Metal-phospholipid complex and its preparation method and application

Abstract

The present invention provides a metal-phospholipid complex and a preparation method and application thereof, and relates to the field of biotechnology. The metal-phospholipid complex provided by the present invention is composed of a phospholipid molecule part, a linker molecule part and a metal ion part. The main function of the metal-phospholipid complex is to adsorb negatively charged drugs, which are self-assembled into metal-phospholipid complex particles (MPP) together with other lipids. While ensuring that the effectiveness is not less than that of LNP based on cationic lipids and / or ionizable lipids, cationic lipids and ionizable lipids are not used. Therefore, compared with LNP, toxicity is greatly reduced, biosafety is significantly improved, and it is more conducive to the transportation of negatively charged drugs in vivo.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure requires that the application number submitted to the China Patent Office on August 9, 2022 is

[0003] 202210950391.8, and the priority of the Chinese patent application entitled “Metal-phospholipid complex and its preparation method and application”, the entire contents of which are incorporated by reference in the present disclosure. Technical Field

[0004] The present invention relates to the field of biotechnology, and in particular to a metal-phospholipid complex and a preparation method and application thereof. Background Art

[0005] With the continuous development of molecular biology technology, the understanding of the relationship between genes and diseases has become more and more in-depth. Nucleic acid drugs refer to artificially synthesized DNA or RNA fragments with disease treatment functions. They have attracted much attention because of their great application potential in disease diagnosis and treatment. Such drugs can directly act on pathogenic target genes or mRNAs and exert therapeutic effects at the genetic level. Compared with traditional small molecule drugs and antibody drugs, nucleic acid drugs are not limited by the drugability of target proteins, can treat a wider range of diseases, and can regulate the expression of pathogenic genes from the root. Nucleic acid drugs also have obvious advantages such as high efficiency, low toxicity and high specificity, and are expected to become the third largest type of drugs after small molecule drugs and antibody drugs.

[0006] However, nucleic acid drugs are easily degraded by nucleases in the body, and due to their large molecular weight and negative charge, they are difficult to pass through the cell membrane to exert their effects. Therefore, finding a safe and effective nucleic acid drug delivery system is a bottleneck problem that needs to be solved in the development of nucleic acid drugs. At present, carriers that can deliver nucleic acid drugs can be mainly divided into viral vectors and non-viral vectors. Viral vectors will cause immune responses after entering the human body and are now less used; the more commonly used non-viral vectors 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 increasing their circulation time in the body. The mechanism of nanoparticle encapsulation of nucleic acids is that cationic lipids with positive charges adsorb negatively charged nucleic acids. However, cationic lipids are highly cytotoxic, and their toxic mechanisms are: ① causing cell atrophy, a decrease in the number of mitosis, and cytoplasmic vacuolation; ② interacting with biological proteins such as protein kinase C to destroy their activity; ③ triggering the secretion of a variety of proinflammatory cytokines and chemokines by activating p38 mitogen-activated protein kinase and nuclear factor κB transcription factor. In addition, ionizable lipids are lipids containing positively charged ionizable amine groups, which are uncharged under physiological conditions (pH = 7.4), but will be 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 the adsorption of nucleic acids. When nanoparticles containing ionizable lipids enter the lysosomes of biological cells, the ionizable lipids become positively charged lipids in the low pH (pH = 4.0-6.5) environment in the lysosomes. Although ionizable lipids reduce the cytotoxic and highly inflammatory effects of some permanently positively charged cationic lipids, their cytotoxicity and immunogenicity are still high. Lipid nanoparticles (LNPs) based on cationic lipids and / or ionizable lipids are currently available clinical nanoparticle nucleic acid drug delivery systems. Cationic lipids and / or ionizable lipids are the main components of LNPs and are responsible for the adsorption of nucleic acids. At the same time, the cytotoxicity and immunogenicity mediated by cationic lipids and / or ionizable lipids are still one of the important reasons for the high toxicity of LNPs.

[0007] Therefore, when using a delivery system to deliver negatively charged drugs (such as nucleic acid drugs, protein drugs, polypeptide drugs, small molecule drugs, etc.), the nanoparticle delivery systems developed based on cationic lipids and / or ionizable lipids cannot fundamentally solve the toxicity problem of the nanoparticle delivery system. There is an urgent need for a liposome delivery system with low toxicity that does not use cationic lipids and / or ionizable lipids.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] The first object of the present invention is to provide a metal-phospholipid complex.

[0010] The second object of the present invention is to provide a method for preparing the above metal-phospholipid complex.

[0011] The third object of the present invention is to provide applications of the above metal-phospholipid complex.

[0012] In order to achieve the above object, the present invention particularly adopts the following technical solutions:

[0013] The metal-phospholipid complex provided by the present invention is composed of a phospholipid molecule part, a linker molecule part and a metal ion part, the phospholipid molecule part is connected to the linker molecule part, the linker molecule part is connected to the metal ion part through a coordination bond, and the metal-phospholipid complex is not a cationic lipid or an ionizable lipid.

[0014] Further, the phospholipid molecule is partially selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), 1-phosphoceramide (SP), phosphatidylinositol (PI), phosphatidylthreonine (PT), sphingomyelin (SM), lysophosphatidylcholine (LPC), lysophosphoethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysosphingomyelin (LSM), 1-sphingosine phosphate (S1P), and a combination of one or more of their derivatives. Among them, the "their" in "and their derivatives" refers to "phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), 1-phosphoceramide (SP), phosphatidylinositol (PI), phosphatidylthreonine (PT), sphingomyelin (SM), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysosphingomyelin (LSM), 1-sphingosine phosphate (S1P)". In the present invention, "and their derivatives" have similar meanings. The phospholipid molecule portion may be, for example but not limited to, phosphatidylcholine (PC), phosphatidylcholine (PC) derivatives, phosphatidylethanolamine (PE), phosphatidylethanolamine (PE) derivatives, phosphatidylglycerol (PG), phosphatidylglycerol (PG) derivatives, phosphatidylglycerol (PG) and phosphatidylcholine (PC), phosphatidylcholine (PC) and phosphatidylcholine (PC) derivatives, and the like.

[0015] Furthermore, the phospholipid molecule is selected from

[0016] Phosphatidylcholine (PC) (Formula 1)

[0017] Phosphatidylethanolamine (PE) (Formula 2) Phosphatidylserine (PS) (Formula 3) Phosphatidic acid (PA) (Formula 4) Phosphatidylglycerol (PG) (Formula 5)

[0018] 1-phosphoceramide (SP) (Formula 6)

[0019]

[0020] Phosphatidylinositol (PI) (Formula 7) Phosphatidylthreonine (PT) (Formula 8)

[0021] Sphingomyelin (SM) (Formula 9) Lysolecithin (LPC) (Formula 10) Lysophosphoethanolamine (LPE) (Formula 11) Lysophosphatidylserine (LPS) (Formula 12) Lysophosphatidic acid (LPA) (Formula 13) Lysophosphatidylglycerol (LPG) (Formula 14)

[0022] Lysophosphatidylinositol (LPI) (Formula 15)

[0023] Lysophosphatidylthreonine (LPT) (Formula 16)

[0024] Lyso-sphingomyelin (LSM) (Formula 17)

[0025] 1-Sphingosine phosphate (S1P) (Formula 18) and combinations of one or more of its derivatives;

[0026] Wherein, R1 and R2 are independently:

[0027] Capryloyl

[0028] Lauroyl

[0029] Myristoyl

[0030] Palmitoyl

[0031] Stearyl

[0032] Oleyl

[0033] Linoleyl

[0034] Erucyl

[0035] Arachidyl or

[0036] Phytanoyl

[0037] Furthermore, the phospholipid molecule is partially selected from phosphatidylcholine (PC) (Formula 1), phosphatidylethanolamine (PE) (Formula 2), phosphatidic acid (PA) (Formula 4), phosphatidylglycerol (PG) (Formula 5), ​​and a combination of one or more of their derivatives.

[0038] Furthermore, the phospholipid molecule is partially selected from distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidic acid (DSPA), distearoylphosphatidylglycerol (DSPG), and a combination of one or more of their derivatives.

[0039] Further, the phospholipid molecule is selected from distearoylphosphatidylcholine (DSPC) (Formula 46)

[0040]

[0041] Distearoylphosphatidylethanolamine (DSPE) (Formula 47)

[0042]

[0043] Distearoylphosphatidic acid (DSPA) (Formula 48)

[0044] Distearoylphosphatidylglycerol

[0045] (DSPG)(Formula 49)

[0046] and combinations of one or more of their derivatives.

[0047] Further, the phospholipid molecule is partially selected from DSPC (Formula 46), DSPE (Formula 47) or DSPA (Formula 48) or DSPG (Formula 49).

[0048] Further, the linker molecule is partially selected from curcumin, chlorogenic acid, anthocyanin, quercetin, dihydromyricetin, hesperetin, naringenin, apigenin, catechin, tea polyphenols, epigallocatechin gallate, ellagic acid, morin, epicatechin gallate, catechin gallate, epigallocatechin gallate or picatechin C, and a combination of one or more of their derivatives.

[0049] Further, the linker molecule is selected from

[0050] Curcumin (Formula 19) Chlorogenic acid (Formula 20) Anthocyanin (Formula 21) R7 and R8 are H, OH or OCH 3 , R3 is H or a glycosyl group, R4, R5 and R6 are OH or a glycosyl group;

[0051] Quercetin (Formula 22) Dihydromyricetin (Formula 23) Hesperidin (Formula 24) Naringenin (Formula 25) Apigenin (Formula 26) Catechin (Formula 27) Tea polyphenols (Formula 28) Epigallocatechin gallate (Formula 29) Ellagic acid (Formula 30) Morin (Formula 31) Epicatechin gallate (Formula 32) Catechin gallate (Formula 33) Epigallocatechin gallate (Formula 34) Pingbei alkaloid C (Formula 35) and combinations of one or more of their derivatives.

[0052] Further, the linker molecule is selected from curcumin (Formula 19),

[0053] Dihydrocurcumin (Formula 36)

[0054] Hexahydrocurcumin (Formula 37)

[0055] Curcumin sulfate (Formula 38)

[0056] Bisdemethoxycurcumin (Formula 39) A combination of one or more of .

[0057] Furthermore, the linker molecule is partially selected from a combination of one or more of curcumin (Formula 19), hesperidin (Formula 24), tea polyphenols (Formula 28), and derivatives thereof.

[0058] Furthermore, the linker molecule is partially selected from curcumin (Formula 19), hesperidin (Formula 24) or tea polyphenols (Formula 28).

[0059] Further, the metal ion moiety is selected from Fe 3+ 、Ag + , Ba 2+ , Ca 2+ 、Cd 2+ , Cu 2+ , Fe 2+ , Mn 2+ Mg 2+ 、Mo 2 + 、Zn 2+ , Pt 2+ 、Au 2+ 、Al 3+ 、Ce 3+ 、Co 3+ Cr 3+ 、Eu 3+ , Gd 3+ 、Ni 3+ , W 3+ 、V 3+ 、Zr 3+ A combination of one or more of .

[0060] Further, the metal ion moiety is selected from Fe 3+ , Ca 2+ 、Al 3+ A combination of one or more of .

[0061] Further, the metal ion moiety is selected from Fe 3+ , Ca 2+ or Al 3+ .

[0062] Furthermore, the metal-phospholipid complex is made of a phospholipid molecule part, a linker molecule part and a metal ion part, wherein the phospholipid molecule part is selected from DSPC, DSPE or DSPA, the linker molecule part is selected from curcumin, hesperidin or tea polyphenols, and the metal ion part is selected from Fe 3+ , Ca 2+ or Al 3+ .

[0063] Further, the metal-phospholipid complex is made of a phospholipid molecule part, a linker molecule part and a metal ion part, the phospholipid molecule part is selected from DSPC (Formula 46), DSPE (Formula 47) or DSPA (Formula 48), the linker molecule part is selected from curcumin (Formula 19), hesperidin (Formula 24) or tea polyphenols (Formula 28), and the metal ion part is selected from Fe 3+ , Ca 2+ or Al 3+ .

[0064] Furthermore, the molar ratio of the phospholipid molecule part, the linker molecule part and the metal ion part is 1:1:(0.5-2).

[0065] Further, the phospholipid molecule portion is DSPC (Formula 46), the linker molecule portion is selected from curcumin (Formula 19), and the metal ion portion is selected from Fe 3+ .

[0066] Furthermore, the molar ratio of the phospholipid molecule part, the linker molecule part and the metal ion part is 1:1:1.

[0067] Furthermore, DSPC, curcumin and Fe 3+ / Al 3+ The molar ratio is 1:1:1.

[0068] Furthermore, DSPC, hesperidin and Fe 3+ / Al 3+ The molar ratio is 1:1:1.

[0069] Furthermore, DSPC, tea polyphenols and Fe 3+ / Al 3+ The molar ratio is 1:1:2.

[0070] Furthermore, Fe 3+ Selected from FeCl 3 , Al 3+ Selected from Al(NO 3 ) 3 9H 2 O.

[0071] The present invention provides a method for preparing a metal-phospholipid complex, comprising the following steps:

[0072] Step 1: Reaction and connection of phospholipid molecules and linker molecules to form phospholipid complexes;

[0073] Step 2: reacting the phospholipid complex prepared in step 1 with metal ions through coordination bonds to form a metal-phospholipid complex.

[0074] Furthermore, in step 1, the phospholipid molecule and the linker molecule are dissolved in ethanol for reaction, and then n-hexane is added for precipitation to obtain the phospholipid complex. Furthermore, the reaction conditions include reacting at 65° C. for 2 hours.

[0075] Furthermore, the molar ratio of the phospholipid molecule to the linker molecule is 1:1.

[0076] Furthermore, in step 2, the phospholipid complex and the metal ion are dissolved in ethanol and reacted to obtain the metal-phospholipid complex. Furthermore, the reaction conditions include reacting at 60° C. for 2 hours.

[0077] Furthermore, the molar ratio of the phospholipid complex to the metal ion is 1:(1-2).

[0078] The present invention provides the use of the above-mentioned metal-phospholipid complex in metal-chelatedphospholipid complex nanoparticles (MPP), wherein the metal-phospholipid complex particles contain (i) the above-mentioned metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, wherein the conjugated lipid that inhibits particle aggregation is not a cationic lipid or an ionizable lipid; and (iii) a non-cationic lipid or a non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation. Wherein, "(iii) a non-cationic lipid or a non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation" can be referred to as "a non-cationic lipid or a non-ionizable lipid".

[0079] Further, the conjugated lipid that inhibits particle aggregation includes polyethylene glycol (PEG)-lipid conjugate and / or PEG-dialkoxypropyl (DAA).

[0080] Further, the PEG-lipid conjugate is selected from phosphatidylethanolamine-polyethylene glycol 2000 (PE-PEG2000) (Formula 42)

[0081]

[0082] Phosphatidylethanolamine-polyethylene glycol 700 (PE-PEG700) (Formula 43)

[0083]

[0084] Phosphatidylethanolamine-polyethylene glycol 1000 (PE-PEG1000) (Formula 44)

[0085]

[0086] Phosphatidylethanolamine-polyethylene glycol 5000 (PE-PEG5000) (Formula 45)

[0087]

[0088] , and a combination of one or more of their derivatives;

[0089] R1 and R2 are independently: capryloyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytanoyl.

[0090] Furthermore, the PEG-lipid conjugate is selected from a combination of one or more of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000 or DSPE-PEG5000.

[0091] Further, the PEG-lipid conjugate is selected from DSPE-PEG2000 (Formula 53)

[0092] DSPE-PEG700 (Formula 50) DSPE-PEG1000 (Formula 51)

[0093] or DSPE-PEG5000 (Formula 52) A combination of one or more of .

[0094] Further, the PEG-lipid conjugate is selected from DSPE-PEG2000 (Formula 53), DSPE-PEG700 (Formula 50), DSPE-PEG1000 (Formula 51) or DSPE-PEG5000 (Formula 52).

[0095] Furthermore, in (iii), the non-cationic lipid or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid for inhibiting particle aggregation is a combination of one or more of cholesterol and its derivatives.

[0096] Furthermore, in (iii), the non-cationic lipid or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid for inhibiting particle aggregation is cholesterol (Formula 40) and combinations of one or more of their derivatives.

[0097] Furthermore, the non-cationic lipid or non-ionizable lipid in (iii) is cholesterol (Formula 40).

[0098] Furthermore, in (iii), in addition to the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation, the non-cationic lipid or non-ionizable lipid, in addition to cholesterol, also includes a combination of one or more selected from phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphoceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysophosphatidylcholine LPC, lysophosphoethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, 1-phosphoceramide S1P, cholesterol sulfate, and their derivatives.

[0099] Furthermore, in (iii), in addition to the metal-phospholipid complex and the conjugated lipid for inhibiting particle aggregation, the non-cationic lipid or non-ionizable lipid, in addition to cholesterol, also includes: phosphatidylcholine (PC) (Formula 1)

[0100]

[0101] Phosphatidylethanolamine (PE) (Formula 2)

[0102] Phosphatidylserine (PS) (Formula 3) Phosphatidic acid (PA) (Formula 4) Phosphatidylglycerol (PG) (Formula 5)

[0103] 1-phosphoceramide (SP) (Formula 6)

[0104]

[0105] Phosphatidylinositol (PI) (Formula 7) Phosphatidylthreonine (PT) (Formula 8) Sphingomyelin (SM) (Formula 9)

[0106] Lysolecithin (LPC) (Formula 10) Lysophosphoethanolamine (LPE) (Formula 11) Lysophosphatidylserine (LPS) (Formula 12) Lysophosphatidic acid (LPA) (Formula 13) Lysophosphatidylglycerol (LPG) (Formula 14) Lysophosphatidylinositol (LPI) (Formula 15)

[0107] Lysophosphatidylthreonine (LPT) (Formula 16)

[0108] Lyso-sphingomyelin (LSM) (Formula 17)

[0109] 1-Sphingosine phosphate (S1P) (Formula 18)

[0110] Cholesterol sulfate (Formula 41) and combinations of one or more of its derivatives;

[0111] Wherein, R1 and R2 are: capryloyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytanoyl.

[0112] Furthermore, the non-cationic lipid or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid for inhibiting particle aggregation in (iii) includes cholesterol, and a combination of one or more selected from DSPC, DSPE, DSPA or DSPG.

[0113] Furthermore, the non-cationic lipid or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid for inhibiting particle aggregation in (iii) includes cholesterol (Formula 40), and a combination of one or more selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48) or DSPG (Formula 49).

[0114] Furthermore, the non-cationic lipid or non-ionizable lipid described in (iii) includes cholesterol (Formula 40) and DSPC (Formula 46).

[0115] Furthermore, the metal-phospholipid complex particles are made of (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation, wherein the metal-phospholipid complex accounts for 10% to 40% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 2% to 10% by mole in the raw material, the cholesterol accounts for 35% to 75% by mole in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol accounts for 0% to 40% by mole in the raw material.

[0116] Further, the metal-phospholipid complex particles are made of (i) metal-phospholipid complex, (ii) conjugated lipids that inhibit particle aggregation, and (iii) non-cationic lipids or non-ionizable lipids, the metal-phospholipid complex accounts for 5% to less than 10% by mole in the raw material, the conjugated lipids that inhibit particle aggregation account for 2% to 10% by mole in the raw material, the cholesterol accounts for 15% to less than 35%, 35% to 75%, or greater than 75% to 80% by mole in the raw material, and the non-cationic lipids or non-ionizable lipids other than cholesterol account for 0% to 40% or greater than 40% to 51% by mole in the raw material. Or

[0117] The molar proportion of the metal-phospholipid complex in the raw material is greater than 40% to 50%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, the molar proportion of cholesterol in the raw material is 15% to less than 35%, 35% to 75% or greater than 75% to 80%, and the molar proportion of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 0% to 40% or greater than 40% to 51%. Or

[0118] The molar proportion of the metal-phospholipid complex in the raw material is 10% to 40%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, the molar proportion of cholesterol in the raw material is 15% to less than 35% or greater than 75% to 80%, and the molar proportion of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 0% to 40% or greater than 40% to 51%.

[0119] Furthermore, the molar proportion of the metal-phospholipid complex in the raw material is 7% to less than 10%, 10% to 30% or 20% to 30%, preferably 25%.

[0120] Furthermore, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10% or 5% to 10%, preferably 10%.

[0121] Furthermore, the molar proportion of cholesterol in the raw material is 15% to less than 35%, 35% to 56% or 35% to 55%, preferably 40%.

[0122] Furthermore, the molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 5% to 30%, 25% to 40%, greater than 40% to 45% or 20% to 25%.

[0123] Furthermore, the molar proportion of the metal-phospholipid complex in the raw material is 15% to 25%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 4% to 10%, the molar proportion of the cholesterol in the raw material is 40% to 46%, the molar proportion of the DSPC in the raw material is 25% to 35%, and the metal ion part of the metal-phospholipid complex is selected from Fe 3+ . .

[0124] Furthermore, the metal-phospholipid complex (the metal ion part is Fe 3+ ) accounts for 15% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 4% by mole in the raw material, the cholesterol accounts for 46% by mole in the raw material, and the DSPC accounts for 35% by mole in the raw material; or the metal-phospholipid complex (the metal ion part is Fe 3+ ) accounts for 25% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 10% by mole in the raw material, the cholesterol accounts for 40% by mole in the raw material, and the DSPC accounts for 25% by mole in the raw material.

[0125] Furthermore, the molar proportion of the metal-phospholipid complex in the raw material is 10% to 30%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%, the molar proportion of the cholesterol in the raw material is 35% to 56%, and the molar proportion of the DSPC in the raw material is 34% to 40%; the metal ion part in the metal-phospholipid complex is selected from Al 3+ .

[0126] Furthermore, the molar proportion of the metal-phospholipid complex in the raw material is 10% to 30%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%, the molar proportion of the cholesterol in the raw material is 35% to 56%, the molar proportion of the DSPC in the raw material is 40% to 45%, and the metal ion part in the metal-phospholipid complex is selected from Al 3+ ;or

[0127] The molar proportion of the metal-phospholipid complex in the raw material is 10% to 30%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%, the molar proportion of the cholesterol in the raw material is 15% to 35%, the molar proportion of the DSPC in the raw material is 34% to 40% or greater than 40% to 45%, and the metal ion part of the metal-phospholipid complex is selected from Al 3+ ;or

[0128] The molar proportion of the metal-phospholipid complex in the raw material is 7% to less than 10%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%, the molar proportion of the cholesterol in the raw material is 15% to less than 35% or 35% to 56%, the molar proportion of the DSPC in the raw material is 34% to 40% or greater than 40% to 45%, and the metal ion part of the metal-phospholipid complex is selected from Al 3+ .

[0129] Furthermore, the metal-phospholipid complex (the metal ion part is Al 3+ ) accounts for 7% by mole in the raw material, the conjugated lipid that inhibits particle aggregation accounts for 3% by mole in the raw material, the cholesterol accounts for 56% by mole in the raw material, and the DSPC accounts for 34% by mole in the raw material.

[0130] Furthermore, the metal-phospholipid complex is DSPC, curcumin and Fe 3+ The molar ratio of DSPE-PEG2000 is 1:1:1, the conjugated lipid that inhibits particle aggregation is DSPE-PEG2000, the non-cationic lipid or non-ionizable lipid is cholesterol and DSPC, the molar proportion of the metal-phospholipid complex in the raw material is 15%, the molar proportion of DSPE-PEG2000 in the raw material is 4%, the molar proportion of cholesterol in the raw material is 46%, and the molar proportion of DSPC in the raw material is 35%.

[0131] Furthermore, the metal-phospholipid complex is DSPC, curcumin and Fe 3+ The molar ratio of DSPE-PEG2000 is 1:1:1, the conjugated lipid that inhibits particle aggregation is DSPE-PEG2000, the non-cationic lipid or non-ionizable lipid is cholesterol and DSPC, the molar proportion of the metal-phospholipid complex in the raw material is 25%, the molar proportion of DSPE-PEG2000 in the raw material is 10%, the molar proportion of cholesterol in the raw material is 40%, and the molar proportion of DSPC in the raw material is 25%.

[0132] Furthermore, the metal-phospholipid complex is DSPC, curcumin and Al 3+ The molar ratio of DSPE-PEG2000 is 1:1:1, the conjugated lipid that inhibits particle aggregation is DSPE-PEG2000, the non-cationic lipid or non-ionizable lipid is cholesterol and DSPC, the molar proportion of the metal-phospholipid complex in the raw material is 7%, the molar proportion of DSPE-PEG2000 in the raw material is 3%, the molar proportion of cholesterol in the raw material is 56%, and the molar proportion of DSPC in the raw material is 34%.

[0133] The present invention provides application of the metal-phospholipid complex in drug-lipid particles, wherein the drug is a molecule with negative charge.

[0134] Furthermore, the drug is encapsulated in the metal-phospholipid complex particles.

[0135] Furthermore, the drug is selected from a combination of one or more of nucleic acids, proteins, polypeptides, small molecules, nucleic acid analogs, protein analogs and polypeptide analogs.

[0136] Furthermore, the nucleic acid is selected from one or more combinations of mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, and artificial nucleic acid.

[0137] Further, the nucleic acid is an mRNA sequence encoding eGFP (Enhanced Green Fluorescent Protein) as shown in SEQ ID No.1, an mRNA sequence encoding the receptor binding domain RBD of the novel coronavirus S1 subunit as shown in SEQ ID No.2, an mRNA sequence encoding NY-ESO-1 (New York esophageal squamous cell carcinoma 1) as shown in SEQ ID No.3, an siRNA sequence of the Bcl-2 gene (B-cell lymphoma / Leukemia-2) whose antisense chain is SEQ ID No.4 and whose sense chain is SEQ ID No.21, an siRNA sequence of the PLK1 gene (Polo-like Kinase 1) whose antisense chain is SEQ ID No.6 and whose sense chain is SEQ ID No.23, an siRNA sequence of the Gal-1 gene as shown in SEQ ID No.8, an ASO sequence of the STAT-3 gene as shown in SEQ ID No.10, and an siRNA sequence of the STAT-3 gene as shown in SEQ ID No.11. The ASO sequence of the α-syn gene (α-synuclein) shown in No. 12, the ASO sequence of the Bcl-2 gene shown in SEQ ID No. 14, the mRNA sequence encoding the wild-type novel coronavirus S protein shown in SEQ ID No. 16, the double-stranded DNA sequence whose antisense chain is SEQ ID No. 17 and whose sense chain is SEQ ID NO. 25, the single-stranded DNA shown in SEQ ID No. 18, or the siRNA sequence of the B7-H4 gene shown in SEQ ID No. 19 and whose antisense chain is SEQ ID No. 26.

[0138] The present invention provides a method for preparing the drug-lipid particles, wherein the drug is encapsulated in metal-phospholipid complex particles to obtain the drug-lipid particles.

[0139] Furthermore, the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic lipid or non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer to form an aqueous phase, and the organic phase and the aqueous phase are mixed to obtain drug-lipid particles.

[0140] Furthermore, the organic compound is ethanol.

[0141] Furthermore, the buffer is enzyme-free PBS buffer.

[0142] Furthermore, the mixing method of the organic phase and the aqueous phase includes a microfluidic chip or ultrasound.

[0143] Further, (a) the drug, (i) the metal-phospholipid complex, (ii) the conjugated lipid for inhibiting particle aggregation, and (iii) the non-cationic lipid or the non-ionizable lipid are mixed to obtain the drug-lipid particle.

[0144] Furthermore, the preparation method comprises the following steps:

[0145] Step 1: Reaction and connection of phospholipid molecules and linker molecules to form phospholipid complexes;

[0146] Step 2: reacting the phospholipid complex prepared in step 1 with metal ions through coordination bonds to form a metal-phospholipid complex;

[0147] Step 3: Mixing the metal-phospholipid complex prepared in step 2, (ii) conjugated lipids for inhibiting particle aggregation, (iii) non-cationic lipids or non-ionizable lipids, and drugs to prepare the drug-lipid particles.

[0148] The present invention provides the use of the metal-phospholipid complex in a nucleic acid delivery system. Further, the nucleic acid delivery system is used to introduce nucleic acid into cells. Further, the nucleic acid is used to silence the expression of a target sequence in a mammalian subject, to deliver a drug in a mammal, to deliver a drug from the body to a mammalian cell, or to treat a disease or condition in a mammal.

[0149] In the above technical scheme, further, the mammal is a human. Further, the disease or condition is associated with the expression of a gene, and the gene contains a target sequence of a drug. Further, the disease or condition includes cancer, viral infection, autoimmune disease, diabetes or Alzheimer's disease. Further, the viral infection includes hepatitis A, hepatitis B, hepatitis C, SARS-Cov-2 (2019 new coronavirus), HIV (AIDS virus), HPV (human papillomavirus), influenza, smallpox or syphilis. Further, the cancer includes liver cancer, glioma, melanoma, lung cancer, pancreatic cancer or breast cancer.

[0150] Further, the nucleic acid delivery system is a vaccine. Further, the administration route of the nucleic acid delivery system includes intrathecal injection, intramuscular administration, intracranial injection, intravenous injection or intratumoral injection.

[0151] A medicament containing the metal-phospholipid complex of the present invention.

[0152] Furthermore, the medicament is a vaccine.

[0153] Furthermore, the vaccine is a new coronavirus vaccine.

[0154] Compared with the prior art, the present invention has the following beneficial effects:

[0155] The metal-phospholipid complex provided by the present invention mainly functions to adsorb negatively charged drugs, and self-assembles with other lipids into metal-phospholipid complex particles (MPP). While ensuring the effectiveness not lower than that of LNP based on cationic lipids and / or ionizable lipids, cationic lipids and ionizable lipids are not used. Therefore, compared with LNP, the toxicity is greatly reduced, the biosafety is significantly improved, and it is more conducive to the transportation of negatively charged drugs in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0156] In order to more clearly illustrate the specific implementation scheme of the present invention or the technical scheme in the prior art, the drawings required for use in the specific implementation scheme or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are some implementation schemes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0157] Figure 1-1 The eGFP-mRNA@MPP(Fe 3+ ) The percentage of eGFP-positive cells caused by transfection of 293T;

[0158] Figure 1-2 RBD-mRNA@MPP(Fe 3+ ) The expression level of RBD caused by transfection of 293T;

[0159] Figure 1-3 RBD-mRNA@MPP(Fe 3+ ) ability to induce humoral immunity;

[0160] Figure 1-4 NY-ESO-1-mRNA@MPP(Fe 3+ ) ability to induce humoral immunity;

[0161] Figure 1-5 RBD-mRNA@MPP(Fe 3+ ) ability to induce cellular immunity;

[0162] Figure 1-6 NY-ESO-1-mRNA@MPP(Fe 3+ ) ability to induce cellular immunity;

[0163] Figure 1-7 eGFP-mRNA@MPP(Al 3+ ) The percentage of eGFP-positive cells caused by transfection of 293T;

[0164] Figure 1-8 RBD-mRNA@MPP(Al 3+ ) The expression level of RBD caused by transfection of 293T;

[0165] Figure 1-9 RBD-mRNA@MPP(Al 3+ ) ability to induce humoral immunity;

[0166] Figure 1-10 NY-ESO-1-mRNA@MPP(Al 3+ ) ability to induce humoral immunity;

[0167] Figure 1-11 RBD-mRNA@MPP(Al 3+ ) ability to induce cellular immunity;

[0168] Figure 1-12 NY-ESO-1-mRNA@MPP(Al 3+ ) ability to induce cellular immunity;

[0169] Figure 1-13 Bcl-2-siRNA@MPP(Fe 3+ ) ability to silence target genes;

[0170] Figure 1-14 PLK1-siRNA@MPP(Fe 3+ ) ability to silence target genes;

[0171] Figure 1-15Gal-1-siRNA@MPP(Fe 3+ ) ability to silence target genes;

[0172] Figure 1-16 Bcl-2-siRNA@MPP(Al 3+ ) ability to silence target genes;

[0173] Figure 1-17 PLK1-siRNA@MPP(Al 3+ ) ability to silence target genes;

[0174] Figure 1-18 Gal-1-siRNA@MPP (Al 3+ ) ability to silence target genes;

[0175] Figure 1-19 STAT3-ASO@MPP(Fe 3+ ) ability to silence cellular target genes;

[0176] Figure 1-20 α-syn-ASO@MPP(Fe 3+ ) ability to silence cellular target genes;

[0177] Figure 1-21 Bcl-2-ASO@MPP(Fe 3+ ) ability to silence cellular target genes;

[0178] Figure 1-22 STAT3-ASO@MPP (Al 3+ ) ability to silence cellular target genes;

[0179] Figure 1-23 α-syn-ASO@MPP (Al 3+ ) ability to silence cellular target genes;

[0180] Figure 1-24 Bcl-2-ASO@MPP(Al 3+ ) ability to silence cellular target genes;

[0181] Figure 1-25 The S-mRNA@MPP(Fe 3+) Expression level of S protein caused by transfection of 293T;

[0182] Figure 1-26 The drug (dsDNA and ssDNA)-lipid particles (Fe 3+ ) function;

[0183] Figure 1-27 S-mRNA@MPP(Al provided in Example 3.8.2 of the present invention 3+ ) Expression level of S protein caused by transfection of 293T;

[0184] Figure 1-28 The drug (dsDNA and ssDNA)-lipid particles (Al 3+ ) function;

[0185] Figure 2-1 The differential scanning calorimetry diagram of the phospholipid complex provided in Example 4.1 of the present invention;

[0186] Figure 2-2 The metal-phospholipid complex (Fe 3+ )’s UV absorption pattern;

[0187] Figure 2-3 The metal-phospholipid complex (Al 3+ )’s UV absorption pattern;

[0188] Figure 2-4 The Fe under low pH (pH=5.0) conditions provided in Example 5 of the present invention 3+ Characterization of shedding from metal-phospholipid complexes;

[0189] Figure 2-5 The drug-metal-phospholipid complex particles (Fe 3+ ) elemental analysis;

[0190] Figure 2-6 The drug-metal-phospholipid complex particles (Al 3+ ) Electron microscopic analysis of MPP;

[0191] Figure 2-7 The efficiency of the drug-lipid particles encapsulating nucleic acids (mRNA and siRNA) provided in Example 7 of the present invention;

[0192] Figure 2-8 siRNA / mRNA@MPP provided in Example 8 of the present invention and

[0193] Nucleic acid lysosomal escape ability of siRNA / mRNA@LNP;

[0194] Figure 2-9 The eGFP-positive cell rates of MPP and LNP provided in Example 9 of the present invention;

[0195] Figure 2-10 Comparison of the ability of MPP and LNP to promote mRNA expression provided in Example 10 of the present invention;

[0196] Figure 2-11 Comparison of the ability of MPP and LNP to promote humoral immunity provided in Example 10 of the present invention;

[0197] Figure 2-12 Comparison of the ability of MPP and LNP to promote cellular immunity provided in Example 10 of the present invention;

[0198] Figure 3-1 This is the effect of intratumoral injection of the drug-metal-phospholipid complex particles provided in Example 13 of the present invention in treating liver cancer. DETAILED DESCRIPTION

[0199] definition

[0200] For ease of explanation, the specific terms recorded in this specification, the embodiments and the appended claims are collectively described here. Unless otherwise defined in this specification, the meaning of the scientific and technical vocabulary used herein is the same as that understood and used by those with ordinary knowledge in the technical field to which the present invention belongs. In addition, unless the context requires otherwise, it should be understood that singular terms should include the same plural form, and plural terms should include the singular. Specifically, unless the context clearly indicates otherwise, the terms "at least one (kind)" and "one (kind) or more" used in this article and the appended claims include one (kind), two (kinds), three (kinds) or more (kinds).

[0201] 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 accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the mean value, depending on the consideration of a person of ordinary skill in the art to which the present invention belongs. Except for the experimental examples, or unless otherwise explicitly stated, it should be understood that all ranges, quantities, values ​​and percentages used herein (for example, to describe the amount of material used, the length of time, temperature, operating conditions, quantitative ratios and the like) are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the attached patent scope are all approximate values ​​and can be changed as needed. At least these numerical parameters should be understood as the number of significant digits indicated and the values ​​obtained by applying the general rounding method.

[0202] The term "lipid" refers to a group of organic compounds, including, but not limited to, lipids of fatty acids. They are usually divided into three categories: "simple lipids", "compound lipids", and "derivative lipids". "Simple lipids" include fats and oils as well as waxes; "compound lipids" include phospholipids and glycolipids; "derivative lipids" such as steroids.

[0203] The term "lipid vesicle" refers to any lipid composition that can be used to deliver a compound, including, but not limited to, liposomes, in which an aqueous volume is encapsulated by an amphiphilic lipid bilayer; or an interior in which lipids encapsulate a macromolecular component, such as mRNA, with a reduced aqueous interior; or lipid aggregates or micelles, in which the encapsulated components are contained in a relatively chaotic lipid mixture. Herein, metal-phospholipid complex particles (MPPs) are "lipid vesicles", and drugs, such as nucleic acid mRNA, are encapsulated in MPPs as encapsulated components, and the "encapsulation" can be full encapsulation and / or partial encapsulation.

[0204] The term "phospholipid" refers to lipids containing phosphate groups, which belong to complex lipids, also known as phospholipids and phospholipids. Phospholipids are the main components of biological membranes and are divided into two major categories: glycerophospholipids and sphingomyelin, which are composed of glycerol and sphingosine respectively. Phospholipids are amphiphilic molecules, with a hydrophilic nitrogen or phosphorus head at one end and a hydrophobic (lipophilic) long hydrocarbon chain at the other end. For this reason, the hydrophilic ends of phospholipid molecules are close to each other, and the hydrophobic ends are close to each other, and they often form a phospholipid bilayer, i.e. the structure of the cell membrane, together with other molecules such as proteins, glycolipids, and cholesterol.

[0205] The phrase "phospholipid molecule portion" herein refers to the structure originally belonging to the phospholipid molecule after the phospholipid molecule reacts with other substances.

[0206] The phrase "linker molecule moiety" herein refers to the structure originally belonging to the linker molecule after the linker molecule reacts with other substances.

[0207] The phrase "metal ion moiety" herein refers to the structure originally belonging to the metal ion after the metal ion moiety reacts with other substances.

[0208] The phrase "phospholipid complex" herein refers to a complex formed by reaction and connection between the above-mentioned phospholipid molecule part having a phosphate group and the above-mentioned linker molecule part.

[0209] The phrase "metal-phospholipid complex" herein refers to a complex composed of the above-mentioned phospholipid molecule part having a phosphate group, the above-mentioned linker molecule part, and the above-mentioned metal ion part, wherein the above-mentioned phospholipid molecule part is connected to the above-mentioned linker molecule part, and the above-mentioned linker part is connected to the above-mentioned metal ion part through a coordination bond, and the metal-phospholipid complex is neither a cationic lipid nor an ionizable lipid.

[0210] 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 lower pH values ​​but is uncharged at physiological pH conditions.

[0211] The term "neutral lipid" refers to any of a number of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0212] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic groups attached to neutral lipids.

[0213] The term "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH. These lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTMA");

[0214] N, N-distearyl-N, N-dimethylammonium bromide ("DDAB"); N-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride ("DOTAP"); 3-(N-(N', N'-dimethylaminoethane)carbamoyl)cholesterol ("DC-Chol") and N-(1,2 dimyristyloxyprop-3-yl)-N, N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"). The following lipids are cationic and have a positive charge below physiological pH: DODAP, DODMA, DMDMA, etc.

[0215] The term "hydrophobic lipid" refers to a compound having a non-polar group, which includes, but is 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, diacylglycerols, dialkylglycerols, NN-dialkylamino, 1,2-diacyloxy-3-aminopropane and 1,2-dialkyl-3-aminopropane.

[0216] The term "non-cationic lipid or non-ionizable lipid" refers to a lipid that is neither a cationic lipid nor a non-ionizable lipid, and may be, for example, an anionic lipid or a neutral lipid.

[0217] Among the components of the metal-phospholipid complex particles, "non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation" in (iii) refers to the lipids remaining in the metal-phospholipid complex particles after excluding the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation.

[0218] The term "fusogenicity" refers to the ability of a liposome, drug-lipid particle or other drug delivery system to fuse with a cell membrane. The membrane may be a plasma membrane or a membrane surrounding a cell organelle, such as an endosome, nucleus, etc.

[0219] In the metal-phospholipid complex particles, non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation are mainly present as vesicle-forming lipids, and the term "vesicle-forming lipids" is intended to include any amphiphilic lipids having a hydrophobic part and a polar head group and which can spontaneously form bilayer vesicles in water by themselves, exemplified by most phospholipids.

[0220] In the metal-phospholipid complex particles, the conjugated lipids that inhibit particle aggregation are primarily present as vesicle-adopting lipids, and the term "vesicle-adopting lipids" is intended to include any amphipathic lipids that stably associate with the lipid bilayer, as well as other amphipathic lipids whose hydrophobic portions are in contact with the inner, hydrophobic regions of the bilayer membrane and whose polar head group portions are oriented toward the outer, polar surface of the membrane. Vesicle-adopting lipids include lipids that are independently capable of adopting a non-lamellar phase and are also capable of adopting a bilayer structure in the presence of a bilayer stabilizing component. Conjugated lipids that inhibit drug-lipid particle aggregation include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid derivatives), peptides, proteins, detergents, lipid derivatives, PEG-lipid derivatives such as PEG coupled to dialkoxypropyl, PEG coupled to diacylglycerol, PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramide (see, U.S. Pat. No. 5,885,613, which is incorporated herein by reference).

[0221] The term "amphiphilic lipid" refers to any suitable material, wherein the hydrophobic portion of the lipid material is oriented toward the hydrophobic phase, and the hydrophilic portion is oriented toward the aqueous phase. Amphiphilic lipids are typically the main components of lipid vesicles. The hydrophilic nature comes from the presence of polar or charged groups such as carbohydrates, phosphates, carboxyls, sulfate radicals, aminos, sulfhydryls, nitros, hydroxyls and other similar groups. Hydrophobicity can be imparted by the inclusion of non-polar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, alicyclic or heterocyclic groups. Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino lipids and sphingolipids. The representative example of phospholipid includes, but is not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus, such as sphingomyelin, sphingolipid family, diacylglycerol and beta-acyloxy acid are also in the group referred to as amphipathic lipid. In addition, above-mentioned amphipathic lipid can be mixed with other lipids, and this lipid comprises triglyceride and sterol.

[0222] The term "diacylglycerol" refers to a compound having a 2-fatty acyl chain, wherein R1 and R2 each independently have 2 to 30 carbon atoms bonded to the 1- and 2-positions of glycerol via ester bonds. The acyl groups may be saturated or have varying degrees of unsaturation. Diacylglycerol has the following formula 54:

[0223]

[0224] The term "diacylglycerol-coupled polyethylene glycol", the conjugated lipid that inhibits particle aggregation in the present invention can be diacylglycerol-coupled polyethylene glycol, that is, diacylglycerol-polyethylene glycol conjugate (DAG-PEG conjugate or PEG-DAG conjugate). In a preferred embodiment, the DAG-PEG conjugate is a dilaurylglycerol (C12)-PEG conjugate, a ditetradecylglycerol (C14)-PEG conjugate (DMG), a dipalmitoylglycerol (C16)-PEG conjugate or a distearylglycerol (C18)-PEG conjugate (DSG). Those skilled in the art will readily understand that other diacylglycerols can be used in the DAG-PEG conjugate of the present invention. Suitable DAG-PEG conjugates used in the present invention and methods of preparing and using them are disclosed in U.S. Application No. 10 / 136,707, published as USPA 2003 / 0077829, and PCT Patent Application No. CA 02 / 00669, the entire contents of each of which are incorporated by reference.

[0225] The term "dialkoxypropyl" refers to a compound having a 2-alkyl chain, wherein R1 and R2 each independently have 2-30 carbons. The alkyl group may be saturated or have varying degrees of unsaturation. Dialkoxypropyl has the following formula 55:

[0226]

[0227] The term "dialkoxypropyl-coupled PEG", the conjugated lipid that inhibits particle aggregation in the present invention can be a dialkoxypropyl-coupled PEG, that is, a dialkoxypropyl conjugate (PEG-DAA conjugate). In a preferred embodiment, the PEG-DAA conjugate has the following formula 56:

[0228] In formula 56, R1 and R2 are independently selected and are long chain alkyl groups having about 10 to about 22 carbon atoms. The long chain alkyl group may be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, lauryl (C12), tetradecyl (C14), hexadecyl (C16), octadecyl (C18) and icosyl (C20). In a preferred embodiment, R1 and R2 are the same, i.e., R1 and R2 are both tetradecyl (i.e., dioctadecyl), R1 and R2 are both octadecyl (i.e., dioctadecyl), etc. In formula 56, PEG is a polyethylene glycol having an average molecular weight of about 550 to about 10,000 daltons and is optionally substituted by an alkyl, alkoxy, acyl, or aryl group at the terminal hydroxyl position. In a preferred embodiment, PEG has an average molecular weight of about 1,000 to about 5,000 daltons, more preferably, an average molecular weight of about 1,000 to about 3,000 daltons and even more preferably, an average molecular weight of about 2,000 daltons. PEG can be optionally substituted with alkyl, alkoxy, acyl or aryl groups. In Formula 56, L is a linker moiety. Any linker moiety suitable for coupling PEG to the dialkoxypropyl backbone can be used. Suitable linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbonate (OC(O)O-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), succinyl (-(O)CCH 2 CH 2 C(O)-), ethers, disulfides and combinations thereof. Other suitable linkers are well known in the art.

[0229] Phosphatidylethanolamine can be conjugated with polyethylene glycol to form a double-layer stabilizing component as a conjugated lipid that inhibits particle aggregation in the present invention, and the phosphatidylethanolamine has various acyl chain groups with different chain lengths and degrees of saturation. These phosphatidylethanolamines are commercially available, or can be separated or synthesized using conventional techniques known to those skilled in the art. Phosphatidylethanolamines containing saturated or unsaturated fatty acids are preferred, and have a carbon chain length within the range of C10-C20. Such phosphatidylethanolamines can also be used, which have a mixture of mono- or di-unsaturated fatty acids and saturated and unsaturated fatty acids. Suitable phosphatidylethanolamines include, but are not limited to, as follows: dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidylethanolamine (DSPE).

[0230] Like phosphatidylethanolamine, ceramide can be coupled with polyethylene glycol to form a double-layer stabilizing component as a conjugated lipid that inhibits particle aggregation in the present invention, and the ceramide has multiple acyl chain groups with different chain lengths and degrees of saturation. It should be clear to those skilled in the art that, compared with phosphatidylethanolamine, ceramide has only one acyl group, which can be easily changed 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 methods and techniques disclosed in U.S. Patent No. 5,820,873, which is incorporated herein by reference. Using the synthetic pathways proposed in the aforementioned applications, ceramides with saturated or unsaturated fatty acids can be prepared, and the fatty acids have carbon chain lengths ranging from C2 to C31.

[0231] The term "ATTA" or "polyamide" refers to, but is not limited to, compounds disclosed in U.S. Patent Nos. 6,320,017 and 6,586,559, both of which are incorporated herein by reference. These compounds include compounds having the following formula 57:

[0232]

[0233] 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 an amino acid side chain; R3 is a member selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, thiol, hydrazine, amino, and NR4R5, wherein R4 and R5 are independently hydrogen or alkyl; n is 4-80; m is 2-6; p is 1-4; and q is 0 or 1. Those skilled in the art will appreciate that other polyamides can be used in the compounds of the present invention.

[0234] The term "congener" refers to an analog that performs the same or similar function, or a derivative of the same parent core that performs the same or similar function.

[0235] As used herein, the terms "mRNA" or "messenger polyribonucleotide" or "messenger RNA" or "messenger RNA" are used interchangeably and refer to a single-stranded polyribonucleotide that is transcribed using one strand of DNA as a template, carries genetic information, and can guide protein synthesis.

[0236] As used herein, the terms "sgRNA" or "small guide RNA" or "guide RNA" or "gRNA" are used interchangeably and are small non-coding RNAs that pair with pre-mRNAs and guide the insertion or deletion of uridine residues into kinetoplastids during RNA editing. gRNA edits RNA molecules, which are approximately 60-80 nucleotides in length and are transcribed from separate genes.

[0237] As used herein, the terms "circRNA" or "circular RNA" or "circular polyribonucleotide" or "circular RNA" are used interchangeably and refer to a polyribonucleotide having no free ends (i.e., no free 3' and / or 5' ends).

[0238] The invention relates to a polyribonucleotide molecule having a polyribonucleotide structure having a ring-shaped or annular structure, for example, a polyribonucleotide molecule having a polyribonucleotide structure having a ring-shaped or annular structure formed by covalent or non-covalent bonds.

[0239] As used herein, the terms "microRNA" or "miRNA" or "microRNA" are used interchangeably and refer to a non-coding single-stranded polyribonucleotide with a length of about 22 nucleotides and free 3' and 5' ends, which can inhibit the translation of target gene protein and thus regulate the biological function of cells by binding to the 3'-untranslated region (3'-UTR) of the mRNA of the target gene.

[0240] As used herein, the terms "ASO" or "antisense oligonucleotide" or "antisense oligonucleotide" are used interchangeably and refer to artificially synthesized nucleic acid fragments that are complementary to a certain segment of a target gene or mRNA, and can bind to the target gene / mRNA through the principle of base complementarity, thereby blocking the expression of the gene, including antisense DNA and antisense RNA.

[0241] As used herein, the terms "siRNA" or "small interfering" or "short interfering" or "silencing RNA" or "small interfering RNA" or "short interfering RNA" or "silencing RNA" are used interchangeably and refer to a class of double-stranded RNA molecules with a length of 20 to 25 nucleotides that can induce degradation of target gene mRNA.

[0242] As used herein, the terms "ecDNA" or "extrachromosomal circular DNA" are used interchangeably and refer to DNA that has fallen off chromosomes and exists in a circular structure outside the chromosomes.

[0243] The term "nucleic acid derivative" refers to the modification or substitution of a nucleic acid sequence, including but not limited to chemical modification of residues, substitution of nucleotides or deoxynucleotides, modification of sequences to increase half-life or stability, and labeling modification. For example, chemical modification includes but is not limited to phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopization of any one or more bases. Substitution of nucleotides or deoxynucleotides includes but is not limited to nucleic acid analogs that replace the sugar-phosphate backbone with polypeptides or other backbones (replacing DNA or RNA with PNA). Modifications to sequences to increase half-life or stability include but are not limited to modification with PEG linkage, fluorine modification. Labeling modification includes but is not limited to linkage with fluorescent groups, amino groups, biotin, digoxin, small peptides, etc.

[0244] The term "artificial nucleic acid" refers to a nucleic acid molecule that has been artificially modified, including but not limited to base modification, ribose modification, PNA, etc.

[0245] The term "nucleic acid" refers to a polymer that contains at least two deoxynucleotides or nucleotides in single or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequences explicitly indicated. 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 substituted by mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);

[0246] Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); and Cassol et al. (1992);

[0247] Rossolini et al., Mol. Cell. Probes 8:91-98 (1994). "Nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are linked by the phosphate group. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that substitute new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and halides. DNA can be present as antisense, plasmid DNA, portions of plasmid DNA, pre-compressed DNA, products of polymerase chain reaction (PCR), vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. The term nucleic acid is used interchangeably with gene, cDNA, mRNA encoded by a gene, and interfering RNA molecules.

[0248] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that includes a partial or full-length coding sequence that is necessary for producing a polypeptide or polypeptide precursor (e.g., a polypeptide or polypeptide precursor from hepatitis A, B, C, D, E, G virus; or herpes simplex virus).

[0249] As used herein, "gene product" refers to the product of a gene such as, including, for example, the transcript of DNA, mRNA.

[0250] The phrase "silencing the expression of a target gene" refers to the ability of the siRNA of the present invention to initiate silencing of a target gene. In order to determine the extent of gene silencing, a sample or assay of cells in a target organism or culture is compared with a control sample, wherein the cells of the target organism or culture express a specific construct and the control does not express the construct. The control sample (lack of expression of the construct) is set to a relative value of 100%. When the test value relative to the control is about 90%, preferably 50%, and more preferably 25%-0%, inhibition of the expression of the target gene is successfully obtained. Suitable assays include, for example, using techniques known to those skilled in the art such as dot stains, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme action, and phenotypic assays known to those skilled in the art to detect protein or mRNA levels.

[0251] A "therapeutically effective amount" or "effective amount" of siRNA is an amount sufficient to produce the desired effect, such as a decrease in expression of the target sequence as compared to normal expression levels detected in the absence of the siRNA.

[0252] As used herein, the term "aqueous solution" refers to a composition comprising wholly or partially water.

[0253] As used herein, the term "organic lipid solution" refers to a composition comprising in whole or in part an organic solvent with lipids.

[0254] When used herein, "systemic delivery" refers to the delivery that causes a compound to be widely biodistributed in an organism. Some techniques of administration can result in systemic delivery of certain compounds, but cannot result in systemic delivery of other compounds. Systemic delivery refers to effective, preferably, therapeutic amounts of compounds in contact with most of the body. In order to obtain extensive biodistribution, a blood survival period is usually required so that the compound is not rapidly degraded or cleared (such as by first passing through an organ (liver, lung, etc.) or by rapid, nonspecific cell binding) before reaching the disease site at the distal end of the site of administration. The systemic delivery of drug-lipid particles can be carried out in any manner known in the art, including, for example, intravenous, subcutaneous, intraperitoneal, and in a preferred embodiment, the systemic delivery of drug-lipid particles is by intravenous delivery.

[0255] As used herein, "local delivery" refers to the delivery of a compound directly to a target site in vivo. 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.

[0256] Metal-phospholipid complex

[0257] In the present invention, the metal-phospholipid complex is composed of a phospholipid molecule portion, a linker molecule portion and a metal ion portion.

[0258] Regarding the phospholipid molecule, it should be noted that the cis-trans isomers of the phospholipid molecule of the present invention will not affect the effects to be achieved by the protection content of the present invention.

[0259] As for the linker molecule part, it is mainly derived from natural plant extracts, such as curcumin, which has a wide range of biological effects, including antibacterial, antiviral, antifungal, antioxidant and anti-inflammatory activities. In addition, it is also an effective immunomodulator that can regulate the activity of various immune cells such as T cells, B cells, macrophages, neutrophils, natural killer cells and dendritic cells, promote the balance of immunity, and enhance the body's immunity. Based on the potential immune enhancement, anti-inflammatory, antioxidant and anti-SARS-CoV-2 effects of curcumin molecules, it is expected to become a potential auxiliary treatment for COVID-19. In addition, the safety of curcumin molecules is extremely high and has been listed in the catalog of food additives and pharmaceutical excipients. Its safety is conducive to the clinical drug registration of the drug-lipid as a whole, shortening the length of clinical drug registration.

[0260] As for the metal ion part, the coordination bond between the linker molecule part and the metal ion part in the metal-phospholipid complex will be broken under the low pH conditions (pH=5.0) of lysosomes, for example, and the metal ion will fall off from the metal-phospholipid complex.

[0261] The proportion of each component in the metal-phospholipid complex can be adjusted according to the structure of the specific metal-phospholipid complex component. The basis for adjusting the proportion is: because the phospholipid molecule and the linker molecule are connected by hydrogen bonds, as long as the phospholipid molecule contains multiple phosphate groups, when synthesizing the phospholipid complex, the proportion of the phospholipid molecule and the linker molecule can be adjusted according to the number of phosphate groups contained in the phospholipid molecule, that is, when the phospholipid molecule contains two phosphate groups, the proportion of the phospholipid molecule and the linker molecule can be adjusted to 1:2; when the phospholipid molecule contains three phosphate groups, the proportion of the phospholipid molecule and the linker molecule can be adjusted to 1:3; because the hydroxyl group of the linker molecule is connected to the metal ion by a coordination bond, as long as the linker molecule contains multiple binding sites, the proportion of the linker molecule and the metal ion can be adjusted according to the number of binding sites contained in the linker.

[0262] First, the phospholipid molecule with a phosphate group is connected to the linker molecule to obtain a phospholipid complex, and then connected to the metal ion to obtain a metal-phospholipid complex. Specifically, the phospholipid molecule and the linker molecule are dissolved in an appropriate amount of ethanol according to a molar ratio, reacted at about 65°C, and then n-hexane is added to precipitate to obtain a phospholipid complex; the phospholipid complex is then connected to the metal ion (such as FeCl 3 etc.) are dissolved in an appropriate amount of ethanol according to a molar ratio and reacted at about 60° C. to obtain a metal-phospholipid complex.

[0263] Metal-chelated phospholipid complex nanoparticles (MPP)

[0264] The principle of loading nucleic acid on metal-phospholipid complex particles assembled by metal-phospholipid complexes is that the linker molecule is bound together with the phospholipid molecule by hydrogen bonds, and the linker molecule is connected to the metal ion by coordination bonds to form a metal-phospholipid complex, and the metal ion of the metal-phospholipid complex is connected to the negatively charged drug by coordination bonds, thereby ensuring that the metal-phospholipid complex and other components (conjugated lipids that inhibit particle aggregation, and non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation) self-assemble into MPP and load the negatively charged drug into the nanoparticle MPP. In this article, the "non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation" refers to component (iii) in the metal-phospholipid complex particles.

[0265] In some embodiments, the conjugated lipids that inhibit particle aggregation refer to conjugated lipids that inhibit drug-lipid particle aggregation, and their main function is to prevent the aggregation of drug-lipid particles, such as PEG coupled to dialkoxypropyl, PEG coupled to diacylglycerol, PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramide, preferably PEG-lipid conjugates. Wherein, the cis-trans isomers of the lipids will not affect the effects to be achieved in the protection content of the present invention.

[0266] In some embodiments, the molar proportion of the metal-phospholipid complex in the raw material is 5% to less than 10%, 10% to 40%, or greater than 40% to 50%, 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%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%.

[0267] In a preferred embodiment, the molar proportion of the metal-phospholipid complex in the raw material is 7% to 40%, for example, it can be 10% to 40%, 7% to 30%, 15% to 25%, 20% to 30%, and more preferably 15%, 25%, 7% or 30%.

[0268] 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%. For example, it can be 3% to 10%, 4% to 10%, 5% to 10%, and more preferably 3%, 4% or 10%.

[0269] In some embodiments, the non-cationic lipid or non-ionizable lipid is cholesterol, and the molar proportion of cholesterol in the raw material is 15% to less than 35%, 35% to 75%, or greater than 75% to 80%, for example, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, %, 9%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%. For example, it can be 35% to 75%, 15% to 56%, 40% to 46% and 35% to 55%, preferably 15%, 40%, 46% or 56%.

[0270] In some embodiments, in addition to cholesterol, the metal-phospholipid complex particles may optionally contain other non-cationic lipids or non-ionizable lipids, which account for 0% to 40% or more than 40% to 51% by mole in the raw material, for example, 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,

[0271] 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or 51%. For example, 5% to 30%, 25% to 35%, 34% to 45%, 20%-25%, preferably 25%, 35%, 34% or 45%.

[0272] Drug-lipid particles

[0273] The drug-lipid particles described herein typically include a drug (a negatively charged molecule, which may be selected from a nucleic acid, a protein, a polypeptide, a small molecule, a nucleic acid analog, a member of a group of one or more combinations of a protein analog, a polypeptide analog, a nucleic acid selected from a mRNA, a siRNA, a circular RNA, a microRNA, a DNA, an ecDNA, a member of a group of one or more combinations of an artificial nucleic acid), a metal-phospholipid 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 aqueous solution.

[0274] In some embodiments, the drug is fully encapsulated inside the metal-phospholipid complex particles to avoid drug degradation, thereby achieving drug delivery into cells.

[0275] In some embodiments, the drug-lipid particles provided by the present invention have a small diameter suitable for systemic delivery, with a particle size of 30 to 400 nm; a surface potential of -10 to 10 mV; stability of at least 3 days, preferably more than 7 days; and a cell delivery efficiency of at least 40%, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

[0276] 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.

[0277] The RNA population can be used to provide long precursor RNAs, or long precursor RNAs with substantial or complete identity to a selected target sequence that can be used to prepare siRNA. The RNAs can be isolated, synthesized, and / or cloned from cells or tissues according to methods well known to those skilled in the art. The RNA can be a mixed population (obtained from cells or tissues, transcribed from cDNA, etc.), or can represent a single target sequence. The RNA can be naturally occurring, such as isolated from a tissue or cell sample, synthesized in vitro, such as using T7 or SP6 polymerase and PCR products or cloned cDNA; or chemically synthesized.

[0278] To form long dsRNA, for synthetic RNAs, the complement can also be transcribed in vitro and hybridized to form dsRNA. If a naturally occurring RNA population is used, for example, by transcribing cDNAs corresponding to the RNA population, or by using RNA polymerase, RNA complements are also provided (e.g., to form dsRNA, which is digested by E. coli RNAse III or Dicer). The precursor RNA is then hybridized to form double-stranded RNAs and digested. The dsRNAs can be directly encapsulated in SNALPs or can be digested in vitro before encapsulation.

[0279] 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 into a sequence that automatically folds into a duplex with a hairpin loop from a DNA template in a plasmid having an RNA polymerase III transcription unit (see, Brummelkamp, ​​et al., Science 296:

[0280] 550 (2002); Donzé, et al., Nucleic Acids Res. 30: e46 (2002); Paddison, et al.,

[0281] 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. 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, which is operably linked to a template for transcription of the desired siRNA sequence, the termination sequence comprising 2-3 uridine residues and a polythymidine (T5) sequence (polyadenylation signal) (Brummelkamp, ​​Science, supra). The selected promoter can provide constitutive or inducible transcription. Compositions and methods of transcription of DNA-guided RNA interference molecules are described in detail in U.S. Patent No. 6,573,099, which is incorporated herein by reference. Preferably, the synthetic or transcribed siRNA has a 3' overhang of about 1-4, preferably about 2-3 nucleotides and a 5' phosphate terminus (Elbashir, et al., Genes Dev. 15: 188 (2001); et al., Cell 107:309 (2001). The transcription unit is incorporated into a plasmid or DNA vector from which the interfering RNA is transcribed. Plasmids suitable for in vivo delivery of genetic material for therapeutic purposes are described in detail in U.S. Pat. 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 clear to those skilled in the art that a plasmid initially designed to express a desired gene sequence can be modified to include a transcription unit cassette that transcribes an siRNA.

[0282] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, preparing and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983);

[0283] Sambrook et al., supra; Ausubel et al., supra), as do 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 disclosing general methods for use 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

[0284] Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

[0285] Applications of metal-phospholipid complexes, metal-phospholipid complex particles, and drug-lipid particles

[0286] In some embodiments, when the drug is a nucleic acid, the metal-phospholipid complex and metal-phospholipid complex particles can be used to promote the lysosomal escape of the drug and promote the expression of the nucleic acid. The metal-phospholipid complex and metal-phospholipid complex particles can also be used to deliver drugs and introduce the drugs into cells, thereby achieving the prevention and treatment of applicable diseases or conditions by the drugs.

[0287] In some embodiments, the present invention provides the application of the drug-lipid particles, for example, for compositions, which can achieve drug delivery or introduce drugs into cells. The composition is, for example, a medicament, which can achieve: silencing the expression of the target sequence in a mammalian subject, delivering drugs in a mammal (drugs are, for example, for treating tumors, imaging agents, etc.), delivering drugs from the body to mammalian cells or treating diseases or disorders of mammals, etc. In the medicament, the drug-lipid particles are the main active ingredients, which can be prepared into different dosage forms according to actual needs, such as solid dosage forms (powders, granules, pills, tablets, gels), semi-solid dosage forms (external ointments, pastes), liquid dosage forms (decoctions, mixtures, syrups, wines, injections), gaseous dosage forms (aerosols, smoke agents), etc.; for example, dosage forms for administration through the gastrointestinal tract, dosage forms for administration through the rectum, and dosage forms for non-gastrointestinal administration, etc.

[0288] In some embodiments, the present invention provides products prepared from the above-mentioned metal-phospholipid complexes, metal-phospholipid complex particles, and drug-lipid particles. The products have the above-mentioned functions and uses of metal-phospholipid complexes, metal-phospholipid complex particles, and drug-lipid particles. The specific types can be, for example, but not limited to, kits, medicines, etc., and the products may optionally contain other excipients.

[0289] For target genes for drug-lipid particle action: Generally, it is desirable to deliver drug-lipid particles so that the translation (i.e., expression) of the target gene product is downregulated or silenced. Suitable classifications of gene products include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., diseases and disorders in which the liver is a target, and liver diseases and disorders), genes associated with tumorigenesis and cell transformation, angiogenic genes, immunomodulator genes such as those associated with inflammation and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.

[0290] Genes associated with viral infection and survival include those expressed by the virus so that it binds, enters and replicates in cells. In particular, viral sequences associated with chronic viral diseases. For example, viral sequences include sequences of hepatitis viruses (Hamasaki, et al., FEBS Lett. 543: 51 (2003); Yokota, et al, EMBO Rep. 4:

[0291] 602(2003); Schlomai, et al., Hepatology 37:764(2003); Wilson, et al.,

[0292] Proc. Natl. Acad. Sci. 100: 2783 (2003); Kapadia, et al., Proc. Natl. Acad. Sci. 100: 2014 (2003); and FIELDSVIROLOGY (Knipe et al. eds. 2001)), human immunodeficiency virus (HIV) (Banerjea, et al., Mol Ther. 8: 62 (2003); Song, et al., J. Virol. 77: 7174 (2003);

[0293] Stephenson JAMA 289: 1494 (2003); Qin, et al., Proc. Natl. Acad. Sci. 100: 183 (2003)), herpes virus (Jia, et al., J. Virol. 77: 3301 (2003)), and human papilloma virus (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 proteases (e.g., NS3 / NS4), helicases (e.g., NS3), polymerases (e.g., NS5B), and envelope proteins (e.g., E1, E2, and p7). Hepatitis A nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_001489; hepatitis B nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_003977; hepatitis C nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_004102; hepatitis D nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_001653; hepatitis E nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_001434; and hepatitis G nucleic acid sequences are referenced, for example, in Genbank Accession No. NC_001710. Silencing sequences encoding genes associated with viral infection and survival can be conveniently used in conjunction with the administration of conventional agents for treating viral diseases.

[0294] Genes associated with metabolic diseases and disorders (e.g., disorders in which the liver is targeted and liver diseases and disorders) include, for example, genes expressed in dyslipidemia (e.g., liver X receptors (e.g., LXRα and LXRβ Genback Accession No. NM_007121)), farnesoid X receptor (FXR) (Genbank Accession No. NM_005123), sterol regulatory element binding protein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl Coenzyme-A reductase (HMG Coenzyme-A reductase), apolipoprotein (ApoB), and apolipoprotein (ApoE)) and diabetes (e.g., glucose-6-phosphate) (see, e.g., Forman et al., Cell 81:687 (1995); Seol et al., Mol. Endocrinol. 9:72 (1995), Zavacki et al., PNAS USA 94: 7909 (1997); Sakai, et al., Cell 85: 1037-1046 (1996); Duncan, et al., J. Biol. Chem. 272: 12778-12785 (1997); Willy, et al., Genes Dev. 9 (9): 1033-45 (1995); Lehmann, et al. al., J. Biol. Chem. 272(6): 3137-3140 (1997); Janowski, et al., Nature 383:728-731 (199; Peet, et al., Cell 93:693-704 (1998)). One skilled in the art will appreciate that genes associated with metabolic diseases and disorders (e.g., diseases and disorders in which the liver is targeted and liver diseases and disorders) include genes expressed in the liver itself as well as genes expressed in other organs and tissues. Silencing sequences encoding genes associated with metabolic diseases and disorders can be conveniently used in conjunction with the administration of conventional agents for treating the disease or disorder.

[0295] Examples of genes associated with tumorigenesis and cell transformation include translocation sequences such as MLL fusion gene, BCR-ABL (Wilda, et al., Oncogene, 21:5716 (2002); Scherr, et al, Blood 101:1566), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO and AML1-MTG8 (Heidenreich, et al., Blood 101:3157 (2003)); overexpressed sequences such as multidrug resistance genes (Nieth, et al., FEBSLett. 545:144 (2003); Wu, et al, Cancer Res. 63:1515 (2003)), cell cycle proteins (Li, et al., Cancer Res. 63:3593 (2003); Zou, et al, Cancer Res. 63:3615 (2003)). al., Genes Dev. 16:2923 (2002)), β-catenin (Verma, et al., Clin Cancer Res. 9:1291 (2003)), telomerase gene (Kosciolek, et al., Mol Cancer Ther. 2:209 (2003)), c-MYC, N-MYC, BCL-2, ERBB1 and ERBB2 (Nagy, et al. Exp. Cell Res. 285:39 (2003)); and mutant sequences such as RAS (reviewed in Tuschl and Borkhardt, Mol. Interventions, 2:158 (2002)). Silencing sequences encoding DNA repair enzymes is used in conjunction with administration of chemotherapeutic agents (Collis, et al., Cancer Res. 63:1550 (2003)). Genes encoding proteins associated with tumor migration are also target sequences of interest, such as integrins, selectins, and metalloproteinases. Any complete or partial gene sequence that is beneficial to or promotes tumorigenesis or cell transformation, tumor growth, or tumor migration can be included as a template sequence.

[0296] Angiogenic genes can promote the formation of new blood vessels, and vascular endothelial growth factor (VEGF) is a key research direction (Reich, et al., Mol. Vis. 9: 210 (2003)).

[0297] Immunomodulator genes are genes that regulate one or more immune responses. Examples of immunomodulator genes include cytokines such as growth factors (e.g., TGF-α, TGF-β, EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12 (Hill, et al., J. Immunol. 171: 691 (2003)), IL-15, IL-18, IL-20, etc.), interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.) and TNF. Fas and Fas ligand genes are also immunomodulator target sequences of interest (Song, et al., Nat. Med. 9: 347 (2003)). 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)).

[0298] Cell receptor ligands include ligands that can bind to cell surface receptors (e.g., insulin receptors, EPO receptors, G-protein coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) to regulate (e.g., inhibit, activate, etc.) physiological pathways involved in the receptors (e.g., glucose level regulation, blood cell development, mitogenesis, etc.). Examples of cell receptor ligands include cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G-protein coupled receptor ligands, etc.). Templates encoding trinucleotide repeat sequence (e.g., CAG repeat sequence) expansions have been found to silence pathogenic sequences in neurodegenerative diseases caused by trinucleotide repeat sequence expansions, such as spinal bulbar muscular atrophy and Huntington's disease (Caplen, et al., Hum. Mol. Genet. 11: 175 (2002)).

[0299] Injectable delivery: In some cases, it is desirable to deliver the drug-lipid particles disclosed herein parenterally, intravenously, intramuscularly, subcutaneously, intradermally or intraperitoneally, as described in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363. The drug-lipid particles can be injected locally to a target site (e.g., a disease site such as inflammation or tumor formation or to a target organ or tissue) or injected systemically for widespread distribution to an 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, and in oil. Optionally, these preparations contain preservatives to prevent the growth of microorganisms. Typically, when administered intravenously, the drug-lipid particle preparation is formulated with a suitable pharmaceutical carrier. Typically, a normal buffered saline solution (135-150 mM NaCl) will be used as a pharmaceutical carrier, but other suitable carriers will be sufficient to meet the requirements. Additional suitable carriers are described, for example,

[0300] REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The phrase "pharmaceutical" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans. The formulation of aqueous compositions is conventionally understood in the art, and the compositions include proteins as active ingredients. Optionally, these compositions are prepared as injections, liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid prior to injection can also be prepared. The formulations can also be emulsified.

[0301] Can be sterilized by conventional liposome sterilization technology, such as filtering to drug-lipid granule.Described drug-lipid granule can comprise pharmaceutical auxiliary substance, and described pharmaceutical auxiliary substance is suitable physiological condition, such as pH adjusting agent and buffer, toxicity adjusting agent, wetting agent etc.Use the technology indicated above to sterilize these compositions, or alternatively, they can produce under aseptic conditions.Can be packaged to obtain aqueous solution to use or filter under aseptic conditions and carry out freeze drying, before using, freeze dried preparation is combined with sterile aqueous solution.

[0302] Preventive and therapeutic treatment: In some embodiments, the drug-lipid particle can be used for the preventive or therapeutic treatment of a subject (e.g., a mammalian subject) suffering from a disease or condition, which is associated with the expression or overexpression of a target sequence. The drug-lipid particle is administered to the subject in an amount sufficient to stimulate the therapeutic response of the patient. An amount sufficient to accomplish this is defined as a "therapeutically effective dose or amount" or "effective dose or amount". In determining the effective amount of the drug-lipid particle to be administered to the treatment or prevention of a disease, the disease is caused by the expression or overexpression of a target gene, and the physician evaluates the circulating plasma levels of the drug-lipid particle, the drug-lipid particle toxicity, and the progression of the disease associated with the expression or overexpression of the target gene. Administration can be accomplished by a single or divided dose.

[0303] For example, the drug-lipid granule can be administered to a subject who is infected by pathogenic microorganisms or has the risk of being infected by pathogenic microorganisms. The drug should preferably correspond to a sequence, and should also be unique (or at least lacking in the natural genome of the patient who experiences treatment) for microorganisms, and the sequence has a key role in the life history of microorganisms. By ex vivo or intravenous injection, the drug-lipid granule is introduced into target cells, tissues or organs with a therapeutically effective dose. The sequence silencing of the gene encoding the gene relevant to pathogenic infection can be conveniently combined with the use of conventional agents for the treatment of pathogenic diseases for use. The treatment can be prophylactically administered to people who are at risk of being infected by pathogenic microorganisms or have been infected by pathogenic microorganisms.

[0304] In a preferred embodiment, the drug-lipid particles of the present invention can be conveniently used to treat cancer, viral infection, 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, for example, FIELDSVIROLOGY, 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 unPEGylated interferon a) (see, for example, Medina et al., Antiviral Res. 60 (2): 135-143 (2003); ribavirin

[0305] (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).

[0306] In another embodiment, medicine-lipid granule of the present invention can be conveniently used for treating the disease and illness characterized by gene or expression of gene group or overexpression.In some respects, medicine-lipid granule of the present invention can be used for treating metabolic diseases and illness (for example, wherein liver is the disease and illness and liver disease and illness of target object) such as, for example, dyslipidemia and diabetes.Those skilled in the art will appreciate that the silence of the gene relevant to metabolic diseases and illness can be combined with the conventional treatment of these diseases. For example, silencing of genes involved in dyslipidemia can be combined with statins, bile acid sequestrants / resins and cholesterol absorption inhibitors such as ezetimibe, plant stanols / sterols, polyphenols, and nutraceuticals such as oat bran, flaxseed and soy protein, phytostanol analogs, squalene synthase inhibitors, bile acid transport inhibitor SREBP cleavage activating protein (SCAP) activating ligand, niacin (nicotinic acid), acipimox, high dose fish oil, antioxidants and policosanol, microsomal triglyceride transport protein (MTP) inhibitors, acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors, gemcabene, rifiberol, pantothenic acid analogs, niacin-receptor agonists, anti-inflammatory agents (such as Lp-PLA(2) antagonists and AGI1067) functional oils, PPAR-α, γ, delta agonists, as well as dual PPAR-α, / γ and 'pan' PPAR-α / γ, / δ agonists, cholesteryl ester transfer protein (CETP) inhibitors (such as torcetrapib), CETP vaccines, upregulators of ATP-binding cassette transporter (ABC) A1, phosphatidylcholine cholesterol acyltransferase (LCAT) and scavenger receptor class B type 1 (SRB1), and synthetic apolipoprotein (Apo) E-related peptides, extended-release niacin / lovastatin, atorvastatin / amlodipine, ezetimibe / simvastatin, atorvastatin / CETP inhibitors, statin / PPAR agonists, extended-release niacin / simvastatin and pravastatin / aspirin in development, and therapeutic combinations of anti-obesity agents (see, e.g., Bays and Stein, Expert Opin. Pharmacother. 4(11):1901-38 (2003)). Likewise, silencing of genes involved in diabetes could be combined with treatment with insulin as well as dietary modifications and exercise.

[0307] Similar methods are used to inhibit the expression of endogenous receptor cell genes, which are related to tumorigenesis and cell transformation, tumor growth and tumor migration; inhibit the expression of angiogenic genes; inhibit the expression of immunomodulator genes, such as those associated with inflammation and autoimmune response; inhibit the expression of ligand receptor genes; inhibit the expression of genes associated with neurodegenerative disorders; and inhibit the expression of additional genes associated with viral infection and survival. The target gene sequences of specific objectives are described above.

[0308] Detection of the particles: The drug-lipid particles herein are detected using any method known in the art. For example, a label is coupled directly or indirectly to a component of the drug-lipid particle or other lipid-based carrier system using methods well known in the art. A wide variety of labels can be used, selected based on the sensitivity required, the ease of conjugation to the drug-lipid particle component, stability requirements, and the availability of tools and processing preparations. Suitable labels include, but are not limited to, spectral labels, such as fluorescent dyes (e.g., (e.g., fluorescein and derivatives, such as fluorescein isothiocyanate (FITC) and Oregon Green™; rhodamine and derivatives, such as Texas Red, tetrarhodimineisothiocynate (TRITC), etc., digoxigenin, biotin, phycoerythrin, AMCA, CyDyes™, etc.; radioactive labels, such as 3H, 125I, 35S, 14C, 32P, 33P, etc.; enzymes such as horseradish peroxidase, alkaline phosphatase, etc.; spectral colorimetric labels such as colloidal gold or colored glass or plastic beads, such as polystyrene, polypropylene, latex, etc.). The label is detected using any means known in the art.

[0309] Detection of Nucleic Acids: The nucleic acids herein are detected and quantified by any of a number of means well known to those skilled in the art. Detection of nucleic acids is performed by methods well known in the art such as Southern blot analysis, Northern blot analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting and affinity chromatography. Additional analytical biochemical methods such as spectrophotometry, X-ray photography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography may also be employed.

[0310] The sensitivity of hybridization assays can be increased by the use of nucleic acid amplification systems that multiply the target nucleic acid being detected. In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques sufficient to guide the skilled artisan by these in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification, and other RNA polymerase-mediated techniques (e.g., NASBA™) are found in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2000, and Ausubel et al.,

[0311] SHORT PROTOCOLSIN MOLECULAR BIOLOGY, eds., Current Protocols,

[0312] a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,

[0313] (2002), and Mullis et al. (1987), U.S. Patent No. 4,683,202;

[0314] 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;

[0315] The Journal Of NIH Research, 3: 81 (1991); (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86: 1173 (1989); Guatelli et al., Proc. Natl. Acad. Sci. USA, 87: 1874 (1990); Lomell et al. al., J. Clin. Chem., 35: 1826 (1989); Landegren et al., Science, 241: 1077 (1988);

[0316] Van Brunt, Biotechnology, 8: 291 (1990); Wu and Wallace, Gene, 4: 560 (1989);

[0317] Barringer et al., Gene, 89:117 (1990), and Sooknanan and Malek, Biotechnology, 13:

[0318] 563 (1995). Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039. Other methods described in the art are nucleic acid sequence-based amplification (NASBA™, Cangene, Mississauga, Ontario) and the Qβ replicase system.

[0319] Oligonucleotides for use, for example, as probes in in vitro amplification methods, as gene probes, or as inhibitor components are chemically synthesized, for example, using an automated synthesizer, as described by Needham VanDevanter et al., Nucleic Acids Res., 12:6159 (1984), typically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetrahedron Letts., 22(20):1859 1862 (1981). If necessary, purification of the oligonucleotides is typically performed by native acrylamide gel electrophoresis or by anion exchange HPLC, as described by Pearson and Regnier, J. Chrom., 255:137149 (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.

[0320] The examples that follow provide illustration but do not limit the claimed invention. Those skilled in the art will readily recognize a variety of noncritical parameters that can produce essentially the same results.

[0321] The drug-lipid particles to be protected by the present invention refer to drug-lipid particles other than those containing cationic / ionizable lipids, namely drug-loaded metal-chelated phospholipid complex nanoparticles (drug@MPP).

[0322] Experimental Example 1: Preparation of drug-metal-phospholipid complex particles

[0323] Example 1: Preparation of phospholipid complex

[0324] Connect the phospholipid molecule with a phosphate group to the linker molecule: add distearoylphosphatidylcholine (DSPC, Formula 46) and curcumin (Formula 19) to the reaction bottle at a molar ratio of 1:1, add an appropriate amount of ethanol to dissolve, react at 65°C for 2h, concentrate, and add n-hexane. The phospholipid complex is precipitated, filtered, and vacuum dried to obtain the phospholipid complex. The structure of the phospholipid complex is shown below:

[0325]

[0326] Result analysis: The yield of the target product obtained by reacting curcumin with DSPC at 65°C for 2 hours was 94%.

[0327] Example 2: Preparation of metal-phospholipid complex

[0328] Example 2.1 Preparation of metal ions: Fe 3+ Metal-phospholipid complex

[0329] The phospholipid complex prepared in Example 1 is connected to the metal ion part: the phospholipid complex and FeCl 3 Add the metal-phospholipid complex to the reaction bottle at a molar ratio of 1:1, add ethanol to dissolve, react at 60°C for 2 hours, and then suspend the reaction solution to dryness, then wash with ultrapure water and vacuum dry to obtain the metal-phospholipid complex. The structure of the metal-phospholipid complex is shown below.

[0330]

[0331] Result analysis: Phospholipid complex and FeCl 3 The reaction was carried out at 60 °C for 2 hours, the phospholipid complex feed concentration was 4.5 mg / mL, and the phospholipid complex and FeCl 3 When the feed ratio is 1:1, the yield of the target product is 95%.

[0332] Example 2.2 Preparation of metal ions as Al 3+ Metal-phospholipid complex

[0333] The difference between this embodiment and embodiment 2.1 is that FeCl 3 Replaced by Al(NO 3 ) 3 9H 2 O. The structure of the prepared metal-phospholipid complex is shown below.

[0334]

[0335] Result analysis: Phospholipid complex and Al(NO 3 ) 3 9H 2 O was reacted at 60 °C for 2 hours, the phospholipid complex feed concentration was 4.5 mg / mL, and the phospholipid complex and Al(NO 3 ) 3 9H 2 When the feed ratio of O was 1:1, the yield of the target product was 95%.

[0336] Example 3. Preparation of mRNA-loaded metal-chelatedphospholipid complex nanoparticles (mRNA@MPP)

[0337] Preparation of metal ions as Fe 3+ mRNA-metal-phospholipid complex particles

[0338] The metal-phospholipid complex was prepared according to the method in Example 2.1, wherein DSPC, curcumin, FeCl 3 According to the feed ratio of 1:1:1, the metal-phospholipid complex and distearoylphosphatidylcholine (DSPC, formula 46, as a non-cationic lipid or non-ionizable lipid), cholesterol (CHOL, formula 40, as a non-cationic lipid or non-ionizable lipid), DSPE-PEG2000 (formula 53, as a conjugated lipid that inhibits particle aggregation) are dissolved in ethanol at different molar proportions as the organic phase. The proportions of metal-phospholipid complex, DSPC, CHOL and DSPE-PEG2000 are 15%, 35%, 46% and 4%, respectively. mRNA is dissolved in enzyme-free PBS buffer at a concentration of 20 μg / mL as the aqueous phase (the components of PBS are 0.137M sodium chloride, 0.0027M potassium chloride, 0.01M disodium hydrogen phosphate and 0.0018M potassium dihydrogen phosphate). The mass of the metal-phospholipid complex and the mass of the mRNA are mixed in a microfluidic chip at a mass ratio of 40:1. The volume ratio of the aqueous phase to the organic phase is 3:1. The flow rate of the organic phase and the aqueous phase in the microfluidic chip is 12 ml / min. Among them, the drug mRNA is an mRNA encoding the fluorescent protein eGFP, and its sequence is SEQ ID NO.1 (720nt). eGFP-mRNA@MPP was prepared. eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of mRNA contained), and the control group was incubated with MPP without drug loading. The cell suspension was collected after 48 hours, and the percentage of eGFP-positive cells was detected by flow cytometry.

[0339] The particle size, surface potential and stability of the eGFP-mRNA@MPP prepared in Example 3 were tested, and the efficiency of nucleic acid encapsulation of the eGFP-mRNA@MPP was calculated.

[0340] Method for detecting particle size and result judgment standard: The particle size of nanoparticles was tested using Malvern Zetasizer, and particle sizes within the range of 30 to 400 nm were considered acceptable.

[0341] Method for detecting surface potential and result judgment standard: The surface potential of nanoparticles was tested using a Malvern Zetasizer laser particle size analyzer, and a potential in the range of -10 to 10 mV was considered acceptable.

[0342] Method for detecting stability and criteria for judging results: Place the nanoparticles at 4°C for 7 days, and use a Malvern Zetasizer to test 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, the stability is considered to be good.

[0343] Method for calculating nucleic acid encapsulation efficiency: Specifically, agarose gel electrophoresis was used. First, the nucleic acid feed amount of each group of lipid nanoparticles was set to 10 μg / mL, and the metal ion was Fe 3+ The mass ratio of the metal-phospholipid complex to the mRNA mass was 40:1. The same concentration of nucleic acid was dissolved in PBS buffer solution as a positive control, and the negative control was PBS buffer solution. The concentration of agarose gel was 1.5%. At this time, the gaps in the gel only allowed free nucleic acids to pass through but did not allow lipid nanoparticles to pass through. The electrophoresis was stopped when the free nucleic acid bands were clearly distinguishable. Image J software was used to count the grayscale values ​​of free nucleic acids in different groups. The positive control group was set as 100%. The ratio of free nucleic acid in each group to the positive control was the relative amount of free nucleic acid. The encapsulation rate of each group was (100-relative amount of free nucleic acid)%. A nucleic acid encapsulation rate of more than 50% was considered acceptable.

[0344] Cell culture method: Human embryonic kidney cell line 293T was cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO. 2 cultured under the conditions of .

[0345] Flow cytometry method for analyzing the percentage of eGFP-positive cells: 293T cells were seeded on a 24-well plate at a seeding density of 5 × 10 5cells / well. When the cell density reached 80%, 1 mL of MPP or eGFP-mRNA@MPP was added to incubate the cells, where the concentration of eGFP-mRNA@MPP was 2 μg / mL. After 48 hours, the cell suspension was collected, 20,000 cells were collected using the FITC channel of the flow cytometer, and the percentage of eGFP-positive cells was analyzed. The calculation formula was: eGFP-positive cell rate calculation formula = number of cells expressing eGFP / total number of cells × 100%. The percentage of eGFP-positive cells above 40% was considered acceptable.

[0346] Preparation of metal ions as Al 3+ mRNA-metal-phospholipid complex particles

[0347] The metal-phospholipid complex was prepared according to the method in Example 2.2, wherein DSPC, curcumin, Al(NO 3 ) 3 9H 2 O was added at a feed ratio of 1:1:1, and the metal-phospholipid complex and distearoylphosphatidylcholine (DSPC, Formula 46, as a non-cationic lipid or non-ionizable lipid), cholesterol (CHOL, Formula 40, as a non-cationic lipid or non-ionizable lipid), DSPE-PEG2000 (Formula 53, as a conjugated lipid that inhibits particle aggregation) were dissolved in ethanol at different molar proportions as the organic phase. The proportions of metal-phospholipid complex, DSPC, CHOL and DSPE-PEG2000 were 7%, 34%, 56% and 3%, respectively. mRNA was dissolved in enzyme-free PBS buffer at a concentration of 20 μg / mL as the aqueous phase (the components of PBS were 0.137M sodium chloride, 0.0027M potassium chloride, 0.01M disodium hydrogen phosphate and 0.0018M potassium dihydrogen phosphate). The mass of the metal-phospholipid complex and the mass of the mRNA were mixed in a microfluidic chip at a mass ratio of 13.3:1. The volume ratio of the aqueous phase to the organic phase is 3:1. The flow rate of the organic phase and the aqueous phase in the microfluidic chip is 12 ml / min. Among them, the drug mRNA is an mRNA encoding the fluorescent protein eGFP, and its sequence is SEQ ID NO.1 (720nt). Prepared eGFP-mRNA@MPP. The eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA), and the control group was incubated with MPP without drug loading. After 48 hours, the cell suspension was collected, and the percentage of eGFP-positive cells was detected by flow cytometry.

[0348] The particle size, surface potential and stability of the eGFP-mRNA@MPP prepared in Example 3 were tested, and the efficiency of nucleic acid encapsulation of the eGFP-mRNA@MPP was calculated.

[0349] Method for detecting particle size and result judgment standard: The particle size of nanoparticles was tested using Malvern Zetasizer, and particle sizes within the range of 30 to 400 nm were considered acceptable.

[0350] Method for detecting surface potential and result judgment standard: The surface potential of nanoparticles was tested using a Malvern Zetasizer laser particle size analyzer, and a potential in the range of -10 to 10 mV was considered acceptable.

[0351] Method for detecting stability and criteria for judging results: Place the nanoparticles at 4°C for 7 days, and use a Malvern Zetasizer to test 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, the stability is considered to be good.

[0352] Method for calculating nucleic acid encapsulation efficiency: Specifically, agarose gel electrophoresis was used. First, the nucleic acid feed amount of each group of lipid nanoparticles was set to 10 μg / mL, and the metal ion was Al 3+ The mass ratio of the metal-phospholipid complex to the mRNA mass was 13.3:1. The same concentration of nucleic acid was dissolved in PBS buffer solution as a positive control, and the negative control was PBS buffer solution. The concentration of agarose gel was 1.5%. At this time, the gaps in the gel only allowed free nucleic acids to pass through but did not allow lipid nanoparticles to pass through. The electrophoresis was stopped when the free nucleic acid bands were clearly distinguishable. Image J software was used to count the grayscale values ​​of free nucleic acids in different groups. The positive control group was set as 100%. The ratio of free nucleic acid in each group to the positive control was the relative amount of free nucleic acid. The encapsulation rate of each group was (100-relative amount of free nucleic acid)%. A nucleic acid encapsulation rate of more than 50% was considered acceptable.

[0353] Cell culture method: Human embryonic kidney cell line 293T was cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO. 2 cultured under the conditions of .

[0354] Flow cytometry method for analyzing the percentage of eGFP-positive cells: 293T cells were seeded on a 24-well plate at a seeding density of 5 × 10 5 cells / well. When the cell density reached 80%, 1 mL of MPP or eGFP-mRNA@MPP was added to incubate the cells, where the concentration of eGFP-mRNA@MPP was 2 μg / mL. After 48 hours, the cell suspension was collected, 20,000 cells were collected using the FITC channel of the flow cytometer, and the percentage of eGFP-positive cells was analyzed. The calculation formula was: eGFP-positive cell rate calculation formula = number of cells expressing eGFP / total number of cells × 100%. The percentage of eGFP-positive cells above 40% was considered acceptable.

[0355] The principle of loading nucleic acid into metal-chelated phospholipid complex nanoparticles (MPP) assembled by metal-phospholipid complexes is that curcumin and DSPC are bound together by hydrogen bonds, while curcumin binds to Fe through coordination bonds. 3+ or Al 3+ connected to form a metal-phospholipid complex, the Fe 3+ or Al 3+ The metal-phospholipid complex is connected to the nucleic acid through a coordination bond, thereby ensuring that the metal-phospholipid complex self-assembles with other lipid components into MPP and loads the nucleic acid into the nanoparticle. There are two possibilities for the contribution of curcumin to MMP loading nucleic acid: ① Curcumin interacts with nucleic acid and assists MPP loading nucleic acid, for example, curcumin assists loading nucleic acid by inserting into the minor groove of nucleic acid; ② Curcumin may not directly interact with nucleic acid.

[0356] Example 3.1. Dosage ratio of components of metal-phospholipid complex

[0357] DSPC, curcumin, FeCl in Example 3 3 According to different delivery ratios (1:1:1, 3:3:

[0358] 2, 2:2:1), and the other steps were the same as in Example 3 to prepare different eGFP-mRNA@MPPs, and their nucleic acid encapsulation rates were detected respectively.

[0359] Result analysis: As shown in Table 1-1, when DSPC, curcumin, FeCl 3 When the ratio of DSPC, curcumin, FeCl 3 When the ratio of DSPC, curcumin, FeCl 3 When the ratio of Fe to Mg was 2:2:1, the eGFP-mRNA encapsulation efficiency of the prepared drug-lipid particles was 60%. 3+ The function of Fe is to connect phospholipid complexes to nucleic acids. 3+ There are at most three complexation sites, so DSPC, curcumin, FeCl 3 The ratio of DSPC, curcumin, FeCl 3When the ratio of DSPC, curcumin, FeCl 3 The dosage ratio ranges from 1:1:1 to 2:2:1, and the nucleic acid encapsulation rate of the drug-lipid particles is above 60%.

[0360] Table 1-1 Component ratio of metal-phospholipid complex and function of drug-lipid particles prepared therefrom

[0361] <![CDATA[The dosing ratios of DSPC, curcumin, and FeCl 3 > mRNA encapsulation efficiency of drug-lipid particles 1:1:1 87％ 3:3:2 70％ 2:2:1 60％

[0362] DSPC, curcumin, Al(NO 3 ) 3 9H 2 O was added according to different addition ratios (1:1:1, 3:3:2, 2:2:1), and the other steps were the same as in Example 3 to prepare different eGFP-mRNA@MPPs, and their nucleic acid encapsulation rates were detected respectively.

[0363] Results analysis: As shown in Table 1-2, when DSPC, curcumin, Al(NO 3 ) 3 9H 2 When the ratio of DSPC, curcumin, Al(NO 3 ) 3 9H 2 When the ratio of O was 3:3:2, the eGFP-mRNA encapsulation efficiency of the prepared metal-phospholipid complex particles was 72%. 3 ) 3 9H 2 When the ratio of O was 2:2:1, the eGFP-mRNA encapsulation efficiency of the prepared metal-phospholipid complex particles was 58%. 3+ The function of Al is to connect the phospholipid complex with nucleic acid. 3+ There are at most three complexation sites, so DSPC, curcumin, Al(NO 3 ) 3 9H 2 The ratio of O in the drug-lipid particles should be 1:1:1 to ensure that the metal-phospholipid complex particles can encapsulate as much nucleic acid as possible. The experimental results also confirmed that when DSPC, curcumin, Al(NO 3 ) 3 9H 2When the ratio of DSPC, curcumin, Al(NO) and Al(NO) was 1:1:1, the eGFP-mRNA encapsulation efficiency of the metal-phospholipid complex particles prepared by the metal-phospholipid complex particles was the highest. 3 ) 3 9H 2 When the ratio of O was from 1:1:1 to 2:2:1, the nucleic acid encapsulation rate of the metal-phospholipid complex particles was above 58%.

[0364] Table 1-2 The proportion of components of metal-phospholipid complexes and the functions of the prepared metal-phospholipid complex particles

[0365] Example 3.2. Ratios of metal-phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000 and cholesterol (CHOL) in the preparation of drug-lipid particles

[0366] Compared with Example 3, the ratio of metal-phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) is as shown in Table 1-3 (the metal ion is Fe 3+ ) and Table 1-4 (metal ions are Al 3+ ), the other conditions are the same.

[0367] Result analysis: As shown in Table 1-3, when the metal-phospholipid complex (metal ion is Fe 3+When the proportion of metal-phospholipid complexes was in the range of (10-40)%, DSPC in the range of (0-40)%, CHOL in the range of (35-75)%, and DSPE-PEG2000 in the range of (2-10)%, the particle size of the drug-lipid particles was in the range of 50-400nm, the surface potential was in the range of -10-10mV, the in vitro stability was more than 3 days, the mRNA encapsulation rate was more than 50%, and the positive expression rate of eGFP protein was more than 70%. Among them, when the metal-phospholipid complex accounted for 15%, distearoylphosphatidylcholine (DSPC) accounted for 35%, cholesterol (CHOL) accounted for 46%, and DSPE-PEG2000 accounted for 4%, the performance of the drug-lipid particles was optimal, that is, the particle size was in the range of 110nm, the surface potential was in the range of -2.04mV, the in vitro stability was more than 7 days, the mRNA encapsulation rate was 87%, and the positive expression rate of eGFP protein was 97%. Because mRNA@MPP mainly relies on metal-phospholipid complexes to adsorb nucleic acids, the proportion of metal-phospholipid complexes cannot be too low; when the DSPC content is in the range of 0-40%, the stability of its nanoparticles is within an acceptable range. When the DSPC content is 0%, the stability of its nanoparticles can be maintained because the metal-phospholipid complex contains DSPC; the role of DSPE-PEG2000 is to prevent nanoparticle aggregation and increase the circulation time in the body, and its performance will be better when its content is in the range of 2-10%; the role of CHOL is to enhance the fluidity of nanoparticles, and maintaining a certain content is beneficial to the stability of nanoparticles.

[0368] The above results suggest that metal-phospholipid complexes (metal ions are Fe 3+ ) accounts for (10-40)%, DSPC accounts for (0-40)%, CHOL accounts for (35-75)%, and DSPE-PEG2000 accounts for (2-10)%, mRNA@MPP has better drug loading performance.

[0369] Table 1-3 eGFP-mRNA@MPP(Fe 3+ )Performance Testing

[0370]

[0371] Results analysis: As shown in Table 1-4, when the metal-phospholipid complex (metal ion is Al 3+When the proportion of metal-phospholipid complexes was in the range of (5-50)%, when the proportion of DSPC was in the range of (0-51)%, when the proportion of CHOL was in the range of (15-80)%, when the proportion of DSPE-PEG2000 was in the range of (2-10)%, the particle size of the drug-lipid particles was in the range of 50-400nm, the surface potential was in the range of -10-10mV, the in vitro stability was more than 3 days, the mRNA encapsulation rate was more than 50%, and the positive expression rate of eGFP protein was more than 70%. Among them, when the metal-phospholipid complex accounted for 7%, distearoylphosphatidylcholine (DSPC) accounted for 34%, cholesterol (CHOL) accounted for 56% and DSPE-PEG2000 accounted for 3%, the performance of the drug-lipid particles was optimal, that is, the particle size was 100nm, the surface potential was -1.57mV, the in vitro stability was more than 7 days, the mRNA encapsulation rate was 92%, and the positive expression rate of eGFP protein was 98%. Because mRNA@MPP mainly relies on metal-phospholipid complexes to adsorb nucleic acids, the proportion of metal-phospholipid complexes cannot be too low; when the DSPC content is in the range of 0-51%, the stability of its nanoparticles is within an acceptable range. When the DSPC content is 0%, the stability of its nanoparticles can be maintained because the metal-phospholipid complex contains DSPC; the role of DSPE-PEG2000 is to prevent nanoparticle aggregation and increase the circulation time in the body, and its performance will be better when its content is in the range of 2-10%; the role of CHOL is to enhance the fluidity of nanoparticles, and maintaining a certain content is beneficial to the stability of nanoparticles.

[0372] The above results suggest that metal-phospholipid complexes (metal ions are Al 3+ ) accounts for (5-50)%, DSPC accounts for (0-51)%, CHOL accounts for (15-80)%, and DSPE-PEG2000 accounts for (2-10)%, mRNA@MPP has better drug loading performance.

[0373] Table 1-4 eGFP-mRNA@MPP(Al 3+ )Performance Testing

[0374]

[0375] Example 3.3: Preparation of non-cationic lipids or non-ionizable lipids in eGFP-mRNA@MPP

[0376] Compared with Example 3, the replacement of distearoylphosphatidylcholine (DSPC) is shown in Table 1-5 and Table 1-6, and the other conditions are the same.

[0377] Results: To explore whether DSPC in eGFP-mRNA@MPP can be replaced by other non-cationic lipids or non-ionizable lipids, we selected three other non-cationic lipids or non-ionizable lipids, namely DSPE, DSPA and DSPG, to replace DSPC. We also tested the particle size, surface potential, stability and mRNA encapsulation efficiency to prove that DSPC in eGFP-mRNA@MPP can be replaced by other non-cationic lipids or non-ionizable lipids, and its function after replacement is equivalent to that of eGFP-mRNA@MPP containing DSPC (Table 1-5 (metal ions are Fe 3+ ) and Table 1-6 (metal ions are Al 3+ )). Since the main function of the non-cationic lipid or non-ionizable lipid DSPC in eGFP-mRNA@MPP is to make the liposome membrane fusion better, more stable and less toxic, and other non-cationic lipids or non-ionizable lipids also have the function of making the liposome membrane fusion better, more stable and less toxic, DSPC in the drug-lipid particles can be replaced by other non-cationic lipids or non-ionizable lipids except the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation, and its efficacy is not affected.

[0378] The structural formulas of three non-cationic lipids or non-ionizable lipids (DSPE, DSPA and DSPG) are shown below.

[0379] DSPE (Formula 47)

[0380]

[0381] DSPA (Equation 48)

[0382] DSPG (Formula 49)

[0383]

[0384] Table 1-5 eGFP-mRNA@MPP(Fe) containing different types of non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation 3+ )

[0385]

[0386] Table 1-6 eGFP-mRNA@MPP (Al) containing different types of non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation 3+ )

[0387]

[0388]

[0389] Example 3.4. Preparation of conjugated lipid species for inhibiting particle aggregation in eGFP-mRNA@MPP

[0390] Compared with Example 3, the substitution of DSPE-PEG2000 (Formula 53) is shown in Table 1-7 (the metal ion is Fe 3+ ) and Table 1-8 (metal ions are Al 3+ ), and the other conditions were the same. Three other conjugated lipids that inhibit particle aggregation are DSPE-PEG700 (Formula 50), DSPE-PEG5000 (Formula 52), and DSPE-PEG1000 (Formula 51).

[0391] Analysis of results: In order to explore whether DSPE-PEG2000 in eGFP-mRNA@MPP can be replaced by other conjugated lipids that inhibit particle aggregation, we selected three other conjugated lipids that inhibit particle aggregation, namely DSPE-PEG700, DSPE-PEG5000 and DSPE-PEG1000 to replace DSPE-PEG2000, respectively, and through the detection of particle size, surface potential, stability and mRNA encapsulation rate, it is proved that DSPE-PEG2000 in eGFP-mRNA@MPP can be replaced by other conjugated lipids that inhibit particle aggregation, and its function after replacement is equivalent to the efficacy of eGFP-mRNA@MPP containing DSPE-PEG2000 (Tables 1-7 and 1-8). Because the main function of DSPE-PEG2000 in eGFP-mRNA@MPP is to inhibit aggregation, and other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation, DSPE-PEG2000 in eGFP-mRNA@MPP can be replaced by other conjugated lipids that inhibit particle aggregation without affecting its efficacy.

[0392] Table 1-7 eGFP-mRNA@MPP(Fe) containing different types of conjugated lipids that inhibit particle aggregation 3+ )

[0393]

[0394] Table 1-8 eGFP-mRNA@MPP (Al) containing different types of conjugated lipids that inhibit particle aggregation 3+ )

[0395]

[0396]

[0397] Example 3.5 Preparation and effect characterization of mRNA@MPP

[0398] Example 3.5.1: Metal ion is Fe 3+ Preparation and effect characterization of mRNA@MPP

[0399] The mRNA in Example 3 was replaced with the other two mRNAs, and three mRNA@MPPs containing different target protein mRNA sequences were prepared according to the method of Example 3. The three different mRNA sequences were:

[0400] ① The mRNA sequence encoding the fluorescent protein eGFP is SEQ ID NO.1 (720nt); ② The mRNA sequence encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit is SEQ ID NO.2 (669nt); ③ The mRNA sequence encoding the tumor antigen NY-ESO-1 is SEQ ID NO.3 (543nt). The preparation process of the remaining drug (mRNA)-lipid particles is the same as that in Example 3, and eGFP-mRNA@MPP, RBD-mRNA@MPP, and NY-ESO-1-mRNA@MPP are obtained respectively.

[0401] eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of mRNA contained). The control group was incubated with MPP. After 48 h, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry. The results are shown in Figure 1-1 ; RBD-mRNA@MPP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA), and the control group was incubated with MPP. After 24 hours, the supernatant was centrifuged and frozen at -20°C for later use. The expression level of the new coronavirus antigen RBD protein on the cells was detected using a commercially available new coronavirus antigen RBD ELISA detection kit. The results are shown in Figure 1-2 .

[0402] ELISA method for detecting RBD expression level:

[0403] 1. Sample collection: Place the cell supernatant at room temperature for 2 hours, centrifuge at 1000×g for 20 minutes, and collect the supernatant;

[0404] 2. Sample addition: Set up blank wells, standard wells, and sample wells on the coated plate. Add 100μL of sample diluent to the blank wells, add graded dilutions of standard wells, and add 100μL of sample wells. Incubate at 37℃ for 60min.

[0405] 3. Discard the liquid in the wells, wash the plate 3 times, soak for 1-2 minutes each time. Add 100 μL of the prepared biotin-labeled anti-RBD antibody working solution to each well, mix well, and incubate at 37°C for 60 minutes;

[0406] 4. Discard the liquid in the wells and wash the plate 3 times, soaking for 1-2 minutes each time;

[0407] 5. Add 100 μL of prepared streptavidin HRP working solution to each well, mix well, and incubate at 37°C for 45 min;

[0408] 6. Discard the liquid in the wells and wash the plate 3 times, soaking for 1-2 minutes each time;

[0409] 7. Add 100 μL of TMB substrate solution (TMB) to each well and incubate at 37°C in the dark for 15 min;

[0410] 8. Add 100 μL of stop solution to each well to terminate the reaction;

[0411] 9. Measure the optical density (OD value) of each well at a wavelength of 450 nm.

[0412] Data analysis: Draw a standard curve with the concentration of the standard as the horizontal axis and the OD value as the vertical axis.

[0413] The experimental animals were randomly divided into two groups (experimental group and control group), with 5 mice in each group. Among them, the RBD-mRNA@MPP animal model was BALB / c mice. Each mouse received the first intramuscular administration on the first day and the second intramuscular administration on the 14th day. The experimental group was injected with RBD-mRNA@MPP, and the control group was injected with metal-phospholipid complex particles (MPP) without mRNA loading. The dose for each administration was 100 μL, and the RBD-mRNA@MPP preparation in the experimental group contained 30 mg of mRNA. The blood of the mice was collected on the 28th day after the first administration, and the serum was separated and gradiently diluted. The total RBD IgG antibodies against the S1 subunit of the new coronavirus produced in the mice were detected by a commercially available ELISA kit. The results are as follows Figure 1-3 shown.

[0414] The animal model of NY-ESO-1-mRNA@MPP is C57BL / 6 mice. Each mouse was intramuscularly administered four times on days 1, 7, 14, and 21. The experimental group was injected with NY-ESO-1-mRNA@MPP, and the control group was injected with metal-phospholipid complex particles (MPP) without mRNA loading. The dose for each administration was 100 μL, and the NY-ESO-1-mRNA@MPP preparation in the experimental group contained 30 mg of mRNA. On the 28th day after the first administration, the blood of the mice was collected, the serum was separated and graded diluted, and the total anti-NY-ESO-1 IgG antibodies produced in the mice were detected by ELISA. The results are shown in the figure. Figure 1-4 shown.

[0415] Method for detecting total anti-NY-ESO-1 IgG antibodies in mice:

[0416] Preparation of reagents used in ELISA method:

[0417] 1. Coating solution: accurately weigh 8.4 g NaHCO 3 Dissolve in 1L distilled water (DDW). After all the solids are dissolved, adjust the pH of the entire solution to 9.6 using 1M NaOH solution. Store the prepared coating solution at 4°C for later use.

[0418] 2. Washing solution: Add 0.5 mL of Tween-20 to 1 L of 0.01 M PBS solution, mix well and place at room temperature.

[0419] 3. Blocking solution: Accurately weigh 20g BSA and add it to 1L 0.01M PBS solution. Ultrasonicate the undissolved BSA powder in the solution. When all the solids in the solution are dissolved and the solution turns light yellow, store it in a 4℃ refrigerator for later use.

[0420] 4. Antibody diluent: Accurately weigh 2.5 g BSA and dissolve it in 250 mL 0.01 M PBS solution. After the solid is completely dissolved, add 1.25 mL Tween-20, mix well, and store at 4°C for later use.

[0421] 5. Color development solution: 0.1M citric acid: 19.2g citric acid added to DDW water to 1000mL (A) 0.2M disodium hydrogen phosphate: 28.4g anhydrous disodium hydrogen phosphate added to DDW water to 1000mL (B) 0.1M citric acid solution (A)

[0422] 24.3mL, 0.2M phosphate buffer (B) 25.7mL, add DDW water 50mL. Add OPD (o-phenylenediamine) 50mg, add 30% H 2 O 2 0.15mL.

[0423] 6. Stop solution: 2M HO 2 SO 4 : Concentrated sulfuric acid 55.5mL, add DDW to 500mL.

[0424] ELISA method to determine the titer of antibodies in mouse serum:

[0425] 1. Coating: Dilute NY-ESO-1 antigen to 1 μg / mL with coating solution, add to 96-well plate, 50 μL / well, and coat overnight at 4°C.

[0426] 2. Blocking: Shake off the coating solution in the well plate, wash with blocking solution 3 times, 5 minutes each time and shake off, add 150 μL blocking solution to each well, and incubate at 37°C for 2 hours.

[0427] 3. Drying: Shake off the blocking solution and incubate at 37°C for 1 to 2 hours until all the liquid at the bottom of the well plate is dry.

[0428] 4. Immunization: The serum samples were initially diluted 1:1000 with antibody diluent, and then serially diluted 1:2. The diluted serum samples were added to a closed 96-well plate, 100 μL / well, and incubated at 37°C for 2 hours; the liquid in the well plate was dried, and the washing solution was added, 300 μL / well, and slowly oscillated for 40 seconds. This step was repeated three times; a 1:1000 diluted biotinylated goat anti-mouse IgG antibody was added to the well plate, 100 μL / well, and incubated at 37°C. 1h; shake off the liquid in the well plate, add washing solution, and repeat the above washing steps; add freshly prepared streptavidin-labeled horseradish peroxidase HRP working solution, 100μL / well, incubate at 37℃ for 1h; shake off the liquid in the well plate, add washing solution, and repeat the above washing steps; add color development solution, 100μL / well, react at room temperature for 5min, and then add stop solution to stop color development, 50μL / well; use an enzyme reader to measure the absorbance at 450nm.

[0429] On the 28th day after administration of RBD-mRNA@MPP, spleens of normal mice were collected and prepared into single cell suspension under sterile conditions. 100,000 spleen cells / well were plated in a cell well plate, 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 determined by ELISA kits. The results are shown in Figure 1-5 shown.

[0430] On the 28th day after administration of NY-ESO-1-mRNA@MPP, spleens of normal mice were collected and prepared into single cell suspension under sterile conditions. 100,000 spleen cells / well were plated in a cell well plate, 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 determined by ELISA kits. The results are shown in Figure 1-6 shown.

[0431] Result analysis: Figure 1-1 As shown, the eGFP-positive cell rate in the eGFP-mRNA@MPP experimental group was 97%, while no eGFP signal was detected in the MPP control group; Figure 1-2As shown in the figure, the RBD protein encoded by the RBD-mRNA encapsulated by MPP was 193.3 ng / mL in the supernatant of 293T cells, while the RBD protein content in the supernatant of 293T cells transfected with the empty vector MPP was 0. The results suggest that mRNA-MPP can encapsulate and deliver any mRNA and directly encode polypeptides in cells. Figure 1-3 As shown in the results 1-4, both RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP can effectively induce humoral immunity in mice and produce high levels of antigen-specific binding antibodies. The IgG antibody titer of mice in the RBD-mRNA@MPP treatment group reached 117268.8; the IgG antibody titer of mice in the NY-ESO-1-mRNA@MPP treatment group reached 5319.52. Figure 1-5 , 1-6, both RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP can effectively induce cellular immunity in mice, that is, activate immune cells and produce a large number of cytokines. Among them, RBD-mRNA@MPP makes the expression of cytokines IFN-γ, IL-2, and IL-4 reach 252.8pg / mL, 207.6pg / mL, and 56.6pg / mL, respectively; NY-ESO-1-mRNA@MPP makes the expression of cytokines IFN-γ, IL-2, and TNF-α reach 70.79pg / mL, 75.29pg / mL, and 75.27pg / mL, respectively. The results suggest that mRNA@MPP can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), and then effectively induce humoral immunity and cellular immunity in mice, produce high levels of antigen-specific binding antibodies and cytokines, and play the role of anti-new coronavirus mRNA vaccines and anti-tumor mRNA vaccines.

[0432] Example 3.5.2: Metal ion is Al 3+ Preparation and effect characterization of mRNA@MPP

[0433] The difference between this embodiment and embodiment 3.5.1 is that the metal ion Fe in embodiment 3.5.1 is 3+ Replace with Al 3 + .

[0434] Result analysis: Figure 1-7 As shown, the eGFP-positive cell rate in the eGFP-mRNA@MPP experimental group was 98.02%, while no eGFP signal was detected in the MPP control group; Figure 1-8As shown in the figure, the RBD protein encoded by the RBD-mRNA encapsulated by MPP was 212.6 ng / mL in the supernatant of 293T cells, while the RBD protein content in the supernatant of 293T cells transfected with the empty vector MPP was 0. The results suggest that mRNA-MPP can encapsulate and deliver any mRNA and directly encode polypeptides in cells. Figure 1-9 As shown in the results of Figures 1-10, both RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP can effectively induce humoral immunity in mice and produce high levels of antigen-specific binding antibodies. The IgG antibody titer in mice treated with RBD-mRNA@MPP reached 129113; the IgG antibody titer in mice treated with NY-ESO-1-mRNA@MPP reached 6507.4. Figure 1-11 As shown in Figures 1-12, both RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP can effectively induce cellular immunity in mice, that is, activate immune cells and produce a large number of cytokines. Among them, RBD-mRNA@MPP makes the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 271.8pg / mL, 234.6pg / mL, and 68.4pg / mL, respectively; NY-ESO-1-mRNA@MPP makes the expression levels of cytokines IFN-γ, IL-2, and TNF-α reach 83.8pg / mL, 98pg / mL, and 97.8pg / mL, respectively. The results suggest that mRNA@MPP can encapsulate and deliver any mRNA, thereby promoting the expression of target proteins (antigens), and then effectively induce humoral immunity and cellular immunity in mice, produce high levels of antigen-specific binding antibodies and cytokines, and play the role of anti-new coronavirus mRNA vaccines and anti-tumor mRNA vaccines.

[0435] Example 3.6. Preparation and Effect of siRNA-loaded metal-chelatedphospholipid complex nanoparticles (siRNA@MPP)

[0436] Example 3.6.1: Metal ion is Fe 3+ Preparation and effect of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPP)

[0437] The mRNA in Example 3 was replaced with siRNA, and three siRNA@MPPs containing different siRNAs were prepared according to the method of Example 3. The genes, sequences and corresponding random control sequences of three different siRNA targeting genes are: ① The sequence of siRNA targeting Bcl-2 gene (Bcl-2-siRNA) is SEQ ID No.4 (antisense chain) and SEQ ID No.21 (sense chain) (19bp), and its random control sequence is SEQ ID No.5 (antisense chain) and SEQ ID No.22 (sense chain) (19bp); ② The sequence of siRNA targeting PLK1 gene (PLK1-siRNA) is SEQ ID NO.6 (antisense chain) and SEQ ID No.23 (sense chain) (21bp), and its random control sequence is SEQ ID NO.7 (antisense chain) and SEQ ID No.24 (sense chain) (19bp); ③ The sequence of siRNA targeting 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@MPP is the same as that of Example 3.

[0438] The sequence of Bcl-2-siRNA is as follows:

[0439] Antisense: 5′-CAGCUUAUAAUGGAUGUAC-3′ (SEQ ID No. 4);

[0440] Sense: 5′-GUACAUCCAUUAUAAGCUG-3′ (SEQ ID No. 21) (19 bp).

[0441] The random control sequence of Bcl-2-siRNA is as follows:

[0442] Antisense: 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID No. 5);

[0443] Sense: 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID No. 22) (19 bp).

[0444] The sequence of PLK1-siRNA is as follows:

[0445] Antisense: 5'-UAAGGAGGGUGAUCUUCUUCA-3' (SEQ ID No. 6);

[0446] Sense: 5'-UGAAGAAGAUCACCCUCCUUA-3'(SEQ ID No.23)

[0447] (21bp).

[0448] The random control sequences of PLK1-siRNA are as follows:

[0449] Antisense: 5'-CUUACGCUGAGUACUUCGA-3' (SEQ ID No. 7);

[0450] Sense: 5'-UCGAAGUACUCAGCGUAAG-3' (SEQ ID No. 24) (19 bp).

[0451] The sequence of Gal-1-siRNA is as follows:

[0452] 5'-GCUGCCAGAUGGAUACGAA-3' (SEQ ID No. 8) (19 bp).

[0453] The random control sequence of Gal-1-siRNA is as follows:

[0454] 5'-GGAAAUCCCCCAACAGUGA-3' (SEQ ID No. 9) (19 bp).

[0455] Cell culture method: U251 human glioblastoma cells were grown in monolayer in high glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin and 2 mm l-glutamine (Bio Industries) culture medium at 37°C and 5% CO. 2 The cells were cultured under the same conditions and passaged twice a week.

[0456] U251 cells were cultured at 1 × 10 6 About 24 hours after the cells were seeded in a 6-well plate at a high density, each well of cells was incubated with siRNA@MPP containing the above siRNA (where the concentration of siRNA was 2 μg / mL) for 72 hours, and then the cells were collected and the total cellular RNA was extracted. The mRNA expression levels of the target genes (Bcl-2, PLK1, Gal-1) were detected by RT-PCR technology, and the ability of siRNA@MPP to silence the target genes in cells was calculated.

[0457] RT-PCR specific process:

[0458] 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 10 minutes, centrifuge at 13000rpm4℃ for 15 minutes to obtain a layered three-phase liquid, in which RNA is 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 10 minutes, centrifuge at 13000rpm 4℃ for 15 minutes to obtain RNA precipitate. Remove the supernatant and add 1mL of 75% newly prepared RNAse-free water to each tube.

[0459] (v / v) ethanol, carefully pipette to lift up the white RNA precipitate at the bottom of the tube, centrifuge at 7500rpm and 4℃ for 10min, remove the supernatant, and try to dry the liquid at the bottom of the tube. Open the lid and dry the RNA precipitate at room temperature, add 50μL enzyme-free water to dissolve, and use an ultra-micro UV-visible spectrophotometer to detect the purity and concentration of RNA.

[0460] cDNA reverse transcription: using Ta Ka Ra Prime Script TM RT reagent Kit with gDNAEraser 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, place it at 37°C for 15 min, then place it at 85°C for 5 sec to terminate the reaction, and then store it at 4°C for later use.

[0461] 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 cDNA of different samples as template. 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 well plate, 10 μL per well, and centrifuged (1000 rpm, 5 min) after sample addition to eliminate bubbles in the liquid wall and reaction solution. Real-time PCR reaction detection was performed using ABIViiA7 real-time fluorescence quantitative PCR instrument, and the reaction program was 95℃, 30sec (1 cycle) → 95℃, 5sec; 55℃, 30sec; 72℃, 30sec (40 cycles) → 60℃-95℃, 2min (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 between the experimental group and the control group was calculated. The control gene was GAPDH. RT-PCR primers are as follows: ①Bcl-2 primer: forward: 5'-AGGATTGTGGCCTTCTTTGAG-3', reverse: 5'-AGACAGCCAGGAGAAATC AAAC-3'; ②PLK1 primer: forward: 5'-ACCAGCACGTCGTAGGATTC-3', reverse: 5'-CAAGCAATTTGCCGTAGG-3'; ③Gal-1 primer: forward: 5'-CAATCATGGCCTGTGGTCTG-3', reverse: 5'-GTG TAGGCACAGGTTGTTGCTG-3'. ④GAPDH primer: forward: 5'-TCAGGGGTTTCACATTTGGCA-3', reverse: 5'-GG AGCGGAA AACCA-3'. The expression level of each target gene was expressed using RQ value (2 -ΔΔCT ). The formula is as follows:

[0462] Fold Change = 2 –ΔΔCt

[0463] Where ΔΔCt=ΔCt 实验组 –ΔCt 对照组 , ΔCt=Ct 目的基因 -Ct 内参基因

[0464] Calculation method of gene silencing efficiency: Calculation method of gene silencing efficiency: 100% - gene expression level of experimental group / gene expression level of control group.

[0465] Result analysis: Figure 1-13, 1-14, 1-15 (where scr siRNA is a random control sequence), the three siRNA@MPPs can significantly interfere with their corresponding target genes. The inhibition rate of Bcl-2-siRNA@MPP on the target gene Bcl-2 reached 76%; the inhibition rate of PLK1-siRNA@MPP on the target gene PLK1 reached 86%; the inhibition rate of Gal-1-siRNA@MPP on the target gene Gal-1 reached 73%. The results suggest that siRNA@MPP can carry any siRNA for intervention therapy of target genes, and play the role of siRNA-carrying drugs, vaccines or other products.

[0466] Example 3.6.2: Metal ion is Al 3+ Preparation and effect of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPP)

[0467] The difference between this embodiment and embodiment 3.6.1 is that the metal ion Fe in embodiment 3.6.1 is replaced by 3+ Replace with Al 3 + .

[0468] Result analysis: Figure 1-16 As shown in Figures 1-17 and 1-18, the three siRNA@MPPs can significantly interfere with their corresponding target genes. The inhibition rate of Bcl-2-siRNA@MPP on the target gene Bcl-2 reached 81%; the inhibition rate of PLK1-siRNA@MPP on the target gene PLK1 reached 90%; and the inhibition rate of Gal-1-siRNA@MPP on the target gene Gal-1 reached 79%. The results suggest that siRNA@MPP can carry any siRNA for intervention therapy of target genes and play the role of siRNA-carrying drugs, vaccines or other products.

[0469] Example 3.7. Preparation and Effect of ASO-loaded metal-chelatedphospholipid complex nanoparticles (ASO@MPP)

[0470] Example 3.7.1: Metal ion is Fe 3+ Preparation and effect of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP)

[0471] The mRNA in Example 3 was replaced with ASO, and three ASO@MPPs containing different ASOs were prepared respectively according to the method of Example 3. The genes, sequences and corresponding random control sequences of the three different ASOs were: ① The sequence of the ASO targeting the STAT3 gene (STAT3-ASO) was SEQ ID No. 10 (17nt), and its random control sequence was SEQ ID NO. 11 (18nt); ② The sequence of the ASO targeting the α-syn gene (α-syn-ASO) was SEQ ID NO. 12 (16nt), and its random control sequence was SEQ ID NO. 13 (16nt); ③ The sequence of the ASO targeting the Bcl-2 gene (Bcl-2-ASO) was SEQ ID NO. 14 (18nt), and its random control sequence was SEQ ID NO. 15 (20nt). The preparation process of the remaining ASO-metal-phospholipid complex particles was the same as that of Example 3. Different ASO@MPPs were incubated with different cells: U251 human glioblastoma cells were incubated with ASO@MPP targeting STAT3 gene; SH-SY5Y human neuroblastoma cells were incubated with ASO@MPP targeting α-syn gene; Daudi human lymphoma cells were incubated with ASO@MPP targeting Bcl-2 gene. 6 About 24 hours after the cells were seeded at a high density in a 6-well plate, each well of cells was incubated with ASO@MPP containing the above-mentioned ASO (where the concentration of ASO was 1 μg / mL) for 48 hours, and then the cells were collected and the total cell RNA was extracted. The mRNA expression levels of the target genes (STAT3, α-syn, Bcl-2) were detected by RT-PCR technology, and the ability of ASO@MPP to silence the cell target genes was calculated.

[0472] The sequence of SEQ ID No.10 (the sequence of STAT3-ASO) is as follows:

[0473] 5'-GCTCCAGCATCTGCTTC-3'(17nt).

[0474] The sequence of SEQ ID No.11 (random control sequence of STAT3-ASO) is as follows:

[0475] 5'-GAAGCAGCAGATGCTGGA-3'(18nt).

[0476] The sequence of SEQ ID No.12 (sequence of α-syn-ASO) is as follows:

[0477] 5'-GCCTCCCTCCACTGTCT-3'(16nt).

[0478] The sequence of SEQ ID No.13 (random control sequence of α-syn-ASO) is as follows:

[0479] 5'-ACTCCCGAACCTGTCT-3'(16nt).

[0480] The sequence of SEQ ID No.14 (sequence of Bcl-2-ASO) is as follows:

[0481] 5'-TCTCCCAGGCGTGCGCCAT-3'(18nt).

[0482] The sequence of SEQ ID No.15 (random control sequence of Bcl-2-ASO) is as follows:

[0483] 5'-CAGCGTGCGCCATCCTTCCC-3'(20nt).

[0484] Cell culture: ①U251 human glioblastoma cells were grown in monolayers in a medium containing high glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (Bio Industries) at 37°C and 5% CO 2 ②SH-SY5Y human neuroblastoma cells were grown in monolayer in a medium containing high glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin and 2 mm l-glutamine (Bio Industries) and incubated at 37°C and 5% CO 2 Daudi human lymphoma cells were cultured under 37°C and 5% CO. 2 The cells were cultured under the same conditions and passaged twice a week.

[0485] RT-PCR specific process:

[0486] 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 10 minutes, centrifuge at 13000rpm4℃ for 15 minutes to obtain a layered three-phase liquid, in which RNA is 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 10 minutes, centrifuge at 13000rpm 4℃ for 15 minutes to obtain RNA precipitate. Remove the supernatant and add 1mL of 75% newly prepared RNAse-free water to each tube.

[0487] (v / v) ethanol, carefully pipette to lift up the white RNA precipitate at the bottom of the tube, centrifuge at 7500rpm and 4℃ for 10min, remove the supernatant, and try to dry the liquid at the bottom of the tube. Open the lid and dry the RNA precipitate at room temperature, add 50μL enzyme-free water to dissolve, and use an ultra-micro UV-visible spectrophotometer to detect the purity and concentration of RNA.

[0488] cDNA reverse transcription: using Ta Ka Ra Prime Script TM RT reagent Kit with gDNAEraser 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.

[0489] 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 cDNA of different samples as template. 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 well plate, 10 μL per well, and centrifuged (1000 rpm, 5 min) after sample addition to eliminate bubbles in the liquid wall and reaction solution. Real-time PCR reaction detection was performed using ABIViiA7 real-time fluorescence quantitative PCR instrument, and the reaction program was 95℃, 30sec (1 cycle) → 95℃, 5sec; 55℃, 30sec; 72℃, 30sec (40 cycles) → 60℃-95℃, 2min (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 between the experimental group and the control group was calculated. The control gene was GAPDH. The sequences of RT-PCR primers are as follows: ①STAT3 primer: forward: 5'-TGATCACCTTTGAGACCGAGG-3', reverse: 5'-GATCACCACAACTGG CAA GG-3'; ②α-syn primer: forward: 5'-TGACGGGTGTGACAGCAGTAG-3', reverse: 5'-CAGTGGCTGCTGCAATG-3'; ③Bcl-2 primer: forward: 5'-AGGATT GTGGCCTTCTTTGAG-3', reverse: 5'-AGACAGCCAGGAGAAATCAAAC-3'④GAPDH primer: forward: 5'-TCAGGGG TTTCACATTTGGCA-3', reverse: 5'-GGAGCGGAA AACCA-3'. The expression level of each target gene was expressed using the RQ value (2 -ΔΔCT ). The formula is as follows:

[0490] Fold Change = 2 –ΔΔCt

[0491] Where ΔΔCt=ΔCt 实验组 –ΔCt 对照组 , ΔCt=Ct 目的基因 -Ct 内参基因

[0492] The calculation method of gene silencing efficiency is: 100% - gene expression level of experimental group / gene expression level of control group.

[0493] Result analysis: Figure 1-19,1-20,1-21 (where scr ASO is a random control sequence), the three ASO@MPPs can significantly interfere with their corresponding target genes, among which STAT3-ASO@MPP has an inhibition rate of 75% on the target gene STAT3; α-syn-ASO@MPP has an inhibition rate of 71% on the target gene α-syn; Bcl-2-ASO@MPP has an inhibition rate of 66% on the target gene Bcl-2. The results suggest that ASO@MPP can carry any ASO for intervention treatment of target genes and play the role of ASO-carrying drugs, vaccines or other products.

[0494] Example 3.7.2: Metal ion is Al 3+ Preparation and effect of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP)

[0495] The difference between this embodiment and embodiment 3.7.1 is that the metal ion Fe in embodiment 3.7.2 is 3+ Replace with Al 3 + .

[0496] Result analysis: Figure 1-22 As shown in Figures 1-23 and 1-24, the three ASO@MPPs can significantly interfere with their corresponding target genes, among which STAT3-ASO@MPP has an inhibition rate of 79% on the target gene STAT3; α-syn-ASO@MPP has an inhibition rate of 80% on the target gene α-syn; and Bcl-2-ASO@MPP has an inhibition rate of 74% on the target gene Bcl-2. The results suggest that ASO@MPP can carry any ASO for target gene intervention therapy and play the role of ASO-carrying drugs, vaccines or other products.

[0497] Example 3.8 Preparation of drug (different types of nucleic acids)-metal-phospholipid complex particles and their effects

[0498] Example 3.8.1: Preparation of metal ions: Fe 3+ Drug (different types of nucleic acids)-metal-phospholipid complex particles and their effects

[0499] The mRNA in Example 3 was replaced by double-stranded RNA (siRNA), single-stranded DNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA. The different types of nucleic acid sequences are:

[0500] ① The sequence of the 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 the single-stranded DNA (STAT3-ASO) is SEQ ID NO.10 (17 nt), and its random control sequence is SEQ ID NO.11 (18 nt); ③ The sequence of the single-stranded RNA (mRNA encoding the wild-type new coronavirus S protein) is SEQ ID No.16 (3822 nt); ④ The sequence of the double-stranded DNA (dsDNA) is SEQ ID NO.17 (antisense strand) and SEQ ID NO.25 (sense strand) (22 bp) (the 3' end of the sequence is labeled with fluorescent probe Cy3); ⑤ The sequence of the single-stranded DNA (ssDNA) is SEQ ID NO.18 (22 nt) (the 3' end of the sequence is labeled with fluorescent probe Cy3). Referring to the method of Example 3, drug-metal-phospholipid complex particles (Bcl-2-siRNA@MPP, STAT3-ASO@MPP, S-mRNA@MPP, dsDNA@MPP, ssDNA@MPP) encapsulating the above-mentioned different types of nucleic acids were prepared respectively, and the preparation process of the remaining drug-lipid particles was the same as that of Example 3.

[0501] The sequence of double-stranded DNA is as follows:

[0502] Antisense: 5'-TAGCTTATCAGACTGATGTTGA-3' (SEQ ID No. 17);

[0503] Sence:5'-TCAACATCAGTCTGATAAGCTA-3'(SEQ ID No.25)

[0504] (22bp).

[0505] The sequence of SEQ ID No.18 (single-stranded DNA sequence) is as follows:

[0506] 5'-TCAACATCAGTCTGATAAGCTA-3'(22nt).

[0507] U251 cells were cultured at 1 × 10 5After the cells were seeded in a 12-well plate at a density of about 24 h, each well of cells was incubated with siRNA@MPP (where the concentration of siRNA was 2 μg / mL) or ASO@MPP (where the concentration of ASO was 2 μg / mL) for 72 h, and the cells were collected and the total RNA of the cells was extracted. The mRNA expression of the target genes (Bcl-2, STAT3) was detected by RT-PCR, and the ability of siRNA@MPP or ASO@MPP to silence the target genes of the cells was calculated. The results are shown in Example 3.6. Figure 1-13 And in Example 3.7 Figure 1-19 shown.

[0508] S-mRNA@MPP was incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA), and the control group was incubated with MPP. After 24 hours, the supernatant was centrifuged and stored at -20°C for later use; the cell pellet was resuspended in 100 μL PBS buffer solution, frozen and thawed twice, and ultrasonicated for 10 minutes before centrifugation to obtain the supernatant. The expression level of S protein in both the cell supernatant and the cell lysate was detected using a commercially available SARS-CoV-2 S protein ELISA detection kit. The results are as follows: Figure 1-25 shown.

[0509] A549 lung cancer cells were incubated with ds-DNA@MPP at a concentration of 100 nM (the concentration of DNA contained) for 2 hours, and then the remaining drug-lipid particles were removed. The cells were washed twice with PBS, and the cell nuclei were stained with Hochest33342 dye for 3 minutes, and then the dye was removed. The cells were washed twice with PBS, and the cells were observed using a high-content imaging system. The efficiency of drug-lipid particle transfection of DNA was calculated. The results are shown in Figure 1-26 .

[0510] HT22 mouse hippocampal neurons were incubated with ss-DNA@MPP at a concentration of 200 nM (the concentration of DNA contained) for 2 hours, and then the remaining drug-lipid particles were removed. After washing the cells twice with PBS, the cells were observed using a high-content imaging system, and the efficiency of drug-lipid particle transfection with DNA was calculated. The results are shown in Figure 1-26 .

[0511] The culture method of human glioblastoma U251 cells is the same as that in Example 3.6.

[0512] The culture method of 293T cells is the same as that in Example 3.

[0513] HT22 mouse hippocampal neurons were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO. 2 under the conditions.

[0514] The RT-PCR method is the same as in Example 3.6.

[0515] ELISA detection of S protein expression level: replace the "anti-RBD antibody working solution" in the ELISA method for detecting RBD in Example 3.5 with "anti-S protein antibody working solution", and the remaining steps are the same as Example 3.5.

[0516] The calculation method of gene silencing efficiency is the same as that in Example 3.6.

[0517] Method for calculating transfection efficiency: Use a high-content imaging system to randomly select 3-5 fields of view, 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 that labels 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 that labels the cell nucleus). Calculate the ratio of the number of cells with Cy3 fluorescence signals in the randomly selected field of view to the number of cells with Hochest33342 fluorescence signals in the same field of view, which is the transfection efficiency.

[0518] Result analysis: As in Example 3.6.1 Figure 1-13 As shown, the inhibition rate of drug (double-stranded RNA)-metal-phospholipid complex particles (Bcl-2-siRNA@MPP) on the target gene Bcl-2 reached 76%; as shown in Example 3.7.1 Figure 1-19 The inhibition rate of drug (single-stranded DNA)-metal-phospholipid complex particles (STAT3-ASO@MPP) on the target gene STAT3 reached 75%; Figure 1-25 As shown, the S protein expression level in the supernatant of 293T cells transfected with drug (single-stranded RNA)-metal-phospholipid complex particles (S-mRNA@MPP) was 161.3 ng / mL, while the S protein content in the supernatant of 293T cells transfected with empty vector MPP was 0; drug (double-stranded DNA)-metal-phospholipid complex particles

[0519] The efficiency of transfection of double-stranded DNA into cells was 100% ( Figure 1-26 ); the efficiency of drug (single-stranded DNA)-metal-phospholipid complex particles (ssDNA@MPP) transfecting single-stranded DNA into cells is 100% ( Figure 1-26 The results suggest that drug-metal-phospholipid complex particles can encapsulate any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and achieve its function, where the length of the nucleic acid ranges from 16-

[0520] 3822nt.

[0521] Example 3.8.2: Preparation of Metal Ions as Al 3+Drug (different types of nucleic acids)-metal-phospholipid complex particles and their effects

[0522] The difference between this embodiment and embodiment 3.8.1 is that the metal ion Fe in embodiment 3.8.1 is replaced by 3+ Replace with Al 3 + .

[0523] Result analysis: As in Example 3.6.2 Figure 1-16 As shown, the inhibition rate of drug (double-stranded RNA)-metal-phospholipid complex particles (Bcl-2-siRNA@MPP) on the target gene Bcl-2 reached 81%; as shown in Example 3.7.2 Figure 1-22 The inhibition rate of drug (single-stranded DNA)-metal-phospholipid complex particles (STAT3-ASO@MPP) on the target gene STAT3 reached 79%; Figure 1-27 As shown, the S protein expression level in the supernatant of 293T cells transfected with drug (single-stranded RNA)-metal-phospholipid complex particles (S-mRNA@MPP) was 178.7 ng / mL, while the S protein content in the supernatant of 293T cells transfected with empty vector MPP was 0; the efficiency of drug (double-stranded DNA)-metal-phospholipid complex particles (dsDNA@MPP) transfecting double-stranded DNA into cells was 100% ( Figure 1-28 ); the efficiency of drug (ssDNA)-metal-phospholipid complex particles (ssDNA@MPP) transfecting single-stranded DNA into cells is 100%

[0524] ( Figure 1-28 The results suggest that drug-metal-phospholipid complex particles can encapsulate any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and achieve its function, wherein the length of the nucleic acid ranges from 16-3822nt.

[0525] Experimental Example 2: Performance Characterization of Drug-Metal-Phospholipid Complex Particles

[0526] Example 4: Synthesis and characterization of metal-phospholipid complexes

[0527] Example 4.1: Metal ion is Fe 3+ Synthesis and characterization of metal-phospholipid complexes

[0528] The characterization method of DSPC and curcumin connection is differential scanning calorimetry. The determination conditions are as follows: weigh 3-5 mg of the test substance, heat up at a rate of 10°C / min, and heat up in a range of 30-300°C. Curcumin, DSPC, and phospholipid complex are scanned respectively. Curves are drawn based on the obtained data. The results are shown in Figure 2-1As can be seen from the spectrum, curcumin has a lattice structure and an obvious melting peak appears at 185°C; DSPC is a mixture, and there are many depressions on the curve, which may be due to the thermal changes of different components under different temperature conditions; there is no peak near the melting peak of curcumin in the phospholipid complex, which is basically a straight line, indicating that curcumin and DSPC are combined and exist in an amorphous form, proving that the phospholipid complex was successfully prepared.

[0529] Phospholipid complex with Fe 3+ Connectivity characterization is spectrophotometric: e.g. Figure 2-2 As shown, the phospholipid complex (CUR-HSPC) and Fe 3+ After binding, its maximum absorption wavelength shifted from 420nm to 375nm, and the conjugated structure of the phospholipid complex changed, proving that Fe 3+ Successfully complexed with curcumin.

[0530] Example 4.2: Metal ion is Al 3+ Synthesis and characterization of metal-phospholipid complexes

[0531] The difference between this embodiment and embodiment 4.1 is that the phospholipid complex and Al 3+ Connectivity characterization is spectrophotometric: e.g. Figure 2-3 As shown, the phospholipid complex (CUR-HSPC) and Al 3+ After binding, its maximum absorption wavelength shifted from 420nm to 433nm, and the conjugated structure of the phospholipid complex changed, proving that Al 3+ Successfully complexed with curcumin.

[0532] Example 5: Metal ion is Fe 3+ At low pH, Fe 3+ Characterization of shedding from metal-phospholipid complexes

[0533] The phospholipid complex in the metal-phospholipid complex binds Fe through coordination bonds 3+ Under the low pH conditions of lysosomes, phospholipid complexes and Fe 3+ The coordination bonds between them will be protonated (absorb hydrogen ions) and broken. 3+ Indeed, it is through the above mechanism that the metal-phospholipid complex is detached. We designed the following experiment: observe the color of the metal-phospholipid complex under physiological pH (pH = 7.4) and lysosomal low pH (pH = 5.0). Figure 2-4 As shown in the figure, the metal-phospholipid complex changes from brown-red to bright yellow under the low pH value (pH = 5.0) of the lysosome, indicating that Fe 3+ The results suggest that under the low pH conditions of lysosomes, Fe3+ Can be detached from metal-phospholipid complexes.

[0534] At low pH, Fe 3+ The principle of shedding from the metal-phospholipid complex is: curcumin and Fe 3+ The coordination bonds between them are protonated under low pH conditions (pH = 5.0), that is, curcumin will bind a large number of protons (H + ), resulting in Fe 3+ The coordination bond between Fe and curcumin is broken, thus allowing Fe 3+ Separation from curcumin eventually leads to Fe 3+ Separation from metal-phospholipid complexes ( Figure 2-4 ).

[0535] Example 6: Elemental Analysis of MPP in Drug-Metal-Phospholipid Complex Particles

[0536] Example 6.1: Metal ion is Fe 3+ Elemental Analysis of MPP in Drug-Metal-Phospholipid Complex Particles

[0537] The mRNA in Example 3 was replaced with thiol-modified siRNA, and the drug-metal-phospholipid complex particles siRNA@MPP were prepared according to the method of Example 3. Elemental analysis was performed using a transmission electron microscope. The results are shown in FIG. Figure 2-5 As shown: C, N, O, and P are common elements, and the Fe element analysis chart shows Fe 3+ Evenly distributed on the lipid nanoparticles, because the siRNA is modified with thiol groups, the S element analysis diagram can specifically indicate the location of the siRNA. It can be seen from the figure that the siRNA is well complexed with Fe 3+ , proving that the drug-lipid nanoparticles successfully encapsulated siRNA.

[0538] Example 6.2: Metal ion is Al 3+ Electron Microscopic Analysis of MPP in Drug-Metal-Phospholipid Complex Particles

[0539] The drug-metal-phospholipid complex particles MPP were prepared according to the method of Example 3. The morphological analysis was performed using a transmission electron microscope. Figure 2-6 As shown: the morphology of MPP in the drug-metal-phospholipid complex particles is standard spherical, with a uniform particle size of about 100 nm.

[0540] Example 7: Metal ion is Fe 3+ Or Al 3+ The efficiency of metal-phospholipid complex particles MPP encapsulating nucleic acids (siRNA and mRNA) and its comparison with LNP.

[0541] The mRNA in Example 3 was replaced with siRNA targeting the Bcl-2 gene (SEQ ID No. 4, 19 bp) and mRNA encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit (SEQ ID No. 2, 669 nt), respectively, to prepare nucleic acid-encapsulated drug-metal-phospholipid complex particles siRNA@MPP and mRNA@MPP, respectively. The preparation process of the remaining drug-metal-phospholipid complex particles was the same as that in Example 3.

[0542] siRNA@LNP and mRNA@LNP were prepared according to the same drug loading amount as siRNA@MPP in Example 3.6 and mRNA@MPP in Example 3.5. The specific method was as follows: an organic phase solution was prepared according to the Onpattro lipid nanoparticle formula, that is, ionizable lipid ALC0315, DSPE-PEG2000, DSPC and cholesterol were mixed at a ratio of 50%:

[0543] 1.5%:10%:38.5% molar ratio was dissolved in ethanol. Bcl-siRNA or RBD-mRNA was added to the aqueous phase

[0544] (0.1M, pH = 4.0 acetic acid-sodium acetate buffer solution). Among them, the ratio of amino lipids to phosphate-containing nucleotides (N / P) is 6:1, and at the same time, the nucleic acid loading amount is ensured to be the same as that of the above-mentioned siRNA@MPP and mRNA@MPP. The aqueous phase and the organic phase are quickly mixed at a flow rate of 14mL / min at a volume ratio of 3:1. After mixing, it is diluted ten times with enzyme-free PBS buffer solution, and the mixed solution is concentrated to one-tenth using a 100kDa ultrafiltration tube. After repeated dilution and concentration operations for 3 times, the ethanol concentration in the mixed solution is reduced to less than 0.0005%, and the pH value of the solution is increased to the normal pH value of the PBS buffer solution (7.2-7.4), that is, siRNA@LNP and mRNA@LNP are prepared respectively.

[0545] Agarose gel electrophoresis was used to detect the encapsulation rate of nucleic acids (siRNA and mRNA) of siRNA@MPP, mRNA@MPP, siRNA@LNP and mRNA@LNP respectively. The encapsulation rate was determined as follows: the amount of nucleic acids (siRNA and mRNA) in each group of lipid nanoparticles was set at 10 μg / mL, the mass ratio of lipid to nucleic acid was 40:1, the nucleic acids were dissolved in PBS buffer solution as the positive control group, and the negative control was a PBS buffer solution without nucleic acids. The concentration of agarose gel was 1.5%, at which time the gaps in the gel only allowed free nucleic acids to pass through but not lipid nanoparticles. When the free nucleic acid bands were electrophoresed to be clearly distinguishable, the electrophoresis was stopped to prevent nucleic acid degradation due to long electrophoresis time. Image J software was used to count the grayscale values ​​of free nucleic acids in different groups, the positive control group was set at 100%, the ratio of free nucleic acids in each group to the positive control was the relative amount of free nucleic acids, and the encapsulation rate of each group was (100-relative amount of free nucleic acids)%.

[0546] Result analysis: Figure 2-7 As shown, MPP(Fe 3+ ) encapsulated siRNA and mRNA with efficiencies of 85.86% and 87.11%, respectively; MPP (Al 3+ The efficiency of siRNA and mRNA encapsulation in MPP was 89.73% and 92.23% respectively; the efficiency of siRNA and mRNA encapsulation in LNP was 84.98% and 79.12% respectively. The results showed that there was no significant difference in the efficiency of nucleic acid encapsulation between MPP and LNP.

[0547] Example 8: Metal ion is Fe 3+ Or Al 3+ Nucleic acid lysosomal escape ability of metal-phospholipid complex particles (MPP) and its comparison with LNP

[0548] The Bcl-2-siRNA (SEQ ID No. 4) of Example 3.6 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@MPP (the concentration of the siRNA contained was 100 nM), and the Bcl-2-siRNA (SEQ ID No. 4) of Example 7 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@LNP (the concentration of the siRNA contained was 100 nM); the eGFP-mRNA (SEQ ID No. 1) of Example 3.5 was replaced with Cy5-labeled eGFP-mRNA to prepare Cy5-mRNA@MPP (the concentration of the mRNA contained was 2 μg / mL), and the RBD-mRNA of Example 7 was replaced with Cy5-labeled RBD-mRNA to prepare Cy5-mRNA@LNP (the concentration of the mRNA contained was 2 μg / mL), and they were respectively mixed with the cell lysosome probe Lysotracker After A549 cells were co-incubated with Cy5 and Lysotracker Green for 3 hours, a high-content imaging system was used to observe the overlap of the fluorescence signal of Cy5 (red) and the fluorescence signal of Lysotracker Green (green) to determine the ability of the drug-lipid particles to promote nucleic acid lysosomal escape.

[0549] The criterion for judging the ability of drug-metal-phospholipid complex particles to promote nucleic acid lysosomal escape: After the drug-lipid nanoparticles were incubated in cells for 3 hours, the high-content imaging system was used to observe the overlap of the Cy5 fluorescence signal (red) and the LysotrackerGreen fluorescence signal (green), and the imageJ software was used to count the overlap rate of the red fluorescence signal and the green fluorescence signal. When the drug-metal-phospholipid complex particles were incubated in cells for 3 hours, the overlap rate of the red fluorescence signal and the green fluorescence signal was less than 50%, indicating that the nucleic acid could escape from the cell lysosome more quickly, and the lipid nanoparticles had a good ability to promote nucleic acid lysosomal escape.

[0550] Result analysis: Figure 2-8 As shown, when Cy5-siRNA@MPP(Fe 3+ ) and Cy5-mRNA@MPP(Fe 3+ ) incubated A549 cells for 3 hours, the overlap rates of red fluorescence signal and green fluorescence signal were 36.05% and 43.07%, that is, the lysosomal escape rates were 63.95% and 56.93%, respectively; when Cy5-siRNA@MPP(Al 3+ ) and Cy5-mRNA@MPP(Al 3+) After incubating A549 cells for 3 hours, the overlap rates of red fluorescence signal and green fluorescence signal were 32.39% and 40.17%, respectively, that is, the lysosomal escape rates were 67.61% and 59.83%, respectively; while after incubating A549 cells for 3 hours with Cy5-siRNA@LNP and Cy5-mRNA@LNP, the overlap rates of red fluorescence signal and green fluorescence signal were 76.89% and 86.87%, respectively, that is, the lysosomal escape abilities were 23.11% and 13.13%, respectively. This indicates that the drug-lipid nanoparticle MPP has a good ability to promote nucleic acid lysosomal escape, and the ability of MPP to promote lysosomal escape is significantly stronger than that of LNP.

[0551] Example 9: Metal ion is Fe 3+ Or Al 3+ The ability of metal-phospholipid complex particles (MPP) to promote nucleic acid expression and its comparison with LNP

[0552] The RBD-mRNA in Example 7 was replaced with mRNA encoding the fluorescent protein eGFP, and the rest of the preparation method was the same as Example 7 to obtain eGFP-mRNA@LNP.

[0553] The eGFP-mRNA@MPP prepared in Example 3 and the above-mentioned eGFP-mRNA@LNP (containing mRNA at a concentration of 2 μg / mL) were incubated with 293T cells, respectively. The control group was incubated with MPP or LNP. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0554] The method for analyzing the eGFP-positive cell rate by flow cytometry is as described in Example 3.

[0555] Result analysis: Figure 2-9 As shown, MPP(Fe 3+ )、MPP(Al 3+ ) and LNP treated 293T cells, the percentages of eGFP positive cells were 97.2%, 98.1% and 63.03% respectively. The results suggest that MPP is better than LNP in promoting nucleic acid expression. The possible reason is that as described in Example 8, MPP has a stronger ability to promote lysosomal escape of nucleic acids than LNP, so more nucleic acids loaded by MPP can be effectively released into the cytoplasm and translated into proteins.

[0556] Example 10: Metal ion is Fe 3+ Or Al 3+ The ability of drug-metal-phospholipid complex particles (MPP) to promote humoral and cellular immunity and its comparison with LNP

[0557] The RBD-mRNA@MPP of Example 3.5 and the RBD-mRNA@LNP of Example 7 were incubated with 293T cells at a concentration of 2 μg / mL (the concentration of the contained mRNA), and the control group was incubated with MPP. After 24 hours, the supernatant was centrifuged and frozen at -20°C for later use; the cell pellet was resuspended in 100 μL PBS buffer solution, frozen and thawed twice, and ultrasonicated for 10 minutes before centrifugation to obtain the supernatant. The expression level of RBD protein in both the cell supernatant and the cell lysate was detected using a commercially available new coronavirus antigen RBD ELISA detection kit. The results are shown in Figure 2-10 .

[0558] The method for detecting RBD expression level by ELISA is as described in Example 3.5.

[0559] The experimental animals were randomly divided into 3 groups (experimental group and control group), with 5 mice in each group. The animal model was BALB / c mice. Each mouse was given the first intramuscular administration on the first day and the second intramuscular administration on the 14th day. The experimental group was injected with RBD-mRNA@MPP(Fe 3+ )、RBD-mRNA@MPP(Al 3+ ) or RBD-mRNA@LNP, and the control group was injected with MPP and LNP without mRNA. The dose of each administration was 100 μL, of which RBD-mRNA@MPP(Fe 3+ )、RBD-mRNA@MPP(Al 3+ ) and RBD-mRNA@LNP preparations each contain 30 mg of mRNA. On the 28th day after the first administration, the blood of mice was collected, the serum was separated and diluted stepwise, and the titer of the total RBD IgG antibody against the S1 subunit of the new coronavirus produced in the mice was detected by a commercially available ELISA kit. The results are as follows Figure 2-11 shown.

[0560] The method for ELISA detection of the titer of total RBD IgG antibodies against the S1 subunit of the new coronavirus is described in Example 3.5.

[0561] In the case of RBD-mRNA@MPP(Fe 3+ )、RBD-mRNA@MPP(Al 3+ ) and RBD-mRNA@LNP, the spleen of normal mice was collected on the 28th day, and a single cell suspension was prepared under sterile conditions. 100,000 spleen cells / well were plated in a cell well plate, 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 determined by ELISA kits. The results are shown in Figure 2-12 shown.

[0562] The method for detecting the expression levels of IFN-γ, IL-2, and IL-4 by ELISA is as described in Example 3.5.

[0563] Result analysis: Figure 2-10 As shown, RBD-mRNA@MPP(Fe 3+ )、RBD-mRNA@MPP(Al 3+ ) and RBD-mRNA@LNP can induce 293T cells to express a certain amount of RBD, but RBD-mRNA@MPP(Al 3+ ) was significantly stronger in inducing cells to express RBD than RBD-mRNA@MPP(Fe 3+ )、RBD-mRNA@MPP(Fe 3+ ) has a stronger ability to induce cells to express RBD than RBD-mRNA@LNP: RBD-mRNA@MPP(Fe 3+ ) treatment group, the expression level of RBD in the cell supernatant was 205 ng / mL, and RBD-mRNA@MPP(Al 3+ The expression level of RBD in the cell supernatant of the ) treatment group was 230 ng / mL, and the expression level of RBD in the cell supernatant of the RBD-mRNA@LNP treatment group was 115.7 ng / mL. Figure 2-11 The results showed that RBD-mRNA@MPP effectively induced humoral immunity in mice and produced high levels of antigen-specific binding antibodies. 3+ ) is clearly superior to RBD-mRNA@MPP(Fe 3+ ), RBD-mRNA@MPP(Fe 3+ ) is clearly superior to RBD-mRNA@LNP in inducing humoral immunity in mice: RBD-mRNA@MPP(Fe 3+ ) treatment group mice IgG antibody titer reached 117233.8; RBD-mRNA@MPP(Al 3+ The IgG antibody titer in the mice treated with ) reached 133116; while the IgG antibody titer in the mice treated with RBD-mRNA@LNP was only 67476. Figure 2-12 As shown, RBD-mRNA@MPP(Al 3+ ) can effectively induce cellular immunity in mice, namely activate immune cells and produce a large number of cytokines, and mRNA@MPP(Al 3+ ) is clearly superior to RBD-mRNA@MPP(Fe 3+ ), RBD-mRNA@MPP(Fe 3+) is clearly superior to RBD-mRNA@LNP in inducing cellular immunity in mice: RBD-mRNA@MPP(Fe 3+ ) made the expression levels of cytokines IFN-γ, IL-2, and IL-4 reach 256.8 pg / mL, 207.6 pg / mL, and 61.8 pg / mL, respectively; RBD-mRNA@MPP(Al 3+ ) made the expression of cytokines IFN-γ, IL-2, and IL-4 reach 298pg / mL, 249pg / mL, and 74.6pg / mL, respectively; while RBD-mRNA@LNP made the expression of cytokines IFN-γ, IL-2, and IL-4 only 104.2pg / mL, 79.2pg / mL, and 27pg / mL. The results suggest that mRNA@MPP(Al 3+ ) is significantly better than RBD-mRNA@MPP(Fe 3+ ), RBD-mRNA@MPP(Fe 3+ ) is clearly superior to RBD-mRNA@LNP in inducing cellular immunity in mice: RBD-mRNA@MPP can more effectively promote cells to express target proteins, and can more effectively activate humoral immunity and cellular immunity in the body. Therefore, the drug (mRNA)-lipid particles are significantly superior to the prior art LNP in terms of the effects of mRNA-carrying drugs, vaccines or other products. The possible reasons are: 1) Compared with LNP, MPP has a stronger ability to promote nucleic acid lysosomal escape; 2) Compared with LNP, MPP has a stronger ability to promote nucleic acid expression into protein (antigen); 3) Compared with LNP, the curcumin in MPP is separated from DSPC in vivo, and as an immune adjuvant (also known as an immunomodulator), it can activate humoral immunity and cellular immunity to enhance the effect of MPP in delivering mRNA vaccines, and can also inhibit the immune factor storm to inhibit excessive and harmful immune responses to the body.

[0564] Example 11: Metal ion is Fe 3+ Or Al 3+ In vivo safety evaluation of metal-chelated phospholipid complex nanoparticles (MPP)

[0565] Using SD rats as research subjects, a 20-day subchronic toxicity study of MPP was conducted, and a 20-day recovery period was set up. The specific experimental methods are as follows:

[0566] 56 SPF SD rats (220±20g), half male and half female, were raised in an environment with a temperature of 25℃, a humidity of 45%-55%, and a light intensity of 12h. After 3-5 days of adaptive feeding, they were randomly divided into the following groups according to gender: 32 rats in the experimental group and 24 rats in the recovery group. There were 14 blank control groups (including 8 rats in the experimental group and 6 rats in the recovery group), half male and half female; there were 14 low-dose MPP groups (8mg / kg) (including 8 rats in the experimental group and 6 rats in the recovery group), half male and half female; there were 14 medium-dose MPP groups (16mg / kg) (including 8 rats in the experimental group and 6 rats in the recovery group), half male and half female; there were 14 high-dose groups (32mg / kg) (including 8 rats in the experimental group and 6 rats in the recovery group), half male and half female. The experimental group (32 rats in total) was dissected after the end of the drug administration, and the recovery group (24 rats in total) was dissected after the end of the drug administration and continued to be raised normally for 20 days.

[0567] Dosage method: The experimental animals were injected with drugs through the tail vein, once every 2 days for a total of 20 days, and the body weight of SD rats was recorded once a week. The prepared MPP was dissolved in DPBS, and the control group was injected with an equal amount of DPBS, and the low-dose MPP group, medium-dose MPP group, and high-dose MPP group were injected with 8 mg / kg, 16 mg / kg, and 32 mg / kg of MPP, respectively.

[0568] The basis for setting the above MPP dosage is: when encapsulating 200μg / kg mRNA (the actual required amount for mRNA animal experiments), the required amount of empty vector MPP is 8mg / kg. In order to fully prove the safety of MPP, we selected 1, 2 and 4 times the actual required amount of MPP animal experiments, namely 8mg / kg, 16mg / kg and 32mg / kg.

[0569] General index detection method: observe the general status of each group of animals after each administration, including survival, diet, appearance, behavior, body weight, and whether there is a local reaction to administration. Perform a gross autopsy during dissection, including timely weighing the wet weight of major organs, such as brain, heart, liver, spleen, lung, and kidney, calculating the organ-to-body ratio and recording the pathological changes of each organ. The organ-to-body ratio = wet weight of rat organs / rat body weight × 100%.

[0570] Acquisition and storage of whole blood and serum of SD rats: 20 days after administration and 20 days of recovery, rats were dissected and blood was collected from the abdominal aorta, that is, SD rats were anesthetized with isoflurane and fixed on the dissection board, the abdomen was disinfected with 75% ethanol, the abdomen of the rat was cut open with sterile ophthalmic scissors, the internal organs were gently pushed aside with cotton balls to expose the abdominal aorta, and whole blood was collected using 500μL negative pressure EDTAK2 anticoagulation blood collection tubes, stored at 4°C for routine blood tests. Whole blood was collected using 5mL negative pressure ordinary blood collection tubes and allowed to stand at room temperature for 30min, centrifuged at 4°C, 1500rpm for 15min, and the supernatant was taken in a 1.5mL centrifuge tube and stored at -20°C for the detection of blood biochemical indicators and immunology-related indicators.

[0571] Blood routine test method: Blood routine indicators include: white blood cell count, lymphocyte count, monocyte count, neutrophil count, lymphocyte percentage, monocyte percentage, neutrophil percentage, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, coefficient of variation of red blood cell distribution width, platelet count, mean platelet volume, platelet distribution width, platelet hematocrit. Gently invert the whole blood sample to mix, take a small amount of whole blood, and use the fully automatic blood cell analyzer to automatically analyze the results.

[0572] Blood biochemical index detection method: Blood biochemical indexes include inorganic ions (Fe 2+ , Na + , K + , Cl - , Ca 2+ ), liver function indicators (ALT, AST, γ-GT, T-BIL, D-BIL, ALP, ALB), renal function indicators (BUN, UA, CR), cardiac function indicators (LDH, CK), glucose metabolism indicators (GSP, GLU, INS), lipid metabolism indicators (CHO, TG, LDL-C, HDL-C). Thaw the serum sample and centrifuge it at 3000rpm for 15min, take the supernatant and pack it for later use, set the corresponding parameters on the automatic biochemical analyzer, add the configured working solution, and then add the serum to be tested, and the automatic biochemical analyzer will automatically determine the results.

[0573] Detection methods of immunology-related indicators: Immunology-related indicators include thyroid function indicators (TT3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN-γ, IFN-α, TNF-α), immunoglobulins (IgG, IgA, IgM), and serum complement (C3, CH50). The above indicators are detected by ELISA method.

[0574] Pathological examination method of the main organs of SD rats: At the end of the drug administration period and the recovery period, the rats in each group were anesthetized, and the main organs of the rats, including the whole brain, heart, liver, spleen, lung, and kidney, were removed with ophthalmic scissors, gently rinsed with 0.9% saline, fixed in 4% paraformaldehyde fixative, routinely paraffin embedded, H&E stained, and optical microscopy was used to observe the histopathological changes of various organs of the rats in the control group and experimental group.

[0575] Results analysis: As shown in Table 2-1, at the end of the administration period and the end of the recovery period, compared with the control group, low, medium and high doses of MPP (Fe3 + ) or MPP(Al 3+ ) group survived well, had normal diet, normal appearance and behavior, and no obvious adverse reactions were observed after administration; compared with the control group, there was no significant difference in the weight gain of male SD rats and female SD rats in the low, medium and high doses of MPP groups; compared with the control group, there was no significant difference in the organ-to-body ratio of the low, medium and high doses of MPP groups.

[0576] At the end of the treatment period and the end of the recovery period, compared with the control group, low, medium, and high doses of MPP (Fe 3+ ) or MPP(Al 3+ ) group had no abnormalities in blood routine indices (white blood cell count, lymphocyte count, monocyte count, neutrophil count, lymphocyte percentage, monocyte percentage, neutrophil percentage, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, coefficient of variation of red blood cell distribution width, platelet count, mean platelet volume, platelet distribution width, platelet hematocrit); compared with the control group, blood biochemical indices in the low, medium and high doses of MPP groups, including inorganic ions (Fe 2+ , Na + , K + , Cl - , Ca 2+ ), liver function indicators (ALT, AST, γ-GT, T-BIL, D-BIL, ALP, ALB), renal function indicators (BUN, UA, CR), cardiac function indicators (LDH, CK), glucose metabolism indicators (GSP, GLU, INS), lipid metabolism indicators (CHO, TG, LDL-C, HDL-C), all showed no abnormalities; compared with the control group, the immunological related indicators of the low, medium and high doses of MPP groups included thyroid function indicators (TT3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN-γ, IFN-α, TNF-α), immunoglobulins (IgG, IgA, IgM), and serum complement (C3, CH50), all showed no abnormalities.

[0577] At the end of the treatment period and the end of the recovery period, compared with the control group, low, medium, and high doses of MPP (Fe 3+ ) or MPP(Al 3+ ) group had intact brain tissue structure, normal tissue staining, intact cell morphology and structure, no nuclear condensation and inflammatory cell infiltration; intact myocardial tissue structure, myocardial cells were arranged neatly, continuously and tightly, cell nuclei were clearly visible, and no obvious cell congestion, edema or necrosis was observed; normal hepatocyte morphology, no inflammatory cell aggregation and necrosis; normal spleen structure, clear boundaries between red and white pulp; intact lung tissue structure, uniform alveolar size, no obvious inflammatory cell aggregation and infiltration; and normal kidney structure.

[0578] The above results suggest that long-term and large-scale injection of MPP (Fe 3+ ) or MPP(Al 3+ ) No obvious chronic toxic reactions were found, indicating that MPP is relatively safe.

[0579] Table 2-1 In vivo safety evaluation of MPP

[0580]

[0581]

[0582] Note: ALT, alanine aminotransferase; AST, aspartate aminotransferase; γ-GT, glutamyl transpeptidase; T-BIL, total bilirubin; D-BIL, direct bilirubin; ALP, alkaline phosphatase; ALB, albumin; BUN, urea nitrogen; UA, uric acid;

[0583] 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

[0584] Example 12: Metal ion is Fe 3+ Or Al 3+ Comparison of the in vivo safety of metal-phospholipid complex particles (MPP) and LNP

[0585] The main toxicity of LNP comes from its main components - cationic lipids and / or ionizable lipids. When LNP is metabolized in the body, the free cationic lipids and / or ionizable lipids will have obvious toxicity to the body. The median lethal dose (IC50) of cationic lipids and / or ionizable lipids to biological cells is 50 ) is an important parameter for evaluating the toxicity of LNP to the body. Metal-chelated phospholipid complex nanoparticles (MPP) replace the cationic lipids / ionizable lipids in LNP with metal-phospholipid complexes. Therefore, we studied the median lethal dose (IC50) of metal-phospholipid complexes and cationic lipids / ionizable lipids on biological cells. 50 ), comparing LNP with MPP (Fe 3+ ) or MPP(Al 3+ ) differences in toxicity.

[0586] The metal-phospholipid complexes of different concentrations (0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3,

[0587] 72.9, 218.7 μM), cationic lipids (DOTAP, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9,

[0588] 218.7 μM, the structural formula is as follows) and ionizable lipids (ALC0315, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM, the structural formula is as follows) were incubated with 293T cells for 48 hours, and the cell activity was detected using a CCK8 activity detection kit. The half lethal dose IC of the metal-phospholipid complex, cationic lipid (DOTAP) and ionizable lipid (ALC0315) on 293T cells was calculated respectively. 50 .

[0589] DOTAP

[0590] ALC0315

[0591] CCK8 detection method:

[0592] 1. Cell culture: Culture cells with DMEM containing 10% FBS and 1% double antibody until the cell density reaches 80%-90% of the culture flask;

[0593] 2. Wash the remaining medium in the culture flask with PBS, add trypsin, and quickly transfer the culture flask to 37℃ with 5% CO 2Place in an incubator. Observe carefully, and when the cells become slightly round, add culture medium to terminate digestion. Transfer to a centrifuge tube, centrifuge at 1500RPM for 5 minutes, and resuspend the cells with fresh culture medium;

[0594] 3. Counting: Dilute the cell suspension to 10,0000 cells per 1mL according to the purpose, 100μL per well in a 96-well plate, at least 5 replicate wells per group. 37℃, 5% CO 2 Drugs were added after 24 h of culture;

[0595] 4. After incubation with drugs for 48 hours, add 10% CCK8, incubate for 1-3 hours, and measure the absorbance at 450nm with a microplate reader;

[0596] 5. Survival rate (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] × 100%.

[0597] IC50 calculation method: With survival rate as the ordinate and drug concentration as the abscissa, use Graphpad to calculate IC using [Inhibitor] vs. normalized response--Variable slope analysis method 50 .

[0598] To compare the in vivo safety of MPP and LNP, MPP (8 mg / kg) and LNP (3.24 mg / kg) capable of carrying the same amount of nucleic acid (200 μg / kg mRNA) were used to conduct in vivo experiments according to the method of Example 11 to evaluate and compare the in vivo toxicity of MPP and LNP.

[0599] Result analysis: As shown in Table 2-2, the IC 50 The results showed that the toxicity of metal-phospholipid complexes was significantly lower than that of cationic lipids and ionizable lipids.

[0600] As shown in Table 2-3, at the end of the treatment period and the end of the recovery period, compared with the control group, MPP (Fe 3+ ) or MPP(Al 3+ There were no significant abnormalities in liver function (ALT, AST, ALP) and cytokines (IL-6, IL-1β) in the LNP group. However, compared with the control group, liver function (ALT, AST, ALP) and cytokines (IL-6, IL-1β) in the LNP group were significantly increased. The results suggest that MPP (Fe 3+ ) or MPP(Al 3+) is safer in vivo than LNP because the core component of LNP is artificially synthesized "cationic lipid / ionizable lipid", which has high cytotoxicity and immunogenicity, and its structure is relatively stable and difficult to decompose and metabolize in vivo; while MPP (Fe 3+ ) or MPP(Al 3+ ) is a metal-phospholipid complex, which is composed of phospholipid molecules, a highly safe natural small molecule substance, curcumin (a food additive and pharmaceutical excipient approved by the FDA), and safe metal ions. After the drug is delivered, it has been decomposed into natural molecules in the body. In summary, because MPP (Fe 3+ ) or MPP(Al 3 + ) components do not contain cationic lipids / ionizable lipids and will not cause toxic side effects related to cationic lipids / ionizable lipids, so MPP(Fe 3+ ) or MPP(Al 3+ ) is safer than LNP.

[0601] Table 2-2 Metal ions are Fe 3+ Or Al 3+ IC of metal-phospholipid complexes with cationic lipids (DOTAP) and ionizable lipids (ALC0315) 50 Compared with

[0602]

[0603] Table 2-3 Comparison of chronic toxicity test indicators of MPP and LNP

[0604]

[0605] Example 3: Clinical application and administration route of drug-metal-phospholipid complex particles

[0606] Example 13: Metal ion is Fe 3+ or Al 3+ Clinical application and administration route of drug-metal-phospholipid complex particles

[0607] The mRNA in Example 3 was replaced with siRNA targeting the B7-H4 gene (B7-H4-siRNA) and its control (scr-siRNA), and mRNA encoding the receptor binding domain (RBD) of the S1 subunit of the new coronavirus (RBD-mRNA).

[0608] The sequences of the above-mentioned different nucleic acids are as follows: ① The sequence of B7-H4-siRNA is SEQ ID No.19 (sense chain) and SEQ ID No.26 (antisense chain) (25bp), and its random control sequence is SEQ ID No.20 (sense chain) and SEQ ID No.27 (antisense chain) (19bp); ② The mRNA sequence encoding the receptor binding domain (RBD) of the novel coronavirus S1 subunit is SEQ ID No.2 (669nt). The drug-metal-phospholipid complex particles (B7-H4-siRNA) containing the above-mentioned different types of nucleic acids were prepared respectively according to the method of Example 3.

[0609] @MPP(Fe 3+ )、RBD-mRNA@MPP(Fe 3+ )、B7-H4-siRNA@MPP(Al 3+ )、RBD-mRNA@MPP(Al 3+ ), the preparation process of the remaining drug-metal-phospholipid complex particles is the same as that in Example 3. The above two different drug-metal-phospholipid complex particles (B7-H4-siRNA@MPP, RBD-mRNA@MPP) are used to treat liver cancer and as mRNA vaccines for the prevention of new coronaviruses.

[0610] The sequence of B7-H4-siRNA is as follows:

[0611] sense 5'-GGG AGA CAC UCC AUC ACA GUC ACU A-3' (SEQ ID No. 19).

[0612] antisense 5'-UAG UGA CUG UGA UGG AGU GUC UCC C-3' (SEQ ID No. 26) (25 bp).

[0613] The random control sequence of B7-H4-siRNA is as follows:

[0614] sense 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID No. 20).

[0615] antisense 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID No. 27) (19 bp).

[0616] To evaluate the B7-H4-siRNA@MPP(Fe 3+ ) and B7-H4-siRNA@MPP(Al3+ ) to treat liver cancer. HepG2 cells were used to make an animal model of liver cancer. When the tumor size increased to about 100 mm 3 The liver cancer mice were randomly divided into 7 groups (5 mice in each group): PBS control group, blank vector MPP (Fe 3+ ) group, blank vector MPP (Al 3+ ) group, Scr-siRNA@MPP(Fe 3+ ) control group, B7-H4-siRNA@MPP(Fe 3+ ) treatment group, Scr-siRNA@MPP(Al 3+ ) control group, B7-H4-siRNA@MPP(Al 3+ ) treatment group. Each group of mice was injected intratumorally with PBS, MPP (Fe 3+ )、MPP(Al 3+ )、Scr-siRNA@MPP(Fe 3+ )、B7-H4 siRNA@MPP(Fe 3+ )、Scr-siRNA@MPP(Al 3+ )、B7-H4 siRNA@MPP(Al 3+ ) once, with a dose of 200 μg siRNA / kg, and 8 injections were performed. The tumor volume was measured and recorded every 3 days. Figure 3-1 shown.

[0617] To evaluate the effect of RBD-mRNA@MPP as an mRNA vaccine to prevent the new coronavirus, the experimental process and experimental methods are as shown in the previous Example 3.5.

[0618] The ELISA detection method is as described in Example 3.5.

[0619] Establishment of liver cancer mouse model: HepG2 cells were collected and cultured at 1×10 7 The cells were resuspended in PBS at a density of 100 μL and kept on ice before inoculation. Then 100 μL of the cell suspension was injected subcutaneously into the back region near the hind legs of female Balb / c nude mice to establish a liver cancer mouse model.

[0620] Result analysis:

[0621] like Figure 3-1 As shown, Scr-siRNA@MPP(Fe 3+ )、Scr-siRNA@MPP(Al 3+ ) had little inhibitory effect on the growth of HepG2 cells, while B7-H4-siRNA@MPP(Fe 3+ ) and B7-H4 siRNA@MPP(Al3+ ) showed a highly effective therapeutic effect and could effectively inhibit the growth of liver cancer tumors. The results suggest that drug-metal-phospholipid complex particles can encapsulate and deliver B7-H4 siRNA, thereby inhibiting the development of liver cancer by inhibiting the expression of target genes.

[0622] As in Example 3.5 Figure 1-3 , Figure 1-5 As shown, RBD-mRNA@MPP(Fe 3+ ) made the expression level of mouse IgG antibody reach 117268.8 ( Figure 1-3 ), the expression levels of cytokines IFN-γ, IL-2, and IL-4 reached 252.8 pg / mL, 207.6 pg / mL, and 56.6 pg / mL, respectively ( Figure 1-5 )。 RBD-mRNA@MPP(Al 3+ ) made the expression level of mouse IgG antibody reach 129113 ( Figure 1-9 ), the expression levels of cytokines IFN-γ, IL-2, and IL-4 reached 271.8 pg / mL, 234.6 pg / mL, and 68.4 pg / mL, respectively ( Figure 1-11 ). The results suggest that RBD-mRNA@MPP can effectively induce humoral immunity in mice and produce high levels of antigen-specific binding antibodies; it can also effectively induce cellular immunity in mice, activating immune cells and producing a large number of cytokines. Therefore, RBD-mRNA@MPP can effectively prevent infection with the new coronavirus.

[0623] like Figure 3-1 As shown, B7-H4-siRNA@MPP can effectively treat liver cancer by intratumoral injection; as shown in Example 3.5 Figure 1-3 , Figure 1-5 , Figure 1-9 and Figure 1-11 As shown, RBD-mRNA@MPP can activate humoral immunity and cellular immunity by intramuscular injection, thereby preventing novel coronavirus infection. The results suggest that drug-metal-phospholipid complex particles can be administered through multiple routes.

[0624] Example 4: DSPC, curcumin, Fe 3+ or Al 3+ Function after being replaced by similar products

[0625] Example 15, DSPC, curcumin, Fe 3+ or Al 3+ Function after being replaced by similar products

[0626] With reference to Example 1, Example 2 and Example 3, DSPC, curcumin, Fe 3+The analogs of DSPC, curcumin, Fe 3+ By substitution, 28 different eGFP-mRNA@MPPs were prepared through different combinations, and the concentration of mRNA contained in each eGFP-mRNA@MPP was 2 μg / mL. 3+ The names and structures of its congeners are shown in Table 4-1. Among the 28 mRNA@MPPs, DSPC, curcumin, Fe 3+ The combination of the same is shown in Table 4-2. The reaction temperature in Example 1 is 65

[0627] ℃, reaction time is 2h, the reaction temperature in Example 2 is 60℃, reaction time is 2h, and other conditions remain unchanged.

[0628] In order to compare the effects of the 28 different eGFP-mRNA@MPPs and eGFP-mRNA@LNPs, we prepared LNPs encapsulating an equal amount of eGFP-mRNA with reference to Example 9 to obtain eGFP-mRNA@LNPs.

[0629] The above 28 different eGFP-mRNA@MPPs and the above eGFP-mRNA@LNPs (the concentration of the mRNA contained was 2 μg / mL) were incubated with 293T cells, respectively. The control group was incubated with MPP or LNP. The cell suspension was collected after 48 hours, and the percentage of eGFP-positive cells was detected by flow cytometry.

[0630] The method for analyzing the eGFP-positive cell rate by flow cytometry is as described in Example 3.

[0631] The main toxicity of LNP comes from its main component - cationic lipids / ionizable lipids. When LNP is metabolized in the body, the free cationic lipids / ionizable lipids will have obvious toxicity to the body. The median lethal dose (IC50) of cationic lipids / ionizable lipids to biological cells is 50 ) is an important parameter for evaluating the toxicity of LNP to the body. Metal-chelated phospholipid complex nanoparticles (MPP) replace the cationic lipids / ionizable lipids in LNP with metal-phospholipid complexes. Therefore, we studied the median lethal dose (IC50) of 28 metal-phospholipid complexes and cationic lipids (DOTAP) / ionizable lipids (ALC0315) on biological cells by studying ... 50 ), and compared the differences in toxicity between LNP and 28 MPPs.

[0632] The calculation method of IC50 is as described in Example 12.

[0633] Results: As shown in Table 4-3, the percentage of eGFP-positive cells in 293T cells treated with 28 different eGFP-mRNA@MPPs was significantly higher than that of eGFP-mRNA@LNP, among which DSPC, curcumin, Fe 3+ The percentage of eGFP-positive cells in the mRNA@MPP composed of DSPC, curcumin, Fe 3+ The mRNA@MPP formed by its homologues is less functional than that formed by DSPC, curcumin, and Fe 3+ The mRNA@MPP composed of mRNA@MPP is superior to mRNA@LNP in function. The possible reason is that as described in Example 8, the ability of MPP to promote lysosomal escape of nucleic acids is stronger than that of LNP, so more nucleic acids loaded by MPP can be effectively released into the cytoplasm and translated into proteins.

[0634] The above results suggest that as long as the following conditions are met, DSPC, curcumin, Fe 3+ The function of the drug-metal-phospholipid complex particles formed after being replaced by its congeners is not affected: ① The congeners of DSPC are amphiphilic phospholipid molecules; ②Fe 3+ The congeners of curcumin are metal ions; ③ The congeners of curcumin and DSPC can form phospholipid complexes and complex with metal ions; ④ Curcumin and Fe 3+ The coordination bonds between them can be broken in response to the low pH environment of the lysosome.

[0635] As shown in Table 4-3, the IC values ​​of 28 metal-phospholipid complexes 50 The toxicity of metal-phospholipid complexes is obviously lower than that of cationic lipids and ionizable lipids, that is, the toxicity of metal-phospholipid complexes composed of DSPC, curcumin, Fe 3+ The safety of lipid nanoparticles (MPP) composed of DSPC and its analogs is higher than that of LNP. The reasons are as follows: the core component of LNP is 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 the body; while the core component of MPP is metal-phospholipid complex, which is composed of phospholipid molecules, highly safe natural small molecules (among which curcumin is a food additive and pharmaceutical excipient approved by the FDA) and safe metal ions, and it has been decomposed into natural molecules in the body after completing drug delivery. In summary, DSPC, curcumin, Fe 3+ The lipid particles (MPP) composed of LNP and its analogs do not contain cationic lipids / ionizable lipids and will not cause toxic side effects related to cationic lipids / ionizable lipids, so the safety of MPP is higher than that of LNP.

[0636] Table 4-1 DSPC, curcumin, Fe 3+ Names and structures of its analogs

[0637]

[0638]

[0639] Table 4-2 DSPC, curcumin, Fe 3+ List of combinations and functions of metal-phospholipid complexes in drug-lipid particles prepared by and analogs thereof

[0640]

[0641]

[0642] Table 4-3 DSPC, curcumin, Fe 3+ IC of metal-phospholipid complexes prepared from metal-phospholipid complexes 50

[0643]

[0644]

[0645]

[0646] Example 16: DSPC, curcumin and its analogs, Fe 3+ Component proportions and functions of drug-metal-phospholipid complex particles prepared therefrom

[0647] According to Example 3, metal-phospholipid complexes were prepared, and curcumin was replaced by its analogs hesperidin (1 molecule of hesperidin contains 4 hydroxyl groups) and tea polyphenols (1 molecule of tea polyphenols contains 8 hydroxyl groups), respectively, to prepare three metal-phospholipid complexes (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP29). When preparing these three metal-phospholipid complexes, DSPC, curcumin or its analogs, FeCl 3 The dosage ratios are: 1:1:1, 1:1:1, 1:1:2. The corresponding drug-metal-phospholipid complex nanoparticles were prepared using these three metal-phospholipid complexes (mRNA@MPP1, mRNA@MPP4, mRNA@MPP29). The mRNA is an mRNA encoding eGFP fluorescent protein, and its sequence is SEQ ID No.1 (720nt). The mRNA encapsulation rate of these four drug-lipid nanoparticles and their ability to promote the expression of eGFP fluorescent protein after treating 293T cells were detected according to the experimental process and experimental methods described in Example 3.5.

[0648] Analysis of results: As shown in Table 4-4, the mRNA encapsulation efficiency and the ability to promote the expression of the target protein of the drug-metal-phospholipid complex particles prepared using different dosage ratios based on the chemical structure of the metal-phospholipid complex components are comparable. The results suggest that the dosage ratio of the metal-phospholipid complex components can be adjusted according to the structure of the specific metal-phospholipid complex components. The basis for adjusting the dosage ratio is: because the DSPC congeners are connected to the curcumin congeners by hydrogen bonds, as long as the DSPC congeners contain multiple phosphate groups, when synthesizing the phospholipid complex, the dosage ratio of the DSPC congeners and the curcumin congeners can be adjusted according to the number of phosphate groups contained in the DSPC congeners, that is, when the DSPC congeners contain two phosphate groups, the dosage ratio of the DSPC congeners and the curcumin congeners can be adjusted to 1:2; when the DSPC congeners contain three phosphate groups, the dosage ratio of the DSPC congeners and the curcumin congeners can be adjusted to 1:3; because the hydroxyl group of the curcumin congeners is bound to Fe 3+ As long as the congeners of curcumin contain multiple binding sites, the congeners of curcumin and Fe 3+ The dosage ratio of the congeners can be adjusted according to the number of binding sites contained in the congeners of curcumin.

[0649] Table 4-4 Component proportions of different metal-phospholipid complexes and functions of the drug-lipid particles prepared therefrom

[0650]

[0651] Example 17: DSPC, curcumin and its analogs, Al 3+ Component proportions and functions of drug-metal-phospholipid complex particles prepared therefrom

[0652] According to Example 3, metal-phospholipid complexes were prepared, and curcumin was replaced by its analogs hesperidin (one hesperidin molecule contains 4 hydroxyl groups) and tea polyphenols (one tea polyphenols molecule contains 8 hydroxyl groups), respectively, to prepare three metal-phospholipid complexes (mRNA@MPP2, mRNA@MPP5, and mRNA@MPP30). When preparing these three metal-phospholipid complexes, DSPC, curcumin or its analogs, Al(NO 3 ) 3 9H 2The dosage ratios of O were 1:1:1, 1:1:1, and 1:1:2. The corresponding drug-metal-phospholipid complex nanoparticles were prepared using these three metal-phospholipid complexes (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP29). The mRNA is an mRNA encoding eGFP fluorescent protein, and its sequence is SEQ ID No.1 (720nt). The mRNA encapsulation rate of these four drug-lipid nanoparticles and their ability to promote the expression of eGFP fluorescent protein after treating 293T cells were detected according to the experimental process and experimental methods described in Example 3.5.

[0653] Analysis of results: As shown in Tables 4-5, the mRNA encapsulation efficiency and the ability to promote the expression of target proteins of drug-metal-phospholipid complex particles prepared using different dosage ratios based on the chemical structure of the metal-phospholipid complex components are comparable. The results suggest that the dosage ratio of the metal-phospholipid complex components can be adjusted according to the structure of the specific metal-phospholipid complex components. The basis for adjusting the dosage ratio is: because the analogs of DSPC and the analogs of curcumin are connected by hydrogen bonds, as long as the analogs of DSPC contain multiple phosphate groups, when synthesizing phospholipid complexes, the dosage ratio of the analogs of DSPC and the analogs of curcumin can be adjusted according to the number of phosphate groups contained in the analogs of DSPC, that is, when the analogs of DSPC contain two phosphate groups, the dosage ratio of the analogs of DSPC and the analogs of curcumin can be adjusted to 1:2; when the analogs of DSPC contain three phosphate groups, the dosage ratio of the analogs of DSPC and the analogs of curcumin can be adjusted to 1:3; because the hydroxyl groups of the analogs of curcumin are bound to Al 3+ As long as the congeners of curcumin contain multiple binding sites, the congeners of curcumin and Al 3+ The dosage ratio of the congeners can be adjusted according to the number of binding sites contained in the congeners of curcumin.

[0654] Table 4-5 Component proportions of different metal-phospholipid complexes and functions of the drug-lipid particles prepared therefrom

[0655]

[0656] The preparation of mRNA@MPP in Example 3 was completed by the research group of Professor Wang Shan from the Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Central South University.

[0657] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. If there is any inconsistency, the meaning described in the present invention or the meaning derived from the content recorded in the present invention shall prevail. In addition, the terms used in this description are only for the purpose of describing the embodiments of the present invention and are not intended to limit the present invention.

[0658] Note that the above are only preferred embodiments of the present invention and the technical principles used. Those skilled in the art will understand that the present invention is not limited to the specific embodiments described herein, and that various obvious changes, readjustments and substitutions can be made by those skilled in the art without departing from the scope of protection of the present invention. Therefore, although the present invention has been described in more detail through the above embodiments, the present invention is not limited to the above embodiments, and may also include more other equivalent embodiments without departing from the technical concept of the present invention, all of which belong to the protection scope of the present invention.

Claims

1. A metal-phospholipid complex, wherein: The metal-phospholipid complex is formed by the reaction of a phospholipid molecule part, a linker molecule part and a metal ion part, the phospholipid molecule part is connected to the linker molecule part, the linker molecule part is connected to the metal ion part through a coordination bond, and the metal-phospholipid complex is not a cationic lipid or an ionizable lipid, and the metal ion of the metal-phospholipid complex is connected to the negatively charged drug through a coordination bond; The phospholipid molecule portion is selected from at least one of DSPC, DSPE, DSPA and DSPG; The linker molecule portion is selected from at least one of curcumin, hesperidin and tea polyphenols; The metal ion moiety is selected from Fe 3+ , Ca 2+ and Al 3+ At least one of; The molar ratio of the phospholipid molecule part, the linker molecule part and the metal ion part is 1:1:(0.5-2); The preparation method of the metal-phospholipid complex comprises the following steps: Step 1: Reaction and connection of phospholipid molecules and linker molecules to form phospholipid complexes; Step 2: reacting the phospholipid complex prepared in step 1 with metal ions through coordination bonds to form a metal-phospholipid complex.

2. The metal-phospholipid complex according to claim 1, wherein The phospholipid molecule is selected from DSPC, and the structural formula is as shown in Formula 46: DSPE, the structural formula is as shown in Formula 47: DSPA, the structural formula is as shown in Formula 48: Or DSPG, the structural formula is as shown in Formula 49: At least one of .

3. The metal-phospholipid complex according to claim 2, wherein The phospholipid molecule portion is selected from DSPC of Formula 46, DSPE of Formula 47 or DSPA of Formula 48.

4. The metal-phospholipid complex according to claim 1, wherein The linker molecule is selected from curcumin, and its structural formula is as shown in Formula 19: Hesperidin, the structural formula is as shown in Formula 24: Or tea polyphenols, the structural formula is as shown in Formula 28: At least one of .

5. The metal-phospholipid complex according to claim 4, wherein The linker molecule is selected from curcumin of Formula 19, hesperidin of Formula 24 or tea polyphenols of Formula 28.

6. The metal-phospholipid complex according to claim 1, wherein The metal ion moiety is selected from Fe 3+ , Ca 2+ or Al 3+ .

7. The metal-phospholipid complex according to claim 1, wherein The metal-phospholipid complex is made of a phospholipid molecule part, a linker molecule part and a metal ion part, wherein the phospholipid molecule part is selected from DSPC, DSPE or DSPA, the linker molecule part is selected from curcumin, hesperidin or tea polyphenols, and the metal ion part is selected from Fe 3+ , Ca 2+ or Al 3+ .

8. The metal-phospholipid complex according to claim 7, wherein The metal-phospholipid complex is made of a phospholipid molecule part, a linker molecule part and a metal ion part, wherein the phospholipid molecule part is selected from DSPC of Formula 46, DSPE of Formula 47 or DSPA of Formula 48, the linker molecule part is selected from curcumin of Formula 19, hesperidin of Formula 24 or tea polyphenols of Formula 28, and the metal ion part is selected from Fe 3+ , Ca 2+ or Al 3+ . 9 . The metal-phospholipid complex according to claim 1 , wherein the molar ratio of the phospholipid molecule portion, the linker molecule portion and the metal ion portion is 1:1:

1.

10. The method for preparing the metal-phospholipid complex according to any one of claims 1 to 9, wherein: The preparation method comprises the following steps: Step 1: Reaction and connection of phospholipid molecules and linker molecules to form phospholipid complexes; Step 2: reacting the phospholipid complex prepared in step 1 with metal ions through coordination bonds to form a metal-phospholipid complex.

11. The preparation method according to claim 10, wherein: In step 1, the phospholipid molecules and the linker molecules are dissolved in ethanol for reaction, and then n-hexane is added for precipitation to obtain the phospholipid complex.

12. The preparation method according to claim 11, wherein The molar ratio of the phospholipid molecules to the linker molecules is 1:

1.

13. The preparation method according to claim 11 or 12, wherein: The reaction conditions included 65°C for 2 hours.

14. The preparation method according to any one of claims 10 to 13, wherein: In step 2, the phospholipid complex is dissolved in ethanol and reacts with the metal ion to obtain the metal-phospholipid complex.

15. The preparation method according to claim 14, wherein: The molar ratio of the phospholipid complex to the metal ion is 1:(1-2).

16. The preparation method according to claim 14 or 15, wherein: The reaction conditions included 60°C for 2 hours.

17. Use of the metal-phospholipid complex according to any one of claims 1 to 9 in preparing metal-phospholipid complex particles, wherein: The metal-phospholipid complex particles contain: (i) Metal-phospholipid complexes; (ii) a conjugated lipid that inhibits particle aggregation, wherein the conjugated lipid that inhibits particle aggregation is not a cationic lipid or an ionizable lipid; as well as (iii) non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation.

18. The use according to claim 17, wherein: The conjugated lipids that inhibit particle aggregation include PEG-lipid conjugates and / or PEG-DAA.

19. The use according to claim 18, wherein: The PEG-lipid conjugate is selected from phosphatidylethanolamine-polyethylene glycol 2000, and the structural formula is as shown in Formula 42: Phosphatidylethanolamine-polyethylene glycol 700, the structural formula is as shown in Formula 43: Phosphatidylethanolamine-polyethylene glycol 1000, the structural formula is as shown in Formula 44: Phosphatidylethanolamine-polyethylene glycol 5000, the structural formula is as shown in Formula 45: and combinations of one or more of its derivatives; R1 and R2 are independently: capryloyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytanoyl.

20. The use according to claim 19, wherein: The PEG-lipid conjugate is selected from a combination of one or more of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000 or DSPE-PEG5000.

21. The use according to claim 20, wherein: The PEG-lipid conjugate is selected from DSPE-PEG2000, and the structural formula is as shown in Formula 53: DSPE-PEG700, the structural formula is as shown in Formula 50: DSPE-PEG1000, the structural formula is as shown in Formula 51: Or DSPE-PEG5000, the structural formula is as shown in Formula 52:

22. The use according to claim 17, wherein: The non-cationic lipid or non-ionizable lipid described in (iii) is a combination of one or more of cholesterol and its derivatives.

23. The use according to claim 22, wherein: The non-cationic lipid or non-ionizable lipid described in (iii) is cholesterol, and its structural formula is as shown in Formula 40:

24. The use according to claim 22, wherein: The non-cationic lipids or non-ionizable lipids described in (iii) also include one or more combinations selected from phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphoceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysophosphatidylcholine LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, 1-phosphoceramide S1P, cholesterol sulfate, and their derivatives.

25. The use according to claim 24, wherein: (iii) The non-cationic lipid or non-ionizable lipid further comprises a combination of one or more selected from phosphatidylcholine PC of formula 1, phosphatidylethanolamine PE of formula 2, phosphatidylserine PS of formula 3, phosphatidic acid PA of formula 4, phosphatidylglycerol PG of formula 5, 1-phosphoceramide SP of formula 6, phosphatidylinositol PI of formula 7, phosphatidylthreonine PT of formula 8, sphingomyelin SM of formula 9, lysophosphatidylcholine LPC of formula 10, lysophosphatidylethanolamine LPE of formula 11, lysophosphatidylserine LPS of formula 12, lysophosphatidic acid LPA of formula 13, lysophosphatidylglycerol LPG of formula 14, lysophosphatidylinositol LPI of formula 15, lysophosphatidylthreonine LPT of formula 16, lysophospholipin LSM of formula 17, 1-phospho-sphingosine S1P of formula 18, cholesterol sulfate of formula 41, and derivatives thereof; Wherein, R1 and R2 are independently: capryloyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytanoyl.

26. The use according to claim 24, wherein: The non-cationic lipid or non-ionizable lipid described in (iii) includes cholesterol and a combination of one or more selected from DSPC, DSPE, DSPA or DSPG.

27. The use according to claim 26, wherein: The non-cationic lipid or non-ionizable lipid described in (iii) includes cholesterol of Formula 40 and DSPC of Formula 46.

28. The use according to claim 24, wherein: The metal-phospholipid complex particles are made of (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid. The molar proportion of the metal-phospholipid complex in the raw material is 10% to 40%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, the molar proportion of cholesterol in the raw material is 35% to 75%, and the molar proportion of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 0% to 40%.

29. The use according to claim 24, wherein: The metal-phospholipid complex particles are made of (i) metal-phospholipid complex, (ii) conjugated lipids that inhibit particle aggregation, and (iii) non-cationic lipids or non-ionizable lipids, wherein the metal-phospholipid complex accounts for 5% to less than 10% by mole in the raw material, the conjugated lipids that inhibit particle aggregation account for 2% to 10% by mole in the raw material, the cholesterol accounts for 15% to less than 35%, 35% to 75%, or greater than 75% to 80% by mole in the raw material, and the non-cationic lipids or non-ionizable lipids other than cholesterol account for 0% to 40% or greater than 40% to 51% by mole in the raw material; or The molar proportion of the metal-phospholipid complex in the raw material is greater than 40% to 50%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, the molar proportion of cholesterol in the raw material is 15% to less than 35%, 35% to 75% or greater than 75% to 80%, and the molar proportion of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 0% to 40% or greater than 40% to 51%; or The molar proportion of the metal-phospholipid complex in the raw material is 10% to 40%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, the molar proportion of cholesterol in the raw material is 15% to less than 35% or greater than 75% to 80%, and the molar proportion of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 0% to 40% or greater than 40% to 51%.

30. The use according to claim 28 or 29, wherein: The molar proportion of the metal-phospholipid complex in the raw material is 7% to less than 10%, 10% to 30% or 20% to 30%, preferably 25%.

31. The use according to claim 28 or 29, wherein: The molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10% or 5% to 10%, preferably 10%.

32. The use according to claim 28 or 29, wherein: The molar proportion of cholesterol in the raw material is 15% to less than 35%, 35% to 56% or 35% to 55%, preferably 40%.

33. The use according to claim 28 or 29, wherein: The molar proportion of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 5% to 30%, 25% to 40%, greater than 40% to 45% or 20% to 25%.

34. The use according to claim 28, wherein: The molar proportion of the metal-phospholipid complex in the raw material is 15% to 25%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 4% to 10%, the molar proportion of cholesterol in the raw material is 40% to 46%, the molar proportion of DSPC in the raw material is 25% to 35%, and the metal ion part of the metal-phospholipid complex is selected from Fe 3+ .

35. The use according to claim 34, wherein: The molar proportion of the metal-phospholipid complex in the raw material is 15%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 4%, the molar proportion of the cholesterol in the raw material is 46%, and the molar proportion of the DSPC in the raw material is 35%; or the molar proportion of the metal-phospholipid complex in the raw material is 25%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 10%, the molar proportion of the cholesterol in the raw material is 40%, and the molar proportion of the DSPC in the raw material is 25%; the metal ion part in the metal-phospholipid complex is selected from Fe 3+ .

36. The use according to claim 28, wherein: The molar proportion of the metal-phospholipid complex in the raw material is 10% to 30%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%, the molar proportion of the cholesterol in the raw material is 35% to 56%, and the molar proportion of the DSPC in the raw material is 34% to 40%; the metal ion part in the metal-phospholipid complex is selected from Al 3+ .

37. The use according to claim 29, wherein: The molar proportion of the metal-phospholipid complex in the raw material is 10% to 30%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%, the molar proportion of the cholesterol in the raw material is 35% to 56%, and the molar proportion of the DSPC in the raw material is 40% to 45%; or The molar proportion of the metal-phospholipid complex in the raw material is 10% to 30%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%, the molar proportion of the cholesterol in the raw material is 15% to 35%, and the molar proportion of the DSPC in the raw material is 34% to 40% or greater than 40% to 45%; or The molar proportion of the metal-phospholipid complex in the raw material is 7% to less than 10%, the molar proportion of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%, the molar proportion of the cholesterol in the raw material is 15% to less than 35% or 35% to 56%, and the molar proportion of the DSPC in the raw material is 34% to 40% or greater than 40% to 45%; the metal ion part of the metal-phospholipid complex is selected from Al 3+ .

38. The use according to claim 37, wherein: The molar proportion of the metal-phospholipid complex in the raw material is 7%, 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 56%, and the molar proportion of DSPC in the raw material is 34%; the metal ion part in the metal-phospholipid complex is selected from Al 3+ .

39. Use of the metal-phospholipid complex according to any one of claims 1 to 9 in preparing a nucleic acid delivery system.

40. The use according to claim 39, wherein: The nucleic acid delivery system is used to introduce nucleic acids into cells.

41. The use according to claim 39, wherein: The nucleic acid is used to silence the expression of a target sequence in a mammalian subject, to deliver a drug in vivo in a mammal, to deliver a drug from the body to a mammalian cell, or to treat a disease or disorder in a mammal.

42. The use according to claim 41, wherein: The mammal is a human.

43. The use according to claim 41, wherein: The disease or disorder is associated with the expression of a gene that contains a target sequence for the drug.

44. The use according to claim 41, wherein: The disease or condition includes cancer, viral infection, diabetes or Alzheimer's disease.

45. The use according to claim 41, wherein: Such diseases or conditions include autoimmune diseases.

46. ​​The use according to claim 44, wherein: The viral infection includes hepatitis A, hepatitis B, hepatitis C, SARS-Cov-2, HIV, HPV, influenza, smallpox or syphilis.

47. The use according to claim 44, wherein: The cancer includes liver cancer, glioma, melanoma, lung cancer, pancreatic cancer or breast cancer.

48. The use according to claim 39, wherein: The nucleic acid delivery system is a vaccine.

49. The use according to claim 39, wherein: The administration routes of the nucleic acid delivery system include intrathecal injection, intramuscular administration, intravenous injection or intratumoral injection.

50. The use according to claim 39, wherein: The administration route of the nucleic acid delivery system includes intracranial injection.

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