Metal-polyphenol composite particles and preparation method and application thereof

Abstract

The present invention relates to the field of biomedicine and specifically provides metal-polyphenol complex particles, their preparation method, and applications. The present invention provides metal-polyphenol complex particles comprising a metal-polyphenol complex, a conjugated lipid that inhibits particle aggregation, and a non-cationic lipid or non-ionizable lipid other than the conjugated lipid that inhibits particle aggregation. These metal-polyphenol complex particles can achieve efficient systemic drug delivery while significantly reducing toxicity compared to LNPs containing cationic or ionizable lipids, enabling safe and effective treatment of diseases or conditions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to Chinese patent application number 202210950180.4 filed with the Patent Office of China on August 9, 2022, entitled “Metal-polyphenol composite particles, preparation method and application thereof”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of biomedicine, and in particular to metal-polyphenol composite particles, a preparation method and application thereof. Background Art

[0004] Nucleic acid drugs are functional DNA or RNA molecules that specifically edit disease-causing genes or proteins through binding, cleavage, knockout, or insertion. The discovery of these nucleic acids not only transcends the traditional notion that nucleic acids only carry genetic information but also provides powerful molecular tools for biomedicine and biosensing.

[0005] Nucleic acid drugs have disadvantages such as being easily rapidly degraded by nucleases in the body, having weak transmembrane ability, and having a short half-life in the blood circulation, which seriously limit their clinical application. Therefore, a safe and effective nucleic acid drug delivery system is one of the hot topics in the development of nucleic acid drugs. At present, carriers that can deliver nucleic acid drugs can be mainly divided into viral vectors and non-viral vectors. Viral vectors (including adenovirus, retrovirus, and lentivirus vectors) will cause an immune response after entering the human body and are now rarely used; the more commonly used non-viral vectors are mainly nanoparticles and small molecule conjugates. Compared with small molecule conjugates directly conjugated to nucleic acid drugs, nanoparticles can more effectively encapsulate nucleic acid drugs, preventing them from being rapidly degraded by nucleases in the body, thereby prolonging their circulation time in the body. The mechanism by which nanoparticles encapsulate nucleic acids is to rely on positively charged cationic lipids to adsorb negatively charged nucleic acids.

[0006] Cationic liposomes are typically composed of a mixture of cationic lipids and co-lipids, such as dioleoylphosphatidylethanolamine (DOPE) and cholesterol, in a specific ratio. Cationic liposomes can be used to deliver genes or drug molecules into target cells. However, during transfection and other processes, cationic liposomes still exhibit a degree of cytotoxicity. While delivering drugs to treat cancer cells, they can also have toxic effects on normal cells, limiting their clinical application. The cascade of reactions induced by cationic liposomes includes the generation of reactive oxygen species, enzyme activation, changes in mitochondrial membrane potential, and apoptosis caused by the release of cytochrome C and caspases.

[0007] In addition, ionizable lipids are lipids containing positively charged ionizable amine groups. They are uncharged under physiological conditions (pH = 7.4), but are protonated and positively charged at lower pH values. Therefore, ionizable lipids can be used to partially or completely replace cationic lipids as the main component of nanoparticles, responsible for adsorbing nucleic acids. When nanoparticles containing ionizable lipids enter the lysosomes of biological cells, the ionizable lipids become positively charged lipids in the low pH environment (pH = 4.0-6.5) within the lysosomes. Although ionizable lipids reduce the cytotoxic and highly inflammatory effects of some permanently positively charged cationic lipids, their cytotoxicity and immunogenicity are still high. Lipid nanoparticles (LNPs) based on cationic lipids and / or ionizable lipids are currently available clinically as nanoparticle nucleic acid drug delivery systems. Cationic lipids and / or ionizable lipids are the main components of LNPs and are responsible for the adsorption of nucleic acids. At the same time, the cytotoxicity and immunogenicity mediated by cationic lipids and / or ionizable lipids are still one of the important reasons for the high toxicity of LNPs.

[0008] Therefore, when using a delivery system to deliver negatively charged drugs (such as nucleic acid drugs, protein drugs, polypeptide drugs, small molecule drugs, etc.), nanoparticle delivery systems developed based on cationic lipids and / or ionizable lipids cannot fundamentally solve the toxicity problem of nanoparticle delivery systems. There is an urgent need for a liposome delivery system with low toxicity that does not use cationic lipids and / or ionizable lipids.

[0009] In view of this, the present invention is proposed. Summary of the Invention

[0010] The object of the present invention is to provide metal-polyphenol composite particles and a preparation method thereof, so as to at least alleviate one technical problem existing in the prior art.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] The present invention provides metal-polyphenol composite particles, wherein the metal-polyphenol composite particles contain:

[0013] (i) a metal-polyphenol complex, which is composed of a polyphenol molecule portion and a metal ion portion reacting with each other, wherein the polyphenol molecule portion and the metal ion portion are connected by a coordination bond;

[0014] (ii) a conjugated lipid that inhibits particle aggregation, wherein the conjugated lipid that inhibits particle aggregation is not a cationic lipid or an ionizable lipid; and

[0015] (iii) Non-cationic lipids or non-ionizable lipids other than conjugated lipids that inhibit particle aggregation.

[0016] In some embodiments, the polyphenol molecule is selected from the group consisting of curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, taxol, brown algae polyphenols, polyflavanol polyphenols, catechins, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, galloylglucose, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate, gallocatechin gallate, and one or more combinations thereof.

[0017] Wherein, the “its” in “and their derivatives” refers to “curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, paclitaxel, brown algae polyphenols, polyflavanol polyphenols, catechin, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, galloylglucose, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate or gallocatechin gallate”. The polyphenol molecular part may be, for example, but not limited to, curcumin, curcumin derivatives, hesperetin, hesperetin derivatives, catechin, catechin derivatives, curcumin and catechin, catechin and catechin derivatives, etc. In the present invention, “and their derivatives” have similar meanings.

[0018] Furthermore, the polyphenol molecular portion is 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), thujafoen (Formula 10), brown algae polyphenols (Formula 11), polyflavanol polyphenols (Formula 12), catechin (Formula 13), ellagic acid (Formula 14), gallic acid (Formula 15), digallic acid (Formula 16), propyl gallate (Formula 17), epigallocatechin gallate (Formula 18), galloylglucose (Formula 19), hydroxyhydroquinone (Formula 20), morin (Formula 21), epicatechin gallate (Formula 22), catechin gallate (Formula 23), gallocatechin gallate (Formula 24), and a combination of one or more of their derivatives.

[0019]

[0020]

[0021]

[0022]

[0023] Furthermore, the polyphenol molecule is partially selected from one or more combinations of curcumin (Formula 1), dihydrocurcumin (Formula 25), hexahydrocurcumin (Formula 26), curcumin sulfate (Formula 27), and bisdemethoxycurcumin (Formula 28).

[0024]

[0025] Furthermore, the polyphenol molecule is partially selected from curcumin (Formula 1), hesperetin (Formula 5) or catechin (Formula 13), and one or more combinations thereof.

[0026] Furthermore, the polyphenol molecule is partially selected from curcumin (Formula 1), hesperetin (Formula 5) or catechin (Formula 13).

[0027] In some embodiments, the metal ion moiety is selected from Fe 3+ 、Ag + 、Ba 2+ , Ca 2+ 、Cd 2+ 、Cu 2+ 、Fe 2+ 、Mn 2+ Mg 2+ 、Mo 2+ 、Zn 2+ , Pt 2+ 、Au 2+ 、Al 3+ 、Ce 3+ 、Co 3+ Cr 3+ 、Eu 3+ 、Gd 3+ 、Ni 3+ 、W 3+ 、V 3+ 、Zr 3+ A combination of one or more of .

[0028] Furthermore, the metal ion is selected from Fe 3+ , Ca 2+ 、Al 3+ A combination of one or more of .

[0029] Furthermore, the metal ion is selected from Fe 3+ , Ca 2+ or Al 3+ .

[0030] In some embodiments, the conjugated lipid that inhibits particle aggregation in (ii) of the metal-polyphenol complex particles comprises polyethylene glycol (PEG)-lipid conjugate and / or PEG-dialkoxypropyl (DAA).

[0031] Furthermore, the PEG-lipid conjugate is 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 a combination of one or more of their derivatives. Wherein, R1 and R2 are independently:

[0032] Capryloyl Lauroyl

[0033] Myristoyl

[0034] Palmitoyl

[0035] Stearyl

[0036] Oleoyl

[0037] Linoleyl

[0038] Erucyl

[0039] Arachidoyl or

[0040] Phytanoyl

[0041]

[0042] Furthermore, the PEG-lipid conjugate is selected from a combination of one or more of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000 or DSPE-PEG5000.

[0043] 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).

[0044]

[0045]

[0046] In some embodiments, the non-cationic lipid or non-ionizable lipid in (iii) of the metal-polyphenol complex particle is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), 1-phosphoceramide (SP), phosphatidylinositol (PI), phosphatidylthreonine (PT), sphingomyelin (SM), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysosphingomyelin (LSM), 1-sphingosine phosphate (S1P), and a combination of one or more of their derivatives.

[0047] Furthermore, in (iii), the non-cationic lipid or non-ionizable lipid is selected from phosphatidylcholine (PC) (Formula 29), phosphatidylethanolamine (PE) (Formula 30), phosphatidylserine (PS) (Formula 31), phosphatidic acid (PA) (Formula 32), phosphatidylglycerol (PG) (Formula 33), 1-phosphoceramide (SP) (Formula 34), phosphatidylinositol (PI) (Formula 35), phosphatidylthreonine (PT) (Formula 36), sphingomyelin (SM) (Formula 37), lysophosphatidylcholine (LPC) (Formula 38), lysophosphatidylethanolamine (LPE) (Formula 39), lysophosphatidylcholine (LPC) (Formula 40), lysophosphatidylethanolamine (LPE) (Formula 41), lysophosphatidylcholine (LPC) (Formula 42), lysophosphatidylethanolamine (LPE) (Formula 43), lysophosphatidylcholine (LPC) (Formula 44), lysophosphatidylethanolamine (LPE) (Formula 45), lysophosphatidylglycerol (PG) (Formula 46), lysophosphatidylcholine (LPC) (Formula 47), lysophosphatidylethanolamine (LPE) (Formula 48), lysophosphatidylcholine (LPC) (Formula 49), lysophosphatidylethanolamine (LPE) (Formula 50), lysophosphatidylglycerol (PG) (Formula 51), lysophosphatidylcholine (SP) (Formula 52), lysophosphatidylcholine (LPC) (Form A combination of one or more of blood phosphatidylserine (LPS) (Formula 40), lysophosphatidic acid (LPA) (Formula 41), lysophosphatidylglycerol (LPG) (Formula 42), lysophosphatidylinositol (LPI) (Formula 43), lysophosphatidylthreonine (LPT) (Formula 44), lyso-sphingomyelin (LSM) (Formula 45), 1-sphingosine phosphate (S1P) (Formula 46), and derivatives thereof; wherein R1 and R2 are independently decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytanoyl.

[0048]

[0049]

[0050]

[0051] It should be noted that, in the present invention, the cis and trans isomers of the components used will not affect the technical effects to be achieved by the protection content of the present invention.

[0052] Furthermore, the non-cationic lipid or non-ionizable lipid in (iii) further comprises at least one of cholesterol and its derivatives.

[0053] In some embodiments, the non-cationic lipid or non-ionizable lipid in (iii) comprises cholesterol and a combination of one or more selected from DSPC, DSPE, DSPA or DSPG.

[0054] Preferably, the cholesterol structural formula is (Formula 59)

[0055] In one embodiment, the non-cationic lipid or non-ionizable lipid in (iii) comprises cholesterol (Formula 59), and a combination of one or more selected from DSPC (Formula 51), DSPE (Formula 52), DSPA (Formula 53) or DSPG (Formula 54);

[0056]

[0057] In some embodiments, the non-cationic lipid or non-ionizable lipid in (iii) comprises cholesterol (Formula 59) and DSPC (Formula 51).

[0058] In some embodiments, the metal-polyphenol complex of the present invention is composed of a polyphenol molecule portion and a metal ion portion, wherein the polyphenol molecule portion is selected from curcumin, hesperidin or catechin, and the metal ion portion is selected from Fe 3+ , Ca 2+ or Al 3+ .

[0059] Furthermore, the metal-polyphenol complex is composed of a polyphenol molecule portion and a metal ion portion, wherein the polyphenol molecule portion is selected from curcumin (Formula 1), hesperidin (Formula 5) or catechin (Formula 13), and the metal ion portion is selected from Fe 3+ , Ca 2+ or Al 3+ .

[0060] Furthermore, the molar ratio of the polyphenol molecule portion to the metal ion portion is 1:(0.5-2).

[0061] Furthermore, the polyphenol molecule portion is curcumin (Formula 1), and the metal ion portion is Fe 3+ .

[0062] Furthermore, curcumin (Formula 1) and Fe 3+ The molar ratio is 1:1.

[0063] Furthermore, hesperetin (Formula 5) and Fe 3+ The molar ratio is 1:1.

[0064] Furthermore, catechin (Formula 13) and Fe 3+ The molar ratio is 1:2.

[0065] Furthermore, the polyphenol molecule portion is curcumin (Formula 1), and the metal ion portion is Al 3+ .

[0066] Furthermore, curcumin (Formula 1) and Al 3+ The molar ratio is 1:1.

[0067] In some embodiments, the metal-polyphenol complex particles are made of (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid (including cholesterol and non-cationic lipids or non-ionizable lipids other than cholesterol), the metal-polyphenol complex has a molar proportion of 10% to 20% in the raw material, the conjugated lipid that inhibits particle aggregation has a molar proportion of 2% to 10% in the raw material, the cholesterol has a molar proportion of 0% to 48% in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol has a molar proportion of 40% to 75% in the raw material.

[0068] In some embodiments, the metal-polyphenol complex particles are made of (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the metal-polyphenol complex accounts for 5% to less than 10% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 2% to 10% by mole in the raw material, the cholesterol accounts for 0% to 48% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 30% to less than 40% or 40% to 75% by mole in the raw material; or

[0069] In some embodiments, the metal-polyphenol complex particles are made of (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the metal-polyphenol complex accounts for 10% to 20% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 2% to 10% by mole in the raw material, the cholesterol accounts for 0% to 48% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 30% to less than 40% by mole in the raw material.

[0070] Furthermore, the metal-polyphenol complex accounts for 5% to less than 10%, 10% to 15% or 15% to 20% by mole in the raw material, preferably 5%, 10% or 15%.

[0071] Furthermore, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 5% or 5% to 10%, preferably 3%, 5% or 10%.

[0072] Furthermore, the molar proportion of cholesterol in the raw material is 10% to 30%, 30% to 47% or 10% to 20%, preferably 10%, 30% or 47%.

[0073] Furthermore, the molar proportion 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%.

[0074] In some embodiments, the metal-polyphenol complex (metal ion portion is selected from Fe 3+ ) accounts for 5% to less than 10% or 10% to 15% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 5% to 10% by mole in the raw material, the cholesterol accounts for 10% to 30% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 60% to 65% by mole in the raw material. Preferably, the polyphenol molecule 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.

[0075] In one embodiment, the metal-polyphenol complex (metal ion portion is selected from Fe 3+ ) accounts for 15% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 10% by mole in the raw material, the cholesterol accounts for 10% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol (such as DSPC, DSPA, DSPE or DSPG) accounts for 65% by mole in the raw material.

[0076] In one embodiment, the metal-polyphenol complex (metal ion portion is selected from Fe 3+ ) has a molar ratio of 5% in the raw material, the conjugated lipid that inhibits particle aggregation has a molar ratio of 5% in the raw material, the cholesterol has a molar ratio of 30% in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol has a molar ratio of 60% in the raw material.

[0077] In some embodiments, the metal-polyphenol complex (metal ion portion is selected from Al 3+ ) accounts for 5% to less than 10% or 10% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 3% to 5% by mole in the raw material, the cholesterol accounts for 30% to 47% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 45% to 55% by mole in the raw material. Preferably, the polyphenol molecule 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.

[0078] In one embodiment, the metal-polyphenol complex (the metal ion portion is selected from Al 3+ ) accounts for 5% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 3% by mole in the raw material, the cholesterol accounts for 47% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 45% by mole in the raw material.

[0079] In one embodiment, the metal-polyphenol complex (the metal ion portion is selected from Al 3+ ) accounts for 10% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 5% by mole in the raw material, the cholesterol accounts for 30% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 55% by mole in the raw material.

[0080] The present invention provides a method for preparing the metal-polyphenol complex particles, comprising mixing (i) a metal-polyphenol complex, (ii) a conjugated lipid for inhibiting particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid to obtain the metal-polyphenol complex particles.

[0081] In some embodiments, the preparation method comprises the following steps:

[0082] Step 1: reacting the polyphenol molecule portion with the metal ion portion through a coordination bond to form a metal-polyphenol complex;

[0083] Step 2: The metal-polyphenol complex prepared in step 1, the conjugated lipid for inhibiting particle aggregation, the non-cationic lipid or the non-ionizable lipid are mixed to prepare the metal-polyphenol complex particles.

[0084] Furthermore, the polyphenol molecules are dissolved in ethanol, and then metal ions are added for reaction; the molar ratio of the polyphenol molecules to the metal ions is 1:(1-2); and the reaction conditions include reacting at 60° C. for 1 hour.

[0085] The present invention provides application of the metal-polyphenol complex particles in drug-lipid particles. The drug-lipid particles are prepared from drugs and metal-polyphenol complex particles.

[0086] Furthermore, the drug is encapsulated in the metal-polyphenol complex particles.

[0087] Furthermore, the drug is selected from one or more combinations of nucleic acids, proteins, polypeptides, small molecules, nucleic acid analogs, protein analogs and polypeptide analogs.

[0088] In some embodiments, the nucleic acid is selected from one or more combinations of mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, and artificial nucleic acid.

[0089] Furthermore, the nucleic acid is 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 novel coronavirus S1 subunit 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) whose antisense chain is SEQ ID No.4 and whose sense chain is SEQ ID No.21, an siRNA sequence of the PLK1 gene (Polo-like Kinase 1) whose antisense chain is SEQ ID No.6 and whose sense chain is SEQ ID No.23, 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, and an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 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 with the antisense chain as SEQ ID No. 17 and the sense chain as SEQ ID NO. 25, the single-stranded DNA shown in SEQ ID No. 18, or the siRNA sequence of the B7-H4 gene with the sense chain as SEQ ID No. 19 and the antisense chain as SEQ ID No. 26.

[0090] The present invention provides a method for preparing the drug-lipid particles, wherein the drug is encapsulated in metal-polyphenol composite particles to obtain the drug-lipid particles.

[0091] In one embodiment, the metal-polyphenol complex particles are 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.

[0092] In one embodiment, 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 lipid or a non-ionizable lipid.

[0093] In one embodiment, a metal-polyphenol complex, a conjugated lipid that inhibits particle aggregation, and a non-cationic lipid or non-ionizable lipid are dissolved in an organic compound to form an organic phase, a drug is dissolved in a buffer to form an aqueous phase, and the organic and aqueous phases are mixed to produce drug-lipid particles. Preferably, the organic compound is ethanol; the buffer is enzyme-free Tris-HCl buffer; and the mixing of the organic and aqueous phases includes microfluidics or ultrasound.

[0094] The present invention provides application of the metal-polyphenol complex particles in a composition, and the composition is used for drug delivery.

[0095] Further, the composition is used to introduce drugs into cells.

[0096] Furthermore, the composition is a medicament.

[0097] Furthermore, the agent is used to silence the expression of a target sequence in a mammalian subject.

[0098] Furthermore, the agent is used to deliver drugs in mammals.

[0099] Furthermore, the agent is used to deliver drugs from within the body to mammalian cells.

[0100] Furthermore, the medicament is used to treat a disease or condition in a mammal.

[0101] Furthermore, the mammal is a human.

[0102] Furthermore, the disease or disorder is associated with the expression of a gene, which comprises a target sequence for the drug.

[0103] Furthermore, the disease or disorder includes cancer, viral infection, autoimmune disease, diabetes or Alzheimer's disease.

[0104] 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.

[0105] Furthermore, the cancer includes liver cancer, glioma, melanoma, lung cancer, pancreatic cancer or breast cancer.

[0106] Furthermore, the agent is a vaccine.

[0107] Furthermore, the administration routes of the agent include intrathecal injection, intramuscular administration, intracranial injection, intravenous injection or intratumoral injection.

[0108] The invention provides a medicament containing the metal-polyphenol composite particles.

[0109] Furthermore, the agent is a vaccine.

[0110] Furthermore, the vaccine is a new coronavirus vaccine.

[0111] Compared with the prior art, the technical effects of the present invention are:

[0112] In the prior art, complexes of metal ions and polyphenol molecules (such as curcumin) are mostly used as active ingredients in the fields of anti-oxidation, anti-inflammatory, and antiviral. However, the author has found through experimental research that the metal-polyphenol complex provided by the present invention can be used in compositions or medicaments. It is mainly used as a carrier of drugs for drug stability, delivery and transportation, etc., and works together with other carriers to achieve effective administration of negatively charged drugs.

[0113] The metal-polyphenol complex particles provided by the present invention have a small diameter suitable for systemic delivery. While ensuring an effectiveness no less than that of LNP, no cationic lipids or ionizable lipids are used. Therefore, compared with LNP, the toxicity of the drug-lipid particles is greatly reduced, the biosafety is significantly improved, and they are more conducive to the delivery of negatively charged drugs in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0115] Figure 1-1 The eGFP-mRNA@MPNP(Fe 3+ ) Statistical graph of the percentage of eGFP-positive cells caused by 293T transfection;

[0116] Figure 1-2 RBD-mRNA@MPNP(Fe 3+ ) Statistical graph of RBD expression caused by 293T transfection;

[0117] Figure 1-3 RBD-mRNA@MPNP(Fe 3+) Statistical graph of the ability to induce humoral immunity;

[0118] Figure 1-4 NY-ESO-1-mRNA@MPNP(Fe 3+ ) Statistical graph of the ability to induce humoral immunity;

[0119] Figure 1-5 RBD-mRNA@MPNP(Fe 3+ ) Statistical graph of the ability to induce cellular immunity;

[0120] Figure 1-6 NY-ESO-1-mRNA@MPNP(Fe 3+ ) Statistical graph of the ability to induce cellular immunity;

[0121] Figure 1-7 The eGFP-mRNA@MPNP (Al provided in Example 2.5.2 of the present invention 3+ ) Statistical graph of the percentage of eGFP-positive cells caused by 293T transfection;

[0122] Figure 1-8 RBD-mRNA@MPNP(Al 3+ ) Statistical graph of RBD expression caused by 293T transfection;

[0123] Figure 1-9 RBD-mRNA@MPNP(Al 3+ ) Statistical graph of the ability to induce humoral immunity;

[0124] Figure 1-10 NY-ESO-1-mRNA@MPNP(Al 3+ ) Statistical graph of the ability to induce humoral immunity;

[0125] Figure 1-11 RBD-mRNA@MPNP(Al 3+ ) Statistical graph of the ability to induce cellular immunity;

[0126] Figure 1-12 NY-ESO-1-mRNA@MPNP(Al 3+ ) Statistical graph of the ability to induce cellular immunity;

[0127] Figure 1-13 Bcl-2-siRNA@MPNP(Fe3+ ) ability to silence target genes;

[0128] Figure 1-14 PLK1-siRNA@MPNP(Fe 3+ ) Statistical graph of the ability to silence target genes;

[0129] Figure 1-15 Gal-1-siRNA@MPNP(Fe 3+ ) Statistical graph of the ability to silence target genes;

[0130] Figure 1-16 Bcl-2-siRNA@MPNP(Al 3+ ) ability to silence target genes;

[0131] Figure 1-17 PLK1-siRNA@MPNP(Al 3+ ) Statistical graph of the ability to silence target genes;

[0132] Figure 1-18 Gal-1-siRNA@MPNP (Al 3+ ) Statistical graph of the ability to silence target genes;

[0133] Figure 1-19 STAT3-ASO@MPNP(Fe 3+ ) Statistical graph of the ability to silence cell target genes;

[0134] Figure 1-20 α-syn-ASO@MPNP(Fe 3+ ) Statistical graph of the ability to silence cell target genes;

[0135] Figure 1-21 Bcl-2-ASO@MPNP(Fe 3+ ) Statistical graph of the ability to silence cell target genes;

[0136] Figure 1-22 STAT3-ASO@MPNP (Al 3+ ) Statistical graph of the ability to silence cell target genes;

[0137] Figure 1-23 α-syn-ASO@MPNP(Al 3+) Statistical graph of the ability to silence cell target genes;

[0138] Figure 1-24 Bcl-2-ASO@MPNP(Al 3+ ) Statistical graph of the ability to silence cell target genes;

[0139] Figure 1-25 For the S-mRNA@MPNP(Fe 3+ ) Statistical graph of S protein expression caused by 293T transfection;

[0140] Figure 1-26 The drug (dsDNA and ssDNA)-metal-polyphenol complex particles (Fe 3 + )’s functional test result diagram;

[0141] Figure 1-27 For S-mRNA@MPNP(Al in Example 2.8.2 of the present invention 3+ ) Statistical graph of S protein expression caused by 293T transfection;

[0142] Figure 1-28 The drug (dsDNA and ssDNA)-metal-polyphenol complex particles (Al 3 + )’s functional test result diagram;

[0143] Figure 2-1 The metal-polyphenol complex (Fe 3+ )’s UV absorption pattern;

[0144] Figure 2-2 The metal-polyphenol complex (Al 3+ )’s UV absorption pattern;

[0145] Figure 2-3 The Fe 3+ Characterization images of shedding from metal-polyphenol complex;

[0146] Figure 2-4 Statistical graph of the efficiency of drug-lipid particles encapsulating nucleic acids (mRNA and siRNA) in Example 5 of the present invention;

[0147] Figure 2-5 The results and statistical graphs of the nucleic acid lysosomal escape ability test of siRNA / mRNA@MPNP and siRNA / mRNA@LNP in Example 6 of the present invention are shown;

[0148] Figure 2-6 This is a statistical graph showing the eGFP-positive cell rate results of MPNP and LNP in Example 7 of the present invention;

[0149] Figure 2-7 This is a statistical graph showing the ability of MPNP and LNP to promote mRNA expression in Example 8 of the present invention;

[0150] Figure 2-8 This is a statistical graph showing the ability of MPNP and LNP to promote humoral immunity in Example 8 of the present invention;

[0151] Figure 2-9 The results of the ability of MPNP and LNP to promote cellular immunity in Example 8 of the present invention are statistically analyzed;

[0152] Figure 3-1 This is a statistical chart showing the results of the intratumoral injection of drug-metal-polyphenol complex particles for the treatment of liver cancer in Example 11 of the present invention. DETAILED DESCRIPTION

[0153] definition

[0154] For ease of explanation, the specific terms described in this specification, the embodiments, and the appended claims are collectively described here. Unless otherwise defined in this specification, the meanings of the scientific and technical terms used herein are the same as those understood and used by those skilled in the art. In addition, unless the context otherwise requires, it should be understood that singular terms should include the same plural forms, and plural terms should include the singular. Specifically, unless the context clearly indicates otherwise, the terms "at least one (kind)" and "one (kind) or more (kinds)" used herein and in the appended claims include one (kind), two (kinds), three (kinds), or more (kinds).

[0155] Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the numerical values ​​of the specific examples are presented herein as precisely as possible. However, any numerical value inherently contains standard deviations that are inherent to the testing methods used. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within an acceptable standard error of the mean, as determined by one of ordinary skill in the art. Except in the experimental examples, or unless otherwise expressly indicated, all ranges, amounts, values, and percentages used herein (e.g., to describe material amounts, time periods, temperatures, operating conditions, quantitative ratios, and the like) are to be understood as modified by the word "about." Therefore, unless otherwise indicated, the numerical parameters disclosed in this specification and the accompanying claims are approximate and are subject to change as needed. At a minimum, these numerical parameters should be understood to include the number of significant digits indicated and to apply normal rounding.

[0156] All ranges provided herein are intended to include each specific range within a given range and combinations of subranges between the given ranges. Furthermore, unless otherwise indicated, all ranges provided herein are inclusive of the endpoints of the ranges. Thus, a range of 1-5 specifically includes 1, 2, 3, 4, and 5, as well as subranges such as 2-5, 3-5, 2-3, 2-4, 1-4, and so on.

[0157] All publications and patent applications cited in this specification are herein incorporated by reference, and each individual publication or patent application is specifically and individually indicated to be incorporated by reference for any and all purposes. In the event of any inconsistency between this document and any publication or patent application incorporated by reference, this document controls.

[0158] The term "lipid" refers to a group of organic compounds, including, but not limited to, lipids of fatty acids. These are generally divided into three categories: "simple lipids," "complex lipids," and "derivative lipids." "Simple lipids" include glycerides, glycerides of fatty acids, waxes, long-chain fatty acids, and long-chain alcohols or sterols; "complex lipids" refer to molecules containing fatty acids and alcohols as well as non-lipid components, including phospholipids and glycolipids; and "derivative lipids" are derived from simple or complex lipids.

[0159] The term "lipid vesicle" refers to any lipid composition that can be used to deliver a compound, including, but not limited to, liposomes, in which an aqueous volume is encapsulated by an amphiphilic lipid bilayer; or in which lipids encapsulate an interior containing macromolecular components, such as mRNA, with a reduced aqueous interior; or lipid aggregates or micelles, in which the encapsulated components are contained in a relatively chaotic lipid mixture. Herein, metal-polyphenol complex particles (MPNPs) are "lipid vesicles," and drugs, such as nucleic acid mRNA, are encapsulated in MPNPs as encapsulated components, and the "encapsulation" can be full encapsulation and / or partial encapsulation.

[0160] The phrase "part of a polyphenol molecule" herein refers to the structure originally belonging to the polyphenol molecule after the polyphenol molecule reacts with other substances.

[0161] The phrase "metal ion moiety" herein refers to the structure originally belonging to the metal ion after the metal ion moiety reacts with other substances.

[0162] The phrase "metal-polyphenol complex" herein is composed of the reaction of the polyphenol molecule portion and the metal ion portion, and the polyphenol portion and the metal ion portion are connected via a coordinate bond.

[0163] The term "ionizable lipid" refers to a lipid containing a positively charged ionizable amine group that can be protonated to become positively charged at lower pH values ​​but is uncharged at physiological pH conditions.

[0164] The term "neutral lipid" refers to any of a number of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0165] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-lauroylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic groups attached to neutral lipids.

[0166] The term "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH. These lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol) and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). The following lipids are cationic and have a positive charge below physiological pH: DODAP, DODMA, DMDMA, etc.

[0167] The term "hydrophobic lipid" refers to a compound having a non-polar group, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and these groups are optionally substituted with one or more aromatic, alicyclic or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerols, dialkylglycerols, NN-dialkylamino, 1,2-diacyloxy-3-aminopropane and 1,2-dialkyl-3-aminopropane.

[0168] The term "non-cationic lipid or non-ionizable lipid" refers to a lipid that is neither a cationic lipid nor a non-ionizable lipid, and may be, for example, an anionic lipid or a neutral lipid.

[0169] Among the components of the metal-polyphenol complex particles, the "non-cationic lipids or non-ionizable lipids other than the conjugated lipids that inhibit particle aggregation" in (iii) means that the non-cationic lipids or non-ionizable lipids in (iii) are the lipids remaining in the metal-polyphenol complex particles after excluding the conjugated lipids that inhibit particle aggregation.

[0170] The term "fusogenicity" refers to the ability of a liposome, drug-lipid particle or other drug delivery system to fuse with a cell membrane. The membrane may be the plasma membrane or the membrane surrounding a cell organelle, such as an endosome, nucleus, or the like.

[0171] In the metal-polyphenol complex particles, non-cationic lipids or non-ionizable lipids other than the conjugated lipids that inhibit particle aggregation are mainly present as vesicle-forming lipids. The term "vesicle-forming lipid" is intended to include any amphiphilic lipid having a hydrophobic part and a polar head group and which can spontaneously form bilayer vesicles in water, exemplified by most phospholipids.

[0172] In the metal-polyphenol complex particles, the conjugated lipids that inhibit particle aggregation are primarily present as vesicle-adopting lipids. The term "vesicle-adopting lipid" is intended to include any amphiphilic lipid that stably associates with the lipid bilayer, as well as other amphiphilic lipids whose hydrophobic portion contacts the inner, hydrophobic region of the bilayer membrane and whose polar head group portion faces the outer, polar surface of the membrane. Vesicle-adopting lipids include lipids that are independently capable of adopting a non-lamellar phase and are also capable of adopting a bilayer structure in the presence of a bilayer-stabilizing component. Conjugated lipids that inhibit drug-lipid particle aggregation include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid derivatives), peptides, proteins, detergents, lipid derivatives, PEG-lipid derivatives such as PEG conjugated to dialkoxypropyl groups, PEG conjugated to diacylglycerols, PEG conjugated to phosphatidylethanolamine, and PEG conjugated to ceramide (see, U.S. Patent No. 5,885,613, incorporated herein by reference).

[0173] The term "amphipathic lipid" refers to any suitable material, wherein the hydrophobic portion of the lipid material is oriented toward the hydrophobic phase, while the hydrophilic portion is oriented toward the hydrophilic phase. Amphipathic lipids are typically the main components of lipid vesicles. The hydrophilic nature comes from the presence of polar or charged groups such as carbohydrates, phosphates, carboxyls, sulfates, aminos, sulfhydryls, nitros, hydroxyls, and other similar groups. Hydrophobicity can be imparted by the inclusion of non-polar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphipathic compounds include, but are not limited to, phospholipids, amino lipids, and sphingolipids. The representative examples of phospholipid include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus are also in the group that is called as amphipathic lipid such as sphingomyelin, sphingosylglycolipid family, diacylglycerol and beta-acyloxy acid. In addition, above-mentioned amphipathic lipid can be mixed with other lipid, and this lipid comprises triglyceride and sterol.

[0174] The term "diacylglycerol" refers to a compound having a 2-fatty acyl chain, wherein R1 and R2 each independently have 2 to 30 carbon atoms bonded to the 1- and 2-positions of glycerol via ester bonds. The acyl groups may be saturated or have varying degrees of unsaturation. Diacylglycerol has the following formula 60:

[0175]

[0176] The term "diacylglycerol-conjugated polyethylene glycol" refers to the conjugated lipid that inhibits particle aggregation in the present invention, which can be a diacylglycerol-conjugated polyethylene glycol, i.e., a diacylglycerol-polyethylene glycol conjugate (DAG-PEG conjugate or PEG-DAG conjugate). In a preferred embodiment, the DAG-PEG conjugate is a dilaurylglycerol (C12)-PEG conjugate, a ditetradecylglycerol (C14)-PEG conjugate (DMG), a dipalmitoylglycerol (C16)-PEG conjugate, or a distearylglycerol (C18)-PEG conjugate (DSG). Those skilled in the art will readily appreciate that other diacylglycerols can be used in the DAG-PEG conjugates of the present invention. Suitable DAG-PEG conjugates for use in the present invention and methods of preparing and using them are disclosed in U.S. Application No. 10 / 136,707, published as USPA 2003 / 0077829, and PCT Patent Application No. CA 02 / 00669, each of which is incorporated by reference in its entirety.

[0177] The term "dialkoxypropyl" refers to a compound having a 2-alkyl chain, wherein R1 and R2 each independently have 2 to 30 carbon atoms. The alkyl group may be saturated or have varying degrees of unsaturation. Dialkoxypropyl has the following formula 61:

[0178]

[0179] The term "dialkoxypropyl-coupled PEG" refers to the conjugated lipid that inhibits particle aggregation in the present invention, which can be a dialkoxypropyl-coupled PEG, i.e., a dialkoxypropyl conjugate (PEG-DAA conjugate). In a preferred embodiment, the PEG-DAA conjugate has the following formula 62:

[0180] In formula 62, R1 and R2 are independently selected and are long-chain alkyl groups having from about 10 to about 22 carbon atoms. The long-chain alkyl group can be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, lauryl (C12), tetradecyl (C14), hexadecyl (C16), octadecyl (C18), and icosyl (C20). In a preferred embodiment, R1 and R2 are the same, i.e., both R1 and R2 are tetradecyl (i.e., dioctadecyl), both R1 and R2 are octadecyl (i.e., dioctadecyl), etc. In formula 62, PEG is a polyethylene glycol having an average molecular weight of from about 550 to about 10,000 daltons and is optionally substituted at the terminal hydroxyl position by an alkyl, alkoxy, acyl, or aryl group. In a preferred embodiment, PEG has an average molecular weight of from about 1,000 to about 5,000 daltons, more preferably, an average molecular weight of from about 1,000 to about 3,000 daltons, and even more preferably, an average molecular weight of about 2,000 daltons. PEG can be optionally substituted with alkyl, alkoxy, acyl, or aryl groups. In Formula 62, L is a linker moiety. Any linker moiety suitable for coupling PEG to the dialkoxypropyl backbone can be used. Suitable linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbonate (OC(O)O-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), succinyl (-(O)CCH2CH2C(O)-), ethers, disulfides, and combinations thereof. Other suitable linkers are well known in the art.

[0181] Phosphatidylethanolamine can be conjugated to polyethylene glycol to form a bilayer stabilizing component as a conjugated lipid that inhibits particle aggregation in the present invention. The phosphatidylethanolamine has various acyl chain groups with different chain lengths and degrees of saturation. These phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. Phosphatidylethanolamines containing saturated or unsaturated fatty acids are preferred, having carbon chain lengths in the range of C10-C20. Phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, the following: dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidylethanolamine (DSPE).

[0182] Like phosphatidylethanolamine, ceramide can be coupled with polyethylene glycol to form a bilayer stabilizing component as a conjugated lipid that inhibits particle aggregation in the present invention. The ceramide has multiple acyl chain groups with different chain lengths and degrees of saturation. It should be clear to those skilled in the art that, compared to phosphatidylethanolamine, ceramide has only one acyl group, which can be easily varied depending on its chain length and degree of saturation. Ceramides suitable for use in accordance with the present invention are commercially available. In addition, ceramides can be isolated, for example, from eggs and brain using well-known separation techniques, or synthesized using the methods and techniques disclosed in U.S. Patent No. 5,820,873, which is incorporated herein by reference. Using the synthetic pathways proposed in the aforementioned applications, ceramides with saturated or unsaturated fatty acids having carbon chain lengths ranging from C2 to C31 can be prepared.

[0183] 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:

[0184]

[0185] wherein: R is a member selected from the group consisting of hydrogen, alkyl, and acyl; R1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R1 and the nitrogen atom to which they are bound form an azido moiety; R2 is a member selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, and an amino acid side chain; R3 is a member selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, thiol, hydrazine, amino, and NR4R5, wherein R4 and R5 are independently hydrogen or alkyl; n is 4-80; m is 2-6; p is 1-4; and q is 0 or 1. It will be apparent to those skilled in the art that other polyamides can be used in the compounds of the present invention.

[0186] The term "congener" refers to an analog that performs the same or similar function, or a derivative of the same parent core that performs the same or similar function.

[0187] As used herein, the terms "mRNA" or "messenger polyribonucleotide" or "messenger RNA" or "messenger RNA" are used interchangeably and refer to a single-stranded polyribonucleotide that is transcribed using one strand of DNA as a template, carries genetic information, and can direct protein synthesis.

[0188] As used herein, the terms "sgRNA," "small guide RNA," "guide RNA," or "gRNA" are used interchangeably and are small noncoding RNAs that pair with pre-mRNAs to guide the insertion or deletion of uridine residues into kinetoplastids during RNA editing. gRNAs edit RNA molecules, approximately 60-80 nucleotides in length, transcribed from separate genes.

[0189] As used herein, the terms "circRNA" or "circular RNA" or "cyclic polyribonucleotide" or "circular RNA" are used interchangeably and refer to a polyribonucleotide molecule having a structure without free ends (i.e., without free 3' and / or 5' ends), such as a polyribonucleotide that forms a ring or annular structure through covalent or non-covalent bonds.

[0190] As used herein, the terms "microRNA" or "miRNA" or "microRNA" are used interchangeably and refer to a non-coding single-stranded polyribonucleotide of approximately 22 nucleotides in length with free 3' and 5' ends, which can inhibit the translation of target gene protein by binding to the 3'-untranslated region (3'-UTR) of the mRNA of the target gene, thereby regulating the biological function of the cell.

[0191] As used herein, the terms "ASO" or "antisense oligonucleotide" or "antisense oligonucleotide" are used interchangeably and refer to artificially synthesized nucleic acid fragments that are complementary to a certain segment of a target gene or mRNA, and can bind to the target gene / mRNA through the principle of base complementarity, thereby blocking the expression of the gene, including antisense DNA and antisense RNA.

[0192] As used herein, the terms "siRNA" or "small interfering" or "short interfering" or "silencing RNA" or "small interfering RNA" or "short interfering RNA" or "silencing RNA" are used interchangeably and refer to a class of double-stranded RNA molecules with a length of 20 to 25 nucleotides that can induce the degradation of target gene mRNA.

[0193] As used herein, the terms "ecDNA" or "extrachromosomal circular DNA" are used interchangeably and refer to DNA that has fallen off chromosomes and exists in a circular structure.

[0194] The term "nucleic acid derivative" refers to modifications or substitutions of nucleic acid sequences, including but not limited to chemical modifications of residues, substitutions of nucleotides or deoxynucleotides, modifications to sequences that increase half-life or stability, and labeling modifications. For example, chemical modifications include but are not limited to phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopization of any one or more bases. Substitutions of nucleotides or deoxynucleotides include but are not limited to nucleic acid analogs that replace the sugar-phosphate backbone with polypeptides or other backbones (replacing DNA or RNA with PNA (peptide nucleic acids)). Modifications to sequences that increase half-life or stability include but are not limited to PEG linkage and fluorine modification. Labeling modifications include but are not limited to attachment of fluorescent groups, amino groups, biotin, digoxigenin, small peptides, and the like.

[0195] The term "artificial nucleic acid" refers to a nucleic acid molecule that has been artificially modified, including but not limited to base modifications, ribose modifications, PNA, etc.

[0196] The term "nucleic acid" refers to a polymer that exists in a single or double-stranded form and comprises at least two deoxynucleotides or nucleotides. Unless specifically limited, the term encompasses nucleic acids comprising known analogs of natural nucleotides that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise noted, specific nucleic acid sequences also implicitly encompass variants (e.g., degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences, as well as sequences clearly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). A "nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are linked via the phosphate group. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that substitute new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. DNA can be present as antisense, plasmid DNA, portions of plasmid DNA, pre-compressed DNA, products of polymerase chain reaction (PCR), vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. The term nucleic acid is used interchangeably with gene, cDNA, mRNA encoded by a gene, and interfering RNA molecules.

[0197] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that includes a partial or full-length coding sequence necessary for the production of a polypeptide or polypeptide precursor (e.g., a polypeptide or polypeptide precursor from hepatitis A, B, C, D, E, G virus; or herpes simplex virus).

[0198] As used herein, "gene product" refers to the product of a gene such as, but not limited to, the transcript of DNA, mRNA.

[0199] The phrase "silencing the expression of the target gene" refers to the ability of the siRNA of the present invention to activate the target gene. In order to determine the degree of gene silencing, a sample or assay of the cells in the target organism or culture is compared with a control sample, wherein the cells of the target organism or culture express a specific construct and the control does not express the construct. The control sample (lacking the expression of the construct) is set to a relative value of 100%. When the test value relative to the control is about 90%, preferably 50%, more preferably 25-0%, the inhibition of the expression of the target gene is successfully obtained. Suitable assays include, for example, using techniques known to those skilled in the art such as dot blots, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme action, and phenotypic assays known to those skilled in the art to detect protein or mRNA levels.

[0200] A "therapeutically effective amount" or "effective amount" of siRNA is an amount sufficient to produce the desired effect, eg, a decrease in expression of the target sequence as compared to normal expression levels detected in the absence of the siRNA.

[0201] As used herein, the term "aqueous solution" refers to a composition that comprises, in whole or in part, water.

[0202] As used herein, the term "organic lipid solution" refers to a composition comprising in whole or in part an organic solvent with lipids.

[0203] As used herein, "systemic delivery" refers to the delivery that causes a compound to be widely biodistributed in an organism. Some techniques of administration can result in systemic delivery of certain compounds, but cannot result in systemic delivery of other compounds. Systemic delivery refers to effective, preferably therapeutic, contact of a compound with a large portion of the body. In order to obtain a wide range of biodistribution, a blood survival period is generally required so that the compound is not rapidly degraded or cleared (such as by initially passing through an organ (liver, lung, etc.) or by rapid, nonspecific cell binding) before reaching the disease site at the distal end of the administration site. Systemic delivery of drug-lipid particles can be carried out in any manner known in the art, including, for example, intravenous, subcutaneous, intraperitoneal, and in a preferred embodiment, systemic delivery of drug-lipid particles is by intravenous delivery.

[0204] As used herein, "local delivery" refers to the delivery of a compound directly to a target site in the body. For example, a compound can be delivered locally by direct injection into a disease site such as a tumor or other target site such as an inflammatory site or a target organ such as the liver, heart, pancreas, kidney, etc.

[0205] The term "phospholipid" refers to lipids containing phosphate groups. These complex lipids are also known as phospholipids or phospholipids. Phospholipids are the primary components of biological membranes and are divided into two main categories: glycerophospholipids and sphingomyelins, composed of glycerol and sphingosine, respectively. Phospholipids are amphiphilic molecules, with a hydrophilic, nitrogen- or phosphorus-containing head at one end and a long, hydrophobic (lipophilic) hydrocarbon chain at the other. Because of this, phospholipid molecules are positioned close together at their hydrophilic and hydrophobic ends, often forming the phospholipid bilayer, the structure of the cell membrane, along with other molecules such as proteins, glycolipids, and cholesterol.

[0206] In the present invention, the polyphenol molecules in the metal-polyphenol complex are mainly derived from natural plant extracts, such as curcumin, which has a wide range of biological effects, including antibacterial, antiviral, antifungal, antioxidant and anti-inflammatory activities. In addition, it is also an effective immunomodulator that can regulate the activity of various immune cells such as T cells, B cells, macrophages, neutrophils, natural killer cells and dendritic cells, promote the balance of immunity, and enhance the body's immunity. Based on the potential immune enhancement, anti-inflammatory, antioxidant and anti-sars-cov-2 effects of curcumin molecules, it is expected to become a potential auxiliary treatment for COVID-19. In addition, the safety of curcumin molecules is extremely high and has been listed in the catalog of food additives and pharmaceutical excipients. Its safety is conducive to the clinical drug registration of the drug-lipid as a whole, shortening the time length of clinical drug registration.

[0207] In the present invention, the coordination bond between the polyphenol molecule and the metal ion in the metal-polyphenol complex is broken under low pH conditions such as lysosomes (pH=5.0), and the metal ion falls off from the metal-polyphenol complex.

[0208] In the present invention, the ratio of each component in the metal-polyphenol complex can be adjusted based on the specific structure of the metal-polyphenol complex components. This adjustment is based on the fact that the hydroxyl groups of the polyphenol molecules are connected to the metal ions via coordination bonds. As long as the polyphenol molecules contain multiple binding sites, the ratio of the polyphenol molecules to the metal ions can be adjusted based on the number of binding sites contained in the polyphenol molecules.

[0209] Metal-chelated polyphenol complex nanoparticles (MPNP)

[0210] The principle of loading nucleic acids into metal-polyphenol complex particles assembled by metal-polyphenol complexes is: polyphenol molecules are connected to metal ions through coordination bonds to form metal-polyphenol complexes, and the metal ions of the metal-polyphenol complex are connected to nucleic acids through coordination bonds, thereby ensuring that the metal-polyphenol complex and other components self-assemble into MPNPs while loading nucleic acids into nanoparticles.

[0211] Herein, the "non-cationic lipid or non-ionizable lipid other than the conjugated lipid that inhibits particle aggregation" refers to component (iii) in the metal-polyphenol complex particles.

[0212] In some embodiments, the conjugated lipid that inhibits particle aggregation refers to a conjugated lipid that inhibits drug-lipid particle aggregation, and its primary function is to prevent drug-lipid particle aggregation, such as PEG coupled to a dialkoxypropyl group, PEG coupled to a diacylglycerol group, PEG coupled to a phosphatidylethanolamine group, and PEG conjugated to a ceramide group, preferably a PEG-lipid conjugate. Cis- and trans-isomers of the lipid do not affect the effects to be achieved by the present invention.

[0213] In some embodiments, the molar proportion 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, it is 5% to less than 10%, 10% to 15%, or 15% to 20%, and more preferably 5%, 10%, or 15%.

[0214] In some embodiments, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, for example 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably 3% to 5% or 5% to 10%, and more preferably 3%, 5% or 10%.

[0215] In some embodiments, the non-cationic lipid or non-ionizable lipid optionally contains cholesterol, and the molar proportion of cholesterol in the raw material is 0% to 48%, for example, 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% or 48%. It is preferably 10% to 30%, 30% to 47% or 10% to 20%, and more preferably 10%, 30% or 47%.

[0216] In some embodiments, in addition to cholesterol, the metal-polyphenol complex particles further contain other non-cationic lipids or non-ionizable lipids, which have a molar proportion of 30% to 75% in the raw material, such as 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%. It is preferably 45% to 55%, 60% to 65% or 50% to 65%, and further preferably 45%, 55%, 60% or 65%.

[0217] Drug-lipid particles

[0218] The drug-lipid particles described herein typically include a drug (which is a negatively charged molecule, which can be selected from the group consisting of one or more combinations of nucleic acids, proteins, polypeptides, small molecules, nucleic acid analogs, protein analogs, polypeptide analogs, and nucleic acids are 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 lipid or a non-ionizable lipid, and a bilayer stabilizing component, such as a conjugated lipid that inhibits particle aggregation. In addition, the nucleic acids encapsulated in the drug-lipid particles of the present invention are resistant to degradation by nucleases in aqueous solution.

[0219] In some embodiments, the drug is fully encapsulated inside the metal-polyphenol complex particles to avoid drug degradation, thereby achieving drug delivery into cells.

[0220] In some embodiments, the drug-lipid particles provided by the present invention 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; stability of at least 3 days, preferably more than 7 days; and a cell delivery efficiency of at least 40%, for example, 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%.

[0221] 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.

[0222] The RNA population can be used to provide long precursor RNAs, or long precursor RNAs having substantial or complete identity to a selected target sequence that can be used to prepare siRNA. The RNAs can be isolated, synthesized, and / or cloned from cells or tissues according to methods well known to those skilled in the art. The RNA can be a mixed population (obtained from cells or tissues, transcribed from cDNA, etc.), or can represent a single target sequence. The RNA can be naturally occurring, for example, isolated from a tissue or cell sample, for example, synthesized in vitro using T7 or SP6 polymerase and PCR products or cloned cDNA; or chemically synthesized.

[0223] To form long dsRNA, for synthetic RNAs, the complement can also be transcribed in vitro and hybridized to form dsRNA. If a naturally occurring RNA population is used, for example, by transcribing cDNAs corresponding to the RNA population, or by using RNA polymerase, RNA complements are also provided (e.g., to form dsRNA, which is digested by E. coli RNAse III or Dicer). The precursor RNA is then hybridized to form double-stranded RNAs and digested. The dsRNAs can be directly encapsulated in SNALPs or can be digested in vitro before encapsulation.

[0224] Alternatively, one or more DNA plasmids encoding one or more siRNA templates can be encapsulated within nucleic acid-lipid particles. For example, siRNAs based on the naturally occurring transcription units of small nuclear RNA U6 or human RNase P RNA H1 can be transcribed as sequences that automatically fold into duplexes with hairpin loops from DNA templates in plasmids harboring RNA polymerase III transcription units (see, Brummelkamp, ​​et al., Science 296:550 (2002); Donzé, et al., Nucleic Acids Res. 30:e46 (2002); Paddison, et al., Genes Dev. 16:948 (2002); Yu, et al., Proc. Natl. Acad. Sci. 99:6047 (2002); Lee, et al., Nat. Biotech. 20:500 (2002); Miyagishi, et al., Nat. Biotech. 20:497 (2002); Paul, et al., Proc. Natl. Acad. Sci. 99:6047 (2002); Lee, et al., Nat. Biotech. 20:500 (2002); Miyagishi, et al., Nat. Biotech. 20:497 (2002); Paul, et al., Proc. Natl. Acad. Sci. 10:1017 (2002); et al., Nat. Biotech. 20: 505 (2002); and Sui, et al., Proc. Natl. Acad. Sci. 99: 5515 (2002). Typically, the transcription unit or cassette will contain an RNA transcription promoter sequence, such as the H1-RNA or U6 promoter, operably linked to a template for transcription of the desired siRNA sequence and a termination sequence comprising 2-3 uridine residues and a polythymidine (T5) sequence (polyadenylation signal) (Brummelkamp, ​​Science, supra). The selected promoter can provide constitutive or inducible transcription. Compositions and methods for transcription of DNA-guided RNA interference molecules are described in detail in U.S. Patent No. 6,573,099, which is incorporated herein by reference. Preferably, the synthetic or transcribed siRNA has a 3' overhang of about 1-4, preferably about 2-3 nucleotides and a 5' phosphate terminus (Elbashir, et al., Genes Dev. 15: 188 (2001); , et al., Cell 107:309 (2001). The transcription unit is incorporated into a plasmid or DNA vector from which the interfering RNA is transcribed. Plasmids suitable for in vivo delivery of genetic material for therapeutic purposes are described in detail in U.S. Patent Nos. 5,962,428 and 5,910,488, both of which are incorporated herein by reference. Selected plasmids can provide transient or stable delivery to target cells. It will be apparent to those skilled in the art that a plasmid initially designed to express a desired gene sequence can be modified to include a transcription unit cassette that transcribes an siRNA.

[0225] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, preparing and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds., 1990)). Expression libraries are also well known to those skilled in the art. Additional basic texts disclosing general methods used in the present invention 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)).

[0226] Applications of metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles

[0227] In some embodiments, when the drug is a nucleic acid, the metal-polyphenol complex and metal-polyphenol complex particles can be used to facilitate the drug's lysosomal escape and promote nucleic acid expression. The metal-polyphenol complex and metal-polyphenol complex particles can also be used to deliver the drug, introducing the drug into cells, thereby preventing and treating the applicable disease or condition.

[0228] In some embodiments, the present invention provides the use of metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles, for example, for compositions that can achieve drug delivery or introduce drugs into cells. The composition is, for example, a medicament that can achieve: silencing the expression of a target sequence in a mammalian subject, delivering drugs in a mammal (for example, drugs for treating tumors, imaging agents, etc.), delivering drugs from the body to mammalian cells, or treating mammalian diseases or conditions, etc. In the medicament, the drug-lipid particles are the main active ingredients and can be prepared into different dosage forms according to actual needs, such as solid dosage forms (powders, granules, pills, tablets, gels), semi-solid dosage forms (external ointments, pastes), liquid dosage forms (decoctions, mixtures, syrups, wines, injections), gaseous dosage forms (aerosols, smoke agents), etc.; for example, dosage forms for gastrointestinal administration, dosage forms for rectal administration, and dosage forms for non-gastrointestinal administration, etc.

[0229] In some embodiments, the present invention provides products prepared from the above-mentioned metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles. The products have the above-mentioned functions and uses of the metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles. The specific types can be, for example, but not limited to, kits, medicines, etc., and the products may optionally contain other excipients.

[0230] For target genes of drug-lipid particles: Generally, it is desirable to deliver drug-lipid particles so that the translation (i.e., expression) of the target gene product is downregulated or silenced. Suitable classifications of gene products include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., diseases and disorders in which the liver is a target, and liver diseases and disorders), genes associated with tumorigenesis and cell transformation, angiogenic genes, immunomodulator genes such as those associated with inflammation and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.

[0231] Genes associated with viral infection and survival include those expressed by viruses to bind, enter, and replicate in cells, particularly viral sequences associated with chronic viral diseases. For example, viral sequences include sequences of hepatitis viruses (Hamasaki, et al., FEBS Lett. 543:51 (2003); Yokota, et al, EMBO Rep. 4:602 (2003); Schlomai, et al, Hepatology 37:764 (2003); Wilson, et al, Proc. Natl. Acad. Sci. 100:2783 (2003); Kapadia, et al, Proc. Natl. Acad. Sci. 100:2014 (2003); and FIELDSVIROLOGY (Knipe et al. eds. 2001)), human immunodeficiency virus (HIV) (Banerjea, et al, Mol Ther. 8:62 (2003); Song, et al, 2003). al., J. Virol. 77:7174 (2003); Stephenson JAMA 289:1494 (2003); Qin, et al., Proc. Natl. Acad. Sci. 100:183 (2003)), herpes virus (Jia, et al., J. Virol. 77:3301 (2003)), and human papillomavirus (HPV) (Hall, et al., J. Virol. 77:6066 (2003); Jiang, et al., Oncogene 21:6041 (2002)). Exemplary hepadnavirus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences involved in transcription and translation (e.g., En1, En2, X, P), nucleic acid sequences encoding structural proteins (e.g., core proteins including C and C-related proteins; capsid and envelope proteins including S, M, and / or L proteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY, 2001, supra). Hepatitis C nucleic acid sequences that can be silenced include, but are not limited to, serine proteases (e.g., NS3 / NS4), helicases (e.g., NS3), polymerases (e.g., NS5B), and envelope proteins (e.g., E1, E2, and p7).Hepatitis A nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_001489; hepatitis B nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_003977; hepatitis C nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_004102; hepatitis D nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_001653; hepatitis E nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_001434; and hepatitis G nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_001710. Silencing sequences encoding genes associated with viral infection and survival can be conveniently used in conjunction with the administration of conventional pharmaceutical agents for treating viral diseases.

[0232] Genes associated with metabolic diseases and disorders (e.g., disorders in which the liver is targeted and liver diseases and disorders) include, for example, genes expressed in dyslipidemia (e.g., liver X receptors (e.g., LXRα and LXRβ Genbank Accession No. NM_007121)), farnesoid X receptor (FXR) (Genbank Accession No. NM_005123), sterol regulatory element binding protein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase (HMG-Coenzyme-A reductase), apolipoprotein (ApoB), and apolipoprotein (ApoE)) and diabetes (e.g., glucose-6-phosphate) (see, e.g., Forman et al., Cell 81:687 (1995); Seol et al., Mol. Endocrinol. 9:72 (1995), Zavackie et al., Mol. Endocrinol. 9:73 (1996)). 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. al., GenesDev. 9(9): 1033-45 (1995); Lehmann, et al., J. Biol. Chem. 272(6): 3137-3140 (1997); Janowski, et al. al., Nature 383:728-731 (199; Peet, et al., Cell 93:693-704 (1998)). Those skilled in the art will understand that genes associated with metabolic diseases and disorders (e.g., diseases and disorders in which the liver is targeted and liver diseases and disorders) include genes expressed in the liver itself as well as genes expressed in other organs and tissues. Silencing sequences encoding genes associated with metabolic diseases and disorders can be conveniently used in conjunction with the administration of conventional pharmaceutical agents for treating the disease or disorder.

[0233] Examples of genes associated with tumorigenesis and cell transformation include translocation sequences such as MLL fusion gene, BCR-ABL (Wilda, et al., Oncogene, 21:5716 (2002); Scherr, et al, Blood 101:1566), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO and AML1-MTG8 (Heidenreich, et al, Blood 101:3157 (2003)); overexpressed sequences such as multidrug resistance genes (Nieth, et al, FEBS Lett. 545:144 (2003); Wu, et al, Cancer Res. 63:1515 (2003)), cell cycle proteins (Li, et al, Cancer Res. 63:3593 (2003); Zou, et al, Genes Dev. 16: 2923 (2002)), β-catenin (Verma, et al., Clin Cancer Res. 9: 1291 (2003)), telomerase gene (Kosciolek, et al., Mol Cancer Ther. 2: 209 (2003)), c-MYC, N-MYC, BCL-2, ERBB1 and ERBB2 (Nagy, et al. Exp. Cell Res. 285: 39 (2003)); and mutant sequences such as RAS (reviewed in Tuschl and Borkhardt, Mol. Interventions, 2: 158 (2002)). Silencing sequences encoding DNA repair enzymes is used in conjunction with administration of chemotherapeutic agents (Collis, et al., Cancer Res. 63: 1550 (2003)). Genes encoding proteins associated with tumor migration, such as integrins, selectins, and metalloproteinases, are also target sequences of interest. Any complete or partial gene sequence that is beneficial to or promotes tumorigenesis or cell transformation, tumor growth, or tumor migration can be included as a template sequence.

[0234] Angiogenic genes can promote the formation of new blood vessels, and vascular endothelial growth factor (VEGF) is a key research direction (Reich, et al., Mol. Vis. 9: 210 (2003)).

[0235] Immunomodulator genes are genes that regulate one or more immune responses. Examples of immunomodulator genes include cytokines such as growth factors (e.g., TGF-α, TGF-β, EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12 (Hill, et al., J. Immunol. 171: 691 (2003)), IL-15, IL-18, IL-20, etc.), interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.) and TNF. Fas and Fas ligand genes are also immunomodulator target sequences of interest (Song, et al., Nat. Med. 9: 347 (2003)). Also included in the present invention are genes encoding secondary signaling molecules in hematopoietic and lymphoid cells, for example, Tec family kinases, such as Bruton's tyrosine kinase (Btk) (Heinonen, et al., FEBS Lett. 527:274 (2002)).

[0236] Cell receptor ligands include ligands that bind to cell surface receptors (e.g., insulin receptors, EPO receptors, G-protein coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) to modulate (e.g., inhibit, activate, etc.) physiological pathways involved in the receptors (e.g., glucose level regulation, blood cell development, mitogenesis, etc.). Examples of cell receptor ligands include cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G-protein coupled receptor ligands, etc.). Templates encoding trinucleotide repeat sequence (e.g., CAG repeat sequence) expansions have been found to be useful for silencing pathogenic sequences in neurodegenerative diseases caused by trinucleotide repeat sequence expansions, such as spinal bulbar muscular atrophy and Huntington's disease (Caplen, et al., Hum. Mol. Genet. 11: 175 (2002)).

[0237] Injectable delivery: In some cases, as described in U.S. Patent No. 5,543,158; U.S. Patent No. 5,641,515 and U.S. Patent No. 5,399,363, it is desirable to deliver the drug-lipid particles disclosed herein parenterally, intravenously, intramuscularly, subcutaneously, intradermally or intraperitoneally. The drug-lipid particles can be injected locally to the target site (e.g., a disease site such as inflammation or tumor formation or to a target organ or tissue) or systemically for widespread distribution to the organism. Solutions of the drug-lipid particles can be prepared in water, suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oils. Optionally, these formulations contain preservatives to prevent the growth of microorganisms. Typically, when administered intravenously, the drug-lipid particle formulation is formulated with a suitable pharmaceutical carrier. Typically, a normal buffered saline solution (135-150 mM NaCl) will be used as a pharmaceutical carrier, but other suitable carriers will suffice. Additional suitable carriers are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a human. The formulation of aqueous compositions comprising a protein as an active ingredient is conventionally understood in the art. Alternatively, these compositions are prepared as injectable solutions, liquid solutions, or suspensions; solid forms suitable for solution or suspension in a liquid prior to injection can also be prepared. The formulations can also be emulsified.

[0238] Can be sterilized by conventional liposome sterilization technology, such as filtering medicine-lipid granule.Described medicine-lipid granule can comprise pharmaceutical auxiliary substance, and described pharmaceutical auxiliary substance is suitable physiological condition, such as pH adjusting agent and buffer agent, toxicity regulator, wetting agent etc.Use the technology indicated above to sterilize these compositions, or alternatively, they can produce under aseptic conditions.Can be packaged to use or filter and carry out lyophilization under aseptic conditions to the aqueous solution obtained, before using, lyophilized preparation is combined together with sterile aqueous solution.

[0239] Prophylactic and therapeutic treatment: In some embodiments, the drug-lipid particle can be used for the prophylactic or therapeutic treatment of a subject (e.g., a mammalian subject) suffering from a disease or condition that is associated with expression or overexpression of a target sequence. The drug-lipid particle is administered to the subject in an amount sufficient to stimulate a therapeutic response in the patient. An amount sufficient to accomplish this is defined as a "therapeutically effective dose or amount" or "effective dose or amount." In determining the effective amount of the drug-lipid particle to be administered for the treatment or prevention of a disease that is due to expression or overexpression of a target gene, the physician evaluates the circulating plasma levels of the drug-lipid particle, the drug-lipid particle toxicity, and the progression of the disease associated with expression or overexpression of the target gene. Administration can be accomplished by single or divided doses.

[0240] In some embodiments, the drug-lipid granule is administered to a subject who is infected by pathogenic microorganisms or who is at risk of being infected by pathogenic microorganisms. The drug should preferably correspond to a sequence, and should also be unique (or at least lacking in the genome of the natural genome of the patient who experiences treatment) for microorganisms, and the sequence has a key role in the life history of microorganisms. By ex vivo or intravenous injection, the drug-lipid granule is introduced into target cells, tissues or organs with therapeutically effective dosage. The sequence silencing of the gene encoding the relevant to pathogenic infection can be easily combined with the use of conventional reagents for the treatment of pathogenic diseases. The treatment can be preventively administered to a subject who is at risk of being infected by pathogenic microorganisms or who has been infected by pathogenic microorganisms.

[0241] In a preferred embodiment, the drug-lipid particles of the present invention can be conveniently used to treat cancer, viral infections, autoimmune diseases, diabetes, and Alzheimer's disease. Viral infections include hepatitis A, hepatitis B, hepatitis C, SARS-CoV-2, HIV, HPV, influenza, smallpox, and syphilis. For example, suitable sites for inhibiting hepatitis B virus include nucleic acid sequences encoding S, C, P, and X proteins, PRE, EnI, and EnII (see, for example, FIELDSVIROLOGY, 2001, supra). Those skilled in the art will appreciate that gene silencing associated with hepatitis infection can be combined with conventional treatments for hepatitis, such as, for example, immunoglobulins, interferons (e.g., PEGylated and non-PEGylated interferon alpha) (see, e.g., Medina et al., Antiviral Res. 60(2):135-143 (2003); ribavirin (see, e.g., Hugle and Cerny, Rev. Med. Virol. 13(6):361-71 (2003); adefovir and lamivudine (see, e.g., Kock et al., Hepatology 38(6):1410-8 (2003); prenylation inhibitors (see, e.g., Bordier et al., J. Clin. Invest. 112(3):407-414 (2003)); famciclovir (see, e.g., Yurdaydin et al., J. Clin. Invest. 113(4):517-529 (2003)); 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).

[0242] In another embodiment, medicine of the present invention-lipid granule can be used to treat easily and be characterized by gene or gene group expression or the disease and illness of overexpression.In some respects, medicine of the present invention-lipid granule can be used to treat metabolic disease and illness (for example, wherein liver is the disease and illness and hepatic disease and illness of target object) such as, for example, dyslipidemia and diabetes.Those skilled in the art will appreciate that the silence of the gene relevant to metabolic disease and illness can be combined with the conventional treatment of these diseases. For example, silencing of genes involved in dyslipidemia can be combined with the use of statins, bile acid sequestrants / resins and cholesterol absorption inhibitors such as ezetimibe, plant stanols / sterols, polyphenols, and nutraceuticals such as oat bran, flaxseed and soy protein, phytostanol analogs, squalene synthase inhibitors, bile acid transport inhibitor SREBP cleavage activating protein (SCAP) activating ligand, nicotinic acid (niacin), acipimox, high-dose fish oil, antioxidants and policosanol, microsomal triglyceride transport protein (MTP) inhibitors, acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors, gemcabene, rifiberol, pantothenic acid analogs, niacin-receptor agonists, anti-inflammatory agents (such as Lp-PLA(2) antagonists and AGI1067) functional oils, PPAR-α, γ, Therapeutic combinations of delta agonists, as well as dual PPAR-α, / γ and 'pan' PPAR-α / γ, / δ agonists, cholesteryl ester transfer protein (CETP) inhibitors (such as torcetrapib), CETP vaccines, upregulators of ATP-binding cassette transporter (ABC) A1, lecithin cholesterol acyltransferase (LCAT) and scavenger receptor class B type 1 (SRB1), and synthetic apolipoprotein (Apo) E-related peptides, extended-release niacin / lovastatin, atorvastatin / amlodipine, ezetimibe / simvastatin, atorvastatin / CETP inhibitors, statin / PPAR agonists, extended-release niacin / simvastatin and pravastatin / aspirin in development, and anti-obesity agents (see, e.g., Bays and Stein, Expert Opin. Pharmacother. 4(11):1901-38 (2003)). Likewise, silencing of genes involved in diabetes can be combined with treatment with insulin, as well as dietary modifications and exercise.

[0243] Similar methods are used to inhibit the expression of endogenous receptor cellular genes that are associated with tumorigenesis and cell transformation, tumor growth and tumor migration; inhibit the expression of angiogenic genes; inhibit the expression of immunomodulator genes, such as those associated with inflammation and autoimmune responses; inhibit the expression of ligand receptor genes; inhibit the expression of genes associated with neurodegenerative disorders; and inhibit the expression of additional genes associated with viral infection and survival. Specific target gene sequences are as described above.

[0244] Detection of the Particles: The drug-lipid particles herein are detected using any method known in the art. For example, a label is coupled directly or indirectly to a component of the drug-lipid particle or other lipid-based carrier system using methods well known in the art. A wide variety of labels can be used, with selection being based on desired sensitivity, ease of conjugation to the drug-lipid particle components, stability requirements, and available tools and processing readiness. Suitable labels include, but are not limited to, spectral labels, such as fluorescent dyes (e.g., (e.g., fluorescein and derivatives, such as fluorescein isothiocyanate (FITC) and Oregon Green™; rhodamine and derivatives, such as Texas Red, tetrarhodimineisothiocynate (TRITC), etc., digoxigenin, biotin, phycoerythrin, AMCA, CyDyes™, etc.; radioactive labels, such as 3H, 125I, 35S, 14C, 32P, 33P, etc.; enzymes such as horseradish peroxidase, alkaline phosphatase, etc.; spectral colorimetric labels such as colloidal gold or colored glass or plastic beads, such as polystyrene, polypropylene, latex, etc.). The label is detected using any means known in the art.

[0245] Detection of Nucleic Acids: Nucleic acids herein can be detected and quantified by any of a number of methods well known to those skilled in the art. Detection of nucleic acids can be performed by methods well known in the art such as Southern blot analysis, Northern blot analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Additional analytical biochemical methods such as spectrophotometry, X-ray photography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography can also be used.

[0246] The sensitivity of hybridization assays can be increased by the use of nucleic acid amplification systems that multiply the amount of target nucleic acid being detected. In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques sufficient to guide the skilled artisan by these in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification, and other RNA polymerase-mediated techniques (e.g., NASBA™) are found in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2000, and Ausubel et al., SHORT PROTOCOLS IN MOLECULARBIOLOGY, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (2002), and Mullis et al. (1987), U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc., San Diego, CA. 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. al., Proc. Natl. Acad. Sci. USA, 87: 1874 (1990); Lomell et al., J. Clin. Chem., 35: 1826 (1989); Landegren et al., Science, 241: 1077 (1988); Van Brunt, Biotechnology, 8: 291 (1990); Wu and Wallace, Gene, 4: 560 (1989); Barringer et al. al., Gene, 89: 117 (1990), and Sooknanan and Malek, Biotechnology, 13: 563 (1995).Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., US Patent No. 5,426,039. Other methods described in the art are nucleic acid sequence-based amplification (NASBA™, Cangene, Mississauga, Ontario) and the Qβ replicase system.

[0247] Oligonucleotides for use as probes in in vitro amplification methods, as gene probes, or as inhibitor components are chemically synthesized, for example, using an automated synthesizer, typically according to the solid-phase phosphoramidite triester method described by Beaucage and Caruthers, Tetrahedron Letts., 22(20):1859-1862 (1981), as described by Needham VanDevanter et al., Nucleic Acids Res., 12:6159 (1984). If necessary, oligonucleotides are typically purified by native acrylamide gel electrophoresis or by anion exchange HPLC, as described by Pearson and Regnier, J. Chrom., 255:137-149 (1983). The sequence of synthetic oligonucleotides can be confirmed using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology, 65:499.

[0248] The following examples provide illustration, but do not limit the claimed invention. Those skilled in the art will readily recognize a variety of noncritical parameters that can produce substantially similar results.

[0249] The drug-lipid particles to be protected by the present invention refer to drug-lipid particles other than those containing cationic / ionizable lipids, namely drug-loaded metal-chelated polyphenol complex nanoparticles (drug@MPNP).

[0250] Experimental Example 1: Preparation of mRNA-loaded metal-chelated polyphenol complex nanoparticles (mRNA@MPNP)

[0251] Example 1. Preparation of Metal-chelated Polyphenol Complex Nanoparticles (MPNP)

[0252] Example 1.1 Preparation of metal ions Fe 3+ Metal-polyphenol complex

[0253] Curcumin (Formula 1) was dissolved in ethanol at 1.5 mg / ml, and anhydrous FeCl3 was added at a molar ratio of 1:1. The reaction was refluxed at 60°C for 1 hour. After the reaction, the solution was suspended to dryness, and the product was dissolved in ultrapure water and filtered. The resulting product, after lyophilization, is the metal-polyphenol complex. The structure of the metal-polyphenol complex is shown below.

[0254]

[0255] Result analysis: Curcumin (Formula 1) and FeCl3 were reacted at 60°C for 1 hour, the curcumin (Formula 1) feed concentration was 1.5 mg / mL, and the feed ratio of curcumin (Formula 1) to FeCl3 was 1:1, and the yield of the target product obtained was 95%.

[0256] Example 1.2 Preparation of metal ions as Al 3+ Metal-polyphenol complex

[0257] The difference between this example and Example 1.1 is that FeCl3 is replaced by Al(NO3)3·9H2O. The structure of the prepared metal-polyphenol composite is shown below.

[0258]

[0259] Result analysis: Curcumin (Formula 1) and Al(NO3)3·9H2O were reacted at 60°C for 1 hour, the curcumin (Formula 1) feed concentration was 1.5 mg / mL, and the feed ratio of curcumin (Formula 1) and Al(NO3)3·9H2O was 1:1, and the yield of the target product obtained was 98%.

[0260] Example 2: Preparation of mRNA-loaded metal-chelated polyphenol complex nanoparticles (mRNA@MPNP)

[0261] The metal ion is Fe 3+ mRNA-metal-polyphenol complex particles

[0262] A metal-polyphenol complex was prepared according to the method described in Example 1.1, wherein curcumin (Formula 1) and FeCl3 were added at a 1:1 feed ratio. The metal-polyphenol complex, 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 conjugated lipid to inhibit particle aggregation) were dissolved in ethanol at varying molar ratios as the organic phase. The metal-polyphenol complex, DSPC (Formula 51), CHOL (Formula 59), and DSPE-PEG2000 (Formula 58) were present in 5%, 60%, 30%, and 5%, respectively. mRNA was dissolved at a concentration of 20 μg / mL in enzyme-free Tris-HCl buffer (0.1 M) at pH 5.0 as the 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 is 3:1. The flow rate of the organic phase and the aqueous phase in the microfluidic chip is 12 ml / min. Among them, the drug mRNA is an mRNA encoding the fluorescent protein eGFP, and its sequence is SEQ ID NO.1 (720nt). eGFP-mRNA@MPNP was prepared. eGFP-mRNA@MPNP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPNP without drug loading. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0263] The particle size, surface potential and stability of the prepared eGFP-mRNA@MPNP were tested, and the efficiency of nucleic acid encapsulation in eGFP-mRNA@MPNP was calculated.

[0264] Method for detecting particle size and result judgment standard: The particle size of nanoparticles was tested using a Malvern Zetasizer laser particle size analyzer, and a particle size within the range of 30 to 400 nm was considered acceptable.

[0265] Method for detecting surface potential and result judgment standard: The surface potential of nanoparticles was tested using a Malvern Zetasizer laser particle size analyzer, and a potential in the range of -10 to 10 mV was considered acceptable.

[0266] Stability testing method and result judgment criteria: The nanoparticles were placed at 4°C for 7 days, and the particle size and surface potential of the nanoparticles were measured using a Malvern Zetasizer. If there was no significant change in the particle size and surface potential within 3-7 days, the stability was considered to be good.

[0267] Method for calculating nucleic acid encapsulation efficiency: Specifically, agarose gel electrophoresis is used. First, the nucleic acid feed amount of each group of lipid nanoparticles is set at 10 μg / mL, and the mass ratio of the metal-polyphenol complex to the nucleic acid is 20:1. Equal concentrations of nucleic acid are dissolved in enzyme-free Tris-HCl buffer as a positive control, and the negative control is enzyme-free Tris-HCl buffer. The concentration of agarose gel is 1.5%. At this time, the gaps in the gel only allow free nucleic acids to pass through, but not lipid nanoparticles. Electrophoresis is stopped when the free nucleic acid bands are clearly distinguishable. ImageJ software is used to calculate the grayscale value of free nucleic acids in different groups. The positive control group is set as 100%. The ratio of free nucleic acid in each group to the positive control is the relative amount of free nucleic acid. The encapsulation rate of each group is (100-relative amount of free nucleic acid)%. A nucleic acid encapsulation rate of more than 50% is considered acceptable.

[0268] Cell culture method: Human embryonic kidney cell line 293T was cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0269] Flow cytometry analysis of eGFP-positive cell percentage: 293T cells were seeded in 12-well plates at a seeding density of 5 × 10 5 Cells were plated at 1 mL of MPNP or eGFP-mRNA@MPNP at a concentration of 2 μg / mL at 80% cell density. After 48 hours, the cell suspension was harvested and 20,000 cells were collected using the FITC channel of a flow cytometer. The percentage of eGFP-positive cells was analyzed using the following formula: eGFP-positive cell percentage = number of eGFP-expressing cells / total number of cells × 100%. An eGFP-positive cell percentage of 40% or higher was considered acceptable.

[0270] Preparation of metal ions as Al 3+ mRNA-metal-polyphenol complex particles

[0271] A metal-polyphenol complex was prepared according to the method described in Example 1.2, wherein curcumin (Formula 1) and Al(NO₃)₃·9H₂O were added at a 1:1 ratio. The metal-polyphenol complex, 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) were dissolved in ethanol at varying molar ratios as the organic phase. The proportions of the 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) at pH 5.0 as the aqueous phase. The mass of the metal-polyphenol complex and the mass of the 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 phase and the aqueous phase in the microfluidic chip was 12 ml / min. Among them, the drug mRNA was an mRNA encoding the fluorescent protein eGFP, and its sequence was SEQ ID NO.1 (720 nt). eGFP-mRNA@MPNP was prepared. eGFP-mRNA@MPNP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA), and the control group was incubated with MPNP. After 48 hours, the cell suspension was collected, and the percentage of eGFP-positive cells was detected by flow cytometry.

[0272] The particle size, surface potential and stability of the prepared eGFP-mRNA@MPNP were tested, and the efficiency of nucleic acid encapsulation in eGFP-mRNA@MPNP was calculated.

[0273] Method for detecting particle size and result judgment standard: The particle size of nanoparticles was tested using a Malvern Zetasizer laser particle size analyzer, and a particle size within the range of 30 to 400 nm was considered acceptable.

[0274] Method for detecting surface potential and result judgment standard: The surface potential of nanoparticles was tested using a Malvern Zetasizer laser particle size analyzer, and a potential in the range of -10 to 10 mV was considered acceptable.

[0275] Stability testing method and result judgment criteria: The nanoparticles were placed at 4°C for 7 days, and the particle size and surface potential of the nanoparticles were measured using a Malvern Zetasizer. If there was no significant change in the particle size and surface potential within 3-7 days, the stability was considered to be good.

[0276] Method for calculating nucleic acid encapsulation efficiency: Specifically, agarose gel electrophoresis is used. First, the nucleic acid feed amount for each group of lipid nanoparticles is set at 10 μg / mL, and the mass ratio of the metal-polyphenol complex to the nucleic acid is 18:1. Equal concentrations of nucleic acid are dissolved in enzyme-free Tris-HCl buffer as a positive control, and the negative control is enzyme-free Tris-HCl buffer. The concentration of the agarose gel is 1.5%. At this time, the gaps in the gel only allow free nucleic acids to pass through, but not lipid nanoparticles. Electrophoresis is stopped when the free nucleic acid bands are clearly distinguishable. ImageJ software is used to calculate the grayscale value of free nucleic acids in different groups. The positive control group is set as 100%. The ratio of free nucleic acid in each group to the positive control is the relative amount of free nucleic acid. The encapsulation efficiency of each group is (100-relative amount of free nucleic acid)%. A nucleic acid encapsulation efficiency of more than 50% is considered acceptable.

[0277] Cell culture method: Human embryonic kidney cell line 293T was cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0278] Flow cytometry analysis of eGFP-positive cell percentage: 293T cells were seeded in 12-well plates at a seeding density of 5 × 10 5 Cells were plated at 1 mL of MPNP or eGFP-mRNA@MPNP at a concentration of 2 μg / mL at 80% cell density. After 48 hours, the cell suspension was harvested and 20,000 cells were collected using the FITC channel of a flow cytometer. The percentage of eGFP-positive cells was analyzed using the following formula: eGFP-positive cell percentage = number of eGFP-expressing cells / total number of cells × 100%. An eGFP-positive cell percentage of 40% or higher was considered acceptable.

[0279] The principle of loading nucleic acid on metal-chelated polyphenol complex nanoparticles (MPNPs) assembled by metal-polyphenol complexes is that curcumin binds to Fe through coordination bonds. 3+ or Al 3+ connected to form a metal-polyphenol complex, the Fe 3+ or Al 3+ The metal-polyphenol complex is linked to the nucleic acid through a coordination bond, ensuring that the metal-polyphenol complex and other components self-assemble into MPNPs while simultaneously loading the nucleic acid into the nanoparticles. There are two possible possibilities for curcumin's contribution to MPNP loading: 1) Curcumin interacts with nucleic acids, assisting MPNP loading with nucleic acids, for example, by inserting into the minor groove of nucleic acids; 2) Curcumin may not directly interact with nucleic acids.

[0280] Example 2.1. Dosage ratio of components of the metal-polyphenol complex

[0281] Curcumin (Formula 1) and FeCl3 in Example 2 were added at different ratios (1:1, 3:2, and 2:1), and the other steps were the same as in Example 2 to prepare different eGFP-mRNA@MPNPs, and their nucleic acid encapsulation efficiencies were tested.

[0282] Result analysis: As shown in Table 1-1, when the ratio of curcumin (Formula 1) and FeCl3 was 1:1, the mRNA encapsulation efficiency of the prepared metal-polyphenol complex particles was 85%; when the ratio of curcumin (Formula 1) and FeCl3 was 3:2, the mRNA encapsulation efficiency of the prepared metal-polyphenol complex particles was 72%; when the ratio of curcumin (Formula 1) and FeCl3 was 2:1, the mRNA encapsulation efficiency of the prepared metal-polyphenol complex particles was 63%. 3+ The function of curcumin is to connect with nucleic acid. 3+ Since there are at most three complexation sites, the curcumin and FeCl₃ ratio in the drug-lipid particles should be 1:1 to ensure maximum nucleic acid loading within the metal-polyphenol complex particles. Our results also confirmed that a 1:1 curcumin:FeCl₃ ratio resulted in the highest mRNA loading efficiency in the metal-polyphenol complex particles. Nucleic acid loading rates of the metal-polyphenol complex particles exceeded 60% when the curcumin:FeCl₃ ratio ranged from 1:1 to 2:1.

[0283] Table 1-1 Metal ions are Fe 3+ The proportion of components of the metal-polyphenol complex and the function of the metal-polyphenol complex particles prepared by the method

[0284] <![CDATA[Dosage ratio of curcumin (Formula 1) and FeCl3]]> mRNA encapsulation efficiency of metal-polyphenol composite particles 1:1 85％ 3:2 72％ 2:1 63％

[0285] Curcumin (Formula 1) and Al(NO3)3·9H2O in Example 2 were added at different ratios (1:1, 3:2, and 2:1). Other steps were the same as in Example 2 to prepare different eGFP-mRNA@MPNPs, and their nucleic acid encapsulation efficiencies were tested.

[0286] Result analysis: As shown in Table 1-2, when the ratio of curcumin (Formula 1) and Al(NO3)3·9H2O was 1:1, the eGFP-mRNA encapsulation efficiency of the prepared metal-polyphenol composite particles was 86%; when the ratio of curcumin (Formula 1) and Al(NO3)3·9H2O was 3:2, the eGFP-mRNA encapsulation efficiency of the prepared metal-polyphenol composite particles was 70%; when the ratio of curcumin (Formula 1) and Al(NO3)3·9H2O was 2:1, the eGFP-mRNA encapsulation efficiency of the prepared metal-polyphenol composite particles was 66%. In the metal-polyphenol composite particles, Al 3+ The function of Al is to connect the phospholipid complex with the nucleic acid. 3+ Since there are a maximum of three complexation sites, the ratio of curcumin and Al(NO3)3·9H2O in the drug-lipid particles should be 1:1 to ensure that the metal-polyphenol complex particles can encapsulate as many nucleic acids as possible. Experimental results also confirmed that when the curcumin and Al(NO3)3·9H2O ratio is 1:1, the metal-polyphenol complex particles prepared with it have the highest eGFP-mRNA encapsulation efficiency. When the curcumin and Al(NO3)3·9H2O ratio ranges from 1:1 to 2:1, the nucleic acid encapsulation efficiency of the metal-polyphenol complex particles exceeds 60%.

[0287] Table 1-2 Metal ions are Al 3+ The proportion of components of the metal-polyphenol complex and the function of the metal-polyphenol complex particles prepared by the method

[0288]

[0289] Example 2.2. Preparation of the Proportions of Metal-Polyphenol Complex, Distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and Cholesterol (CHOL) in Drug-Lipid Particles

[0290] Compared with Example 2, the ratio of metal-polyphenol complex, distearoylphosphatidylcholine (DSPC, Formula 51), DSPE-PEG2000 (Formula 58), and cholesterol (CHOL, Formula 59) is as shown in Table 1-3 (the metal ion is Fe 3+ ) and Table 1-4 (metal ions are Al 3+ ), the other conditions are the same.

[0291] Result analysis: As shown in Table 1-3, when the metal-polyphenol complex (metal ion is Fe 3+When the metal-polyphenol complex accounted for 5% (5-20%), DSPC accounted for 40-75% (40-75%), CHOL accounted for 0-48% (0-48%), and DSPE-PEG2000 accounted for 2-10%, the drug-lipid particle size was in the range of 30-400 nm, the surface potential was in the range of -10-10 mV, the in vitro stability was ≥3 days, the mRNA encapsulation efficiency was >50%, and the eGFP protein positive cell rate was above 65%. Among them, when the metal-polyphenol complex accounted for 5%, distearoylphosphatidylcholine (DSPC) accounted for 60%, cholesterol (CHOL) accounted for 30%, and DSPE-PEG2000 accounted for 5%, the drug-lipid particle performance was optimal, namely the particle size was in the range of 120 nm, the surface potential was in the range of -1.99 mV, the in vitro stability was >7 days, the mRNA encapsulation efficiency was 85%, and the eGFP protein positive cell rate was 97%. Because the metal-chelated polyphenol complex nanoparticles (MPNP) mainly rely on the metal-polyphenol complex to adsorb nucleic acids, the proportion of the metal-polyphenol complex cannot be too low; the role of DSPC is to maintain the stability of the nanoparticle structure, and its performance will be better when its content is in the range of 40%-75%; the role of DSPE-PEG2000 is to prevent nanoparticle aggregation and prolong the circulation time in the body, and its performance will be better when its content is in the range of 2%-10%; when the CHOL content is 0%, according to experimental results, the stability of its drug-lipid particles is within an acceptable range. When the CHOL content is greater than 0% and less than 48%, CHOL has the effect of enhancing the fluidity of the nanoparticles, which is beneficial to maintaining the stability of the nanoparticles.

[0292] The above results suggest that metal-polyphenol complexes (metal ions are Fe 3+ When the proportion of ) is in the range of (5-20)%, the proportion of DSPC is in the range of (40-75)%, the proportion of CHOL is in the range of (0-48)%, and the proportion of DSPE-PEG2000 is in the range of (2-10)%, the drug-metal-polyphenol complex particles (mRNA@MPNP) have better drug loading performance.

[0293] Table 1-3 Metal ions are Fe 3+ The ratio of each component in the drug-metal-polyphenol composite particles (mRNA@MPNP)

[0294]

[0295] Result analysis: As shown in Table 1-4, when the metal-polyphenol complex (metal ion is Al 3+When the metal-polyphenol complexes accounted for 5% of the total metal-polyphenol complex, 45% of distearoylphosphatidylcholine (DSPC), 47% of cholesterol (CHOL), and 3% of DSPE-PEG2000, the particle size ranged from 30 to 400 nm, the surface potential ranged from -10 to 10 mV, the in vitro stability was ≥3 days, the mRNA loading efficiency was >50%, and the eGFP protein positive expression rate was above 65%. The metal-polyphenol complexes exhibited the best performance when the metal-polyphenol complexes accounted for 5%, distearoylphosphatidylcholine (DSPC) accounted for 45%, cholesterol (CHOL) accounted for 47%, and 3% of DSPE-PEG2000, with a particle size range of 100 nm, a surface potential range of -2.74 mV, in vitro stability for >7 days, an mRNA loading efficiency of 86%, and an eGFP protein positive cell rate of 97%. Because mRNA@MPNP mainly relies on metal-polyphenol complexes to adsorb nucleic acids, the proportion of metal-polyphenol complexes cannot be too low; when the DSPC content is in the range of (30-75)%, the stability of its nanoparticles is within an acceptable range; the function of DSPE-PEG2000 is to prevent nanoparticle aggregation and increase the circulation time in the body, and its performance will be better when its content is in the range of (2-10)%; the function of CHOL is to enhance the fluidity of nanoparticles, and maintaining a certain content is beneficial to the stability of nanoparticles.

[0296] The above results suggest that metal-polyphenol complexes (metal ions are Al 3+ ) accounts for (5-20)%, when DSPC accounts for (30-75)%, when CHOL accounts for (0-48)%, when DSPE-PEG2000 accounts for (2-10)%, mRNA@MPNP has better drug loading performance.

[0297] Table 1-4 Metal ions are Al 3+ The ratio of each component in the drug-metal-polyphenol composite particles (mRNA@MPNP)

[0298]

[0299]

[0300] Example 2.3: Types of non-cationic lipids or non-ionizable lipids in preparing eGFP-mRNA@MPNP

[0301] Compared with Example 2, the replacement of distearoylphosphatidylcholine (DSPC) is shown in Table 1-5 and Table 1-6, and the other conditions are the same.

[0302] Results: To explore whether DSPC in eGFP-mRNA@MPNP can be replaced by other non-cationic lipids or non-ionizable lipids in addition to the conjugated lipids that inhibit particle aggregation, DSPE, DSPA and DSPG were selected to replace DSPC, and the particle size, surface potential, stability and mRNA encapsulation efficiency were tested to prove that DSPC in eGFP-mRNA@MPNP can be replaced by other non-cationic lipids or non-ionizable lipids. After replacement, its function is equivalent to that of eGFP-mRNA@MPNP containing DSPC (Table 1-5 (metal ions are Fe 3+ ) and Table 1-6 (metal ions are Al 3+ Because the main function of the non-cationic lipid DSPC in eGFP-mRNA@MPNP is to improve liposome membrane fusion, increase stability, and reduce toxicity, and other non-cationic lipids or non-ionizable lipids also have the function of improving liposome membrane fusion, increasing stability, and reducing toxicity, DSPC in the drug-lipid particle can be replaced by other non-cationic lipids or non-ionizable lipids without affecting its efficacy.

[0303] Table 1-5 Metal ions are Fe 3+ Performance of non-cationic lipids other than conjugated lipids in drug-metal-polyphenol composite particles (mRNA@MPNP) to inhibit particle aggregation

[0304]

[0305] Table 1-6 Metal ions are Al 3+ Performance of non-cationic lipids other than conjugated lipids in drug-metal-polyphenol composite particles (mRNA@MPNP) to inhibit particle aggregation

[0306]

[0307] Example 2.4: Preparation of conjugated lipids to inhibit particle aggregation in eGFP-mRNA@MPNPs

[0308] Compared with Example 2, the substitution of DSPE-PEG2000 is shown in Table 1-7 (metal ion is Fe 3+ ) and Table 1-8 (metal ions are Al 3+ ), the other conditions are the same.

[0309] Analysis of results: In order to explore whether DSPE-PEG2000 in eGFP-mRNA@MPNP can be replaced by other conjugated lipids that inhibit particle aggregation, three other conjugated lipids that inhibit particle aggregation, namely DSPE-PEG700, DSPE-PEG5000 and DSPE-PEG1000, were selected to replace DSPE-PEG2000 respectively. By testing the particle size, surface potential, stability and mRNA encapsulation rate, it was proved that DSPE-PEG2000 in eGFP-mRNA@MPNP can be replaced by other conjugated lipids that inhibit particle aggregation. After replacement, its function is equivalent to that of eGFP-mRNA@MPNP containing DSPE-PEG2000 (Table 1-7 and Table 1-8). Because the main function of DSPE-PEG2000 in eGFP-mRNA@MPNP is to inhibit aggregation, and other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation, DSPE-PEG2000 in eGFP-mRNA@MPNP can be replaced by other conjugated lipids that inhibit particle aggregation without affecting its efficacy.

[0310] Table 1-7 Metal ions are Fe 3+ Conjugated lipid species for inhibiting particle aggregation in drug-metal-polyphenol complex particles (mRNA@MPNP)

[0311]

[0312] Table 1-8 Metal ions are Al 3+ Conjugated lipid species for inhibiting particle aggregation in drug-metal-polyphenol complex particles (mRNA@MPNP)

[0313]

[0314] Example 2.5 Preparation and Effect Characterization of mRNA@MPNP

[0315] Example 2.5.1: Metal ion is Fe 3+ Preparation and effect characterization of mRNA@MPNP

[0316] The mRNA in Example 2 was replaced with two other mRNAs, and three mRNA@MPNPs containing different target protein mRNA sequences were prepared according to the method of Example 2. The three different mRNA sequences are: ① The mRNA sequence encoding the fluorescent protein eGFP is SEQ ID NO.1 (720nt); ② The mRNA sequence encoding the receptor binding domain (RBD) of the new coronavirus S1 subunit is SEQ ID NO.2 (669nt); ③ The mRNA sequence encoding the tumor antigen NY-ESO-1 is SEQ ID NO.3 (543nt). The preparation process of the remaining drug (mRNA)-lipid particles is the same as that in Example 2, and eGFP-mRNA@MPNP, RBD-mRNA@MPNP, and NY-ESO-1-mRNA@MPNP are obtained respectively.

[0317] eGFP-mRNA@MPNP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPNP. After 48 h, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry. The results are shown in Figure 1-1 ; RBD-mRNA@MPNP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPNP. After 24 hours, the supernatant was centrifuged and frozen at -20°C for later use. The expression level of the new coronavirus antigen RBD protein on the cells was detected using a commercially available new coronavirus antigen RBD ELISA detection kit. The results are shown in Figure 1-2 .

[0318] ELISA method for detecting RBD expression level:

[0319] 1. Sample collection: Place the cell supernatant at room temperature for 2 hours, centrifuge at 1000×g for 20 minutes, and collect the supernatant;

[0320] 2. Sample addition: Set up blank wells, standard wells, and test sample wells on the coated plate. Add 100 μL of sample diluent to the blank wells, add serially diluted standards to the standard wells, and add 100 μL of the test sample wells. Incubate at 37°C for 60 minutes.

[0321] 3. Discard the liquid in the wells and wash the plate 3 times, soaking for 1-2 minutes each time. Add 100 μL of the prepared biotinylated anti-RBD antibody working solution to each well, mix well, and incubate at 37°C for 60 minutes.

[0322] 4. Discard the liquid in the wells and wash the plate 3 times, soaking for 1-2 minutes each time;

[0323] 5. Add 100 μL of prepared streptavidin HRP working solution to each well, mix well, and incubate at 37°C for 45 minutes;

[0324] 6. Discard the liquid in the wells and wash the plate 3 times, soaking for 1-2 minutes each time;

[0325] 7. Add 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution to each well and incubate at 37°C in the dark for 15 minutes.

[0326] 8. Add 100 μL of stop solution to each well to terminate the reaction;

[0327] 9. Measure the optical density (OD) of each well at a wavelength of 450 nm.

[0328] Data analysis: Draw a standard curve with the concentration of the standard as the horizontal axis and the OD value as the vertical axis.

[0329] The experimental animals were randomly divided into two groups (experimental group and control group), with 5 animals in each group. Among them, the RBD-mRNA@MPNP animal model was BALB / c mice. Each mouse received the first intramuscular administration on the first day and the second intramuscular administration on the 14th day. The experimental group was injected with RBD-mRNA@MPNP, and the control group was injected with metal-polyphenol complex particles (MPNP) that were not loaded with mRNA. The dose for each administration was 100 μL, and the RBD-mRNA@MPNP preparation in the experimental group contained 30 mg of mRNA. On the 28th day after the first administration, the blood of the mice was collected, the serum was separated and gradiently diluted, and the total RBD IgG antibodies against the new coronavirus S1 subunit produced in the mice were detected by a commercially available ELISA kit. The results are as follows: Figure 1-3 shown.

[0330] The animal model of NY-ESO-1-mRNA@MPNP was C57BL / 6 mice. Each mouse received four intramuscular injections on days 1, 7, 14, and 21. The experimental group was injected with NY-ESO-1-mRNA@MPNP, while the control group was injected with metal-polyphenol complex particles (MPNP) without mRNA loading. The dose of each administration was 100 μL, and the mRNA@MPNP preparation in the experimental group contained 30 mg of mRNA. On the 28th day after the first administration, the blood of the mice was collected, the serum was separated and serially diluted, and the total anti-NY-ESO-1 IgG antibodies produced in the mice were detected by ELISA. The results are shown in the figure below. Figure 1-4 shown.

[0331] Method for detecting total anti-NY-ESO-1 IgG antibodies in mice:

[0332] Preparation of reagents used in ELISA method:

[0333] 1. Coating solution: Accurately weigh 8.4 g of NaHCO3 and dissolve it in 1 L of distilled water (DDW). After the solid is completely dissolved, use 1 M NaOH solution to adjust the pH of the entire solution to 9.6. Store the prepared coating solution at 4°C until use.

[0334] 2. Wash solution: Add 0.5 mL of Tween-20 to 1 L of 0.01 M PBS solution, mix well and place at room temperature.

[0335] 3. Blocking solution: Accurately weigh 20g of BSA and add it to 1L of 0.01M PBS solution. Ultrasonicate the undissolved BSA powder in the solution. When all the solids in the solution are dissolved and the solution turns light yellow, place it in a refrigerator at 4℃ for future use.

[0336] 4. Antibody diluent: Accurately weigh 2.5 g BSA and dissolve it in 250 mL 0.01 M PBS solution. After the solid is completely dissolved, add 1.25 mL Tween-20, mix well, and store at 4°C until ready to use.

[0337] 5. Color development solution: 0.1M citric acid: Add 19.2g of citric acid to 1000mL of DDW water (A). 0.2M disodium hydrogen phosphate: Add 28.4g of anhydrous disodium hydrogen phosphate to 1000mL of DDW water (B). 24.3mL of 0.1M citric acid solution (A), 25.7mL of 0.2M phosphate buffer (B), and 50mL of DDW water. Add 50mg of OPD (o-phenylenediamine) immediately before use, and 0.15mL of 30% H2O2.

[0338] 6. Stop solution: 2MH2SO4: concentrated sulfuric acid 55.5mL, add DDW to 500mL.

[0339] ELISA method was used to determine the titer of antibodies in mouse serum:

[0340] 1. Coating: Dilute NY-ESO-1 antigen to 1 μg / mL with coating solution, add 50 μL / well to a 96-well plate, and coat overnight at 4°C.

[0341] 2. Blocking: Dry the coating solution in the well plate, wash with blocking solution 3 times, 5 minutes each time and dry, add 150 μL blocking solution to each well, and incubate at 37°C for 2 hours.

[0342] 3. Drying: Shake off the blocking solution and incubate at 37°C for 1-2 hours until the liquid at the bottom of the plate is completely dry.

[0343] 4. Immunization: The serum sample was initially diluted 1:1000 with antibody diluent, and then serially diluted 1:2. The diluted serum sample was added to a closed 96-well plate at 100 μL / well and incubated at 37°C for 2 hours. The liquid in the well plate was shaken dry, and the washing solution was added at 300 μL / well. The plate was shaken slowly for 40 seconds and this step was repeated three times. A 1:1000 diluted biotinylated goat anti-mouse IgG antibody was added at 100 μL / well and incubated at 37°C. 1h; shake dry the liquid in the well plate, add washing solution, and repeat the above washing steps; add freshly prepared streptavidin-labeled horseradish peroxidase HRP working solution, 100μL / well, incubate at 37℃ for 1h; shake dry the liquid in the well plate, add washing solution, and repeat the above washing steps; add color development solution, 100μL / well, react at room temperature for 5min, and then add stop solution, 50μL / well, to stop color development; use a microplate reader to measure the absorbance at 450nm.

[0344] On the 28th day after administration of RBD-mRNA@MPNP, spleens of normal mice were collected and prepared into single cell suspensions under sterile conditions. 100,000 spleen cells / well were plated in a cell plate, and RBD protein was added at a final concentration of 10 mg / mL and cultured for 48 hours. The supernatant was removed by centrifugation, and the expression levels of IFN-γ, IL-2, and IL-4 were determined by ELISA kits. The results are shown in Figure 3. Figure 1-5 shown.

[0345] On the 28th day after administration of NY-ESO-1-mRNA@MPBP, spleens of normal mice were collected and prepared into single cell suspensions under sterile conditions. 100,000 spleen cells / well were plated in a cell plate, NY-ESO-1 protein was added at a final concentration of 10 mg / mL, and cultured for 48 hours. The supernatant was removed by centrifugation, and the expression levels of IFN-γ, IL-2, and TNF-α were determined by ELISA kits. The results are shown in Figure 3. Figure 1-6 shown.

[0346] Result analysis: Figure 1-1 As shown, 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; Figure 1-2 As shown in the figure, the RBD protein encoded by the MPNP-encapsulated RBD-mRNA was 166 ng / mL in the supernatant of 293T cells, while the RBD protein content in the supernatant of 293T cells transfected with the empty vector MPNP was 0. The results suggest that the drug-metal-polyphenol complex particles (mRNA-MPNP) can encapsulate and deliver any mRNA and directly encode polypeptides in cells. Figure 1-3As shown in the results 1-4, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP can effectively induce humoral immunity in mice and produce high levels of antigen-specific binding antibodies. The IgG antibody titer in mice treated with RBD-mRNA@MPNP reached 84363.4; the IgG antibody titer in mice treated with NY-ESO-1-mRNA@MPNP reached 4283.56. Figure 1-5 , as shown in Figures 1-6, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP can effectively induce cellular immunity in mice, that is, activate immune cells and produce a large amount of cytokines. Among them, RBD-mRNA@MPNP makes the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 271.8pg / mL, 269.6pg / mL, and 75.8pg / mL, respectively; NY-ESO-1-mRNA@MPNP makes the expression levels of cytokines IFN-γ, IL-2, and TNF-α reach 76.38pg / mL, 74.56pg / mL, and 69.31pg / mL, respectively. The results suggest that the drug-metal-polyphenol complex particles (mRNA@MPNP) can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), thereby effectively inducing humoral immunity and cellular immunity in mice, producing high levels of antigen-specific binding antibodies and cytokines, and playing the role of anti-new coronavirus mRNA vaccines and anti-tumor mRNA vaccines.

[0347] Example 2.5.2: Metal ion is Al 3+ Preparation and effect characterization of mRNA@MPNP

[0348] The difference between this embodiment and embodiment 2.5.1 is that the metal ion Fe in embodiment 2.5.1 is replaced by 3+ Replaced by Al 3+ .

[0349] Result analysis: Figure 1-7 As shown, 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; Figure 1-8 As shown in the figure, the RBD protein encoded by the MPNP-encapsulated RBD-mRNA was 207 ng / mL in the supernatant of 293T cells, while the RBD protein content in the supernatant of 293T cells transfected with the empty vector MPNP was 0. The results suggest that mRNA-MPNP can encapsulate and deliver any mRNA and directly encode polypeptides in cells. Figure 1-9As shown in the results 1-10, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP can effectively induce humoral immunity in mice and produce high levels of antigen-specific binding antibodies. The IgG antibody titer in mice treated with RBD-mRNA@MPNP reached 94828.6; the IgG antibody titer in mice treated with NY-ESO-1-mRNA@MPNP reached 5848.02. Figure 1-11 As shown in Figures 1-12, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP can effectively induce cellular immunity in mice, that is, activate immune cells and produce a large amount of cytokines. Among them, RBD-mRNA@MPNP makes the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 306.2pg / mL, 289.6pg / mL, and 88.2pg / mL, respectively; NY-ESO-1-mRNA@MPNP makes the expression levels of cytokines IFN-γ, IL-2, and TNF-α reach 91.88pg / mL, 85.32pg / mL, and 80.22pg / mL, respectively. The results suggest that mRNA@MPNP can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), and then effectively inducing humoral immunity and cellular immunity in mice, producing high levels of antigen-specific binding antibodies and cytokines, and playing the role of anti-new coronavirus mRNA vaccines and anti-tumor mRNA vaccines.

[0350] Example 2.6 Preparation and Effect of siRNA-Metal-Polyphenol Composite Particles (siRNA@MPNP)

[0351] Example 2.6.1: Metal ion is Fe 3+ Preparation and effect of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPNP)

[0352] The mRNA in Example 2 was replaced with siRNA, and three siRNA@MPNPs containing different siRNAs were prepared according to the method of Example 2. The genes, sequences, and corresponding random control sequences of the three different siRNA targeting genes are as follows: ① The sequence of the siRNA targeting the Bcl-2 gene (Bcl-2-siRNA) is SEQ ID NO.4 (antisense strand) and SEQ ID No.21 (sense strand) (19 bp), and its random control sequence is SEQ ID NO.5 (antisense strand) and SEQ ID No.22 (sense strand) (19 bp); ② The sequence of the siRNA targeting the PLK1 gene (PLK1-siRNA) is SEQ ID NO.6 (antisense strand) and SEQ ID No.23 (sense strand) (21 bp), and its random control sequence is SEQ ID NO.7 (antisense strand) and SEQ ID No.24 (sense strand) (19 bp); ③ The sequence of the siRNA targeting the Gal-1 gene (Gal-1-siRNA) is SEQ ID NO.8 (19 bp); its random control sequence is SEQ ID NO.9 (19 bp). The preparation process of the remaining siRNA@MPNP is the same as that in Example 2.

[0353] The sequence of Bcl-2-siRNA is as follows:

[0354] Antisense: 5′-CAGCUUAUAAUGGAUGUAC-3′ (SEQ ID No. 4);

[0355] Sense: 5′-GUACAUCCAUUAUAAGCUG-3′ (SEQ ID No. 21) (19 bp).

[0356] The random control sequences of Bcl-2-siRNA are as follows:

[0357] Antisense: 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID No. 5);

[0358] Sense: 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID No. 22) (19 bp).

[0359] The sequence of PLK1-siRNA is as follows:

[0360] Antisense: 5'-UAAGGAGGGUGAUCUUCUUCA-3' (SEQ ID No. 6);

[0361] Sense: 5'-UGAAGAAGAUCACCCUCCUUA-3' (SEQ ID No. 23) (21 bp).

[0362] The random control sequences of PLK1-siRNA are as follows:

[0363] Antisense: 5'-CUUACGCUGAGUACUUCGA-3' (SEQ ID No. 7);

[0364] Sense: 5'-UCGAAGUACUCAGCGUAAG-3' (SEQ ID No. 24) (19 bp).

[0365] The sequence of Gal-1-siRNA is as follows:

[0366] 5'-GCUGCCAGAUGGAUACGAA-3' (SEQ ID No. 8) (19 bp).

[0367] The random control sequence of Gal-1-siRNA is as follows:

[0368] 5'-GGAAAUCCCCCAACAGUGA-3' (SEQ ID No. 9) (19 bp).

[0369] Cell culture methods: U251 human glioblastoma cells were grown as monolayers in high-glucose (4.5 g / L) DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (Bio Industries) at 37°C and 5% CO2, and passaged twice a week.

[0370] U251 cells were cultured at 1 × 10 6 After the cells were seeded in a 6-well plate at a high density for approximately 24 hours, each well of cells was incubated with siRNA@MPNP containing the above-mentioned siRNA (where the concentration of siRNA was 2 μg / mL) for 72 hours. The cells were then collected and total RNA was extracted. The mRNA expression levels of the target genes (Bcl-2, PLK1, and Gal-1) were detected by RT-PCR, and the ability of siRNA@MPNP to silence the target genes in the cells was calculated.

[0371] RT-PCR specific process:

[0372] Total RNA extraction: Discard the culture medium from the six-well plate, rinse three times with PBS buffer, and lyse the cells by adding 1 mL of Trizol to each well. Add 200 μL of chloroform, shake thoroughly, and let stand at room temperature for 10 minutes. Centrifuge at 13,000 rpm and 4°C for 15 minutes to obtain a three-phase liquid, with RNA dissolved in the upper aqueous phase. Aspirate the upper aqueous phase and transfer it to a new enzyme-free 1.5 mL centrifuge tube. Add 500 μL of isopropanol, let stand at room temperature for 10 minutes, and centrifuge at 13,000 rpm and 4°C for 15 minutes to obtain an RNA precipitate. Remove the supernatant and add 1 mL of 75% (v / v) ethanol, freshly prepared with RNase-free water, to each tube. Carefully pipette to dissolve the white RNA precipitate at the bottom of the tube. Centrifuge at 7,500 rpm and 4°C for 10 minutes. Remove the supernatant and aspirate as much of the liquid as possible. Air the RNA precipitate at the bottom of the tube with the lid open at room temperature. Dissolve it in 50 μL of enzyme-free water. Assay the RNA purity and concentration using an ultra-micro UV-visible spectrophotometer.

[0373] cDNA reverse transcription: using Ta Ka Ra Prime Script TM The RT reagent kit with gDNAEraser reverse transcribes RNA into cDNA. Removing genomic DNA (gDNA) prior to 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 I, 1 μL RT Primer Mix, 4 μL 5× Prime Script Buffer 2, and 4 μL RNase-free dH2O. After preparing the reaction mixture, incubate at 37°C for 15 minutes, then terminate the reaction by incubating at 85°C for 5 seconds. Store at 4°C until ready to use.

[0374] RT-PCR Procedure: This detection method uses the SYBR Green dye method and does not require a probe. Specifically, real-time PCR reactions were performed using cDNA from different samples as templates. The reaction solution was prepared on ice: 5 μL SYBR Premix DimerEraser (2×), 0.3 μL PCR Forward Primer (10 μM), 0.3 μL PCR Reverse Primer (10 μM), 0.2 μL ROX Reference Dye II (50×), 1 μL cDNA template obtained in the previous step, and 3.2 μL dH2O. Samples were added to the well plate, with 10 μL per well. After addition, the sample was centrifuged (1000 rpm, 5 min) to eliminate liquid adhering to the wall and any bubbles in the reaction solution. Real-time PCR was performed using an ABIViiA7 real-time fluorescence quantitative PCR instrument. The reaction procedure was: 95°C, 30 seconds (1 cycle) → 95°C, 5 seconds; 55°C, 30 seconds; 72°C, 30 seconds (40 cycles) → 60°C-95°C, 2 minutes (1 cycle). The experiment was repeated three times, and the average Ct value was calculated for each group. The fold difference in expression between the experimental and control groups was calculated. The control gene was GAPDH. RT-PCR primers were as follows: ①Bcl-2 primer: forward: 5'-AGGATTGTGGCCTTCTTTGAG-3', reverse: 5'-AGACAGCCAGGAGAAATCAAAC-3'; ②PLK1 primer: forward: 5'-ACCAGCACGTCGTAGGATTC-3', reverse: 5'-CAAGCAATTTGCCGTAGG-3'; ③Gal-1 primer: forward: 5'-CAATCATGGCCTGTGGTCTG-3', reverse: 5'-GTGTAGGCACAGGTTGTTGCTG-3'. ④GAPDH primer: forward: 5'-TCAGGGGTTTCACATTTGGCA-3', reverse: 5'-GGAGCGGAAAACCA-3'. The expression level of each target gene was expressed using the RQ value (2 -ΔΔCT ). The formula is as follows:

[0375] Fold Change = 2 –ΔΔCt

[0376] Where ΔΔCt=ΔCt 实验组 –ΔCt 对照组 , ΔCt=Ct 目的基因 -Ct 内参基因

[0377] The calculation method of gene silencing efficiency is: 100% - gene expression level of experimental group / gene expression level of control group.

[0378] Result analysis: Figure 1-13 As shown in Figures 1-14 and 1-15 (where scrsiRNA is a random control sequence), the three drug-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP, PLK1-siRNA@MPNP, and Gal-1-siRNA@MPNP) all significantly interfered with their corresponding target genes. Bcl-2-siRNA@MPNP achieved a 67% inhibition rate on the target gene Bcl-2; PLK1-siRNA@MPNP achieved an 87% inhibition rate on the target gene PLK1; and Gal-1-siRNA@MPNP achieved a 64% inhibition rate on the target gene Gal-1. These results suggest that siRNA@MPNP can carry any siRNA for target gene intervention therapy, potentially acting as a carrier of siRNA drugs, vaccines, or other products.

[0379] Example 2.6.2: Metal ion is Al 3+ Preparation and effect of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPNP)

[0380] The difference between this embodiment and embodiment 2.6.1 is that the metal ion Fe in embodiment 3.6.1 is replaced by 3+ Replaced by Al 3 + .

[0381] Result analysis: Figure 1-16 As shown in Figures 1-17 and 1-18, all three siRNA@MPNPs significantly interfered with their corresponding target genes. Bcl-2-siRNA@MPNP achieved a 72% inhibition rate against the target gene Bcl-2; PLK1-siRNA@MPNP achieved an 88.07% inhibition rate against the target gene PLK1; and Gal-1-siRNA@MPNP achieved a 70.11% inhibition rate against the target gene Gal-1. These results suggest that siRNA@MPNPs can carry any siRNA for target gene intervention therapy, potentially maximizing the potential of siRNA-loaded drugs, vaccines, or other products.

[0382] Example 2.7 Preparation and Effect of ASO-Metal-Polyphenol Composite Particles (ASO@MPNP)

[0383] Example 2.7.1: Metal ion is Fe 3+Preparation and effect of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPNP)

[0384] The mRNA in Example 2 was replaced with ASO, and three drug-metal-polyphenol composite particles (ASO@MPNP) containing different ASOs were prepared according to the method of Example 2. The genes, sequences, and corresponding random control sequences of the three different ASOs were as follows: ① The ASO targeting the STAT3 gene (STAT3-ASO) sequence was SEQ ID NO.10 (17 nt), and its random control sequence was SEQ ID NO.11 (18 nt); ② The 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); ③ The 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 preparation process of the remaining drug (ASO)-metal-polyphenol composite particles was the same as in Example 2. Different ASO@MPNPs were incubated with different cells: ASO@MPNPs targeting STAT3 gene were incubated with U251 human glioblastoma cells; ASO@MPNPs targeting α-syn gene were incubated with SH-SY5Y human neuroblastoma cells; ASO@MPNPs targeting Bcl-2 gene were incubated with Daudi human lymphoma cells. 6 After the cells were seeded in a 6-well plate at a high density for approximately 24 hours, the cells in each well were incubated with drug-metal-polyphenol complex particles (ASO@MPNP) containing the above-mentioned ASO (wherein the concentration of ASO was 1 μg / mL) for 48 hours. The cells were then collected and total RNA was extracted. The mRNA expression levels of the target genes (STAT3, α-syn, Bcl-2) were detected by RT-PCR technology, and the ability of ASO@MPNP to silence the target genes in the cells was calculated.

[0385] The sequence of SEQ ID No. 10 (SEQ ID No. 10 of STAT3-ASO) is as follows:

[0386] 5'-GCTCCAGCATCTGCTTC-3'(17nt).

[0387] The sequence of SEQ ID No.11 (random control sequence of STAT3-ASO) is as follows:

[0388] 5'-GAAGCAGCAGATGCTGGA-3'(18nt).

[0389] The sequence of SEQ ID No. 12 (sequence of α-syn-ASO) is as follows:

[0390] 5'-GCCTCCCTCCACTGTCT-3'(16nt).

[0391] The sequence of SEQ ID No. 13 (random control sequence of α-syn-ASO) is as follows:

[0392] 5'-ACTCCCGAACCTGTCT-3'(16nt).

[0393] The sequence of SEQ ID No. 14 (sequence of Bcl-2-ASO) is as follows:

[0394] 5'-TCTCCCAGGCGTGCGCCAT-3'(18nt).

[0395] The sequence of SEQ ID No. 15 (random control sequence of Bcl-2-ASO) is as follows:

[0396] 5'-CAGCGTGCGCCATCCTTCCC-3'(20nt).

[0397] Cell culture: ①U251 human glioblastoma cells were grown as monolayers in a medium containing high glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (BioIndustries) and cultured at 37°C in 5% CO2. They were passaged twice a week. ②SH-SY5Y human neuroblastoma cells were grown in a medium containing high glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (BioIndustries). ③ Daudi human lymphoma cells were grown in a monolayer in a medium containing RPMI1640 + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin and 2 mm l-glutamine (BioIndustries), cultured at 37°C, 5% CO2, and passaged twice a week.

[0398] RT-PCR specific process:

[0399] Extraction of total RNA: Discard the culture medium from the six-well plate, rinse three times with PBS buffer, and add 1 mL of Trizol to each well to lyse the cells. Add 200 μL of chloroform, shake thoroughly, let stand at room temperature for 10 minutes, and centrifuge at 13,000 rpm at 4°C for 15 minutes to obtain a three-phase liquid, in which the RNA is dissolved in the upper aqueous phase. Pipette the upper aqueous phase into a new enzyme-free 1.5 ml centrifuge tube, add 500 μL of isopropanol, let stand at room temperature for 10 minutes, and centrifuge at 13,000 rpm at 4°C for 15 minutes to obtain the RNA precipitate. Remove the supernatant and add 1 mL of 75% (v / v) ethanol freshly prepared with RNase-free water to each tube. Carefully pipette to lift the white RNA precipitate at the bottom of the tube. Centrifuge at 7,500 rpm at 4°C for 10 minutes, remove the supernatant, and aspirate as much liquid as possible at the bottom of the tube. Open the lid and air dry the RNA precipitate at the bottom of the tube at room temperature, add 50 μL of enzyme-free water to dissolve it, and use an ultra-micro UV-visible spectrophotometer to detect the purity and concentration of the extracted RNA.

[0400] cDNA reverse transcription: using Ta Ka Ra Prime Script TM The RT reagent kit with gDNAEraser reverse transcribes RNA into cDNA. Removing genomic DNA (gDNA) prior to 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 I, 1 μL RT Primer Mix, 4 μL 5× Prime Script Buffer 2, and 4 μL RNase-free dH2O. After preparing the reaction mixture, incubate at 37°C for 15 minutes, then terminate the reaction by incubating at 85°C for 5 seconds. Store at 4°C until ready to use.

[0401] RT-PCR Procedure: This detection method uses the SYBR Green dye method and does not require a probe. Specifically, real-time PCR reactions were performed using cDNA from different samples as templates. The reaction solution was prepared on ice: 5 μL SYBR Premix DimerEraser (2×), 0.3 μL PCR Forward Primer (10 μM), 0.3 μL PCR Reverse Primer (10 μM), 0.2 μL ROX Reference Dye II (50×), 1 μL cDNA template obtained in the previous step, and 3.2 μL dH2O. Samples were added to the well plate, with 10 μL per well. After addition, the sample was centrifuged (1000 rpm, 5 min) to eliminate liquid adhering to the wall and any bubbles in the reaction solution. Real-time PCR was performed using an ABIViiA7 real-time fluorescence quantitative PCR instrument. The reaction procedure was: 95°C, 30 seconds (1 cycle) → 95°C, 5 seconds; 55°C, 30 seconds; 72°C, 30 seconds (40 cycles) → 60°C-95°C, 2 minutes (1 cycle). The experiment was repeated three times, and the average Ct value was calculated for each group. The fold difference in expression between the experimental and control groups was calculated. The control gene was GAPDH. The RT-PCR primer sequences are as follows: ①STAT3 primer: forward: 5'-TGATCACCTTTGAGACCGAGG-3', reverse: 5'-GATCACCACAACTGGCAAGG-3'; ②α-syn primer: forward: 5'-TGACGGGTGTGACAGCAGTAG-3', reverse: 5'-CAGTGGCTGCTGCAATG-3'; ③Bcl-2 primer: forward: 5'-AGGATT GTGGCCTTCTTTGAG-3', reverse: 5'-AGACAGCCAGGAGAAATCAAAC-3'; ④GAPDH primer: forward: 5'-TCAGGGG TTTCACATTTGGCA-3', reverse: 5'-GGAGCGGAA AACCA-3'. The expression levels of each target gene were analyzed using the RQ value (2 -ΔΔCT ). The formula is as follows:

[0402] Fold Change = 2 –ΔΔCt

[0403] Where ΔΔCt=ΔCt 实验组 –ΔCt 对照组 , ΔCt=Ct 目的基因 -Ct 内参基因

[0404] The calculation method of gene silencing efficiency is: 100% - gene expression level of experimental group / gene expression level of control group.

[0405] Result analysis: Figure 1-19 As shown in Figures 1-20 and 1-21 (where scrASO is a random control sequence), all three ASO@MPNPs significantly interfered with their corresponding target genes. STAT3-ASO@MPNP achieved a 72% inhibition rate on the target gene STAT3; α-syn-ASO@MPNP achieved a 78% inhibition rate on the target gene α-syn; and Bcl-2-ASO@MPNP achieved a 62% inhibition rate on the target gene Bcl-2. These results suggest that the drug-metal-polyphenol complex particles (ASO@MPNP) can carry any ASO for target gene intervention therapy, potentially acting as ASO-carrying drugs, vaccines, or other products.

[0406] Example 2.7.2: Metal ion is Al 3+ Preparation and effect of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPNP)

[0407] The difference between this embodiment and embodiment 2.7.1 is that the metal ion Fe in embodiment 2.7.2 is replaced by 3+ Replaced by Al 3 + .

[0408] Result analysis: Figure 1-22 As shown in Figures 1-23 and 1-24, all three ASO@MPNPs significantly interfered with their corresponding target genes. The STAT3-ASO@MPNP achieved a 75.4% inhibition rate against the target gene STAT3; the α-syn-ASO@MPNP achieved an 80.87% inhibition rate against the target gene α-syn; and the Bcl-2-ASO@MPNP achieved a 67.91% inhibition rate against the target gene Bcl-2. These results suggest that ASO@MPNPs can carry any ASO for target gene intervention therapy, potentially leveraging the potential of ASO-carrying drugs, vaccines, or other products.

[0409] Example 2.8 Preparation of drug (different types of nucleic acids)-metal-polyphenol complex particles and their effects

[0410] Example 2.8.1: Preparation of Metal Ions: Fe 3+ Drug (different types of nucleic acids)-metal-polyphenol complex particles and their effects

[0411] The mRNA in Example 2 was replaced by double-stranded RNA (siRNA), single-stranded DNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively. The different types of nucleic acid sequences are: ① The sequence of double-stranded RNA (Bcl-2-siRNA) is SEQ ID NO.4 (antisense chain) and SEQ ID No.21 (sense chain) (19 bp), and its random control sequence is SEQ ID NO.5 (antisense chain) and SEQ ID No.22 (sense chain) (19 bp); ② The sequence of single-stranded DNA (STAT3-ASO) is SEQ ID NO.10 (17 nt), and its random control sequence is SEQ ID NO.11 (18 nt); ③ The sequence of single-stranded RNA (mRNA encoding wild-type SARS-CoV-2 S protein) is SEQ ID NO.16 (3822 nt); ④ The sequence of double-stranded DNA (dsDNA) is SEQ ID NO.17 (antisense chain) and SEQ ID NO.25 (sense chain) (22 bp) (the 3' end of the sequence is labeled with fluorescent probe Cy3); ⑤ The sequence of single-stranded DNA (ssDNA) is SEQ ID NO.18 (22 nt) (the 3' end of the sequence is labeled with fluorescent probe Cy3). The drug-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP, STAT3-ASO@MPNP, S-mRNA@MPNP, dsDNA@MPNP, ssDNA@MPNP) containing the above-mentioned different types of nucleic acids were prepared according to the method of Example 2. The preparation process of the remaining drug-lipid particles was the same as that in Example 2.

[0412] U251 cells were cultured at 1 × 10 6 After about 24 hours of cell seeding in a 6-well plate, each well of cells was incubated with siRNA@MPNP (wherein the concentration of siRNA was 2 μg / mL) or ASO@MPNP (wherein the concentration of ASO was 2 μg / mL) for 72 hours, and then the cells were collected and total RNA was extracted. The mRNA expression levels of target genes (Bcl-2, STAT3) were detected by RT-PCR, and the ability of siRNA@MPNP or ASO@MPNP to silence the target genes in cells was calculated. The results are as shown in Example 2.6. Figure 1-13 And in Example 2.7 Figure 1-19 shown.

[0413] S-mRNA@MPNP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPNP. After 24 hours, the supernatant was centrifuged and stored at -20°C for later use. The cell pellet was resuspended in 100 μL PBS buffer solution, frozen and thawed twice, and sonicated for 10 minutes before centrifugation to obtain the supernatant. The expression levels of S protein in both the cell supernatant and the cell lysate were detected using a commercially available SARS-CoV-2 S protein ELISA kit. The results are as follows: Figure 1-25 shown.

[0414] A549 lung cancer cells were incubated with ds-DNA@MPNP at a concentration of 100 nM (the concentration of DNA contained) for 2 hours, and then the drug-lipid particles were removed. The cells were washed twice with PBS, and the cell nuclei were stained with Hochest33342 dye for 3 minutes, after which the dye was removed. The cells were washed twice with PBS, and the cells were observed using a high-content imaging system. The efficiency of drug-lipid particle transfection with DNA was calculated. The results are shown in Figure 1-26 .

[0415] HT22 mouse hippocampal neurons were incubated with ss-DNA@MPNP at a concentration of 200 nM (the concentration of DNA contained) for 2 hours, and then the drug-lipid particles were removed. After washing the cells twice with PBS, the cells were observed using a high-content imaging system, and the efficiency of drug-lipid particle transfection DNA was calculated. The results are shown in Figure 1-26 .

[0416] The culture method of human glioblastoma U251 cells is the same as that in Example 2.6.

[0417] The culture method of 293T cells is the same as that in Example 2.5.

[0418] Culture method of HT22 mouse hippocampal neurons: culture in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0419] The RT-PCR method is the same as in Example 2.6.

[0420] ELISA detection of S protein expression level: replace the "anti-RBD antibody working solution" in the ELISA method for detecting RBD in Example 2.5 with "anti-S protein antibody working solution", and the remaining steps are the same as Example 2.5.

[0421] The calculation method of gene silencing efficiency is the same as Example 2.6.

[0422] Method for calculating transfection efficiency: Use a high-content imaging system to randomly select 3-5 fields of view to obtain cell morphology under ordinary light source, fluorescence signal under excitation / emission light of 550nm / 570nm (excitation light of the fluorescent dye Cy3 that labels DNA) and fluorescence signal under excitation / emission light of 352nm / 461nm (excitation light of the fluorescent dye Hoechst33342 that labels the cell nucleus) in the same field of view. The ratio of the number of cells with Cy3 fluorescent signal in the randomly selected field of view to the number of cells with Hoechst33342 fluorescent signal in the same field of view is calculated as the transfection efficiency.

[0423] Result analysis: As in Example 2.6 Figure 1-13 As shown in Example 2.7, the inhibition rate of drug (double-stranded RNA)-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP) on the target gene Bcl-2 reached 67%; Figure 1-19 The inhibition rate of drug (single-stranded DNA)-metal-polyphenol complex particles (STAT3-ASO@MPNP) on the target gene STAT3 reached 72%; Figure 1-25 As shown in the figure, 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 vector MPNP was 0; the efficiency of drug (double-stranded DNA)-metal-polyphenol complex particles (dsDNA@MPNP) transfection of double-stranded DNA into cells was 100% ( Figure 1-26 ); the efficiency of drug (ssDNA)-metal-polyphenol complex particles (ssDNA@MPNP) transfection of single-stranded DNA into cells was 100% ( Figure 1-26 The results suggest that the drug-metal-polyphenol composite particles can encapsulate any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and achieve its function, wherein the length of the nucleic acid ranges from 16-3822nt.

[0424] Example 2.8.2: Preparation of Metal Ions as Al 3+ Drug (different types of nucleic acids)-metal-polyphenol complex particles and their effects

[0425] The difference between this embodiment and embodiment 2.8.1 is that the metal ion Fe in embodiment 2.8.1 is replaced by 3+ Replaced by Al 3 + .

[0426] Result analysis: As in Example 3.6.2 Figure 1-16As shown in Example 3.7.2, the inhibition rate of drug (double-stranded RNA)-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP) on the target gene Bcl-2 reached 72%; Figure 1-22 The inhibition rate of drug (single-stranded DNA)-metal-polyphenol complex particles (STAT3-ASO@MPNP) on the target gene STAT3 reached 75.4%; Figure 1-27 As shown in the figure, 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 vector MPNP was 0; the efficiency of drug (double-stranded DNA)-metal-polyphenol complex particles (dsDNA@MPNP) transfection of double-stranded DNA into cells was 100% ( Figure 1-28 ); the efficiency of drug (ssDNA)-metal-polyphenol complex particles (ssDNA@MPNP) transfection of single-stranded DNA into cells was 100% ( Figure 1-28 The results suggest that the drug-metal-polyphenol composite particles can encapsulate any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and achieve its function, where the length of the nucleic acid ranges from 16-3822nt.

[0427] Experimental Example 2: Performance Characterization of Drug-Metal-Polyphenol Composite Particles

[0428] Example 3: Synthesis and characterization of metal-polyphenol complexes

[0429] Example 3.1: Metal ion is Fe 3+ Synthesis and characterization of metal-polyphenol complexes

[0430] Curcumin and Fe 3+ Connectivity characterization is spectrophotometric: e.g. Figure 2-1 As shown, curcumin and Fe 3+ After binding, its maximum absorption wavelength shifted from 420nm to 372nm, and the conjugated structure of the metal-polyphenol complex changed, proving that curcumin successfully bound to Fe 3+ Complexation.

[0431] Example 3.2: Metal ion is Al 3+ Synthesis and characterization of metal-polyphenol complexes

[0432] Curcumin and Al 3+ Connectivity characterization is spectrophotometric: e.g. Figure 2-2 As shown, curcumin and Al 3+ After binding, its maximum absorption wavelength shifted from 420nm to 433nm, and the conjugated structure of the metal-polyphenol complex changed, proving that curcumin successfully bound to Al3+ Complexation.

[0433] Example 4: Fe under low pH conditions 3+ Characterization of shedding from metal-polyphenol complexes

[0434] Curcumin in the metal-polyphenol complex binds Fe via coordination bonds 3+ Under the low pH conditions of lysosomes, curcumin and Fe 3+ The coordination bonds between the metal and polyphenols are protonated (absorb hydrogen ions) and broken. 3+ Indeed, it is through the above mechanism that the metal-polyphenol complex is detached from the lipid complex. We designed the following experiment: the color of the metal-polyphenol complex was observed under physiological pH (pH = 7.4) and lysosomal low pH (pH = 5.0). Figure 2-3 As shown in the figure, the metal-polyphenol complex changes from brown-red to bright yellow under the low pH value (pH = 5.0) of the lysosome, indicating that Fe 3+ The results suggest that under the low pH conditions of lysosomes, Fe 3+ Can be detached from the metal-polyphenol complex.

[0435] At low pH, Fe 3+ The principle of shedding from the metal-polyphenol complex is: curcumin and Fe 3+ The coordination bonds between them are protonated under low pH conditions (pH = 5.0), that is, curcumin binds a large number of protons (H + ), leading to Fe 3+ The coordination bond between Fe and curcumin is broken, which makes Fe 3+ Separation from curcumin, ultimately leading to Fe 3+ Separation from metal-polyphenol complexes ( Figure 2-3 ).

[0436] Example 5: Metal ion is Fe 3+ Or Al 3+ The efficiency of drug-metal-polyphenol complex particles MPNP encapsulating nucleic acids (siRNA and mRNA) and its comparison with LNP.

[0437] The mRNA in Example 2 was replaced with siRNA targeting the Bcl-2 gene (SEQ ID NO. 4, 21 bp) and mRNA encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit (SEQ ID NO. 2, 669 nt), respectively, to prepare nucleic acid-encapsulated drug-metal-polyphenol complex particles siRNA@MPNP and mRNA@MPNP, respectively. The preparation process of the remaining drug-metal-polyphenol complex particles was the same as in Example 2.

[0438] siRNA@LNPs and mRNA@LNPs were prepared with the same drug loading as in Example 2.6 for Bcl-2-siRNA@MPNPs and Example 2.5 for RBD-mRNA@MPNPs. The organic phase solution was prepared according to the Onpattro lipid nanoparticle formulation, with the ionizable lipid ALC0315, DSPE-PEG2000, DSPC, and cholesterol dissolved in ethanol at a molar ratio of 50%:1.5%:10%:38.5%. Bcl-siRNA or RBD-mRNA was added to the aqueous phase (0.1 M acetic acid-sodium acetate buffer, pH 4.0). The amino lipid to phosphate nucleotide ratio (N / P) was 6:1, ensuring the same nucleic acid loading as in the siRNA@MPNPs and mRNA@MPNPs described above. The aqueous and organic phases were rapidly mixed at a volume ratio of 3:1 at a flow rate of 14 mL / min. After mixing, the mixture was diluted tenfold with pH 7.4 Tris-HCl buffer solution and concentrated to one-tenth using a 100kDa ultrafiltration tube. After repeating the dilution and concentration operation three times, the ethanol concentration in the mixture was reduced to below 0.0005%, and the pH value of the solution was increased to the normal pH value of Tris-HCl buffer solution (7.2-7.4), thus producing siRNA@LNP and mRNA@LNP, respectively.

[0439] Agarose gel electrophoresis was used to test the nucleic acid (siRNA and mRNA) loading efficiency of siRNA@MPNP, mRNA@MPNP, siRNA@LNP, and mRNA@LNP. The loading efficiency was determined as follows: the nucleic acid (siRNA and mRNA) loading amount of each lipid nanoparticle group was set at 10 μg / mL, the lipid to nucleic acid mass ratio was 10 μg / mL, and the metal in the metal-polyphenol complex was Fe. 3+ When the mass ratio is 20:1, the metal in the metal-polyphenol complex is Al 3+The nucleic acids were dissolved in a pH 5.0 Tris-HCl buffer solution at a mass ratio of 18:1 as a positive control group, and a nucleic acid-free PBS buffer solution was used as a negative control. The concentration of the agarose gel was 1.5%. At this point, the gaps in the gel only allow free nucleic acids to pass through, but not lipid nanoparticles. Electrophoresis was stopped when the free nucleic acid bands were clearly distinguishable to prevent nucleic acid degradation due to prolonged electrophoresis. ImageJ software was used to calculate the grayscale value of free nucleic acids in different groups. The positive control group was set as 100%. The ratio of free nucleic acid in each group to the positive control was the relative amount of free nucleic acid. The encapsulation rate of each group was (100-relative amount of free nucleic acid)%.

[0440] Result analysis: Figure 2-4 As shown, MPNP(Fe 3+ ) encapsulated siRNA and mRNA with efficiencies of 87.78% and 83.17% respectively; MPNP (Al 3+ The efficiency of MPNP encapsulation of siRNA and mRNA was 89.65% and 82.53%, respectively; the efficiency of LNP encapsulation of siRNA and mRNA was 87.32% and 79.94%, respectively. The results suggest that there is no significant difference in the efficiency of nucleic acid encapsulation between MPNP and LNP.

[0441] Example 6: Metal ion is Fe 3+ Or Al 3+ Nucleic acid lysosomal escape ability of drug-metal-polyphenol complex particles MPNP and its comparison with LNP

[0442] The Bcl-2-siRNA (SEQ ID NO.4) in Example 2.6 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@MPNP (the concentration of the siRNA contained was 100 nM); the Bcl-2-siRNA (SEQ ID NO.4) in Example 5 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@LNP (the concentration of the siRNA contained was 100 nM); the eGFP-mRNA (SEQ ID NO.1) in Example 2.5 was replaced with Cy5-labeled mRNA (Cy5-mRNA) to prepare Cy5-mRNA@MPNP (the concentration of the mRNA contained was 2 μg / mL); the RBD-mRNA (SEQ ID NO. NO.2) was replaced with Cy5-labeled mRNA (Cy5-mRNA) to prepare Cy5-mRNA@LNP (containing an mRNA concentration of 2 μg / mL). After incubating A549 cells with the cell lysosomal probe LysotrackerGreen for 3 hours, a high-content imaging system was used to observe the overlap of the Cy5 fluorescence signal (red) and the LysotrackerGreen fluorescence signal (green) to determine the ability of the drug-lipid particles to promote nucleic acid lysosomal escape.

[0443] The ability of drug-metal-polyphenol complex particles to promote nucleic acid lysosomal escape was determined by incubating cells with the drug-metal-polyphenol complex nanoparticles for 3 hours. The overlap between the Cy5 fluorescence signal (red) and the LysotrackerGreen fluorescence signal (green) was observed using a high-content imaging system, and the overlap ratio between the red and green fluorescence signals was calculated using imageJ software. When the overlap ratio between the red and green fluorescence signals was less than 50% after incubating cells with the drug-metal-polyphenol complex nanoparticles for 3 hours, this indicated that nucleic acids could escape from the cell lysosomes more quickly, indicating that the drug-metal-polyphenol complex nanoparticles had a strong ability to promote nucleic acid lysosomal escape.

[0444] Result analysis: Figure 2-5 As shown, when Cy5-siRNA@MPNP(Fe 3+ ) and Cy5-mRNA@MPNP(Fe 3+ ) incubated A549 cells for 3 hours, the overlap rates of red fluorescence signal and green fluorescence signal were 39.20% ± 8.89% and 44.96% ± 3.85%, respectively, that is, the lysosomal escape rates were 60.80% ± 8.89% and 55.04% ± 3.85%, respectively; when Cy5-siRNA@MPNP(Al 3+ ) and Cy5-mRNA@MPNP(Al 3+After incubation of A549 cells with Cy5-siRNA@LNP for 3 hours, the overlap rates of the red and green fluorescence signals were 34.70% ± 4.98% and 39.10% ± 4.43%, respectively, indicating lysosomal escape rates of 65.30% ± 4.98% and 60.90% ± 4.43%, respectively. In contrast, after incubation of A549 cells with Cy5-siRNA@LNP and Cy5-mRNA@LNP for 3 hours, the overlap rates of the red and green fluorescence signals were 76.02% ± 7.90% and 85.33% ± 4.87%, respectively, indicating lysosomal escape abilities of 23.98% ± 7.90% and 14.67% ± 4.87%, respectively. This suggests that the drug-lipid nanoparticle MPNP has a good ability to promote nucleic acid lysosomal escape, and the ability of MPNP to promote lysosomal escape is significantly stronger than that of LNP.

[0445] Example 7: Metal ion is Fe 3+ Or Al 3+ The ability of drug-metal-polyphenol composite particles MPNP to promote nucleic acid expression and its comparison with LNP

[0446] The RBD-mRNA (SEQ ID NO. 2) in Example 5 was replaced with mRNA encoding the fluorescent protein eGFP, and the rest of the preparation method was the same as that in Example 5 to obtain eGFP-mRNA@LNP.

[0447] The eGFP-mRNA@MPNP prepared in Example 2.5 and the above-mentioned eGFP-mRNA@LNP (containing mRNA at a concentration of 2 μg / mL) were incubated with 293T cells, respectively. The control group was incubated with MPNP or LNP. After 48 h, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0448] The method for analyzing the eGFP-positive cell rate by flow cytometry is as described in Example 2.

[0449] Result analysis: Result analysis: Figure 2-6 As shown, MPNP(Fe 3+ )、MPNP(Al 3+ After treatment of 293T cells with MPNP and LNP, the percentages of eGFP-positive cells were 93.47%, 97.06%, and 63.09%, respectively. These results suggest that MPNP is superior to LNP in promoting nucleic acid expression. This may be due to the fact that, as described in Example 6, MPNP's ability to promote lysosomal escape of nucleic acids is stronger than LNP, allowing more nucleic acids loaded with MPNP to be effectively released into the cytoplasm and subsequently translated into protein.

[0450] Example 8: Ability of drug-metal-polyphenol composite particles MPNP to promote humoral and cellular immunity and comparison with LNP

[0451] The RBD-mRNA@MPNP in Example 2.5 and the RBD-mRNA@LNP in Example 5 were incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). The control group was incubated with MPNP. After 24 hours, the supernatant was centrifuged and frozen at -20°C for later use. The cell pellet was resuspended in 100 μL PBS buffer solution and then frozen and thawed twice and sonicated for 10 minutes before centrifugation to obtain the supernatant. The expression level of RBD protein in both the cell supernatant and the cell lysate was detected using a commercially available new coronavirus antigen RBD ELISA detection kit. The results are shown in FIG. Figure 2-7 .

[0452] The ELISA method for detecting RBD expression levels is as described in Example 2.5.

[0453] The experimental animals were randomly divided into 3 groups (experimental group and control group), with 5 mice in each group. The animal model was BALB / c mice. Each mouse was injected intramuscularly for the first time on the first day and for the second time on the 14th day. The experimental group was injected with RBD-mRNA@MPNP(Fe 3+ )、RBD-mRNA@MPNP(Al 3+ ) or RBD-mRNA@LNP, and the control group was injected with MPNP and LNP without mRNA. The dose of each administration was 100 μL, of which RBD-mRNA@MPNP(Fe 3+ )、RBD-mRNA@MPNP(Al 3+ ) and RBD-mRNA@LNP formulations each contain 30 mg of mRNA. On the 28th day after the first dose, mouse blood was collected, serum was separated and serially diluted, and the titer of total RBD IgG antibodies against the novel coronavirus S1 subunit produced in the mice was detected using a commercially available ELISA kit. The results are as follows: Figure 2-8 shown.

[0454] The ELISA method for detecting the titer of total RBD IgG antibodies against the novel coronavirus S1 subunit is as described in Example 2.5.

[0455] In the case of RBD-mRNA@MPNP(Fe 3+ )、RBD-mRNA@MPNP(Al 3+ ) and RBD-mRNA@LNP, the spleens of normal mice were collected 28 days after the incubation and single-cell suspension was prepared under sterile conditions. 100,000 spleen cells / well were plated in a cell plate, and RBD protein was added at a final concentration of 10 mg / mL and cultured for 48 h. The supernatant was removed by centrifugation, and the expression levels of IFN-γ, IL-2, and IL-4 were determined by ELISA kits. The results are shown in the figure. Figure 2-9 shown.

[0456] The method for detecting the expression levels of IFN-γ, IL-2, and IL-4 by ELISA is as described in Example 2.5.

[0457] Result analysis: Figure 2-7 As shown, RBD-mRNA@MPNP(Fe 3+ )、RBD-mRNA@MPNP(Al 3+ ) and RBD-mRNA@LNP can induce 293T cells to express a certain amount of RBD, but RBD-mRNA@MPNP(Al 3+ ) was significantly more effective in inducing cell expression of RBD than RBD-mRNA@MPNP(Fe 3+ ), RBD-mRNA@MPNP(Fe 3+ ) was significantly stronger in inducing cell expression of RBD than RBD-mRNA@LNP: RBD-mRNA@MPNP(Fe 3+ ) treatment group, the expression level of RBD in the cell supernatant was 178 ng / mL, and RBD-mRNA@MPNP(Al 3+ The expression level of RBD in the cell supernatant of the ) treatment group was 215 ng / mL, and the expression level of RBD in the cell supernatant of the RBD-mRNA@LNP treatment group was 115.67 ng / mL. Figure 2-8 The results showed that RBD-mRNA@MPNP effectively induced humoral immunity in mice and produced high levels of antigen-specific binding antibodies. 3+ ) is clearly superior to RBD-mRNA@MPNP(Fe 3+ ), RBD-mRNA@MPNP(Fe 3+ ) is clearly superior to RBD-mRNA@LNP in inducing humoral immunity in mice: RBD-mRNA@MPNP(Fe 3+ ) treatment group mice IgG antibody titer reached 84975; RBD-mRNA@MPNP(Al 3+ The IgG antibody titer in the mice treated with RBD-mRNA@LNP was 96418, while that in the mice treated with RBD-mRNA@LNP was only 67476. Figure 2-9 As shown in Figure 2, RBD-mRNA@MPNP can effectively induce cellular immunity in mice, namely, activate immune cells and produce a large amount of cytokines, and mRNA@MPNP (Al 3+ ) is clearly superior to RBD-mRNA@MPNP(Fe 3+ ), RBD-mRNA@MPNP(Fe 3+) is clearly superior to RBD-mRNA@LNP in inducing cellular immunity in mice: RBD-mRNA@MPNP(Fe 3+ ) made the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 274.4 pg / mL, 254.2 pg / mL, and 77.4 pg / mL, respectively; RBD-mRNA@MPNP(Al 3+ ) made the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 309pg / mL, 299pg / mL, and 91.2pg / mL, respectively; while RBD-mRNA@LNP made the expression levels of cytokines IFN-γ, IL-2, and IL-4 only 104.2pg / mL, 79.2pg / mL, and 27pg / mL. The results suggest that mRNA@MPNP(Al 3+ ) is significantly better than RBD-mRNA@MPNP(Fe 3+ ), RBD-mRNA@MPNP(Fe 3+ ) is clearly superior to RBD-mRNA@LNP in inducing cellular immunity in mice: RBD-mRNA@MPNP can more effectively promote cell expression of target proteins and can more effectively activate humoral immunity and cellular immunity in the body. Therefore, the drug (mRNA)-lipid particles are significantly superior to the existing technology LNP in terms of the effects of mRNA-loaded drugs, vaccines or other products. The possible reasons are: 1) Compared with LNP, MPNP has a stronger ability to promote nucleic acid lysosomal escape; 2) Compared with LNP, MPNP has a stronger ability to promote nucleic acid expression into protein (antigen); 3) Compared with LNP, after the curcumin in MPNP is released, it acts as an immune adjuvant (also known as an immunomodulator), which can activate humoral immunity and cellular immunity to enhance the effect of MPNP in delivering mRNA vaccines, and can also suppress the immune factor storm to suppress excessive and harmful immune responses to the body.

[0458] Example 9: In vivo safety evaluation of metal-polyphenol composite particles (MPNPs)

[0459] SD rats were used as research subjects to investigate the effect of MPNP (Fe 3+ or Al 3+ ) conducted a 20-day subchronic toxicity study with a 20-day recovery period. The specific experimental methods are as follows:

[0460] Fifty-six SPF Sprague-Dawley rats (220 ± 20 g), half male and half female, were housed at 25°C, 45%-55% humidity, and 12 h of light. After 3-5 days of acclimatization, they were randomly divided into the following sex groups: an experimental group (n=32) and a recovery group (n=24). The blank control group consisted of 14 rats (8 in the experimental group and 6 in the recovery group), half male and half female; the low-dose MPNP group (25 mg / kg) consisted of 14 rats (8 in the experimental group and 6 in the recovery group), half male and half female; the medium-dose MPNP group (50 mg / kg) consisted of 14 rats (8 in the experimental group and 6 in the recovery group), half male and half female; and the high-dose group (100 mg / kg) consisted of 14 rats (8 in the experimental group and 6 in the recovery group), half male and half female. The experimental group (32 rats) was autopsied after the end of drug administration. The recovery group (24 rats) was autopsied after 20 days of continued normal feeding.

[0461] Administration: Experimental animals were administered via tail vein injection every two days for 20 days, and the body weight of SD rats was recorded weekly. The prepared MPNPs were dissolved in pH 7.4 Tris-HCl buffer. The control group was injected with an equal volume of Tris-HCl buffer, while the low-dose MPNP group, medium-dose MPNP group, and high-dose MPNP group were injected with 8 mg / kg, 16 mg / kg, and 32 mg / kg of MPNP, respectively.

[0462] The MPNP dosages described above were based on the following assumptions: To encapsulate 200 μg / kg of mRNA (the actual dosage required for mRNA animal studies), the required dosage of empty MPNP vector is 8 mg / kg. To fully demonstrate MPNP safety, we selected doses of 1, 2, and 4 times the actual dosage required for animal studies: 8 mg / kg, 16 mg / kg, and 32 mg / kg.

[0463] General Index Testing Methods: After each administration, the general condition of each group of animals was observed, including survival, diet, appearance, behavior, body weight, and any local reactions to the administration. A gross autopsy was performed, including timely weighing of the wet weights of major organs, such as the brain, heart, liver, spleen, lungs, and kidneys. Organ-to-body ratios were calculated, and pathological changes in each organ were recorded. Organ-to-body ratio = organ wet weight / body weight × 100%.

[0464] Collection and storage of whole blood and serum from SD rats: 20 days after administration and a 20-day recovery period, rats were dissected and blood was collected from the abdominal aorta. The rats were anesthetized with isoflurane and fixed on a dissecting board. The abdomen was disinfected with 75% ethanol. Sterile ophthalmic scissors were used to cut the rat's abdomen open. The internal organs were gently parted with cotton balls to expose the abdominal aorta. Whole blood was collected using a 500μL negative pressure EDTAK2 anticoagulant blood collection tube and stored at 4°C for routine blood testing. Whole blood was collected using a 5mL negative pressure conventional blood collection tube and allowed to stand at room temperature for 30 minutes. The supernatant was collected in a 1.5mL centrifuge tube and stored at -20°C for the detection of blood biochemical and immunological indicators.

[0465] Routine blood test method: Routine blood test parameters include: white blood cell count, lymphocyte count, monocyte count, neutrophil count, lymphocyte percentage, monocyte percentage, neutrophil percentage, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, coefficient of variation of red blood cell distribution width, platelet count, mean platelet volume, platelet distribution width, and platelet hematocrit. Gently invert the whole blood sample to mix thoroughly. A small amount of whole blood is then collected and analyzed using an automated hematology analyzer.

[0466] Blood biochemical index detection method: Blood biochemical index includes inorganic ions (Fe 2+ , Na + , K + , Cl - , Ca 2+ ), liver function indicators (ALT, AST, γ-GT, T-BIL, D-BIL, ALP, ALB), renal function indicators (BUN, UA, CR), cardiac function indicators (LDH, CK), glucose metabolism indicators (GSP, GLU, INS), lipid metabolism indicators (CHO, TG, LDL-C, HDL-C). Thaw serum samples and centrifuge at 3000 rpm for 15 minutes. Remove the supernatant and aliquot it for later use. Set the corresponding parameters on the automatic biochemical analyzer, add the prepared working solution, and then add the serum to be tested. The automatic biochemical analyzer will automatically determine the results.

[0467] Immunological indicators include thyroid function markers (TT3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN-γ, IFN-α, TNF-α), immunoglobulins (IgG, IgA, IgM), and serum complement (C3, CH50). These indicators are measured using ELISA.

[0468] Pathological examination method of the main organs of SD rats: At the end of the administration period and the recovery period, the rats in each group were anesthetized, and the main organs of the rats, including the whole brain, heart, liver, spleen, lungs, and kidneys, were removed by ophthalmic scissors. The organs were gently rinsed with 0.9% saline, fixed in 4% paraformaldehyde fixative, routinely paraffin-embedded, and stained with H&E. The histopathological changes of various organs of the rats in the control group and the experimental group were observed under an optical microscope.

[0469] Analysis of results: As shown in Table 2-1, at the end of the dosing period and the end of the recovery period, compared with the control group, the rats in the low-, medium- and high-dose MPNP groups survived well, had normal diet, normal appearance and behavioral activities, and no obvious adverse reactions were observed after administration; compared with the control group, there was no significant difference in the weight gain values ​​of male SD rats and female SD rats in the low-, medium- and high-dose MPNP groups; compared with the control group, there was no significant difference in the organ-to-body ratios of the low-, medium- and high-dose MPNP groups.

[0470] At the end of the administration period and the end of the recovery period, compared with the control group, the blood routine indicators (white blood cell count, lymphocyte count, monocyte count, neutrophil count, lymphocyte percentage, monocyte percentage, neutrophil percentage, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, coefficient of variation of red blood cell distribution width, platelet count, mean platelet volume, platelet distribution width, platelet hematocrit) in the low, medium and high dose MPNP groups were normal. Compared with the control group, the blood biochemical indicators, including inorganic ions (Fe 2+ , Na + , K + , Cl - , Ca 2+ ), liver function indicators (ALT, AST, γ-GT, T-BIL, D-BIL, ALP, ALB), renal function indicators (BUN, UA, CR), cardiac function indicators (LDH, CK), glucose metabolism indicators (GSP, GLU, INS), lipid metabolism indicators (CHO, TG, LDL-C, HDL-C), all showed no abnormalities; compared with the control group, the immunological related indicators of the low, medium and high doses of MPNP groups included thyroid function indicators (TT3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN-γ, IFN-α, TNF-α), immunoglobulins (IgG, IgA, IgM), and serum complement (C3, CH50), all showed no abnormalities.

[0471] At the end of the administration period and the end of the recovery period, compared with the control group, the brain tissue structure of rats in the low-, medium-, and high-dose MPNP groups was intact, with normal tissue staining, intact cell morphology and structure, and no nuclear pyknosis or inflammatory cell infiltration; the myocardial tissue structure was intact, with myocardial cells arranged neatly, continuously, and tightly, and the cell nuclei were clearly visible, without obvious cell congestion, edema, or necrosis; the hepatocyte morphology was normal, without inflammatory cell aggregation and necrosis; the spleen structure was normal, with clear boundaries between red and white pulp; the lung tissue structure was intact, with alveoli of uniform size, and no obvious inflammatory cell aggregation or infiltration; and the kidney structure was normal.

[0472] The above results suggest that long-term and large-scale injection of MPNP (Fe 3+ or Al 3+ ) No obvious chronic toxicity was found, indicating that MPNP is relatively safe.

[0473] Table 2-1 In vivo safety evaluation of MPNP

[0474]

[0475]

[0476] Note: ALT, alanine aminotransferase; AST, aspartate aminotransferase; γ-GT, glutamyl transpeptidase; T-BIL, total bilirubin; D-BIL, direct bilirubin; ALP, alkaline phosphatase; ALB, albumin; BUN, blood urea nitrogen; UA, uric acid; CR, creatinine; LDH, lactate dehydrogenase; CK, creatine phosphokinase; GSP, fructosamine; GLU, glucose; INS, insulin; CHO, cholesterol; TG, triglycerides; LDL-C, low-density lipoprotein; HDL-C, high-density lipoprotein; TT3, triiodothyronine; TT4, tetraiodothyronine; TSH, thyroid-stimulating hormone; IL-1, interleukin-1; IL-2, interleukin-2; IL-4, interleukin-4; IFN-γ, interferon-γ; IFN-α, interferon-α; TNF-α, tumor necrosis factor-α; IgG, immunoglobulin G; IgA, immunoglobulin A; IgM, immunoglobulin M; C3, complement C3; CH50, total complement CH50

[0477] Example 10: Comparison of in vivo safety of metal-polyphenol composite particles (MPNP) and LNP

[0478] The main toxicity of LNP comes from its main components - cationic lipids and / or ionizable lipids. When LNP is metabolized in the body, the free cationic lipids and / or ionizable lipids will produce significant toxicity to the body. The median lethal dose (IC50) of cationic lipids and / or ionizable lipids to biological cells is 50) is an important parameter for evaluating the toxicity of LNP to the body. The drug-metal-polyphenol complex particles (MPNP) use metal-polyphenol complexes to replace the cationic lipids / ionizable lipids in LNPs. Therefore, we studied the median lethal dose (IC50) of metal-polyphenol complexes and cationic lipids / ionizable lipids on biological cells. 50 ), and compared the differences in toxicity between LNP and MPNP.

[0479] 293T cells were incubated with different concentrations of the metal-polyphenol complex (0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM), cationic lipid (DOTAP, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM) and ionizable lipid (ALC0315, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM) for 48 hours, and then the cell viability was detected using a CCK8 activity detection kit. The median lethal dose (IC50) of the metal-polyphenol complex, cationic lipid (DOTAP) and ionizable lipid (ALC0315) on 293T cells was calculated. 50 .

[0480] CCK8 detection method:

[0481] Cell culture: Culture cells in DMEM containing 10% FBS and 1% double-antibody until the cell density reaches 80%-90% of the culture flask;

[0482] Wash the remaining culture medium from the culture flask with PBS, add trypsin, and quickly transfer the flask to a 37°C incubator with 5% CO2. Observe carefully until the cells have slightly rounded, then add culture medium to terminate the digestion. Transfer the cells to a centrifuge tube, centrifuge at 1500 RPM for 5 minutes, and resuspend the cells in fresh culture medium.

[0483] Counting: Dilute the cell suspension to 10,000 cells per mL according to the intended use. Add 100 μL per well of a 96-well plate, with at least five replicates per group. Incubate at 37°C, 5% CO2 for 24 hours before adding the drug.

[0484] After drug incubation for 48 h, 10% CCK8 was added and incubated for 1-3 h. The absorbance was measured at 450 nm using a microplate reader.

[0485] Survival rate (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] x 100%.

[0486] IC 50IC calculation method: With survival rate as the vertical axis and drug concentration as the horizontal axis, IC was calculated using Graphpad using the [Inhibitor] vs. normalized response--Variable slope analysis method. 50 .

[0487] To compare the in vivo safety of MPNP and LNP, MPNP (8 mg / kg) and LNP (3.24 mg / kg) capable of carrying an equal amount of nucleic acid (200 μg / kg mRNA) were used in an in vivo experiment according to the method of Example 9 to evaluate and compare the in vivo toxicity of MPNP and LNP.

[0488] Result analysis: As shown in Table 2-2, the IC 50 The toxicity of the metal-polyphenol complex is significantly lower than that of cationic lipids (DOTAP) and ionizable lipids (ALC0315).

[0489] As shown in Table 2-3, at the end of the administration period and the end of the recovery period, MPNP (Fe 3+ ) or MPNP(Al 3+ ) group showed no significant abnormalities in liver function indicators ALT, AST, ALP and cytokine IL-6, IL-1β expression levels. However, compared with the control group, the liver function indicators ALT, AST, ALP and cytokine IL-6, IL-1β expression levels in the LNP group were significantly increased. The results suggest that MPNP (Fe 3+ ) or MPNP(Al 3+ ) is safer in vivo than LNP. The reasons are: the core component of LNP is artificially synthesized "cationic lipid / ionizable lipid", which has high cytotoxicity and immunogenicity, and its structure is relatively stable and difficult to decompose and metabolize in the body; while MPNP (Fe 3+ ) or MPNP(Al 3+ The core component of MPNP (Fe) is a metal-polyphenol complex, which is composed of a highly safe natural small molecule curcumin (a food additive and pharmaceutical excipient approved by the FDA) and safe metal ions. After the drug is delivered, it is decomposed into natural molecules in the body. 3+ ) or MPNP(Al 3+ ) components do not contain cationic lipids / ionizable lipids and will not cause toxic side effects related to cationic lipids / ionizable lipids, so MPNP (Fe 3+ ) or MPNP(Al 3+ ) is safer than LNP.

[0490] The structural formula of DOTAP

[0491] The structural formula of ALC0315

[0492]

[0493] Table 2-2 Metal ions are Fe 3+ Or Al 3+ IC of metal-polyphenol complexes with cationic lipids (DOTAP) and ionizable lipids (ALC0315) 50 Comparison

[0494]

[0495]

[0496] Table 2-3 Metal ions are Fe 3+ Or Al 3+ Comparison of chronic toxicity test indicators of MPNP and LNP at

[0497]

[0498] Example 3 Clinical Application and Administration Route of Drug-Metal-Polyphenol Composite Particles MPNP

[0499] Example 11: Metal ion is Fe 3+ or Al 3+ Clinical application and administration route of drug-metal-polyphenol composite particles MPNP

[0500] The mRNA in Example 2 was replaced with siRNA targeting the B7-H4 gene (B7-H4-siRNA) and its control (scr-siRNA), and mRNA encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit (RBD-mRNA).

[0501] The sequences of the above-mentioned different nucleic acids are as follows: ① The sequence of B7-H4-siRNA is SEQ ID No.19 (sense chain) and SEQ ID No.26 (antisense chain) (25 bp), and its random control sequence is SEQ ID No.20 (sense chain) and SEQ ID No.27 (antisense chain) (19 bp); ② The mRNA sequence encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit is SEQ ID No.2 (669 nt). Referring to the method of Example 2, drug-metal-polyphenol composite particles (B7-H4-siRNA@MPNP (Fe3+ )、RBD-mRNA@MPNP(Fe 3+ )、B7-H4-siRNA@MPNP(Al 3+ )、RBD-mRNA@MPNP(Al 3+ ), and the preparation process of the remaining drug-metal-polyphenol complex particles was the same as that in Example 2. The above two different drug-metal-polyphenol complex particles (B7-H4-siRNA@MPNP and RBD-mRNA@MPNP) were used to treat liver cancer and as mRNA vaccines for the prevention of the new coronavirus, respectively.

[0502] The sequence of B7-H4-siRNA is as follows:

[0503] sense 5'-GGGAGA CAC UCC AUC ACA GUC ACU A-3' (SEQ ID No. 19).

[0504] antisense 5'-UAG UGA CUG UGA UGG AGU GUC UCC C-3' (SEQ ID No. 26) (25 bp).

[0505] The random control sequence of B7-H4-siRNA is as follows:

[0506] sense5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID No. 20).

[0507] antisense 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID No. 27) (19 bp).

[0508] To evaluate the B7-H4-siRNA@MPNP(Fe 3+ ) and B7-H4-siRNA@MPNP(Al 3+ ) to treat liver cancer. Animal models of liver cancer were established using HepG2 cells. When the tumor size increased to about 100 mm 3 The mice with liver cancer were randomly divided into 7 groups (5 mice in each group): Tris-HCl buffer solution control group, blank carrier MPNP (Fe 3+ ) group, blank carrier MPNP (Al 3+ ) group, Scr-siRNA@MPNP(Fe 3+ ) 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. Each group of mice was intratumorally injected with pH 7.4 Tris-HCl buffer solution, MPNP (Fe 3+ )、MPNP(Al 3+ )、Scr-siRNA@MPNP(Fe 3+ )、B7-H4siRNA@MPNP(Fe 3+ )、Scr-siRNA@MPNP(Al 3+ )、B7-H4siRNA@MPNP(Al 3+ ) once, with a dose of 200 μg siRNA / kg, and 8 injections were performed. The tumor volume was measured and recorded every 3 days. Figure 3-1 shown.

[0509] To evaluate the effect of RBD-mRNA@MPNP as an mRNA vaccine in preventing the new coronavirus, the experimental process and experimental methods are as shown in the previous Example 2.5.

[0510] The ELISA detection method is as described in Example 2.5.

[0511] Establishment of liver cancer mouse model: HepG2 cells were collected and cultured at 1×10 7 The cells were resuspended in PBS at a density of 100 μL and kept on ice before inoculation. 100 μL of the cell suspension was then injected subcutaneously into the dorsal region near the hind legs of female Balb / c nude mice to establish a liver cancer mouse model.

[0512] Result analysis:

[0513] like Figure 3-1 As shown, Scr-siRNA@MPNP(Fe 3+ )、Scr-siRNA@MPNP(Al 3+ ) had almost no inhibitory effect on the growth of HepG2 cells, while B7-H4-siRNA@MPNP(Fe 3+ ) and B7-H4siRNA@MPNP(Al 3+ ) showed a highly effective therapeutic effect, effectively inhibiting the growth of liver cancer tumors. The results suggest that drug-metal-polyphenol complex particles can encapsulate and deliver B7-H4 siRNA, inhibiting the expression of the target gene, thereby inhibiting the development of liver cancer.

[0514] As in Example 2.5, Figure 1-3 , Figure 1-5 As shown, RBD-mRNA@MPNP(Fe 3+ ) made the expression level of mouse IgG antibody reach 84363.4 ( Figure 1-3 ), and 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 ( Figure 1-5 ). RBD-mRNA@MPNP(Al 3+ ) made the expression level of mouse IgG antibody reach 94828.6 ( Figure 1-17 ), and 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 ( Figure 1-19 The results suggest that RBD-mRNA@MPNP can effectively induce humoral immunity in mice, producing high levels of antigen-specific binding antibodies. It can also effectively induce cellular immunity in mice, activating immune cells and producing large amounts of cytokines. Therefore, RBD-mRNA@MPNP can effectively prevent infection with the novel coronavirus.

[0515] like Figure 3-1 As shown, B7-H4-siRNA@MPNP can effectively treat liver cancer by intratumoral injection; as shown in Example 2.5, Figure 1-3 、 1-5 As shown in Figures 1-17 and 1-19, RBD-mRNA@MPNP administered via intramuscular injection can activate humoral and cellular immunity, thereby preventing novel coronavirus infection. These results suggest that drug-metal-polyphenol complex particles can be administered via multiple routes.

[0516] Example 4: Curcumin, Fe 3+ Function after being replaced by similar products

[0517] Example 12, Curcumin, Fe 3+ Function after being replaced by similar products

[0518] With reference to Example 1, curcumin, Fe 3+ The analogs of curcumin, Fe 3+ Instead, 9 different drug-metal-polyphenol composite particles (eGFP-mRNA@MPNP) were prepared by different combinations according to Example 2, wherein the concentration of mRNA contained in each eGFP-mRNA@MPNP was 2 μg / mL. 3+ The names and structures of curcumin, Fe 3+ The combination of the same is shown in Table 4-2. In Example 1, the reaction temperature is 60°C, the reaction time is 2 hours, and other conditions remain unchanged.

[0519] To compare the effects of the nine different eGFP-mRNA@MPNPs and eGFP-mRNA@LNPs, we prepared LNPs encapsulating equal amounts of eGFP mRNA with reference to Example 5 to obtain eGFP-mRNA@LNPs.

[0520] The above nine different eGFP-mRNA@MPNPs and the above eGFP-mRNA@LNPs (all containing mRNA at a concentration of 2 μg / mL) were incubated with 293T cells, respectively. The control group was incubated with MPNP or LNP. After 48 h, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0521] The method for analyzing the eGFP-positive cell rate by flow cytometry is as described in Example 2.

[0522] The main toxicity of LNP comes from its main component - cationic lipids / ionizable lipids. When LNP is metabolized in the body, the free cationic lipids / ionizable lipids will produce significant toxicity to the body. The median lethal dose (IC50) of cationic lipids / ionizable lipids to biological cells is 50 ) is an important parameter for evaluating the toxicity of LNP to the body. The metal-polyphenol complex particles (MPNP) replace the cationic lipid / ionizable lipid in LNP with a metal-polyphenol complex. Therefore, we studied the median lethal dose (IC50) of 9 metal-polyphenol complexes and cationic lipid (DOTAP) / ionizable lipid (ALC0315) on biological cells by Table 4-2. 50 ), and compared the differences in toxicity between LNP and 9 MPNPs.

[0523] The calculation method of IC50 is as described in Example 10.

[0524] Results analysis: As shown in Table 4-3, the percentage of eGFP-positive cells in 293T cells treated with 9 different eGFP-mRNA@MPNPs was significantly higher than that of eGFP-mRNA@LNP, among which mRNA@MPNP1 had the highest percentage of eGFP-positive cells. 3+ The function of mRNA@MPNP formed after being replaced by its analog is inferior to that of mRNA@MPNP1, but slightly better than that of mRNA@LNP. The possible reason is that, as described in Example 5, MPNP has a stronger ability to promote lysosomal escape of nucleic acids than LNP, so more nucleic acids loaded by MPNP can be effectively released into the cytoplasm and translated into proteins.

[0525] The above results suggest that as long as the following conditions are met, curcumin, Fe 3+The function of the drug-metal-polyphenol composite particles formed after being replaced by its congeners is not affected: ① The congeners of curcumin are hydrophobic polyphenols that can complex with metals; ② Fe 3+ The congeners are metal ions; ③ Curcumin and Fe 3+ The coordination bonds between them can be broken in response to the low pH environment of the lysosome.

[0526] As shown in Table 4-3, the IC values ​​of 9 metal-polyphenol complexes 50 The toxicity of metal-polyphenol complex is significantly lower than that of cationic lipid (DOTAP) and ionizable lipid (ALC0315). 3+ The safety of lipid particles (MPNP) composed of and its analogs is higher than that of LNP. The reasons are as follows: the core component of LNP is artificially synthesized "cationic lipid / ionizable lipid", which has high cytotoxicity and immunogenicity, and its structure is relatively stable and difficult to decompose and metabolize in the body; while the core component of MPNP is metal-polyphenol complex, which is composed of non-cationic lipid, natural small molecule with high safety (of which curcumin is a food additive and pharmaceutical excipient approved by the FDA) and safe metal ions, and it has been decomposed into natural molecules in the body after completing drug delivery. In summary, curcumin, Fe 3+ The components of MPNP composed of LNP and its analogs do not contain cationic lipids / ionizable lipids and will not cause toxic side effects related to cationic lipids / ionizable lipids, so the safety of MPNP is higher than that of LNP.

[0527] Table 4-1 Curcumin, Fe 3+ The names and structures of its analogs

[0528]

[0529]

[0530] Table 4-2 Curcumin, Fe 3+ List of combinations and functions of metal-polyphenol complexes in drug-lipid nanoparticles prepared from its analogs

[0531]

[0532] Table 4-3 Curcumin, Fe 3+ IC of metal-polyphenol complexes prepared from its analogs 50

[0533]

[0534]

[0535] Example 13, Curcumin, Fe 3+ Component ratio and function of drug-metal-polyphenol composite particles prepared therefrom

[0536] Example 13.1 Metal ion is Fe 3+ Polyphenols, Fe 3+ Component ratio and function of drug-metal-polyphenol composite particles prepared therefrom

[0537] According to Example 2.1, metal-polyphenol complexes were prepared, and curcumin was replaced by its analogs hesperetin (1 hesperetin molecule contains 4 hydroxyl groups) and catechin (1 catechin molecule contains 5 hydroxyl groups), respectively, to prepare three metal-polyphenol complexes (mRNA@MPNP1, mRNA@MPNP4, mRNA@MPNP7). When preparing these three metal-polyphenol complexes, curcumin or its analogs were mixed with Fe 3+ The dosage ratios were 1:1, 1:1, and 1:2, respectively. Corresponding drug-metal-polyphenol complex particles were prepared using these three metal-polyphenol complexes (mRNA@MPNP1, mRNA@MPNP4, and mRNA@MPNP7). The mRNA encoding the eGFP fluorescent protein, whose sequence is SEQ ID NO. 1 (720 nt), was used. The mRNA encapsulation efficiency of these three drug-lipid particles and their ability to promote eGFP fluorescent protein expression after treatment in 293T cells were tested according to the experimental procedures and methods described in Example 2.5.

[0538] Results analysis: As shown in Table 4-4, the mRNA encapsulation efficiency and the ability to promote target protein expression (i.e., positive cell rate) of drug-metal-polyphenol complex particles prepared using different dosage ratios based on the chemical structure of the metal-polyphenol complex components are comparable. The results suggest that the dosage ratio of the metal-polyphenol complex components can be adjusted according to the structure of the specific metal-polyphenol complex components. The basis for adjusting the dosage ratio is: because the hydroxyl group of curcumin analogs reacts with Fe 3+ As long as the congeners of curcumin contain multiple binding sites, the congeners of curcumin and Fe 3+ The dosage ratio of the congeners can be adjusted according to the number of binding sites contained in the curcumin congeners.

[0539] Table 4-4 Metal ions are Fe 3+ Function of drug-lipid particles prepared by different ratios of components of metal-polyphenol complexes

[0540] Example 13.2 When the metal ion is Al3+, the ratio of polyphenol and Al3+ components in different metal-polyphenol complexes and the function of the prepared drug-metal-polyphenol complex particles

[0541] According to Example 2.2, metal-polyphenol complexes were prepared, and curcumin was replaced by its analogs hesperetin (1 hesperetin molecule contains 4 hydroxyl groups) and catechin (1 catechin molecule contains 5 hydroxyl groups), respectively, to prepare three metal-polyphenol complexes (mRNA@MPNP3, mRNA@MPNP6, and mRNA@MPNP9). When preparing these three metal-polyphenol complexes, curcumin or its analogs were mixed with Al 3+ The dosage ratios were 1:1, 1:1, and 1:2, respectively. Corresponding drug-metal-polyphenol complex particles were prepared using these three metal-polyphenol complexes (mRNA@MPNP3, mRNA@MPNP6, and mRNA@MPNP9). The mRNA encoding the eGFP fluorescent protein, whose sequence is SEQ ID NO. 1 (720 nt), was used. The mRNA encapsulation efficiency of these three drug-lipid particles and their ability to promote eGFP fluorescent protein expression after treatment in 293T cells were tested according to the experimental procedures and methods described in Example 2.5.

[0542] Results analysis: As shown in Tables 4-5, the mRNA encapsulation efficiency and the ability to promote target protein expression of drug-metal-polyphenol complex particles prepared using different dosage ratios based on the chemical structure of the metal-polyphenol complex components are comparable. The results suggest that the dosage ratio of the metal-polyphenol complex components can be adjusted according to the structure of the specific metal-polyphenol complex components. The basis for adjusting the dosage ratio is that the hydroxyl groups of curcumin analogs react with Al 3+ As long as the congeners of curcumin contain multiple binding sites, the congeners of curcumin and Al 3+ The dosage ratio of the congeners can be adjusted according to the number of binding sites contained in the curcumin congeners.

[0543] Table 4-5 Metal ions are Al 3+ Function of drug-lipid particles prepared by different ratios of components of metal-polyphenol complexes

[0544]

[0545] The preparation of mRNA@MPNP in Example 2 was completed by Professor Wang Shan's research group at the Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Central South University.

[0546] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In the event of any inconsistency, the meanings described in the present invention or derived from the contents described in the present invention shall prevail. In addition, the terms used in this specification are only for the purpose of describing the embodiments of the present invention and are not intended to limit the present invention.

[0547] Note that the above are only preferred embodiments of the present invention and the technical principles employed. Those skilled in the art will understand that the present invention is not limited to the specific embodiments described herein, and that various obvious changes, readjustments, and substitutions can be made by those skilled in the art without departing from the scope of protection of the present invention. Therefore, although the present invention has been described in more detail through the above embodiments, the present invention is not limited to the above embodiments, and may include many other equivalent embodiments without departing from the technical concept of the present invention, all of which fall within the scope of protection of the present invention.

Claims

1. A drug-lipid particle, wherein The drug-lipid particles contain: Drug and metal-polyphenol complex particles, wherein the drug is a negatively charged nucleic acid; The metal-polyphenol composite particles contain: (i) a metal-polyphenol complex, which is composed of a polyphenol molecule portion and a metal ion portion reacting with each other, wherein the polyphenol molecule portion and the metal ion portion are connected by a coordination bond; The polyphenol molecule portion is selected from at least one of curcumin, hesperetin or catechin; The metal ion portion is selected from Fe 3+ , Ca 2+ or Al 3+ At least one of; The molar ratio of the polyphenol molecule portion to the metal ion portion is 1:(0.5-2); (ii) a conjugated lipid that inhibits particle aggregation, wherein the conjugated lipid that inhibits particle aggregation is a PEG-lipid conjugate; as well as (iii) non-cationic lipids or non-ionizable lipids other than conjugated lipids that inhibit particle aggregation; The preparation method of the drug-lipid particles comprises the following steps: Step 1: reacting the polyphenol molecule portion with the metal ion portion through a coordination bond to form a metal-polyphenol complex; Step 2: The metal-polyphenol complex prepared in step 1, the conjugated lipid that inhibits particle aggregation, the non-cationic lipid or the non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer solution to form an aqueous phase, and the organic phase and the aqueous phase are mixed to obtain drug-lipid particles.

2. The drug-lipid particle according to claim 1, wherein The polyphenol molecule is selected from curcumin formula 1, hesperidin formula 5 or catechin formula 13; Formula 1; Formula 5; Formula 13.

3. The drug-lipid particle according to claim 1, wherein The metal ion portion is selected from Fe 3+ , Ca 2+ or Al 3+ .

4. The drug-lipid particle according to claim 1, wherein The PEG-lipid conjugate is selected from one or more of phosphatidylethanolamine-polyethylene glycol 2000 (Formula 47), phosphatidylethanolamine-polyethylene glycol 700 (Formula 48), phosphatidylethanolamine-polyethylene glycol 1000 (Formula 49), and phosphatidylethanolamine-polyethylene glycol 5000 (Formula 50); Wherein, R1 and R2 are independently: Capryloyl , lauroyl 、 Myristoyl 、 Palmitoyl , stearoyl 、 Oleoyl 、 Linoleyl , erucyl 、 Arachidoyl or phytanoyl ; Formula 47; Formula 48; Formula 49; Formula 50.

5. The drug-lipid particle according to claim 1, wherein The PEG-lipid conjugate is selected from a combination of one or more of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000 and DSPE-PEG5000.

6. The drug-lipid particle according to claim 1, 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; Formula 58; Formula 55; Formula 56; Formula 57.

7. The drug-lipid particle according to claim 1, wherein The non-cationic lipid or non-ionizable lipid described in (iii) is selected from one or more combinations of phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphoceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysophosphatidylcholine LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, and 1-sphingosine 1-phosphate S1P.

8. The drug-lipid particle according to claim 1, wherein The non-cationic lipid or non-ionizable lipid described in (iii) is selected from phosphatidylcholine PC (Formula 29), phosphatidylethanolamine PE (Formula 30), phosphatidylserine PS (Formula 31), phosphatidic acid PA (Formula 32), phosphatidylglycerol PG (Formula 33), 1-phosphoceramide SP (Formula 34), phosphatidylinositol PI (Formula 35), phosphatidylthreonine PT (Formula 36), sphingomyelin SM (Formula 37), lysophosphatidylcholine LPC (Formula 38), lysophosphatidylethanolamine LPE (Formula 39), lysophosphatidylserine LPS (Formula 40), lysophosphatidic acid LPA (Formula 41), lysophosphatidylglycerol LPG (Formula 42), lysophosphatidylinositol LPI (Formula 43), lysophosphatidylthreonine LPT (Formula 44), lysophospholipid LSM (Formula 45), 1-phospho-sphingosine S1P (Formula 46), A combination of one or more of Formula 46; wherein R1 and R2 are independently decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl, or phytanoyl; Formula 29; Formula 30; Formula 31; Formula 32; Formula 33; Formula 34; Formula 35; Formula 36; Formula 37; Formula 38; Formula 39; Formula 40; Formula 41; Formula 42; Formula 43; Formula 44; Formula 45; Formula 46.

9. The drug-lipid particle according to claim 7, wherein The non-cationic lipid or non-ionizable lipid described in (iii) further includes cholesterol.

10. The drug-lipid particle according to claim 9, wherein The non-cationic lipid or non-ionizable lipid described in (iii) includes cholesterol and a combination of one or more selected from DSPC, DSPE, DSPA or DSPG.

11. The drug-lipid particle according to claim 10, wherein The non-cationic lipid or non-ionizable lipid described in (iii) includes cholesterol (Formula 59) and a combination of one or more selected from DSPC (Formula 51), DSPE (Formula 52), DSPA (Formula 53) or DSPG (Formula 54); Formula 59; Formula 51; Formula 52; Formula 53; Formula 54.

12. The drug-lipid particle according to claim 11, wherein The non-cationic lipids or non-ionizable lipids described in (iii) include cholesterol (Formula 59) and DSPC (Formula 51).

13. The drug-lipid particle according to claim 1, wherein The metal-polyphenol complex is composed of a polyphenol molecule portion and a metal ion portion, wherein the polyphenol molecule portion is selected from curcumin, hesperidin or catechin, and the metal ion portion is selected from Fe 3+ , Ca 2+ or Al 3+ .

14. The drug-lipid particle according to claim 13, wherein The metal-polyphenol complex is formed by the reaction of a polyphenol molecule part and a metal ion part, wherein the polyphenol molecule part is selected from curcumin formula 1, hesperidin formula 5 or catechin formula 13, and the metal ion part is selected from Fe 3+ , Ca 2+ or Al 3+ .

15. The drug-lipid particle according to claim 1, wherein The polyphenol molecule part is curcumin formula 1, and the metal ion part is Fe 3+ .

16. The drug-lipid particle according to claim 15, wherein Curcumin formula 1 and Fe 3+ The molar ratio is 1:

1.

17. The drug-lipid particle according to claim 1, wherein The polyphenol molecule part is curcumin formula 1, and the metal ion part is Al 3+ .

18. The drug-lipid particle according to claim 17, wherein Curcumin formula 1 and Al 3+ The molar ratio is 1:

1.

19. The drug-lipid particle according to claim 9, wherein The metal-polyphenol complex particles are made of (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid. The molar proportion of the metal-polyphenol complex in the raw material is 10% to 20%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, the molar proportion of cholesterol in the raw material is 0% to 48%, and the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 40% to 75%; the total molar proportion of (i) the metal-polyphenol complex, (ii) the conjugated lipid that inhibits particle aggregation, and (iii) the non-cationic lipid or non-ionizable lipid in the raw material is 100%.

20. The drug-lipid particle according to claim 9, wherein The metal-polyphenol complex particles are made of (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the metal-polyphenol complex accounts for 5% to less than 10% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 2% to 10% by mole in the raw material, the cholesterol accounts for 0% to 48% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 30% to less than 40% or 40% to 75% by mole in the raw material; or The metal-polyphenol complex particles are made of (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the metal-polyphenol complex accounts for 10% to 20% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 2% to 10% by mole in the raw material, the cholesterol accounts for 0% to 48% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 30% to less than 40% by mole in the raw material; The total molar proportion of (i) the metal-polyphenol complex, (ii) the conjugated lipid for inhibiting particle aggregation, and (iii) the non-cationic lipid or non-ionizable lipid in the starting material is 100%.

21. The drug-lipid particle according to claim 19 or 20, wherein The metal-polyphenol complex accounts for 5% to less than 10%, 10% to 15%, or 15% to 20% by mole in the raw materials.

22. The drug-lipid particle according to claim 21, wherein The metal-polyphenol complex accounts for 5%, 10% or 15% by mole in the raw materials.

23. The drug-lipid particle according to claim 19 or 20, wherein The molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 5% or 5% to 10%.

24. The drug-lipid particle according to claim 23, wherein The conjugated lipids that inhibit particle aggregation were present at 3%, 5%, or 10% by mole in the starting material.

25. The drug-lipid particle according to claim 19 or 20, wherein The molar proportion of cholesterol in the raw material is 10% to 30% or 30% to 47%.

26. The drug-lipid particle according to claim 25, wherein The molar proportion of cholesterol in the raw material is 10% to 20%.

27. The drug-lipid particle according to claim 25, wherein The molar proportion of cholesterol in the raw material is 10%, 30% or 47%.

28. The drug-lipid particle according to claim 19 or 20, wherein The molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 45% to 55% or 60% to 65%.

29. The drug-lipid particle according to claim 28, wherein The molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 50% to 65%.

30. The drug-lipid particle according to claim 28, wherein The molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 45%, 55%, 60% or 65%.

31. The drug-lipid particle according to claim 19 or 20, wherein The molar proportion of the metal-polyphenol complex in the raw material is 5% to less than 10% or 10% to 15%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 5% to 10%, the molar proportion of cholesterol in the raw material is 10% to 30%, and the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 60% to 65%, and the metal ion portion of the metal-polyphenol complex is selected from Fe 3+ The total molar proportion of (i) the metal-polyphenol complex, (ii) the conjugated lipid for inhibiting particle aggregation, and (iii) the non-cationic lipid or non-ionizable lipid in the raw material is 100%.

32. The drug-lipid particle according to claim 31, wherein The molar proportion of the metal-polyphenol complex in the raw material is 15%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 10%, the molar proportion of cholesterol in the raw material is 10%, and the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 65%. The metal ion part of the metal-polyphenol complex is selected from Fe 3+ .

33. The drug-lipid particle according to claim 31, wherein The molar proportion of the metal-polyphenol complex in the raw material is 5%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 5%, the molar proportion of cholesterol in the raw material is 30%, and the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 60%. The metal ion part of the metal-polyphenol complex is selected from Fe 3+ .

34. The drug-lipid particle according to claim 19 or 20, wherein The molar proportion of the metal-polyphenol complex in the raw material is 5% to less than 10% or 10%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 5%, the molar proportion of cholesterol in the raw material is 30% to 47%, and the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 45% to 55%. The metal ion part of the metal-polyphenol complex is selected from Al 3+ The total molar proportion of (i) the metal-polyphenol complex, (ii) the conjugated lipid for inhibiting particle aggregation, and (iii) the non-cationic lipid or non-ionizable lipid in the raw material is 100%.

35. The drug-lipid particle according to claim 34, wherein The metal-polyphenol complex accounts for 5% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 3% by mole in the raw material, the cholesterol accounts for 47% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 45% by mole in the raw material; the metal ion part in the metal-polyphenol complex is selected from Al 3+ ;or The metal-polyphenol complex accounts for 10% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 5% by mole in the raw material, the cholesterol accounts for 30% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 55% by mole in the raw material; the metal ion part in the metal-polyphenol complex is selected from Al 3+ .

36. The method for preparing the drug-lipid particles according to any one of claims 1 to 35, wherein: Step 1: reacting the polyphenol molecule portion with the metal ion portion through a coordination bond to form a metal-polyphenol complex; Step 2: The metal-polyphenol complex prepared in step 1, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer solution to form an aqueous phase, and the organic phase and the aqueous phase are mixed to obtain drug-lipid particles.

37. The preparation method according to claim 36, wherein The polyphenol molecules are dissolved in ethanol, and then metal ions are added to react to obtain the metal-polyphenol complex.

38. The preparation method according to claim 37, wherein The molar ratio of polyphenol molecules to metal ions is 1:(1~2).

39. The preparation method according to claim 37, wherein The reaction conditions included reaction at 60°C for 1 hour.

40. The preparation method according to claim 36, wherein The organic compound is ethanol.

41. The preparation method according to claim 36, wherein The buffer is enzyme-free Tris-HCl buffer.

42. The preparation method according to claim 36, wherein The mixing methods of the organic phase and the aqueous phase include microfluidic chip or ultrasound.

43. Use of the drug-lipid particle described in any one of claims 1 to 35 in preparing a composition for the delivery of a drug.

44. The use according to claim 43, wherein The composition is used to introduce the drug into cells.

45. The use according to claim 43, wherein The composition is a medicament.

46. ​​The use according to claim 45, wherein The medicament is used for silencing the expression of a target sequence in a mammalian subject, for delivering a drug in a mammal, for delivering a drug from the body to a mammalian cell, or for treating a disease or disorder in a mammal.

47. The use according to claim 46, wherein The mammal is a human.

48. The use according to claim 46, wherein The disease or condition is associated with the expression of a gene that contains a target sequence for the drug.

49. The use according to claim 46, wherein The disease or condition is cancer or viral infection, and the virus is SARS-Cov-2.

50. The use according to claim 49, wherein The cancer includes liver cancer, glioma or lung cancer.

51. The use according to claim 45, wherein The medicament is a vaccine.

52. The use according to claim 45, wherein The administration routes of the agent include intrathecal injection, intramuscular administration, intracranial injection, intravenous injection or intratumoral injection.

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