Drug-lipid particles, preparation method and application thereof

Abstract

The present invention relates to the field of biopharmaceutical technologies, and specifically provides a drug-lipid particle, a preparation method thereof, and an application thereof. The drug-lipid particle provided by the present invention comprises a drug and a metal-polyphenol complex particle, wherein the metal-polyphenol complex particle contains a metal-polyphenol complex, a conjugated lipid that inhibits particle aggregation, and a non-cationic lipid or a non-ionizable lipid other than the conjugated lipid that inhibits particle aggregation. The drug-lipid particle can achieve efficient systemic delivery of the drug, and at the same time, compared with LNP containing cationic lipids or ionizable lipids, the toxicity is significantly reduced, realizing safe and effective treatment of diseases or disorders.

Description

[0001] Cross-reference to related applications

[0002] This invention claims the priority of a Chinese patent application with the application number 202210950179.1, titled "Drug-lipid particles and their preparation methods and applications", which was filed with the Chinese Patent Office on August 9, 2022. The entire content is incorporated herein by reference. Technical field

[0003] This invention relates to the field of biomedical technology, and specifically relates to a drug-lipid particle and its preparation methods and applications. Background art

[0004] Nucleic acid drugs refer to functional DNA or RNA that specifically edits pathogenic genes or proteins through binding, cleavage, knockout, insertion, etc. The discovery of such nucleic acids has not only broken through the traditional idea that nucleic acids only carry genetic information, but also provided a powerful molecular tool for biomedicine and biosensing.

[0005] Nucleic acid drugs have disadvantages such as being easily degraded rapidly by nucleases in the body, having weak transmembrane ability, and short half-life in blood circulation, which severely limit their clinical applications. Therefore, a safe and effective nucleic acid drug delivery system is one of the research hotspots in nucleic acid drug development. Currently, the carriers that can deliver nucleic acid drugs can be mainly divided into viral carriers and non-viral carriers. Viral carriers (including adenovirus, retrovirus, and lentiviral vectors) can cause immune responses after entering the human body and are now less used; among non-viral carriers, the more commonly used ones are mainly nanoparticles and small molecule conjugates. Compared with small molecule conjugates directly conjugated with nucleic acid drugs, nanoparticles can more effectively encapsulate nucleic acid drugs to prevent them from being rapidly degraded by nucleases in the body, thereby prolonging their in vivo circulation time. The mechanism of nanoparticles encapsulating nucleic acids is that cationic lipids with positive charges adsorb nucleic acids with negative charges.

[0006] Cationic liposomes are usually formed by mixing cationic lipids with co-lipids such as dioleoyl phosphatidylethanolamine (DOPE), cholesterol, etc. in a certain proportion. Cationic liposomes can be used to transport gene or drug molecules into target cells. However, during their transfection and other processes, cationic liposomes still contain certain cytotoxicity, and they will produce certain toxic effects on normal cells while transporting drugs to treat cancer cells. Therefore, there are certain limitations to their clinical application research. The cascade reactions caused by cationic liposomes include the generation of reactive oxygen species, enzyme activation reactions, mitochondrial membrane potential changes, apoptosis caused by the release of cytochrome C and caspases, etc.

[0007] In addition, an ionizable lipid is a lipid containing a positively charged ionizable amine group, which is uncharged under physiological conditions (pH = 7.4), but becomes 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, in the low pH environment (pH = 4.0 - 6.5) within the lysosomes, the ionizable lipids become positively charged lipids. Although ionizable lipids reduce the cytotoxic effects and high pro-inflammatory effects of some permanently positively charged cationic lipids, their cytotoxicity and immunogenicity are still relatively high. Lipid nanoparticles (LNPs) based on cationic lipids and / or ionizable lipids are currently nanoparticle nucleic acid drug delivery systems available for clinical use. Among them, cationic lipids and / or ionizable lipids, as the main components of LNPs, are responsible for adsorbing 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 relatively high toxicity of LNPs.

[0008] Therefore, when delivering negatively charged drugs (such as nucleic acid drugs, protein drugs, polypeptide drugs, small molecule drugs, etc.) using a delivery system, nanoparticle delivery systems developed relying 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 that does not use cationic lipids and / or ionizable lipids and has low toxicity. Summary of the Invention

[0009] The purpose of the present invention is to provide drug-lipid particles and a preparation method to at least alleviate one technical problem existing in the prior art.

[0010] To achieve the above purpose, the present invention adopts the following technical solutions:

[0011] The present invention provides a drug-lipid particle, which includes:

[0012] (a) A drug, where the drug is a negatively charged molecule; and

[0013] (b) A metal-polyphenol complex particle, which contains:

[0014] (i) A metal-polyphenol complex, which is formed by the reaction of a polyphenol molecule part and a metal ion part, and the polyphenol molecule part and the metal ion part are connected by a coordination bond;

[0015] (ii) A conjugated lipid that inhibits particle aggregation, where the conjugated lipid that inhibits particle aggregation is not a cationic lipid or an ionizable lipid; and

[0016] (iii) Non-cationic or non-ionizable lipids other than the conjugated lipids that inhibit particle aggregation.

[0017] In some embodiments, the polyphenol molecular moiety is selected from one or more combinations of curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, taxifolin, phlorotannins, polyflavanol polyphenols, catechins, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, glucogallin, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate, gallocatechin gallate, and their derivatives.

[0018] Among them, "its" in "and its derivatives" refers to "curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, taxifolin, phlorotannins, polyflavanol polyphenols, catechins, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, glucogallin, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate, or gallocatechin gallate". The polyphenol molecular moiety can be, for example but not limited to, curcumin, curcumin derivatives, hesperetin, hesperetin derivatives, catechins, catechin derivatives, curcumin and catechins, catechins and catechin derivatives, etc. In the present invention, "and its derivatives" has a similar meaning.

[0019] Furthermore, the polyphenol molecular moiety is selected from one or more combinations of curcumin (Formula 1), quercetin (Formula 2), kaempferol (Formula 3), rutin (Formula 4), hesperetin (Formula 5), naringenin (Formula 6), eriodictyol (Formula 7), luteolin (Formula 8), apigenin (Formula 9), taxifolin (Formula 10), phlorotannins (Formula 11), polyflavanol polyphenols (Formula 12), catechins (Formula 13), ellagic acid (Formula 14), gallic acid (Formula 15), digallic acid (Formula 16), propyl gallate (Formula 17), epigallocatechin gallate (Formula 18), glucogallin (Formula 19), hydroxyhydroquinone (Formula 20), morin (Formula 21), epicatechin gallate (Formula 22), catechin gallate (Formula 23), gallocatechin gallate (Formula 24), and their derivatives.

[0020]

[0021]

[0022]

[0023]

[0024] Further, the polyphenol molecular moiety is selected from one or more combinations of curcumin (Formula 1), dihydrocurcumin (Formula 25), hexahydrocurcumin (Formula 26), curcumin sulfate (Formula 27), bisdemethoxycurcumin (Formula 28).

[0025]

[0026] Further, the polyphenol molecular moiety is selected from one or more combinations of curcumin (Formula 1), hesperetin (Formula 5), or catechin (Formula 13), and their derivatives.

[0027] Further, the polyphenol molecular moiety is selected from curcumin (Formula 1), hesperetin (Formula 5), or catechin (Formula 13).

[0028] 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+ in one or more combinations.

[0029] Further, the metal ion moiety is selected from Fe 3+ 、Ca 2+ 、Al 3+ in one or more combinations.

[0030] Further, the metal ion moiety is selected from Fe 3+ 、Ca 2+ or Al 3+ 。

[0031] In some embodiments, the conjugated lipid that inhibits particle aggregation in (ii) of the metal-polyphenol complex particles comprises a polyethylene glycol (PEG)-lipid conjugate and / or PEG-dialkoxypropyl (DAA).

[0032] Furthermore, the PEG-lipid conjugate is selected from one or a combination of more than one of phosphatidylethanolamine-polyethylene glycol 2000 (Formula 47), phosphatidylethanolamine-polyethylene glycol 700 (Formula 48), phosphatidylethanolamine-polyethylene glycol 1000 (Formula 49), phosphatidylethanolamine-polyethylene glycol 5000 (Formula 50), and their derivatives. Among them, R1 and R2 are both independently:

[0033] Caproyl Lauroyl

[0034] Myristoyl

[0035] Palmitoyl

[0036] Stearoyl

[0037] Oleoyl

[0038] Linoleoyl

[0039] Erucoyl

[0040] Arachidyl or

[0041] Phytanyl

[0042]

[0043] Furthermore, the PEG-lipid conjugate is selected from one or a combination of more than one of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000 or DSPE-PEG5000.

[0044] Furthermore, the PEG-lipid conjugate is selected from DSPE-PEG2000 (Formula 58), DSPE-PEG700 (Formula 55), DSPE-PEG1000 (Formula 56) or DSPE-PEG5000 (Formula 57).

[0045]

[0046]

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

[0048] Furthermore, in (iii), the non-cationic lipid or non-ionizable lipid is selected from one or more combinations of lecithin (PC) (Formula 29), phosphatidylethanolamine (PE) (Formula 30), phosphatidylserine (PS) (Formula 31), phosphatidic acid (PA) (Formula 32), phosphatidylglycerol (PG) (Formula 33), ceramide 1-phosphate (SP) (Formula 34), phosphatidylinositol (PI) (Formula 35), phosphatidylthreonine (PT) (Formula 36), sphingomyelin (SM) (Formula 37), 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), lysosphingomyelin (LSM) (Formula 45), sphingosine 1-phosphate (S1P) (Formula 46), and derivatives thereof; wherein, R1 and R2 are each independently decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucoyl, arachidoyl, or phytanoyl.

[0049]

[0050]

[0051]

[0052] It should be noted that in the present invention, the cis-trans isomers of the components used do not affect the technical effects to be achieved by the protected content of the present invention.

[0053] Furthermore, in (iii), the non-cationic lipid or non-ionizable lipid further includes at least one of cholesterol and its derivatives.

[0054] 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.

[0055] Preferably, the structural formula of cholesterol is (Formula 59)

[0056] 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);

[0057]

[0058] In some embodiments, the non-cationic lipid or non-ionizable lipid in (iii) comprises cholesterol (Formula 59) and DSPC (Formula 51).

[0059] In some embodiments, the metal-polyphenol complex of the present invention is formed by the reaction of a polyphenol molecular moiety and a metal ion moiety. The polyphenol molecular moiety is selected from curcumin, hesperetin, or catechin, and the metal ion moiety is selected from Fe 3+ , Ca 2+ or Al 3+ .

[0060] Furthermore, the metal-polyphenol complex is formed by the reaction of a polyphenol molecular moiety and a metal ion moiety. The polyphenol molecular moiety is selected from curcumin (Formula 1), hesperetin (Formula 5), or catechin (Formula 13), and the metal ion moiety is selected from Fe 3+ , Ca 2+ or Al 3+ .

[0061] Furthermore, the molar ratio of the polyphenol molecular moiety to the metal ion moiety is 1:(0.5 - 2).

[0062] Furthermore, the polyphenol molecular moiety is curcumin (Formula 1), and the metal ion moiety is Fe 3+ .

[0063] Furthermore, the molar ratio of curcumin (Formula 1) to Fe 3+ is 1:1.

[0064] Furthermore, the polyphenol molecular moiety is curcumin (Formula 1), and the metal ion moiety is Al 3+ .

[0065] Furthermore, the molar ratio of curcumin (Formula 1) to Al 3+ is 1:1.

[0066] 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 accounts for 10% to 20% in molar ratio in the raw materials, the conjugated lipid that inhibits particle aggregation accounts for 2% to 10% in molar ratio in the raw materials, cholesterol accounts for 0% to 48% in molar ratio in the raw materials, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 40% to 75% in molar ratio in the raw materials.

[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. The metal-polyphenol complex accounts for 5% to less than 10% in molar ratio in the raw materials, the conjugated lipid that inhibits particle aggregation accounts for 2% to 10% in molar ratio in the raw materials, cholesterol accounts for 0% to 48% in molar ratio in the raw materials, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 30% to less than 40% or 40% to 75% in molar ratio in the raw materials; or

[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. The metal-polyphenol complex accounts for 10% to 20% in molar ratio in the raw materials, the conjugated lipid that inhibits particle aggregation accounts for 2% to 10% in molar ratio in the raw materials, cholesterol accounts for 0% to 48% in molar ratio in the raw materials, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 30% to less than 40% in molar ratio in the raw materials.

[0069] Further, the metal-polyphenol complex accounts for 5% to less than 10%, 10% to 15%, or 15% to 20% in molar ratio in the raw materials, preferably 5%, 10%, or 15%.

[0070] Further, the conjugated lipid that inhibits particle aggregation accounts for 3% to 5% or 5% to 10% in molar ratio in the raw materials, preferably 3%, 5%, or 10%.

[0071] Further, cholesterol accounts for 10% to 30%, 30% to 47%, or 10% to 20% in molar ratio in the raw materials, preferably 10%, 30%, or 47%.

[0072] Furthermore, the molar ratio of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 45% - 55%, 60% - 65% or 50% - 65%, preferably 45%, 55%, 60% or 65%.

[0073] In some embodiments, the metal-polyphenol complex (the metal ion part is selected from Fe 3+ ) has a molar ratio of 5% - less than 10% or 10% - 15% in the raw material, the conjugated lipid that inhibits particle aggregation has a molar ratio of 5% - 10% in the raw material, the cholesterol has a molar ratio of 10% - 30% in the raw material, and the non-cationic lipids or non-ionizable lipids other than cholesterol have a molar ratio of 60% - 65% in the raw material. Preferably, the polyphenol molecular part is selected from curcumin (Formula 1), the conjugated lipid that inhibits particle aggregation is DSPE-PEG2000, and the non-cationic lipids or non-ionizable lipids are cholesterol and DSPC.

[0074] In one embodiment, the metal-polyphenol complex (the metal ion part is selected from Fe 3+ ) has a molar ratio of 15% in the raw material, the conjugated lipid that inhibits particle aggregation has a molar ratio of 10% in the raw material, the cholesterol has a molar ratio of 10% in the raw material, and the non-cationic lipids or non-ionizable lipids (such as DSPC, DSPA, DSPE or DSPG) other than cholesterol have a molar ratio of 65% in the raw material.

[0075] In one embodiment, the metal-polyphenol complex (the metal ion part 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 lipids or non-ionizable lipids other than cholesterol have a molar ratio of 60% in the raw material.

[0076] In some embodiments, the metal-polyphenol complex (the metal ion part is selected from Al 3+ ) has a molar ratio of 5% - less than 10% or 10% in the raw material, the conjugated lipid that inhibits particle aggregation has a molar ratio of 3% - 5% in the raw material, the cholesterol has a molar ratio of 30% - 47% in the raw material, and the non-cationic lipids or non-ionizable lipids other than cholesterol have a molar ratio of 45% - 55% in the raw material. Preferably, the polyphenol molecular part is selected from curcumin (Formula 1), the conjugated lipid that inhibits particle aggregation is DSPE-PEG2000, and the non-cationic lipids or non-ionizable lipids are cholesterol and DSPC.

[0077] In one embodiment, the metal-polyphenol complex (the metal ion part is selected from Al 3+)It has a molar proportion of 5% in the raw material. The conjugated lipid that inhibits particle aggregation has a molar proportion of 3% in the raw material. The cholesterol has a molar proportion of 47% in the raw material. The non-cationic lipid or non-ionizable lipid other than cholesterol has a molar proportion of 45% in the raw material.

[0078] In one embodiment, the metal-polyphenol complex (the metal ion part is selected from Al 3+ )It has a molar proportion of 10% in the raw material. The conjugated lipid that inhibits particle aggregation has a molar proportion of 5% in the raw material. The cholesterol has a molar proportion of 30% in the raw material. The non-cationic lipid or non-ionizable lipid other than cholesterol has a molar proportion of 55% in the raw material.

[0079] In some embodiments, the drug is encapsulated in the metal-polyphenol complex particles.

[0080] 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.

[0081] 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 acids.

[0082] Furthermore, the nucleic acid is the mRNA sequence encoding eGFP (Enhanced Green Fluorescent Protein) shown in SEQ ID No.1, the mRNA sequence encoding the receptor-binding domain RBD of the S1 subunit of the novel coronavirus shown in SEQ ID No.2, the mRNA sequence encoding NY-ESO-1 (New York esophageal squamous cell carcinoma 1) shown in SEQ ID No.3, the siRNA sequence of the Bcl-2 gene (B-cell lymphoma / Leukemia-2) with the antisense strand shown in SEQ ID No.4 and the sense strand shown in SEQ ID No.21, the siRNA sequence of the PLK1 gene (Polo-like Kinase 1) with the antisense strand shown in SEQ ID No.6 and the sense strand shown in SEQ ID No.23, the siRNA sequence of the Gal-1 gene shown in SEQ ID No.8, the ASO sequence of the STAT-3 gene shown in SEQ ID No.10, the ASO sequence of the α-syn gene (α-synuclein) shown in SEQ ID No.12, the ASO sequence of the Bcl-2 gene shown in SEQ ID No.14, the mRNA sequence encoding the wild-type S protein of the novel coronavirus shown in SEQ ID No.16, the double-stranded DNA sequence with the antisense strand shown in SEQ ID No.17 and the sense strand shown in 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 strand shown in SEQ ID No.19 and the antisense strand shown in SEQ ID No.26.

[0083] The present invention provides a method for preparing the above-mentioned drug-lipid particles, wherein the drug is encapsulated in metal-polyphenol complex particles to obtain the drug-lipid particles.

[0084] In one embodiment, (i) the metal-polyphenol complex, (ii) the conjugated lipid that inhibits particle aggregation, and (iii) the non-cationic lipid or non-ionizable lipid are mixed to obtain the metal-polyphenol complex particles.

[0085] In one embodiment, (a) the drug, (i) the metal-polyphenol complex, (ii) the conjugated lipid that inhibits particle aggregation, and (iii) the non-cationic lipid or non-ionizable lipid are mixed to obtain the drug-lipid particles.

[0086] In some embodiments, the preparation method comprises the following steps:

[0087] Step 1: Reacting a polyphenol molecule moiety with a metal ion moiety through a coordination bond reaction to form a metal-polyphenol complex;

[0088] Step 2: Mixing the metal-polyphenol complex prepared in Step 1, a conjugated lipid that inhibits particle aggregation, a non-cationic lipid or a non-ionizable lipid, and a drug to prepare the metal-polyphenol complex particles.

[0089] Furthermore, the polyphenol molecule is dissolved in ethanol, and then a metal ion is added for reaction; the molar ratio of the polyphenol molecule to the metal ion is 1:(1 - 2); the reaction conditions include reacting at 60°C for 1 hour.

[0090] In one embodiment, the metal-polyphenol complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or non-ionizable lipid are dissolved in an organic compound to form an organic phase, and the drug is dissolved in a buffer solution to form an aqueous phase. The organic phase and the aqueous phase are mixed to obtain drug-lipid particles. Preferably, the organic compound is ethanol; the buffer solution is an enzyme-free Tris-HCl buffer solution; the mixing method of the organic phase and the aqueous phase includes a microfluidic chip or ultrasound.

[0091] The present invention provides the application of the drug-lipid particles in a composition, and the composition is used for drug delivery.

[0092] In some embodiments, the composition is used for introducing a drug into cells.

[0093] In some embodiments, the composition is a medicament.

[0094] In some embodiments, the medicament is used for silencing the expression of a target sequence in a mammalian subject.

[0095] In some embodiments, the medicament is used for delivering a drug in a mammalian body.

[0096] In some embodiments, the medicament is used for delivering a drug from the body to mammalian cells.

[0097] In some embodiments, the medicament is used for treating a disease or disorder in a mammalian.

[0098] In some embodiments, the mammalian is a human.

[0099] In some embodiments, the disease or disorder is related to the expression of a gene, and the gene contains a target sequence of the drug.

[0100] In some embodiments, the disease or disorder includes cancer, viral infection, autoimmune disease, diabetes or Alzheimer's disease.

[0101] In some embodiments, the viral infections include hepatitis A, hepatitis B, hepatitis C, SARS-CoV-2 (novel coronavirus 2019), HIV (human immunodeficiency virus), HPV (human papillomavirus), influenza, smallpox or syphilis.

[0102] In some embodiments, the cancer includes liver cancer, glioma, melanoma, lung cancer, pancreatic cancer or breast cancer.

[0103] In some embodiments, the medicament is a vaccine.

[0104] In some embodiments, the administration routes of the medicament include intrathecal injection, intramuscular administration, intracranial injection, intravenous injection or intratumoral injection.

[0105] The present invention provides a medicament containing the drug-lipid particles.

[0106] In some embodiments, the medicament is a vaccine.

[0107] In some embodiments, the vaccine is a novel coronavirus vaccine.

[0108] Compared with the prior art, the technical effects of the present invention are as follows:

[0109] In the prior art, complexes of metal ions and polyphenol molecules (such as curcumin, etc.) are mostly used as active ingredients in the fields of antioxidant, anti-inflammatory, antiviral, etc. However, the applicant has found through experimental research that the metal-polyphenol complex provided by the present invention can be used in a composition or a medicament, and it is mainly used as a carrier of a drug for drug stability, delivery and transportation, etc., and acts together with other carriers to achieve the effective administration of a negatively charged drug.

[0110] The drug-lipid particles provided by the present invention have a small diameter suitable for systemic delivery. Without using cationic lipids or ionizable lipids while ensuring the effectiveness not lower than that of LNP, the drug-lipid particles have a significantly reduced toxicity and a remarkable improvement in biosafety compared with LNP, and are more conducive to the transport of negatively charged drugs in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0112] Figure 1-1 For Example 2.5.1 of the present invention, eGFP-mRNA@MPNP (Fe3+ ) Statistical chart of the percentage of eGFP-positive cells caused by transfection of 293T;

[0113] Figure 1-2 This is for RBD-mRNA@MPNP(Fe) in Example 2.5.1 of the present invention 3+ ) Statistical chart of the expression level of RBD caused by transfection of 293T;

[0114] Figure 1-3 This is for RBD-mRNA@MPNP(Fe) in Example 2.5.1 of the present invention 3+ ) Statistical chart of the ability to induce humoral immunity;

[0115] Figure 1-4 This is for NY-ESO-1-mRNA@MPNP(Fe) in Example 2.5.1 of the present invention 3+ ) Statistical chart of the ability to induce humoral immunity;

[0116] Figure 1-5 This is for RBD-mRNA@MPNP(Fe) in Example 2.5.1 of the present invention 3+ ) Statistical chart of the ability to induce cellular immunity;

[0117] Figure 1-6 This is for NY-ESO-1-mRNA@MPNP(Fe) in Example 2.5.1 of the present invention 3+ ) Statistical chart of the ability to induce cellular immunity;

[0118] Figure 1-7 This is for eGFP-mRNA@MPNP(Al) provided in Example 2.5.2 of the present invention 3+ ) Statistical chart of the percentage of eGFP-positive cells caused by transfection of 293T;

[0119] Figure 1-8 This is for RBD-mRNA@MPNP(Al) in Example 2.5.2 of the present invention 3+ ) Statistical chart of the expression level of RBD caused by transfection of 293T;

[0120] Figure 1-9 This is for RBD-mRNA@MPNP(Al) in Example 2.5.2 of the present invention 3+ ) Statistical chart of the ability to induce humoral immunity;

[0121] Figure 1-10 This is for NY-ESO-1-mRNA@MPNP(Al) in Example 2.5.2 of the present invention 3+ ) Statistical chart of the ability to induce humoral immunity;

[0122] Figure 1-11Statistical chart of the ability of RBD-mRNA@MPNP(Al in Example 2.5.2 of the present invention 3+ ) to induce cellular immunity;

[0123] Figure 1-12 Statistical chart of the ability of NY-ESO-1-mRNA@MPNP(Al in Example 2.5.2 of the present invention 3+ ) to induce cellular immunity;

[0124] Figure 1-13 Ability of Bcl-2-siRNA@MPNP(Fe in Example 2.6.1 of the present invention 3+ ) to silence the target gene;

[0125] Figure 1-14 Statistical chart of the ability of PLK1-siRNA@MPNP(Fe in Example 2.6.1 of the present invention 3+ ) to silence the target gene;

[0126] Figure 1-15 Statistical chart of the ability of Gal-1-siRNA@MPNP(Fe in Example 2.6.1 of the present invention 3+ ) to silence the target gene;

[0127] Figure 1-16 Ability of Bcl-2-siRNA@MPNP(Al in Example 2.6.2 of the present invention 3+ ) to silence the target gene;

[0128] Figure 1-17 Statistical chart of the ability of PLK1-siRNA@MPNP(Al in Example 2.6.2 of the present invention 3+ ) to silence the target gene;

[0129] Figure 1-18 Statistical chart of the ability of Gal-1-siRNA@MPNP(Al in Example 2.6.2 of the present invention 3+ ) to silence the target gene;

[0130] Figure 1-19 Statistical chart of the ability of STAT3-ASO@MPNP(Fe in Example 2.7.1 of the present invention 3+ ) to silence the cellular target gene;

[0131] Figure 1-20 Statistical chart of the ability of α-syn-ASO@MPNP(Fe in Example 2.7.1 of the present invention 3+ ) to silence the cellular target gene;

[0132] Figure 1-21Statistical chart of the ability of Bcl-2-ASO@MPNP(Fe 3+ ) to silence the target gene in cells;

[0133] Figure 1-22 Statistical chart of the ability of STAT3-ASO@MPNP(Al 3+ ) to silence the target gene in cells;

[0134] Figure 1-23 Statistical chart of the ability of α-syn-ASO@MPNP(Al 3+ ) to silence the target gene in cells;

[0135] Figure 1-24 Statistical chart of the ability of Bcl-2-ASO@MPNP(Al 3+ ) to silence the target gene in cells;

[0136] Figure 1-25 Statistical chart of the expression level of S protein caused by transfection of 293T with S-mRNA@MPNP(Fe 3+ );

[0137] Figure 1-26 Functional detection result chart of the drug (dsDNA and ssDNA)-metal-polyphenol complex particles (Fe 3 + ) in Example 2.8.1 of the present invention;

[0138] Figure 1-27 Statistical chart of the expression level of S protein caused by transfection of 293T with S-mRNA@MPNP(Al 3+ ) in Example 2.8.2 of the present invention;

[0139] Figure 1-28 Functional detection result chart of the drug (dsDNA and ssDNA)-metal-polyphenol complex particles (Al 3 + ) in Example 2.8.2 of the present invention;

[0140] Figure 2-1 Ultraviolet absorption graph of the metal-polyphenol complex (Fe 3+ ) in Example 3.1 of the present invention;

[0141] Figure 2-2 Ultraviolet absorption graph of the metal-polyphenol complex (Al 3+ ) in Example 3.2 of the present invention;

[0142] Figure 2-3In Example 4 of the present invention, Fe under the condition of low pH value (pH = 5.0) 3+ Characterization pictures of the detachment of 3+ from the metal-polyphenol complex;

[0143] 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;

[0144] Figure 2-5 Detection results and statistical graph of the nucleic acid lysosomal escape ability of siRNA / mRNA@MPNP and siRNA / mRNA@LNP in Example 6 of the present invention;

[0145] Figure 2-6 Statistical graph of the results of the eGFP positive cell rate of MPNP and LNP in Example 7 of the present invention;

[0146] Figure 2-7 Statistical graph of the results of the ability of MPNP and LNP to promote mRNA expression in Example 8 of the present invention;

[0147] Figure 2-8 Statistical graph of the results of the ability of MPNP and LNP to promote humoral immunity in Example 8 of the present invention;

[0148] Figure 2-9 Statistics of the results of the ability of MPNP and LNP to promote cellular immunity in Example 8 of the present invention;

[0149] Figure 3-1 Statistical graph of the results of treating liver cancer by intratumoral injection of drug-metal-polyphenol complex particles in Example 11 of the present invention. Detailed implementation manners

[0150] Definitions

[0151] For the convenience of description, specific terms recorded in this specification, examples and the appended patent application scope are integrally explained herein. Unless otherwise defined in this specification, the meanings of scientific and technical terms used herein are the same as those understood and commonly used by those skilled in the art. Additionally, 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 states otherwise, the terms "at least one" and "one or more" used in this text and the appended patent application scope include one, two, three or more.

[0152] Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate values, the relevant numerical values in the specific embodiments have been presented as precisely as possible herein. However, any numerical value inherently and inevitably contains standard deviations resulting from individual testing methods. 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" represents that the actual value falls within the acceptable standard error of the mean value, depending on the consideration of those of ordinary skill in the art to which the present invention pertains. Except for experimental examples or unless otherwise explicitly stated, it is to be understood that all ranges, amounts, numerical values, and percentages (such as those used to describe material amounts, time durations, temperatures, operating conditions, quantity ratios, and others similar) used herein are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the accompanying patent application scope are approximate values and can be varied as needed. At a minimum, these numerical parameters should be understood as the values indicated by the significant digits and obtained by applying ordinary rounding methods.

[0153] In this document, all ranges provided are intended to include every specific range within the given range and combinations of sub-ranges between the given ranges. Additionally, unless otherwise stated, all ranges provided herein include the endpoints of the stated ranges. Thus, the range 1 - 5 specifically includes 1, 2, 3, 4, and 5, as well as sub-ranges such as 2 - 5, 3 - 5, 2 - 3, 2 - 4, 1 - 4, etc.

[0154] All publications and patent applications cited in this specification are hereby incorporated by reference herein, and for any and all purposes, each individual publication or patent application is specifically and individually indicated to be incorporated by reference herein. In the event of any inconsistency between this document and any publication or patent application incorporated by reference herein, this document shall prevail.

[0155] The term "lipid" refers to a group of organic compounds that includes, but is not limited to, esters of fatty acids. They are generally divided into three categories: "simple lipids", "compound lipids", and "derived lipids". "Simple lipids" include glycerides, glycerol esters of fatty acids, waxes, long-chain fatty acids and long-chain alcohols or sterols; "compound lipids" refer to those that, in addition to containing fatty acids and alcohols in their molecules, also contain non-lipid components, which include phospholipids and glycolipids; "derived lipids" are derived from simple lipids or compound lipids.

[0156] The term "lipid vesicle" refers to any lipid composition that can be used to deliver a compound, including, but not limited to, liposomes, where an aqueous volume is encapsulated by an amphiphilic lipid bilayer; or where the lipid coating encloses an interior that includes a macromolecular component, such as including mRNA, with a reduced aqueous interior; or lipid aggregates or micelles, where the encapsulated components are contained within a relatively disordered lipid mixture. In this article, the metal-polyphenol complex particle (MPNP) is a "lipid vesicle", and a drug, such as nucleic acid mRNA, is encapsulated in the MPNP as an encapsulated component, and this "encapsulation" can be full encapsulation and / or partial encapsulation.

[0157] In this article, the phrase "polyphenol molecular moiety" refers to the structure that originally belonged to the polyphenol molecule after the polyphenol molecule reacted with other substances.

[0158] In this article, the phrase "metal ion moiety" refers to the structure that originally belonged to the metal ion after the metal ion moiety reacted with other substances.

[0159] In this article, the phrase "metal-polyphenol complex" is composed of the above-mentioned polyphenol molecular moiety and the above-mentioned metal ion moiety reacting, and the above-mentioned polyphenol moiety and the above-mentioned metal ion moiety are connected by a coordination bond.

[0160] The term "ionizable lipid" refers to a lipid containing a positively charged ionizable amine group that can be protonated to carry a positive charge at a lower pH value and is uncharged under physiological pH conditions.

[0161] The term "neutral lipid" refers to any one of many lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol.

[0162] The term "anionic lipid" refers to any lipid that carries a negative charge at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacyl phosphatidylserine, diacyl phosphatidic acid, N-dodecanoyl phosphatidylethanolamine, N-succinyl phosphatidylethanolamine, N-glutaroyl phosphatidylethanolamine, lysyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylglycerol (POPG), and other anionic groups linked to neutral lipids.

[0163] 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-dioleyldimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyldimethylammonium 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-dimyristyloxypropyl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Lipids such as DODAP, DODMA, DMDMA, etc. are cationic and have a positive charge at a pH below physiological pH.

[0164] The term "hydrophobic lipid" refers to compounds having nonpolar groups, which include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and these groups are optionally substituted by one or more aromatic, alicyclic or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane and 1,2-dialkyl-3-aminopropane.

[0165] 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 can be, for example, an anionic lipid or a neutral lipid.

[0166] In the components of the metal-polyphenol complex particles, the "non-cationic lipid or non-ionizable lipid other than the conjugated lipid that inhibits particle aggregation" in (iii) means that the non-cationic lipid or non-ionizable lipid in (iii) is the lipid remaining in the metal-polyphenol complex particles after excluding the conjugated lipid that inhibits particle aggregation.

[0167] The term "fusogenicity" refers to the ability of liposomes, drug-lipid particles or other drug delivery systems to fuse with cell membranes. This membrane can be the plasma membrane or the membrane surrounding organelles, such as endosomes, nuclei, etc.

[0168] In the metal-polyphenol complex particles, the non-cationic lipid or non-ionizable lipid other than the conjugated lipid that inhibits particle aggregation mainly exists as the lipid forming vesicles. The term "lipid forming vesicles" tends to include any amphiphilic lipid having a hydrophobic part and a polar head group and which can spontaneously form bilayer vesicles in water by itself, exemplified by most phospholipids.

[0169] In the metal-polyphenol complex particles, the conjugated lipids that inhibit particle aggregation are mainly present as lipids adopting vesicles. The term "lipids adopting vesicles" tends to include any amphiphilic lipids that are stable in binding to the lipid bilayer, as well as other amphiphilic lipids whose hydrophobic part contacts the hydrophobic region of the inner bilayer membrane and whose polar head group part faces the polar surface of the outer membrane. Lipids adopting vesicles include lipids that can independently adopt non-lamellar phases and can also adopt bilayer structures in the presence of bilayer-stabilizing components. The conjugated lipids that inhibit the aggregation of drug-lipid particles 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 with dialkoxypropyl, PEG conjugated with diacylglycerol, PEG conjugated with phosphatidylethanolamine, and PEG conjugated with ceramide (see, U.S. Patent No. 5,885,613, which is incorporated herein by reference).

[0170] The term "amphiphilic lipid" refers to any suitable material in which the hydrophobic part of the lipid material faces the hydrophobic phase and the hydrophilic part faces the hydrophilic phase. Amphiphilic lipids are usually the main components of lipid vesicles. The hydrophilic nature comes from the presence of polar or charged groups such as carbohydrates, phosphates (esters), carboxyl groups, sulfates, amino groups, mercapto groups, nitro groups, hydroxyl groups, and other similar groups. Hydrophobicity can be imparted by the inclusion of non-polar groups, which include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphiphilic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, distearoyl phosphatidylcholine, or dilinoleoyl phosphatidylcholine. Other compounds lacking phosphorus, such as sphingomyelin, the glycosphingolipid family, diacylglycerol, and β-acyl oxy acids, are also in the group called amphiphilic lipids. Additionally, the above-mentioned amphiphilic lipids can be mixed with other lipids, which include triglycerides and sterols.

[0171] The term "diacylglycerol" refers to a compound having two fatty acyl chains, where R1 and R2 both independently have 2-30 carbon atoms bonded to the 1- and 2-positions of glycerol through ester bonds. The acyl groups can be saturated or have different degrees of unsaturation. Diacylglycerol has the following formula 60:

[0172]

[0173] The term "diacylglycerol-coupled polyethylene glycol", the conjugated lipid that inhibits particle aggregation in the present invention can be diacylglycerol-coupled polyethylene glycol, i.e., diacylglycerol-polyethylene glycol conjugate (DAG-PEG conjugate or PEG-DAG conjugate). In a preferred embodiment, the DAG-PEG conjugate is a dilauroylglycerol (C12)-PEG conjugate, a ditetradecylglycerol (C14)-PEG conjugate (DMG), a dipalmitoylglycerol (C16)-PEG conjugate or a distearoylglycerol (C18)-PEG conjugate (DSG). Those skilled in the art will readily understand that other diacylglycerols can be used in the DAG-PEG conjugates of the present invention. Suitable DAG-PEG conjugates for use in the present invention and methods for their preparation and use are disclosed in U.S. Application No. 10 / 136,707, published as U.S.P.A 2003 / 0077829, and PCT Patent Application No. CA 02 / 00669, the entire contents of each of which are incorporated herein by reference.

[0174] The term "dialkoxypropyl" refers to a compound having a 2-alkyl chain, wherein both R1 and R2 independently have 2-30 carbons. The alkyl groups can be saturated or have varying degrees of unsaturation. Dialkoxypropyl has the following formula 61:

[0175]

[0176] The term "dialkoxypropyl-coupled PEG", the conjugated lipid that inhibits particle aggregation in the present invention can be dialkoxypropyl-coupled PEG, i.e., dialkoxypropyl conjugate (PEG-DAA conjugate). In a preferred embodiment, the PEG-DAA conjugate has the following formula 62:

[0177] 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 groups can be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, lauryl (C12), myristyl (C14), cetyl (C16), stearyl (C18), and icosyl (C20). In a preferred embodiment, R1 and R2 are the same, i.e., both R1 and R2 are myristyl (i.e., dimyristyl), both R1 and R2 are stearyl (i.e., distearyl), etc. In Formula 62, PEG is 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 with 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 from about 1,000 to about 3,000 daltons, and even more preferably about 2,000 daltons. PEG can optionally be substituted with an alkyl, alkoxy, acyl, or aryl group. In Formula 62, L is a linker moiety. Any linker moiety suitable for conjugating PEG to 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 (O-C(O)O-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), succinyl (-(O)CCH 2 CH 2 C(O)-), ether, disulfide, and combinations thereof. Other suitable linkers are well known in the art.

[0178] Phosphatidylethanolamine can be conjugated with polyethylene glycol to form a conjugated lipid that inhibits particle aggregation in the present invention and forms a bilayer stabilizing component. The phosphatidylethanolamine has various acyl chain groups with different chain lengths and degrees of saturation. These phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. Phosphatidylethanolamines containing saturated or unsaturated fatty acids are preferred, having a carbon chain length in the range of C10 - C20. Phosphatidylethanolamines having 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: dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dioleoyl phosphatidylethanolamine (DOPE), and distearoyl phosphatidylethanolamine (DSPE).

[0179] Ceramides, such as phosphatidylethanolamine, can be conjugated with polyethylene glycol to serve as conjugated lipids that inhibit particle aggregation in the present invention, forming bilayer-stabilizing components. The ceramides have multiple acyl chain groups with different chain lengths and degrees of saturation. It should be clear to those skilled in the art that, compared with phosphatidylethanolamine, ceramides have only one acyl group, and the acyl group can be easily varied according to 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 brains using well-known separation techniques, or synthesized using the methods and techniques disclosed in U.S. Patent No. 5,820,873, which is incorporated herein by reference. Using the synthetic routes set forth in the foregoing applications, ceramides with saturated or unsaturated fatty acids can be prepared, and the fatty acids have carbon chain lengths in the range of C2-C31.

[0180] The term "ATTA" or "polyamide" refers to, but is not limited to, the compounds disclosed in U.S. Patent Nos. 6,320,017 and 6,586,559, which are both incorporated herein by reference. These compounds include compounds having the following formula 63:

[0181]

[0182] 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 attached form an azido moiety; R2 is a member selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, and amino acid side chains; R3 is a member selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, mercapto, hydrazino, 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 clear to those skilled in the art that other polyamides can be used in the compounds of the present invention.

[0183] The term "analogue" refers to analogues that perform the same or similar functions, or derivatives of the same parent nucleus that perform the same or similar functions.

[0184] As used herein, the terms "mRNA" or "messenger polynucleotide" or "messenger RNA" or "messengerRNA" are used interchangeably and mean a single-stranded polynucleotide transcribed from one strand of DNA, carrying genetic information, and capable of directing protein synthesis.

[0185] As used herein, the terms "sgRNA" or "small guide RNA" or "guide RNA" or "gRNA" are used interchangeably and direct the insertion or deletion of uridine residues into kinetoplastids during the process of RNA editing. It belongs to a type of small non-coding RNA and can pair with pre-mRNA. gRNAs edit RNA molecules, which are approximately 60 - 80 nucleotides in length and are transcribed from separate genes.

[0186] As used herein, the terms "circRNA" or "circular RNA" or "circular polynucleotide" or "circular RNA" are used interchangeably and refer to a polynucleotide molecule having a structure without free ends (i.e., no free 3' and / or 5' ends), such as a polynucleotide that forms a circular or ring structure through covalent or non-covalent bonds.

[0187] As used herein, the terms "microRNA" or "miRNA" or "microRNA" are used interchangeably and refer to a non-coding single-stranded polynucleotide approximately 22 nucleotides in length with free 3' and 5' ends, which can inhibit the translation of the target gene protein by binding to the 3'-untranslated region (3'-UTR) of the mRNA of the target gene and thus regulate the biological functions of cells.

[0188] As used herein, the terms "ASO" or "antisense oligonucleotide" or "antisense oligonucleotide" are used interchangeably and refer to an artificially synthesized nucleic acid fragment complementary to a certain segment of a target gene or mRNA, which can bind to the target gene / mRNA through the principle of base complementarity, thereby blocking the expression of the gene, a single-stranded poly(deoxy)ribonucleotide, including antisense DNA and antisense RNA.

[0189] As used herein, the terms "siRNA" or "small interfering" or "short interfering" or "silencing RNA" or "small interfering RNA" or "short interfering RNA" or "silencing RNA" are used interchangeably and refer to a class of double-stranded RNA molecules that are 20 to 25 nucleotides in length and can induce the degradation of the mRNA of the target gene.

[0190] As used herein, the terms "ecDNA" or "extrachromosomal circular DNA" are used interchangeably and refer to DNA that has detached from the chromosome and exists in a circular structure outside the chromosome.

[0191] 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 increase the half-life or stability of the sequence, and labeling modifications. For example, chemical modifications include but are not limited to phosphorylation, methylation, amination, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopic labeling of any one or more bases. Substitutions of nucleotides or deoxynucleotides include but are not limited to nucleic acid analogs in which the sugar-phosphate backbone is replaced with a polypeptide or other backbone (replacing DNA or RNA with PNA (peptide nucleic acids)). Modifications to increase the half-life or stability of the sequence include but are not limited to modifications by linking with PEG, fluorine modifications. Labeling modifications include but are not limited to linking fluorescent groups, amino groups, biotin, digoxin, small peptides, etc.

[0192] The term "artificial nucleic acid": nucleic acid molecules that have been artificially modified, including but not limited to base modifications, ribose modifications, PNA, etc.

[0193] The term "nucleic acid" refers to a polymer present in single- or double-stranded form and containing at least two deoxynucleotides or nucleotides. Unless specifically restricted, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequences. Specifically, degenerate codon substitutions can be obtained by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue (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)). "Nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked by phosphate groups. "Base" includes 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, which include, but are not limited to, modifications substituting new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates (esters), and haloalkanes. DNA can exist as antisense, plasmid DNA, a portion of plasmid DNA, precompressed DNA, the product of a polymerase chain reaction (PCR), a vector (P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or a derivative of these groups. The term nucleic acid is used interchangeably with gene, cDNA, mRNA encoded by a gene, and interfering RNA molecules.

[0194] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that includes a partial-length or full-length coding sequence that is necessary to produce a polypeptide or polypeptide precursor (e.g., a polypeptide or polypeptide precursor from hepatitis virus types A, B, C, D, E, G; or herpes simplex virus).

[0195] As used herein, "gene product" refers to the product of a gene such as, for example, a transcript of DNA, mRNA.

[0196] The phrase "expression silencing of the target gene" refers to the ability of the siRNA of the present invention to initiate silencing of the target gene. To determine the degree of gene silencing, a sample or assay of cells in a target organism or culture is compared with a control sample, where the cells of the target organism or culture express a specific construct and the control does not express the construct. The control sample (lacking expression of the construct) is set to a relative value of 100%. Inhibition of the expression of the target gene is successfully achieved when the test value relative to the control is about 90%, preferably 50%, more preferably 25 - 0%. Suitable assays include, for example, detecting protein or mRNA levels using techniques known to those skilled in the art such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme activity, and phenotypic assays known to those skilled in the art.

[0197] A "therapeutically effective amount" or "effective amount" of siRNA is an amount sufficient to produce a desired effect, which is, for example, a reduction in the expression of the target sequence compared to the normal expression level detected in the absence of siRNA.

[0198] As used herein, the term "aqueous solution" refers to a composition that contains water, either in whole or in part.

[0199] As used herein, the term "organic lipid solution" refers to a composition that contains an organic solvent with lipids, either in whole or in part.

[0200] As used herein, "systemic delivery" refers to delivery that results in the widespread biodistribution of a compound in an organism. Some administration techniques can result in the systemic delivery of certain compounds, but not others. Systemic delivery means that an effective, preferably therapeutic, amount of the compound contacts a substantial portion of the body. To achieve widespread biodistribution, a blood survival period is generally required so that the compound is not rapidly degraded or cleared (such as by first-pass organs (liver, lung, etc.)) or by rapid, non-specific cell binding before reaching a disease site distal to the site of administration. Systemic delivery of drug-lipid particles can be carried out in any manner known in the art, which includes, for example, intravenous, subcutaneous, intraperitoneal, and in a preferred embodiment, systemic delivery of drug-lipid particles is by intravenous delivery.

[0201] As used herein, "local delivery" refers to the delivery of a compound directly to a target site within an organism. For example, a compound can be delivered locally by direct injection into a disease site such as a tumor or other target site such as an inflammatory site or a target organ such as the liver, heart, pancreas, kidney, etc.

[0202] In the term, "phospholipid" refers to lipids containing phosphate groups, belonging to compound lipids, also known as phospholipids and phosphatides. Phospholipids are the main components of biological membranes and are divided into two major categories: glycerophospholipids and sphingomyelins, which are composed of glycerol and sphingosine respectively. Phospholipids are amphipathic molecules, with one end being a hydrophilic nitrogen- or phosphorus-containing head and the other end being a hydrophobic (lipophilic) long hydrocarbon chain. For this reason, the hydrophilic ends of phospholipid molecules approach each other, and the hydrophobic ends approach each other, often jointly constituting a phospholipid bilayer together with other molecules such as proteins, glycolipids, and cholesterol, that is, the structure of the cell membrane.

[0203] In the present invention, the polyphenol molecular part in the metal-polyphenol complex mainly comes 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 activities 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 immunity of the body. Based on the potential immune-enhancing, anti-inflammatory, antioxidant, and anti-SARS-CoV-2 effects of curcumin molecules, it is expected to become a potential adjuvant treatment for COVID-19. Moreover, the safety of curcumin molecules is extremely high, and it has been listed in the catalog of food additives and pharmaceutical excipients, and its safety is conducive to the clinical drug registration of the drug-lipid as a whole, shortening the time length of clinical drug registration.

[0204] In the present invention, the coordination bond between the polyphenol molecular part and the metal ion part in the metal-polyphenol complex will break under conditions of low pH value (pH = 5.0) such as in lysosomes, and the metal ions will fall off from the metal-polyphenol complex.

[0205] In the present invention, the dosing ratios of the components in the metal-polyphenol complex can be adjusted according to the structure of the specific metal-polyphenol complex components. The basis for the adjustable dosing ratios is that since the hydroxyl groups of polyphenol molecules are connected to metal ions by coordination bonds, as long as the polyphenol molecules contain multiple binding sites, the dosing ratios of polyphenol molecules and metal ions can be adjusted according to the number of binding sites contained in the polyphenol molecules.

[0206] Metal-chelated polyphenol complex nanoparticles (MPNP)

[0207] The principle of loading nucleic acids into metal-polyphenol complex nanoparticles assembled from metal-polyphenol complexes is that polyphenol molecules are connected to metal ions through coordination bonds to form a metal-polyphenol complex, and the metal ions of the metal-polyphenol complex are connected to nucleic acids through coordination bonds, thereby ensuring that while the metal-polyphenol complex self-assembles with other components into MPNP, the nucleic acids are loaded into the nanoparticles.

[0208] As used 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.

[0209] In some embodiments, the conjugated lipid that inhibits particle aggregation refers to the conjugated lipid that inhibits the aggregation of drug-lipid particles, and its main function is to prevent the aggregation of drug-lipid particles, such as PEG conjugated with dialkoxypropyl, PEG conjugated with diacylglycerol, PEG conjugated with phosphatidylethanolamine, and PEG conjugated with ceramide, preferably a PEG-lipid conjugate. Among them, the cis-trans isomers of the lipid do not affect the effects to be achieved by the protected content of the present invention.

[0210] In some embodiments, the molar proportion of the metal-polyphenol complex in the raw material is 5% to 30%, such as 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%. Preferably 5% to less than 10%, 10% to 15% or 15% to 20%, and more preferably 5%, 10% or 15%.

[0211] In some embodiments, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, such as 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%.

[0212] 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%, such as 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%. Preferably 10% to 30%, 30% to 47% or 10% to 20%, and more preferably 10%, 30% or 47%.

[0213] In some embodiments, in addition to cholesterol, the metal-polyphenol complex particles further contain other non-cationic lipids or non-ionizable lipids, which account for 30% to 75% in molar ratio in the raw materials, 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%. Preferably, it is 45% to 55%, 60% to 65% or 50% to 65%, and further preferably 45%, 55%, 60% or 65%.

[0214] Drug-lipid particles

[0215] The drug-lipid particles described herein typically include a drug (which is a negatively charged molecule, and the molecule can be selected from one or more combinations of nucleic acids, proteins, polypeptides, small molecules, nucleic acid analogs, protein analogs, polypeptide analogs, and the nucleic acid is selected from one or more combinations of mRNA, siRNA, circular RNA, microRNA, sgRNA, DNA, ecDNA, 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 acid encapsulated in the drug-lipid particles of the present invention is resistant to degradation by nucleases in an aqueous solution.

[0216] In some embodiments, the drug is fully encapsulated inside the metal-polyphenol complex particles to avoid degradation of the drug and achieve delivery of the drug into cells.

[0217] 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; a stability of at least 3 days, preferably up to more than 7 days; and a cell delivery efficiency of at least 40%, such as 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%.

[0218] 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.

[0219] RNA populations can be used to provide long precursor RNAs, or long precursor RNAs having substantial or complete identity to selected target sequences that can be used to prepare siRNAs. The RNAs can be isolated, synthesized, and / or cloned from cells or tissues by 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, e.g., isolated from a tissue or cell sample, synthesized in vitro using, for example, T7 or SP6 polymerase and a PCR product or cloned cDNA; or chemically synthesized.

[0220] To form long dsRNAs, for synthetic RNAs, the complement can also be transcribed in vitro and hybridized to form dsRNAs. If naturally occurring RNA populations are used, e.g., by transcribing cDNAs corresponding to the RNA populations, or by using RNA polymerases, RNA complements are also provided (e.g., to form dsRNAs that are digested by Escherichia coli (E. coli) RNase III or Dicer). The precursor RNAs are then hybridized to form double-stranded RNAs for digestion. The dsRNAs can be directly encapsulated in SNALPs or can be digested in vitro prior to encapsulation.

[0221] Alternatively, one or more DNA plasmids encoding one or more siRNA templates can be encapsulated in nucleic acid-lipid particles. For example, based on the naturally occurring transcription units of small nuclear RNA U6 or human RNase P RNA H1, siRNA can be transcribed from a DNA template in a plasmid into a sequence that automatically folds into a duplex with a hairpin loop, the plasmid having an RNA polymerase III transcription unit (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., Nat. Biotech. 20:505 (2002); and Sui, et al., Proc. Natl. Acad. Sci. 99:5515 (2002)). Typically, the transcription unit or cassette will contain an RNA transcription promoter sequence, such as an H1-RNA or U6 promoter and a termination sequence, the promoter sequence being operably linked to a template for the siRNA sequence required for transcription, the termination sequence including 2-3 uridine residues and a poly-thymidine (T5) sequence (polyadenylation signal) (Brummelkamp, Science, supra). The selected promoter can provide constitutive or inducible transcription. The composition and method for transcription of DNA-directed 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. The selected plasmid can provide transient or stable delivery to target cells. It will be apparent to those skilled in the art that plasmids initially designed to express a desired gene sequence can be modified to contain a transcription unit cassette for transcribing siRNA.

[0222] Methods for separating 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), and so are PCR methods (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Expression libraries are also well known to those skilled in the art. Additional basic books that disclose 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)).

[0223] Applications of metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles

[0224] In some embodiments, when the drug is a nucleic acid, both metal-polyphenol complexes and metal-polyphenol complex particles can be used for promoting drug lysosomal escape and promoting nucleic acid expression. Metal-polyphenol complexes and metal-polyphenol complex particles can also be used for delivering drugs and introducing drugs into cells, thereby achieving the prevention and treatment of applicable diseases or disorders by the drugs.

[0225] In some embodiments, the present invention provides the use of metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles, for example, in a composition that enables drug delivery or the introduction of a drug into cells. The composition is, for example, a medicament that can achieve: silencing the expression of a target sequence in a mammalian subject, delivering a drug (such as a therapeutic for tumors, a contrast agent, etc.) in a mammalian body, delivering a drug from the body to mammalian cells, or treating a disease or disorder in a mammalian. In the medicament, the drug-lipid particle is the main active ingredient, and according to actual needs, it can be prepared into different dosage forms through different pharmaceutically acceptable excipients or preparation processes, such as solid dosage forms (powders, granules, pills, tablets, glue agents), semi-solid dosage forms (external ointments, pastes), liquid dosage forms (decoctions, mixtures, syrups, medicinal wines, injections), gas dosage forms (aerosols, smokes), etc.; for example, dosage forms for administration via the gastrointestinal tract, dosage forms for rectal administration, dosage forms for non-gastrointestinal administration, etc.

[0226] In some embodiments, the present invention provides a product prepared from the above metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles. The product has the above functions and uses of the metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles. The specific types can, for example, but are not limited to, be kits, medicaments, etc. The product optionally further contains other excipients.

[0227] For the target genes on which the drug-lipid particles act: Generally, it is desirable to deliver the drug-lipid particles to downregulate or silence the translation (i.e., expression) of the target gene product. Suitable classifications of gene products include, but are not limited to, genes related to viral infection and survival, genes related to metabolic diseases and disorders (such as diseases and disorders where the liver is the target, and liver diseases and disorders), genes related to tumorigenesis and cell transformation, angiogenic genes, immunomodulator genes such as those related to inflammation and autoimmune responses, ligand receptor genes, and genes related to neurodegenerative disorders.

[0228] Genes related to viral infection and survival include those that are expressed by the virus to bind, enter, and replicate in cells. In particular, viral sequences associated with chronic viral diseases. For example, viral sequences include those of hepatitis viruses (Hamasaki, et al., FEBS Lett. 543:51 (2003); Yokota, et al, EMBO Rep. 4:602 (2003); Schlomai, et al., Hepatology 37:764 (2003); Wilson, et al., Proc. Natl. Acad. Sci. 100:2783 (2003); Kapadia, et al., Proc. Natl. Acad. Sci. 100:2014 (2003); and FIELDS VIROLOGY (Knipe et al. eds. 2001)), human immunodeficiency virus (HIV) (Banerjea, et al., Mol Ther. 8:62 (2003); Song, et al., J. Virol. 77:7174 (2003); Stephenson JAMA 289:1494 (2003); Qin, et al., Proc. Natl. Acad. Sci. 100:183 (2003)), herpesviruses (Jia, et al., J. Virol. 77:3301 (2003)), and human papillomavirus (HPV) (Hall, et al., J. Virol. 77:6066 (2003); Jiang, et al., Oncogene 21:6041 (2002)). Exemplary hepatitis virus nucleic acid sequences that can be silenced include, but are not limited to: nucleic acid sequences involved in transcription and translation (e.g., En1, En2, X, P), nucleic acid sequences encoding structural proteins (e.g., core proteins including C and C-related proteins; capsid and envelope proteins including S, M, and / or L proteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY, 2001, supra). Hepatitis C nucleic acid sequences that can be silenced include, but are not limited to: serine protease (e.g., NS3 / NS4), helicase (e.g., NS3), polymerase (e.g., NS5B), and envelope proteins (e.g., E1, E2, and p7).The nucleic acid sequences of hepatitis A are mentioned, for example, in Genbank accession number NC_001489; the nucleic acid sequences of hepatitis B are mentioned, for example, in Genbank accession number NC_003977; the nucleic acid sequences of hepatitis C are mentioned, for example, in Genbank accession number NC_004102; the nucleic acid sequences of hepatitis D are mentioned, for example, in Genbank accession number NC_001653; the nucleic acid sequences of hepatitis E are mentioned, for example, in Genbank accession number NC_001434; and the nucleic acid sequences of hepatitis G are mentioned, for example, in Genbank accession number NC_001710. Silencing the sequences encoding genes related to viral infection and survival can be conveniently used in combination with the administration of conventional agents for treating viral diseases.

[0229] Genes associated with metabolic diseases and disorders (e.g., diseases and disorders in which the liver is targeted, as well as liver diseases and disorders) include, for example, genes expressed in dyslipidemia (e.g., liver X receptor (e.g., LXRα and LXRβ Genbank accession number NM_007121), farnesoid X receptor (FXR) (Genbank accession number NM_005123), sterol regulatory element-binding protein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG coenzyme-A reductase), apolipoprotein (ApoB), and apolipoprotein (ApoE)) and diabetes (e.g., glucose-6-phosphate) (see, e.g., Forman et al., Cell 81:687 (1995); Seol et al., Mol. Endocrinol. 9:72 (1995), Zavacki et al., PNAS USA 94:7909 (1997); Sakai, et al., Cell 85:1037-1046 (1996); Duncan, et al., J. Biol. Chem. 272:12778-12785 (1997); Willy, et al., Genes Dev. 9(9):1033-45 (1995); Lehmann, et al., J. Biol. Chem. 272(6):3137-3140 (1997); Janowski, et al., Nature 383:728-731 (199; Peet, et al., Cell 93:693-704 (1998)). Those skilled in the art will understand that genes associated with metabolic diseases and disorders (e.g., diseases and disorders in which the liver is targeted, as well as liver diseases and disorders) include genes expressed in the liver itself as well as genes expressed in other organs and tissues. Silencing the sequences encoding genes associated with metabolic diseases and disorders can be conveniently used in combination with the administration of conventional agents for treating the disease or disorder.

[0230] Examples of genes associated with tumorigenesis and cell transformation include translocation sequences such as MLL fusion genes, BCR-ABL (Wilda, et al., Oncogene, 21:5716 (2002); Scherr, et al, Blood 101:1566), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO and AML1-MTG8 (Heidenreich, et al., Blood 101:3157 (2003)); overexpressed sequences such as multidrug resistance genes (Nieth, et al., FEBSLett. 545:144 (2003); Wu, et al, Cancer Res. 63:1515 (2003)), cyclins (Li, et al., CancerRes. 63:3593 (2003); Zou, et al., Genes Dev. 16:2923 (2002)), β-catenin (Verma, etal., Clin Cancer Res. 9:1291 (2003)), telomerase gene (Kosciolek, et al., MolCancer Ther. 2:209 (2003)), c-MYC, N-MYC, BCL-2, ERBB1 and ERBB2 (Nagy, et al.Exp.CellRes. 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 combination with the administration of chemotherapeutic agents (Collis, et al., CancerRes. 63:1550 (2003)). Genes encoding proteins associated with tumor migration are also target sequences, such proteins, for example, integrins, selectins and metalloproteinases. Any complete or partial gene sequence that favors or promotes tumorigenesis or cell transformation, tumor growth or tumor migration can be included as a template sequence.

[0231] Angiogenic genes are capable of promoting the formation of new blood vessels. Vascular endothelial growth factor (VEGF) is a major area of research (Reich, et al., Mol.Vis. 9:210 (2003)).

[0232] Immune modulator genes are genes that regulate one or more immune responses. Examples of immune modulator genes include cytokines such as growth factors (e.g., TGF-α, TGF-β, EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12 (Hill, et al., J. Immunol. 171:691 (2003)), IL-15, IL-18, IL-20, etc.), interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.) and TNF. Fas and Fas ligand genes are also target immune modulator target sequences (Song, et al., Nat. Med. 9:347 (2003)). Genes encoding secondary signaling molecules in hematopoietic and lymphoid cells are also included in the present invention, for example, Tec family kinases such as Bruton's tyrosine kinase (Btk) (Heinonen, et al., FEBS Lett. 527:274 (2002)).

[0233] Cell receptor ligands include ligands that can bind to cell surface receptors (e.g., insulin receptor, EPO receptor, G-protein coupled receptor, receptor with tyrosine kinase activity, cytokine receptor, growth factor receptor, etc.) to regulate (e.g., inhibit, activate, etc.) the physiological pathways involved by the receptor (e.g., glucose level regulation, blood cell development, mitosis occurrence, etc.). Examples of cell receptor ligands include cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G-protein coupled receptor ligands, etc.). Templates encoding the expansion of trinucleotide repeats (e.g., CAG repeats) are found to be useful for silencing pathogenic sequences in neurodegenerative diseases caused by the expansion of trinucleotide repeats, such as spinal and bulbar muscular atrophy and Huntington's disease (Caplen, et al., Hum. Mol. Genet. 11:175 (2002)).

[0234] Injectable Delivery: In certain instances, as described in U.S. Patent 5,543,158; U.S. Patent 5,641,515 and U.S. Patent 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 into a target site (e.g., a disease site such as inflammation or tumor formation or into a target organ or tissue) or systemically injected for widespread distribution throughout the organism. Solutions of the drug-lipid particles can be prepared in water, which is 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. Generally, when administered intravenously, the drug-lipid particle formulation is formulated with a suitable pharmaceutical carrier. Generally, normal buffered salt solutions (135 - 150 mM NaCl) will be used as the 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, etc. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The formulation of aqueous compositions is a conventional understanding in the art, the compositions containing a protein as an active ingredient. Optionally, these compositions are prepared as injectable solutions, liquid solutions or suspensions; solid forms suitable for solutions or suspensions in a liquid prior to injection can also be prepared. The formulations can also be emulsified.

[0235] The drug-lipid particles can be sterilized by conventional liposome sterilization techniques, such as filtration. The drug-lipid particles can contain pharmaceutical adjuvants, which are suitable for physiological conditions, such as pH regulators and buffers, toxicity modifiers, wetting agents, etc. These compositions can be sterilized using the techniques referred to above, or alternatively, they can be produced under aseptic conditions. The resulting aqueous solution can be packaged for use or filtered under aseptic conditions and lyophilized, and the lyophilized formulation is combined with a sterile aqueous solution prior to administration.

[0236] Prophylactic and therapeutic treatment: In some embodiments, the drug-lipid particles can be used for prophylactic or therapeutic treatment of a subject (e.g., a mammalian subject) having a disease or disorder associated with the expression or overexpression of a target sequence. The drug-lipid particles are administered to the subject in an amount sufficient to elicit a therapeutic response in the patient. The 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 particles to be administered in the treatment or prevention of a disease, which is caused by the expression or overexpression of a target gene, the physician evaluates the circulating plasma levels of the drug-lipid particles, the drug-lipid particle toxicity, and the progression of the disease associated with the expression or overexpression of the target gene. The administration can be accomplished by a single or divided dose.

[0237] For example, the drug-lipid particles can be administered to a subject who is infected or at risk of being infected by a pathogenic microorganism. The drug should preferably correspond to a sequence and should also be unique to the microorganism (or at least absent from the genome of the natural genome of the patient undergoing treatment), and the sequence has a key role in the life history of the microorganism. The drug-lipid particles are introduced into target cells, tissues or organs in a therapeutically effective dose by ex vivo or intravenous injection. Silencing the sequence encoding the gene associated with the pathogenic infection can be conveniently used in combination with the administration of conventional reagents for treating pathogenic diseases. The treatment can be administered prophylactically to a person at risk of being infected by a pathogenic microorganism or who has already been infected by a pathogenic microorganism.

[0238] In a preferred embodiment, the pharmaceutical-lipid particles of the present invention can be conveniently used for the treatment of cancer, viral infections, autoimmune diseases, diabetes, Alzheimer's disease. Viral infections include hepatitis A, hepatitis B, hepatitis C, SARS-Cov-2, HIV, HPV, influenza, smallpox, 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, e.g., FIELDS VIROLOGY, 2001, supra). Those skilled in the art will understand 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 a) (see, e.g., Medina et al., Antiviral Res. 60(2):135-143 (2003)); ribavirin (see, e.g., Hugle and Cerny, Rev. Med. Virol. 13(6):361-71 (2003)); adefovir and lamivudine (see, e.g., Kock et al., Hepatology 38(6):1410-8 (2003)); prenylation inhibitors (see, e.g., Bordier et al., J. Clin. Invest. 112(3):407-414 (2003)); famciclovir (see, e.g., Yurdaydin et al., J Hepatol. 37(2):266-71 (2002); and saikosaponins c and d (see, e.g., Chiang et al., Planta Med. 69(8):705-9 (2003).

[0239] In another embodiment, the pharmaceutical-lipid particles of the invention can be conveniently used to treat diseases and disorders characterized by the expression or overexpression of a gene or group of genes. In some aspects, the pharmaceutical-lipid particles of the invention can be used to treat metabolic diseases and disorders (e.g., diseases and disorders where the liver is the target and liver diseases and disorders) such as, for example, dyslipidemia and diabetes. Those skilled in the art will understand that the silencing of genes associated with metabolic diseases and disorders can be combined with the conventional treatment of these diseases. For example, the silencing of genes involved in dyslipidemia can be combined with treatment with statins, bile acid sequestrants / resins, and cholesterol absorption inhibitors such as ezetimibe, plant sterols / stanols, polyphenols, and nutritional products such as oat bran, flaxseed, and soy protein, phytostanol analogs, squalene synthase inhibitors, bile acid transport inhibitors, SREBP cleavage-activating protein (SCAP) activating ligands, niacin (nicotinic acid), acipimox, high-dose fish oil, antioxidants, and sugarcane fatty alcohols, microsomal triglyceride transfer protein (MTP) inhibitors, acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors, gemcabene, lofibrol, pantothenic acid analogs, niacin-receptor agonists, anti-inflammatory agents (such as Lp-PLA(2) antagonists and AGI1067), functional oils, PPAR-α, γ, δ agonists, and 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 inhibitor, statin / PPAR agonist, 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)). Similarly, the silencing of genes involved in diabetes can be combined with treatment with insulin and dietary modification and exercise.

[0240] Similar methods are used to inhibit the expression of endogenous recipient cell genes, said endogenous recipient genes being related to tumorigenesis and cell transformation, tumor growth and tumor migration; to inhibit the expression of angiogenic genes; to inhibit the expression of immunomodulator genes, such as those related to inflammation and autoimmune responses; to inhibit the expression of ligand receptor genes; to inhibit the expression of genes related to neurodegenerative disorders; and to inhibit the expression of additional genes related to viral infection and survival. The target gene sequences for specific targets are as described above.

[0241] Detecting the particles: Detect the drug-lipid particles herein using any method known in the art. For example, label is directly or indirectly conjugated to the components of the drug-lipid particles or other lipid-based carrier systems using methods well known in the art. A wide variety of labels can be used, and the selection is made based on the required sensitivity, ease of conjugation to the components of the drug-lipid particles, stability requirements, and available tools and handling preparations. Suitable labels include, but are not limited to, spectroscopic labels such as fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon GreenTM; rhodamine and derivatives such as Texas Red, tetrarhodimineisothiocynate (TRITC), etc., digoxin, biotin, phycoerythrin, AMCA, CyDyesTM, etc.); radioactive labels such as 3H, 125I, 35S, 14C, 32P, 33P, etc.; enzymes such as horseradish peroxidase, alkaline phosphatase, etc.; spectroscopic colorimetric labels such as colloidal gold or colored glass or plastic beads such as polystyrene, polypropylene, latex, etc.). Detect the label using any manner known in the art.

[0242] Detection of nucleic acids: Detect and quantify the nucleic acids herein by any one of a number of ways well known to those skilled in the art. Detection of nucleic acids is carried out by methods well known in the art such as Southern blot analysis, Northern blot analysis, gel electrophoresis, PCR, radioactive labeling, scintillation counting, and affinity chromatography. Additional analytical biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography can also be applied.

[0243] The sensitivity of hybridization assays can be enhanced by applying nucleic acid amplification systems that multiply the target nucleic acids being detected. In vitro amplification techniques are known that are suitable for amplifying sequences used as molecular probes or for generating nucleic acid fragments for subsequent subcloning. 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., NASBATM), technical examples sufficient to guide a person skilled in the art can be found in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2000, and Ausubel et al., SHORT PROTOCOLS IN MOLECULAR BIOLOGY, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (2002), as well as Mullis et al. (1987), U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990), C&EN 36; The Journal Of NIH Research, 3:81 (1991); (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989); Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990); Lomell et al., 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., Gene, 89:117 (1990), and Sooknanan and Malek, Biotechnology, 13:563 (1995).Improved methods for the in vitro amplification of nucleic acids are described in Wallace et al., U.S. Patent No. 5,426,039. Other methods described in the art are based on the amplification of nucleic acid sequences (NASBATM, Cangene, Mississauga, Ontario) and the Qβ replicase system.

[0244] As described by Needham VanDevanter et al., Nucleic Acids Res., 12: 6159 (1984), oligonucleotides are typically chemically synthesized according to the solid-phase phosphoramidite triester method described by Beaucage and Caruthers, Tetrahedron Letts., 22(20): 1859 - 1862 (1981), for example using an automated synthesizer. The oligonucleotides are used as probes in in vitro amplification methods, for example, as gene probes, or as inhibitor components. If necessary, oligonucleotide purification is typically carried out 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.

[0245] The following examples are provided by way of illustration, but do not limit the claimed invention. Those skilled in the art will readily recognize various non-critical parameters that can produce substantially similar results.

[0246] The pharmaceutical-lipid particles to be protected by the present invention refer to pharmaceutical-lipid particles other than those containing cationic / ionizable lipids, that is, drug-loaded metal-chelated polyphenol complex nanoparticles (drug@MPNP).

[0247] Experimental Example 1. Preparation of drug-loaded metal-chelated polyphenol complex nanoparticles (mRNA@MPNP)

[0248] Example 1. Preparation of metal-chelated polyphenol complex nanoparticles (MPNP)

[0249] Example 1.1 Preparation of Metal-Polyphenol Complex with Metal Ion as Fe 3+ Metal-polyphenol complex when the metal ion is Fe

[0250] Dissolve curcumin (Formula 1) in ethanol at 1.5 mg / ml, and then add anhydrous FeCl 3 , where the molar ratio of curcumin (Formula 1) to anhydrous FeCl 3 is 1:1, and reflux the reaction for 1 hour at 60 °C. After the reaction is completed, dry the solution to dryness, dissolve the product with ultrapure water and filter it. The product obtained after freeze-drying is the metal-polyphenol complex. The structure of the metal-polyphenol complex is shown below.

[0251]

[0252] Result analysis: Curcumin (Formula 1) and FeCl 3 react for 1 hour at 60 °C. The yield of the target product obtained when the feeding concentration of curcumin (Formula 1) is 1.5 mg / mL and the feeding ratio of curcumin (Formula 1) to FeCl 3 is 1:1 is 95%.

[0253] Example 1.2 Preparation of Metal-Polyphenol Complex with Metal Ion as Al 3+ Metal-polyphenol complex when the metal ion is Al

[0254] The difference between this example and Example 1.1 is that FeCl 3 is replaced by Al(NO 3 ) 3 ·9H 2 O.

[0255] The structure of the prepared metal-polyphenol complex is shown below.

[0256]

[0257] Result analysis: Curcumin (Formula 1) and Al(NO 3 ) 3 ·9H 2 O react for 1 hour at 60 °C. The yield of the target product obtained when the feeding concentration of curcumin (Formula 1) is 1.5 mg / mL and the feeding ratio of curcumin (Formula 1) to Al(NO 3 ) 3 ·9H 2 O is 98%.

[0258] Example 2. Preparation of mRNA-loaded metal-chelated polyphenol complex nanoparticles (mRNA@MPNP)

[0259] Prepare the mRNA-loaded metal-chelated polyphenol complex nanoparticles with Fe as the metal ion 3+

[0260] Prepare the metal-chelated polyphenol complex according to the method in Example 1.1, in which curcumin (Formula 1) and FeCl 3 are added in a feeding ratio of 1:1, and the metal-chelated polyphenol complex, distearoylphosphatidylcholine (DSPC, Formula 51, as a non-cationic lipid or non-ionizable lipid), cholesterol (CHOL, Formula 59, as a non-cationic lipid or non-ionizable lipid), and DSPE-PEG2000 (Formula 58, as a conjugated lipid for inhibiting particle aggregation) are dissolved in ethanol in different molar ratios as the organic phase. The proportions of the metal-chelated polyphenol complex, DSPC (Formula 51), CHOL (Formula 59), and DSPE-PEG2000 (Formula 58) are 5%, 60%, 30%, and 5% respectively. mRNA is dissolved in an enzyme-free Tris-HCl buffer (molar concentration 0.1 M) with pH 5.0 at a concentration of 20 μg / mL as the aqueous phase. The metal-chelated polyphenol complex and mRNA are 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 rates of the organic phase and the aqueous phase in the microfluidic chip are 12 ml / min. Among them, the drug mRNA is the mRNA encoding the fluorescent protein eGFP, and its sequence is SEQ ID NO.1 (720 nt). eGFP-mRNA@MPNP is prepared. eGFP-mRNA@MPNP is incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA), and the control group is incubated with MPNP without drug loading. After 48 h, the cell suspension is collected, and the percentage of eGFP-positive cells is detected by flow cytometry.

[0261] Detect the particle size, surface potential and stability of the prepared eGFP-mRNA@MPNP, and calculate the nucleic acid encapsulation efficiency of eGFP-mRNA@MPNP.

[0262] Method for detecting particle size and result judgment criteria: Use a Malvern laser particle size analyzer Zetasizer to measure the particle size of the nanoparticles. The particle size within the range of 30 - 400 nm is considered acceptable.

[0263] ​Method for detecting surface potential and result judgment criteria: Use a Malvern laser particle size analyzer Zetasizer to measure the surface potential of nanoparticles. A potential in the range of -10 to 10 mV is considered acceptable.

[0264] Method for detecting stability and result judgment criteria: Place the nanoparticles at 4 °C for 7 days. Use a Malvern laser particle size analyzer Zetasizer to measure the particle size and surface potential of the nanoparticles. If there is no significant change in the particle size and surface potential within 3 - 7 days, it is considered to have good stability.

[0265] Method for calculating nucleic acid encapsulation efficiency: Specifically, the agarose gel electrophoresis method is used. First, set the nucleic acid input amount of each group of lipid nanoparticles to 10 μg / mL. The mass ratio of the metal-polyphenol complex to the nucleic acid is 20:1. Dissolve the nucleic acid at the same concentration in enzyme-free Tris-HCl buffer as the 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 pores of the gel only allow free nucleic acid to pass through and do not allow lipid nanoparticles to pass through. Stop electrophoresis when the free nucleic acid band migrates to a clearly distinguishable position. Use Image J software to statistically analyze the gray value of free nucleic acid 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. Then the encapsulation rate of each group is (100 - relative amount of free nucleic acid)%. A nucleic acid encapsulation rate above 50% is considered an acceptable range.

[0266] Cell culture method: Human embryonic kidney cell line 293T is cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37 °C, 5% CO 2 conditions.

[0267] Method for analyzing the percentage of eGFP-positive cells by flow cytometry: Seed 293T cells in a 12-well plate at an inoculation density of 5×10 5 cells / well. When the cell density reaches 80%, add 1 mL of MPNP or eGFP-mRNA@MPNP to incubate the cells, where the concentration of eGFP-mRNA@MPNP is 2 μg / mL. After 48 h, collect the cell suspension, collect 20,000 cells using the FITC channel of a flow cytometer, and analyze the percentage of eGFP-positive cells. The calculation formula is: eGFP positive cell rate calculation formula = number of cells expressing eGFP / total number of cells × 100%. A percentage of eGFP-positive cells above 40% is considered an acceptable range.

[0268] Preparation of mRNA-metal-polyphenol complex particles when the metal ion is Al 3+

[0269] ​Prepare the metal-polyphenol complex according to the method in Example 1.2, where curcumin (Formula 1), Al(NO 3 ) 3 ·9H 2 O are put in at a feeding ratio of 1:1, and the metal-polyphenol complex, distearoylphosphatidylcholine (DSPC, Formula 51, non-cationic lipid or non-ionizable lipid), cholesterol (CHOL, Formula 59, non-cationic lipid or non-ionizable lipid), and DSPE-PEG2000 (Formula 58, conjugated lipid that inhibits particle aggregation) are dissolved in ethanol at different molar ratios as the organic phase. The proportions of the metal-polyphenol complex, DSPC (Formula 51), CHOL (Formula 59), and DSPE-PEG2000 (Formula 58) are 5%, 45%, 47%, and 3% respectively. The mRNA is dissolved in an enzyme-free Tris-HCl buffer (molar concentration of 0.1 M) with a pH of 5.0 at a concentration of 20 μg / mL as the aqueous phase. The mass ratio of the mass of the metal-polyphenol complex to the mass of the mRNA is 18:1 and they are mixed in a microfluidic chip. The volume ratio of the aqueous phase to the organic phase is 3:1. The flow rates of the organic phase and the aqueous phase in the microfluidic chip are 12 ml / min. Among them, the drug mRNA is the mRNA encoding the fluorescent protein eGFP, and its sequence is SEQ ID NO.1 (720 nt). eGFP-mRNA@MPNP is prepared. Incubate eGFP-mRNA@MPNP with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA), and incubate the control group with MPNP. After 48 h, collect the cell suspension, and detect the percentage of eGFP-positive cells by flow cytometry.

[0270] Detect the particle size, surface potential and stability of the prepared eGFP-mRNA@MPNP, and calculate the efficiency of eGFP-mRNA@MPNP encapsulating nucleic acid.

[0271] Method for detecting particle size and result judgment criterion: Use a Malvern laser particle size analyzer Zetasizer to test the particle size of the nanoparticles. The particle size within the range of 30 - 400 nm is considered acceptable.

[0272] Method for detecting surface potential and result judgment criterion: Use a Malvern laser particle size analyzer Zetasizer to test the surface potential of the nanoparticles. The potential within the range of -10 - 10 mV is considered acceptable.

[0273] Method for detecting stability and result judgment criterion: Place the nanoparticles at 4°C for 7 days, use a Malvern laser particle size analyzer Zetasizer to test the particle size and surface potential of the nanoparticles. When there is no obvious change in its particle size and surface potential within 3 - 7 days, it is considered to have good stability.

[0274] Method for calculating nucleic acid encapsulation efficiency: Specifically, the agarose gel electrophoresis method is adopted. First, the nucleic acid dosage of each group of lipid nanoparticles is set at 10 μg / mL, and the mass ratio of the metal-polyphenol complex to nucleic acid is 18:1. Nucleic acid with the same concentration is dissolved in enzyme-free Tris-HCl buffer as the 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 pores of the gel only allow free nucleic acid to pass through and do not allow lipid nanoparticles to pass through. When the free nucleic acid band migrates to a position where it can be clearly distinguished, the electrophoresis is stopped. The gray values of free nucleic acid in different groups are statistically analyzed using Image J software. 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. Then, the encapsulation rate of each group is (100 - relative amount of free nucleic acid)%. A nucleic acid encapsulation rate above 50% is considered an acceptable range.

[0275] Cell culture method: Human embryonic kidney cell line 293T is cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37 °C and 5% CO 2 .

[0276] Method for analyzing the percentage of eGFP-positive cells by flow cytometry: 293T cells are seeded in a 12-well plate at a seeding density of 5×10 5 cells / well. When the cell density reaches 80%, 1 mL of MPNP or eGFP-mRNA@MPNP is added to incubate the cells, and the concentration of eGFP-mRNA@MPNP is 2 μg / mL. After 48 h, the cell suspension is collected. 20,000 cells are collected using the FITC channel of a flow cytometer, and the percentage of eGFP-positive cells is analyzed. The calculation formula is: eGFP positive cell rate calculation formula = number of cells expressing eGFP / total number of cells × 100%. A percentage of eGFP-positive cells above 40% is considered an acceptable range.

[0277] The principle of loading nucleic acid by metal-polyphenol complex nanoparticles (MPNP) assembled from metal-polyphenol complexes is as follows: Curcumin is connected to Fe 3+ or Al 3+ through coordination bonds to form a metal-polyphenol complex. The Fe 3+ or Al 3+It is connected to nucleic acid through a coordination bond, so as to ensure that while the metal-polyphenol complex self-assembles with other components into MPNP, the nucleic acid is loaded into the nanoparticles. There are two possibilities for the contribution of curcumin in the loading of nucleic acid by MPNP: ① Curcumin interacts with nucleic acid to assist MPNP in loading nucleic acid. For example, curcumin assists in loading nucleic acid by inserting into the minor groove of nucleic acid; ② Curcumin may not directly interact with nucleic acid.

[0278] Example 2.1, Dosage ratio of components of metal-polyphenol complex

[0279] The curcumin (Formula 1) and FeCl in Example 2 3 were added in different dosage ratios (1:1, 3:2, 2:1), and the other steps were the same as those in Example 2 to prepare different eGFP-mRNA@MPNP, and their nucleic acid encapsulation rates were detected respectively.

[0280] Result analysis: As shown in Result Table 1-1, when the dosage ratio of curcumin (Formula 1) and FeCl 3 was 1:1, the mRNA encapsulation efficiency of the prepared metal-polyphenol complex particles was 85%; when the dosage ratio of curcumin (Formula 1) and FeCl 3 was 3:2, the mRNA encapsulation efficiency of the prepared metal-polyphenol complex particles was 72%; when the dosage ratio of curcumin (Formula 1) and FeCl 3 was 2:1, the mRNA encapsulation rate of the prepared metal-polyphenol complex particles was 63%. In the drug-lipid particles, the function of Fe 3+ is to connect curcumin and nucleic acid. Each Fe 3+ has at most three coordination sites. Therefore, the dosage ratio of curcumin and FeCl 3 in the drug-lipid particles should be 1:1 to ensure that the metal-polyphenol complex particles can encapsulate as much nucleic acid as possible. Our results also confirm that when the dosage ratio of curcumin and FeCl 3 is 1:1, the mRNA encapsulation rate of the metal-polyphenol complex particles prepared with it is the highest. When the dosage ratio of curcumin and FeCl 3 is in the range from 1:1 to 2:1, the nucleic acid encapsulation rate of its metal-polyphenol complex particles is above 60%.

[0281] Table 1-1 Dosage ratio of components of metal-polyphenol complex when metal ion is Fe 3+ and the function of the prepared metal-polyphenol complex particles

[0282] <![CDATA[Curcumin (Formula 1), FeCl 3 Dosage ratio]]> mRNA Encapsulation Efficiency of Metal-Polyphenol Complex Particles 1:1 85％ 3:2 72％ 2:1 63％

[0283] The curcumin (Formula 1) and Al(NO 3) 3 ·9H 2 O was added at different ratios (1:1, 3:2, 2:1), and other steps were the same as in Example 2 to prepare different eGFP-mRNA@MPNP, and their nucleic acid encapsulation rates were detected respectively.

[0284] Result analysis: As shown in Result Table 1-2, when the addition ratio of curcumin (Formula 1), Al(NO 3 ) 3 ·9H 2 O was 1:1, the eGFP-mRNA encapsulation efficiency of the prepared metal-polyphenol complex particles was 86%; when the addition ratio of curcumin (Formula 1), Al(NO 3 ) 3 ·9H 2 O was 3:2, the eGFP-mRNA encapsulation efficiency of the prepared metal-polyphenol complex particles was 70%; when the addition ratio of curcumin (Formula 1), Al(NO 3 ) 3 ·9H 2 O was 2:1, the eGFP-mRNA encapsulation efficiency of the prepared metal-polyphenol complex particles was 66%. The function of Al 3+ in the metal-polyphenol complex particles is to connect the phospholipid complex and the nucleic acid. Each Al 3+ has at most three complexation sites. Therefore, the addition ratio of curcumin, Al(NO 3 ) 3 ·9H 2 O in the drug-lipid particles should be 1:1 to ensure that the metal-polyphenol complex particles can encapsulate as much nucleic acid as possible. The experimental results also confirmed that when the addition ratio of curcumin, Al(NO 3 ) 3 ·9H 2 O was 1:1, the eGFP-mRNA encapsulation rate of the metal-polyphenol complex particles prepared with it was the highest. When the addition ratio of curcumin, Al(NO 3 ) 3 ·9H 2 O was in the range of 1:1 to 2:1, the nucleic acid encapsulation rate of its metal-polyphenol complex particles was above 60%.

[0285] Table 1-2 Component addition ratios of metal-polyphenol complexes with metal ion Al 3+ and functions of the prepared metal-polyphenol complex particles

[0286]

[0287] Example 2.2. Proportion of metal-polyphenol complex, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), DSPE-PEG2000 and cholesterol (CHOL) in the preparation of drug-lipid particles

[0288] Compared with Example 2, the proportions of metal-polyphenol complex, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, formula 51), DSPE-PEG2000 (formula 58), and cholesterol (CHOL, formula 59) are shown in Table 1-3 (metal ion is Fe 3+ ) and Table 1-4 (metal ion is Al 3+ ), and the remaining conditions are the same.

[0289] Result analysis: As shown in Result Table 1-3, when the proportion of metal-polyphenol complex (metal ion is Fe 3+ ) 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 particle size of the drug-lipid particles is in the range of 30-400 nm, the surface potential is in the range of -10 to 10 mV, the in vitro stability is ≥ 3 days, the mRNA encapsulation rate > 50%, and the positive cell rate of eGFP protein is more than 65%. Among them, when the proportion of metal-polyphenol complex is 5%, the proportion of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is 60%, the proportion of cholesterol (CHOL) is 30%, and the proportion of DSPE-PEG2000 is 5%, the performance of the drug-lipid particles is optimal, that is, the particle size is in the range of 120 nm, the surface potential is in the range of -1.99 mV, the in vitro stability > 7 days, the mRNA encapsulation rate is 85%, and the positive cell rate of eGFP protein is 97%. Since the metal-chelated polyphenol complex nanoparticles (MPNP) mainly adsorb nucleic acids by the metal-polyphenol complex, 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 content in the range of 40%-75% will have better performance; the role of DSPE-PEG2000 is to prevent nanoparticle aggregation and extend the in vivo circulation time, and its content in the range of 2%-10% will have better performance; when the CHOL content is 0%, from the 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.

[0290] The above results suggest that the metal-polyphenol complex (metal ion is Fe 3+)When the proportion 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 good drug-loading performance.

[0291] Table 1-3 The metal ion is Fe 3+ The proportions of each component in the drug-metal-polyphenol complex particles (mRNA@MPNP)

[0292]

[0293]

[0294] Result analysis: As shown in Result Table 1-4, when the proportion of the metal-polyphenol complex (the metal ion is Al 3+ ) is in the range of (5 - 20)%, the proportion of DSPC is in the range of (30 - 75)%, the proportion of CHOL is in the range of (0 - 48)%, and the proportion of DSPE-PEG2000 is in the range of (2 - 10)%, the particle size of the metal-polyphenol complex particles is in the range of 30 - 400 nm, the surface potential is in the range of -10 - 10 mV, the in vitro stability is ≥ 3 days, the mRNA encapsulation rate is > 50%, and the positive expression rate of eGFP protein is above 65%. Among them, when the proportion of the metal-polyphenol complex is 5%, the proportion of distearoyl phosphatidylcholine (DSPC) is 45%, the proportion of cholesterol (CHOL) is 47%, and the proportion of DSPE-PEG2000 is 3%, the performance of the metal-polyphenol complex particles is optimal, that is, the particle size is in the range of 100 nm, the surface potential is in the range of -2.74 mV, the in vitro stability is > 7 days, the mRNA encapsulation rate is 86%, and the positive cell rate of eGFP protein is 97%. Because mRNA@MPNP mainly adsorbs nucleic acids by the metal-polyphenol complex, the proportion of the metal-polyphenol complex cannot be too low; when the content of DSPC is in the range of (30 - 75)%, the stability of its nanoparticles is within an acceptable range; the role of DSPE-PEG2000 is to prevent nanoparticle aggregation and increase the in vivo circulation time, and its performance is better when the content is in the range of (2 - 10)%; the role of CHOL is to enhance the fluidity of nanoparticles, and maintaining a certain content is beneficial to the stability of nanoparticles.

[0295] The above results suggest that when the proportion of the metal-polyphenol complex (the metal ion is Al 3+ ) is in the range of (5 - 20)%, the proportion of DSPC is in the range of (30 - 75)%, the proportion of CHOL is in the range of (0 - 48)%, and the proportion of DSPE-PEG2000 is in the range of (2 - 10)%, mRNA@MPNP has good drug-loading performance.

[0296] Table 1-4 Metal ion is Al 3+ Proportion of each component in the drug-metal-polyphenol complex particles (mRNA@MPNP)

[0297]

[0298] Example 2.3, Preparation of the types of non-cationic lipids or non-ionizable lipids in eGFP-mRNA@MPNP

[0299] Compared with Example 2, the substitution of distearoyl phosphatidylcholine (DSPC) is shown in Table 1-5 and Table 1-6, and the other conditions are the same.

[0300] Result analysis: To explore whether DSPC in eGFP-mRNA@MPNP can be replaced by other non-cationic lipids or non-ionizable lipids except the conjugated lipids that inhibit particle aggregation, DSPE, DSPA and DSPG were respectively selected to replace DSPC, and by detecting the particle size, surface potential, stability and mRNA encapsulation rate, it was proved that DSPC in eGFP-mRNA@MPNP can be replaced by other non-cationic lipids or non-ionizable lipids, and its function is equivalent to that of eGFP-mRNA@MPNP containing DSPC (Table 1-5 (metal ion is Fe 3 +) and Table 1-6 (metal ion is Al 3 +). Because the main role of the non-cationic lipid DSPC in eGFP-mRNA@MPNP is to make the liposome membrane have better fusion, higher stability and lower toxicity, and other non-cationic lipids or non-ionizable lipids also have the functions of making the liposome membrane fuse better, having higher stability and lower toxicity, so, DSPC in the drug-lipid particles can be replaced by other non-cationic lipids or non-ionizable lipids, and its efficacy is not affected.

[0301] Table 1-5 Metal ion is Fe 3 Performance of other non-cationic lipids except the conjugated lipids that inhibit particle aggregation in the drug-metal-polyphenol complex particles (mRNA@MPNP)

[0302]

[0303] Table 1-6 Metal ion is Al 3 Performance of other non-cationic lipids except the conjugated lipids that inhibit particle aggregation in the drug-metal-polyphenol complex particles (mRNA@MPNP)

[0304]

[0305] Example 2.4. Types of conjugated lipids that inhibit particle aggregation in the preparation of eGFP-mRNA@MPNP

[0306] 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 ion is Al 3 +), and the other conditions are the same.

[0307] Result analysis: 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 detecting particle size, surface potential, stability, and mRNA encapsulation efficiency, it was proved that DSPE-PEG2000 in eGFP-mRNA@MPNP can be replaced by other conjugated lipids that inhibit particle aggregation, and its function after replacement is equivalent to the efficacy of eGFP-mRNA@MPNP containing DSPE-PEG2000 (Table 1-7 and Table 1-8). Because the main role 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, so DSPE-PEG2000 in eGFP-mRNA@MPNP can be replaced by other conjugated lipids that inhibit particle aggregation, and its efficacy is not affected.

[0308] Table 1-7 Types of conjugated lipids that inhibit particle aggregation in drug-metal-polyphenol complex particles (mRNA@MPNP) with metal ion Fe 3+

[0309]

[0310] Table 1-8 Types of conjugated lipids that inhibit particle aggregation in drug-metal-polyphenol complex particles (mRNA@MPNP) with metal ion Al 3+

[0311]

[0312] Example 2.5. Preparation and effect characterization of mRNA@MPNP

[0313] Example 2.5.1. Preparation and effect characterization of mRNA@MPNP with metal ion Fe 3+

[0314] ​​​Replace the mRNA in Example 2 with two other mRNAs, and prepare three kinds of mRNA@MPNP containing different target protein mRNA sequences respectively according to the method of Example 2. The three different mRNA sequences are as follows: ① The mRNA sequence encoding the fluorescent protein eGFP is SEQ ID NO.1 (720 nt); ② The mRNA sequence encoding the receptor binding domain (RBD) of the S1 subunit of the novel coronavirus is SEQ ID NO.2 (669 nt); ③ The mRNA sequence encoding the tumor antigen NY-ESO-1 is SEQ ID NO.3 (543 nt). The preparation processes of the remaining drug (mRNA)-lipid particles are the same as those in Example 2, and eGFP-mRNA@MPNP, RBD-mRNA@MPNP, and NY-ESO-1-mRNA@MPNP are obtained respectively.

[0315] Incubate eGFP-mRNA@MPNP with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA), and incubate the control group with MPNP. After 48 h, collect the cell suspension, and detect the percentage of eGFP-positive cells by flow cytometry. The results are shown in Figure 1-1 ; Incubate RBD-mRNA@MPNP with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA), and incubate the control group with MPNP. After 24 h, centrifuge and freeze the supernatant at -20 °C for later use. Use a commercially available new coronavirus antigen RBD ELISA detection kit to detect the expression level of the new coronavirus antigen RBD protein in the cell supernatant. The results are shown in Figure 1-2 .

[0316] Method for detecting the RBD expression level by ELISA:

[0317] 1. Sample collection: Let the cell supernatant stand at room temperature for 2 hours, centrifuge at 1000×g for 20 min, and take the supernatant;

[0318] 2. Sample addition: Set blank wells, standard wells, and wells for samples to be tested on the coated plate. Add 100 μL of sample diluent to the blank well, add serially diluted standards to the standard wells in turn, and add 100 μL of the sample to be tested to the wells for samples to be tested. Incubate at 37 °C for 60 min;

[0319] 3. Discard the liquid in the wells, wash the plate 3 times, and soak each time for 1 - 2 min. Add 100 μL of the prepared working solution of biotin-labeled anti-RBD antibody to each well, mix well, and incubate at 37 °C for 60 min;

[0320] 4. Discard the liquid in the wells, wash the plate 3 times, and soak each time for 1 - 2 min;

[0321] 5. Add 100 μL of the prepared streptavidin HRP working solution to each well, mix well, and incubate at 37 °C for 45 min;

[0322] 6. Discard the liquid in the wells and wash the plate 3 times, soaking for 1 - 2 min each time;

[0323] 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 min;

[0324] 8. Add 100 μL of stop solution to each well to terminate the reaction;

[0325] 9. Measure the optical density (OD) value of each well at a wavelength of 450 nm.

[0326] Data analysis: Using the concentration of the standard product as the abscissa and the OD value as the ordinate, plot the standard curve.

[0327] Randomly divide the experimental animals into 2 groups (experimental group and control group), with 5 animals in each group. Among them, the RBD-mRNA@MPNP animal model is BALB / c mice. Each mouse is given the first intramuscular injection on day 1 and the second intramuscular injection on day 14. The experimental group is injected with RBD-mRNA@MPNP, and the control group is injected with metal-polyphenol complex particles (MPNP) without loaded mRNA. The dose of each injection is 100 μL, and the RBD-mRNA@MPNP preparation in the experimental group contains 30 mg of mRNA. Collect the blood of the mice on day 28 after the first injection, separate the serum and dilute it in gradients, and detect the total IgG antibody against the RBD of the S1 subunit of the novel coronavirus in the mice using a commercially available ELISA kit. The results are as Figure 1-3 shown.

[0328] The animal model of NY-ESO-1-mRNA@MPNP is C57BL / 6 mice. Each mouse is given four intramuscular injections on days 1, 7, 14, and 21. The experimental group is injected with NY-ESO-1-mRNA@MPNP, and the control group is injected with metal-polyphenol complex particles (MPNP) without loaded mRNA. The dose of each injection is 100 μL, and the mRNA@MPNP preparation in the experimental group contains 30 mg of mRNA. Collect the blood of the mice on day 28 after the first injection, separate the serum and dilute it in gradients, and detect the total IgG antibody against NY-ESO-1 in the mice by ELISA. The results are as Figure 1-4 shown.

[0329] Method for detecting the total IgG antibody against NY-ESO-1 in mice:

[0330] Reagent Preparation for ELISA Method:

[0331] 1. Coating Buffer: Weigh 8.4 g of NaHCO 3 and dissolve it in 1 L of distilled water (DDW). After all the solids are dissolved, adjust the pH of the entire solution to 9.6 using 1 M NaOH solution. Store the prepared coating buffer at 4°C for later use.

[0332] 2. Wash Buffer: Add 0.5 mL of Tween - 20 to 1 L of 0.01 M PBS solution, mix well, and store at room temperature.

[0333] 3. Blocking Buffer: Weigh 20 g of BSA and add it to 1 L of 0.01 M PBS solution. Ultrasonically treat the undissolved BSA powder in the solution. After all the solids in the solution are dissolved and the solution turns light yellow, store it in a 4°C refrigerator for later use.

[0334] 4. Antibody Diluent: Weigh 2.5 g of BSA and dissolve it in 250 mL of 0.01 M PBS solution. After the solids are completely dissolved, add 1.25 mL of Tween - 20 to it, mix well, and store at 4°C for later use.

[0335] 5. Chromogenic Solution: 0.1 M Citric Acid: Add 19.2 g of citric acid to DDW to make 1000 mL (A); 0.2 M Disodium Hydrogen Phosphate: Add 28.4 g of anhydrous disodium hydrogen phosphate to DDW to make 1000 mL (B); 24.3 mL of 0.1 M citric acid solution (A), 25.7 mL of 0.2 M phosphate buffer solution (B), and add 50 mL of DDW. Add 50 mg of OPD (o - phenylenediamine) immediately before use, and then add 0.15 mL of 30% H 2 O 2 0.15 mL.

[0336] 6. Stop Solution: 2 M H 2 SO 4 : Add 55.5 mL of concentrated sulfuric acid to DDW to make 500 mL.

[0337] Determination of Antibody Titer in Mouse Serum by ELISA Method:

[0338] 1. Coating: Dilute NY - ESO - 1 antigen with coating buffer to 1 μg / mL, add it to a 96 - well plate, 50 μL / well, and coat overnight at 4°C.

[0339] 2. Blocking: Discard the coating buffer in the wells by centrifugation, wash 3 times with blocking buffer, 5 min each time and then discard the liquid by centrifugation. Add 150 μL of blocking buffer to each well and incubate at 37°C for 2 h.

[0340] 3. Drying: Discard the blocking buffer by centrifugation, incubate at 37°C for 1 - 2 h until all the liquid at the bottom of the well plate is dry.

[0341] 4. Immunity: The serum sample was initially diluted 1:1000 with antibody diluent, and then serially diluted 1:2 successively. The diluted serum sample was added to a sealed 96-well plate, 100 μL / well, and incubated at 37 °C for 2 h; the liquid in the well plate was spun dry, washing solution was added, 300 μL / well, and slowly shaken for 40 s, and this step was repeated three times; biotinylated goat anti-mouse IgG antibody diluted 1:1000 was added to the well plate, 100 μL / well, and incubated at 37 °C for 1 h; the liquid in the well plate was spun dry, washing solution was added, and the above-mentioned plate washing steps were repeated; freshly prepared streptavidin-labeled horseradish peroxidase HRP working solution was added, 100 μL / well, and incubated at 37 °C for 1 h; the liquid in the well plate was spun dry, washing solution was added, and the above-mentioned plate washing steps were repeated; chromogenic solution was added under light-proof conditions, 100 μL / well, and after reacting at room temperature for 5 min, stop solution was added to terminate chromogenesis, 50 μL / well; the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0342] On the 28th day after administration of RBD-mRNA@MPNP, the spleens of normal mice were collected, and single-cell suspensions were prepared under sterile conditions. They were plated in cell well plates at 100,000 spleen cells / well, and RBD protein with a final concentration of 10 mg / mL was added and cultured for 48 h. The supernatant was removed by centrifugation, and the expression levels of IFN-γ, IL-2, and IL-4 were measured using an ELISA kit. The results are as Figure 1-5 shown.

[0343] On the 28th day after administration of NY-ESO-1-mRNA@MPBP, the spleens of normal mice were collected, and single-cell suspensions were prepared under sterile conditions. They were plated in cell well plates at 100,000 spleen cells / well, and NY-ESO-1 protein with a final concentration of 10 mg / mL was added and cultured for 48 h. The supernatant was removed by centrifugation, and the expression levels of IFN-γ, IL-2, and TNF-α were measured using an ELISA kit. The results are as Figure 1-6 shown.

[0344] Result analysis: As Figure 1-1 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; as Figure 1-2 shown, the RBD protein encoded by RBD-mRNA encapsulated by MPNP was 166 ng / mL in the supernatant of 293T cells, while the content of RBD protein 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 intracellularly. As Figure 1-3, as shown in Results 1-4, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP can effectively induce humoral immunity in mice, generating high levels of antigen-specific binding antibodies. Among them, the IgG antibody titer in the mice treated with RBD-mRNA@MPNP reached 84363.4; the IgG antibody titer in the mice treated with NY-ESO-1-mRNA@MPNP reached 4283.56. As Figure 1-5 , as shown in 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 increased the expression levels of cytokines IFN-γ, IL-2, and IL-4 to 271.8 pg / mL, 269.6 pg / mL, and 75.8 pg / mL respectively; NY-ESO-1-mRNA@MPNP increased the expression levels of cytokines IFN-γ, IL-2, and TNF-α to 76.38 pg / mL, 74.56 pg / mL, and 69.31 pg / mL respectively. The results suggest that the drug-metal-polyphenol complex particles (mRNA@MPNP) can encapsulate and deliver any mRNA, thereby promoting the expression of the target protein (antigen), and then effectively inducing humoral immunity and cellular immunity in mice, generating high levels of antigen-specific binding antibodies and cytokines, and playing the role of anti-SARS-CoV-2 mRNA vaccine and anti-tumor mRNA vaccine.

[0345] Example 2.5.2. The metal ion is Al 3+ Preparation and effect characterization of mRNA@MPNP

[0346] The difference between this example and Example 2.5.1 is that the metal ion Fe in Example 2.5.1 3+ is replaced with Al 3 + .

[0347] Result analysis: As Figure 1-7 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; as Figure 1-8 shown, the RBD protein encoded by RBD-mRNA encapsulated by MPNP 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. As Figure 1-9, as shown in 1-10, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP can effectively induce the humoral immunity of mice and produce high levels of antigen-specific binding antibodies. Among them, the IgG antibody titer in the mice treated with RBD-mRNA@MPNP reached 94828.6; the IgG antibody titer in the mice treated with NY-ESO-1-mRNA@MPNP reached 5848.02. As Figure 1-11 , as shown in 1-12, both RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP can effectively induce the cellular immunity of mice, that is, activate immune cells and produce a large number of cytokines. Among them, the expression levels of cytokines IFN-γ, IL-2, and IL-4 in RBD-mRNA@MPNP were 306.2 pg / mL, 289.6 pg / mL, and 88.2 pg / mL respectively; the expression levels of cytokines IFN-γ, IL-2, and TNF-α in NY-ESO-1-mRNA@MPNP were 91.88 pg / mL, 85.32 pg / mL, and 80.22 pg / mL respectively. The results suggest that mRNA@MPNP can encapsulate and deliver any mRNA, thereby promoting the expression of the target protein (antigen), and then effectively inducing the humoral immunity and cellular immunity of mice, producing high levels of antigen-specific binding antibodies and cytokines, and playing the role of an anti-SARS-CoV-2 mRNA vaccine and an anti-tumor mRNA vaccine.

[0348] Example 2.6. Preparation and effect of siRNA-metal-polyphenol complex particles (siRNA@MPNP)

[0349] Example 2.6.1. When the metal ion is Fe 3+ Preparation and effect of siRNA-metal-polyphenol complex particles (siRNA-loaded metal-chelated phospholipid complex nanoparticles, siRNA@MPNP)

[0350] Replace the mRNA in Example 2 with siRNA, and prepare three siRNA@MPNP containing different siRNAs respectively according to the method of Example 2. The genes, sequences and their corresponding random control sequences targeted by the three different siRNAs are as follows: ① The sequence of siRNA targeting the Bcl-2 gene (Bcl-2-siRNA) is SEQ ID NO.4 (antisense strand) and SEQ ID No.21 (sense strand) (19bp), and its random control sequence is SEQ ID NO.5 (antisense strand) and SEQ ID No.22 (sense strand) (19bp); ② The sequence of siRNA targeting the PLK1 gene (PLK1-siRNA) is SEQ ID NO.6 (antisense strand) and SEQ ID No.23 (sense strand) (21bp), and its random control sequence is SEQ ID NO.7 (antisense strand) and SEQ ID No.24 (sense strand) (19bp); ③ The sequence of siRNA targeting the Gal-1 gene (Gal-1-siRNA) is SEQ ID NO.8 (19bp); its random control sequence is SEQ ID NO.9 (19bp). The preparation process of the remaining siRNA@MPNP is the same as that in Example 2.

[0351] The sequence of Bcl-2-siRNA is as follows:

[0352] Antisense: 5′-CAGCUUAUAAUGGAUGUAC-3′ (SEQ ID No.4);

[0353] Sense: 5′-GUACAUCCAUUAUAAGCUG-3′ (SEQ ID No.21) (19bp).

[0354] The random control sequence of Bcl-2-siRNA is as follows:

[0355] Antisense: 5’-ACGUGACACGUUCGGAGAA-3’ (SEQ ID No.5);

[0356] Sense: 5’-UUCUCCGAACGUGUCACGU-3’ (SEQ ID No.22) (19bp).

[0357] The sequence of PLK1-siRNA is as follows:

[0358] Antisense: 5’-UAAGGAGGGUGAUCUUCUUCA-3’ (SEQ ID No.6);

[0359] Sense: 5’-UGAAGAAGAUCACCCUCCUUA-3’ (SEQ ID No.23) (21bp).

[0360] The random control sequence of PLK1-siRNA is as follows:

[0361] Antisense: 5’-CUUACGCUGAGUACUUCGA-3’ (SEQ ID No.7);

[0362] Sense: 5’-UCGAAGUACUCAGCGUAAG-3’ (SEQ ID No.24) (19bp).

[0363] The sequence of Gal-1-siRNA is as follows:

[0364] 5’-GCUGCCAGAUGGAUACGAA-3’ (SEQ ID No.8) (19bp).

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

[0366] 5’-GGAAAUCCCCCAACAGUGA-3’ (SEQ ID No.9) (19bp).

[0367] Cell culture method: U251 human glioblastoma cells grow in monolayers in high-glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mmol l-glutamine (Bio Industries) medium, and are cultured at 37 °C and 5% CO 2 and subcultured twice a week.

[0368] U251 cells were seeded at a cell density of 1×10 6 per well in 6-well plates. After about 24 h, each well of cells was incubated with siRNA@MPNP containing the above siRNA (where the concentration of siRNA was 2 μg / mL) for 72 h, then the cells were collected, total cellular RNA was extracted, and the mRNA expression levels of the target genes (Bcl-2, PLK1, Gal-1) were detected by RT-PCR technology to statistically analyze the ability of siRNA@MPNP to silence the target genes in cells.

[0369] The specific procedure of RT-PCR:

[0370] Extraction of total RNA: discard the culture medium from the six-well plate, rinse 3 times with PBS buffer, and add 1mL Trizol to each well to lyse the cells. Add 200μL chloroform, shake well, let stand at room temperature for 10min, centrifuge at 13000rpm 4℃ for 15min, and obtain a three-phase liquid with RNA dissolved in the upper aqueous phase. Pipette the upper aqueous phase into a new enzyme-free 1.5ml centrifuge tube, add 500μL isopropanol, let stand at room temperature for 10min, centrifuge at 13000rpm 4℃ for 15min to obtain RNA precipitation. Remove the supernatant, add 1mL of 75% (v / v) ethanol newly prepared with RNase-free water to each tube, blow carefully to blow up the white RNA precipitate at the bottom of the tube, centrifuge at 7500rpm 4℃ for 10min, remove the supernatant, and try to dry the liquid 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 RNA.

[0371] cDNA reverse transcription: using TaKa RaPrime Script TM RT reagent Kit with g DNA Eraser Kit reverse transcribes RNA into cDNA, and removes genomic DNA (gDNA) before the reverse transcription step to make the results more accurate and reliable. Prepare the total RNA reverse transcription reaction system on ice: 1μL Prime Script RT Enzyme MixⅠ, 1μL RT Primer Mix, 4μL 5× Prime Script Buffer2, 4μL RNase Free dH 2 After the reaction mixture is prepared, it is placed at 37°C for 15 min, then placed at 85°C for 5 sec to terminate the reaction, and then stored at 4°C for later use.

[0372] RT-PCR operation: This detection method is SYBR Green dye method, and no probe is required. Specifically, Real-time PCR reaction is performed based on the cDNA of different samples as templates. Prepare the reaction solution on ice: 5μL SYBR Premix DimerEraser (2×), 0.3μL PCR Forward primer (10μM), 0.3μL PCR Reverse primer (10μM), 0.2μL ROXReference DyeⅡ (50×), 1μL cDNA template obtained in the previous step and 3.2μL dH 2O. Samples were added to the wells of the orifice plate at 10 μL per well. After sample addition, centrifugation (1000 rpm, 5 min) was performed to eliminate liquid wall adhesion and air bubbles in the reaction solution. Real-time PCR reactions were detected using an ABI ViiA7 real-time fluorescence quantitative PCR instrument. The reaction program was 95 °C, 30 sec (1 cycle) → 95 °C, 5 sec; 55 °C, 30 sec; 72 °C, 30 sec (40 cycles) → 60 °C - 95 °C, 2 min (1 cycle). The experiment was repeated three times, and the average Ct value of each group was obtained to calculate the expression difference multiple between the experimental group and the control group. The reference gene was GAPDH. The RT-PCR primers were as follows: ① Bcl-2 primers: forward: 5’-AGGATTGTGGCCTTCTTTGAG-3’, reverse: 5’-AGACAGCCAGGAGAAATC AAAC-3’; ② PLK1 primers: forward: 5’-ACCAGCACGTCGTAGGATTC-3’, reverse: 5’-CAAGCAATTTGCCGTAGG-3’; ③ Gal-1 primers: forward: 5’-CAATCATGGCCTGTGGTCTG-3’, reverse: 5’-GTG TAGGCACAGGTTGTTGCTG-3’. ④ GAPDH primers: forward: 5’-TCAGGGGTTTCACATTTGGCA-3’, reverse: 5’-GG AGCGGAAAACCA-3’. The expression levels of each target gene were represented by the RQ value (2 -ΔΔCT ) as follows. The formula was as follows:

[0373] Fold Change = 2 –ΔΔCt

[0374] where ΔΔCt = ΔCt 实验组 –ΔCt 对照组 , ΔCt = Ct 目的基因 -Ct 内参基因

[0375] Calculation method for gene silencing efficiency: 100% - expression level of the experimental group / expression level of the control group.

[0376] Result analysis: As Figure 1-13, as shown in Figures 1-14 and 1-15 (wherein, scr siRNA is a random control sequence), all three drug-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP, PLK1-siRNA@MPNP, Gal-1-siRNA@MPNP) can significantly interfere with their corresponding target genes. The inhibition rate of Bcl-2-siRNA@MPNP on the target gene Bcl-2 reaches 67%; the inhibition rate of PLK1-siRNA@MPNP on the target gene PLK1 reaches 87%; the inhibition rate of Gal-1-siRNA@MPNP on the target gene Gal-1 reaches 64%. The results suggest that siRNA@MPNP can carry any siRNA for the intervention treatment of target genes and play the role of siRNA-loaded drugs, vaccines or other products.

[0377] Example 2.6.2. The metal ion is Al 3+ Preparation and effect of siRNA-metal-polyphenol complex nanoparticles (siRNA-loaded metal-chelated phospholipid complex nanoparticles, siRNA@MPNP) when

[0378] The difference between this example and Example 2.6.1 is that the metal ion Fe in Example 3.6.1 3+ is replaced by Al 3 + .

[0379] Result analysis: As Figure 1-16 , shown in Figures 1-17 and 1-18, all three siRNA@MPNP can significantly interfere with their corresponding target genes. The inhibition rate of Bcl-2-siRNA@MPNP on the target gene Bcl-2 reaches 72%; the inhibition rate of PLK1-siRNA@MPNP on the target gene PLK1 reaches 88.07%; the inhibition rate of Gal-1-siRNA@MPNP on the target gene Gal-1 reaches 70.11%. The results suggest that siRNA@MPNP can carry any siRNA for the intervention treatment of target genes and play the role of siRNA-loaded drugs, vaccines or other products.

[0380] Example 2.7. Preparation and effect of ASO-metal-polyphenol complex particles (ASO@MPNP)

[0381] Example 2.7.1. The metal ion is Fe 3+ Preparation and effect of ASO-metal-polyphenol complex nanoparticles (ASO-loaded metal-chelated phospholipid complex nanoparticles, ASO@MPNP) when

[0382] Replace the mRNA in Example 2 with ASO, and prepare three kinds of drug-metal-polyphenol complex particles (ASO@MPNP) containing different ASOs respectively according to the method of Example 2. The genes, sequences and their corresponding random control sequences targeted by the three different ASOs are as follows: ① The sequence of ASO targeting STAT3 gene (STAT3-ASO) is SEQ ID NO.10 (17nt), and its random control sequence is SEQ ID NO.11 (18nt); ② The sequence of ASO targeting α-syn gene (α-syn-ASO) is SEQ ID NO.12 (16nt), and its random control sequence is SEQ ID NO.13 (16nt); ③ The sequence of ASO targeting Bcl-2 gene (Bcl-2-ASO) is SEQ ID NO.14 (18nt), and its random control sequence is SEQ ID NO.15 (20nt). The preparation process of the remaining drug (ASO)-metal-polyphenol complex particles is the same as that in Example 2. Incubate different ASO@MPNP with different cells: ASO@MPNP targeting STAT3 gene is incubated with U251 human glioblastoma cells; ASO@MPNP targeting α-syn gene is incubated with SH-SY5Y human neuroblastoma cells; ASO@MPNP targeting Bcl-2 gene is incubated with Daudi human lymphoma cells. At a cell density of 1×10 6 After inoculating in a 6-well plate for about 24 h, each well of cells is incubated with the drug-metal-polyphenol complex particles (ASO@MPNP) containing the above ASO (the concentration of ASO is 1 μg / mL) for 48 hours, then the cells are collected, total cellular RNA is extracted, and the mRNA expression levels of the target genes (STAT3, α-syn, Bcl-2) are detected by RT-PCR technology respectively to calculate the ability of ASO@MPNP to silence the target genes of cells.

[0383] The sequence of SEQ ID No.10 (the sequence of STAT3-ASO) is as follows:

[0384] 5’-GCTCCAGCATCTGCTTC-3’ (17nt).

[0385] The sequence of SEQ ID No.11 (the random control sequence of STAT3-ASO) is as follows:

[0386] 5’-GAAGCAGCAGATGCTGGA-3’ (18nt).

[0387] The sequence of SEQ ID No.12 (the sequence of α-syn-ASO) is as follows:

[0388] 5’-GCTCCCTCCACTGTCT-3’ (16 nt).

[0389] The sequence of SEQ ID No.13 (random control sequence of α-syn-ASO) is as follows:

[0390] 5’-ACTCCCGAACCTGTCT-3’ (16 nt).

[0391] The sequence of SEQ ID No.14 (sequence of Bcl-2-ASO) is as follows:

[0392] 5’-TCTCCCAGCGTGCGCCAT-3’ (18 nt).

[0393] The sequence of SEQ ID No.15 (random control sequence of Bcl-2-ASO) is as follows:

[0394] 5’-CAGCGTGCGCCATCCTTCCC-3’ (20 nt).

[0395] Cell culture: ① U251 human glioblastoma cells grow in a monolayer in a medium of high-glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mmol l-glutamine (Bio Industries), and are cultured at 37 °C and 5% CO 2 conditions, with subculture twice a week; ② SH-SY5Y human neuroblastoma cells grow in a monolayer in a medium of high-glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mmol l-glutamine (Bio Industries), and are cultured at 37 °C and 5% CO 2 conditions, with subculture twice a week; ③ Daudi human lymphoma cells grow in a medium of RPMI1640 + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mmol l-glutamine (Bio Industries), and are cultured at 37 °C and 5% CO 2 conditions, with subculture twice a week.

[0396] Specific procedure of RT-PCR:

[0397] Extraction of total RNA: discard the culture medium from the six-well plate, rinse 3 times with PBS buffer, and add 1mL Trizol to each well to lyse the cells. Add 200μL chloroform, shake well, let stand at room temperature for 10min, centrifuge at 13000rpm 4℃ for 15min, and obtain a three-phase liquid with RNA dissolved in the upper aqueous phase. Pipette the upper aqueous phase into a new enzyme-free 1.5ml centrifuge tube, add 500μL isopropanol, let stand at room temperature for 10min, centrifuge at 13000rpm 4℃ for 15min to obtain RNA precipitation. Remove the supernatant, add 1mL of 75% (v / v) ethanol newly prepared with RNase-free water to each tube, blow carefully to blow up the white RNA precipitate at the bottom of the tube, centrifuge at 7500rpm 4℃ for 10min, remove the supernatant, and try to dry the liquid 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.

[0398] cDNA reverse transcription: using TaKa RaPrime Script TM RT reagent Kit with g DNA Eraser Kit reverse transcribes RNA into cDNA, and removes genomic DNA (gDNA) before the reverse transcription step to make the results more accurate and reliable. Prepare the total RNA reverse transcription reaction system on ice: 1μL Prime Script RT Enzyme MixⅠ, 1μL RT Primer Mix, 4μL 5× Prime Script Buffer 2, 4μL RNase Free dH 2 After the reaction mixture is prepared, it is placed at 37°C for 15 min, then placed at 85°C for 5 sec to terminate the reaction, and then stored at 4°C for later use.

[0399] RT-PCR operation: This detection method is SYBR Green dye method, and no probe is required. Specifically, Real-time PCR reaction is performed based on the cDNA of different samples as templates. Prepare the reaction solution on ice: 5μL SYBR Premix DimerEraser (2×), 0.3μL PCR Forward primer (10μM), 0.3μL PCR Reverse primer (10μM), 0.2μL ROXReference DyeⅡ (50×), 1μL cDNA template obtained in the previous step and 3.2μL dH 2O. Samples were added into the orifice plate at 10 μL per well. After sample addition, centrifugation (1000 rpm, 5 min) was performed to eliminate liquid wall adhesion and air bubbles in the reaction solution. Real-time PCR reaction detection was carried out using an ABI ViiA7 real-time fluorescence quantitative PCR instrument. The reaction program was 95°C, 30 sec (1 cycle) → 95°C, 5 sec; 55°C, 30 sec; 72°C, 30 sec (40 cycles) → 60°C - 95°C, 2 min (1 cycle). The experiment was repeated three times, and the average value was taken to obtain the Ct value of each group, and the expression difference multiple between the experimental group and the control group was calculated. The reference gene was GAPDH. The RT-PCR primer sequences were as follows: ① STAT3 primers: forward: 5’-TGATCACCTTTGAGACCGAGG-3’, reverse: 5’-GATCACCACAACTGG CAA GG-3’; ② α-syn primers: forward: 5’-TGACGGGTGTGACAGCAGTAG-3’, reverse: 5’-CAGTGGCTGCTGCAATG-3’; ③ Bcl-2 primers: forward: 5’-AGGATT GTGGCCTTCTTTGAG-3’, reverse: 5’-AGACAGCCAGGAGAAATCAAAC-3’ ④ GAPDH primers: forward: 5’-TCAGGGG TTTCACATTTGGCA-3’, reverse: 5’-GGAGCGGAAAACCA-3’. The expression level of each target gene was represented by the RQ value (2 -ΔΔCT ) as follows:

[0400] Fold Change = 2 –ΔΔCt

[0401] where ΔΔCt = ΔCt 实验组 –ΔCt 对照组 , ΔCt = Ct 目的基因 -Ct 内参基因

[0402] Calculation method of gene silencing efficiency: 100% - expression level of the experimental group / expression level of the control group.

[0403] Result analysis: As Figure 1-19, as shown in 1-20, 1-21 (where scrASO is the random control sequence), all three ASO@MPNP can significantly interfere with their corresponding target genes. Among them, the inhibition rate of STAT3-ASO@MPNP on the target gene STAT3 reaches 72%; the inhibition rate of α-syn-ASO@MPNP on the target gene α-syn reaches 78%; the inhibition rate of Bcl-2-ASO@MPNP on the target gene Bcl-2 reaches 62%. The results suggest that the drug-metal-polyphenol complex particles (ASO@MPNP) can carry any ASO for the intervention treatment of target genes and play the role of carrying ASO drugs, vaccines or other products.

[0404] Example 2.7.2. The metal ion is Al 3+ Preparation and effects of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPNP) when

[0405] The difference between this example and Example 2.7.1 is that the metal ion Fe in Example 2.7.2 3+ is replaced by Al 3 + .

[0406] Result analysis: As Figure 1-22 , 1-23, 1-24 shown, all three ASO@MPNP can significantly interfere with their corresponding target genes. Among them, the inhibition rate of STAT3-ASO@MPNP on the target gene STAT3 reaches 75.4%; the inhibition rate of α-syn-ASO@MPNP on the target gene α-syn reaches 80.87%; the inhibition rate of Bcl-2-ASO@MPNP on the target gene Bcl-2 reaches 67.91%. The results suggest that ASO@MPNP can carry any ASO for the intervention treatment of target genes and play the role of carrying ASO drugs, vaccines or other products.

[0407] Example 2.8. Preparation of drug (different types of nucleic acid)-metal-polyphenol complex particles and their effects

[0408] Example 2.8.1. Preparation of drug (different types of nucleic acid)-metal-polyphenol complex particles and their effects when the metal ion is Fe 3+

[0409] ​Replace the mRNA in Example 2 with double-stranded RNA (siRNA), single-stranded DNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively. The sequences of different types of nucleic acids are as follows: ① The sequence of double-stranded RNA (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 single-stranded DNA (STAT3-ASO) is SEQ IDNO.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 strand) and SEQ ID NO.25 (sense strand) (22 bp) (the 3' end of its sequence is labeled with the fluorescent probe Cy3); ⑤ The sequence of single-stranded DNA (ssDNA) is SEQ ID NO.18 (22 nt) (the 3' end of its sequence is labeled with the fluorescent probe Cy3). Refer to the method of Example 2 to prepare drug-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP, STAT3-ASO@MPNP, S-mRNA@MPNP, dsDNA@MPNP, ssDNA@MPNP) encapsulating the above different types of nucleic acids, and the preparation process of the remaining drug-lipid particles is the same as that of Example 2.

[0410] U251 cells were seeded in a 6-well plate at a cell density of 1×10 6 After about 24 h, each well of cells was incubated with siRNA@MPNP (where the concentration of siRNA was 2 μg / mL) or ASO@MPNP (where the concentration of ASO was 2 μg / mL) for 72 hours, and then the cells were collected. Total cellular RNA was extracted, and the mRNA expression levels of the target genes (Bcl-2, STAT3) were detected by RT-PCR technology, and the ability of siRNA@MPNP or ASO@MPNP to silence the target genes in cells was calculated. The results are as shown in Figure 1-13 Example 2.6 and Figure 1-19 Example 2.7.

[0411] Incubate S-mRNA@MPNP with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA). Incubate the control group with MPNP. After 24 h, centrifuge and freeze the supernatant at -20 °C for later use. Resuspend the cell pellet in 100 μL of PBS buffer solution, perform 2 cycles of freeze-thawing and sonicate for 10 min, then centrifuge and take the supernatant. Use a commercially available ELISA kit for detecting the SARS-CoV-2 S protein to detect the expression levels of the S protein in both the cell supernatant and cell lysate. The results are as Figure 1-25 shown.

[0412] Incubate ds-DNA@MPNP with A549 lung cancer cells at a concentration of 100 nM (the concentration of the contained DNA) for 2 h, then remove the drug-lipid particles. Wash the cells twice with PBS, stain the cell nuclei with Hochest33342 dye for 3 min, then remove the dye. Wash the cells twice with PBS, observe the cells using a high-content imaging system, and calculate the transfection efficiency of the drug-lipid particles with DNA. The results are shown in Figure 1-26 .

[0413] Incubate ss-DNA@MPNP with HT22 mouse hippocampal neurons at a concentration of 200 nM (the concentration of the contained DNA) for 2 h, then remove the drug-lipid particles. Wash the cells twice with PBS, observe the cells using a high-content imaging system, and calculate the transfection efficiency of the drug-lipid particles with DNA. The results are shown in Figure 1-26 .

[0414] The culture method of human glioblastoma U251 cells is the same as that in Example 2.6.

[0415] The culture method of 293T cells is the same as that in Example 2.5.

[0416] The culture method of HT22 mouse hippocampal neurons: Culture in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37 °C, 5% CO 2 conditions.

[0417] The method of RT-PCR is the same as that in Example 2.6.

[0418] ELISA for detecting the expression level of the S protein: Replace the "anti-RBD antibody working solution" in the method of ELISA for detecting RBD in Example 2.5 with the "anti-S protein antibody working solution", and the remaining steps are the same as those in Example 2.5.

[0419] The calculation method of gene silencing efficiency is the same as that in Example 2.6.

[0420] Calculation method of transfection efficiency: Randomly select 3 - 5 fields of view with a high - content imaging system to obtain the cell morphology under ordinary light source, the fluorescence signal when the excitation / emission light is 550nm / 570nm (excitation light of the fluorescent dye Cy3 labeled with DNA) in the same field of view, and the fluorescence signal when the excitation / emission light is 352nm / 461nm (excitation light of the fluorescent dye Hoechst33342 labeled with the nucleus). Calculate the proportion of the number of cells with Cy3 fluorescence signal in the randomly selected fields of view to the number of cells with Hoechst33342 fluorescence signal in the same fields of view, which is the transfection efficiency.

[0421] Result analysis: As in Example 2.6 Figure 1-13 shown, the inhibition rate of the drug (double - stranded RNA) - metal - polyphenol complex particles (Bcl - 2 - siRNA@MPNP) on the target gene Bcl - 2 reached 67%; as in Example 2.7 Figure 1-19 the inhibition rate of the drug (single - stranded DNA) - metal - polyphenol complex particles (STAT3 - ASO@MPNP) on the target gene STAT3 reached 72%; as Figure 1-25 shown, the expression level of S protein in the supernatant of 293T cells transfected with the drug (single - stranded RNA) - metal - polyphenol complex particles (S - mRNA@MPNP) was 134 ng / mL, while the content of S protein in the supernatant of 293T cells transfected with the empty vector MPNP was 0; the transfection efficiency of the drug (double - stranded DNA) - metal - polyphenol complex particles (dsDNA@MPNP) for double - stranded DNA into cells was 100% ( Figure 1-26 ); the transfection efficiency of the drug (single - stranded DNA) - metal - polyphenol complex particles (ssDNA@MPNP) for single - stranded DNA into cells was 100% ( Figure 1-26 ). The results suggest that the drug - metal - polyphenol complex particles can encapsulate any nucleic acid (double - stranded RNA, single - stranded RNA, double - stranded DNA, single - stranded DNA) and realize its function, where the length of the nucleic acid varies from 16 - 3822 nt.

[0422] Example 2.8.2. Preparation of drug (different types of nucleic acid) - metal - polyphenol complex particles with metal ion Al 3+ and its effects

[0423] The difference between this example and Example 2.8.1 is that the metal ion Fe in Example 2.8.1 3+ is replaced by Al 3 + .

[0424] Result analysis: As in Example 3.6.2 Figure 1-16As shown, the inhibition rate of the drug (double-stranded RNA)-metal-polyphenol complex particles (Bcl-2-siRNA@MPNP) against the target gene Bcl-2 reached 72%; as in Example 3.7.2 Figure 1-22 The inhibition rate of the drug (single-stranded DNA)-metal-polyphenol complex particles (STAT3-ASO@MPNP) against the target gene STAT3 reached 75.4%; as Figure 1-27 shown, the S protein expression level in the supernatant of 293T cells transfected with the 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 the empty vector MPNP was 0; the efficiency of the drug (double-stranded DNA)-metal-polyphenol complex particles (dsDNA@MPNP) in transfecting double-stranded DNA into cells was 100% ( Figure 1-28 ); the efficiency of the drug (single-stranded DNA)-metal-polyphenol complex particles (ssDNA@MPNP) in transfecting single-stranded DNA into cells was 100% ( Figure 1-28 ). The results suggest that the drug-metal-polyphenol complex particles can encapsulate any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and achieve its function, where the length of the nucleic acid varies from 16 - 3822 nt.

[0425] Experimental Example 2. Performance Characterization of Drug-Metal-Polyphenol Complex Particles

[0426] Example 3. Synthesis and Characterization of Metal-Polyphenol Complexes

[0427] Example 3.1. Synthesis and Characterization of Metal-Polyphenol Complexes When the Metal Ion is Fe 3+

[0428] The connection characterization of curcumin and Fe 3+ was by spectrophotometry: as Figure 2-1 shown, after curcumin was combined with Fe 3+ , its maximum absorption wavelength shifted from 420 nm to 372 nm, and the conjugated structure of the metal-polyphenol complex changed, proving that curcumin was successfully complexed with Fe 3+ .

[0429] Example 3.2. Synthesis and Characterization of Metal-Polyphenol Complexes When the Metal Ion is Al 3+

[0430] The connection characterization of curcumin and Al 3+ was by spectrophotometry: as Figure 2-2 shown, after curcumin was combined with Al 3+ , its maximum absorption wavelength shifted from 420 nm to 433 nm, and the conjugated structure of the metal-polyphenol complex changed, proving that curcumin was successfully complexed with Al​​3+ Complexation

[0431] Example 4. Characterization of the detachment of Fe 3+ from metal-polyphenol complexes under low pH conditions

[0432] Curcumin in the metal-polyphenol complex binds to Fe through coordination bonds 3+ , and under the low pH conditions of lysosomes, the coordination bond between curcumin and Fe 3+ will be protonated (absorbing hydrogen ions) and broken. To prove that the Fe 3+ in the metal-polyphenol complex is indeed detached from the lipid complex through the above mechanism, we designed the following experiment: Observe the color of the metal-polyphenol complex under physiological pH (pH = 7.4) and lysosomal low pH (pH = 5.0) conditions respectively. As Figure 2-3 shown, the metal-polyphenol complex changed from brownish red to bright yellow under the lysosomal low pH (pH = 5.0) condition, indicating that Fe 3+ has detached from the complex. The results suggest that: under the low pH conditions of lysosomes, Fe 3+ can be detached from the metal-polyphenol complex

[0433] The principle of the detachment of Fe 3+ from the metal-polyphenol complex under low pH conditions is that the coordination bond between curcumin and Fe 3+ is protonated under low pH conditions (pH = 5.0), that is, curcumin will bind a large number of protons (H + ) from the solution, resulting in the breakage of the coordination bond between Fe 3+ and curcumin, thereby separating Fe 3+ from curcumin, and ultimately causing Fe 3+ to be separated from the metal-polyphenol complex ( Figure 2-3 )

[0434] Example 5. Efficiency of drug-metal-polyphenol complex particles MPNP loaded with nucleic acids (siRNA and mRNA) when the metal ion is Fe 3+ or Al 3+ and its comparison with LNP

[0435] The mRNA in Example 2 was replaced with siRNA targeting the Bcl-2 gene (SEQ ID NO.4, 21bp) and mRNA encoding the receptor binding domain (RBD) of the S1 subunit of the novel coronavirus (SEQ ID NO.2, 669nt), respectively, to prepare drug-metal-polyphenol complex particles encapsulating nucleic acids, siRNA@MPNP and mRNA@MPNP. The preparation process of the remaining drug-metal-polyphenol complex particles was the same as that in Example 2.

[0436] siRNA@LNP and mRNA@LNP were prepared with the same drug loading amounts as Bcl-2-siRNA@MPNP in Example 2.6 and RBD-mRNA@MPNP in Example 2.5, respectively. The specific method was as follows: an organic phase solution was prepared according to the Onpattro lipid nanoparticle formulation, that is, the ionizable lipid ALC0315, DSPE-PEG2000, DSPC, and cholesterol were dissolved in ethanol at a molar ratio of 50%:1.5%:10%:38.5%. Bcl-siRNA or RBD-mRNA was added to the aqueous phase (0.1M, pH = 4.0 acetic acid-sodium acetate buffer solution). Among them, the ratio of amino lipid to phosphate-containing nucleotide (N / P) was 6:1, and at the same time, it was ensured that the nucleic acid drug loading amount was the same as that of the above siRNA@MPNP and mRNA@MPNP. The aqueous phase and the organic phase were rapidly mixed at a volume ratio of 3:1 and a flow rate of 14 mL / min. After mixing, it was diluted tenfold with pH 7.4 Tris-Hcl buffer solution, and the mixture was concentrated to one-tenth using a 100 kDa ultrafiltration tube. After repeating the dilution and concentration operations 3 times, the ethanol concentration in the mixture was reduced to less than 0.0005%, and the solution pH value was increased to the normal pH value (7.2 - 7.4) of the Tris-Hcl buffer solution, thus obtaining siRNA@LNP and mRNA@LNP, respectively.

[0437] The encapsulation rates of siRNA@MPNP, mRNA@MPNP, siRNA@LNP, and mRNA@LNP for nucleic acids (siRNA and mRNA) were detected by agarose gel electrophoresis. The method for measuring the encapsulation rate was as follows: the nucleic acid (siRNA and mRNA) feeding amount of each group of lipid nanoparticles was set at 10 μg / mL, and the mass ratio of lipid to nucleic acid was 20:1 when the metal in the drug-metal-polyphenol complex was Fe 3+ and 20:1 when the metal in the drug-metal-polyphenol complex was Al 3+At a mass ratio of 18:1, dissolve the nucleic acid in a Tris-HCl buffer solution with pH 5.0 as the positive control group, and the negative control is the PBS buffer solution without nucleic acid. The concentration of the agarose gel is 1.5%. At this time, the pores of the gel only allow free nucleic acids to pass through and do not allow lipid nanoparticles to pass through. Stop electrophoresis when the free nucleic acid band can be clearly resolved to prevent nucleic acid degradation due to too long electrophoresis time. Use Image J software to statistically analyze the gray values of free nucleic acids in different groups. The positive control group is set as 100%. The ratio of free nucleic acids in each group relative to the positive control is the relative amount of free nucleic acids. Then the encapsulation efficiency of each group is (100 - relative amount of free nucleic acids)%.

[0438] Result analysis: As Figure 2-4 shown, the efficiencies of MPNP(Fe 3+ ) for encapsulating siRNA and mRNA are 87.78% and 83.17% respectively; the efficiencies of MPNP(Al 3+ ) for encapsulating siRNA and mRNA are 89.65% and 82.53% respectively; the efficiencies of LNP for encapsulating siRNA and mRNA are 87.32% and 79.94% respectively. The results suggest that there is no significant difference in the efficiency of MPNP and LNP for encapsulating nucleic acids.

[0439] Example 6. Nucleic acid lysosomal escape ability of the drug-metal-polyphenol complex particles MPNP when the metal ion is Fe 3+ or Al 3+ and its comparison with LNP

[0440] Replace the Bcl-2-siRNA (SEQ ID NO.4) in Example 2.6 with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@MPNP (the concentration of the contained siRNA is 100 nM); replace the Bcl-2-siRNA (SEQ ID NO.4) in Example 5 with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@LNP (the concentration of the contained siRNA is 100 nM); replace the eGFP-mRNA (SEQ ID NO.1) in Example 2.5 with Cy5-labeled mRNA (Cy5-mRNA) to prepare Cy5-mRNA@MPNP (the concentration of the contained mRNA is 2 μg / mL); replace the RBD-mRNA (SEQ ID NO.2) in Example 5 with Cy5-labeled mRNA (Cy5-mRNA) to prepare Cy5-mRNA@LNP (the concentration of the contained mRNA is 2 μg / mL). After co-incubating them with the cell lysosome probe Lysotracker Green for 3 hours in A549 cells, observe the overlapping situation of the Cy5 fluorescence signal (red) and the Lysotracker Green fluorescence signal (green) using a high-content imaging system, and judge and explore the ability of the drug-lipid particles to promote nucleic acid lysosomal escape.

[0441] Criterion for judging the ability of drug-metal-polyphenol complex particles to promote nucleic acid lysosomal escape: After incubating drug-metal-polyphenol complex nanoparticles with cells for 3 hours, observe the overlapping situation of the Cy5 fluorescence signal (red) and the Lysotracker Green fluorescence signal (green) using a high-content imaging system, and use imageJ software to calculate the overlapping rate of the red fluorescence signal and the green fluorescence signal. When the overlapping rate of the red fluorescence signal and the green fluorescence signal is less than 50% after incubating drug-metal-polyphenol complex particles with cells for 3 hours, it indicates that nucleic acid can escape from cell lysosomes relatively quickly, and the drug-metal-polyphenol complex particles have good ability to promote nucleic acid lysosomal escape.

[0442] Result analysis: As Figure 2-5 shown, when Cy5-siRNA@MPNP (Fe 3+ ) and Cy5-mRNA@MPNP (Fe 3+ ) are incubated with A549 cells for 3 hours, the overlapping rates of the red fluorescence signal and the green fluorescence signal are 39.20% ± 8.89% and 44.96% ± 3.85% respectively, that is, the lysosomal escape rates are 60.80% ± 8.89% and 55.04% ± 3.85% respectively; when Cy5-siRNA@MPNP (Al 3+ ) and Cy5-mRNA@MPNP (Al 3+)After incubating A549 cells for 3 hours, the overlap rates of red fluorescence signal and green fluorescence signal were 34.70% ± 4.98% and 39.10% ± 4.43% respectively, that is, the lysosomal escape rates were 65.30% ± 4.98% and 60.90% ± 4.43% respectively; while after incubating A549 cells with Cy5-siRNA@LNP and Cy5-mRNA@LNP for 3 hours, the overlap rates of red fluorescence signal and green fluorescence signal were 76.02% ± 7.90% and 85.33% ± 4.87% respectively, that is, the lysosomal escape abilities were 23.98% ± 7.90% and 14.67% ± 4.87% respectively. It is indicated that the drug-lipid nanoparticle MPNP has good ability to promote nucleic acid lysosomal escape, and the ability of MPNP to promote lysosomal escape is significantly stronger than that of LNP.

[0443] Example 7. The metal ion is Fe 3+ or Al 3+ The ability of the drug-metal-polyphenol complex particle MPNP to promote nucleic acid expression and its comparison with LNP

[0444] Replace the RBD-mRNA (SEQ ID NO.2) in Example 5 with the mRNA encoding the fluorescent protein eGFP, and the remaining preparation methods are the same as those in Example 5 to obtain eGFP-mRNA@LNP.

[0445] Incubate the eGFP-mRNA@MPNP prepared in Example 2.5 and the above eGFP-mRNA@LNP (the concentration of the contained mRNA is 2 μg / mL) with 293T cells respectively. The control group is incubated with MPNP or LNP. After 48 h, collect the cell suspension and detect the percentage of eGFP-positive cells by flow cytometry.

[0446] The method for analyzing the eGFP-positive cell rate by flow cytometry is as described in Example 2.

[0447] Result analysis: As Figure 2-6 shown, the percentages of eGFP-positive cells after treating 293T cells with MPNP (Fe 3+ ), MPNP (Al 3+ ) and LNP are 93.47%, 97.06% and 63.09% respectively. The results indicate that the function of MPNP to promote nucleic acid expression is better than that of LNP. The possible reason is that, as described in Example 6, the ability of MPNP to promote nucleic acid lysosomal escape is stronger than that of LNP, so more nucleic acids loaded by MPNP can be effectively released into the cytoplasm and thus be translated into proteins.

[0448] Example 8. The ability of the drug-metal-polyphenol complex particle MPNP to promote humoral immunity and cellular immunity and its comparison with LNP

[0449] 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 h, the supernatant was taken after centrifugation and stored at -20 °C for later use; the cell pellet was resuspended with 100 μL of PBS buffer solution, frozen and thawed twice, sonicated for 10 min, and then centrifuged to obtain the supernatant. The expression levels of the RBD protein in both the cell supernatant and the cell lysate were detected using a commercially available new coronavirus antigen RBD ELISA detection kit. The results are shown in Figure 2-7 .

[0450] The method for detecting the RBD expression level by ELISA was as described in Example 2.5.

[0451] The experimental animals were randomly divided into 3 groups (experimental group and control group), with 5 animals in each group. The animal model was BALB / c mice. Each mouse was given the first intramuscular injection on the 1st day and the second intramuscular injection 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 loaded mRNA. The dose for each injection was 100 μL. Among them, the RBD-mRNA@MPNP (Fe 3+ ), RBD-mRNA@MPNP (Al 3+ ), and RBD-mRNA@LNP preparations in the experimental group each contained 30 mg of mRNA. Blood was collected from the mice on the 28th day after the first injection, the serum was separated and serially diluted, and the titers of the total IgG antibodies against the RBD of the S1 subunit of the new coronavirus in the mice were detected using a commercially available ELISA kit. The results are as Figure 2-8 shown.

[0452] The method for detecting the titers of the total IgG antibodies against the RBD of the S1 subunit of the new coronavirus by ELISA was as described in Example 2.5.

[0453] On the 28th day after administration of RBD-mRNA@MPNP (Fe 3+ ), RBD-mRNA@MPNP (Al 3+ ), and RBD-mRNA@LNP, spleens of normal mice were collected, and single-cell suspensions were prepared under sterile conditions. They were plated in cell culture plates at a density of 100,000 spleen cells / well, and cultured with RBD protein at a final concentration of 10 mg / mL for 48 h. The supernatant was removed by centrifugation, and the expression levels of IFN-γ, IL-2, and IL-4 were determined using an ELISA kit. The results are as Figure 2-9 shown.

[0454] The method for detecting the expression levels of IFN-γ, IL-2, and IL-4 by ELISA is as described in Example 2.5.

[0455] Result analysis: As Figure 2-7 shown, RBD-mRNA@MPNP(Fe 3+ ), RBD-mRNA@MPNP(Al 3+ ), and RBD-mRNA@LNP can all induce a certain amount of RBD expression in 293T cells, but the ability of RBD-mRNA@MPNP(Al 3+ ) to induce RBD expression in cells is significantly stronger than that of RBD-mRNA@MPNP(Fe 3+ ), and the ability of RBD-mRNA@MPNP(Fe 3+ ) to induce RBD expression in cells is significantly stronger than that of RBD-mRNA@LNP: The expression level of RBD in the cell supernatant of the RBD-mRNA@MPNP(Fe 3+ ) treatment group is 178 ng / mL, the expression level of RBD in the cell supernatant of the RBD-mRNA@MPNP(Al 3+ ) treatment group is 215 ng / mL, and the expression level of RBD in the cell supernatant of the RBD-mRNA@LNP treatment group is 115.67 ng / mL. As Figure 2-8 shown by the results, RBD-mRNA@MPNP effectively induces the humoral immunity of mice, generating high levels of antigen-specific binding antibodies, and the ability of RBD-mRNA@MPNP(Al 3+ ) to induce the humoral immunity of mice is clearly superior to that of RBD-mRNA@MPNP(Fe 3+ ), and the ability of RBD-mRNA@MPNP(Fe 3+ ) to induce the humoral immunity of mice is clearly superior to that of RBD-mRNA@LNP: The IgG antibody titer in the mice of the RBD-mRNA@MPNP(Fe 3+ ) treatment group reaches 84975; the IgG antibody titer in the mice of the RBD-mRNA@MPNP(Al 3+ ) treatment group reaches 96418; while the IgG antibody titer in the mice of the RBD-mRNA@LNP treatment group is only 67476. As Figure 2-9 shown, RBD-mRNA@MPNP can effectively induce the cellular immunity of mice, that is, activate immune cells and produce a large amount of cytokines, and the ability of mRNA@MPNP(Al 3+ ) to induce the cellular immunity of mice is clearly superior to that of RBD-mRNA@MPNP(Fe 3+ ), and the ability of RBD-mRNA@MPNP(Fe 3+) The ability to induce cellular immunity in mice is clearly superior to RBD-mRNA@LNP: RBD-mRNA@MPNP(Fe 3+ ) The expression levels of cytokines IFN-γ, IL-2, and IL-4 reached 274.4 pg / mL, 254.2 pg / mL, and 77.4 pg / mL, respectively; RBD-mRNA@MPNP(Al 3+ ) The expression levels of cytokines IFN-γ, IL-2, and IL-4 reached 309 pg / mL, 299 pg / mL, and 91.2 pg / mL, respectively; while for RBD-mRNA@LNP, the expression levels of cytokines IFN-γ, IL-2, and IL-4 were only 104.2 pg / mL, 79.2 pg / mL, and 27 pg / mL. The results suggest that the ability of mRNA@MPNP(Al 3+ ) to deliver any mRNA and achieve its function is significantly superior to RBD-mRNA@MPNP(Fe 3+ ), and the ability of RBD-mRNA@MPNP(Fe 3+ ) to induce cellular immunity in mice is clearly superior to RBD-mRNA@LNP: RBD-mRNA@MPNP can more effectively promote the expression of the target protein in cells, can more effectively activate the humoral immunity and cellular immunity in vivo. Therefore, the described drug (mRNA)-lipid particles are significantly superior to the prior art LNP in terms of the functions of drugs, vaccines, or other products carrying mRNA. The possible reasons are as follows: 1) Compared with LNP, MPNP has a stronger ability to promote the escape of nucleic acids from lysosomes; 2) Compared with LNP, MPNP has a stronger ability to promote the expression of nucleic acids into proteins (antigens); 3) Compared with LNP, after curcumin in MPNP is released, as an immune adjuvant (also known as an immunomodulator), it can not only activate humoral immunity and cellular immunity to enhance the effect of MPNP delivering mRNA vaccines, but also inhibit the cytokine storm to inhibit excessive and harmful immune responses to the body.

[0456] Example 9. In vivo safety evaluation of metal-polyphenol complex particles (MPNP)

[0457] Using SD rats as the research object, a sub-chronic toxicity study of MPNP(Fe 3+ or Al 3+ ) was carried out for 20 days, and a 20-day recovery period was set up. The specific experimental methods are as follows:

[0458] Fifty-six SPF-grade SD rats (220 ± 20 g), with an equal number of males and females, were housed in an environment with a temperature of 25 °C, a humidity of 45% - 55%, and a 12-hour light cycle. After 3 - 5 days of adaptive feeding, they were randomly divided into groups by gender: 32 rats in the experimental group and 24 rats in the recovery group. Fourteen rats in the blank control group (Control) (including 8 rats from the experimental group and 6 rats from the recovery group), with an equal number of males and females; 14 rats in the low-dose MPNP group (25 mg / kg) (including 8 rats from the experimental group and 6 rats from the recovery group), with an equal number of males and females; 14 rats in the medium-dose MPNP group (50 mg / kg) (including 8 rats from the experimental group and 6 rats from the recovery group), with an equal number of males and females; 14 rats in the high-dose group (100 mg / kg) (including 8 rats from the experimental group and 6 rats from the recovery group), with an equal number of males and females. The rats in the experimental group (32 in total) were sacrificed for sample collection after the administration, and the rats in the recovery group (24 in total) were sacrificed for sample collection 20 days after normal feeding continued after the administration ended.

[0459] Administration method: The experimental animals were administered by tail vein injection every 2 days for a total of 20 days, and the body weight of the SD rats was recorded once a week. The prepared MPNP was dissolved in pH 7.4 Tris-Hcl buffer solution. The control group was injected with an equal volume of Tris-Hcl buffer solution, and 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.

[0460] Basis for setting the above MPNP administration doses: When encapsulating 200 μg / kg of mRNA (the actual requirement for mRNA animal experiments), the required amount of the empty vector MPNP is 8 mg / kg. To fully prove the safety of MPNP, we selected doses of 1, 2, and 4 times the actual requirement for MPNP animal experiments, namely 8 mg / kg, 16 mg / kg, and 32 mg / kg.

[0461] General index detection method: After each administration, the general status of the animals in each group was observed, including survival, diet, appearance characteristics, behavioral activities, body weight, and whether there were local reactions at the administration site. Gross autopsy was performed during dissection, including timely weighing of the wet weights of major organs such as the brain, heart, liver, spleen, lungs, and kidneys, calculating the organ-body ratio, and recording the pathological changes of each organ. Among them, the organ-body ratio = wet weight of rat organ / rat body weight × 100%.

[0462] Obtaining and preserving whole blood and serum of SD rats: After 20 days of drug administration and 20 days of recovery period, the rats were dissected. Blood was collected from the abdominal aorta. That is, the SD rats were anesthetized with isoflurane and fixed on the dissection board. The abdomen was disinfected with 75% ethanol. The abdomen of the rats was cut open with a sterile ophthalmic scissors. The internal organs were gently pushed aside with a cotton ball to expose the abdominal aorta. Whole blood was collected with a 500 μL negative pressure EDTAK2 anticoagulant blood collection tube and stored at 4°C for routine blood test. Whole blood was collected with a 5 mL negative pressure ordinary blood collection tube and left to stand at room temperature for 30 min, then centrifuged at 4°C and 1500 rpm for 15 min. The supernatant was taken and placed in a 1.5 mL centrifuge tube and stored at -20°C for the detection of blood biochemical indexes and immunology-related indexes.

[0463] Method for routine blood test: Routine blood indexes include: number of white blood cells, number of lymphocytes, number of monocytes, number of neutrophils, percentage of lymphocytes, percentage of monocytes, percentage of neutrophils, number of red blood cells, hemoglobin, red blood cell volume, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, coefficient of variation of red blood cell distribution width, number of platelets, mean platelet volume, platelet distribution width, plateletcrit. The whole blood specimen was gently inverted and mixed evenly. A small amount of whole blood was taken and the results were automatically analyzed using an automatic blood cell analyzer.

[0464] Method for detecting blood biochemical indexes: Blood biochemical indexes include inorganic ions (Fe 2+ , Na + , K + , Cl-, Ca 2+ ), liver function indexes (ALT, AST, γ-GT, T-BIL, D-BIL, ALP, ALB), kidney function indexes (BUN, UA, CR), heart function indexes (LDH, CK), glucose metabolism indexes (GSP, GLU, INS), lipid metabolism indexes (CHO, TG, LDL-C, HDL-C). The thawed serum sample was centrifuged at 3000 rpm for 15 min, and the supernatant was taken and aliquoted for use. The corresponding parameters were set on the automatic biochemical analyzer, the prepared working solution was added, and then the serum to be tested was added. The automatic biochemical analyzer automatically determined the results.

[0465] Method for detecting immunology-related indexes: Immunology-related indexes include thyroid function indexes (TT3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN-γ, IFN-α, TNF-α), immunoglobulins (IgG, IgA, IgM), serum complement (C3, CH50). The above indexes were detected by ELISA method.

[0466] Main organ pathological examination method for SD rats: At the end of the dosing period and the end of the recovery period, anesthetize the rats in each group, and use ophthalmic scissors to remove the main organs of the rats, including the whole brain, heart, liver, spleen, lungs, and kidneys. Gently rinse them with 0.9% normal saline, fix them in 4% paraformaldehyde fixative, embed them in paraffin routinely, stain with H&E, and observe the histopathological changes of each organ of the control group and the experimental group of rats under an optical microscope.

[0467] Result analysis: 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 behavior, and no obvious adverse reactions were observed after dosing; compared with the control group, there were no significant differences in the body weight gain values of male and female SD rats in the low-, medium-, and high-dose MPNP groups; compared with the control group, there were no significant differences in the organ-body ratios of the low-, medium-, and high-dose MPNP groups.

[0468] At the end of the dosing period and the end of the recovery period, compared with the control group, the blood routine indexes (number of white blood cells, number of lymphocytes, number of monocytes, number of neutrophils, percentage of lymphocytes, percentage of monocytes, percentage of neutrophils, number of red blood cells, hemoglobin, red blood cell packing, mean red blood cell volume, mean red blood cell hemoglobin content, mean red blood cell hemoglobin concentration, coefficient of variation of red blood cell distribution width, number of platelets, mean platelet volume, platelet distribution width, platelet hematocrit) of the low-, medium-, and high-dose MPNP groups were normal; compared with the control group, the blood biochemical indexes of the low-, medium-, and high-dose MPNP groups, including inorganic ions (Fe 2+ , Na + , K + , Cl - , Ca 2+ ), liver function indexes (ALT, AST, γ-GT, T-BIL, D-BIL, ALP, ALB), kidney function indexes (BUN, UA, CR), heart function indexes (LDH, CK), glucose metabolism indexes (GSP, GLU, INS), lipid metabolism indexes (CHO, TG, LDL-C, HDL-C), were all normal; compared with the control group, the immunology-related indexes of the low-, medium-, and high-dose MPNP groups, including thyroid function indexes (TT3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN-γ, IFN-α, TNF-α), immunoglobulins (IgG, IgA, IgM), serum complements (C3, CH50), were all normal.

[0469] At the end of the dosing 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, the tissue staining was normal, the cell morphological structure was intact, and there was no karyopyknosis or inflammatory cell infiltration; the myocardial tissue structure was intact, the myocardial cells were arranged neatly, continuously, and tightly, the cell nuclei were clearly visible, and there was no obvious cell congestion, edema or necrosis; the hepatocyte morphology was normal, and there was no aggregation or necrosis of inflammatory cells; the spleen structure was normal, and the boundaries between red and white pulp were clear; the lung tissue structure was intact, the alveoli were of the same size, and there was no obvious aggregation or infiltration of inflammatory cells; the kidney structure was normal.

[0470] The above results suggest that long-term and high-dose injection of MPNP (Fe 3+ or Al 3+ ) into SD rats did not show obvious chronic toxic reactions, indicating that MPNP has relatively high safety.

[0471] Table 2-1 In vivo safety evaluation of MPNP

[0472]

[0473]

[0474] Note: ALT, alanine aminotransferase; AST, aspartate aminotransferase; γ-GT, gamma-glutamyl transpeptidase; T-BIL, total bilirubin; D-BIL, direct bilirubin; ALP, alkaline phosphatase; ALB, albumin; BUN, urea nitrogen; UA, uric acid; CR, creatinine; LDH, lactate dehydrogenase; CK, creatine phosphokinase; GSP, fructosamine; GLU, glucose; INS, insulin; CHO, cholesterol; TG, triglyceride; LDL-C, low-density lipoprotein; HDL-C, high-density lipoprotein; TT3, triiodothyronine; TT4, tetraiodothyronine; TSH, thyroid-stimulating hormone; IL-1, interleukin-1; IL-2, interleukin-2; IL-4, interleukin-4; IFN-γ, interferon-γ; IFN-α, interferon-α; TNF-α, tumor necrosis factor-α; IgG, immunoglobulin G; IgA, immunoglobulin A; IgM, immunoglobulin M; C3, complement C3; CH50 total complement CH50

[0475] Example 10. In vivo safety comparison between metal-polyphenol complex particles (MPNP) and LNP

[0476] The main toxicity of LNP comes from its main components - cationic lipids and / or ionizable lipids. When LNP metabolizes in the body, the free cationic lipids and / or ionizable lipids will produce obvious toxicity to the body. The median lethal dose (IC 50) is an important parameter for evaluating the toxicity of LNP to the body. The drug-metal-polyphenol complex particles (MPNP) replace the cationic lipid / ionizable lipid in LNP with a metal-polyphenol complex. Therefore, we compared the toxicity differences between LNP and MPNP by studying the median lethal dose (IC 50 ) of the metal-polyphenol complex and the cationic lipid / ionizable lipid to biological cells.

[0477] After incubating 293T cells 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 respectively, the cell viability was detected using a CCK8 activity detection kit, and the median lethal dose IC of the metal-polyphenol complex, cationic lipid (DOTAP), and ionizable lipid (ALC0315) to 293T cells was calculated respectively. 50 .

[0478] Detection method of CCK8:

[0479] Cell culture: Cells were cultured in DMEM medium containing 10% FBS and 1% double antibody. Wait until the cell density reaches 80%-90% of the culture flask for use;

[0480] Wash the remaining medium in the culture flask with PBS, add trypsin, and quickly transfer the culture flask to an incubator at 37°C containing 5% CO 2 . Observe carefully. After the cells become slightly round, add culture medium to terminate digestion. Transfer to a centrifuge tube, centrifuge at 1500 RPM for 5 min, and resuspend the cells with fresh culture medium;

[0481] Counting: Dilute the cell suspension to 100,000 cells per 1 mL according to the purpose, add 100 μL to each well of a 96-well plate, with at least 5 replicates in each group. Incubate at 37°C and 5% CO 2 for 24 h and then add the drug;

[0482] After incubating the drug for 48 h, add 10% CCK8 and incubate for 1-3 h. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay reader;

[0483] Survival rate (%) = [A (drug added) - A (blank)] / [A (0 drug added) - A (blank)] × 100%.

[0484] IC 50Calculation method: Taking the survival rate as the ordinate and the drug concentration as the abscissa, use Graphpad to calculate the IC by [Inhibitor] vs. normalized response--Variable slope analysis method 50 .

[0485] To compare the in vivo safety of MPNP and LNP, take MPNP (8 mg / kg) and LNP (3.24 mg / kg) that can carry an equal amount of nucleic acid (200 μg / kg mRNA), and perform in vivo experiments according to the method of Example 9 to evaluate and compare the in vivo toxicity of MPNP and LNP.

[0486] Result analysis: As shown in Table 2-2, the IC of the metal-polyphenol complex 50 is significantly greater than that of the cationic lipid (DOTAP) and the ionizable lipid (ALC0315). It is suggested that the toxicity of the metal-polyphenol complex is significantly less than that of the cationic lipid and the ionizable lipid, and the effect is remarkable.

[0487] As shown in Table 2-3, at the end of drug administration and the end of recovery, compared with the control group, the expression levels of liver function indexes ALT, AST, ALP and cytokines IL-6, IL-1β in the MPNP (Fe 3+ ) or MPNP (Al 3+ ) group were not significantly abnormal. However, compared with the control group, the expression levels of liver function indexes ALT, AST, ALP and cytokines IL-6, IL-1β in the LNP group were significantly increased. The results suggest that the in vivo safety of MPNP (Fe 3+ ) or MPNP (Al 3+ ) is higher than that of LNP. The reason is that: the core component of LNP is the 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 vivo; while the core component of MPNP (Fe 3+ ) or MPNP (Al 3+ ) is the metal-polyphenol complex, and its metal-polyphenol complex is composed of the natural small molecule curcumin with high safety (a food additive and pharmaceutical excipient approved by the FDA) and safe metal ions, and it has been decomposed into natural molecules in vivo after completing drug delivery. In summary, because there is no cationic lipid / ionizable lipid in the composition of MPNP (Fe 3+ ) or MPNP (Al 3+ ), it will not cause the toxic and side reactions related to cationic lipid / ionizable lipid, so the safety of MPNP (Fe 3+ ) or MPNP (Al 3+ ) is higher than that of LNP.

[0488] Structural formula of DOTAP

[0489] Structural formula of ALC0315

[0490]

[0491] Table 2-2 Metal ion is Fe 3+ or Al 3+ IC of metal-polyphenol complex, cationic lipid (DOTAP) and ionizable lipid (ALC0315) when 50 Comparison

[0492]

[0493] Table 2-3 Metal ion is Fe 3+ or Al 3+ Comparison of chronic toxicity test indexes of MPNP and LNP when

[0494]

[0495] Example 3 Clinical application and administration route of drug-metal-polyphenol complex particle MPNP

[0496] Example 11 Metal ion is Fe 3+ or Al 3+ Clinical application and administration route of drug-metal-polyphenol complex particle MPNP when

[0497] Replace the mRNA in Example 2 with siRNA targeting B7-H4 gene (B7-H4-siRNA) and its control (scr-siRNA), and mRNA encoding the receptor binding domain (RBD) of the S1 subunit of the novel coronavirus.

[0498] The sequences of the above different nucleic acids are as follows: ① The sequence of B7-H4-siRNA is SEQ ID No.19 (sense strand) and SEQ ID No.26 (antisense strand) (25bp), and its random control sequence is SEQ ID No.20 (sense strand) and SEQ ID No.27 (antisense strand) (19bp); ② The mRNA sequence encoding the receptor binding domain (RBD) of the S1 subunit of the novel coronavirus is SEQ ID No.2 (669nt). Prepare drug-metal-polyphenol complex particles (B7-H4-siRNA@MPNP(Fe 3+ )) and RBD-mRNA@MPNP(Fe 3+)、B7-H4-siRNA@MPNP(Al 3+ )、RBD-mRNA@MPNP(Al 3+ )),The preparation processes of the remaining drug-metal-polyphenol complex particles are the same as those in Example 2. The above two different drug-metal-polyphenol complex particles (B7-H4-siRNA@MPNP, RBD-mRNA@MPNP) are used to treat liver cancer and as an mRNA vaccine to prevent novel coronavirus respectively.

[0499] The sequence of B7-H4-siRNA is as follows:

[0500] sense 5’-GGGAGACAC UCCAUC ACAGUCACUA-3’(SEQ ID No.19).

[0501] antisense 5’-UAG UGACUG UGAUGGAGU GUC UCC C-3’(SEQ ID No.26)(25bp).

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

[0503] sense 5’-UUCUCCGAACGUGUCACGU-3’(SEQ ID No.20).

[0504] antisense 5’-ACGUGACACGUUCGGAGAA-3’(SEQ ID No.27)(19bp).

[0505] To evaluate the effects of B7-H4-siRNA@MPNP(Fe 3+ ) and B7-H4-siRNA@MPNP(Al 3+ ) in treating liver cancer, a liver cancer animal model was made with HepG2 cells. When the tumor size increased to about 100mm 3 , the liver cancer mice were randomly divided into 7 groups (5 mice in each group): Tris-Hcl buffer solution control group, blank vector MPNP(Fe 3+ ) group, blank vector 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 pH7.4 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-H4 siRNA@MPNP(Al 3+ ) once every 3 days, with a dose of 200 μg siRNA / kg, for 8 injections. The tumor volume was measured and recorded every 3 days. The results are as Figure 3-1 shown.

[0506] To evaluate the effect of RBD-mRNA@MPNP as an mRNA vaccine against SARS-CoV-2, the experimental process and methods were as shown in Example 2.5 above.

[0507] The ELISA detection method was as described in Example 2.5.

[0508] Establishment of a liver cancer mouse model: HepG2 cells were collected, resuspended in PBS at a density of 1×10 7 / mL, and stored on ice before inoculation. Then, 100 μL of the cell suspension was subcutaneously injected into the dorsal area near the hind legs of female Balb / c nude mice to establish a liver cancer mouse model.

[0509] Result analysis:

[0510] As Figure 3-1 shown, Scr-siRNA@MPNP(Fe 3+ ), Scr-siRNA@MPNP(Al 3+ ) had little inhibitory effect on the growth of liver cancer HepG2 cells, while B7-H4-siRNA@MPNP(Fe 3+ ) and B7-H4 siRNA@MPNP(Al 3+ ) showed highly efficient therapeutic effects and could effectively inhibit the growth of liver cancer tumors. The results suggest that the drug-metal-polyphenol complex particles can encapsulate and deliver B7-H4 siRNA, and inhibit the development of liver cancer by inhibiting the expression of target genes.

[0511] As in Example 2.5 above, Figure 1-3 , Figure 1-5 shown, RBD-mRNA@MPNP(Fe 3+ ) increased the expression level of mouse IgG antibody to 84363.4( Figure 1-3), such that the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 271.8 pg / mL, 269.6 pg / mL, and 75.8 pg / mL respectively( Figure 1-5 ). RBD-mRNA@MPNP(Al 3+ ) enables the expression level of mouse IgG antibody to reach 94828.6( Figure 1-17 ), such that the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 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 the humoral immunity of mice, producing high levels of antigen-specific binding antibodies; at the same time, it can effectively induce the cellular immunity of mice, that is, activate immune cells and produce a large number of cytokines. Therefore, RBD-mRNA@MPNP can effectively prevent the infection of novel coronavirus.

[0512] As Figure 3-1 shown, B7-H4-siRNA@MPNP can effectively treat liver cancer by intratumoral injection; as shown in Example 2.5, Figure 1-3 , 1-5 , 1-17 and 1-19, RBD-mRNA@MPNP can activate humoral immunity and cellular immunity by intramuscular injection, thus playing a role in preventing the infection of novel coronavirus. The results suggest that drug-metal-polyphenol composite particles can be administered through multiple routes.

[0513] Example 4: The functions after curcumin and Fe 3+ are replaced by homologs

[0514] Example 12, curcumin, Fe 3+ The functions after being replaced by homologs

[0515] Referring to Example 1, curcumin and Fe 3+ homologs are used to replace curcumin and Fe 3+ respectively. Referring to Example 2, 9 different drug-metal-polyphenol composite particles (eGFP-mRNA@MPNP) are prepared through different combinations, and the concentration of mRNA contained in each eGFP-mRNA@MPNP is 2 μg / mL. The names and structures of curcumin, Fe 3+ and their homologs are shown in Table 4-1, and the combination methods of curcumin, Fe 3+ and their homologs in 9 kinds of mRNA@MPNP are shown in Table 4-2. Among them, the reaction temperature in Example 1 is 60 °C and the reaction time is 2 h, and other conditions remain unchanged.

[0516] To compare the effects of the nine different eGFP-mRNA@MPNPs and eGFP-mRNA@LNP, we prepared LNP encapsulating an equal amount of eGFP mRNA with reference to Example 5 to obtain eGFP-mRNA@LNP.

[0517] The above nine different eGFP-mRNA@MPNPs and the above eGFP-mRNA@LNP (the concentration of the contained mRNA was 2 μg / mL) were respectively incubated with 293T cells. 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.

[0518] The method for analyzing the eGFP-positive cell rate by flow cytometry was as described in Example 2.

[0519] The main toxicity of LNP comes from its main component, cationic lipid / ionizable lipid. When LNP metabolizes in the body, the free cationic lipid / ionizable lipid will produce obvious toxicity to the body. The half-lethal dose (IC 50 ) of cationic lipid / ionizable lipid to biological cells is an important parameter for evaluating the toxicity of LNP to the body. The metal-polyphenol complex particle (MPNP) replaces the cationic lipid / ionizable lipid in LNP with a metal-polyphenol complex. Therefore, we studied the half-lethal dose (IC 50 ) of the nine metal-polyphenol complexes and the cationic lipid (DOTAP) / ionizable lipid (ALC0315) to biological cells in Table 4-2 to compare the toxicity differences between LNP and the nine MPNPs.

[0520] The calculation method of IC50 was as described in Example 10.

[0521] Result analysis: As shown in Table 4-3, the percentage of eGFP-positive cells after treating 293T cells with the nine different eGFP-mRNA@MPNPs was significantly higher than that of eGFP-mRNA@LNP, and the percentage of eGFP-positive cells of mRNA@MPNP1 was the highest. The results suggest that the function of mRNA@MPNP formed after curcumin and Fe 3+ were replaced by their analogs was inferior to that of mRNA@MPNP1, but slightly better than that of mRNA@LNP. The possible reason is that, as described in Example 5, the ability of MPNP to promote nucleic acid lysosomal escape is stronger than that of LNP. Therefore, more nucleic acids loaded by MPNP can be effectively released into the cytoplasm and thus be translated into proteins.

[0522] The above results suggest that as long as the following conditions are met, then curcumin and Fe 3+The function of the drug-metal-polyphenol complex particles formed after being replaced by their analogs is not affected: ① The analog of curcumin is a hydrophobic polyphenol that can complex with metals; ② The analog of Fe 3+ is a metal ion; ③ The coordination bond between curcumin and Fe 3+ can break in response to the low pH environment of lysosomes.

[0523] As shown in Table 4-3, the IC 50 of the 9 metal-polyphenol complexes is significantly greater than that of cationic lipids (DOTAP) and ionizable lipids (ALC0315). It is suggested that the toxicity of metal-polyphenol complexes is significantly less than that of cationic lipids and ionizable lipids, that is, the safety of the lipid particles (MPNP) composed of curcumin, Fe 3+ and its analogs is higher than that of LNP. The reason is that: the core component of LNP is artificially synthesized "cationic lipid / ionizable lipid", which has high cytotoxicity and immunogenicity, and its structure is relatively stable and difficult to decompose and metabolize in the body; while the core component of MPNP is a metal-polyphenol complex, and its metal-polyphenol complex is composed of non-cationic lipids, natural small molecule substances with high safety (curcumin among them is a food additive and pharmaceutical excipient approved by the FDA) and safe metal ions, and it has been decomposed into natural molecules in the body after completing drug delivery. In summary, the lipid particles (MPNP) composed of curcumin, Fe 3+ and its analogs do not contain cationic lipids / ionizable lipids in their composition and will not cause toxic and side reactions related to cationic lipids / ionizable lipids, so the safety of MPNP is higher than that of LNP.

[0524] Table 4-1 Names and structures of curcumin, Fe 3+ and its analogs

[0525]

[0526] Table 4-2 List of combination methods and functions of metal-polyphenol complexes in drug-lipid nanoparticles prepared from curcumin, Fe 3+ and its analogs

[0527]

[0528] Table 4-3 IC 3+ of metal-polyphenol complexes prepared from curcumin, Fe 50

[0529]

[0530] Example 13 Proportion of curcumin and Fe 3+ components in different metal-polyphenol complexes and functions of the prepared drug-metal-polyphenol complex particles

[0531] Example 13.1 The metal ion is Fe 3+ The polyphenols and Fe of different metal-polyphenol complexes when 3+ Component dosing ratios and the functions of the drug-metal-polyphenol complex particles prepared therefrom

[0532] According to Example 2.1, metal-polyphenol complexes were prepared, and curcumin was replaced with its analogues hesperetin (1 molecule of hesperetin contains 4 hydroxyl groups) and catechin (1 molecule of catechin contains 5 hydroxyl groups) respectively, to obtain three metal-polyphenol complexes (mRNA@MPNP1, mRNA@MPNP4, mRNA@MPNP7). When preparing these three metal-polyphenol complexes, the dosing ratios of curcumin or its analogues to Fe 3+ were respectively: 1:1, 1:1, 1:2. And the corresponding drug-metal-polyphenol complex particles were prepared using these three metal-polyphenol complexes (mRNA@MPNP1, mRNA@MPNP4, mRNA@MPNP7). Among them, mRNA is the mRNA encoding the eGFP fluorescent protein, and its sequence is SEQ ID NO.1 (720 nt). According to the experimental procedures and methods described in Example 2.5, the mRNA encapsulation efficiency of these three drug-lipid particles and their ability to promote the expression of the eGFP fluorescent protein after treating 293T cells were detected.

[0533] Result analysis: As shown in Table 4-4, the mRNA encapsulation efficiency and the ability to promote the expression of the target protein (i.e., the positive cell rate) of the drug-metal-polyphenol complex particles prepared using different dosing ratios according to the chemical structures of the metal-polyphenol complex components are comparable. The results suggest that the dosing ratio of the metal-polyphenol complex components can be adjusted according to the specific structure of the metal-polyphenol complex components. The basis for the adjustable dosing ratio is that: since the hydroxyl groups of the analogues of curcumin are connected to the analogues of Fe 3+ by coordination bonds, as long as the analogues of curcumin contain multiple binding sites, then the dosing ratio of the analogues of curcumin and Fe 3+ analogues can be adjusted according to the number of binding sites contained in the analogues of curcumin.

[0534] Table 4-4 The polyphenol and Al dosing ratios of different metal-polyphenol complexes when the metal ion is Fe 3+ and the functions of the drug-lipid particles prepared therefrom

[0535]

[0536] Example 13.2 The metal ion is Al 3+ The polyphenols and Al of different metal-polyphenol complexes when 3+Component dosing ratio and functions of the drug-metal-polyphenol complex particles prepared therefrom

[0537] According to Example 2.2, metal-polyphenol complexes were prepared, and curcumin was replaced with its analogues hesperetin (1 molecule of hesperetin contains 4 hydroxyl groups) and catechin (1 molecule of catechin contains 5 hydroxyl groups) respectively, to obtain three metal-polyphenol complexes (mRNA@MPNP3, mRNA@MPNP6, mRNA@MPNP9). When preparing these three metal-polyphenol complexes, the dosing ratios of curcumin or its analogues to Al 3+ were respectively: 1:1, 1:1, 1:2. And the corresponding drug-metal-polyphenol complex particles were prepared using these three metal-polyphenol complexes (mRNA@MPNP3, mRNA@MPNP6, mRNA@MPNP9). Among them, mRNA is the mRNA encoding the eGFP fluorescent protein, and its sequence is SEQ ID NO.1 (720 nt). According to the experimental procedures and methods described in Example 2.5, the mRNA encapsulation rates of these three drug-lipid particles and their abilities to promote the expression of eGFP fluorescent protein after treating 293T cells were detected.

[0538] Result analysis: As shown in Table 4-5, the mRNA encapsulation efficiency and the ability to promote the expression of the target protein of the drug-metal-polyphenol complex particles prepared using different dosing ratios according to the chemical structures of the components of the metal-polyphenol complexes are comparable. The results suggest that the dosing ratio of the components of the metal-polyphenol complex can be adjusted according to the structure of the specific metal-polyphenol complex components. The basis for the adjustable dosing ratio is that: since the hydroxyl groups of the analogues of curcumin are connected to the analogues of Al 3+ by coordination bonds, as long as the analogues of curcumin contain multiple binding sites, then the dosing ratio of the analogues of curcumin and the analogues of Al 3+ can be adjusted according to the number of binding sites contained in the analogues of curcumin.

[0539] Table 4-5 Component dosing ratios of different metal-polyphenol complexes and functions of the drug-lipid particles prepared therefrom when the metal ion is Al 3+

[0540]

[0541]

[0542] The preparation of mRNA@MPNP in Example 2 above was completed by the research group of Professor Wang Shan of the Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Central South University.

[0543] ​Unless otherwise defined, all technical and scientific terms used throughout this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of inconsistency, the meaning set forth in this invention or the meaning derived from the content recorded throughout this invention shall prevail. Additionally, the terms used in this specification are for the purpose of describing embodiments of this invention only and are not intended to limit this invention.

[0544] Note that the above is only a preferred embodiment of the present invention and the technical principles applied. Those skilled in the art will understand that the present invention is not limited to the specific embodiments described herein, and various obvious changes, re-adjustments, and substitutions can be made by those skilled in the art without departing from the protection scope of the present invention. Therefore, although the present invention has been described in detail through the above embodiments, the present invention is not limited to the above embodiments only. Without departing from the technical concept of the present invention, more other equivalent embodiments can be included, all of which fall within the protection scope of the present invention.

Claims

1. A drug-lipid particle, wherein, the drug-lipid particle comprises: a drug, wherein the drug is a negatively charged molecule selected from one or a combination of more than one of mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, double-stranded DNA, and single-stranded DNA; and a metal-polyphenol complex particle, which contains: (i) a metal-polyphenol complex, which is formed by the reaction of a polyphenol molecular moiety and a metal ion moiety, and the polyphenol molecular moiety and the metal ion moiety are connected by a coordination bond; the polyphenol molecular moiety is selected from at least one of curcumin, hesperetin, or catechin; The metal ion part is selected from at least one of Fe 3+ , Ca 2+ , Al 3+ ; the molar ratio of the polyphenol molecular moiety to the metal ion moiety is 1:(0.5-2); (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 the conjugated lipid that inhibits particle aggregation is a PEG-lipid conjugate; and (iii) a non-cationic lipid or a non-ionizable lipid other than the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or the non-ionizable lipid further includes cholesterol; wherein the drug is encapsulated in the metal-polyphenol complex particle; the preparation method of the drug-lipid particle comprises the following steps: Step 1: React the polyphenol molecular moiety with the metal ion moiety through a coordination bond to form a metal-polyphenol complex; Step 2: Dissolve the metal-polyphenol complex prepared in Step 1, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or the non-ionizable lipid in an organic compound to form an organic phase, dissolve the drug in a buffer solution to form an aqueous phase, and mix the organic phase and the aqueous phase to obtain a drug-lipid particle; the molar proportion of the metal-polyphenol complex in the raw materials is 10%-20%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw materials is 2%-10%, the molar proportion of cholesterol in the raw materials is 0%-48%, and the molar proportion of the non-cationic lipid or the non-ionizable lipid other than cholesterol in the raw materials is 40%-75%; or the molar proportion of the metal-polyphenol complex in the raw materials is 5%-less than 10%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw materials is 2%-10%, the molar proportion of cholesterol in the raw materials is 0%-48%, and the molar proportion of the non-cationic lipid or the non-ionizable lipid other than cholesterol in the raw materials is 40%-75%; or the molar proportion of the metal-polyphenol complex in the raw materials is 10%-20%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw materials is 2%-10%, the molar proportion of cholesterol in the raw materials is 0%-48%, and the molar proportion of the non-cationic lipid or the non-ionizable lipid other than cholesterol in the raw materials is 30%-less than 40%; (i) the molar proportion sum of the metal-polyphenol complex, (ii) the conjugated lipid that inhibits particle aggregation, and (iii) the non-cationic lipid or the non-ionizable lipid in the raw materials is 100%.

2. The drug-lipid particle according to claim 1, wherein, The polyphenol molecular moiety is selected from curcumin, hesperetin or catechin.

3. The drug-lipid particle according to claim 2, wherein, the catechin is Formula 13.

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

5. The drug-lipid particle according to claim 1, wherein, the PEG-lipid conjugate is selected from one or a combination of more than one of phosphatidylethanolamine-polyethylene glycol 2000 (Formula 47), phosphatidylethanolamine-polyethylene glycol 700 (Formula 48), phosphatidylethanolamine-polyethylene glycol 1000 (Formula 49), phosphatidylethanolamine-polyethylene glycol 5000 (Formula 50); wherein, R1 and R2 are both independently: Capryloyl 、Lauryl 、 Myristoyl 、Palmitoyl 、 Stearyl 、oleoyl 、 Linoleoyl 、Erucoyl 、 arachidonoyl or phytanoyl ; Formula 47; Formula 48; Formula 49; Formula 50.

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

7. The drug-lipid particle according to claim 6, 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.

8. The drug-lipid particle according to claim 1, wherein, the non-cationic lipid or non-ionizable lipid in (iii) is selected from one or a combination of more than one of lecithin PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, phosphatidylthreonine PT.

9. The drug-lipid particle according to claim 8, wherein, the non-cationic lipid or non-ionizable lipid in (iii) is selected from one or a combination of more than one of lecithin (PC) (Formula 29), phosphatidylethanolamine (PE) (Formula 30), phosphatidylserine (PS) (Formula 31), phosphatidic acid (PA) (Formula 32), phosphatidylglycerol (PG) (Formula 33), phosphatidylthreonine (PT) (Formula 36); wherein, R1 and R2 are both independently caproyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucoyl, arachidoyl or phytanoyl; Formula 29; Formula 30; Formula 31; Formula 32; Formula 33; Formula 36.

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

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

12. The drug-lipid particle according to claim 1, wherein, The metal-polyphenol complex is composed of the reaction of a polyphenol molecular part and a metal ion part. The polyphenol molecular part is selected from curcumin, hesperetin or catechin, and the metal ion part is selected from Fe 3+ , Ca 2+ or Al 3+ .

13. The drug-lipid particle according to claim 12, wherein, the catechin is Formula 13.

14. The drug-lipid particle according to claim 12, wherein, The polyphenol molecular moiety is curcumin, and the metal ion moiety is Fe 3+ .

15. The drug-lipid particle according to claim 14, Among them, The molar ratio of curcumin and Fe 3+ is 1:

1.

16. The drug-lipid particle according to claim 12, wherein, The polyphenol molecular moiety is curcumin, and the metal ion moiety is Al 3+ .

17. The drug-lipid particle according to claim 16, wherein, Curcumin and Al 3+ have a molar ratio of 1:

1.

18. The drug-lipid particle according to claim 1, wherein, the molar proportion of the metal-polyphenol complex in the raw material is 5% to less than 10%, 10% to 15%, or 15% to 20%.

19. The drug-lipid particle according to claim 18, wherein, the molar proportion of the metal-polyphenol complex in the raw material is 5%, 10%, or 15%.

20. The drug-lipid particle according to claim 1, wherein, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 5% or 5% to 10%.

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

22. The drug-lipid particle according to claim 1, wherein, the molar proportion of the cholesterol in the raw material is 10% to 30%, 30% to 47%, or 10% to 20%.

23. The drug-lipid particle according to claim 22, wherein, the molar proportion of the cholesterol in the raw material is 10%, 30%, or 47%.

24. The drug-lipid particle according to claim 1, wherein, the molar proportion of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 45% to 55%, 60% to 65%, or 50% to 65%.

25. The drug-lipid particle according to claim 24, wherein, the molar proportion of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 45%, 55%, 60%, or 65%.

26. The drug-lipid particle according to claim 1, 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%. The metal ion part in the metal-polyphenol complex is selected from Fe 3+ .

27. The drug-lipid particle according to claim 26, 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 in the metal-polyphenol complex is selected from Fe 3+ .

28. The drug-lipid particle according to claim 26, 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%, the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 60%, and the metal ion part in the metal-polyphenol complex is selected from Fe 3+ .

29. The drug-lipid particle according to claim 1, 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%, the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 45% to 55%, and the metal ion part in the metal-polyphenol complex is selected from Al 3+ .

30. The drug-lipid particle according to claim 29, 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 3%, the molar proportion of cholesterol in the raw material is 47%, and the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 45%; the metal ion part in the metal-polyphenol complex is selected from Al 3+ ; or The molar proportion of the metal-polyphenol complex in the raw material is 10%, 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 55%; the metal ion part in the metal-polyphenol complex is selected from Al 3+ .

31. The drug-lipid particle according to claim 1, wherein, The drug is the mRNA sequence encoding eGFP shown in SEQ ID No.1, the mRNA sequence encoding the receptor-binding domain RBD of the novel coronavirus S1 subunit shown in SEQ ID No.2, the mRNA sequence encoding NY-ESO-1 shown in SEQ ID No.3, the siRNA sequence of the Bcl-2 gene with the antisense strand shown in SEQ ID No.4 and the sense strand shown in SEQ ID No.21, the siRNA sequence of the PLK1 gene with the antisense strand shown in SEQ ID No.6 and the sense strand shown in SEQ ID No.23, the siRNA sequence of the Gal-1 gene shown in SEQ ID No.8, the ASO sequence of the STAT-3 gene shown in SEQ ID No.10, the ASO sequence of the α-syn gene shown in SEQ ID No.12, the ASO sequence of the Bcl-2 gene shown in SEQ ID No.14, the mRNA sequence encoding the wild-type novel coronavirus S protein shown in SEQ ID No.16, the double-stranded DNA sequence with the antisense strand shown in SEQ ID No.17 and the sense strand shown in 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 strand shown in SEQ ID No.19 and the antisense strand shown in SEQ ID No.

26.

32. A method for preparing the drug-lipid particles according to any one of claims 1 to 31, wherein, Step 1: React the polyphenol molecule part with the metal ion part through a coordination bond reaction to form a metal-polyphenol complex; Step 2: Dissolve the metal-polyphenol complex prepared in Step 1, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or non-ionizable lipid in an organic compound to form an organic phase, dissolve the drug in a buffer solution to form an aqueous phase, and mix the organic phase and the aqueous phase to obtain the drug-lipid particles.

33. The preparation method according to claim 32, wherein, The polyphenol molecule is dissolved in ethanol, and then a metal ion is added for reaction to obtain the metal-polyphenol complex.

34. The preparation method according to claim 33, wherein, The molar ratio of the polyphenol molecule to the metal ion is 1:(1~2).

35. The preparation method according to claim 33, wherein, The reaction conditions include reacting at 60°C for 1 hour.

36. The preparation method according to claim 32, wherein, The organic compound is ethanol.

37. The preparation method according to claim 32, wherein, The buffer solution is an enzyme-free Tris-HCl buffer solution.

38. The preparation method according to claim 32, wherein, The mixing method of the organic phase and the aqueous phase includes a microfluidic chip or ultrasound.

39. Use of the drug-lipid particles according to any one of claims 1 to 31 in the preparation of a composition for drug delivery.

40. The use according to claim 39, wherein, The composition is used to introduce the drug into cells.

41. The application according to claim 39, wherein, the composition is a medicament.

42. The application according to claim 41, wherein, the medicament is used to silence the expression of a target sequence in a mammalian subject, to deliver a drug in a mammalian body, to deliver a drug from the body to mammalian cells, or to treat a disease or disorder in a mammalian.

43. The application according to claim 42, wherein, the mammalian is a human.

44. The application according to claim 42, wherein, the disease or disorder is related to the expression of a gene, and the gene contains a target sequence of a drug.

45. The application according to claim 42, wherein, the disease or disorder is cancer or a viral infection, and the virus is SARS-Cov-2.

46. The application according to claim 45, wherein, the cancer includes liver cancer, glioma or lung cancer.

47. The application according to claim 41, wherein, the medicament is a vaccine.

48. The application according to claim 41, wherein, the administration route of the medicament includes intrathecal injection, intramuscular administration, intracranial injection, intravenous injection or intratumoral injection.

49. A medicament comprising the drug-lipid particles according to any one of claims 1 to 31.

50. The medicament according to claim 49, wherein, the medicament is a vaccine.

51. The medicament according to claim 50, wherein, the vaccine is a novel coronavirus vaccine.

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