, CR creatinine, LDH lactate dehydrogenase, CK creatine kinase, GSP glycosylated serum protein, GLU glucose, INS insulin, CHO cholesterol, TG triglyceride, 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

[0444] Example 10: Comparison of in vivo safety between metal-polyphenol complex particles (MPNPs) and LNPs

[0445] The main toxicity of LNPs comes from their main components, cationic lipids and / or ionizable lipids. During metabolism of LNPs in the body, free cationic lipids and / or ionizable lipids produce obvious toxicity to organisms. The median lethal dose (IC50) of cationic lipids and / or ionizable lipids to living cells is 50) is an important parameter for evaluating the degree of toxicity of LNP to organisms. The drug-metal-polyphenol complex particles (MPNPs) are prepared by replacing the cationic lipids / ionizable lipids in LNPs with metal-polyphenol complexes, and therefore, the median lethal dose (IC50) of the metal-polyphenol complexes and cationic lipids / ionizable lipids to living cells is evaluated. 50 ) to compare the differences in toxicity between LNPs and MPNPs.

[0446] The metal-polyphenol complexes (0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM), cationic lipids (DOTAP, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM), and ionizable lipids (ALC0315, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM) were incubated with 293T cells for 48 hours, and cell viability was detected using a CCK8 activity detection kit. The median lethal dose (IC50) of the metal-polyphenol complexes, cationic lipids (DOTAP), and ionizable lipids (ALC0315) on 293T cells was calculated. 50 Calculate each.

[0447] The method for detecting CCK8 is as follows:

[0448] Culture the cells in DMEM medium containing 10% FBS and 1% double antibody until the cell density reaches 80%-90% of the culture flask, then set aside for use.

[0449] Wash the remaining medium from the culture flask with PBS, add pancreatin, and quickly transfer the culture flask to a 37°C incubator containing 5% CO2. Carefully observe the cells and, when they become slightly rounded, add culture medium to terminate the digestion. Transfer the cells to a centrifuge tube, centrifuge at 1500 RPM for 5 minutes, and resuspend the cells in fresh medium.

[0450] For counting purposes, the cell suspension is diluted to 10,000 cells per mL, 100 μL per well of a 96-well plate, and at least 5 duplicate wells per group. After 24 hours of incubation at 37°C, 5% CO2, drugs are added.

[0451] After incubating the drug for 48 hours, 10% CCK8 is added and incubated for 1 to 3 hours, and the absorbance at 450 nm is measured using a microplate reader.

[0452] Viability (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] × 100%.

[0453] I C 50 The IC was calculated using Graphpad with the [Inhibitor] vs. normalized response -- Variable slope analysis method, with survival rate on the vertical axis and drug concentration on the horizontal axis. 50 Calculate.

[0454] To compare the in vivo safety of MPNPs and LNPs, MPNPs (8 mg / kg) and LNPs (3.24 mg / kg) capable of carrying the same amount of nucleic acid (200 μg / kg mRNA) were used, and in vivo experiments were performed according to the method in Example 9 to evaluate and compare the in vivo toxicity of MPNPs and LNPs.

[0455] The results were analyzed and shown in Table 2-2. 50 is significantly greater than that of the cationic lipid (DOTAP) and the ionizable lipid (ALC0315), suggesting that the toxicity of the metal-polyphenol complex is significantly less than that of the cationic lipid and the ionizable lipid, and that its effect is significant.

[0456] As shown in Table 2-3, at the end of the administration period and the end of the recovery period, the MPNP (Fe 3+) or MPNP(Al 3+ ) group, no obvious abnormalities were found in the expression levels of liver function indicators ALT, AST, ALP, and cytokines IL-6 and IL-1β. However, compared with the control group, the expression levels of liver function indicators ALT, AST, ALP, and cytokines IL-6 and IL-1β expression levels were significantly increased in the LNP group. This result is indicative of the MPNP (Fe 3+ ) or MPNP(Al 3+ ) suggests that the in vivo safety of MPNPs (Fe) is higher than that of LNPs. This is because the core component of LNPs is an artificially synthesized "cationic lipid / ionizable lipid," which has relatively high cytotoxicity and immunogenicity, and its structure is relatively stable and is not easily degraded or metabolized in the body. 3+ ) or MPNP(Al 3+ The core component of MPNPs (Fe) is a metal-polyphenol complex, which consists of a highly safe natural small molecule substance, curcumin (an FDA-approved food additive and pharmaceutical excipient), and safe metal ions, and is already decomposed into natural molecules in the body after drug delivery is completed. 3+ ) or MPNP(Al 3+ ) does not contain cationic lipids / ionizable lipids, which prevents the toxic side reactions associated with cationic lipids / ionizable lipids, and therefore, the MPNPs (Fe 3+ ) or MPNP(Al 3+ ) is safer than LNP. [ka] [Table 10] Table 2-2 Metal ions are Fe 3+ or Al 3+ IC of metal-polyphenol complexes with cationic lipid (DOTAP) and ionizable lipid (ALC0315) when 50 Comparison of [Table 11] Table 2-3 Metal ions are Fe 3+ or Al 3+ Comparison of experimental chronic toxicity indicators of MPNP and LNP when

[0457] Example 3 Clinical application and administration route of drug-metal-polyphenol complex particles MPNP

[0458] Example 11 Metal ion is Fe 3+ or Al 3+ Clinical application and administration route of drug-metal-polyphenol complex particles MPNP in the case

[0459] The mRNA in Example 2 was replaced with siRNA targeting the B7-H4 gene (B7-H4-siRNA) and its control (scr-siRNA), as well as mRNA encoding the receptor binding domain (RBD) of the S1 subunit of the novel coronavirus (RBD-mRNA).

[0460] The sequences of the above different nucleic acids are as follows: (1) The sequences of B7-H4-siRNA are SEQ ID No. 19 (sense strand) and SEQ ID No. 26 (antisense strand) (25 bp), and its random control sequence is SEQ ID No. 20 (sense strand) and SEQ ID No. 27 (antisense strand) (19 bp). (2) The mRNA sequence encoding the receptor binding domain (RBD) of the S1 subunit of the novel coronavirus is SEQ ID No. 2 (669 nt). Drug-metal-polyphenol complex particles (B7-H4-siRNA @MPNP(Fe)) encapsulating the above different types of nucleic acids were prepared according to the method of Example 2. 3+ ), RBD-mRNA@MPNP(Fe 3+ ), B7-H4-siRNA @MPNP (Al 3+ ), RBD-mRNA@MPNP(Al 3+The remaining drug-metal-polyphenol complex particle manufacturing processes were the same as in Example 2. The above two different drug-metal-polyphenol complex particles (B7-H4-siRNA@MPNP, RBD-mRNA@MPNP) are used for the treatment of liver cancer and as an mRNA vaccine to prevent the new coronavirus, respectively.

[0461] The sequence of B7-H4-siRNA is as follows:

[0462] sense 5'-GGG AGA CAC UCC AUC ACA GUC ACU A -3'(SEQ ID No.19).

[0463] antisense 5'-UAG UGA CUG UGA UGG AGU GUC UCC C-3'(SEQ ID No.26)(25bp).

[0464] The random control sequence of B7-H4-siRNA is as follows:

[0465] sense 5'-UUCUCCGAACGUGUCACGU-3'(SEQ ID No.20).

[0466] antisense 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID No. 27) (19bp).

[0467] B7-H4-siRNA@MPNP(Fe) in the treatment of liver cancer 3+ ) and B7-H4-siRNA@MPNP(Al 3+ To evaluate the role of IL-1 in liver cancer, we use HepG2 cells to create an animal model of liver cancer. The tumor size is approximately 100 mm. 3 Once the tumor growth rate reached 100%, the mice were randomly divided into seven groups (5 mice per group): a Tris-HCl buffer control group, a blank carrier MPNP (Fe 3+ ) group, blank carrier MPNP(Al 3+ ) group, Scr-siRNA@MPNP(Fe3+ ) control group, B7-H4-siRNA@MPNP(Fe 3+ ) treatment group, Scr-siRNA@MPNP(Al 3+ ) control group, B7-H4-siRNA@MPNP(Al 3+ ) treatment group. Mice in each group were treated every 3 days with pH 7.4 Tris-HCl buffer, MPNP (Fe 3+ ), MPNP(Al 3+ ), Scr-siRNA@MPNP(Fe 3+ ), B7-H4 siRNA@MPNP(Fe 3+ ), Scr-siRNA@MPNP(Al 3+ ), B7-H4 siRNA@MPNP(Al 3+ ) was injected intratumorally once, with a dose of 200 μg siRNA / kg for 8 injections. Tumor volume was measured and recorded every 3 days. The results are shown in Figure 3-1.

[0468] To evaluate the role of RBD-mRNA@MPNPs as an mRNA vaccine in preventing novel coronavirus, the experimental process and method were as described in the previous Example 2.5.

[0469] The ELISA detection method is as described in Example 2.5.

[0470] Construction of liver cancer mouse model: Collect 1 x 10 HepG2 cells. 7 Resuspend the cells in PBS at a density of 100 µL / mL and store on ice before inoculation. Then, inject 100 µL of the cell suspension subcutaneously into the back area near the hind leg of female Balb / c nude mice to establish a liver cancer mouse model.

[0471] The result analysis is

[0472] As shown in Figure 3-1, Scr-siRNA@MPNP(Fe 3+ ) and Scr-siRNA@MPNP(Al 3+ ) had little inhibitory effect on the growth of liver cancer HepG2 cells, whereas B7-H4-siRNA@MPNP(Fe 3+) and B7-H4 siRNA@MPNP(Al 3+ ) showed an efficient therapeutic effect and could effectively inhibit the growth of liver cancer tumors. These results suggest that the drug-metal-polyphenol complex particles could encapsulate and deliver B7-H4 siRNA and inhibit the development of liver cancer by inhibiting the expression of the target gene.

[0473] As shown in the previous Example 2.5, Figures 1-3 and 1-5, RBD-mRNA@MPNP(Fe 3+ ) induced the expression level of mouse IgG antibody to reach 84363.4 (Figure 1-3), and the expression levels of cytokines IFN-γ, IL-2, and IL-4 to reach 271.8 pg / mL, 269.6 pg / mL, and 75.8 pg / mL, respectively (Figure 1-5). 3+ ) induced mouse IgG antibody expression levels of 94828.6 (Figure 1-17), and cytokine expression levels of IFN-γ, IL-2, and IL-4 reached 306.2 pg / mL, 289.6 pg / mL, and 88.2 pg / mL, respectively (Figure 1-19). These results suggest that RBD-mRNA@MPNPs can effectively induce humoral immunity in mice, producing high levels of antigen-specific binding antibodies, while also effectively inducing cellular immunity in mice, activating immune cells and producing large amounts of cytokines. Therefore, RBD-mRNA@MPNPs can effectively prevent novel coronavirus infection.

[0474] As shown in Figure 3-1, B7-H4-siRNA@MPNPs can effectively treat liver cancer via intratumoral injection. As shown in Example 2.5 and Figures 1-3, 1-5, 1-17, and 1-19, RBD-mRNA@MPNPs can activate humoral and cellular immunity via intramuscular injection, thereby preventing COVID-19 infection. These results suggest that drug-metal-polyphenol complex particles can be administered via multiple routes.

[0475] Example 4 Curcumin, Fe 3+ Function after replacement by its analogue

[0476] Example 12 Curcumin, Fe 3+ Function after replacement by its analogue

[0477] See Example 1 for curcumin, Fe 3+ Curcumin and Fe, respectively 3+ Using the same compounds as those listed in Example 2, nine different drug-metal-polyphenol complex particles (eGFP-mRNA@MPNPs) were prepared using different combinations, with the mRNA concentration of each eGFP-mRNA@MPNP being 2 μg / mL. 3+ The names and structures of the compounds and their analogues are shown in Table 4-1. 3+ The combinations of the compounds and the like are shown in Table 4-2. The reaction temperature in Example 1 is 60°C, the reaction time is 2 hours, and the other conditions are the same.

[0478] To compare the effects of the nine different eGFP-mRNA@MPNPs and eGFP-mRNA@LNPs, LNPs containing the same amount of eGFP mRNA were prepared with reference to Example 5 to obtain eGFP-mRNA@LNPs.

[0479] The nine different eGFP-mRNA@MPNPs and the eGFP-mRNA@LNPs (each containing 2 μg / mL of mRNA) were incubated with 293T cells, respectively. Control groups were incubated with MPNPs or LNPs. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0480] The method for analyzing the proportion of eGFP-positive cells by flow cytometry is as described in Example 2.

[0481] The main toxicity of LNP comes from its main component, cationic lipids / ionizable lipids. During metabolism of LNP in the body, free cationic lipids / ionizable lipids cause obvious toxicity to organisms. The median lethal dose (IC50) of cationic lipids / ionizable lipids to living cells is 50 ) is an important parameter for evaluating the degree of toxicity of LNP to organisms. The metal-polyphenol complex particles (MPNPs) are prepared by replacing the cationic lipids / ionizable lipids in LNPs with metal-polyphenol complexes. Therefore, the median lethal dose (IC50) of the nine metal-polyphenol complexes and cationic lipid (DOTAP) / ionizable lipid (ALC0315) in Table 4-2 to living cells was evaluated. 50 ) to compare the differences in toxicity between LNPs and nine types of MPNPs.

[0482] The method for calculating IC50 is as described in Example 10.

[0483] As shown in Table 4-3, after 293T cells were treated with nine different eGFP-mRNA@MPNPs, the percentage of eGFP-positive cells was significantly higher than that of eGFP-mRNA@LNPs, and the percentage of eGFP-positive cells was highest in mRNA@MPNP1. This result was consistent with the results of curcumin, Fe 3+ This suggests that the function of mRNA@MPNPs formed after the replacement of mRNA@MPNPs by their analogs is inferior to that of mRNA@MPNP1 but slightly superior to that of mRNA@LNPs. A possible reason for this is that, as described in Example 5, MPNPs have a stronger ability than LNPs to promote lysosomal escape of nucleic acids, so that more nucleic acids loaded by MPNPs are effectively released into the cytoplasm and can be translated into proteins.

[0484] The above results suggest that as long as the following conditions are met, curcumin, Fe 3+These results suggest that the functionality of the drug-metal-polyphenol complex particles formed after substitution of Fe with their congeners is not affected. (1) Curcumin congeners are hydrophobic polyphenols that can be conjugated with metals. (2) Fe 3+ (3) Curcumin and Fe 3+ The coordination bond between can be cleaved in response to the low pH environment of the lysosome.

[0485] As shown in Table 4-3, the IC values of nine metal-polyphenol complexes 50 The toxicity of metal-polyphenol complexes is significantly lower than that of cationic lipids (DOTAP) and ionizable lipids (ALC0315). This indicates that the toxicity of metal-polyphenol complexes is significantly lower than that of cationic lipids and ionizable lipids, i.e., curcumin, Fe 3+ It has been suggested that lipid particles (MPNPs) composed of hydroxybenzoates and their analogues are safer than LNPs. This is because the core component of LNPs is an artificially synthesized "cationic lipid / ionizable lipid," which has relatively high cytotoxicity and immunogenicity, a relatively stable structure, and is not easily degraded or metabolized in the body. On the other hand, the core component of MPNPs is a metal-polyphenol complex, which is composed of a non-cationic lipid, a highly safe natural small molecule substance (curcumin is an FDA-approved food additive and pharmaceutical excipient), and a safe metal ion, and is already degraded into natural molecules in the body after drug delivery is complete. Therefore, it is possible to confirm the safety of curcumin, Fe, and other similar lipids. 3+ The components of lipid particles (MPNPs) made of LNPs and their analogues do not contain cationic lipids / ionizable lipids, and therefore do not cause toxic side reactions associated with cationic lipids / ionizable lipids, making MPNPs safer than LNPs. [Table 12] Table 4-1 Curcumin, Fe 3+ and the names and structures of the same [Table 13] Table 4-2 Curcumin, Fe 3+ List of combination methods and functions of metal-polyphenol complexes in drug-lipid nanoparticles prepared from these and their analogues [Table 14] Table 4-3 Curcumin, Fe 3+ IC of metal-polyphenol complexes produced from the same and their analogues 50

[0486] Example 13 Different metal-polyphenol complexes: curcumin, Fe 3+ Ingredient input ratio and function of drug-metal-polyphenol complex particles produced therefrom

[0487] Example 13.1 Metal ion is Fe 3+ Polyphenols of different metal-polyphenol complexes when Fe 3+ Ingredient input ratio and function of drug-metal-polyphenol complex particles produced therefrom

[0488] Metal-polyphenol complexes were prepared according to Example 2.1, and three types of metal-polyphenol complexes (mRNA@MPNP1, mRNA@MPNP4, and mRNA@MPNP7) were prepared by replacing curcumin with its analogues, hesperetin (one molecule of hesperetin contains four hydroxyl groups) and catechin (one molecule of catechin contains five hydroxyl groups). When preparing these three types of metal-polyphenol complexes, curcumin or its analogues and Fe 3+The input ratios of these three types of metal-polyphenol complexes (mRNA@MPNP1, mRNA@MPNP4, and mRNA@MPNP7) were used to produce the corresponding drug-metal-polyphenol complex particles. The mRNA encodes the eGFP fluorescent protein, and its sequence is SEQ ID NO. 1 (720 nt). The experimental procedures and methods described in Example 2.5 were used to detect the mRNA encapsulation rates of these three types of drug-lipid particles and their ability to promote eGFP fluorescent protein expression after treatment of 293T cells.

[0489] Analysis of the results, as shown in Table 4-4, showed that the mRNA encapsulation efficiency and ability to promote protein expression (i.e., positive cell rate) of drug-metal-polyphenol complex particles manufactured at different dosage ratios based on the chemical structure of the metal-polyphenol complex components were comparable. This result suggests that the dosage ratio of the metal-polyphenol complex components can be adjusted according to the structure of the specific metal-polyphenol complex components. The reasons for adjusting the dosage ratio are as follows: the hydroxyl group of curcumin analogues and Fe 3+ Congeners of curcumin are linked by coordination bonds, so if curcumin congeners contain multiple binding sites, the interaction between curcumin congeners and Fe 3+ The input ratio of the congeners can be adjusted based on the number of binding sites contained in the curcumin congener. [Table 15] Table 4-4 Metal ions are Fe 3+ Ingredient input ratios of different metal-polyphenol complexes and the function of drug-lipid particles prepared therefrom

[0490] Example 13.2 Metal ion is Al 3+ Polyphenols of different metal-polyphenol complexes when Al 3+ Ingredient input ratio and function of drug-metal-polyphenol complex particles produced therefrom

[0491] According to Example 2.2, metal-polyphenol complexes were prepared, and curcumin was replaced with its analogues, hesperetin (one molecule of hesperetin contains four hydroxyl groups) and catechin (one molecule of catechin contains five hydroxyl groups), to produce three types of metal-polyphenol complexes (mRNA@MPNP3, mRNA@MPNP6, and mRNA@MPNP9). When preparing these three types of metal-polyphenol complexes, curcumin or its analogues and Al 3+ The input ratios of these three types of metal-polyphenol complexes (mRNA@MPNP3, mRNA@MPNP6, and mRNA@MPNP9) were 1:1, 1:1, and 1:2, respectively. The corresponding drug-metal-polyphenol complex particles were then prepared using these three types of metal-polyphenol complexes (mRNA@MPNP3, mRNA@MPNP6, and mRNA@MPNP9). The mRNA encodes eGFP fluorescent protein, and its sequence is SEQ ID NO. 1 (720 nt). The experimental procedures and methods described in Example 2.5 were used to determine the mRNA encapsulation rate of these three types of drug-lipid particles and their ability to promote the expression of eGFP fluorescent protein after treatment of 293T cells.

[0492] Analysis of the results, as shown in Tables 4-5, showed that the mRNA encapsulation efficiency and ability to promote target protein expression of drug-metal-polyphenol complex particles prepared at different dosage ratios based on the chemical structure of the metal-polyphenol complex components were comparable. This result suggests that the dosage ratio of the metal-polyphenol complex components can be adjusted according to the structure of the specific metal-polyphenol complex components. The reasons for adjusting the dosage ratio are as follows: the hydroxyl group of curcumin analogues and Al 3+ Congeners of curcumin are linked by coordination bonds, so if the congener contains multiple binding sites, the interaction between the congener and Al 3+ The input ratio of the congeners can be adjusted based on the number of binding sites contained in the curcumin congener. [Table 16] Table 4-5 Metal ions are Al 3+Ingredient input ratios of different metal-polyphenol complexes and the function of drug-lipid particles prepared therefrom

[0493] The preparation of mRNA@MPNPs in Example 2 above was completed by the research group of Professor Wang Shan of the Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Central South University.

[0494] Unless otherwise defined, all technical and scientific terms used throughout this disclosure have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of any discrepancy, the meaning described in the entire disclosure or the meaning derived from the contents described in the entire disclosure shall prevail. Furthermore, the terms used herein are intended to describe the embodiments of this disclosure and are not intended to limit this disclosure.

[0495] It should be noted that the above are merely preferred embodiments of the present disclosure and the technical principles utilized. Those skilled in the art will understand that the present disclosure is not limited to the specific embodiments described herein, and that various obvious modifications, rearrangements, and substitutions can be made by those skilled in the art without departing from the scope of the present disclosure. Therefore, although the present disclosure has been described in relatively detail through the above embodiments, the present disclosure is not limited to the above embodiments, and may include other equivalent embodiments without departing from the technical spirit of the present disclosure, all of which are within the protection scope of the present disclosure.

Claims

1. The application of a metal-polyphenol complex in a nucleic acid delivery system, The metal-polyphenol complex is formed by a reaction between a polyphenol molecule portion and a metal ion portion, and the polyphenol molecule portion and the metal ion portion are linked by a coordinate bond.

2. 2. The application of claim 1, wherein the polyphenol molecule moiety is one or a combination of two or more selected from the group consisting of curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, taxifolin, brown algae polyphenols, polyflavanol polyphenols, catechin, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, glucogallin, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate, gallocatechin gallate, and derivatives thereof.

3. The polyphenol molecule portion may be selected from the group consisting of curcumin (formula 1), quercetin (formula 2), kaempferol (formula 3), rutin (formula 4), hesperetin (formula 5), naringenin (formula 6), eriodictyol (formula 7), luteolin (formula 8), apigenin (formula 9), taxifolin (formula 10), brown algae polyphenols (formula 11), polyflavanol polyphenols (formula 12), catechin (formula 13), ellagic acid (formula 14), 3. The application of claim 2, wherein the compound is one or a combination of compounds selected from the group consisting of gallic acid (Formula 15), digallic acid (Formula 16), propyl gallate (Formula 17), epigallocatechin gallate (Formula 18), glucogallin (Formula 19), hydroxyhydroquinone (Formula 20), morin (Formula 21), epicatechin gallate (Formula 22), catechin gallate (Formula 23), gallocatechin gallate (Formula 24), and derivatives thereof. 【Chemical 1】 【change】 【change】 【change】

4. 4. The method of claim 3, wherein the polyphenol moiety is one or a combination of two or more selected from the group consisting of curcumin (Formula 1), dihydrocurcumin (Formula 25), hexahydrocurcumin (Formula 26), curcumin sulfate (Formula 27), and bisdemethoxycurcumin (Formula 28). 【Chemistry 2】

5. 4. The application of claim 3, wherein the polyphenol molecule moiety is one or a combination of two or more selected from the group consisting of curcumin (Formula 1), hesperetin (Formula 5), and catechin (Formula 13), and derivatives thereof.

6. 6. The method of claim 5, wherein the polyphenol moiety is selected from curcumin (formula 1), hesperetin (formula 5) or catechin (formula 13).

7. The metal ion moiety is 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+ The application according to any one of claims 1 to 6, wherein the application is one or a combination of several selected from the following:

8. The metal ion moiety is Fe 3+ , Ca 2+ , Al 3+ The application according to claim 7, wherein the compound is one or a combination of compounds selected from the following:

9. The metal ion moiety is Fe 3+ , Ca 2+ or Al 3+ The application according to claim 8, wherein the application is selected from the group consisting of:

10. The metal-polyphenol complex is formed by the reaction of a polyphenol molecule portion with a metal ion portion, the polyphenol molecule portion being selected from curcumin, hesperetin, and catechin, and the metal ion portion being Fe 3+ , Ca 2+ or Al 3+ 8. The application according to claim 2 or 7, selected from:

11. The metal-polyphenol complex is formed by the reaction of a polyphenol molecular portion with a metal ion portion, the polyphenol molecular portion being selected from curcumin (formula 1), hesperetin (formula 5), or catechin (formula 13), and the metal ion portion being Fe. 3+ , Ca 2+ or Al 3+ The application according to claim 10, selected from the group consisting of:

12. The application of claim 11, wherein the molar ratio of the polyphenol molecule moiety to the metal ion moiety is 1:(0.5-2).

13. The polyphenol molecule portion is curcumin (Formula 1), and the metal ion portion is Fe 3+ 13. The application of claim 12, wherein:

14. Curcumin (Formula 1) and Fe 3+ 14. The application of claim 13, wherein the molar ratio of is 1:

1.

15. The polyphenol molecule portion is curcumin (Formula 1), and the metal ion portion is Al 3+ 13. The application of claim 12, wherein:

16. Curcumin (Formula 1) and Al 3+ 16. The application of claim 15, wherein the molar ratio of is 1:

1.

17. The application of any one of claims 1 to 16, wherein the nucleic acid delivered by the nucleic acid delivery system is one or a combination of multiple nucleic acids selected from mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, and artificial nucleic acids.

18. The nucleic acids include an mRNA sequence encoding eGFP (Enhanced Green Fluorescent Protein) shown in SEQ ID No. 1, an mRNA sequence encoding the receptor-binding domain (RBD) of the S1 subunit of the novel coronavirus shown in SEQ ID No. 2, an mRNA sequence encoding NY-ESO-1 (tumor antigen) shown in SEQ ID No. 3, an siRNA sequence of the Bcl-2 gene (B-cell lymphoma / leukemia-2 gene) whose antisense strand is shown in SEQ ID No. 4 and whose sense strand is shown in SEQ ID No. 21, an siRNA sequence of the PLK1 gene (Polo-like kinase 1) whose antisense strand is shown in SEQ ID No. 6 and whose sense strand is shown in SEQ ID No. 23, an siRNA sequence of the Gal-1 gene shown in SEQ ID No. 8, an ASO sequence of the STAT-3 gene shown in SEQ ID No. 10, an ASO sequence of the α-syn gene (α-synuclein) shown in SEQ ID No. 12, an siRNA sequence of the α-syn gene (α-synuclein) shown in SEQ ID No. 13, an siRNA sequence of the STAT-3 gene shown in SEQ ID No. 14, an siRNA sequence of the α-syn gene (α-synuclein) shown in SEQ ID No. 15, an siRNA sequence of the STAT-3 gene shown in SEQ ID No. 16, an siRNA sequence of the α-syn gene (α-synuclein) shown in SEQ ID No. 17, an siRNA sequence of the STAT-3 gene shown in SEQ ID No. 18, an siRNA sequence of the α-syn gene shown in SEQ ID No. 19, an siRNA sequence of the α-syn gene shown in SEQ ID No. 20, an siRNA sequence of the α-syn gene shown in SEQ ID No. 21, an siRNA sequence of the α-syn gene shown in SEQ ID The application of claim 17, wherein the ASO sequence of the Bcl-2 gene is shown in SEQ ID No. 14, the mRNA sequence encoding the wild-type novel coronavirus S protein is shown in SEQ ID No. 16, the double-stranded DNA sequence of which the antisense strand is SEQ ID No. 17 and the sense strand is shown in SEQ ID No. 25, the single-stranded DNA sequence of which is SEQ ID No. 18, or the siRNA sequence of the B7-H4 gene is shown in SEQ ID No. 19 and the antisense strand is shown in SEQ ID No.

26.

19. 19. The application of any one of claims 1 to 18, wherein the nucleic acid delivery system is used to introduce a nucleic acid into a cell.

20. 20. The application of claim 19, wherein the nucleic acid is used to silence expression of a target sequence in a mammalian subject or to treat a mammalian disease or condition.

21. 21. The application of claim 20, wherein the mammal is a human.

22. 21. The application of claim 20, wherein the disease or condition is associated with expression of a gene, and the gene contains a target sequence for a drug.

23. 23. The application of claim 22, wherein the disease or condition comprises cancer, a viral infection, an autoimmune disease, diabetes or Alzheimer's disease.

24. 24. The application of claim 23, wherein the viral infection comprises Hepatitis A, Hepatitis B, Hepatitis C, SARS-Cov-2, HIV, HPV, influenza, smallpox or syphilis.

25. 24. The application of claim 23, wherein the cancer comprises liver cancer, glioma, melanoma, lung cancer, pancreatic cancer or breast cancer.

26. The application of claim 1 , wherein the nucleic acid delivery system is used in the manufacture of a vaccine.

27. 27. The application of claim 26, wherein the vaccine is a novel coronavirus vaccine.

28. 1. A metal-polyphenol complex particle comprising: The metal-polyphenol complex particles are (i) a metal-polyphenol complex, which is formed by reacting a polyphenol molecular portion with a metal ion portion, and the polyphenol molecular portion and the metal ion portion are linked by a coordinate bond; (ii) a conjugated lipid that inhibits particle aggregation, wherein the conjugated lipid that inhibits particle aggregation is not a cationic lipid or an ionizable lipid; (iii) Metal-polyphenol complex particles containing a non-cationic or non-ionizable lipid other than the conjugated lipid that inhibits particle aggregation.

29. 29. The metal-polyphenol complex particle of claim 28, wherein the polyphenol molecule portion is one or a combination of multiple polyphenols selected from the group consisting of curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, taxifolin, brown algae polyphenols, polyflavanol polyphenols, catechin, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, glucogallin, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate, gallocatechin gallate, and derivatives thereof.

30. The polyphenol molecule portion may be selected from curcumin (Formula 1), quercetin (Formula 2), kaempferol (Formula 3), rutin (Formula 4), hesperetin (Formula 5), naringenin (Formula 6), eriodictyol (Formula 7), luteolin (Formula 8), apigenin (Formula 9), taxifolin (Formula 10), brown algae polyphenols (Formula 11), polyflavanol polyphenols (Formula 12), catechin (Formula 13), ellagic acid (Formula 14), gallic acid (Formula 15), and the like. 5), digallic acid (Formula 16), propyl gallate (Formula 17), epigallocatechin gallate (Formula 18), glucogallin (Formula 19), hydroxyhydroquinone (Formula 20), morin (Formula 21), epicatechin gallate (Formula 22), catechin gallate (Formula 23), gallocatechin gallate (Formula 24), and derivatives thereof.

31. 31. The metal-polyphenol complex particle of claim 30, wherein the polyphenol molecule portion is one or a combination of more selected from curcumin (Formula 1), dihydrocurcumin (Formula 25), hexahydrocurcumin (Formula 26), curcumin sulfate (Formula 27), and bisdemethoxycurcumin (Formula 28).

32. 31. The metal-polyphenol complex particle of claim 30, wherein the polyphenol molecule portion is one or a combination of two or more selected from curcumin (Formula 1), hesperetin (Formula 5), or catechin (Formula 13), and derivatives thereof.

33. 33. The metal-polyphenol complex particle of claim 32, wherein the polyphenol molecule moiety is selected from curcumin (Formula 1), hesperetin (Formula 5), or catechin (Formula 13).

34. The metal ion moiety is 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+ 34. The metal-polyphenol complex particles according to any one of claims 28 to 33, which are one or a combination of two or more selected from the following:

35. The metal ion moiety is Fe 3+ , Ca 2+ , Al 3+ 35. The metal-polyphenol complex particles according to claim 34, which are one or a combination of two or more selected from the following:

36. The metal ion moiety is Fe 3+ , Ca 2+ or Al 3+ 36. The metal-polyphenol complex particles of claim 35, selected from the group consisting of:

37. 37. The metal-polyphenol complex particles of any one of claims 28 to 36, wherein the lipid of the conjugation that inhibits particle aggregation comprises a PEG-lipid conjugate and / or a PEG-DAA.

38. the PEG-lipid conjugate is one or more selected from the group consisting of phosphatidylethanolamine-polyethylene glycol 2000 (Formula 47), phosphatidylethanolamine-polyethylene glycol 700 (Formula 48), phosphatidylethanolamine-polyethylene glycol 1000 (Formula 49), phosphatidylethanolamine-polyethylene glycol 5000 (Formula 50), and derivatives thereof; The metal-polyphenol complex particle of claim 37, wherein R1 and R2 are each independently as follows: 【Chemistry 3】 【change】 【change】

39. 39. The metal-polyphenol complex particles of claim 38, wherein the PEG-lipid conjugate is one or a combination of two or more selected from DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000, and DSPE-PEG5000.

40. 40. The metal-polyphenol complex particle of claim 39, wherein the PEG-lipid conjugate is selected from DSPE-PEG2000 (Formula 58), DSPE-PEG700 (Formula 55), DSPE-PEG1000 (Formula 56) or DSPE-PEG5000 (Formula 57). 【Chemistry 4】 【change】 【change】

41. 41. The metal-polyphenol complex particle according to any one of claims 28 to 40, wherein the non-cationic or non-ionizable lipid described in (iii) is one or a combination of more than one selected from the group consisting of lecithin PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, ceramide-1-phosphate SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysolecithin LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, sphingosine-1-phosphate S1P, and derivatives thereof.

42. The non-cationic lipid or non-ionizable lipid according to (iii) may be lecithin (PC) (formula 29), phosphatidylethanolamine (PE) (formula 30), phosphatidylserine (PS) (formula 31), phosphatidic acid (PA) (formula 32), phosphatidylglycerol (PG) (formula 33), ceramide-1-phosphate (SP) (formula 34), phosphatidylinositol (PI) (formula 35), phosphatidylthreonine (PT) (formula 36), sphingomyelin (SM) (formula 37), lysolecithin (LPC) (formula 38), lysophosphatidylethanolamine (LPE) (formula 39), lysophosphatidylserine (LPS) (formula 40), lysophosphatidyl 42. The metal-polyphenol complex particle according to claim 41, wherein the polyphenol is one or a combination of two or more selected from the group consisting of lysophosphatidyl phosphonate (LPA) (formula 41), lysophosphatidylglycerol (LPG) (formula 42), lysophosphatidylinositol (LPI) (formula 43), lysophosphatidylthreonine (LPT) (formula 44), lysosphingomyelin (LSM) (formula 45), sphingosine-1-phosphate (S1P) (formula 46), and derivatives thereof, and R1 and R2 are each independently a capryl group, a lauroyl group, a tetradecanol group, a palmitoyl group, a stearoyl group, an oleoyl group, a linoleoyl group, an erucoyl group, an arachidonoyl group, or a diphytanoyl group. 【Chemistry 5】 【change】 【change】

43. 43. The metal-polyphenol complex particles of claim 41 or 42, wherein the non-cationic or non-ionizable lipid described in (iii) comprises at least one of cholesterol and its derivatives.

44. 44. The metal-polyphenol complex particle of claim 43, wherein the non-cationic or non-ionizable lipid described in (iii) comprises a combination of cholesterol and one or more selected from DSPC, DSPE, DSPA, or DSPG.

45. 45. The metal-polyphenol complex particle of claim 44, wherein the non-cationic or non-ionizable lipid described in (iii) comprises cholesterol (Formula 59) and one or more combinations selected from DSPC (Formula 51), DSPE (Formula 52), DSPA (Formula 53) or DSPG (Formula 54). 【Chemistry 6】 【change】

46. 46. The metal-polyphenol complex particle of claim 45, wherein the non-cationic or non-ionizable lipid described in (iii) comprises cholesterol (Formula 59) and DSPC (Formula 51).

47. The metal-polyphenol complex is formed by the reaction of a polyphenol molecule portion with a metal ion portion, the polyphenol molecule portion being selected from curcumin, hesperetin, and catechin, and the metal ion portion being Fe 3+ , Ca 2+ or Al 3+ 35. The metal-polyphenol complex particles according to claim 29 or 34, selected from the group consisting of:

48. The metal-polyphenol complex is formed by the reaction of a polyphenol molecular portion with a metal ion portion, the polyphenol molecular portion being selected from curcumin (formula 1), hesperetin (formula 5), or catechin (formula 13), and the metal ion portion being Fe. 3+ , Ca 2+ or Al 3+ 48. The metal-polyphenol complex particles of claim 47, selected from the group consisting of:

49. 49. The metal-polyphenol complex particles of claim 48, wherein the molar ratio of the polyphenol molecule portion to the metal ion portion is 1:(0.5-2).

50. The polyphenol molecule portion is curcumin (Formula 1), and the metal ion portion is Fe 3+ 50. The metal-polyphenol complex particle of claim 49, wherein

51. Curcumin (Formula 1) and Fe 3+ 51. The metal-polyphenol complex particles of claim 50, wherein the molar ratio of is 1:

1.

52. The polyphenol molecule portion is curcumin (Formula 1), and the metal ion portion is Al 3+ 50. The metal-polyphenol complex particle of claim 49, wherein

53. Curcumin (Formula 1) and Al 3+ 53. The metal-polyphenol complex particles of claim 52, wherein the molar ratio of is 1:

1.

54. 44. The metal-polyphenol complex particles of claim 43, wherein the metal-polyphenol complex particles comprise (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the molar fraction of the metal-polyphenol complex in the feedstock is 10% to 20%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the feedstock is 2% to 10%, the molar fraction of the cholesterol in the feedstock is 0% to 48%, and the molar fraction of the non-cationic lipid or the non-ionizable lipid other than cholesterol in the feedstock is 40% to 75%.

55. the metal-polyphenol complex particles comprise (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the molar fraction of the metal-polyphenol complex in the feedstock is 5% or more and less than 10%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the feedstock is 2% to 10%, the molar fraction of the cholesterol in the feedstock is 0% to 48%, and the molar fraction of the non-cationic lipid or non-ionizable lipid other than cholesterol in the feedstock is 30% or more and less than 40%, or 40% to 75%; or 44. The metal-polyphenol complex particles according to claim 43, wherein the metal-polyphenol complex particles comprise (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the molar fraction of the metal-polyphenol complex in the raw material is 10% to 20%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, the molar fraction of the cholesterol in the raw material is 0% to 48%, and the molar fraction of the non-cationic lipid or the non-ionizable lipid other than cholesterol in the raw material is 30% or more but less than 40%.

56. 56. The metal-polyphenol complex particles according to claim 54 or 55, wherein the molar fraction of the metal-polyphenol complex in the raw material is 5% or more but less than 10%, 10% to 15%, or 15% to 20%.

57. 57. The metal-polyphenol complex particles of claim 56, wherein the molar fraction of the metal-polyphenol complex in the raw material is 5%, 10%, or 15%.

58. 56. The metal-polyphenol complex particles of claim 54 or 55, wherein the mole fraction of lipid in the conjugation that inhibits particle aggregation in the raw material is 3% to 5% or 5% to 10%.

59. 59. The metal-polyphenol complex particles of claim 58, wherein the mole fraction of lipid in the conjugation that inhibits particle aggregation in the raw material is 3%, 5%, or 10%.

60. 56. The metal-polyphenol complex particles of claim 54 or 55, wherein the mole fraction of cholesterol in the raw material is 10% to 30%, 30% to 47%, or 10% to 20%.

61. 61. The metal-polyphenol complex particles of claim 60, wherein the mole fraction of cholesterol in the raw material is 10%, 30%, or 47%.

62. 56. The metal-polyphenol complex particles of claim 54 or 55, wherein the molar fraction of non-cationic or non-ionizable lipids other than cholesterol in the raw material is 45% to 55%, 60% to 65%, or 50% to 65%.

63. 63. The metal-polyphenol complex particles of claim 62, wherein the molar fraction of non-cationic or non-ionizable lipids other than cholesterol in the raw material is 45%, 55%, 60% or 65%.

64. The molar fraction of the metal-polyphenol complex in the raw material is 5% or more but less than 10% or 10% to 15%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 5% to 10%, the molar fraction of the cholesterol in the raw material is 10% to 30%, the molar fraction of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 60% to 65%, and the metal ion moiety in the metal-polyphenol complex is Fe 3+ 56. The metal-polyphenol complex particle according to claim 54 or 55, selected from the group consisting of:

65. The molar fraction of the metal-polyphenol complex in the raw material is 15%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 10%, the molar fraction of the cholesterol in the raw material is 10%, the molar fraction of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 65%, and the metal ion moiety in the metal-polyphenol complex is Fe 3+ 65. The metal-polyphenol complex particle of claim 64, selected from the group consisting of:

66. The molar fraction of the metal-polyphenol complex in the raw material is 5%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 5%, the molar fraction of the cholesterol in the raw material is 30%, the molar fraction of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 60%, and the metal ion moiety in the metal-polyphenol complex is Fe 3+ 65. The metal-polyphenol complex particle of claim 64, selected from the group consisting of:

67. The molar fraction of the metal-polyphenol complex in the raw material is 5% or more but less than 10%, or 10%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 5%, the molar fraction of the cholesterol in the raw material is 30% to 47%, the molar fraction of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 45% to 55%, and the metal ion portion in the metal-polyphenol complex is Al. 3+ 56. The metal-polyphenol complex particle according to claim 54 or 55, selected from the group consisting of:

68. The molar fraction of the metal-polyphenol complex in the raw material is 5%, the molar fraction of the particle aggregation-inhibiting conjugated lipid in the raw material is 3%, the molar fraction of the cholesterol in the raw material is 47%, the molar fraction of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 45%, and the metal ion moiety in the metal-polyphenol complex is Al 3+ or The molar fraction of the metal-polyphenol complex in the raw material is 10%, the molar fraction of the particle aggregation-inhibiting conjugated lipid in the raw material is 5%, the molar fraction of the cholesterol in the raw material is 30%, and the molar fraction of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 55%, and the metal ion moiety in the metal-polyphenol complex is Al 3+ 68. The metal-polyphenol complex particle of claim 67, selected from the group consisting of:

69. 69. A method for producing the metal-polyphenol composite particles according to any one of claims 28 to 68, comprising: The method for producing the metal-polyphenol complex particles is obtained by mixing (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid.

70. Step 1: reacting a polyphenol molecule portion with a metal ion portion through a coordinate bond to form a metal-polyphenol complex; Step 2: mixing the metal-polyphenol complex prepared in Step 1 with a conjugation lipid that inhibits particle aggregation, a non-cationic lipid, or a non-ionizable lipid to prepare the metal-polyphenol complex particles; 70. The method of claim 69, comprising:

71. The method of claim 70, wherein polyphenol molecules are dissolved in ethanol, and then metal ions are added to react with the polyphenol molecules to obtain the metal-polyphenol complex.

72. The method of claim 71, wherein the molar ratio of polyphenol molecules to metal ions is 1: (1 to 2).

73. 72. The method of claim 71, wherein the reaction conditions include reacting at 60°C for 1 hour.

74. A drug-lipid particle, the drug-lipid particle comprising: (a) a drug, which is a negatively charged molecule; (b) a metal-polyphenol complex particle according to any one of claims 28 to 68. A drug-lipid particle comprising:

75. 72. The drug-lipid particle of claim 71, wherein the drug is encapsulated in the metal-polyphenol complex particle.

76. 76. The drug-lipid particle of claim 75, wherein the drug is one or a combination of more selected from nucleic acids, proteins, polypeptides, small molecules, nucleic acid analogs, protein analogs, and polypeptide analogs.

77. The drug-lipid particle of claim 76, wherein the nucleic acid is one or a combination of more selected from mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, and artificial nucleic acids.

78. The nucleic acids include an mRNA sequence encoding eGFP shown in SEQ ID No. 1, an mRNA sequence encoding the receptor-binding domain (RBD) of the S1 subunit of the novel coronavirus shown in SEQ ID No. 2, an mRNA sequence encoding NY-ESO-1 shown in SEQ ID No. 3, an siRNA sequence of the Bcl-2 gene shown in SEQ ID No. 4 as an antisense strand and SEQ ID No. 21 as a sense strand, an siRNA sequence of the PLK1 gene shown in SEQ ID No. 6 as an antisense strand and SEQ ID No. 23 as a sense strand, an siRNA sequence of the Gal-1 gene shown in SEQ ID No. 8, an ASO sequence of the STAT-3 gene shown in SEQ ID No. 10, an ASO sequence of the α-syn gene shown in SEQ ID No. 12, an ASO sequence of the Bcl-2 gene shown in SEQ ID No. 14, an mRNA sequence encoding the wild-type novel coronavirus S protein shown in SEQ ID No. 16, a double-stranded DNA sequence shown in SEQ ID No. 17 as an antisense strand and SEQ ID No. 25 as a sense strand, an siRNA sequence of the STAT-3 gene shown in SEQ ID No. 8, an ASO sequence of the α-syn gene shown in SEQ ID No. 12, an ASO sequence of the Bcl-2 gene shown in SEQ ID No. 14, an mRNA sequence encoding the wild-type novel coronavirus S protein shown in SEQ ID No. 16, and a double-stranded DNA sequence shown in SEQ ID No. 17 as an antisense strand and SEQ ID No. 25 as a sense strand. The drug-lipid particle of claim 77, wherein the single-stranded DNA shown in SEQ ID No. 18, or the siRNA sequence of the B7-H4 gene, the sense strand of which is SEQ ID No. 19 and the antisense strand of which is SEQ ID No. 26, is a single-stranded DNA shown in SEQ ID No. 18, or the siRNA sequence of the B7-H4 gene, the sense strand of which is SEQ ID No. 19 and the antisense strand of which is SEQ ID No.

26.

79. The method for producing drug-lipid particles according to any one of claims 74 to 78, wherein the drug is encapsulated in the metal-polyphenol complex particles to obtain the drug-lipid particles.

80. 39. The method of claim 38, wherein the drug-lipid particles are obtained by mixing (a) a drug, (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic or non-ionizable lipid.

81. The method of claim 80, wherein the metal-polyphenol complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic or non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer to form an aqueous phase, and the organic and aqueous phases are uniformly mixed to obtain the drug-lipid particles.

82. 82. The method of claim 81, wherein the organic compound is ethanol.

83. 82. The method of claim 81, wherein the buffer is an enzyme-free Tris-HCl buffer.

84. 82. The method of claim 81, wherein the mixing method of the organic phase and the aqueous phase comprises a microfluidic chip or ultrasound.

85. 69. The application of the metal-polyphenol complex particles according to any one of claims 28 to 68 in drug-lipid particles.

86. 20. The application of the metal-polyphenol complex particles of any one of claims 28 to 68 or the drug-lipid particles of any one of claims 74 to 78 in a composition, comprising: The composition is used in the delivery of a drug application.

87. 87. The application of claim 86, wherein the composition is used to introduce a drug into a cell.

88. 87. The application of claim 86, wherein the composition is a drug.

89. 89. The application of claim 88, wherein the agent is used to silence expression of a target sequence in a mammalian subject, to deliver a drug within a mammalian body, to deliver a drug from the body to mammalian cells, or to treat a disease or condition in a mammal.

90. 90. The application of claim 89, wherein the mammal is a human.

91. 90. The application of claim 89, wherein the disease or condition is associated with expression of a gene, and the gene contains a target sequence for a drug.

92. 90. The application of claim 89, wherein the disease or condition comprises cancer, a viral infection, an autoimmune disease, diabetes, or Alzheimer's disease.

93. 93. The application of claim 92, wherein the viral infection comprises Hepatitis A, Hepatitis B, Hepatitis C, SARS-Cov-2, HIV, HPV, influenza, smallpox or syphilis.

94. 93. The application of claim 92, wherein the cancer comprises liver cancer, glioma, melanoma, lung cancer, pancreatic cancer or breast cancer.

95. 89. The application of claim 88, wherein the agent is a vaccine.

96. 89. The application of claim 88, wherein the route of administration of the agent comprises intrathecal injection, intramuscular injection, intracranial injection, intravenous injection, or intratumoral injection.

97. A pharmaceutical comprising a metal-polyphenol complex particle described in any one of claims 28 to 68 or a drug-lipid particle described in any one of claims 74 to 78.

98. 98. The agent of claim 97, wherein the agent is a vaccine.

99. The method of claim 98, wherein the vaccine is a novel coronavirus vaccine.

100. 8. Use of the metal-polyphenol complex particles of any one of claims 28 to 68 or the drug-lipid particles of any one of claims 74 to 78 in the prevention and / or treatment of a mammalian disease or condition.

101. 101. The application of claim 100, wherein the mammal is a human.

102. The application of claim 100, wherein the disease or condition is associated with expression of a gene, and the gene contains a target sequence for a drug.

103. 101. The application of claim 100, wherein the disease or condition comprises cancer, a viral infection, an autoimmune disease, diabetes, or Alzheimer's disease.

104. 104. The application of claim 103, wherein the viral infection comprises Hepatitis A, Hepatitis B, Hepatitis C, SARS-Cov-2, HIV, HPV, influenza, smallpox or syphilis.

105. 104. The application of claim 103, wherein the cancer comprises liver cancer, glioma, melanoma, lung cancer, pancreatic cancer or breast cancer.

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