Metal-phospholipid complex, metal-phospholipid complex particle, drug-lipid particle, and method for producing the same and application thereof

Abstract

The present disclosure provides metal-phospholipid complexes, metal-phospholipid complex particles, and drug-lipid particles, as well as their manufacturing methods and applications, and relates to the field of biotechnology. Metal-phospholipid complexes are formed by the reaction of a phospholipid molecular moiety, a linker molecular moiety, and a metal ion moiety. Metal-phospholipid complex particles contain a metal-phospholipid complex, a conjugated lipid that inhibits particle aggregation, and a non-cationic or non-ionizable lipid. Drug-lipid particles include drugs and metal-phospholipid complex particles. Metal-phospholipid complexes are used to adsorb negatively charged drugs and self-assemble with other components into metal-phospholipid complex particles (MPPs). While these MPPs are no less effective than LNPs based on cationic and / or ionizable lipids, they do not use cationic or ionizable lipids and, compared to LNPs, offer significantly reduced toxicity, significantly improved biological safety, and easier delivery of negatively charged drugs within living organisms.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This disclosure claims priority to three Chinese patent applications: application number 202210951941.8, filed on August 9, 2022, with the Patent Office of China, entitled "Drug-Lipid Particles, and Their Preparation Methods and Applications"; application number 202210950392.2, filed on August 9, 2022, with the Patent Office of China, entitled "Metal-Phospholipid Complex Particles, and Their Preparation Methods and Applications"; and application number 202210950391.8, filed on August 9, 2022, with the Patent Office of China, entitled "Metal-Phospholipid Complexes, and Their Preparation Methods and Applications," the contents of which are incorporated herein by reference in their entireties. [Technical Field]

[0002] The present disclosure relates to the field of biotechnology, and in particular to metal-phospholipid complexes, metal-phospholipid complex particles, and drug-lipid particles, as well as methods for producing and applications thereof. [Background technology]

[0003] With the continuous development of molecular biology technology, the correlation between genes and diseases is becoming increasingly understood. Nucleic acid drugs are artificially synthesized fragments of DNA or RNA with therapeutic functions, attracting attention due to their great potential for application in disease diagnosis and treatment. These drugs act directly on disease-causing target genes or mRNA, exerting their therapeutic role at the genetic level. Compared with traditional small molecule drugs and antibody drugs, nucleic acid drugs are not limited by the druggability of target proteins, can treat a wider range of diseases, and can fundamentally control the expression of disease-causing genes. Nucleic acid drugs have clear advantages such as high efficiency, low toxicity, and high specificity, and are expected to become the third largest type of drug after small molecule drugs and antibody drugs.

[0004] However, nucleic acid drugs are easily degraded by nucleases in the body, and their relatively large molecular weight and negative charge make them difficult to penetrate cell membranes and act. Therefore, the search for a safe and effective nucleic acid drug delivery system is a bottleneck in nucleic acid drug development that must be resolved as soon as possible. Currently, nucleic acid drug delivery carriers can be mainly divided into viral and non-viral carriers. Viral carriers induce an immune response upon entry into the human body and are therefore not widely used. Among non-viral carriers, nanoparticles and small molecule conjugates are the most commonly used. Compared with small molecule conjugates directly conjugated to nucleic acid drugs, nanoparticles can more effectively encapsulate nucleic acid drugs, prevent their rapid degradation by nucleases in the body, and increase their circulation time in the body. The mechanism of nucleic acid encapsulation in nanoparticles relies on the adsorption of negatively charged nucleic acids by positively charged cationic lipids. However, cationic lipids are relatively cytotoxic, and their toxic mechanism is as follows: (1) They cause cell atrophy, reduce the number of mitoses, and vacuolate the cytoplasm. (2) They interact with biological proteins such as protein kinase C, thereby disrupting their activity. (3) They induce the secretion of various proinflammatory cytokines and chemokines by activating p38 mitogen-activated protein kinase and nuclear factor-κB transcription factors. Ionizable lipids are lipids containing positively charged ionizable amine groups that are uncharged under physiological conditions (pH = 7.4) but become protonated and positively charged at relatively low pH values. Therefore, ionizable lipids can be used to partially or completely replace cationic lipids as the main component of nanoparticles responsible for nucleic acid adsorption. After nanoparticles containing ionizable lipids enter the lysosomes of living cells, the ionizable lipids become positively charged in the low pH (pH = 4.0-6.5) environment within the lysosome. Although ionizable lipids reduce the cytotoxic and highly pro-inflammatory effects of some permanently positively charged cationic lipids, their cytotoxicity and immunogenicity remain relatively high.Lipid nanoparticles (LNPs) based on cationic and / or ionizable lipids are currently available as nanoparticle nucleic acid drug delivery systems for clinical use. Cationic and / or ionizable lipids are the main components of LNPs and are responsible for nucleic acid adsorption. At the same time, cytotoxicity and immunogenicity mediated by cationic and / or ionizable lipids remain an important reason for the relatively high toxicity of LNPs.

[0005] Therefore, when using a delivery system to deliver negatively charged drugs (e.g., nucleic acid drugs, protein drugs, polypeptide drugs, small molecule drugs, etc.), none of the nanoparticle delivery systems developed relying on cationic lipids and / or ionizable lipids can fundamentally solve the toxicity problem of nanoparticle delivery systems. There is an urgent need for a liposome delivery system with low toxicity that does not use cationic lipids and / or ionizable lipids.

[0006] An object of the present disclosure is to provide a metal-phospholipid complex and a method for producing the same, so as to solve at least one of the technical problems existing in the prior art.

[0007] In order to achieve the above object, the present disclosure employs the following inventions.

[0008] The metal-phospholipid complex provided by the present disclosure comprises a phospholipid molecular portion, a linker molecular portion, and a metal ion portion, the phospholipid molecular portion and the linker molecular portion being linked by a coordinate bond, and the metal-phospholipid complex is not a cationic lipid or an ionizable lipid.

[0009] Furthermore, the phospholipid molecule portion is one or a combination of multiple molecules selected from lecithin (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), ceramide-1-phosphate (SP), phosphatidylinositol (PI), phosphatidylthreonine (PT), sphingomyelin (SM), lysolecithin (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysosphingomyelin (LSM), sphingosine-1-phosphate (S1P), and derivatives thereof. The term "these" in "and derivatives thereof" refers to "lecithin (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), ceramide-1-phosphate (SP), phosphatidylinositol (PI), phosphatidylthreonine (PT), sphingomyelin (SM), lysolecithin (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysosphingomyelin (LSM), and sphingosine-1-phosphate (S1P)." In the present disclosure, "and derivatives thereof" all have the same meaning. The phospholipid molecule portion may be, for example, but is not limited to, lecithin (PC), a lecithin (PC) derivative, phosphatidylethanolamine (PE), a phosphatidylethanolamine (PE) derivative, phosphatidylglycerol (PG), a phosphatidylglycerol (PG) derivative, phosphatidylglycerol (PG) and lecithin (PC), lecithin (PC) and a lecithin (PC) derivative, etc.

[0010] Furthermore, the phospholipid molecule portion is [ka] JPEG2025527283000003.jpg243170JPEG2025527283000004.jpg220170JPEG2025527283000005.jpg210170JPEG2025527283000006.jpg128170

[0011] Furthermore, the phospholipid molecule portion is one or a combination of more selected from lecithin (PC) (Formula 1), phosphatidylethanolamine (PE) (Formula 2), phosphatidic acid (PA) (Formula 4), phosphatidylglycerol (PG) (Formula 5), and derivatives thereof.

[0012] Furthermore, the phospholipid molecule portion is one or a combination of two or more selected from distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidic acid (DSPA), distearoylphosphatidylglycerol (DSPG), and derivatives thereof.

[0013] Furthermore, the phospholipid molecule portion is [ka]

[0014] Furthermore, the phospholipid molecule portion is selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48), or DSPG (Formula 49).

[0015] Furthermore, the linker molecule portion is one or a combination of two or more 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 pinbeimin C, and derivatives thereof.

[0016] Furthermore, the linker molecule portion is [ka] JPEG2025527283000009.jpg199170JPEG2025527283000010.jpg210170JPEG2025527283000011.jpg162169

[0017] Furthermore, the linker molecule portion is [ka]

[0018] Furthermore, the linker molecule portion is one or a combination of two or more selected from curcumin (formula 19), hesperetin (formula 24), tea polyphenol (formula 28), and derivatives thereof.

[0019] Further, the linker molecule portion is selected from curcumin (Formula 19), hesperetin (Formula 24) or tea polyphenols (Formula 28).

[0020] Furthermore, the metal ion moiety is Fe 3+ , Ag + , Ba 2+ , Ca 2+ , Cd 2+ , Cu 2+ , Fe 2+ , Mn 2+ , Mg 2+ , Mo 2+ , Zn 2+ , Pt 2+ , Au 2+ , Al 3+ , Ce 3+ , Co 3+ , Cr3+, Eu 3+ , Gd 3+ , Ni 3+ , W 3+ , V 3+ , Zr 3+ It is one or a combination of several selected from the above.

[0021] Furthermore, the metal ion moiety is Fe 3+, Ca 2+ , Al 3+ It is one or a combination of several selected from the above.

[0022] Furthermore, the metal ion moiety is Fe 3+ , Ca 2+ or Al 3+ is selected from.

[0023] Furthermore, the metal-phospholipid complex comprises a phospholipid molecular portion, a linker molecular portion, and a metal ion portion, the phospholipid molecular portion being selected from DSPC, DSPE, or DSPA, the linker molecular portion being selected from curcumin, hesperetin, or tea polyphenol, and the metal ion portion being Fe 3+ , Ca 2+ or Al 3+ is selected from.

[0024] Furthermore, the metal-phospholipid complex comprises a phospholipid molecular portion, a linker molecular portion, and a metal ion portion, the phospholipid molecular portion being selected from DSPC (Formula 46), DSPE (Formula 47), or DSPA (Formula 48), the linker molecular portion being selected from curcumin (Formula 19), hesperetin (Formula 24), or tea polyphenol (Formula 28), and the metal ion portion being Fe 3+ , Ca 2+ or Al 3+ is selected from.

[0025] Furthermore, the molar ratio of the phospholipid molecule portion, the linker molecule portion and the metal ion portion is 1:1:(0.5 to 2).

[0026] Furthermore, the phospholipid molecule portion is DSPC (Formula 46), the linker molecule portion is selected from curcumin (Formula 19), and the metal ion portion is Fe 3+ is selected from.

[0027] Furthermore, the molar ratio of the phospholipid molecule portion, the linker molecule portion, and the metal ion portion is 1:1:1.

[0028] Furthermore, DSPC, curcumin and Fe 3+ / Al 3+ The molar ratio is 1:1:1.

[0029] In addition, DSPC, hesperetin and Fe 3+ / Al 3+ The molar ratio is 1:1:1.

[0030] In addition, DSPC, tea polyphenols and Fe 3+ / Al 3+ The molar ratio is 1:1:2.

[0031] Furthermore, Fe 3+ is selected from FeCl3, and Al 3+ is selected from Al(NO3)3·9H2O.

[0032] The present disclosure provides a method for producing a metal-phospholipid complex, the method comprising:

[0033] Step 1: reacting and connecting phospholipid molecules with linker molecules to form a phospholipid complex;

[0034] and Step 2, in which the phospholipid complex produced in Step 1 is reacted with a metal ion through a coordinate bond to form a metal-phospholipid complex.

[0035] In step 1, the phospholipid molecule and the linker molecule are dissolved in ethanol to react, and then precipitated by adding n-hexane to obtain the phospholipid complex. The reaction conditions include reacting at 65°C for 2 hours.

[0036] Furthermore, the molar ratio of the phospholipid molecules to the linker molecules is 1:1.

[0037] In step 2, the phospholipid complex and the metal ion are dissolved in ethanol and reacted to obtain the metal-phospholipid complex, and the reaction conditions include reacting at 60°C for 2 hours.

[0038] Furthermore, the molar ratio of the phospholipid complex to the metal ion is 1:(1 to 2).

[0039] The present disclosure provides metal-chelated phospholipid complex nanoparticles (MPPs). The metal-phospholipid complex particles contain (i) the metal-phospholipid complex described above, (ii) a conjugated lipid that inhibits particle aggregation and is not a cationic or ionizable lipid, and (iii) a non-cationic or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation. "(iii) a non-cationic or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation" may be simply referred to as "non-cationic or non-ionizable lipid."

[0040] Additionally, the particle aggregation inhibiting conjugation lipids include polyethylene glycol (PEG)-lipid conjugates and / or PEG-dialkyloxypropyl (DAA).

[0041] Furthermore, the PEG-lipid conjugate may be [ka] and a combination of one or more selected from the group consisting of derivatives thereof, R1 and R2 are each independently a decanoyl group, a lauroyl group, a myristoyl group, a palmitoyl group, a stearoyl group, an oleyl group, a linoleoyl group, an erucyl group, an arachidonoyl group, or a phytanoyl group.

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

[0043] Furthermore, the PEG-lipid conjugate may be [ka] JPEG2025527283000015.jpg178170

[0044] 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).

[0045] Furthermore, the non-cationic or non-ionizable lipid other than the metal-phospholipid complex and the lipid of the conjugation that inhibits particle aggregation in (iii) is a combination of one or more of cholesterol and its derivatives.

[0046] Furthermore, the non-cationic or non-ionizable lipid other than the metal-phospholipid complex and the lipid of the conjugation that inhibits particle aggregation in (iii) is [ka] and combinations of one or more of the derivatives thereof.

[0047] Furthermore, the non-cationic or non-ionizable lipid in (iii) is cholesterol (Formula 40).

[0048] Furthermore, in (iii), the non-cationic or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation further includes, in addition to cholesterol, one or more combinations selected from lecithin PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, ceramide-1-phosphate SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysolecithin LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, sphingosine-1-phosphate S1P, cholesterol sulfate, and derivatives thereof.

[0049] Further, the non-cationic or non-ionizable lipid other than the metal-phospholipid complex and the lipid of the conjugation that inhibits particle aggregation in (iii) may be, in addition to cholesterol, [ka] JPEG2025527283000018.jpg212168JPEG2025527283000019.jpg242167JPEG2025527283000020.jpg109169 and derivatives thereof, R1 and R2 are decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleyl, linoleoyl, erucyl, arachidonoyl, or phytanoyl groups.

[0050] Furthermore, in (iii), the non-cationic or non-ionizable lipid other than the metal-phospholipid complex and the lipid of the conjugation that inhibits particle aggregation includes a combination of cholesterol and one or more selected from DSPC, DSPE, DSPA, and DSPG.

[0051] Furthermore, in (iii), the non-cationic or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation includes a combination of cholesterol (Formula 40) and one or more selected from DSPC (Formula 46), DSPE (Formula 47), DSPA (Formula 48), or DSPG (Formula 49).

[0052] Further, the non-cationic or non-ionizable lipids described in (iii) include cholesterol (formula 40) and DSPC (formula 46).

[0053] Furthermore, the metal-phospholipid complex particles comprise (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation, wherein the molar fraction of the metal-phospholipid complex in the raw material is 10% to 40%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, the molar fraction of the cholesterol in the raw material is 35% to 75%, and the molar fraction of the non-cationic lipid or the non-ionizable lipid other than cholesterol in the raw material is 0% to 40%.

[0054] Furthermore, the metal-phospholipid complex particles comprise (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the molar fraction of the metal-phospholipid complex in the raw material is 5% or more and less than 10%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, the molar fraction of the cholesterol in the raw material is 15% or more and less than 35%, 35% to 75%, or more than 75% to 80%, and the molar fraction of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 0% to 40%, or more than 40% to 51%. Alternatively,

[0055] The molar fraction of the metal-phospholipid complex in the raw material is greater than 40% to 50%, the molar fraction of the particle aggregation-inhibiting conjugated lipid in the raw material is 2% to 10%, the molar fraction of the cholesterol in the raw material is 15% or greater but less than 35%, 35% to 75%, or greater than 75% to 80%, and the molar fraction of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 0% to 40%, or greater than 40% to 51%. Alternatively,

[0056] The molar fraction of the metal-phospholipid complex in the raw material is 10% to 40%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 2% to 10%, the molar fraction of the cholesterol in the raw material is 15% or more but less than 35% or more than 75% to 80%, and the molar fraction of non-cationic lipids or non-ionizable lipids other than cholesterol in the raw material is 0% to 40% or more than 40% to 51%.

[0057] Furthermore, the molar fraction of the metal-phospholipid complex in the raw material is 7% or more and less than 10%, 10% to 30%, or 20% to 30%, and preferably 25%.

[0058] Furthermore, the molar fraction of conjugated lipids that inhibit particle aggregation in the raw material is 3% to 10% or 5% to 10%, and preferably 10%.

[0059] Furthermore, the molar fraction of cholesterol in the raw material is 15% or more and less than 35%, 35% to 56%, or 35% to 55%, and preferably 40%.

[0060] Furthermore, the molar fraction of non-cationic 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%.

[0061] Furthermore, the molar fraction of the metal-phospholipid complex in the raw material is 15% to 25%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 4% to 10%, the molar fraction of the cholesterol in the raw material is 40% to 46%, the molar fraction of the DSPC in the raw material is 25% to 35%, and the metal ion moiety in the metal-phospholipid complex is Fe 3+ is selected from.

[0062] Furthermore, the metal-phospholipid complex (metal ion portion is Fe 3+ The molar fraction of the metal-phospholipid complex (metal ion moiety is Fe) in the raw material is 15%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 4%, the molar fraction of the cholesterol in the raw material is 46%, and the molar fraction of the DSPC in the raw material is 35%. 3+ The molar fraction of the particle aggregation-inhibiting conjugated lipid in the feedstock is 10%, the molar fraction of the cholesterol in the feedstock is 40%, and the molar fraction of the DSPC in the feedstock is 25%.

[0063] Furthermore, the molar fraction of the metal-phospholipid complex in the raw material is 10% to 30%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%, the molar fraction of the cholesterol in the raw material is 35% to 56%, the molar fraction of the DSPC in the raw material is 34% to 40%, and the metal ion moiety in the metal-phospholipid complex is Al 3+ is selected from.

[0064] Furthermore, the molar fraction of the metal-phospholipid complex in the raw material is 10% to 30%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%, the molar fraction of the cholesterol in the raw material is 35% to 56%, the molar fraction of the DSPC in the raw material is 40% to 45%, and the metal ion moiety in the metal-phospholipid complex is Al 3+ Alternatively,

[0065] The molar fraction of the metal-phospholipid complex in the raw material is 10% to 30%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%, the molar fraction of the cholesterol in the raw material is 15% to 35%, the molar fraction of the DSPC in the raw material is 34% to 40% or more than 40% to 45%, and the metal ion moiety in the metal-phospholipid complex is Al 3+ Alternatively,

[0066] The molar fraction of the metal-phospholipid complex in the raw material is 7% or more and less than 10%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 3% to 10%, the molar fraction of the cholesterol in the raw material is 15% or more and less than 35% or 35% to 56%, the molar fraction of the DSPC in the raw material is 34% to 40%, or more than 40% to 45%, and the metal ion moiety in the metal-phospholipid complex is Al 3+ is selected from.

[0067] Furthermore, the metal-phospholipid complex (metal ion portion is Al 3+ The molar fraction of lipids that inhibit particle aggregation in the feedstock is 7%, the molar fraction of conjugated lipids that inhibit particle aggregation in the feedstock is 3%, the molar fraction of cholesterol in the feedstock is 56%, and the molar fraction of DSPC in the feedstock is 34%.

[0068] Furthermore, the metal-phospholipid complexes were composed of DSPC, curcumin, and Fe 3+ The conjugated lipid that inhibits particle aggregation is DSPE-PEG2000, the non-cationic or non-ionizable lipid is cholesterol and DSPC, the molar fraction of the metal-phospholipid complex in the raw material is 15%, the molar fraction of DSPE-PEG2000 in the raw material is 4%, the molar fraction of cholesterol in the raw material is 46%, and the molar fraction of DSPC in the raw material is 35%.

[0069] Furthermore, the metal-phospholipid complexes were composed of DSPC, curcumin, and Fe 3+The conjugated lipid that inhibits particle aggregation is DSPE-PEG2000, the non-cationic or non-ionizable lipid is cholesterol and DSPC, the molar fraction of the metal-phospholipid complex in the raw material is 25%, the molar fraction of DSPE-PEG2000 in the raw material is 10%, the molar fraction of cholesterol in the raw material is 40%, and the molar fraction of DSPC in the raw material is 25%.

[0070] Furthermore, the metal-phospholipid complexes were composed of DSPC, curcumin, and Al. 3+ The conjugated lipid that inhibits particle aggregation is DSPE-PEG2000, the non-cationic or non-ionizable lipid is cholesterol and DSPC, the molar fraction of the metal-phospholipid complex in the raw material is 7%, the molar fraction of DSPE-PEG2000 in the raw material is 3%, the molar fraction of cholesterol in the raw material is 56%, and the molar fraction of DSPC in the raw material is 34%.

[0071] The present disclosure provides a method for producing the above-mentioned metal-phospholipid complex particles, comprising mixing (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation to obtain the metal-phospholipid complex particles.

[0072] The present disclosure provides:

[0073] (a) a drug, which is a negatively charged molecule;

[0074] (b) A drug-lipid particle containing metal-chelated phospholipid complex nanoparticles (MPP), which are the metal-phospholipid complex particles of the present disclosure, is provided.

[0075] Furthermore, the drug is encapsulated in the metal-phospholipid complex particles.

[0076] Furthermore, the drug is a combination of one or more selected from nucleic acids, proteins, polypeptides, small molecules, nucleic acid analogs, protein analogs, and polypeptide analogs.

[0077] Furthermore, the nucleic acid is one or a combination of multiple types selected from mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, and artificial nucleic acids.

[0078] Further, the nucleic acid may be an mRNA sequence encoding eGFP (Enhanced Green Fluorescent Protein) as shown in SEQ ID No. 1, an mRNA sequence encoding the receptor-binding domain RBD of the S1 subunit of the novel coronavirus as shown in SEQ ID No. 2, an mRNA sequence encoding NY-ESO-1 (New York esophageal squamous cell carcinoma-1) as shown in SEQ ID No. 3, an siRNA sequence of the Bcl-2 gene (B-cell lymphoma / Leukemia-2 gene) as shown in SEQ ID No. 4 as an antisense strand and SEQ ID No. 21 as a sense strand, an siRNA sequence of the PLK1 gene (Polo-like Kinase 1) as shown in SEQ ID No. 6 as an antisense strand and SEQ ID No. 23 as a sense strand, an siRNA sequence of the Gal-1 gene as shown in SEQ ID No. 8, an ASO sequence of the STAT-3 gene as shown in SEQ ID No. 10, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 11, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 12, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 13, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 14, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 15, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 16, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 17, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 18, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 19, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 20, an siRNA sequence of the STAT-3 gene as shown in SEQ ID No. 21, an siRNA sequence of the The ASO sequence of the α-syn gene (α-synuclein) shown in No. 12, the ASO sequence of the Bcl-2 gene shown in SEQ ID No. 14, the mRNA sequence encoding the wild-type novel coronavirus S protein shown in SEQ ID No. 16, the double-stranded DNA sequence whose antisense strand is SEQ ID No. 17 and whose sense strand is SEQ ID No. 25, the single-stranded DNA shown in SEQ ID No. 18, or the siRNA sequence of the B7-H4 gene whose sense strand is SEQ ID No. 19 and whose antisense strand is SEQ ID No. 26.

[0079] The present disclosure provides a method for producing the drug-lipid particles, which are obtained by encapsulating a drug in a metal-phospholipid complex particle.

[0080] Furthermore, the metal-phospholipid complex, the conjugated lipid that inhibits particle aggregation, and the non-cationic or non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer to form an aqueous phase, and the organic phase and the aqueous phase are uniformly mixed to obtain drug-lipid particles.

[0081] Furthermore, the organic compound is ethanol.

[0082] Furthermore, the buffer is an enzyme-free PBS buffer.

[0083] Additionally, methods for mixing the organic and aqueous phases include microfluidic chips or ultrasound.

[0084] Further, (a) a drug, (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic or non-ionizable lipid are mixed to obtain the drug-lipid particles.

[0085] The present disclosure provides an application of the above-mentioned metal-phospholipid complex in a nucleic acid delivery system. The nucleic acid delivery system is further used to introduce a nucleic acid into a cell. The nucleic acid is further used to silence the expression of a target sequence in a mammalian subject, to deliver a drug within a mammalian body, to deliver a drug from the body to mammalian cells, or to treat a disease or condition in a mammal.

[0086] The present disclosure provides the use of the above-described metal-phospholipid complex particles or the above-described drug-lipid particles in a composition used for drug delivery. Furthermore, the composition is used to introduce a drug into a cell. The composition is a pharmaceutical agent. Furthermore, the pharmaceutical agent is used to silence expression of a target sequence in a mammalian subject, to deliver a drug within a mammalian body, to deliver a drug from the body to mammalian cells, or to treat a disease or condition in a mammal.

[0087] The present disclosure provides the application of the above metal-phospholipid complexes, or metal-phospholipid complex particles, or drug-lipid particles or drugs in the prevention / treatment of mammalian diseases or conditions.

[0088] In the above invention, the mammal may be a human. The disease or condition may be associated with the expression of a gene containing a target sequence for the drug. The disease or condition may include cancer, viral infection, autoimmune disease, diabetes, or Alzheimer's disease. The viral infection may include hepatitis A, hepatitis B, hepatitis C, SARS-CoV-2 (2019 novel coronavirus), HIV (AIDS virus), HPV (human papillomavirus), influenza, smallpox, or syphilis. The cancer may include liver cancer, glioma, melanoma, lung cancer, pancreatic cancer, or breast cancer.

[0089] Furthermore, the agent is a vaccine. Furthermore, the route of administration of the agent includes intrathecal injection, intramuscular injection, intracranial injection, intravenous injection, or intratumoral injection.

[0090] A pharmaceutical agent comprising the metal-phospholipid complex or metal-phospholipid complex particle or drug-lipid particle of the present disclosure.

[0091] Additionally, the agent is a vaccine.

[0092] Furthermore, the vaccine is a COVID-19 vaccine.

[0093] Compared with the prior art, the advantageous effects of the present disclosure are as follows:

[0094] The primary role of the metal-phospholipid complexes provided by the present disclosure is to adsorb negatively charged drugs. When self-assembled with other lipids into metal-phospholipid complex particles (MPPs), they achieve efficacy not lower than that of LNPs based on cationic and / or ionizable lipids. The absence of cationic and ionizable lipids significantly reduces toxicity compared to LNPs, significantly improving biological safety and facilitating the delivery of negatively charged drugs in living organisms. [Brief explanation of the drawings]

[0095] In order to more clearly describe the invention according to the specific embodiments of the present disclosure or the prior art, the drawings necessary for describing the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present disclosure. Those skilled in the art can obtain other drawings based on these drawings without creative work.

[0096] [Figure 1-1] 1 shows the percentage of eGFP-positive cells caused by transfection of 293T with eGFP-mRNA@MPP(Fe 3+ ), as provided in Example 3.5.1 of the present disclosure.

[0097] [Figure 1-2] 1 shows the expression levels of RBD caused by transfection of 293T with RBD-mRNA@MPP(Fe3+), as provided in Example 3.5.1 of the present disclosure.

[0098] [Figure 1-3] Provided in Example 3.5.1 of the present disclosure shows the ability of RBD-mRNA@MPP(Fe3+) to induce humoral immunity.

[0099] [Figure 1-4] Provided in Example 3.5.1 of the present disclosure shows the ability of NY-ESO-1-mRNA @MPP(Fe3+) to induce humoral immunity.

[0100] [Figure 1-5] Provided in Example 3.5.1 of the present disclosure shows the ability of RBD-mRNA@MPP(Fe3+) to induce cellular immunity.

[0101] [Figure 1-6] Provided in Example 3.5.1 of the present disclosure shows the ability of NY-ESO-1-mRNA @MPP(Fe3+) to induce cellular immunity.

[0102] [Figure 1-7] 10 shows the percentage of eGFP-positive cells caused by transfection of 293T with eGFP-mRNA@MPP(Al3+) provided in Example 3.5.2 of the present disclosure.

[0103] [Figure 1-8] 10 shows the expression levels of RBD caused by transfection of 293T with RBD-mRNA@MPP(Al3+) as provided in Example 3.5.2 of the present disclosure.

[0104] [Figure 1-9] Provided in Example 3.5.2 of the present disclosure is the ability of RBD-mRNA@MPP(Al3+) to induce humoral immunity.

[0105] [Figure 1-10] 1 shows the ability of NY-ESO-1-mRNA @MPP(Al3+) to induce humoral immunity, as provided in Example 3.5.2 of the present disclosure.

[0106] [Figure 1-11] Provided in Example 3.5.2 of the present disclosure is the ability of RBD-mRNA@MPP(Al3+) to induce cellular immunity.

[0107] [Figure 1-12] Provided in Example 3.5.2 of the present disclosure shows the ability of NY-ESO-1-mRNA @MPP(Al3+) to induce cellular immunity.

[0108] [Figure 1-13] 3 shows the ability of Bcl-2-siRNA@MPP(Fe3+) to silence target genes, as provided in Example 3.6.1 of the present disclosure.

[0109] [Figure 1-14]3 shows the ability of PLK1-siRNA@MPP(Fe3+) to silence target genes, as provided in Example 3.6.1 of the present disclosure.

[0110] [Figure 1-15] 3 shows the ability of Gal-1-siRNA@MPP(Fe3+) to silence target genes, as provided in Example 3.6.1 of the present disclosure.

[0111] [Figure 1-16] 3 shows the ability of Bcl-2-siRNA@MPP(Al3+) to silence target genes, as provided in Example 3.6.2 of the present disclosure.

[0112] [Figure 1-17] 3 shows the ability of PLK1-siRNA@MPP(Al 3+ ) to silence target genes, as provided in Example 3.6.2 of the present disclosure.

[0113] [Figure 1-18] 10 shows the ability of Gal-1-siRNA@MPP(Al 3+ ) to silence target genes, as provided in Example 3.6.2 of the present disclosure.

[0114] [Figure 1-19] Provided in Example 3.7.1 of the present disclosure is an illustration of the ability of STAT3-ASO@MPP(Fe3+) to silence cellular target genes.

[0115] [Figure 1-20] Provided in Example 3.7.1 of the present disclosure is an illustration of the ability of α-syn-ASO@MPP(Fe3+) to silence cellular target genes.

[0116] [Figure 1-21] Provided in Example 3.7.1 of the present disclosure is an illustration of the ability of Bcl-2-ASO@MPP(Fe3+) to silence cellular target genes.

[0117] [Figure 1-22] Provided in Example 3.7.2 of the present disclosure is an illustration of the ability of STAT3-ASO@MPP(Al3+) to silence cellular target genes.

[0118] [Figure 1-23] Provided in Example 3.7.2 of the present disclosure is an illustration of the ability of α-syn-ASO@MPP(Al3+) to silence cellular target genes.

[0119] [Figure 1-24] Provided in Example 3.7.2 of the present disclosure is an illustration of the ability of Bcl-2-ASO@MPP(Al3+) to silence cellular target genes.

[0120] [Figure 1-25] 3 shows the expression level of S protein induced by S-mRNA@MPP(Fe3+) in 293T, as provided in Example 3.8.1 of the present disclosure.

[0121] [Figure 1-26] The function of drug (dsDNA and ssDNA)-lipid particles (Fe3+) is shown, as provided in Example 3.8.1 of the present disclosure.

[0122] [Figure 1-27] 1 shows the expression levels of S protein caused by transfection of 293T with S-mRNA@MPP(Al 3+ ), as provided in Example 3.8.2 of the present disclosure.

[0123] [Figure 1-28] The function of drug (dsDNA and ssDNA)-lipid particles (Al 3+ ) provided in Example 3.8.2 of the present disclosure is shown.

[0124] [Figure 2-1] FIG. 4 is a differential scanning calorimetry diagram of the phospholipid complex provided in Example 4.1 of the present disclosure.

[0125] [Figure 2-2] FIG. 1 is an ultraviolet absorption diagram of a metal-phospholipid complex (Fe3+) provided in Example 4.1 of the present disclosure.

[0126] [Figure 2-3] FIG. 1 is an ultraviolet absorption diagram of a metal-phospholipid complex (Al 3+ ) provided in Example 4.2 of the present disclosure.

[0127] [Figure 2-4] 1 shows the characterization of Fe3+ released from metal-phospholipid complexes under low pH (pH=5.0) conditions, provided in Example 5 of the present disclosure.

[0128] [Figure 2-5] 6 shows the elemental analysis of the drug-metal-phospholipid complex particles (Fe3+) provided in Example 6.1 of the present disclosure.

[0129] [Figure 2-6] 6 shows an electron microscopy analysis of MPP in drug-metal-phospholipid complex particles (Al 3+ ) provided in Example 6.2 of the present disclosure.

[0130] [Figure 2-7] 1 shows the efficiency of drug-lipid particles encapsulating nucleic acids (mRNA and siRNA) provided in Example 7 of the present disclosure.

[0131] [Figure 2-8] 1 shows the nucleic acid lysosomal escape ability of siRNA / mRNA@MPP and siRNA / mRNA@LNP provided in Example 8 of the present disclosure.

[0132] [Figure 2-9] 1 shows the eGFP-positive cell rates of MPPs and LNPs provided in Example 9 of the present disclosure.

[0133] [Figure 2-10] 1 shows a comparison of the ability of MPPs and LNPs to promote mRNA expression, as provided in Example 10 of the present disclosure.

[0134] [Figure 2-11] 1 shows a comparison of the ability of MPPs and LNPs to promote humoral immunity, as provided in Example 10 of the present disclosure.

[0135] [Figure 2-12] 1 shows a comparison of the ability of MPPs and LNPs to promote cell-mediated immunity as provided in Example 10 of the present disclosure.

[0136] [Figure 3-1] Provided in Example 13 of the present disclosure is the role of tumor injection of drug-metal-phospholipid complex particles in the treatment of liver cancer. DETAILED DESCRIPTION OF THE INVENTION

[0137] definition

[0138] For convenience of explanation, certain terms used in the specification, examples, and appended claims are collected here. Unless otherwise defined herein, scientific and technical terms used herein have the same meaning as commonly understood and commonly used by those of ordinary skill in the art to which this disclosure belongs. Furthermore, unless the context requires otherwise, singular terms should be understood to include the plural form of the same, and plural terms should be understood to include the singular form. Specifically, unless the context clearly dictates otherwise, the terms "at least one" and "one or more" as used in the specification and appended claims include one, two, three, or more species.

[0139] Although all numerical ranges and parameters used to define the broad scope of the present disclosure are approximations, the relevant numerical values in the specific examples herein are presented as precisely as possible. However, any numerical value inherently contains standard deviations resulting from the particular testing method employed. Herein, the term "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of the particular numerical value or range. Alternatively, the term "about" may mean that the actual numerical value falls within an acceptable standard error of the mean, as determined by a person of ordinary skill in the art to which the present disclosure pertains. Except in experimental examples, or unless otherwise expressly stated, all ranges, quantities, values, and percentages used herein (e.g., those used to describe amounts of materials, lengths of time, temperatures, operating conditions, proportions of quantities, and other similar terms) are understood to be modified by the term "about." Therefore, unless specifically stated to the contrary, all numerical parameters disclosed in this specification and the appended claims are approximations and can be modified as necessary. At the very least, these numerical parameters should be understood to mean the number of significant digits represented and the resulting values obtained by applying ordinary rounding techniques.

[0140] The term "lipid" refers to a group of organic compounds, including but not limited to fatty acid lipids. These are typically divided into three categories: "simple lipids," "compound lipids," and "derived lipids." Simple lipids include fats, oils, and waxes; compound lipids include phospholipids and glycolipids; and derived lipids, such as steroids.

[0141] 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 the aqueous volume is encapsulated by an amphiphilic lipid bilayer, or in which the lipid coating contains an interior containing a large molecular component such as mRNA and the aqueous interior is reduced, or lipid aggregates or micelles in which the encapsulated components are contained in a relatively chaotic lipid mixture. As used herein, metal-phospholipid complex particles (MPPs) are "lipid vesicles" in which a drug, such as a nucleic acid mRNA, is encapsulated in the MPP as an encapsulated component. The "encapsulation" can be complete and / or partial encapsulation.

[0142] The term "phospholipid" refers to lipids containing phosphate groups, which belong to the complex lipid family and are also called phospholipids or phospholipids. Phospholipids are the main components of biological membranes and are divided into two categories: glycerophospholipids and sphingomyelins, each composed of glycerol and sphingosine. Phospholipids are amphipathic molecules with a hydrophilic nitrogen- or phosphorus-containing head group at one end and a long hydrophobic (lipophilic) hydrocarbyl chain at the other end. Therefore, the hydrophilic ends of phospholipid molecules are close to each other, and the hydrophobic ends are close to each other, forming phospholipid bilayers, i.e., the structure of cell membranes, together with other molecules such as proteins, glycolipids, and cholesterol.

[0143] As used herein, the phrase "phospholipid molecule portion" refers to the structure that originally belonged to the phospholipid molecule after the phospholipid molecule has reacted with another substance.

[0144] As used herein, the phrase "linker molecule portion" refers to the structure originally belonging to the linker molecule after the linker molecule has reacted with another substance.

[0145] As used herein, the phrase "metal ion moiety" refers to the structure originally belonging to the metal ion after the metal ion moiety has reacted with another substance.

[0146] The term "phospholipid complex" as used herein refers to a complex formed by reacting and linking the above-mentioned phospholipid molecular moiety having a phosphate group with the above-mentioned linker molecular moiety.

[0147] The term "metal-phospholipid complex" as used herein refers to a complex formed by the reaction of the phospholipid molecular moiety having a phosphate group, the linker molecular moiety, and the metal ion moiety, wherein the phospholipid molecular moiety and the linker molecular moiety are linked together, and the linker molecular moiety and the metal ion moiety are linked together by a coordinate bond, and the metal-phospholipid complex is neither a cationic lipid nor an ionizable lipid.

[0148] The term "ionizable lipid" refers to a lipid containing a positively charged, ionizable amine group that can be protonated and positively charged at relatively low pH values, but is uncharged at physiological pH values.

[0149] The term "neutral lipid" refers to any one of a number of lipid species that are uncharged or exist in a neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0150] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH, including, but not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic groups linked to neutral lipids.

[0151] The term "cationic lipid" refers to any one of a number of lipid species that are positively charged at a selected pH, such as physiological pH. These lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"), N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTMA"), N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"), N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride ("DOTAP"), 3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol ("DC-Chol"), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"). The following lipids are cationic, i.e., DODAP, DODMA, DMDMA, etc., which carry a positive charge at sub-physiological pH:

[0152] The term "hydrophobic lipid" refers to a compound having a non-polar group, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, which are optionally substituted with one or more aromatic, alicyclic or heterocyclic groups.Suitable examples include but are not limited to diacylglycerol, dialkylglycerol, N-N-dialkylamino group, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0153] The term "non-cationic or non-ionizable lipid" refers to a lipid that is neither a cationic lipid nor a non-ionizable lipid, and can be, for example, an anionic lipid, a neutral lipid.

[0154] Among the components of the metal-phospholipid complex particles, the "non-cationic lipid or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation" in (iii) refers to the lipid that remains in the metal-phospholipid complex particles after removing the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation.

[0155] The term "fusogenic" refers to the ability of a liposome, drug-lipid particle, or other drug delivery system to fuse with a cell membrane, which may be the plasma membrane or a membrane surrounding an organelle such as an endosome, nucleus, etc.

[0156] In metal-phospholipid complex particles, non-cationic or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation are present primarily as vesicle-forming lipids, and the term "vesicle-forming lipid" tends to include any amphipathic lipid that has a hydrophobic portion and a polar head group and that can itself spontaneously form bilayer vesicles in water, examples of which are most phospholipids.

[0157] In metal-phospholipid complex particles, conjugated lipids that inhibit particle aggregation are primarily present as vesicle-adopting lipids. The term "vesicle-adopting lipids" tends to include any amphipathic lipid that stabilizes lipid bilayer binding, as well as other amphipathic lipids, whose hydrophobic portion is in contact with the interior, hydrophobic region of the bilayer membrane, and whose polar head group is oriented toward the exterior, polar surface of the membrane. Vesicle-adopting lipids include lipids that are independently suitable for adopting a nonlamellar phase and can also adopt a bilayer structure in the presence of a bilayer-stabilizing component. Conjugated lipids that inhibit drug-lipid particle aggregation include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid derivatives), peptides, proteins, decontaminants, lipid derivatives, and PEG-lipid derivatives such as dialkyloxypropyl-conjugated PEG, diacylglycerol-conjugated PEG, phosphatidylethanolamine-conjugated PEG, and ceramide-conjugated PEG (see U.S. Pat. No. 5,885,613, incorporated herein by reference).

[0158] The term "amphipathic lipid" refers to any suitable lipid material in which the hydrophobic portion of the lipid material faces the hydrophobic phase and the hydrophilic portion faces the aqueous phase. Amphipathic lipids are typically the main components of lipid vesicles. Hydrophilic properties arise from the presence of polar or charged groups such as carbohydrates, phosphate (ester), carboxyl, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other similar groups. Hydrophobicity can be imparted by the inclusion of nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine.Other compounds lacking phosphorus, such as sphingomyelin, glycosphingolipid family, diacylglycerol, and β-acyloxyacid, are also included in the group known as amphipathic lipids.In addition, the above amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.

[0159] The term "diacylglycerol" refers to a compound in which R1 and R2 each independently have a 2-fatty acyl chain of 2 to 30 carbon atoms attached to positions 1 and 2 of glycerol via an ester bond. The acyl group may be saturated or may have varying degrees of unsaturation. Diacylglycerol has the following formula: [ka]

[0160] In the term "diacylglycerol-conjugated polyethylene glycol," the lipid conjugated to inhibit particle aggregation in the present disclosure can be diacylglycerol-conjugated polyethylene glycol, i.e., diacylglycerol-polyethylene glycol conjugate (DAG-PEG conjugate or PEG-DAG conjugate). In a preferred embodiment, the DAG-PEG conjugate is a dilaurylglycerol (C12)-PEG conjugate, a dimyristylglycerol (C14)-PEG conjugate (DMG), a dipalmitoylglycerol (C16)-PEG conjugate, or a disterylglycerol (C18)-PEG conjugate (DSG). Those skilled in the art will readily understand that other diacylglycerols can be used in the DAG-PEG conjugates of the present disclosure. DAG-PEG conjugates suitable for use in the present disclosure, as well as methods for making and using them, are disclosed in U.S. Application No. 10 / 136,707, published as USPA 2003 / 0077829, and PCT Patent Application No. CA 02 / 00669, the entire contents of each of which are incorporated by reference.

[0161] The term "dialkyloxypropyl" refers to a compound in which R1 and R2 each independently have a 2-alkyl chain having 2 to 30 carbons. The alkyl groups may be saturated or have varying degrees of unsaturation. Dialkyloxypropyl has the following formula: [ka]

[0162] In the term "dialkyloxypropyl-conjugated PEG," the lipid of the conjugation that inhibits particle aggregation in the present disclosure can be a dialkyloxypropyl-conjugated PEG, i.e., a dialkyloxypropyl conjugate (PEG-DAA conjugate). In a preferred embodiment, the PEG-DAA complex has the following formula 56: [ka]

[0163] In Formula 56, R1 and R2 are independently selected long-chain alkyl groups having from about 10 to about 22 carbon atoms. The long-chain alkyl groups may be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, dodecyl (C12), tetradecyl (C14), hexadecyl (C16), octadecyl (C18), and icosyl (C20). In preferred embodiments, R1 and R2 are the same; i.e., R1 and R2 are both tetradecyl (i.e., ditetradecyl) and R1 and R2 are both octadecyl (i.e., dioctadecyl). In Formula 56, PEG is polyethylene glycol having an average molecular weight of from about 550 to about 10,000 daltons, optionally substituted at the terminal hydroxyl position with an alkyl, alkoxy, acyl, or aryl group. In a preferred embodiment, the PEG has an average molecular weight of about 1,000 to about 5,000 daltons, more preferably about 1,000 to about 3,000 daltons, and even more preferably about 2,000 daltons. The PEG may be optionally substituted with an alkyl, alkoxy, acyl, or aryl group. In Formula 56, L is a linker moiety. Any linker moiety suitable for conjugating PEG to a dialkyloxypropyl backbone may be used. Suitable linker moieties include, but are not limited to, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbonate (OC(O)O-), urethane (-NHC(O)O-), urea (-NHC(O)NH-), succinyl (-(O)CCHCHC(O)-), ether, disulfide, and combinations thereof. Other suitable linkers are well known in the art.

[0164] Phosphatidylethanolamines can be conjugated to polyethylene glycol to form bilayer-stabilizing components as conjugated lipids that inhibit particle aggregation in the present disclosure. The phosphatidylethanolamines have a variety of acyl chain groups with different chain lengths and degrees of saturation. These phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths ranging from C10 to C20 are preferred. Also, such phosphatidylethanolamines containing monounsaturated or diunsaturated fatty acids, as well as mixtures of saturated and unsaturated fatty acids, can be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoylphosphatidylethanolamine (DSPE).

[0165] Similar to phosphatidylethanolamine, ceramide can be conjugated with polyethylene glycol to form a bilayer-stabilizing component as a conjugated lipid to inhibit particle aggregation in the present disclosure. The ceramide has multiple acyl chain groups of varying chain length and saturation. It should be apparent to those skilled in the art that, compared to phosphatidylethanolamine, ceramide has only one acyl group. The acyl group can be easily varied depending on its chain length and saturation. Ceramides suitable for application in accordance with the present disclosure are commercially available. Ceramides can be isolated from, for example, eggs and brains using well-known isolation techniques, or synthesized using the methods and techniques disclosed in U.S. Patent No. 5,820,873 (which is incorporated herein by reference). Using the synthetic route proposed in the aforementioned application, ceramides with saturated or unsaturated fatty acids with carbon chain lengths ranging from C2 to C31 can be prepared.

[0166] 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: [ka]

[0167] R is a member selected from the group consisting of hydrogen, an alkyl group, and an acyl group. R1 is a member selected from the group consisting of hydrogen and an alkyl group. Alternatively, optionally, R and R1 and the nitrogen atom to which they are attached form an azide group moiety. R2 is a member selected from the group consisting of hydrogen, an optionally substituted alkyl group, an optionally substituted aryl group, and an amino acid side chain. R3 is a member selected from the group consisting of hydrogen, halogen, hydroxyl, an alkoxy group, a mercapto group, a hydrazine group, an amino group, and NRR5, where R4 and R5 are independently hydrogen or an alkyl group, n is 4 to 80, m is 2 to 6, p is 1 to 4, and q is 0 or 1. It will be apparent to one skilled in the art that other polyamides may be used in the compounds of the present disclosure.

[0168] The term "congener" refers to an analog that fulfills the same or a similar function, or a derivative of the same parent nucleus that fulfills the same or a similar function.

[0169] As used herein, the terms "mRNA" or "messenger polyribonucleotide" or "messenger RNA" or "messenger RNA" may be used interchangeably and refer to a single-stranded polyribonucleotide that is transcribed using a single strand of DNA as a template, carries genetic information, and can direct protein synthesis.

[0170] As used herein, the terms "sgRNA" or "small guide RNA" or "guide RNA" or "gRNA" may be used interchangeably and refer to small non-coding RNAs that can pair with pre-mRNA and guide the insertion or deletion of uridine residues in kinetoplastids during RNA editing. gRNAs edit RNA molecules approximately 60-80 nucleotides in length that are transcribed by individual genes.

[0171] As used herein, the terms "circRNA" or "circular RNA" or "circular polyribonucleotide" or "circular RNA" may be used interchangeably and refer to a structure that has no free ends (i.e., no free 3' and / or 5' ends), e.g., a polyribonucleotide that forms a circular or ring structure by covalent or non-covalent bonds.

[0172] As used herein, the terms "microRNA" or "miRNA" or "microRNA" may be used interchangeably and refer to a non-coding, single-stranded polyribonucleotide that is approximately 22 nucleotides in length and has free 3' and 5' ends, which can inhibit the translation of target gene proteins by binding to the mRNA 3'-untranslated region (3'-UTR) of a target gene, thereby regulating the biological functions of cells.

[0173] As used herein, the terms "ASO" or "antisense oligonucleotide" or "antisense oligonucleotide" may be used interchangeably and refer to an artificially synthesized nucleic acid fragment complementary to a segment of a target gene or mRNA, and is a single-stranded poly(deoxy)ribonucleotide that can bind to a target gene / mRNA through the principle of base complementarity, thereby blocking gene expression, and includes antisense DNA and antisense RNA.

[0174] As used herein, the terms "siRNA" or "small interfering" or "short interfering" or "silencing RNA" or "small interfering RNA" or "short interfering RNA" or "silencing RNA" may be used interchangeably and refer to a type of double-stranded RNA molecule that is 20 to 25 nucleotides in length and can induce degradation of target gene mRNA.

[0175] As used herein, the terms "ecDNA" or "ectopic in vitro circular DNA" may be used interchangeably and refer to DNA that has been shed from a chromosome and exists in a circular structure away from the chromosome.

[0176] The term "nucleic acid derivative" refers to modifications or substitutions of nucleic acid sequences, including, but not limited to, chemical modifications of residues, nucleotide or deoxynucleotide substitutions, sequence modifications to enhance half-life or stability, and marking modifications. For example, chemical modifications include, but are not limited to, phosphorylation, methylation, amination, thiolation, sulfur substitution for oxygen, selenium substitution for oxygen, or isotope substitution of any one or more bases. Nucleotide or deoxynucleotide substitutions include, but are not limited to, nucleic acid analogs in which the sugar-phosphate backbone is replaced with a polypeptide or other backbone (e.g., replacing DNA or RNA with PNA). Sequence modifications to enhance half-life or stability include, but are not limited to, PEG conjugation and fluorine modification. Marking modifications include, but are not limited to, the attachment of fluorescent groups, amino groups, biotin, digoxin, small peptides, etc.

[0177] The term "artificial nucleic acid" refers to an artificially modified nucleic acid molecule, including but not limited to base modifications, ribose modifications, PNA, etc.

[0178] The term "nucleic acid" refers to a polymer containing at least two deoxynucleotides or nucleotides, present in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). A "nucleotide" includes a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are attached via the phosphate group. "Bases" include purines and pyrimidines, as well as the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that substitute new reactive groups. Such reactive groups include, but are not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. DNA can exist as antisense, plasmid DNA, portions of plasmid DNA, pre-packaged DNA, polymerase chain reaction (PCR) products, carriers (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. The term nucleic acid can be used interchangeably to refer to genes, cDNAs, mRNAs encoded by genes, and interfering RNA molecules.

[0179] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence comprising a partial or full-length coding sequence necessary for the production of a polypeptide or polypeptide precursor (e.g., a polypeptide or polypeptide precursor from hepatitis A, B, C, D, E, G virus, or herpes simplex virus).

[0180] As used herein, "gene product" refers to a gene product, including, for example, a DNA transcript, mRNA, and the like.

[0181] The phrase "silencing expression of a target gene" refers to the ability of the siRNA of the present disclosure to initiate silencing of a target gene. To determine the degree of gene silencing, a sample or measurement of cells in a target organism or culture expressing a particular construct is compared to a control sample not expressing the construct. The control sample (lacking expression of the construct) is set to a relative value of 100%. Successful inhibition of target gene expression is achieved when the test value relative to the control is approximately 90%, preferably 50%, and more preferably 25% to 0%. Suitable measurements include, for example, detection of protein or mRNA levels using techniques known to those skilled in the art, such as dotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzymatic action, and phenotypic measurements known to those skilled in the art.

[0182] A "therapeutically effective amount" or "effective amount" of an siRNA is an amount sufficient to produce a desired effect, such as a decrease in target sequence expression compared to normal expression levels detected in the absence of the siRNA.

[0183] As used herein, the term "aqueous solution" refers to a composition that comprises, in whole or in part, water.

[0184] As used herein, the term "organic lipid solution" refers to a composition comprising an organic solvent, in whole or in part, with a lipid.

[0185] As used herein, "systemic delivery" refers to delivery that results in widespread biodistribution of a compound within an organism. Some administration techniques can result in systemic delivery of some compounds but not others. Systemic delivery means that an effective, preferably therapeutic, amount of a compound comes into contact with most of the body. Achieving widespread biodistribution typically requires a blood survival period to prevent the compound from being rapidly degraded or eliminated (e.g., by first-pass organ (e.g., liver, lung) or rapid nonspecific cellular binding) before reaching disease sites distal to the administration site. Systemic delivery of drug-lipid particles can be achieved by any method known in the art, including, for example, intravenous, subcutaneous, or intraperitoneal delivery; in a preferred embodiment, the systemic delivery of drug-lipid particles is intravenous.

[0186] As used herein, "local delivery" refers to the direct delivery of a compound to a target site within an organism. For example, a compound can be delivered locally by direct injection into a disease site such as a tumor, or other target site such as a site of inflammation, or into a target organ such as the liver, heart, pancreas, or kidney.

[0187] Metal-phospholipid complex

[0188] In the present disclosure, a metal-phospholipid complex is composed of a phospholipid molecule portion, a linker molecule portion, and a metal ion portion.

[0189] In the case of the phospholipid molecule portion, it should be explained that the cis-trans isomers of the phospholipid molecules of the present disclosure do not affect the effects achieved by the protective subject matter of the present disclosure.

[0190] The linker molecule moiety, derived primarily from natural plant extracts such as curcumin, possesses a wide range of biological effects, including antibacterial, antiviral, antifungal, antioxidant, and anti-inflammatory activities. It also regulates the activity of various immune cells, including T cells, B cells, macrophages, neutrophil granulocytes, natural killer cells, and dendritic cells, promoting immune balance and enhancing the immune system. Curcumin's potential immune-enhancing, anti-inflammatory, antioxidant, and anti-SARS-CoV-2 effects suggest it could potentially serve as an adjunct treatment for COVID-19. Furthermore, curcumin is highly safe and is listed in food additive and pharmaceutical excipient catalogs. Its safety benefits contribute to the clinical drug registration of the entire drug-lipid complex, shortening the clinical drug registration period.

[0191] In the case of a metal ion moiety, the coordination bond between the linker molecule and the metal ion in the metal-phospholipid complex is cleaved under the low pH condition (pH = 5.5) of the lysosome, for example, and the metal ion is released from the metal-phospholipid complex.

[0192] The ratio of each component in a metal-phospholipid complex can be adjusted depending on the structure of the specific metal-phospholipid complex component. The reason for this adjustment is as follows: Because the phospholipid molecules and the linker molecules are connected by hydrogen bonds, as long as the phospholipid molecules contain multiple phosphate groups, the ratio of the phospholipid molecules to the linker molecules when synthesizing a phospholipid complex can be adjusted depending on the number of phosphate groups contained in the phospholipid molecules. That is, if the phospholipid molecules contain two phosphate groups, the ratio of the phospholipid molecules to the linker molecules can be adjusted to 1:2. If the phospholipid molecules contain three phosphate groups, the ratio of the phospholipid molecules to the linker molecules can be adjusted to 1:3. Because the hydroxyl group of the linker molecule and the metal ion are connected by a coordinate bond, as long as the linker molecule contains multiple binding sites, the ratio of the linker molecules to the metal ions can be adjusted based on the number of binding sites contained in the linker.

[0193] First, phospholipid molecules having phosphate groups are linked to linker molecules to obtain a phospholipid complex, which is then linked to a metal ion to obtain a metal-phospholipid complex. Specifically, the phospholipid molecules and linker molecules are dissolved in an appropriate amount of ethanol according to the molar ratio, reacted at approximately 65°C, and n-hexane is added to precipitate the phospholipid complex. The phospholipid complex and metal ions (e.g., FeCl3, etc.) are dissolved in an appropriate amount of ethanol according to the molar ratio, and reacted at approximately 60°C to obtain a metal-phospholipid complex.

[0194] Metal-chelated phospholipid complex nanoparticles (MPP)

[0195] The principle of loading nucleic acids into metal-phospholipid complex particles assembled from metal-phospholipid complexes is as follows: Linker molecules and phospholipid molecules are bonded by hydrogen bonds, and at the same time, the linker molecules are linked to metal ions by coordinate bonds to form metal-phospholipid complexes. The metal ions of the metal-phospholipid complexes are linked to negatively charged drugs by coordinate bonds, thereby ensuring that the metal-phospholipid complexes self-assemble into MPPs with other components (injugation lipids that inhibit particle aggregation, and non-cationic or non-ionizable lipids other than the metal-phospholipid complexes and injugation lipids that inhibit particle aggregation), thereby loading the negatively charged drug into the nanoparticle MPPs. In this specification, the "non-cationic or non-ionizable lipids other than the metal-phospholipid complexes and injugation lipids that inhibit particle aggregation" refers to component (iii) in the metal-phospholipid complex particles.

[0196] In some embodiments, the conjugation lipid that inhibits particle aggregation refers to a conjugation lipid that inhibits drug-lipid particle aggregation. Its main function is to prevent the aggregation of drug-lipid particles, such as PEG conjugated with dialkyloxypropyl, PEG conjugated with diacylglycerol, PEG conjugated with phosphatidylethanolamine, and PEG bound with ceramide, preferably PEG-lipid conjugates. The cis-trans isomers of the lipid do not affect the effect achieved by the protected subject matter of the present disclosure.

[0197] In some embodiments, the molar fraction of the metal-phospholipid complex in the feedstock is greater than or equal to 5% but less than 10%, between 10% and 40%, or greater than 40% to 50%, e.g., 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%.

[0198] In a preferred embodiment, the molar fraction of the metal-phospholipid complex in the raw material is 7% to 40%, and may be, for example, 10% to 40%, 7% to 30%, 15% to 25%, or 20% to 30%, and more preferably 15%, 25%, 7%, or 30%.

[0199] In some embodiments, the molar fraction of conjugated lipids that inhibit particle aggregation in the feedstock is 2% to 10%, e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, e.g., 3% to 10%, 4% to 10%, 5% to 10%, and more preferably 3%, 4%, or 10%.

[0200] In some embodiments, the non-cationic or non-ionizable lipid is cholesterol, and the mole fraction of cholesterol in the feedstock is greater than or equal to 15% but less than 35%, between 35% and 75%, or greater than 75% and 80%, e.g., 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%, 116%, 117%, 118%, 119 %, 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%, or 80%. For example, it may be 35% to 75%, 15% to 56%, 40% to 46%, or 35% to 55%, and preferably 15%, 40%, 46%, or 56%.

[0201] In some embodiments, in addition to cholesterol, the metal-phospholipid complex particles may optionally further contain other non-cationic or non-ionizable lipids, the molar fraction of which in the raw material is 0% to 40% or greater than 40% to 51%, for example, 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, It is 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, it is 5% to 30%, 25% to 35%, 34% to 45%, or 20% to 25%, and preferably 25%, 35%, 34%, or 45%.

[0202] Drug-lipid particles

[0203] The drug-lipid particles described herein typically contain a drug (a negatively charged molecule that may be selected from the group consisting of one or more combinations of nucleic acids, proteins, polypeptides, small molecules, nucleic acid analogs, protein analogs, and polypeptide analogs; the nucleic acid may be selected from the group consisting of one or more combinations of mRNA, siRNA, circular RNA, microRNA, DNA, ecDNA, and artificial nucleic acids), 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. The nucleic acids encapsulated in the drug-lipid particles of the present disclosure are resistant to degradation by nucleases in aqueous solution.

[0204] In some embodiments, the drug is sufficiently encapsulated within the metal-phospholipid complex particles, thereby avoiding drug degradation and achieving drug delivery to cells.

[0205] In some embodiments, the drug-lipid particles provided by the present disclosure have a small diameter suitable for systemic delivery, with a particle size of 30 to 400 nm, a surface potential of -10 to 10 mV, a stability of at least 3 days, preferably 7 days or more, and a cellular delivery efficiency of at least 40%, e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0206] 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.

[0207] A population of RNA can be used to provide long precursor RNAs, or long precursor RNAs with basic or complete identity to a selected target sequence, which can be used to produce siRNA. The RNA can be isolated from cells or tissues, synthesized, and / or cloned according to methods well known to those of skill in the art. The RNA can be a mixed population (obtained from cells or tissues, transcribed from cDNA, etc.) or can represent a single target sequence. The RNA can be naturally occurring, e.g., isolated from a tissue or cell sample, synthesized in vitro using, e.g., T7 or SP6 polymerase and PCR products or cloned cDNA, or synthesized by chemical methods.

[0208] To form long dsRNA, in the case of synthetic RNA, the complement may be transcribed in vitro and hybridized to form dsRNA. When a naturally occurring RNA population is used, an RNA complement (e.g., to form dsRNA that can be digested by E. coli RNAse III or other enzymes) is further provided, for example, by transcribing cDNA corresponding to the RNA population or by using RNA polymerase. Precursor RNA is then hybridized to form double-stranded RNA, which then undergoes digestion. The dsRNA can be directly encapsulated in SNALP or can be digested in vitro before encapsulation.

[0209] Alternatively, one or more DNA plasmids encoding one or more siRNA templates can be encapsulated in the nucleic acid-lipid particles. For example, based on the naturally occurring transcription units of the small nuclear RNA U6 or human RNase P RNA H1, siRNA can be transcribed from a DNA template in a plasmid into a sequence that automatically folds into a double-stranded form with a hairpin loop, and the plasmid contains an RNA polymerase III transcription unit (Brummelkamp et al., Science 296:550 (2002); Donze et al., Nucleic Acids Res. 30:e46 (2002); Paddison et al., Genes 2002). Dev. (Genetics Dev.) 16:948 (2002); Yu et al., Proc. Natl. Acad. Sci. (Proceedings of the National Academy of Sciences) 99:6047 (2002); Lee et al., Nat. Biotech. (National Biotechnology) 20:500 (2002); Miyagishi et al., Nat. Biotech. (National Biotechnology) 20:497 (2002); Paul et al., Nat. Biotech. (National Biotechnology) 20:505 (2002); and Sui et al., Proc. Natl. Acad. Sci. (Proceedings of the National Academy of Sciences) 99:5515 (2002). Typically, a transcription unit or cassette includes an RNA transcription promoter sequence, such as an H1-RNA or U6 promoter, and a termination sequence. The promoter sequence is operably linked to a template for transcribing the desired siRNA sequence, and the termination sequence includes two to three uridine residues and a polythymidine (T5) sequence (polyadenylation signal) (Brummelkamp, Science, supra). The selected promoter can provide constitutive or inducible transcription. Compositions and methods for transcribing DNA-guided RNA interference molecules are described in detail in U.S. Patent No. 6,573,099, incorporated herein by reference.Preferably, the synthetic or transcribed siRNA has a 3' overhang of about 1-4 nucleotides, preferably about 2-3 nucleotides, and a 5' phosphate terminus (Elbashir et al., Genes Dev. 15:188 (2001); Nykaenen et al., Cell 107:309 (2001)). The transcription unit is attached to a plasmid or DNA carrier, and the interfering RNA is transcribed from the plasmid or DNA vector. Plasmids suitable for delivering genetic material into the body for therapeutic purposes are described in detail in U.S. Patent Nos. 5,962,428 and 5,910,488, both of which are incorporated herein by reference. The selected plasmid can provide instantaneous or stable delivery to target cells. It will be apparent to those skilled in the art that a plasmid originally designed to express a desired gene sequence can be modified to contain a transcription unit cassette that transcribes siRNA.

[0210] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, preparing and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds., 1990)). Expression libraries are also well known to those of skill in the art. Additional basic books that disclose the general methods used in this disclosure include: Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed., 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

[0211] Applications of metal-phospholipid complexes, metal-phospholipid complex particles, and drug-lipid particles

[0212] In some embodiments, when the drug is a nucleic acid, the metal-phospholipid complex or metal-phospholipid complex particle can be used to promote lysosomal escape of the drug and promote nucleic acid expression. The metal-phospholipid complex or metal-phospholipid complex particle can also be used to deliver the drug and introduce the drug into cells, thereby achieving the prevention and treatment of the corresponding disease or condition.

[0213] In some embodiments, the present disclosure provides applications of the drug-lipid particles in compositions capable of, for example, delivering drugs or introducing drugs into cells. The compositions are, for example, pharmaceuticals capable of silencing the expression of a target sequence in a mammalian subject, delivering drugs (e.g., drugs for treating tumors, imaging agents, etc.) into the mammalian body, delivering drugs from the body to mammalian cells, or treating mammalian diseases or conditions. In pharmaceuticals, the drug-lipid particles are the main active ingredient. Depending on actual needs, different dosage forms can be prepared using different pharmaceutically acceptable excipients or manufacturing processes, such as solid dosage forms (powders, granules, pills, tablets, gels), semisolid dosage forms (ointments for external use, pastes), liquid dosage forms (decoctions, mixtures, syrups, medicinal liquors, injections), and gaseous dosage forms (aerosols, smoke). Examples include dosage forms for gastrointestinal administration, rectal administration, and parenteral administration.

[0214] In some embodiments, the present disclosure provides products made from the above-described metal-phospholipid complexes, metal-phospholipid complex particles, and drug-lipid particles. The products have the above-described functions and uses of the metal-phospholipid complexes, metal-phospholipid complex particles, and drug-lipid particles. Specific examples include, but are not limited to, kits, pharmaceuticals, etc. The products may optionally further contain other excipients.

[0215] Regarding target genes for drug-lipid particle action, it is generally desirable to deliver the drug-lipid particles so that the translation (i.e., expression) of the target gene product is downregulated or silenced. Suitable classes of gene products include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and conditions (e.g., diseases and conditions targeting the liver, and liver diseases and conditions), genes associated with tumorigenesis and cell transformation, angiogenesis genes, immunomodulator genes such as those associated with inflammation and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative diseases.

[0216] Genes associated with viral infection and survival include those expressed by viruses to bind to, enter, and replicate within cells, particularly viral sequences associated with chronic viral diseases. For example, viral sequences include those of hepatitis viruses (Hamasaki et al., FEBS Lett. 543:51 (2003); Yokota et al., EMBO Rep. 4:602 (2003); Schlomai et al., Hepatology 37:764 (2003); Wilson et al., Proc. Natl. Acad. Sci. 100:2783 (2003); Kapadia et al., Proc. Natl. Acad. Sci. 100:2014 (2003); FIELDS VIROLOGY (Knipe et al., eds., 2001)), human immunodeficiency virus (HIV) (Banerjea et al., Mol Ther. (Journal of Molecular Therapy) 8:62 (2003); Song et al., J. Virol. (Journal of Virology) 77:7174 (2003); Stephenson JAMA (Stephenson JAMA) 289:1494 (2003); Qin et al., Proc. Natl. Acad. Sci. (Proceedings of the National Academy of Sciences of the United States of America) 100:183 (2003)), herpesviruses (Jia et al., J. Virol. (Journal of Virology) 77:3301 (2003)), and human papillomavirus (HPV) (Hall et al., J. Virol. (Journal of Virology) 77:6066 (2003); Jiang et al., Oncogene 21:6041 (2002)). Exemplary hepatitis virus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences involved in transcription and translation (e.g., En1, En2, X, P), nucleic acid sequences encoding structural proteins (e.g., core proteins, including C and C-related proteins, capsid and envelope proteins, including S, M, and / or L proteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY, 2001, supra).Hepatitis C nucleic acid sequences that can be silenced include, but are not limited to, serine proteases (e.g., NS3 / NS), helicases (e.g., NS3), polymerases (e.g., NS5B), and envelope proteins (e.g., E1, E2, and p7). Hepatitis A nucleic acid sequences are, for example, referenced in Genbank accession number NC_001489, hepatitis B nucleic acid sequences are, for example, referenced in Genbank accession number NC_003977, hepatitis C nucleic acid sequences are, for example, referenced in Genbank accession number NC_004102, hepatitis D nucleic acid sequences are, for example, referenced in Genbank accession number NC_001653, hepatitis E nucleic acid sequences are, for example, referenced in Genbank accession number NC_001434, and hepatitis G nucleic acid sequences are, for example, referenced in Genbank accession number NC_001710. Silencing of sequences encoding genes associated with viral infection and survival can be advantageously used in combination with the administration of conventional drugs used to treat viral diseases.

[0217] Genes associated with metabolic diseases and conditions (e.g., diseases targeting the liver and diseases and conditions of the liver) include, for example, genes expressed in dyslipidemia (e.g., liver X receptors (e.g., LXRα and LXRβ Genbank Accession No. NM_007121)), farnesoid X receptor (FXR) (Genbank Accession No. NM_005123), sterol regulatory element-binding protein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG coenzyme A reductase), apolipoprotein (ApoB), apolipoprotein (ApoE), and diabetes (e.g., glucose-6-phosphate) (e.g., Forman et al., Cell 81:687 (1995); Seol et al., Mol. Endocrinol. 9:72 (1995); Zavacki et al., PNAS USA 94:7909(1997), Sakai et al., Cell 85:1037-1046(1996), Duncan et al., J. Biol. Chem. 272:12778-12785(1997), Willy et al., Genes Dev. 9(9):1033-45(1995), Lehmann et al., J. Biol. Chem. 272(6):3137-3140(1997), Janowski et al., Nature 383:728-731 (1999; see Peet et al., Cell 93:693-704 (1998)). Those skilled in the art will understand that genes associated with metabolic diseases and conditions (e.g., diseases and conditions targeted at the liver, and liver diseases and conditions) include genes expressed in the liver itself as well as genes expressed in other organs and tissues. Silencing of sequences encoding genes associated with metabolic diseases and conditions can be advantageously used in combination with the administration of conventional drugs used to treat the disease or condition.

[0218] Examples of genes involved in tumorigenesis and cell transformation include MLL fusion genes, translocation sequences such as BCR-ABL (Wilda et al., Oncogene 21:5716 (2002); Scherr et al., Blood 101:1566), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO, and AML1-MTG8 (Heidenreich et al., Blood 101:3157 (2003)), overexpressed sequences such as multidrug resistance genes (Nieth et al., FEBS Lett. 545:144 (2003); Wu et al., Cancer Res. 63:1515 (2003)), cyclins (Li et al., Cancer Res. 63:3593 (2003); Zou et al., Genes Dev. (Genetics Development) 16:2923 (2002)), beta-catenin (Verma et al., Clin Cancer Res. (Clinical Cancer Research) 9:1291 (2003)), the telomerase gene Kosciolek et al., Mol Cancer Ther. (Molecular Cancer Ther.) 2:209 (2003)), c-MYC, N-MYC, BCL-2, ERBB1, and ERBB2 (Nagy et al., Exp. Cell Res. (Experimental Cell Research) 285:39 (2003)), and mutated sequences such as RAS (reviewed in Tuschl and Borkhardt, Mol. Interventions (Molecular Interventions) 2:158 (2002)). Silencing of sequences encoding DNA repair enzymes is used in combination with the administration of chemotherapeutic agents (Collis et al., Cancer Res. 63:1550 (2003)). Genes encoding proteins associated with tumor migration, such as integrins, selectins, metalloproteases, etc., are also suitable target sequences. Any complete or partial gene sequence that favors or promotes tumorigenesis or cell transformation, tumor growth, or tumor migration may be included as a template sequence.

[0219] Angiogenic genes can promote the formation of new blood vessels. Vascular endothelial growth factor (VEGF) is an important research direction (Reich et al., Mol. Vis. 9:210 (2003)).

[0220] Immunomodulatory genes are genes that regulate one or more immune responses. Examples of immunomodulatory genes include cytokines such as growth factors (e.g., TGF-α, TGF-β, EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12 (Hill et al., J. Immunol. 171:691 (2003)), IL-15, IL-18, IL-20, etc.), interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.), and TNF. Fas and Fas ligand genes are also immunomodulatory target sequences (Song et al., Nat. Med. 9:347 (2003)). Genes encoding secondary signaling molecules in hematopoietic and lymphoid cells, for example, Tec family kinases such as Bruton's tyrosine kinase (Btk), are also included in the present disclosure (Heinonen et al., FEBS Lett. 527:274 (2002)).

[0221] Cell receptor ligands include ligands that bind to cell surface receptors (e.g., insulin receptors, EPO receptors, G protein-coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) and regulate (e.g., inhibit, activate, etc.) physiological pathways in which the receptor is involved (e.g., glucose level regulation, blood cell development, mitogenesis, etc.). Examples of cell receptor ligands include cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G protein-coupled receptor ligands, etc. Templates encoding trinucleotide repeat (e.g., CAG repeat) expansions have been found to be useful for silencing pathogenic sequences in neurodegenerative diseases, such as spinal-bulbar muscular atrophy and Huntington's disease (Caplen et al., Hum. Mol. Genet. (Human Molecular Genetics) 11:175 (2002)).

[0222] Injectable delivery, as described in U.S. Patent Nos. 5,543,158, 5,641,515, and 5,399,363, is desirable in some cases, including parenteral, intravenous, intramuscular, subcutaneous, intradermal, or intraperitoneal delivery of the drug-lipid particles disclosed herein. The drug-lipid particles can be injected locally at a target site (e.g., a disease site, such as inflammation or tumor formation, or a target organ or tissue) or systemically for widespread distribution throughout the organism. Solutions of the drug-lipid particles can be prepared in water in appropriate mixtures with surfactants. Dispersions can also be prepared in glycerin, liquid polyethylene glycol, and mixtures thereof, as well as oils. Optionally, these formulations contain a preservative to prevent microbial growth. For intravenous administration, drug-lipid particle formulations are typically prepared with a suitable pharmaceutical carrier. Buffered saline (135-150 mM NaCl) is typically used as the pharmaceutical carrier, although other suitable carriers may also suffice. Further suitable carriers are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The term "medicinal" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans. The preparation of aqueous compositions containing proteins as active ingredients is conventionally understood in the art. Alternatively, these compositions may be prepared as injectables, liquid solutions or suspensions, and further, solid forms suitable for solution or suspension in liquid prior to injection. Additionally, the preparations may be emulsified.

[0223] The drug-lipid particles can be sterilized by conventional liposome sterilization techniques such as filtration. The drug-lipid particles can contain pharmaceutical auxiliary substances that create appropriate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and wetting agents. These compositions can be sterilized using the techniques described above, or alternatively, can be produced under aseptic conditions. The resulting aqueous solution can be packaged for use, or filtered and lyophilized under sterile conditions, and the lyophilized preparation can be combined with a sterile aqueous solution before administration.

[0224] Prophylactic and Therapeutic Treatment: In some embodiments, drug-lipid particles can be used for the prophylactic or therapeutic treatment of a subject (e.g., a mammalian subject) suffering from a disease or condition associated with the expression or overexpression of a target sequence. The drug-lipid particles are administered to the subject in an amount sufficient to elicit a therapeutic response in the patient. An amount sufficient to achieve this is defined as a "therapeutically effective dose or amount" or "effective dose or amount." When determining the effective amount of drug-lipid particles to be administered to treat or prevent a disease caused by the expression or overexpression of a target gene, a physician will evaluate the circulating plasma levels of the drug-lipid particles, the toxicity of the drug-lipid particles, and the progression of the disease associated with the expression or overexpression of the target gene. Administration can be accomplished by single or divided dose administration.

[0225] For example, the drug-lipid particles can be administered to subjects infected with or at risk of infection by pathogenic microorganisms. The drug preferably corresponds to a sequence that plays an important role in the life cycle of the microorganism and should be unique to the microorganism (or at least absent from the native genome of the patient being treated). The drug-lipid particles are introduced into target cells, tissues, or organs in a therapeutically effective dose by ex vivo or intravenous injection. Silencing of sequences encoding genes associated with pathogenic infections can be advantageously used in combination with the administration of conventional reagents used to treat pathogenic diseases. The treatment can be administered prophylactically to individuals at risk of infection with pathogenic microorganisms or those already infected with pathogenic microorganisms.

[0226] In a preferred embodiment, the drug-lipid particles of the present disclosure can be advantageously used to treat cancer, viral infections, autoimmune diseases, diabetes, and Alzheimer's disease. Viral infections include hepatitis A, hepatitis B, hepatitis C, SARS-CoV-2, HIV, HPV, influenza, smallpox, and syphilis. For example, suitable sites for inhibiting hepatitis B virus include nucleic acid sequences encoding the S, C, P, and X proteins, PRE, EnI, and EnII (see, e.g., FIELDSVIROLOGY, 2001, supra). Those skilled in the art will appreciate that gene silencing associated with hepatitis infection may be achieved by conventional treatments for hepatitis, such as immunoglobulins, interferons (e.g., PEGylated and non-PEGylated interferon alpha) (see, e.g., Medina et al., Antiviral Res. 60(2):135-143 (2003)), ribavirin (see, e.g., Hugle and Cerny, Rev. Med. Virol. 13(6):361-71 (2003)), adefovir and lamivudine (see, e.g., Kock et al., Hepatology 38(6):1410-8 (2003)), prenylation inhibitors (see, e.g., Bordier et al., J. Clin. Invest. 112(3):407-414 (2003)), famciclovir (see, e.g., Yurdaydin et al., J. Hepatol. 37(2):266-71 (2002)), and saikosaponins c and d (see, e.g., Chiang et al., Planta Med. 69(8):705-9 (2003)).

[0227] In another embodiment, the drug-lipid particles of the present disclosure can be advantageously used to treat diseases and conditions characterized by the expression or overexpression of a gene or a group of genes. In some aspects, the drug-lipid particles of the present disclosure can be used to treat metabolic diseases and conditions (e.g., liver-targeted diseases and conditions, and liver diseases and conditions), such as dyslipidemia and diabetes. Those skilled in the art will understand that silencing genes associated with metabolic diseases and conditions can be combined with conventional treatments for these diseases.For example, silencing of genes involved in dyslipidemia has been demonstrated with statins, bile acid sequestrants / resins, cholesterol absorption inhibitors such as ezetimibe, phytostanols / sterols, polyphenols, dietary supplements such as oat bran, flaxseed, and soy protein, phytostanol analogs, squalene synthase inhibitors, bile acid transport inhibitors, SREBP cleavage activating protein (SCAP) activating ligands, niacin (nicotinic acid), acipimox, high-dose fish oil, antioxidants, and sugarcane fatty alcohols, microsomal triacylglycerol transporter (MTP) inhibitors, fatty acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors, gemcabene, lifevero, pantothenic acid analogs, niacin receptor agonists, anti-inflammatory agents (such as Lp-PLA(2) antagonists and AGI1067), and functional oils. , PPAR-α, gamma, delta agonists, dual PPAR-α, gamma, and "pan" PPAR-α / γ / δ agonists, cholesteryl ester transfer protein (CETP) inhibitors (such as torcetrapib), CETP vaccines, upregulators of ATP-binding cassette transport protein (ABC) A1, lecithin cholesterol acyltransferase (LCAT) and scavenger receptor class B type 1 (SRB1), and synthetic apolipoprotein (Apo) E-related peptides, sustained-release niacin / lovastatin, atorvastatin / amlodipine, ezetimibe / simvastatin, atorvastatin / CETP inhibitors, statins / PPAR agonists, developmental delayed-release niacin / simvastatin and pravastatin / aspirin, and anti-obesity agents (e.g., Bays and Stein, Expert See Opin. Pharmacother. 4(11):1901-38 (2003). Similarly, silencing of genes involved in diabetes can be combined with insulin treatment and dietary modification and exercise.

[0228] Similar methods are used to inhibit the expression of endogenous receptor cell genes associated with tumorigenesis and cell transformation, tumor growth and tumor migration, to inhibit the expression of angiogenic genes, to inhibit the expression of genes associated with inflammation and autoimmune responses, to inhibit the expression of ligand receptor genes, to inhibit the expression of genes associated with neurodegenerative diseases, and to inhibit the expression of additional genes associated with viral infection and survival. Specific targeted gene sequences are described above.

[0229] The drug-lipid particles of the present invention can be detected by any method known in the art. For example, a label can be directly or indirectly conjugated to a component of the drug-lipid particle or other lipid-based carrier system using a method well known in the art. A wide variety of labels can be used, selected based on the required sensitivity, ease of conjugation to the drug-lipid particle component, stability requirements, tool availability, processing readiness, etc. Suitable labels include, but are not limited to, spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives such as Texas Red, tetramethylrhodamine isothiocyanate (TRITC), digitoxin, biotin, phycoerythrin, AMCA, CyDyes™, radioactive labels such as 3H, 125I, 35S, 14C, 32P, 33P, enzymes such as horseradish peroxidase, alkaline phosphatase, and spectrocolorimetric labels such as colloidal gold, colored glass, plastic beads such as polystyrene, polypropylene, latex, etc.). Labels are detected using any means known in the art.

[0230] Nucleic acids herein can be detected and quantified by any of a number of means well known to those skilled in the art. Nucleic acids can be detected by methods well known in the art, such as Southern blot analysis, Northern blot analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Additional analytical biochemical techniques can also be applied, such as spectrophotometry, X-ray radiography, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and hyperdiffusion chromatography.

[0231] The sensitivity of hybridization assays can be improved by using nucleic acid width systems that double the amount of target nucleic acid detected. In vitro amplification techniques are known that are suitable for amplifying sequences to be used as molecular probes or for generating nucleic acid fragments for subsequent subcloning. Examples of in vitro amplification methods sufficient to guide one of skill in the art, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ replicase amplification, and other RNA polymerase-mediated techniques (e.g., NASBA™), include Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2000; Ausubel et al., SHORT PROTOCOLS IN MOLECULAR BIOLOGY (eds.), Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (2002); Mullis et al., SHORT PROTOCOLS IN MOLECULAR BIOLOGY (eds.), Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (2002); et al., (1987), U.S. Patent No. 4,683,202, A Guide to PCR Protocol Methods and Applications (Innis et al., eds.), Academic Press Inc., San Diego, CA (1990) (Innis), Arnheim & Levinson (October 1, 1990), C&EN 36, (The Journal of NIH Research), 3:81 (1991), (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989); Guatelli et al., Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences), 87:1874 (1990), Lomell et al., J. Clin. Chem.(Journal of the Japanese Society of Clinical Chemistry, 35:1826 (1989); Landegren et al., Science, 241:1077 (1988); Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene, 4:560 (1989); Barringer et al., Gene, 89:117 (1990); Sooknanan and Malek, Biotechnology, 13:563 (1995). Improved methods for in vitro amplification and cloning of nucleic acids are described in U.S. Patent No. 5,426,039 to Wallace et al. Other methods described in the art are nucleic acid sequence-based amplification (NASBA™, Cangene, Mississauga, Ontario) and the Qβ replicase system.

[0232] As described in Needham VanDevanter et al., Nucleic Acids Res., 12:6159 (1984), this typically follows the solid-phase phosphoramidite method described by Beaucage and Caruthers, Tetrahedron Letts., 22(20):1859-1862 (1981). For example, automated synthesizers are used to chemically synthesize oligonucleotides for use as probes in in vitro amplification procedures, gene probes, or inhibitor components. If necessary, oligonucleotides are typically purified by native acrylamide gel electrophoresis or anion-exchange HPLC, as described in Pearson and Regnier, J. Chrom., 255:137-149 (1983). The sequence of synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.), Academic Press, New York, Methods in Enzymology, 65:499.

[0233] The following examples are provided to illustrate, but not to limit, the claimed disclosure. Those of skill in the art will readily identify a variety of non-critical parameters that can yield substantially the same results.

[0234] The drug-lipid particles sought to be protected in this disclosure refer to drug-lipid particles other than those containing cationic / ionizable lipids, i.e., drug-loaded metal-chelated phospholipid complex nanoparticles (drug@MPP).

[0235] Experimental Example 1: Preparation of drug-metal-phospholipid complex particles

[0236] Example 1 Preparation of phospholipid complex

[0237] Phospholipid molecules bearing phosphate groups are linked to linker molecules. Distearoylphosphatidylcholine (DSPC, Formula 46) and curcumin (Formula 19) are added to a reaction bottle in a molar ratio of 1:1, dissolved with an appropriate amount of ethanol, and reacted at 65°C for 2 hours. The mixture is then concentrated and n-hexane is added. The phospholipid complex is precipitated, filtered, and vacuum-dried to obtain the phospholipid complex. The structure of the phospholipid complex is as follows: [ka]

[0238] The result analysis showed that the yield of the target product obtained by reacting curcumin with DSPC at 65°C for 2 hours was 94%.

[0239] Example 2 Preparation of metal-phospholipid complexes

[0240] Example 2.1 Metal ion is Fe 3+ Preparation of metal-phospholipid complexes when

[0241] The phospholipid complex prepared in Example 1 is partially linked to a metal ion. The phospholipid complex and FeCl3 were added to a reaction bottle in a molar ratio of 1:1, dissolved in ethanol, and reacted at 60°C for 2 hours. The reaction solution was then suspended and dried, immediately washed with ultrapure water, and vacuum dried to obtain the metal-phospholipid complex. The structure of the metal-phospholipid complex is shown below. [ka]

[0242] The results showed that the phospholipid complex and FeCl3 were reacted at 60°C for 2 hours, the input concentration of the phospholipid complex was 4.5 mg / mL, and the input ratio of the phospholipid complex to FeCl3 was 1:1, and the yield of the target product was 95%.

[0243] Example 2.2 Metal ion is Al 3+ Preparation of metal-phospholipid complexes when

[0244] The difference between this example and Example 2.1 is that FeCl3 was replaced with Al(NO3)3·9H2O. The structure of the prepared metal-phospholipid complex is shown below. [ka]

[0245] The results showed that the phospholipid complex and Al(NO3)3·9H2O were reacted at 60°C for 2 hours, and the yield of the target product was 95% when the input concentration of the phospholipid complex was 4.5 mg / mL and the input ratio of the phospholipid complex to Al(NO3)3·9H2O was 1:1.

[0246] Example 3: Preparation of mRNA-loaded metal-chelated phospholipid complex nanoparticles (mRNA@MPP)

[0247] Metal ion is Fe3+ Preparation of mRNA-metal-phospholipid complex particles when

[0248] Metal-phospholipid complexes were prepared according to the method in Example 2.1, with DSPC, curcumin, and FeCl3 added in a 1:1:1 ratio. The metal-phospholipid complexes, distearoylphosphatidylcholine (DSPC, Formula 46, as a non-cationic or non-ionizable lipid), cholesterol (CHOL, Formula 40, as a non-cationic or non-ionizable lipid), and DSPE-PEG2000 (Formula 53, as a conjugation lipid to inhibit particle aggregation) were dissolved in ethanol at different molar fractions as the organic phase. The proportions of the metal-phospholipid complexes, DSPC, CHOL, and DSPE-PEG2000 were 15%, 35%, 46%, and 4%, respectively. The mRNA was dissolved in enzyme-free PBS buffer at a concentration of 20 μg / mL as the aqueous phase (PBS components: 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.0018 M potassium dihydrogen phosphate). The metal-phospholipid complex mass and mRNA mass were mixed in a microfluidic chip at a mass ratio of 40:1. The volume ratio of the aqueous phase to the organic phase was 3:1. The flow rate of the organic and aqueous phases in the microfluidic chip was 12 ml / min. The drug mRNA was mRNA encoding the fluorescent protein eGFP, and its sequence was SEQ ID NO. 1 (720 nt). eGFP-mRNA@MPP was produced. eGFP-mRNA@MPPs were incubated with 293T at a concentration of 2 μg / mL (the concentration of the mRNA contained), and a control group was incubated with MPPs without drug loading. After 48 hours, the cell suspension was collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0249] The particle size, surface potential, and stability of the eGFP-mRNA@MPP produced in Example 3 were detected, and the efficiency of the eGFP-mRNA@MPP encapsulating nucleic acids was calculated.

[0250] The particle size detection method and criteria for the results are as follows: the particle size of nanoparticles is tested using a Malvern laser particle size analyzer, Zetasizer; if the particle size is in the range of 30 to 400 nm, it is considered acceptable.

[0251] The method for detecting the surface potential and the criteria for judging the results are as follows: the surface potential of nanoparticles is measured using a Malvern laser particle size analyzer, Zetasizer; if the potential is in the range of -10 to 10 mV, it is considered acceptable.

[0252] The method for detecting stability and the criteria for judging the results are as follows: nanoparticles are left at 4°C for 7 days, and the particle size and surface potential of the nanoparticles are measured using a Malvern Zetasizer laser particle size analyzer; if the particle size and surface potential do not change significantly within 3 to 7 days, the stability is considered to be relatively good.

[0253] The nucleic acid encapsulation efficiency was calculated by agarose gel electrophoresis. First, the amount of nucleic acid added to each lipid nanoparticle was set to 10 μg / mL, and the metal ion content was set to Fe. 3+ In this case, the mass ratio of the metal-phospholipid complex to the mRNA was 40:1. An equal concentration of nucleic acid was dissolved in PBS buffer as a positive control, and PBS buffer was used as a negative control. The agarose gel concentration was 1.5%, and the gap in the gel allowed only free nucleic acid to pass through, but not lipid nanoparticles. Electrophoresis was stopped when the free nucleic acid bands were clearly distinguishable. Image J software was used to calculate the gray values of free nucleic acid for each group. The positive control group was set to 100%, and the ratio of free nucleic acid in each group to the positive control was used as the relative amount of free nucleic acid. The encapsulation rate for each group was calculated as (100 - relative amount of free nucleic acid)%. A nucleic acid encapsulation rate of 50% or higher was considered acceptable.

[0254] The cell culture method involved culturing the human embryonic kidney cell line 293T in DMEM medium containing 10% FBS and 1% penicillin-streptomycin under conditions of 37°C and 5% CO2.

[0255] Analysis of the percentage of eGFP-positive cells by flow cytometry was performed using 293T cells at 5 x 10 5 Cells were seeded into 24-well plates at a seeding density of 100 cells / well. When cell density reached 80%, 1 mL MMPP or eGFP-mRNA@MPP was added and incubated at a concentration of 2 μg / mL. After 48 hours, the cell suspension was collected and 20,000 cells were collected using the FITC channel of a flow cytometer. The percentage of eGFP-positive cells was analyzed using the formula: eGFP-expressing cells / total cells × 100%. A percentage of eGFP-positive cells of 40% or higher was considered acceptable.

[0256] Metal ion is Al 3+ Preparation of mRNA-metal-phospholipid complex particles when

[0257] Metal-phospholipid complexes were prepared according to the method described in Example 2.2. DSPC, curcumin, and Al(NO3)3·9H2O were added in a 1:1:1 ratio. The metal-phospholipid complexes, distearoylphosphatidylcholine (DSPC, Formula 46, as a non-cationic or non-ionizable lipid), cholesterol (CHOL, Formula 40, as a non-cationic or non-ionizable lipid), and DSPE-PEG2000 (Formula 53, as a conjugated lipid to inhibit particle aggregation) were dissolved in ethanol at different molar fractions as the organic phase. The proportions of the metal-phospholipid complex, DSPC, CHOL, and DSPE-PEG2000 were 7%, 34%, 56%, and 3%, respectively. The mRNA was dissolved in enzyme-free PBS buffer at a concentration of 20 μg / mL as the aqueous phase (PBS components: 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.0018 M potassium dihydrogen phosphate). The metal phospholipid complex mass and mRNA mass 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 was 3:1. The flow rate of the organic and aqueous phases in the microfluidic chip was 12 ml / min. The drug mRNA was mRNA encoding the fluorescent protein eGFP, and its sequence is SEQ ID NO. 1 (720 nt). eGFP-mRNA@MPP was produced. 293T cells were incubated with eGFP-mRNA@MPP at a concentration of 2 μg / mL (the concentration of the mRNA contained), while a 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.

[0258] The particle size, surface potential, and stability of the eGFP-mRNA@MPP prepared in Example 3 are detected, and the efficiency of the eGFP-mRNA@MPP encapsulating nucleic acids is calculated.

[0259] The particle size detection method and criteria for the results are as follows: nanoparticle particle size is tested using a Malvern laser particle size analyzer, Zetasizer; if the particle size is in the range of 30 to 400 nm, it is considered acceptable.

[0260] The method for detecting the surface potential and the criteria for judging the results are as follows: the surface potential of nanoparticles is tested using a Malvern laser particle size analyzer, Zetasizer; if the potential is in the range of -10 to 10 mV, it is considered acceptable.

[0261] The method for detecting stability and the criteria for judging the results are as follows: nanoparticles are left at 4°C for 7 days, and the particle size and surface potential of the nanoparticles are tested using a Malvern laser particle size analyzer, Zetasizer. If the particle size and surface potential do not change significantly within 3 to 7 days, the stability is considered to be relatively good.

[0262] Specifically, the nucleic acid encapsulation efficiency was calculated using agarose gel electrophoresis. First, the amount of nucleic acid added to each group of lipid nanoparticles was set to 10 μg / mL, and the metal ion was Al. 3+ The ratio of metal-phospholipid complex mass to mRNA mass was 13.3:1. An equal concentration of nucleic acid was dissolved in PBS buffer as a positive control, and PBS buffer was used as a negative control. The agarose gel concentration was 1.5%, and the gap in the gel allowed only free nucleic acid to pass through, but not lipid nanoparticles. Electrophoresis was stopped when the free nucleic acid band was clearly visible. Image J software was used to calculate the gray values of free nucleic acid for each group. The positive control group was set to 100%, and the ratio of free nucleic acid in each group to the positive control was used as the relative amount of free nucleic acid. The encapsulation rate for each group was calculated as (100 - relative amount of free nucleic acid). A nucleic acid encapsulation rate of 50% or higher was considered acceptable.

[0263] The cell culture method involves culturing the human embryonic kidney cell line 293T in DMEM medium containing 10% FBS and 1% penicillin-streptomycin under conditions of 37°C and 5% CO2.

[0264] The percentage of eGFP-positive cells was analyzed by flow cytometry using 293T cells at 5 x 10 5Cells were seeded into 24-well plates at a seeding density of 100 cells / well. When cell density reached 80%, 1 mL of MPP or eGFP-mRNA@MPP was added and incubated. The eGFP-mRNA@MPP concentration was 2 μg / mL. After 48 hours, the cell suspension was collected and 20,000 cells were collected using the FITC channel of a flow cytometer. The percentage of eGFP-positive cells was analyzed using the formula: eGFP-positive cell rate = number of eGFP-expressing cells / total number of cells × 100%. A percentage of eGFP-positive cells of 40% or higher was considered acceptable.

[0265] The principle of loading nucleic acids into metal-chelated phospholipid complex nanoparticles (MPPs) organized by metal-phospholipid complexes is that curcumin and DSPC are bound by hydrogen bonds, while curcumin is bound to Fe via coordination bonds. 3+ or Al 3+ to form a metal-phospholipid complex, and the Fe 3+ or Al 3+ is linked to nucleic acids by a coordinate bond, which ensures that the metal-phospholipid complex loads nucleic acids onto the nanoparticles while self-assembling with other lipid components into MPPs. There are two possibilities for the contribution of curcumin to MMP-mediated nucleic acid loading: (1) curcumin interacts with nucleic acids and aids MPP loading of nucleic acids, e.g., curcumin aids nucleic acid loading by inserting into the minor groove of nucleic acids; or (2) curcumin may not directly interact with nucleic acids.

[0266] Example 3.1: Metal-phospholipid complex component loading ratios

[0267] The DSPC, curcumin, and FeCl3 in Example 3 were added at different ratios (1:1:1, 3:3:2, and 2:2:1), and the other steps were the same as in Example 3. Different eGFP-mRNA@MPPs were prepared and their nucleic acid encapsulation rates were measured.

[0268] Analysis of the results, as shown in Table 1-1, shows that when the input ratio of DSPC, curcumin, and FeCl3 is 1:1:1, the eGFP-mRNA encapsulation efficiency of the drug-lipid particles produced is 97%. When the input ratio of DSPC, curcumin, and FeCl3 is 3:3:2, the eGFP-mRNA encapsulation efficiency of the drug-lipid particles produced is 70%. When the input ratio of DSPC, curcumin, and FeCl3 is 2:2:1, the eGFP-mRNA encapsulation efficiency of the drug-lipid particles produced is 60%. Fe in the drug-lipid particles 3+ The function of each Fe is to link the phospholipid complex with the nucleic acid. 3+ Since DSPC has up to three conjugation sites, the input ratio of DSPC, curcumin, and FeCl3 in the drug-lipid particles should be 1:1:1 to ensure that the drug-lipid particles can encapsulate as much nucleic acid as possible. Experimental results also demonstrated that when the input ratio of DSPC, curcumin, and FeCl3 is 1:1:1, the drug-lipid particles produced using this method have the highest eGFP-mRNA encapsulation rate. When the input ratio of DSPC, curcumin, and FeCl3 is between 1:1:1 and 2:2:1, the nucleic acid encapsulation rate of the drug-lipid particles is above 60%.

[0269] [Table 1]

[0270] The DSPC, curcumin, and Al(NO3)3·9H2 of Example 3 were added in different ratios (1:1:1, 3:3:2, 2:2:1), and the other steps were the same as in Example 3 to produce different eGFP-mRNA@MPPs and detect their nucleic acid encapsulation rates.

[0271] Analysis of the results shows that, as shown in Table 1-2, when the input ratio of DSPC, curcumin, and Al(NO3)3·9H2O is 1:1:1, the eGFP-mRNA entrapment efficiency of the prepared metal-phospholipid complex particles is 98%. When the input ratio of DSPC, curcumin, and Al(NO3)3·9H2O is 3:3:2, the eGFP-mRNA entrapment efficiency of the prepared metal-phospholipid complex particles is 72%. When the input ratio of DSPC, curcumin, and Al(NO3)3·9H2O is 2:2:1, the eGFP-mRNA entrapment efficiency of the prepared metal-phospholipid complex particles is 58%. 3+ The function of each Al is to link the phospholipid complex with the nucleic acid. 3+ Because DSPC has a maximum of three conjugation sites, the input ratio of DSPC, curcumin, and Al(NO3)3·9H2O in the drug-lipid particles should be 1:1:1 to ensure that the metal-phospholipid complex particles can encapsulate as much nucleic acid as possible. Experimental results also demonstrated that the highest eGFP-mRNA encapsulation rate was achieved in metal-phospholipid complex particles prepared using a 1:1:1 input ratio of DSPC, curcumin, and Al(NO3)3·9H2O. When the input ratio of DSPC, curcumin, and Al(NO3)3·9H2O ranged from 1:1:1 to 2:2:1, the nucleic acid encapsulation rate of the metal-phospholipid complex particles was always above 58%.

[0272] [Table 2]

[0273] Example 3.2 Proportions of metal-phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) in the prepared drug-lipid particles

[0274] Compared with Example 3, the ratios of metal-phospholipid complex, distearoylphosphatidylcholine (DSPC), DSPE-PEG2000, and cholesterol (CHOL) are shown in Table 1-3 (metal ion is Fe 3+) and Table 1-4 (metal ion is Al 3+ (where ∇ is the same as ∇ ...

[0275] The results of the analysis are shown in Table 1-3. 3+ When the proportion of DSPE-PEG2000 in the drug-lipid particles is in the range of (10-40)%, the proportion of DSPC in the range of (0-40)%, the proportion of CHOL in the range of (35-75)%, and the proportion of DSPE-PEG2000 in the range of (2-10)%, the particle size of the drug-lipid particles is in the range of 50-400 nm, the surface potential is in the range of -10-10 mV, the in vitro stability is 3 days or more, the mRNA encapsulation rate is 50% or more, and the positive expression rate of eGFP protein is 70% or more. Among these, the drug-lipid particle performance was best when the metal-phospholipid complex content was 15%, distearoylphosphatidylcholine (DSPC) content was 35%, cholesterol (CHOL) content was 46%, and DSPE-PEG2000 content was 4%. That is, the particle diameter was in the range of 110 nm, the surface potential was in the range of -2.04 mV, the in vitro stability was >7 days, the mRNA encapsulation rate was 87%, and the positive expression rate of eGFP protein was 97%. Because mRNA@MPP mainly relies on the metal-phospholipid complex for nucleic acid adsorption, the metal-phospholipid complex content should not be too low. When the DSPC content was in the range of 0-40%, the nanoparticle stability was within an acceptable range. When the DSPC content was 0%, the stability of the nanoparticles was maintained due to the presence of DSPC in the metal-phospholipid complex. The role of DSPE-PEG2000 is to prevent nanoparticle aggregation and prolong circulation time in the body, and its performance is relatively excellent when its content is in the range of 2-10%. The role of CHOL is to increase the fluidity of nanoparticles, and maintaining a constant content is beneficial to the stability of nanoparticles.

[0276] The above results indicate that the metal-phospholipid complex (metal ion is Fe 3+These results suggest that mRNA@MPP has relatively good drug loading performance when the ratio of DSPC is in the range of (10–40)%, the ratio of DSPC is in the range of (0–40)%, the ratio of CHOL is in the range of (35–75)%, and the ratio of DSPE-PEG2000 is in the range of (2–10)%.

[0277] [Table 3]

[0278] The results of the analysis are shown in Table 1-4. The metal-phospholipid complex (metal ion is Al 3+When the ratio of DSPC to DSPE-PEG2000 is in the range of (5-50)%, the ratio of DSPC is in the range of (0-51), the ratio of CHOL is in the range of (15-80), and the ratio of DSPE-PEG2000 is in the range of (2-10), the particle size of the drug-lipid particles is in the range of 50-400 nm, the surface potential is in the range of -10-10 mV, the in vitro stability is more than 3 days, the mRNA encapsulation rate is more than 50%, and the positive expression rate of eGFP protein is more than 70%. When the metal-phospholipid complex content was 7%, distearoylphosphatidylcholine (DSPC) content was 34%, cholesterol (CHOL) content was 56%, and DSPE-PEG2000 content was 3%, the drug-lipid particle performance was optimal, i.e., particle diameter was 100 nm, surface potential was -1.57 mV, in vitro stability was >7 days, mRNA entrapment rate was 92%, and eGFP protein positive expression rate was 98%. Because mRNA@MPP mainly relies on the metal-phospholipid complex for nucleic acid adsorption, the metal-phospholipid complex content should not be too low. When the DSPC content was in the range of 0-51%, nanoparticle stability was within an acceptable range. When the DSPC content was 0%, nanoparticle stability was maintained due to the presence of DSPC in the metal-phospholipid complex. The role of DSPE-PEG2000 was to prevent nanoparticle aggregation and prolong circulation time in the body, and when its content was in the range of 2-10%, performance was relatively good. The role of CHOL is to increase the fluidity of the nanoparticles, and maintaining a constant content is beneficial to the stability of the nanoparticles.

[0279] The above results indicate that the metal-phospholipid complex (metal ion haAl 3+ This suggests that mRNA@MPP has relatively good drug loading performance when the ratio of DSPC is in the range of (5-50)%, the ratio of DSPC is in the range of (0-51)%, the ratio of CHOL is in the range of (15-80)%, and the ratio of DSPE-PEG2000 is in the range of (2-10)%.

[0280] [Table 4] JPEG2025527283000032.jpg66156

[0281] Example 3.3: Types of non-cationic or non-ionizable lipids in the prepared eGFP-mRNA@MPP

[0282] Compared with Example 3, the substitution of distearoylphosphatidylcholine (DSPC) is as shown in Tables 1-5 and 1-6, and the other conditions are the same.

[0283] To explore whether DSPC in eGFP-mRNA@MPP could be replaced with other non-cationic or non-ionizable lipids, three other non-cationic or non-ionizable lipids, namely DSPE, DSPA, and DSPG, were selected instead of DSPC. The particle size, surface potential, stability, and mRNA encapsulation rate were measured, proving that DSPC in eGFP-mRNA@MPP could be replaced with other non-cationic or non-ionizable lipids. The functionality of the replaced eGFP-mRNA@MPP was comparable to that of eGFP-mRNA@MPP containing DSPC (Tables 1-5). The metal ion was Fe. 3+ ) and Table 1-6 (metal ions are Al 3+ The main role of the non-cationic or non-ionizable lipid DSPC in eGFP-mRNA@MPP is to improve liposome membrane fusion, increase stability, and reduce toxicity. However, other non-cationic or non-ionizable lipids also have the functions of improving liposome membrane fusion, increasing stability, and reducing toxicity. Therefore, DSPC in the drug-lipid particles can be replaced with other non-cationic or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation, without affecting their efficacy.

[0284] The structural formulas of three non-cationic or non-ionizable lipids (DSPE, DSPA, and DSPG) are shown below: [ka] [Table 5] [Table 6]

[0285] Example 3.4: Types of conjugated lipids that inhibit particle aggregation in the prepared eGFP-mRNA@MPP

[0286] Compared to Example 3, the substitution of DSPE-PEG2000 (Formula 53) is shown in Table 1-7 (metal ion is Fe 3+ ) and Table 1-8 (metal ions are Al 3+ (Formula 50), other conditions being the same. Three other conjugated lipids that inhibit particle aggregation are DSPE-PEG700 (Formula 50), DSPE-PEG5000 (Formula 52), and DSPE-PEG1000 (Formula 51).

[0287] To investigate whether the DSPE-PEG2000 in eGFP-mRNA@MPP could be replaced with other conjugated lipids that inhibit particle aggregation, three other conjugated lipids that inhibit particle aggregation, namely, DSPE-PEG700, DSPE-PEG5000, and DSPE-PEG1000, were selected instead of SPE-PEG2000. Measurements of particle size, surface potential, stability, and mRNA encapsulation rate demonstrated that the DSPE-PEG2000 in eGFP-mRNA@MPP could be replaced with other conjugated lipids that inhibit particle aggregation, and the performance of the replacement lipids was comparable to that of eGFP-mRNA@MPP containing DSPE-PEG2000 (Tables 1-7 and 1-8). Although the main role of DSPE-PEG2000 in eGFP-mRNA@MPP is to inhibit aggregation, other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation. Therefore, DSPE-PEG2000 in eGFP-mRNA@MPP can be replaced with other conjugated lipids that inhibit particle aggregation without affecting its efficacy. [Table 7] [Table 8]

[0288] Example 3.5 Preparation and Characterization of mRNA@MPP

[0289] Example 3.5.1 Metal ion is Fe 3+ Preparation and characterization of the effect of mRNA@MPP

[0290] By replacing the mRNA in Example 3 with two other mRNAs and following the method of Example 3, three types of mRNA@MPP containing mRNA sequences for different target proteins were prepared. The three different mRNA sequences are as follows: (1) The mRNA sequence encoding the fluorescent protein eGFP is SEQ ID NO. 1 (720 nt); (2) The mRNA sequence encoding the receptor binding domain (RBD) of the S1 subunit of the novel coronavirus is SEQ ID NO. 2 (669 nt); and (3) The mRNA sequence encoding the tumor antigen NY-ESO-1 is SEQ ID NO. 3 (543 nt). The remaining drug (mRNA)-lipid particle preparation process was the same as in Example 3, resulting in eGFP-mRNA@MPP, RBD-mRNA@MPP, and NY-ESO-1-mRNA@MPP, respectively.

[0291] 293T cells were incubated with eGFP-mRNA@MPP at a concentration of 2 μg / mL (mRNA concentration) or with MPP alone as a control. After 48 hours, the cell suspension was harvested and the percentage of eGFP-positive cells was detected by flow cytometry. The results are shown in Figure 1-1. 293T cells were incubated with RBD-mRNA@MPP at a concentration of 2 μg / mL (mRNA concentration) or with MPP alone as a control. After 24 hours, the supernatant was centrifuged and frozen at -20°C for later use. The expression level of the novel coronavirus antigen RBD protein in the cell supernatant was detected using a commercially available novel coronavirus antigen RBD ELISA detection kit. The results are shown in Figure 1-2.

[0292] The method for detecting RBD expression levels by ELISA is as follows:

[0293] 1. To collect the sample, let the cell supernatant stand at room temperature for 2 hours, then centrifuge it at 1000 xg for 20 minutes and collect the supernatant.

[0294] 2. To add samples, prepare blank wells, standard wells, and sample wells to be tested on the coated plate. Add 100 μL of sample dilution to the blank wells, add serially diluted standard solutions to the standard wells, and add 100 μL of test sample to the sample wells to be tested. Incubate at 37°C for 60 minutes.

[0295] 3. Discard the liquid in the wells and wash the plate three times, soaking for 1-2 minutes each time. Add 100 μL of the prepared biotin-labeled anti-RBD antibody working solution to each well, mix evenly, and incubate at 37°C for 60 minutes.

[0296] 4. Discard the liquid in the wells and wash the plate three times, soaking for 1-2 minutes each time.

[0297] 5. Add 100 μL of the prepared streptavidin-HRP working solution to each well, mix evenly, and incubate at 37°C for 45 minutes.

[0298] 6. Discard the liquid in the wells and wash the plate three times, soaking for 1-2 minutes each time.

[0299] 7. Add 100 μL of TMB substrate solution (TMB) to each well and incubate at 37°C for 15 minutes away from light.

[0300] 8. Stop the reaction by adding 100 μL of stop solution to each well.

[0301] 9. Measure the optical density (OD value) of each well at a wavelength of 450 nm.

[0302] For data analysis, a standard curve is drawn with the concentration of the standard on the horizontal axis and the OD value on the vertical axis.

[0303] The experimental animals were randomly divided into two groups (experimental and control), with five mice in each group. The RBD-mRNA@MPP animal model was performed using BALB / c mice. Each mouse received a first intramuscular injection on day 1 and a second intramuscular injection on day 14. The experimental group received RBD-mRNA@MPP, while the control group received metal-phospholipid particles (MPPs) without mRNA. Each injection was 100 μL, and the RBD-mRNA@MPP formulation in the experimental group contained 30 mg of mRNA. Blood was collected 28 days after the first injection, and serum was separated and gradient diluted. A commercially available ELISA kit was used to detect RBD total IgG antibodies against the S1 subunit of the novel coronavirus (COVID-19). The results are shown in Figures 1–3.

[0304] The animal model for NY-ESO-1-mRNA@MPP was C57BL / 6 mice. Each mouse was intramuscularly administered four times on days 1, 7, 14, and 21. The experimental group received NY-ESO-1-mRNA@MPP injections, while the control group received metal-phospholipid particles (MPPs) without mRNA. Each injection was 100 μL, and the NY-ESO-1-mRNA@MPP formulation in the experimental group contained 30 mg of mRNA. On day 28 after the first injection, blood was collected from the mice, and serum was separated and gradient diluted. ELISA was performed to detect anti-NY-ESO-1 total IgG antibodies produced in the mice. The results are shown in Figures 1–4.

[0305] The method for detecting anti-NY-ESO-1 total IgG antibodies in mice is as follows:

[0306] The preparation of reagents used in the ELISA method is as follows:

[0307] 1. To prepare the coating solution, accurately weigh 8.4 g of NaHCO3 and dissolve it in 1 L of distilled water (DDW). After all the solids have dissolved, adjust the pH of the entire solution to 9.6 using 1 M NaOH solution. Store the prepared coating solution at 4°C for later use.

[0308] 2. For the washing solution, add 0.5 mL of Tween-20 to 1 L of 0.01 M PBS solution, mix evenly, and leave at room temperature.

[0309] 3. To prepare the blocking solution, accurately weigh 20 g of BSA and add it to 1 L of 0.01 M PBS solution. Sonicate to remove any undissolved BSA powder. Once all solids have dissolved and the solution has turned pale yellow, store it in a 4°C refrigerator for later use.

[0310] 4. To prepare the antibody diluent, accurately weigh 2.5 g of BSA and dissolve it in 250 mL of 0.01 M PBS solution. Once the solid is completely dissolved, add 1.25 mL of Tween-20, mix thoroughly, and store at 4°C for later use.

[0311] 5. For the color developing solution, add 19.2 g of citric acid to DDW water to make 1000 mL. (A) For 0.2 M disodium hydrogen phosphate, add 28.4 g of anhydrous disodium hydrogen phosphate to DDW water to make 1000 mL. (B) Add 24.3 mL of 0.1 M citric acid solution (A), 25.7 mL of 0.2 M phosphate buffer (B), and 50 mL of DDW water. At the time of use, add 50 mg of OPD (o-phenylenediamine) and 20.15 mL of 30% HO.

[0312] 6. To prepare the stop solution, add 55.5 mL of 2 M H2SO4: concentrated sulfuric acid and DDW to make a 500 mL solution.

[0313] Measurement of antibody titers in mouse serum by ELISA.

[0314] 1. For coating, dilute the NY-ESO-1 antigen in coating solution to 1 μg / mL, add 50 μL / well to a 96-well plate, and coat overnight at 4°C.

[0315] 2. For blocking, dehydrate the coating solution in the well plate, wash with blocking solution three times, once every 5 minutes, and dehydrate. Add 150 μL of blocking solution to each well and incubate at 37°C for 2 hours.

[0316] 3. Drying: Dehydrate the blocking solution and incubate at 37°C for 1-2 hours until all the liquid at the bottom of the well plate has dried.

[0317] 4. For immunization, serum samples were initially diluted 1:1000 in antibody diluent, then serially diluted 1:2. The diluted serum samples were added to a sealed 96-well plate at 100 μL per well and incubated at 37°C for 2 hours. The wells were then drained, 300 μL of washing solution was added per well, and the plate was gently shaken for 40 seconds. This step was repeated three times. Biotinylated goat anti-mouse IgG antibody diluted 1:1000 was added to the wells at 100 μL per well and incubated at 37°C for 1 hour. The wells were then drained, washing solution was added, and the plate washing step was repeated. Freshly prepared streptavidin-labeled horseradish peroxidase (HRP) working solution was added at 100 μL per well and incubated at 37°C for 1 hour. The wells were then drained, washing solution was added, and the plate washing step was repeated. Add 100 μL of color-developing solution to each well under dark conditions, incubate at room temperature for 5 minutes, then add 50 μL of stop solution to each well to stop the color development. Measure the absorbance at 450 nm using a microplate reader.

[0318] On the 28th day after administration of RBD-mRNA@MPP, the spleens of normal mice were collected, and single cell suspensions were prepared under sterile conditions. 100,000 splenocytes / well were seeded onto cell well plates, and 10 mg / mL of RBD protein was added to the plate for a final concentration of 10 mg / mL. The mixture was then cultured for 48 hours. The supernatant was removed by centrifugation, and the expression levels of IFN-γ, IL-2, and IL-4 were measured using ELISA kits. The results are shown in Figures 1-5.

[0319] On the 28th day after administration of NY-ESO-1-mRNA@MPP, normal mouse spleens were collected, and single cell suspensions were prepared under sterile conditions. These were then seeded into cell well plates at 100,000 spleen cells / well. NY-ESO-1 protein was added at a final concentration of 10 mg / mL and cultured for 48 hours. The supernatant was then centrifuged and the expression levels of IFN-γ, IL-2, and TNF-α were measured using ELISA kits. The results are shown in Figures 1-6.

[0320] As shown in Figure 1-1, the eGF-positive cell rate in the eGFP-mRNA@MPP experimental group was 97%, while no eGFP signal was detected in the MPP control group. As shown in Figure 1-2, the RBD protein encoded by the RBD-mRNA encapsulated in MPPs was 193.3 ng / mL in the supernatant of 293T cells, whereas the RBD protein content in the supernatant of 293T cells transfected with empty carrier MPPs was 0. These results suggest that mRNA-MPPs can encapsulate and deliver any mRNA and directly encode polypeptides within cells. As shown in Figures 1-3 and 1-4, both RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP effectively induced humoral immunity in mice, generating high levels of antigen-specific binding antibodies. The IgG antibody titer in mice treated with RBD-mRNA@MPP reached 117,268.8 pg / mL, while the IgG antibody titer in mice treated with NY-ESO-1-mRNA@MPP reached 5,319.52 pg / mL. As shown in Figures 1-5 and 1-6, both RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP effectively induced cellular immunity in mice, activating immune cells and producing large amounts of cytokines. RBD-mRNA@MPP induced the expression levels of IFN-γ, IL-2, and IL-4 at 252.8 pg / mL, 207.6 pg / mL, and 56.6 pg / mL, respectively. NY-ESO-1-mRNA@MPP induced the expression levels of IFN-γ, IL-2, and TNF-α at 70.79 pg / mL, 75.29 pg / mL, and 75.27 pg / mL, respectively. These results suggest that mRNA@MPP, by encapsulating and delivering any mRNA, can promote the expression of the target protein (antigen), thereby effectively inducing humoral and cellular immunity in mice, producing high levels of antigen-specific binding antibodies and cytokines, and playing a role as an anti-coronavirus mRNA vaccine and an anti-tumor mRNA vaccine.

[0321] Example 3.5.2 Metal ion is Al 3+ Preparation and characterization of the effect of mRNA@MPP

[0322] The difference between this example and Example 3.5.1 is that the metal ion Fe in Example 3.5.1 3+ Al 3+ is to be replaced by

[0323] As shown in Figure 1-7, 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. As shown in Figure 1-8, the RBD protein encoded by the RBD-mRNA encapsulated in MPP was 212.6 ng / mL in the supernatant of 293T cells, whereas the RBD protein content in the supernatant of 293T cells transfected with empty carrier MPP was 0. These results suggest that mRNA-MPP can encapsulate and deliver any mRNA to directly encode polypeptides within cells. As shown in Figures 1-9 and 1-10, both RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP effectively induced humoral immunity in mice, generating high levels of antigen-specific binding antibodies. The IgG antibody titers of mice treated with RBD-mRNA@MPP reached 129,113 pg / mL, while those of mice treated with NY-ESO-1-mRNA@MPP reached 6,507.4 pg / mL. As shown in Figures 1-11 and 1-12, both RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP effectively induced cellular immunity in mice, activating immune cells and producing large amounts of cytokines. RBD-mRNA@MPP induced the expression levels of IFN-γ, IL-2, and IL-4 at 271.8 pg / mL, 234.6 pg / mL, and 68.4 pg / mL, respectively. NY-ESO-1-mRNA@MPP induced the expression levels of IFN-γ, IL-2, and TNF-α at 83.8 pg / mL, 98 pg / mL, and 97.8 pg / mL, respectively. These results suggest that mRNA@MPP, by encapsulating and delivering any mRNA, can promote the expression of target proteins (antigens), thereby effectively inducing humoral and cellular immunity in mice, producing high levels of antigen-specific binding antibodies and cytokines, and playing a role as an anti-coronavirus mRNA vaccine and an anti-tumor mRNA vaccine.

[0324] Example 3.6 Preparation and effects of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPP)

[0325] Example 3.6.1 Metal ion is Fe 3+ Preparation and effect of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPP)

[0326] Using the same method as in Example 3, three siRNA@MPPs containing different siRNAs were prepared, replacing the mRNA in Example 3 with siRNA. The target genes, sequences, and corresponding random control sequences of the three different siRNAs were as follows: (1) The sequences of the siRNA targeting the Bcl-2 gene (Bcl-2-siRNA) were SEQ ID No. 4 (antisense strand) and SEQ ID No. 21 (sense strand) (19 bp), and its random control sequences were SEQ ID No. 5 (antisense strand) and SEQ ID No. 22 (sense strand) (19 bp). (2) The sequences of the siRNA targeting the PLK1 gene (PLK1-siRNA) were SEQ ID No. 6 (antisense strand) and SEQ ID No. 23 (sense strand) (21 bp), and its random control sequences were SEQ ID No. 7 (antisense strand) and SEQ ID No. 24 (sense strand) (19 bp). (3) The sequence of siRNA targeting the Gal-1 gene (Gal-1-siRNA) is SEQ ID NO. 8 (19 bp), and its random control sequence is SEQ ID NO. 9 (19 bp). The remaining siRNA@MPP preparation process was the same as in Example 3.

[0327] The sequence of Bcl-2-siRNA is as follows:

[0328] Antisense: 5′-CAGCUUAUAAUGGAUGUAC-3′ (SEQ ID No. 4)

[0329] The sense sequence is 5'-GUACAUCCAUUAUAAGCUG-3' (SEQ ID No. 21) (19 bp).

[0330] The random control sequence of Bcl-2-siRNA is as follows:

[0331] Antisense: 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID No. 5)

[0332] The sense sequence is 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID No. 22) (19 bp).

[0333] The sequence of PLK1-siRNA is as follows:

[0334] Antisense: 5'-UAAGGAGGGUGAUCUUCUUCA-3' (SEQ ID No. 6)

[0335] The sense sequence is 5'-UGAAGAAGAUCACCCUCCUUA-3' (SEQ ID No. 23) (21 bp).

[0336] The random control sequence of PLK1-siRNA is as follows:

[0337] Antisense: 5'-CUUACGCUGAGUACUUCGA-3' (SEQ ID No. 7)

[0338] The sense sequence is 5'-UCGAAGUACUCAGCGUAAG-3' (SEQ ID No. 24) (19 bp).

[0339] The sequence of Gal-1-siRNA is as follows:

[0340] 5'-GCUGCCAGAUGGAUACGAA-3' (SEQ ID No. 8) (19bp).

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

[0342] 5'-GGAAAUCCCCAACAGUGA-3' (SEQ ID No. 9) (19bp).

[0343] For cell culture, U251 human brain glioblastoma cells were grown in monolayer in high-glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 m m l -glutamine (Bio Industries) medium at 37°C and 5% CO and passaged twice weekly.

[0344] 1 × 10 U251 cells per well 6 The cells were seeded into a 6-well plate at a cell density of 100x for approximately 24 hours. After incubating the cells in each well with siRNA@MPP containing the above siRNA (the siRNA concentration was 2 μg / mL) for 72 hours, the cells were collected, total cellular RNA was extracted, and the mRNA expression levels of the target genes (Bcl-2, PLK1, Gal-1) were detected using RT-PCR technology to determine the ability of the siRNA@MPP to silence the cellular target genes.

[0345] The specific process of RT-PCR is as follows:

[0346] To extract total RNA, discard the medium from the 6-well plate, rinse three times with PBS buffer, and add 1 mL of Trizol to each well to lyse the cells. Add 200 μL of chloroform, shake thoroughly, and incubate at room temperature for 10 minutes. Then, centrifuge at 13,000 rpm and 4°C for 15 minutes to obtain a three-phase liquid with the RNA dissolved in the upper aqueous phase. Aspirate the upper aqueous phase and place it in a new 1.5 mL centrifuge tube without enzymes. Add 500 μL of isopropyl alcohol, incubate at room temperature for 10 minutes, and centrifuge at 13,000 rpm and 4°C for 15 minutes to obtain the RNA precipitate. Remove the supernatant, add 1 mL of 75% (v / v) ethanol freshly prepared with RNase-free water to each tube, carefully blow off the white RNA precipitate at the bottom of the tube, and centrifuge at 7,500 rpm and 4°C for 10 minutes. Remove the supernatant and aspirate as much liquid as possible from the bottom of the tube. Open the lid, dry the RNA precipitate at the bottom of the tube at room temperature, add 50 µL of enzyme-free water to dissolve it, and detect the purity and concentration of the RNA using an ultra-microvolume UV-visible spectrophotometer.

[0347] cDNA reverse transcription was performed using Ta Ka Ra Prime Script TM The RT Reagent Kit and gDNA Eraser Kit are used to reverse transcribe RNA to cDNA, respectively. Removing genomic DNA (gDNA) before the reverse transcription step ensures more accurate and reliable results. Prepare the total RNA reverse transcription reaction on ice: 1 μL Prime Script RT Enzyme Mix I, 1 μL RT Primer Mix, 4 μL 5x Prime Script Buffer II, and 4 μL RNase-free dH2O. After preparing the reaction mixture, incubate at 37°C for 15 minutes, then heat at 85°C for 5 seconds to terminate the reaction. Store at 4°C for further use.

[0348] The RT-PCR procedure uses the SYBR Green dye method, which does not require a probe for detection. Specifically, real-time PCR reactions were performed using cDNA from different samples as templates. The reaction mixture was prepared on ice: 5 μL SYBR Premix Dimer Eraser (2x), 0.3 μL PCR Forward Primer (10 μM), 0.3 μL PCR Reverse Primer (10 μM), 0.2 μL ROX Reference Dye II (50x), 1 μL of the cDNA template obtained in the previous step, and 3.2 μL dH2O. The sample was added to a well plate at 10 μL per well. After sample loading was complete, the mixture was centrifuged (1000 rpm, 5 minutes) to remove any liquid residue and any air bubbles in the reaction mixture. The ABI ViiA7 real-time fluorescent quantitative PCR system was used for real-time PCR detection. The reaction procedure was as follows: 95°C, 30 seconds (1 cycle) → 95°C, 5 seconds, 55°C, 30 seconds, 72°C, 30 seconds (40 cycles) → 60°C - 95°C, 2 minutes (1 cycle). The experiment was repeated three times, and the average value was calculated to determine the Ct value for each group, and the fold difference in expression between the experimental and control groups was calculated. The control gene was GAPDH. The RT-PCR primers were as follows: (1) Bcl-2 primer: forward 5'-AGGATTGTGGCCTTCTTTGAG-3', reverse 5'-AGACAGCCAGGAGAAATC AAAC-3'; (2) PLK1 primer: forward 5'-ACCAGCACGTCGTAGGATTC-3', reverse 5'-CAAGCAATTTGCCGTAGG-3'; (3) Gal-1 primer: forward 5'-CAATCAT GGCCTGTGGTCTG-3', reverse 5'-GTG TAGGCACAGGTTGTTGCTG-3'; (4) GAPDH primer: forward 5'-TCAGGGGTTTCACATTTGGCA-3', reverse 5'-GG AGCGGAA AACCA-3'. The expression level of each target gene was measured using the RQ value (2 -ΔΔCT ) The formula is as follows: Fold Change = 2 - ΔΔCt In the formula, ΔΔCt = ΔCt 実験群 - ΔCt 対照群 , ΔCt = Ct 標的遺伝子 -Ct 内部参照遺伝子

[0349] The gene silencing efficiency was calculated as follows: 100% - gene expression level in the experimental group / gene expression level in the control group.

[0350] Analysis of the results, as shown in Figures 1-13, 1-14, and 1-15 (scr siRNA was a random control sequence), showed that all three siRNA@MPPs significantly inhibited their corresponding target genes. The Bcl-2-siRNA@MPP inhibited the target gene Bcl-2 by 76%, the PLK1-siRNA@MPP inhibited the target gene PLK1 by 86%, and the Gal-1-siRNA@MPP inhibited the target gene Gal-1 by 73%. These results suggest that siRNA@MPPs can deliver any siRNA for targeted gene intervention and can serve as siRNA-carrying drugs, vaccines, or other products.

[0351] Example 3.6.2 Metal ion is Al 3+ Preparation and effect of siRNA-loaded metal-chelated phospholipid complex nanoparticles (siRNA@MPP)

[0352] The difference between this example and Example 3.6.1 is that the metal ion Fe in Example 3.6.1 3+ Al 3+ is to be replaced by

[0353] Analysis of the results, as shown in Figures 1-16, 1-17, and 1-18, showed that all three siRNA@MPPs significantly inhibited their corresponding target genes. The Bcl-2-siRNA@MPP inhibited the target gene Bcl-2 by 81%, the PLK1-siRNA@MPP inhibited the target gene PLK1 by 90%, and the Gal-1-siRNA@MPP inhibited the target gene Gal-1 by 79%. These results suggest that siRNA@MPPs can deliver any siRNA for targeted gene intervention and can serve as siRNA-carrying drugs, vaccines, or other products.

[0354] Example 3.7 Preparation and effects of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP)

[0355] Example 3.7.1 Metal ion is Fe 3+ Preparation and effects of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP)

[0356] Three types of ASO@MPP containing different ASOs were prepared by replacing the mRNA in Example 3 with ASO and following the method of Example 3. The target genes, sequences, and corresponding random control sequences of the three different ASOs are as follows: (1) ASO targeting the STAT3 gene (STAT3-ASO) sequence is SEQ ID No. 10 (17 nt), and its random control sequence is SEQ ID No. 11 (18 nt); (2) ASO targeting the α-syn gene (α-syn-ASO) sequence is SEQ ID No. 12 (16 nt), and its random control sequence is SEQ ID No. 13 (16 nt); (3) ASO targeting the Bcl-2 gene (Bcl-2-ASO) sequence is SEQ ID No. 14 (18 nt), and its random control sequence is SEQ ID No. 15 (20 nt). The remaining ASO-metal-phospholipid complex particle preparation process was the same as in Example 3. Different ASO@MPPs were incubated with different cells. ASO@MPPs targeting the STAT3 gene were incubated with U2511 human brain glioblastoma cells, ASO@MPPs targeting the α-syn gene were incubated with SH-SY5Y human neuroblastoma cells, and ASO@MPPs targeting the Bcl-2 gene were incubated with Daudi human lymphoma cells. 1 × 10 per well. 6 After seeding the cells into a 6-well plate at a density of 0.1 μg / mL for approximately 24 hours, the cells in each well were incubated with the ASO@MPP containing the above ASO (the concentration of ASO was 1 μg / mL) for 48 hours, after which the cells were collected, total cellular RNA was extracted, and the mRNA expression levels of the target genes (STAT3, α-syn, Bcl-2) were detected using RT-PCR technology to calculate the ability of the ASO@MPP to silence the cellular target genes.

[0357] The sequence of SEQ ID No. 10 (the sequence of STAT3-ASO) is as follows:

[0358] 5'-GCTCCAGCATCTGCTTC-3' (17nt).

[0359] The sequence of SEQ ID No. 11 (random control sequence of STAT3-ASO) is as follows:

[0360] 5'-GAAGCAGCAGATGCTGGA-3'(18nt).

[0361] The sequence of SEQ ID No. 12 (the sequence of α-syn-ASO) is as follows:

[0362] 5'-GCTCCCTCCACTGTCT-3'(16nt).

[0363] The sequence of SEQ ID No. 13 (random control sequence of α-syn-ASO) is as follows:

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

[0365] The sequence of SEQ ID No. 14 (the sequence of Bcl-2-ASO) is as follows:

[0366] 5'-TCTCCCAGCGTGCGCCAT-3' (18nt).

[0367] The sequence of SEQ ID No. 15 (random control sequence of Bcl-2-ASO) is as follows:

[0368] 5'-CAGCGTGCGCCATCCTTCCC-3' (20nt).

[0369] Cell culture was as follows: (1) U251 human brain glioblastoma cells were grown in monolayers in high-glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (Bio Industries) at 37°C, 5% CO2, and passaged twice weekly. (2) SH-SY5Y human neuroblastoma cells were grown in monolayers in high-glucose (4.5 g / L) DMEM + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (Bio Industries) at 37°C, 5% CO2, and passaged twice weekly. (3) Daudi human lymphoma cells were grown in RPMI 1640 + 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mm l-glutamine (Bio Industries) medium at 37°C, 5% CO2, and passaged twice a week.

[0370] The specific process of RT-PCR is as follows:

[0371] To extract total RNA, discard the medium from the 6-well plate, rinse three times with PBS buffer, and add 1 mL of Trizol to each well to lyse the cells. Add 200 μL of chloroform, shake thoroughly, and incubate at room temperature for 10 minutes. Then, centrifuge at 13,000 rpm and 4°C for 15 minutes to obtain a three-phase liquid with the RNA dissolved in the upper aqueous phase. Aspirate the upper aqueous phase and place it in a new 1.5 mL centrifuge tube without enzymes. Add 500 μL of isopropyl alcohol, incubate at room temperature for 10 minutes, and centrifuge at 13,000 rpm and 4°C for 15 minutes to obtain the RNA precipitate. Remove the supernatant, add 1 mL of 75% (v / v) ethanol freshly prepared with RNase-free water to each tube, carefully blow off the white RNA precipitate at the bottom of the tube, and centrifuge at 7,500 rpm and 4°C for 10 minutes. Remove the supernatant and aspirate as much liquid as possible from the bottom of the tube. Open the lid, dry the RNA precipitate at the bottom of the tube at room temperature, add 50 µL of enzyme-free water to dissolve it, and detect the purity and concentration of the RNA using an ultra-microvolume UV-visible spectrophotometer.

[0372] cDNA reverse transcription was performed using Ta Ka Ra Prime Script TM Using the RT Reagent Kit with the gDNA Eraser Kit to reverse transcribe RNA to cDNA, respectively, removes genomic DNA (gDNA) prior to the reverse transcription step, resulting in more accurate and reliable results. Prepare the total RNA reverse transcription reaction on ice: 1 μL Prime Script RT Enzyme Mix I, 1 μL RT Primer Mix, 4 μL 5x Prime Script Buffer 2, and 4 μL RNase-free dH2O. After preparing the reaction mixture, incubate at 37°C for 15 minutes, then heat at 85°C for 5 seconds to terminate the reaction. Store at 4°C for further use.

[0373] The RT-PCR procedure uses the SYBR Green dye method, which does not require a probe for detection. Specifically, real-time PCR reactions were performed using cDNA from different samples as templates. The reaction mixture was prepared on ice: 5 μL SYBR Premix Dimer Eraser (2x), 0.3 μL PCR Forward Primer (10 μM), 0.3 μL PCR Reverse Primer (10 μM), 0.2 μL ROX Reference Dye II (50x), 1 μL of the cDNA template obtained in the previous step, and 3.2 μL dH2O. The sample was added to a well plate at 10 μL per well. After sample loading was complete, the mixture was centrifuged (1000 rpm, 5 minutes) to remove any liquid residue and any air bubbles in the reaction mixture. The ABI ViiA7 real-time fluorescent quantitative PCR system was used for real-time PCR detection. The reaction procedure was as follows: 95°C, 30 seconds (1 cycle) → 95°C, 5 seconds, 55°C, 30 seconds, 72°C, 30 seconds (40 cycles) → 60°C - 95°C, 2 minutes (1 cycle). The experiment was repeated three times, and the average value was used to calculate the Ct value for each group, and the fold difference in expression between the experimental and control groups was calculated. The control gene was GAPDH. The RT-PCR primer sequences are as follows: (1) STAT3 primer, forward 5'-TGATCACCTTTGAGACCGAGG-3', reverse 5'-GATCACCACAACTGG CAA GG-3'; (2) α-syn primer, forward 5'-TGACGGTGTGACAGCAGTAG-3', reverse 5'-CAGTGGCTGCTGCAATG-3'; (3) Bcl-2 primer, forward 5'-AGGATT GTG GCCTTCTTTGAG-3', reverse 5'-AGACAGCCAGGAGAAATCAAAC-3'; (4) GAPDH primer, forward 5'-TCAGGGG TTTCACATTTGGCA-3', reverse 5'-GGAGCGGAA AACCA-3'. The expression level of each target gene was calculated using the RQ value (2 -ΔΔCT ) The formula is as follows: Fold Change = 2 - ΔΔCt In the formula, ΔΔCt = ΔCt 実験群 - ΔCt 対照群 , ΔCt = Ct 標的遺伝子 -Ct 内部参照遺伝子

[0374] The gene silencing efficiency was calculated as follows: 100% - gene expression level in the experimental group / gene expression level in the control group.

[0375] Analysis of the results, as shown in Figures 1-19, 1-20, and 1-21 (scr ASO was a random control sequence), showed that all three ASO@MPPs significantly inhibited their corresponding target genes. The TAT3-ASO@MPP inhibited the target gene STAT3 by 75%, the α-syn-ASO@MPP inhibited the target gene α-syn by 71%, and the Bcl-2-ASO@MPP inhibited the target gene Bcl-2 by 66%. These results suggest that ASO@MPPs can deliver any ASO for targeted gene intervention and can serve as ASO-carried drugs, vaccines, or other products.

[0376] Example 3.7.2 Metal ion is Al 3+ Preparation and effects of ASO-loaded metal-chelated phospholipid complex nanoparticles (ASO@MPP)

[0377] The difference between this example and Example 3.7.1 is that the metal ion Fe in Example 3.7.2 3 Al 3+ It has been replaced by.

[0378] Analysis of the results, as shown in Figures 1-22, 1-23, and 1-24, showed that all three ASO@MPPs significantly inhibited their corresponding target genes. The STAT3-ASO@MPP inhibited the target gene STAT3 by 79%, the α-syn-ASO@MPP inhibited the target gene α-syn by 80%, and the Bcl-2-ASO@MPP inhibited the target gene Bcl-2 by 74%. These results suggest that ASO@MPPs can deliver any ASO for targeted gene intervention and can serve as ASO-carried drugs, vaccines, or other products.

[0379] Example 3.8 Preparation of drug (different types of nucleic acid)-metal-phospholipid complex particles and their effects

[0380] Example 3.8.1 Metal ion is Fe 3+ Preparation of drug (different kinds of nucleic acid)-metal-phospholipid complex particles and their effects when

[0381] The mRNA in Example 3 was replaced with double-stranded RNA (siRNA), single-stranded DNA (ASO), single-stranded RNA (mRNA), double-stranded DNA, and single-stranded DNA, respectively. The different nucleic acid sequences were as follows: (1) The double-stranded RNA (Bcl-2-siRNA) sequences were SEQ ID NO. 4 (antisense strand) and SEQ ID NO. 21 (sense strand) (19 bp), and its random control sequences were SEQ ID NO. 5 (antisense strand) and SEQ ID NO. 22 (sense strand) (19 bp). (2) The single-stranded DNA (STAT3-ASO) sequence was SEQ ID NO. 10 (17 nt), and its random control sequence was SEQ ID NO. 11 (18 nt). (3) The single-stranded RNA (mRNA encoding the wild-type novel coronavirus S protein) sequence was SEQ ID NO. 16 (3822 nt). (4) The double-stranded DNA (dsDNA) sequences were SEQ ID NO. 17 (antisense strand) and SEQ ID NO. 25 (sense strand) (22 bp) (the 3' end of the sequence was labeled with the fluorescent probe Cy3). (5) The single-stranded DNA (ssDNA) sequence was SEQ ID NO. 18 (22 nt) (the 3' end of the sequence was labeled with the fluorescent probe Cy3). Using the method of Example 3, the drug-metal-phospholipid complex particles encapsulating the above different types of nucleic acids (Bcl-2-siRNA@MPP, STAT3-ASO@MPP, S-mRNA@MPP, dsDNA@MPP, ssDNA@MPP) were prepared, respectively. The remaining drug-lipid particle preparation processes were the same as in Example 3.

[0382] The sequence of the double-stranded DNA is as follows:

[0383] Antisense: 5'-TAGCTTATCAGACTGATGTTGA-3' (SEQ ID No. 17)

[0384] The sequence is 5'-TCAACATCAGTCTGATAAGCTA-3' (SEQ ID No. 25) (22 bp).

[0385] The sequence of SEQ ID No. 18 (single-stranded DNA sequence) is as follows:

[0386] 5'-TCAACATCAGTCTGATAAGCTA-3' (22nt).

[0387] 1 × 10 U251 cells per well 5 Approximately 24 hours after seeding the cells into a 12-well plate at a density of 1000 x g / mL, the cells in each well were incubated with siRNA@MPP (siRNA concentration: 2 μg / mL) or ASO@MPP (ASO concentration: 2 μg / mL) for 72 hours, respectively. The cells were then harvested, total cellular RNA was extracted, and the mRNA expression of target genes (Bcl-2, STAT3) was detected using RT-PCR technology, respectively. The ability of siRNA@MPP or ASO@MPP to silence cellular target genes was calculated, and the results are shown in Figures 1-13 in Example 3.6 and 1-19 in Example 3.7.

[0388] 293T cells were incubated with S-mRNA @MPP at a concentration of 2 μg / mL (mRNA concentration), while the control group was incubated without MPP. After 24 hours, the supernatant was centrifuged and frozen at -20°C for later use. The cell pellet was resuspended in 100 μL of PBS buffer, frozen and thawed twice, sonicated for 10 minutes, and then centrifuged to remove the supernatant. The expression levels of S protein in both the cell supernatant and cell lysate were detected using a commercially available novel coronavirus S protein ELISA detection kit. The results are shown in Figures 1-25.

[0389] After incubating ds-DNA@MPP with A549 lung cancer cells at a concentration of 100 nM (concentration of DNA contained) for 2 hours, the remaining drug-lipid particles were removed and the cells were washed twice with PBS. After staining the cell nuclei with Hochest33342 dye for 3 minutes, the dye was removed and the cells were washed twice with PBS. The cells were then observed using a high-content imaging system and the transfection efficiency of the drug-lipid particles into DNA was calculated. The results are shown in Figures 1-26.

[0390] After incubating HT22 mouse hippocampal neurons with ss-DNA@MPP at a concentration of 200 nM (concentration of DNA) for 2 hours, the remaining drug-lipid particles were removed and the cells were washed twice with PBS. The cells were then observed using a high-content imaging system and the transfection efficiency of the drug-lipid particles into DNA was calculated. The results are shown in Figures 1-26.

[0391] The method for culturing human brain glioblastoma U251 cells was the same as in Example 3.6.

[0392] The method for culturing 293T cells was the same as in Example 3.

[0393] HT22 mouse hippocampal neurons are cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin under conditions of 37°C and 5% CO2.

[0394] The RT-PCR method was the same as in Example 3.6.

[0395] To detect the expression level of S protein by ELISA, replace the "anti-RBD antibody working solution" in the RBD detection method by ELISA in Example 3.5 with the "anti-S protein antibody working solution," and the remaining steps are the same as in Example 3.5.

[0396] The method for calculating gene silencing efficiency is the same as in Example 3.6.

[0397] The transfection efficiency was calculated by randomly selecting 3 to 5 fields using a high-content imaging system, observing cell morphology under a normal light source, and obtaining the fluorescent signals in the same fields when the excitation light / emission light was 550 nm / 570 nm (excitation light for the fluorescent dye Cy3 that labels DNA), and when the excitation light / emission light was 352 nm / 461 nm (excitation light for the fluorescent dye Hoechst33342 that labels cell nuclei). The transfection efficiency was calculated as the ratio of the number of cells with Cy3 fluorescent signals in the randomly selected fields to the number of cells with Hoechst33342 fluorescent signals in the same fields.

[0398] As shown in Figure 1-13 in Example 3.6.1, the drug (double-stranded RNA)-metal-phospholipid complex particles (Bcl-2-siRNA@MPP) inhibited the target gene Bcl-2 by 76%. As shown in Figure 1-19 in Example 3.7.1, the drug (single-stranded DNA)-metal-phospholipid complex particles (STAT3-ASO@MPP) inhibited the target gene STAT3 by 75%. As shown in Figure 1-25, 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 carrier MPP was 0. The drug (double-stranded DNA)-metal-phospholipid complex particles (dsDNA@MPP) transfected double-stranded DNA into cells with 100% efficiency (Figure 1-26). The transfection efficiency of single-stranded DNA in drug (ssDNA)-metal-phospholipid complex particles (ssDNA@MPP) into cells can be 100% (Figure 1-26). This result suggests that drug-metal-phospholipid complex particles can encapsulate any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and realize its function, with the length of the nucleic acid varying from 16 to 3822 nt.

[0399] Example 3.8.2 Metal ion is Al 3+Preparation of drug (different kinds of nucleic acid)-metal-phospholipid complex particles and their effects when

[0400] The difference between this example and Example 3.8.1 is that the metal ion Fe in Example 3.8.1 3+ Al 3+ is to be replaced by

[0401] As shown in Figure 1-16 in Example 3.6.2, the drug (double-stranded RNA)-metal-phospholipid complex particles (Bcl-2-siRNA@MPP) inhibited the target gene Bcl-2 by 81%. As shown in Figure 1-22 in Example 3.7.2, the drug (single-stranded DNA)-metal-phospholipid complex particles (STAT3-ASO@MPP) inhibited the target gene STAT3 by 79%. As shown in Figure 1-27, 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 carrier MPP was 0. The drug (double-stranded DNA)-metal-phospholipid complex particles (dsDNA@MPP) transfected double-stranded DNA into cells with 100% efficiency (Figure 1-28). The transfection efficiency of single-stranded DNA into cells using drug (ssDNA)-metal-phospholipid complex particles (ssDNA@MPP) was 100% (Figure 1-28). This result suggests that drug-metal-phospholipid complex particles can encapsulate any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA) and realize its function, with the length of the nucleic acid varying from 16 to 3822 nt.

[0402] Experimental Example 2: Performance Characterization of Drug-Metal-Phospholipid Complex Particles

[0403] Example 4 Synthesis and Characterization of Metal-Phospholipid Conjugates

[0404] Example 4.1 Metal ion is Fe 3+ Synthesis and characterization of metal-phospholipid complexes in the case

[0405] The method for characterizing the DSPC-curcumin bond was differential scanning calorimetry (DSC), with the following measurement conditions: 3-5 mg of sample was weighed, and the temperature was increased at a rate of 10°C / min over a temperature range of 30-300°C. Curcumin, DSPC, and the phospholipid complex were scanned separately. The resulting curves were plotted and shown in Figure 2-1. The spectrum indicated that curcumin possessed a lattice structure and exhibited a clear melting peak at 185°C. DSPC was a mixture with many dips in the curve, suggesting that different components may exhibit caloric changes under different temperature conditions. The phospholipid complex exhibited no peaks near the curcumin melting peak, forming a nearly linear line. This indicated that curcumin was bound to DSPC and existed in an amorphous form, proving the successful preparation of the phospholipid complex.

[0406] Phospholipid complexes and Fe 3+ The binding of CUR-HSPC to Fe was characterized by spectrophotometry. 3+ After binding with Fe, its maximum absorption wavelength shifted from 420 nm to 375 nm, and the conjugated structure of the phospholipid complex changed, resulting in the formation of Fe. 3+ has been shown to be successfully conjugated with curcumin.

[0407] Example 4.2 Metal ion is Al 3+ Synthesis and characterization of metal-phospholipid complexes in the case

[0408] The difference between this example and Example 4.1 is the phospholipid complex and Al 3+ The binding of Al was characterized by spectrophotometry. As shown in Figure 2-3, the phospholipid complex (CUR-HSPC) was 3+ After binding with Al, its maximum absorption wavelength shifted from 420 nm to 433 nm, and the conjugated structure of the phospholipid complex changed, resulting in Al 3+ has been shown to be successfully conjugated with curcumin.

[0409] Example 5 Metal ion is Fe 3+ If the Fe is released from the metal-phospholipid complex at low pH, 3+ Characterization

[0410] The phospholipid complex in the metal-phospholipid complex is linked to Fe by coordination bonding. 3+ and binds to phospholipid complexes and Fe under the low pH conditions of the lysosome. 3+ The coordination bond between the Fe and the metal is protonated (absorbs a hydrogen ion) and broken. 3+ To prove that Fe is actually released from the lipid complexes through the above mechanism, the following experiment was designed. The color of the metal-phospholipid complexes was observed under physiological pH (pH = 7.4) and lysosomal low pH (pH = 5.0) conditions. As shown in Figures 2-4, the metal-phospholipid complexes changed from reddish brown to bright yellow under lysosomal low pH (pH = 5.0), and Fe 3+ This result indicates that Fe is already released from the complex under the low pH conditions of the lysosome. 3+ This suggests that the metal-phospholipid complex may be shed.

[0411] Fe under low pH conditions 3+ The mechanism by which Fe is released from the metal-phospholipid complex is as follows. 3+ The coordinate bond between the two is protonated under low pH conditions (pH = 5.0), i.e., curcumin has many protons (H + ) and Fe 3+ The coordination bond between Fe and curcumin is broken, thus 3+ was isolated from curcumin and ultimately Fe 3+ is separated from the metal-phospholipid complex (Figure 2-4).

[0412] Example 6 Elemental analysis of MPP in drug-metal-phospholipid complex particles

[0413] Example 6.1 Metal ion is Fe 3+ Elemental analysis of MPP in drug-metal-phospholipid complex particles when

[0414] The mRNA in Example 3 was replaced with sulfhydryl-modified siRNA, and the drug-metal-phospholipid complex particle siRNA@MPP was prepared according to the method of Example 3. Elemental analysis was performed using a transmission electron microscope. The results are shown in Figures 2-5. C, N, O, and P are common elements, and the Fe elemental analysis diagram shows Fe 3+ The S elemental analysis diagram shows that the siRNA is uniformly distributed on the lipid nanoparticles. Because the siRNA is modified with sulfhydryl groups, the S elemental analysis diagram can specifically show the location of the siRNA. From this diagram, it can be seen that the siRNA is 3+ Conjugation was observed in the vicinity of the target site, demonstrating that the drug-lipid nanoparticles successfully encapsulated siRNA.

[0415] Example 6.2 Metal ion is Al 3+ Electron microscopic analysis of MPP in drug-metal-phospholipid complex particles when

[0416] Drug-metal-phospholipid complex particles (MPP) were prepared according to the method of Example 3. Morphological analysis was performed using a transmission electron microscope. The results are shown in Figures 2-6. The MPP in the drug-metal-phospholipid complex particles had a standard spherical shape, a uniform particle diameter of approximately 100 nm.

[0417] Example 7 Metal ion is Fe 3+ or Al 3+ Nucleic acid (siRNA and mRNA) encapsulation efficiency of metal-phospholipid complex particles MPP in the presence of phospholipids and its comparison with LNP

[0418] The mRNA in Example 3 was replaced with siRNA (SEQ ID No. 4, 19 bp) targeting the Bcl-2 gene and mRNA (SEQ ID No. 2, 669 nt) encoding the receptor binding domain (RBD) of the S1 subunit of the novel coronavirus, respectively, to prepare nucleic acid-encapsulated drug-metal-phospholipid complex particles siRNA@MPP and mRNA@MPP, respectively. The remaining manufacturing process of the drug-metal-phospholipid complex particles was the same as in Example 3.

[0419] siRNA@LNPs and mRNA@LNPs were prepared with the same drug loading as the siRNA@MPPs in Example 3.6 and mRNA@MPPs in Example 3.5. The specific method is as follows: An organic phase solution was prepared according to the Onpattro lipid nanoparticle formulation: the ionizable lipid ALC0315, DSPE-PEG2000, DSPC, and cholesterol were dissolved in ethanol in a molar ratio of 50%:1.5%:10%:38.5%. Bcl-siRNA or RBD-mRNA was added to the aqueous phase (0.1 M acetic acid-sodium acetate buffer, pH 4.0). The ratio of amino lipids to phosphate-containing nucleotides (N / P) was 6:1, ensuring that the nucleic acid drug loading was the same as that of the siRNA@MPPs and mRNA@MPPs described above. The aqueous and organic phases were rapidly mixed at a volume ratio of 3:1 at a flow rate of 14 mL / min. After mixing, the mixture was diluted 10-fold with enzyme-free PBS buffer and concentrated to 1 / 10 using a 100 kDa ultrafiltration tube. After repeating the dilution and concentration procedure three times, the ethanol concentration in the mixture was adjusted to 0.0005% or less, and the pH value of the solution was raised to the normal pH value of PBS buffer (7.2–7.4), to obtain siRNA@LNP and mRNA@LNP, respectively.

[0420] Agarose gel electrophoresis was used to measure the encapsulation rates of nucleic acids (siRNA and mRNA) in siRNA@MPP, mRNA@MPP, siRNA@LNP, and mRNA@LNP. The encapsulation rates were measured as follows: The input amount of nucleic acid (siRNA and mRNA) in each lipid nanoparticle group was 10 μg / mL, and the lipid:nucleic acid mass ratio was 40:1. The positive control group consisted of nucleic acid dissolved in PBS buffer, while the negative control was PBS buffer without nucleic acid. The agarose gel concentration was 1.5%, and the voids in the gel allowed only free nucleic acid to pass through, but not lipid nanoparticles. Electrophoresis was stopped once the free nucleic acid bands were clearly distinguishable to prevent degradation of the nucleic acid due to prolonged electrophoresis. Image J software was used to calculate the gray values of free nucleic acid for each group. The positive control group was set to 100%, and the ratio of free nucleic acid in each group to the positive control was the relative amount of free nucleic acid. The encapsulation rate for each group was calculated as (100 - relative amount of free nucleic acid)%.

[0421] The results were analyzed as shown in Figures 2-7. 3+ The encapsulation efficiency of siRNA and mRNA by MPP (Al 3+ The encapsulation efficiencies of siRNA and mRNA by MPP were 89.73% and 92.23%, respectively, and those of LNP were 84.98% and 79.12%, respectively. These results suggest that there is no significant difference in the encapsulation efficiency of nucleic acids between MPP and LNP.

[0422] Example 8 Metal ion is Fe 3+ or Al 3+ Lysosomal escape ability of metal-phospholipid complex particles (MPP) in the presence of phospholipids and comparison with LNP

[0423] The Bcl-2-siRNA (SEQ ID No. 4) in Example 3.6 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@MPP (the siRNA concentration was 100 nM). The Bcl-2-siRNA (SEQ ID No. 4) in Example 7 was replaced with Cy5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@LNP (the siRNA concentration was 100 nM). The eGFP-mRNA (SEQ ID No. 1) in Example 3.5 was replaced with Cy5-labeled eGFP-mRNA to prepare Cy5-mRNA@MPP (the mRNA concentration was 2 μg / mL). The RBD-mRNA in Example 7 was replaced with Cy5-labeled RBD-mRNA to prepare Cy5-mRNA@LNP (the mRNA concentration was 2 μg / mL). These were then used for the lysosomal probe Lysotracker. After incubating A549 cells with Lysotracker Green for 3 hours, a high-content imaging system was used to observe the overlap of Cy5 fluorescent signals (red) and Lysotracker Green fluorescent signals (green) to determine and explore the ability of the drug-lipid particles to promote lysosomal escape.

[0424] The ability of drug-metal-phospholipid complex particles to promote lysosomal escape of nucleic acids was determined by incubating cells with drug-lipid nanoparticles for 3 hours, then observing the overlap of the Cy5 fluorescent signal (red) and the Lysotracker Green fluorescent signal (green) using a high-content imaging system and calculating the overlap rate between the red and green fluorescent signals using ImageJ software. After incubating cells with drug-metal-phospholipid complex particles for 3 hours, the overlap rate between the red and green fluorescent signals was less than 50%, indicating that nucleic acids were able to escape from cellular lysosomes relatively quickly and that the lipid nanoparticles had a relatively good ability to promote lysosomal escape of nucleic acids.

[0425] The results were analyzed using Cy5-siRNA@MPP(Fe 3+ ) and Cy5-mRNA@MPP(Fe 3+After incubating A549 cells with Cy5-siRNA@MPP(Al) for 3 hours, the overlap rates of red and green fluorescent signals were 36.05% and 43.07%, respectively, i.e., the lysosomal escape rates were 63.95% and 56.93%, respectively. 3+ ) and Cy5-mRNA@MPP(Al 3+ After incubating A549 cells with Cy5-siRNA@LNP for 3 hours, the overlap rates of red and green fluorescent signals were 32.39% and 40.17%, respectively, i.e., the lysosomal escape rates were 67.61% and 59.83%, respectively. Meanwhile, after incubating A549 cells with Cy5-siRNA@LNP and Cy5-mRNA@LNP for 3 hours, the overlap rates of red and green fluorescent signals were 76.89% and 86.87%, respectively, i.e., the lysosomal escape capacities were 23.11% and 13.13%, respectively. The drug-lipid nanoparticle MPP has a relatively superior ability to promote nucleic acid lysosomal escape, suggesting that the ability of MPP to promote lysosomal escape is significantly stronger than that of LNP.

[0426] Example 9 Metal ion is Fe 3+ or Al 3+ Nucleic acid expression promoting ability of metal-phospholipid complex particles MPP in the presence of phospholipids and its comparison with LNP

[0427] The RBD-mRNA in Example 7 was replaced with mRNA encoding the fluorescent protein eGFP, and the remaining production methods were the same as in Example 7 to obtain eGFP-mRNA@LNP.

[0428] The eGFP-mRNA@MPP prepared in Example 3 and the above-mentioned eGFP-mRNA@LNP (containing 2 μg / mL of mRNA) 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.

[0429] The method for analyzing the eGFP-positive cell rate by flow cytometry is as described in Example 3.

[0430] The results were analyzed as shown in Figure 2-9. 3+ ), MPP(Al 3+ After treating 293T cells with MPP and LNP, the percentage of eGFP-positive cells was 97.2%, 98.1%, and 63.03%, respectively. This result suggests that the function of MPP in promoting lysosomal expression is superior to that of LNP. A possible reason for this is that, as described in Example 8, MPP's ability to promote nucleic acid escape from lysosomes is stronger than that of LNP, so that more nucleic acid loaded by MPP is effectively released into the cytoplasm and can be translated into protein.

[0431] Example 10 Metal ion is Fe 3+ or Al 3+ Ability of drug-metal-phospholipid complex particles MPP to stimulate humoral and cellular immunity when compared with LNP

[0432] 293T cells were incubated with the RBD-mRNA@MPP from Example 3.5 and the RBD-mRNA@LNP from Example 7 at a concentration of 2 μg / mL (mRNA concentration), while the control group was incubated with MPP. After 24 hours, the cells were centrifuged to collect the supernatant and frozen at -20°C for later use. The cell pellet was resuspended in 100 μL of PBS buffer, frozen and thawed twice, sonicated for 10 minutes, and centrifuged to collect the supernatant. The expression levels of RBD protein in both the cell supernatant and cell lysate were detected using a commercially available novel coronavirus antigen RBD ELISA detection kit. The results are shown in Figures 2-10.

[0433] The method for detecting RBD expression levels by ELISA is as described in Example 3.5.

[0434] The experimental animals were randomly divided into three groups (experimental group and control group), with five animals in each group. The animal model was BALB / c mice, and each mouse received the first intramuscular injection on day 1 and the second intramuscular injection on day 14. The experimental group received RBD-mRNA@MPP (Fe 3+ ), RBD-mRNA@MPP(Al 3+ ) or RBD-mRNA@LNP, respectively, and the control group was injected with MPP and LNP without mRNA loading. The administration volume per injection was 100 μL. 3+ ), RBD-mRNA@MPP(Al 3+ The RBD-mRNA@LNP formulations each contained 30 mg of mRNA. Blood samples were collected from the mice 28 days after the first administration, and serum samples were separated and gradient diluted. The titers of RBD total IgG antibodies against the S1 subunit of the novel coronavirus produced in the mice were detected using a commercially available ELISA kit. The results are shown in Figure 2-11.

[0435] The method for detecting the titer of RBD total IgG antibodies against the S1 subunit of the novel coronavirus by ELISA is as described in Example 3.5.

[0436] RBD-mRNA@MPP (Fe 3+ ), RBD-mRNA@MPP(Al 3+ On day 28 after administration of RBD-mRNA@LNP, spleens from normal mice were collected and prepared into single-cell suspensions under sterile conditions. 100,000 spleen cells / well were plated in a well plate. RBD protein was added to the LNP at a final concentration of 10 mg / mL. The cells were then cultured for 48 hours. The supernatant was removed by centrifugation, and the expression levels of IFN-γ, IL-2, and IL-4 were measured using ELISA kits. The results are shown in Figure 2-12.

[0437] The method for detecting the expression levels of IFN-γ, IL-2, and IL-4 by ELISA is as described in Example 3.5. The results were analyzed using RBD-mRNA@MPP(Fe3+ ), RBD-mRNA@MPP(Al 3+ Both RBD-mRNA@MPP (Al) and RBD-mRNA@LNP can induce a certain level of RBD expression in 293T cells. 3+ The ability of RBD-mRNA@MPP(Fe) to induce RBD expression in cells was 3+ ) and RBD-mRNA@MPP(Fe 3+ ) has a significantly stronger ability to induce RBD expression in cells than RBD-mRNA@LNP. 3+ The expression level of RBD in the cell supernatant of the )-treated group was 205ng / mL, and the RBD-mRNA@MPP(Al 3+ The expression level of RBD in the cell supernatant of the RBD-mRNA@MPP (Al)-treated group was 230 ng / mL, while the expression level of RBD in the cell supernatant of the RBD-mRNA@LNP-treated group was 115.7 ng / mL. As shown in Figure 2-11, RBD-mRNA@MPP effectively induced humoral immunity in mice, producing high levels of antigen-specific binding antibodies. 3+ ) ability of RBD-mRNA@MPP(Fe 3+ ) and showed a clear advantage over RBD-mRNA@MPP(Fe 3+ ) is clearly superior to RBD-mRNA@LNP. 3+ The IgG antibody titer in mice treated with RBD-mRNA@MPP(Al 3+ The IgG antibody titer in the mice treated with RBD-mRNA@MPP(Al) reached 133116, while the IgG antibody titer in the mice treated with RBD-mRNA@LNP was only 67476. As shown in Figure 2-12, 3+ ) can effectively induce cellular immunity in mice, that is, activate immune cells to produce a large amount of cytokines, and induce cellular immunity in mice. 3+ ) ability of RBD-mRNA@MPP(Fe 3+ ) and showed a clear advantage over RBD-mRNA@MPP(Fe3+ ) ability is clearly superior to that of RBD-mRNA@LNP. RBD-mRNA@MPP(Fe 3+ ) induced the expression levels of cytokines IFN-γ, IL-2, and IL-4 to reach 256.8 pg / mL, 207.6 pg / mL, and 61.8 pg / mL, respectively. 3+ ) induced the expression levels of cytokines IFN-γ, IL-2, and IL-4 to reach 298 pg / mL, 249 pg / mL, and 74.6 pg / mL, respectively. On the other hand, the expression levels of cytokines IFN-γ, IL-2, and IL-4 were only 104.2 pg / mL, 79.2 pg / mL, and 27 pg / mL, respectively, with RBD-mRNA@LNP. This result is in line with mRNA@MPP(Al 3+ ) delivers any mRNA and realizes its function. 3+ ) and RBD-mRNA@MPP(Fe 3+ These results suggest that the ability of RBD-mRNA@MPP to induce cellular immunity in mice is significantly superior to that of RBD-mRNA@LNP. RBD-mRNA@MPP can more effectively promote the expression of target proteins in cells and more effectively activate humoral and cellular immunity in the body, making the drug (mRNA)-lipid particles significantly superior to conventional LNPs in the role of mRNA-carrying drugs, vaccines, or other products. Possible reasons for this include: 1) MPPs have a stronger ability to promote lysosomal escape of nucleic acids compared to LNPs. 2) MPPs have a stronger ability to promote the expression of nucleic acids into proteins (antigens) compared to LNPs. 3) Compared to LNPs, curcumin in MPPs, after being separated from DSPC in the body, can function as an immune adjuvant (also known as an immunomodulator) to activate humoral and cellular immunity, thereby not only enhancing the delivery effect of MPP mRNA vaccines, but also suppressing the storm of immune factors, thereby suppressing excessive and harmful immune responses against organisms.

[0438] Example 11 Metal ion is Fe 3+ or Al 3+In vivo safety evaluation of metal-chelated phospholipid complex nanoparticles (MPPs) when

[0439] A 20-day subchronic toxicity test of MPP was conducted on SD rats, followed by a 20-day recovery period. The specific experimental method is as follows:

[0440] Fifty-six SPF-grade SD rats (220 ± 20 g), half male and half female, were housed at 25°C, 45%–55% humidity, and 12-hour light exposure. After 3–5 days of acclimation, they were randomly assigned to groups according to sex: 32 experimental rats and 24 recovery rats. Fourteen rats (eight experimental rats and six recovery rats) were assigned to the blank control group (control). Fourteen rats (eight experimental rats and six recovery rats) were assigned to the low-dose MPP group (8 mg / kg), half male and half female. Fourteen rats (eight experimental rats and six recovery rats) were assigned to the medium-dose MPP group (16 mg / kg), half male and half female. Fourteen rats (eight experimental rats and six recovery rats) were assigned to the high-dose group (32 mg / kg), half male and half female. The experimental group (32 animals in total) was dissected and sampled after administration was completed, and the recovery group (24 animals in total) was kept in normal care for 20 days after administration was completed, and then dissected and sampled.

[0441] The experimental animals were administered the compound via tail vein injection every two days for a total of 20 days, and the SD rats' weights were recorded weekly. The prepared MPP was dissolved in DPBS, and the control group was injected with the same amount of DPBS. 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.

[0442] The basis for the MPP dosage setting above is that when 200 μg / kg of mRNA (the amount actually required in mRNA animal experiments) is contained, the required amount of blank MPP is 8 mg / kg. To fully demonstrate the safety of MPP, doses 1, 2, and 4 times the actual dose required in animal experiments, i.e., 8 mg / kg, 16 mg / kg, and 32 mg / kg, were selected and administered.

[0443] The general indicator detection method involves observing the general condition of each group of animals after each administration, including their survival status, feeding status, external characteristics, behavioral activity, body weight, and whether or not there is any local reaction to the administration. During autopsy, a macroscopic autopsy is performed, including timely weighing of the wet weights of major organs such as the brain, heart, liver, spleen, lungs, and kidneys, calculating the organ-to-body ratio, and recording pathological changes in each organ. Organ-to-body ratio = rat organ wet weight / rat body weight × 100%.

[0444] After 20 days of treatment and a 20-day recovery period, SD rats were anesthetized with isoflurane, fixed to a dissection board, and their abdomens were disinfected with 75% ethanol. The rat's abdomen was incised with sterile ophthalmic scissors, and the internal organs were gently pried open using a cotton ball to expose the abdominal aorta. Whole blood was collected using a 500 μL negative-pressure EDTAK2 anticoagulant blood collection tube and stored at 4°C for routine blood testing. Whole blood was collected using a 5 mL negative-pressure standard blood collection tube, allowed to stand at room temperature for 30 minutes, and centrifuged at 1500 rpm for 15 minutes at 4°C. The supernatant was transferred to a 1.5 mL centrifuge tube and stored at -20°C for the detection of blood biochemistry and immunological indices.

[0445] Routine blood testing methods include white blood cell count, lymphocyte count, monocyte count, neutrophil count, lymphocyte percentage, monocyte percentage, neutrophil percentage, red blood cell count, hemoglobin, hematocrit, mean red blood cell volume, mean red blood cell hemoglobin content, mean red blood cell hemoglobin concentration, red blood cell distribution width coefficient of variation, platelet count, mean platelet volume, platelet distribution width, and plateletcrit. A small amount of whole blood is collected by gently inverting and mixing the whole blood sample, and the results are automatically analyzed using a fully automated blood cell analyzer.

[0446] The blood biochemical index detection method is as follows: 2+ , Na + , K. + , Cl - , Ca 2+ ), liver function indicators (ALT, AST, γ-GT, T-BIL, D-BIL, ALP, ALB), kidney function indicators (BUN, UA, CR), cardiac function indicators (LDH, CK), glucose metabolism indicators (GSP, GLU, INS), and lipid metabolism indicators (CHO, TG, LDL-C, HDL-C). Serum samples are thawed and centrifuged at 3000 rpm for 15 minutes, the supernatant is collected, and aliquoted for later use. The corresponding parameters are set on the automated biochemistry instrument, the formulated working fluid is added, followed by the serum to be tested, and the results are automatically measured by the automated biochemistry instrument.

[0447] Immunological indicators include thyroid function indicators (TT3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN-γ, IFN-α, TNF-α), immunoglobulins (IgG, IgA, IgM), and serum complements (C3, CH50). ELISA is used to detect the above indicators.

[0448] The pathological examination method for the major organs of SD rats was as follows: at the end of the administration period and the end of the recovery period, rats in each group were anesthetized, and then the major organs of the rats, including the whole brain, heart, liver, spleen, lungs, and kidneys, were removed with ophthalmic scissors, rinsed lightly with 0.9% saline, fixed in 4% paraformaldehyde fixative, embedded in standard paraffin, and stained with H&E. Histopathological changes in each organ of the rats in the control and experimental groups were observed under an optical microscope.

[0449] As shown in Table 2-1, the results of the analysis showed that the low, medium, and high doses of MPP (Fe 3+ ) or MPP(Al 3+The rats in the 2000-MPP group survived well, showed normal feeding, normal appearance, and normal behavioral activity, and showed no obvious adverse reactions after administration. Compared with the control group, there was no significant difference in body weight gain in male and female SD rats in the low-, medium-, and high-dose MPP groups. Compared with the control group, there was no significant difference in organ-to-body ratio in the low-, medium-, and high-dose MPP groups.

[0450] At the end of the administration period and the end of the recovery period, the low, medium and high doses of MPP(Fe 3+ ) or MPP(Al 3+ No abnormalities were found in routine blood indices (white blood cell count, lymphocyte count, monocyte count, neutrophil count, lymphocyte percentage, monocyte percentage, neutrophil percentage, red blood cell count, hemoglobin, hematocrit, mean red blood cell volume, mean red blood cell hemoglobin content, mean red blood cell hemoglobin concentration, red blood cell distribution width coefficient of variation, platelet count, mean platelet volume, platelet distribution width, plateletcrit) in the low, medium, and high dose MPP groups compared with the control group. 2+ , Na + , K. + , Cl - , Ca 2+ No abnormalities were found in blood biochemistry indices, including liver function indices (ALT, AST, γ-GT, T-BIL, D-BIL, ALP, ALB), kidney function indices (BUN, UA, CR), cardiac function indices (LDH, CK), glucose metabolism indices (GSP, GLU, INS), and lipid metabolism indices (CHO, TG, LDL-C, HDL-C).Compared to the control group, no abnormalities were found in immunology-related indices, including thyroid function indices (TT3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN-γ, IFN-α, TNF-α), immunoglobulins (IgG, IgA, IgM), and serum complements (C3, CH50), in the low, medium, and high-dose MPP groups.

[0451] At the end of the administration period and the end of the recovery period, the low, medium and high doses of MPP(Fe 3+ ) or MPP(Al 3+) group, the brain tissue structure of the rats was completely organized, the tissue staining was normal, the cell morphology and structure were completely organized, and there was no nuclear pyknosis or inflammatory cell infiltration. The myocardial tissue structure was completely organized, the myocardial cells were neatly, continuously and firmly arranged, the cell nuclei were clearly visible, and there was no obvious cell congestion, edema or necrosis. The hepatic cell morphology was normal, and there was no inflammatory cell aggregation or necrosis. The spleen structure was normal, and there was a clear boundary between the red pulp and the white pulp. The lung tissue structure was completely organized, the alveoli were consistent in size, and there was no obvious inflammatory cell aggregation or infiltration. The kidney structure was normal.

[0452] The above results show that MPP(Fe 3+ ) or MPP(Al 3+ This suggests that no obvious chronic toxic reactions were found in long-term, large-dose injections of MPP, suggesting that the safety of MPP is relatively high. [Table 9] JPEG2025527283000039.jpg233153JPEG2025527283000040.jpg107150Note: ALT alanine aminotransferase, AST glutamic oxaloacetic transaminase, γ-GT glutamyl transpeptidase, T-BIL total bilirubin, D-BIL direct bilirubin, ALP alkaline phosphatase, ALB albumin, BUN urea nitrogen, UA uric acid, CR creatinine, LDH lactate dehydrogenase, CK creatine phosphokinase, GSP glycosylated serum protein, GLU glucose, INS insulin, CHO cholesterol, TG triglyceride, LDL-C low-density lipoprotein, HDL-C high-density lipoprotein, TT3 triiodothyronine, TT4 tetraiodothyronine, TSH thyroid-stimulating hormone, IL-1 Interleukin 1, IL-2 Interleukin 2, IL-4 Interleukin 4, IFN-γ Interferon gamma, 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

[0453] Example 12 Metal ion is Fe 3+ or Al 3+ Comparison of the in vivo safety of metal-phospholipid complex particles (MPP) and LNP when

[0454] The main toxicity of LNPs comes from their main components, cationic lipids and / or ionizable lipids. During metabolism of LNPs in the body, free cationic lipids and / or ionizable lipids produce obvious toxicity to organisms. The median lethal dose (IC50) of cationic lipids and / or ionizable lipids to living cells is 50) is an important parameter for evaluating the degree of toxicity of LNP to organisms. Metal-chelated phospholipid complex nanoparticles (MPPs) are made by replacing the cationic lipids / ionizable lipids in LNPs with metal-phospholipid complexes, and therefore the median lethal dose (IC50) of metal-phospholipid complexes and cationic lipids / ionizable lipids to living cells is significantly higher than that of MPPs. 50 ) and LNP and MPP (Fe 3+ ) or MPP(Al 3+ ) to compare the differences in toxicity.

[0455] The metal-phospholipid complexes (0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM), cationic lipids (DOTAP, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7 μM; structural formula: ...) were incubated with 293T cells for 48 hours. The cell viability was then detected using a CCK 50 Calculate each.

[0456] [ka]

[0457] [ka]

[0458] The method for detecting CCK8 is as follows:

[0459] 1. Culture the cells in DMEM medium containing 10% FBS and 1% double antibody until the cell density reaches 80-90% of the culture flask, then store for use.

[0460] 2. Wash the remaining medium in the culture flask with PBS, add pancreatin, and quickly transfer the culture flask to a 37°C incubator containing 5% CO2. Carefully observe the cells and, when they become slightly rounded, add culture medium to terminate the digestion. Transfer the cells to a centrifuge tube, centrifuge at 1500 RPM for 5 minutes, and resuspend the cells in fresh medium.

[0461] 3. Count the cells according to your needs by diluting the cell suspension to 10,000 cells per mL, 100 μL per well of a 96-well plate, and at least 5 duplicate wells per group. Incubate at 37°C, 5% CO2 for 24 hours before adding the drug.

[0462] 4. After incubating the drug for 48 hours, add 10% CCK8, incubate for 1-3 hours, and measure the absorbance at 450 nm using a microplate reader.

[0463] 5. Viability (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] × 100%.

[0464] I C 50 The IC was calculated using Graphpad with the [Inhibitor] vs. normalized response -- Variable slope analysis method, with survival rate on the vertical axis and drug concentration on the horizontal axis. 50 Calculate.

[0465] To compare the in vivo safety of MPP and LNP, MPP (8 mg / kg) and LNP (3.24 mg / kg) were used, each capable of carrying the same amount of nucleic acid (200 μg / kg mRNA). In vivo experiments were performed according to the method described in Example 11 to evaluate and compare the in vivo toxicity of MPP and LNP.

[0466] The results were analyzed and shown in Table 2-2. 50 is significantly greater than that of the cationic lipid (DOTAP) and the ionizable lipid (ALC0315). This result indicates that the toxicity of the metal-phospholipid complex is significantly lower than that of the cationic lipid and the ionizable lipid.

[0467] As shown in Tables 2 and 3, at the end of the administration period and the end of the recovery period, MPP (Fe 3+ ) or MPP(Al 3+ ) group showed no obvious abnormalities in liver function (ALT, AST, ALP) and cytokines (IL-6, IL-1β). However, compared with the control group, liver function (ALT, AST, ALP) and cytokines (IL-6, IL-1β) in the LNP group were clearly increased. This result is consistent with the MPP (Fe 3+ ) or MPP(Al 3+ ) is suggested to have a higher in vivo safety than LNP. The reasons are as follows: The core component of LNP is an artificially synthesized "cationic lipid / ionizable lipid" that has relatively high cytotoxicity and immunogenicity, is relatively stable in structure, and is not easily degraded or metabolized in the body, whereas MPP (Fe 3+ ) or MPP(Al 3+ The core component of MPP (Fe) is a metal-phospholipid complex, which is composed of phospholipid molecules, the highly safe natural small molecule substance curcumin (an FDA-approved food additive and pharmaceutical excipient), and safe metal ions, and is decomposed into natural molecules in the body after drug delivery is complete. 3+ ) or MPP(Al 3+) does not contain cationic lipids / ionizable lipids, so it does not cause toxic side reactions associated with cationic lipids / ionizable lipids. 3+ ) or MPP(Al 3+ ) is safer than LNP. [Table 10] [Table 11]

[0468] Example 3 Clinical Applications and Administration Routes of Drug-Metal-Phospholipid Complex Particles

[0469] Example 13 Metal ion is Fe 3+ or Al 3+ Clinical application and administration route of drug-metal-phospholipid complex particles in the case of

[0470] The mRNA in Example 3 was replaced with siRNA targeting the B7-H4 gene (B7-H4-siRNA) and its control (scr-siRNA), as well as mRNA encoding the receptor binding domain (RBD) of the S1 subunit of the novel coronavirus (RBD-mRNA).

[0471] The sequences of the above different nucleic acids are as follows: (1) The sequences of B7-H4-siRNA are SEQ ID No. 19 (sense strand) and SEQ ID No. 26 (antisense strand) (25 bp), and its random control sequence is SEQ ID No. 20 (sense strand) and SEQ ID No. 27 (antisense strand) (19 bp). (2) The mRNA sequence encoding the receptor binding domain (RBD) of the S1 subunit of the novel coronavirus is SEQ ID No. 2 (669 nt). Referring to the method of Example 3, the drug-metal-phospholipid complex particles (B7-H4-siRNA @MPP(Fe 3+), RBD-mRNA@MPP(Fe 3+ ), B7-H4-siRNA @MPP (Al 3+ ), RBD-mRNA@MPP(Al 3+ ) were prepared, and the remaining drug-metal-phospholipid complex particle preparation processes were the same as in Example 3. The above two different drug-metal-phospholipid complex particles (B7-H4-siRNA@MPP, RBD-mRNA@MPP) are used for the treatment of liver cancer and as an mRNA vaccine to prevent the new coronavirus, respectively.

[0472] The sequence of B7-H4-siRNA is as follows:

[0473] sense 5'-GGG AGA CAC UCC AUC ACA GUC ACU A -3'(SEQ ID No.19).

[0474] antisense 5'-UAG UGA CUG UGA UGG AGU GUC UCC C-3'(SEQ ID No.26)(25bp).

[0475] The random control sequence of B7-H4-siRNA is as follows:

[0476] sense 5'-UUCUCCGAACGUGUCACGU-3'(SEQ ID No.20).

[0477] antisense 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID No. 27) (19bp).

[0478] B7-H4-siRNA@MPP(Fe) in the treatment of liver cancer 3+ ) and B7-H4-siRNA@MPP(Al 3+ To evaluate the efficacy of chemoattractant therapy, an animal model of liver cancer was created using HepG2 cells, with tumors measuring approximately 100 mm 3 When the concentration of ATP increased to 1000kJ / kg, the liver cancer mice were treated with PBS control group, blank carrier MPP (Fe 3+) group, blank support 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+ The mice were randomly divided into seven treatment groups (five mice per group). Mice in each group were treated every three days 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+ ) was injected intratumorally once, followed by eight injections at a dose of 200 μg siRNA / kg. Tumor volume was measured and recorded every three days. The results are shown in Figure 3-1.

[0479] To evaluate the role of RBD-mRNA@MPP as an mRNA vaccine in preventing novel coronavirus, the experimental process and methods were as described in Example 3.5 above.

[0480] The ELISA detection method is as described in Example 3.5.

[0481] Construction of liver cancer mouse model: Collect 1 x 10 HepG2 cells. 7 The cells were resuspended in PBS at a density of 100 µL / mL and stored on ice before inoculation. 100 µL of the cell suspension was then subcutaneously injected into the back area near the hind limb of female Balb / c nude mice to establish a liver cancer mouse model.

[0482] The result analysis is

[0483] As shown in Figure 3-1, Scr-siRNA@MPP(Fe 3+ ), Scr-siRNA@MPP(Al 3+) had little inhibitory effect on the growth of liver cancer HepG2 cells, whereas B7-H4-siRNA@MPP(Fe 3+ ) and B7-H4 siRNA@MPP(Al 3+ ) showed an efficient therapeutic effect and could effectively inhibit the growth of liver cancer tumors. These results suggest that the drug-metal-phospholipid complex particles can encapsulate and deliver B7-H4 siRNA, inhibiting the expression of the target gene and thereby inhibiting the progression of liver cancer.

[0484] As shown in Figures 1-3 and 1-5 in the previous Example 3.5, RBD-mRNA@MPP(Fe 3+ ) induced the expression level of mouse IgG antibody to reach 117268.8 pg / mL (Figure 1-3), and the expression levels of cytokines IFN-γ, IL-2, and IL-4 to reach 252.8 pg / mL, 207.6 pg / mL, and 56.6 pg / mL, respectively (Figure 1-5). 3+ ) induced mouse IgG antibody expression levels of 129113 (Figure 1-9), and cytokine expression levels of 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). These results suggest that RBD-mRNA@MPP can effectively induce humoral immunity in mice, producing high levels of antigen-specific binding antibodies, while also effectively inducing cellular immunity in mice, i.e., activating immune cells to produce large amounts of cytokines. Therefore, RBD-mRNA@MPP can effectively prevent novel coronavirus infection.

[0485] As shown in Figure 3-1, B7-H4-siRNA@MPP can effectively treat liver cancer using intratumoral injection. As shown in Figures 1-3, 1-5, 1-9, and 1-11 in Example 3.5, RBD-mRNA@MPP can activate humoral and cellular immunity via intramuscular injection, thereby preventing COVID-19 infection. These results suggest that drug-metal-phospholipid complex particles can be administered via multiple routes.

[0486] Example 4 DSPC, curcumin, Fe 3+ or Al 3+ Function after replacement by its analogue

[0487] Example 15 DSPC, curcumin, Fe 3+ or Al 3+ Function after replacement by its analogue

[0488] With reference to Examples 1, 2 and 3, DSPC, curcumin, Fe 3+ DSPC, curcumin, and Fe, respectively. 3+ The mRNA concentration of each eGFP-mRNA@MPP was 2 μg / mL. 3+ The names and structures of the compounds and their analogues are shown in Table 4-1. 3+ The combination methods of the compounds and their analogues are shown in Table 4-2. In Example 1, the reaction temperature was 65°C and the reaction time was 2 hours, and in Example 2, the reaction temperature was 60°C and the reaction time was 2 hours, and the other conditions were the same.

[0489] To compare the effects of the 28 different eGFP-mRNA@MPPs and eGFP-mRNA@LNPs, LNPs encapsulating the same amount of eGFP-mRNA were prepared with reference to Example 9 to obtain eGFP-mRNA@LNPs.

[0490] The 28 different eGFP-mRNA@MPPs and the eGFP-mRNA@LNPs (each containing 2 μg / mL of mRNA) were incubated with 293T cells, respectively. Control groups were incubated with MPPs or LNPs. After 48 hours, the cell suspensions were collected and the percentage of eGFP-positive cells was detected by flow cytometry.

[0491] The method for analyzing the rate of eGFP-positive cells by flow cytometry is as described in Example 3.

[0492] The main toxicity of LNP comes from its main components, cationic lipids / ionizable lipids. During metabolism of LNP in the body, free cationic lipids / ionizable lipids cause obvious toxicity to organisms. The median lethal dose (IC50) of cationic lipids / ionizable lipids to living cells is 50 ) is an important parameter for evaluating the degree of toxicity of LNP to organisms. Metal-chelated phospholipid complex nanoparticles (MPPs) are produced by replacing the cationic lipids / ionizable lipids in LNPs with metal-phospholipid complexes. Therefore, the median lethal dose (IC50) of the 28 metal-phospholipid complexes and cationic lipid (DOTAP) / ionizable lipid (ALC0315) in Table 4-2 to living cells was calculated. 50 ) to compare the differences in toxicity between LNP and 28 types of MPP.

[0493] The method for calculating IC50 is as described in Example 12.

[0494] As shown in Table 4-3, after treating 293T cells with 28 types of eGFP-mRNA@MPP, the percentage of eGFP-positive cells was significantly higher than that of eGFP-mRNA@LNP. 3+ The percentage of eGFP-positive cells was highest in the mRNA@MPP composed of DSPC, curcumin, and Fe. 3+ The function of mRNA@MPP formed after replacing with their analogues was confirmed by DSPC, curcumin, and Fe. 3+ This suggests that the function of the MPP is inferior to that of the mRNA@MPP composed of α- and β-actin, but superior to that of the mRNA@LNP. A possible reason for this is that, as described in Example 8, the ability of the MPP to promote lysosomal escape of nucleic acids is stronger than that of the LNP, so that more nucleic acids loaded by the MPP are effectively released into the cytoplasm and can be translated into proteins.

[0495] The above results suggest that DSPC, curcumin, and Fe can be effectively used as long as the following conditions are met: 3+ These results suggest that the function of the drug-metal-phospholipid complex particles formed after substitution of Fe with its analogues is not affected. (1) Analogues of DSPC are amphiphilic phospholipid molecules. (2) Fe 3+ (3) Curcumin analogues can form phospholipid complexes with DSPC analogues and simultaneously conjugate with metal ions. (4) Curcumin and Fe 3+ The coordination bond between can be cleaved in response to the low pH environment of the lysosome.

[0496] As shown in Table 4-3, the IC values of 28 metal-phospholipid complexes 50 The toxicity of the metal-phospholipid complexes was significantly lower than that of the cationic lipids (DOTAP) and the ionizable lipids (ALC0315). 3+ It has been suggested that lipid nanoparticles (MPPs) composed of DSPC and their analogues are safer than LNPs. The reason for this is that the core component of LNPs is an artificially synthesized "cationic lipid / ionizable lipid" that has relatively high cytotoxicity and immunogenicity, a relatively stable structure, and is not easily degraded or metabolized in the body, whereas the core component of MPPs is a metal-phospholipid complex, which is composed of phospholipid molecules, a highly safe natural small molecule substance (curcumin is an FDA-approved food additive and pharmaceutical excipient), and a safe metal ion, and is already degraded into natural molecules in the body after drug delivery is complete. Therefore, the metal-phospholipid complexes composed of DSPC, curcumin, Fe, and their analogues are more stable and are more easily degraded in the body. 3+ The components of lipid particles (MPPs) composed of these and their analogues do not contain cationic lipids / ionizable lipids, and therefore do not cause toxic side reactions associated with cationic lipids / ionizable lipids, making MPPs safer than LNPs. [Table 12] JPEG2025527283000046.jpg119170 [Table 13] JPEG2025527283000048.jpg233150JPEG2025527283000049.jpg212152 [Table 14] JPEG2025527283000051.jpg234153JPEG2025527283000052.jpg212152

[0497] Example 16 Different metal-phospholipid complexes of DSPC, curcumin and their congeners, Fe 3+ The component ratio and the function of the drug-metal-phospholipid complex particles produced therefrom

[0498] Metal-phospholipid complexes were prepared according to Example 3, with curcumin replaced by its analog, hesperetin (one molecule of hesperetin contains four hydroxyl groups), or tea polyphenol (one molecule of tea polyphenol contains eight hydroxyl groups). Three metal-phospholipid complexes (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP29) were prepared. The input ratios of DSPC, curcumin or its analog, and FeCl3 were 1:1:1, 1:1:1, and 1:1:2, respectively. These three metal-phospholipid complexes (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP29) were used to prepare the corresponding drug-metal-phospholipid complex nanoparticles. The mRNA was encoding eGFP fluorescent protein, and its sequence is SEQ ID No. 1 (720 nt). The experimental procedures and methods described in Example 3.5 were followed to detect the mRNA encapsulation rates of the four drug-lipid nanoparticles and their ability to promote the expression of eGFP fluorescent protein after treatment of 293T cells.

[0499] Analysis of the results, as shown in Table 4-4, showed that the mRNA encapsulation efficiency and target protein expression promotion ability of drug-metal-phospholipid complex particles prepared at different input ratios based on the chemical structure of the metal-phospholipid complex components were comparable. This suggests that the input ratio of the metal-phospholipid complex components can be adjusted depending on the specific structure of the metal-phospholipid complex components. The rationale for this adjustment is as follows: Because the DSPC analog and the curcumin analog are connected by hydrogen bonds, as long as the DSPC analog contains multiple phosphate groups, the input ratio of the DSPC analog to the curcumin analog during phospholipid complex synthesis can be adjusted based on the number of phosphate groups contained in the DSPC analog. That is, if the DSPC analog contains two phosphate groups, the input ratio of the DSPC analog to the curcumin analog can be adjusted to 1:2. If the DSPC analog contains three phosphate groups, the input ratio of the DSPC analog to the curcumin analog can be adjusted to 1:3. Hydroxyl groups and Fe in curcumin congeners 3+ Since the congeners are linked by coordinate bonds, as long as the congeners contain multiple binding sites, the congeners of curcumin and Fe 3+ The input ratio of the congeners can be adjusted based on the number of binding sites contained in the congener of curcumin. [Table 15]

[0500] Example 17 Different metal-phospholipid complexes of DSPC, curcumin and its congeners, Al 3+ Component ratio and function of drug-metal-phospholipid complex particles produced from it

[0501] Metal-phospholipid complexes were prepared according to Example 3. Curcumin was replaced with its analog, hesperetin (one molecule of hesperetin contains four hydroxyl groups), and tea polyphenol (one molecule of tea polyphenol contains eight hydroxyl groups). Three metal-phospholipid complexes (mRNA@MPP2, mRNA@MPP5, and mRNA@MPP30) were prepared by replacing curcumin with its analog, hesperetin (one molecule of hesperetin contains four hydroxyl groups), and tea polyphenol (one molecule of tea polyphenol contains eight hydroxyl groups). The input ratios of DSPC, curcumin or its analog, and Al(NO3)3·9H2O were 1:1:1, 1:1:1, and 1:1:2, respectively. These three metal-phospholipid complexes (mRNA@MPP1, mRNA@MPP4, and mRNA@MPP29) were used to prepare the corresponding drug-metal-phospholipid complex nanoparticles. The mRNA was encoding the eGFP fluorescent protein, and its sequence is SEQ ID No. 1 (720 nt). According to the experimental process and method described in Example 3.5, the mRNA encapsulation rate of these four kinds of drug-lipid nanoparticles and their ability to promote the expression of eGFP fluorescent protein after treatment of 293T cells are detected.

[0502] Analysis of the results, as shown in Tables 4-5, showed that the mRNA encapsulation efficiency and target protein expression promotion ability of drug-metal-phospholipid complex particles prepared at different input ratios based on the chemical structure of the metal-phospholipid complex components were comparable. This suggests that the input ratio of the metal-phospholipid complex components can be adjusted depending on the structure of the specific metal-phospholipid complex components. The rationale for this adjustment is as follows: Because DSPC analogs and curcumin analogs are connected by hydrogen bonds, if the DSPC analog contains multiple phosphate groups, the input ratio of the DSPC analog to the curcumin analog during phospholipid complex synthesis can be adjusted depending on the number of phosphate groups contained in the DSPC analog. That is, if the DSPC analog contains two phosphate groups, the input ratio of the DSPC analog to the curcumin analog can be adjusted to 1:2. If the DSPC analog contains three phosphate groups, the input ratio of the DSPC analog to the curcumin analog can be adjusted to 1:3. Hydroxyl groups and Al in curcumin congeners 3+Since the congeners are linked by coordinate bonds, as long as the congeners contain multiple binding sites, the congeners of curcumin and Al 3+ The input ratio of the congeners can be adjusted based on the number of binding sites contained in the curcumin congener. [Table 16]

[0503] The preparation of mRNA@MPP in Example 3 above was completed by the research group of Professor Wang Shan of the Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Central South University.

[0504] Unless otherwise defined, all technical and scientific terms used throughout this disclosure have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of any discrepancy, the meaning described in the entire disclosure or the meaning derived from the contents described in the entire disclosure shall prevail. Furthermore, the terms used herein are intended to describe the embodiments of this disclosure and are not intended to limit this disclosure.

[0505] It should be noted that the above are merely preferred embodiments of the present disclosure and the technical principles utilized. Those skilled in the art will understand that the present disclosure is not limited to the specific embodiments described herein, and that various obvious modifications, rearrangements, and substitutions can be made by those skilled in the art without departing from the scope of the present disclosure. Therefore, although the present disclosure has been described in relatively detail through the above embodiments, the present disclosure is not limited to the above embodiments, and may include other equivalent embodiments without departing from the technical spirit of the present disclosure, all of which are within the protection scope of the present disclosure.

Claims

1. A metal-phospholipid complex, the metal-phospholipid complex is formed by the reaction of a phospholipid molecular portion, a linker molecular portion, and a metal ion portion, the phospholipid molecular portion and the linker molecular portion are linked together, and the linker molecular portion and the metal ion portion are linked together by a coordinate bond, and the metal-phospholipid complex is not a cationic lipid or an ionizable lipid.

2. 2. The metal-phospholipid complex according to claim 1, wherein the phospholipid molecule portion is one or a combination of a plurality of molecules selected from the group consisting of lecithin (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), ceramide-1-phosphate (SP), phosphatidylinositol (PI), phosphatidylthreonine (PT), sphingomyelin (SM), lysolecithin (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysosphingomyelin (LSM), sphingosine-1-phosphate (S1P), and derivatives thereof.

3. The phospholipid molecule portion is 【Chemical 1】 【change】 【change】 and a combination of one or more selected from the group consisting of derivatives thereof, R1 and R2 are both independent, 【Chemistry 2】 【change】 3. The metal-phospholipid complex of claim 2, wherein

4. 4. The metal-phospholipid complex according to claim 3, wherein the phospholipid molecule portion is one or a combination of two or more selected from the group consisting of lecithin (PC, Formula 1), phosphatidylethanolamine (PE, Formula 2), phosphatidic acid (PA, Formula 4), phosphatidylglycerol (PG, Formula 5), and derivatives thereof.

5. 5. The metal-phospholipid complex according to claim 4, wherein the phospholipid molecule portion is one or a combination of a plurality of phospholipid molecules selected from DSPC, DSPE, DSPA, DSPG, and derivatives thereof.

6. The phospholipid molecule portion is 【Chemistry 3】 6. The metal-phospholipid complex according to claim 5, which is one or a combination of a plurality of compounds selected from the group consisting of phospholipids, ...

7. 7. The metal-phospholipid complex of claim 6, wherein the phospholipid molecule portion is selected from DSPC (Formula 46), DSPE (Formula 47), or DSPA (Formula 48).

8. The metal-phospholipid complex according to any one of claims 1 to 7, wherein the linker molecule portion is one or a combination of multiple 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 pinbeimin C, and derivatives thereof.

9. The linker molecule portion is 【Chemistry 4】 【change】 【change】 【change】 9. The metal-phospholipid complex according to claim 8, which is one or a combination of a plurality of compounds selected from the group consisting of phospholipids, ...

10. The linker molecule portion is Curcumin (Formula 19), 【Chemistry 5】 10. The metal-phospholipid complex according to claim 9, wherein the metal-phospholipid complex is one or a combination of two or more selected from the following:

11. 10. The metal-phospholipid complex of claim 9, wherein the linker molecule portion is one or a combination of two or more selected from the group consisting of curcumin (Formula 19), hesperetin (Formula 24), tea polyphenol (Formula 28), and derivatives thereof.

12. 12. The metal-phospholipid complex of claim 11, wherein the linker molecule moiety is selected from curcumin (Formula 19), hesperetin (Formula 24), or tea polyphenol (Formula 28).

13. The metal ion moiety is Fe 3+ , Ag + , Ba 2+ , Ca 2+ , Cd 2+ , Cu 2+ , Fe 2+ , Mn 2+ , Mg 2+ , Mo 2+ , Zn 2+ , Pt 2+ , Au 2+ , Al 3+ , Ce 3+ , Co 3+ , Cr 3+ ,EU 3+ , Gd 3+ , Ni 3+ , W 3+ , V 3+ , Zr 3+ 13. The metal-phospholipid complex according to claim 1, wherein the metal-phospholipid complex is one or a combination of two or more selected from the following:

14. The metal ion moiety is Fe 3+ , Ca 2+ , Al 3+ 14. The metal-phospholipid complex according to claim 13, wherein the metal-phospholipid complex is one or a combination of two or more selected from the following:

15. The metal ion moiety is Fe 3+ , Ca 2+ or Al 3+ 15. The metal-phospholipid complex of claim 14, selected from the group consisting of:

16. The metal-phospholipid complex comprises a phospholipid molecule portion, a linker molecule portion, and a metal ion portion, the phospholipid molecule portion being selected from DSPC, DSPE, or DSPA, the linker molecule portion being selected from curcumin, hesperetin, or tea polyphenol, and the metal ion portion being Fe 3+ , Ca 2+ or Al 3+ 16. The metal-phospholipid complex according to any one of claims 1 to 15, wherein the metal-phospholipid complex is selected from the group consisting of:

17. The metal-phospholipid complex comprises a phospholipid molecule portion, a linker molecule portion, and a metal ion portion, the phospholipid molecule portion being selected from DSPC (Formula 46), DSPE (Formula 47), or DSPA (Formula 48), the linker molecule portion being selected from curcumin (Formula 19), hesperetin (Formula 24), or tea polyphenol (Formula 28), and the metal ion portion being Fe 3+ , Ca 2+ or Al 3+ 17. The metal-phospholipid complex of claim 16, selected from the group consisting of:

18. 18. The metal-phospholipid complex according to claim 16 or 17, wherein the molar ratio of the phospholipid molecule portion, the linker molecule portion, and the metal ion portion is 1:1:(0.5 to 2).

19. 20. The metal-phospholipid complex of claim 18, wherein the molar ratio of the phospholipid molecule portion, the linker molecule portion, and the metal ion portion is 1:1:

1.

20. 20. A method for producing a metal-phospholipid complex according to any one of claims 1 to 19, comprising: Step 1: reacting and connecting phospholipid molecules with linker molecules to form a phospholipid complex; Step 2: reacting the phospholipid complex produced in Step 1 with a metal ion through a coordinate bond to form a metal-phospholipid complex; A manufacturing method comprising:

21. 21. The method of claim 20, wherein in step 1, the phospholipid molecule and the linker molecule are dissolved in ethanol to react with each other, and then n-hexane is added to precipitate the phospholipid complex.

22. 22. The method of claim 21, wherein the molar ratio of the phospholipid molecules to the linker molecules is 1:

1.

23. 23. The method according to claim 21 or 22, wherein the reaction conditions include reacting at 65°C for 2 hours.

24. 24. The method according to claim 20, wherein in step 2, the phospholipid complex and the metal ion are dissolved in ethanol and reacted to obtain the metal-phospholipid complex.

25. The method of claim 24, wherein the molar ratio of the phospholipid complex to the metal ion is 1:(1 to 2).

26. 26. The method according to claim 24 or 25, wherein the reaction conditions include reacting at 60°C for 2 hours.

27. A metal-phospholipid complex particle, comprising: The metal-phospholipid complex particles are (i) a metal-phospholipid complex, which is a metal-phospholipid complex according to any one of claims 1 to 19; (ii) a conjugated lipid that inhibits particle aggregation, and that is not a cationic or ionizable lipid; and (iii) containing a non-cationic or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation.

28. 28. The metal-phospholipid complex particle of claim 27, wherein the particle aggregation-inhibiting conjugation lipid comprises a PEG-lipid conjugate and / or a PEG-DAA.

29. The PEG-lipid conjugate is 【Chemistry 6】 and a combination of one or more selected from the group consisting of derivatives thereof, 29. The metal-phospholipid complex particle according to claim 28, wherein R1 and R2 are each independently a decanoyl group, a lauroyl group, a myristoyl group, a palmitoyl group, a stearoyl group, an oleyl group, a linoleoyl group, an erucyl group, an arachidonoyl group, or a phytanoyl group.

30. 30. The metal-phospholipid complex particle according to claim 29, wherein the PEG-lipid conjugate is one or a combination of two or more selected from DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000, and DSPE-PEG5000.

31. The PEG-lipid conjugate is 【Chemistry 7】 【change】 31. The metal-phospholipid complex particle according to claim 30, selected from the group consisting of:

32. 32. The metal-phospholipid complex particle according to any one of claims 27 to 31, wherein the non-cationic or non-ionizable lipid according to (iii) is a combination of one or more of cholesterol and its derivatives.

33. The non-cationic or non-ionizable lipid according to (iii) 【Chemistry 8】 33. The metal-phospholipid complex particle according to claim 32, wherein

34. 33. The metal-phospholipid complex particle of claim 32, wherein the non-cationic or non-ionizable lipid described in (iii) further comprises one or more combinations selected from the group consisting of lecithin PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, ceramide-1-phosphate SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysolecithin LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, sphingosine-1-phosphate S1P, cholesterol sulfate, and derivatives thereof.

35. The non-cationic or non-ionizable lipids described in (iii) include lecithin (PC, Formula 1), phosphatidylethanolamine (PE, Formula 2), phosphatidylserine (PS, Formula 3), phosphatidic acid (PA, Formula 4), phosphatidylglycerol (PG, Formula 5), ceramide-1-phosphate (SP, Formula 6), phosphatidylinositol (PI, Formula 7), phosphatidylthreonine (PT, Formula 8), sphingomyelin (SM, Formula 9), lysolecithin (LYS, Formula 10 ...serine (PHS, Formula 11), phosphatidylserine (PHS, Formula 12), phosphatidylserine (PHS, Formula 13), phosphatidylserine (PHS, Formula 14), phosphatidylserine (PHS, Formula 15), phosphatidylserine (PHS, Formula 16), phosphatidylserine (PHS, Formula 17), phosphatidylserine (PHS, Formula 18), phosphatidylserine (PHS, Formula 19), phosphatidylserine (PHS, Formula 20), phosphatidylserine (PHS, Formula 21), Lysophosphatidylcholine (LPC, Formula 10), lysophosphatidylethanolamine (LPE, Formula 11), lysophosphatidylserine (LPS, Formula 12), lysophosphatidic acid (LPA, Formula 13), lysophosphatidylglycerol (LPG, Formula 14), lysophosphatidylinositol (LPI, Formula 15), lysophosphatidylthreonine (LPT, Formula 16), lysosphingomyelin (LSM, Formula 17), sphingosine-1-phosphate (S1P, Formula 18), 【Chemistry 9】 35. The metal-phospholipid complex particle of claim 34, further comprising a combination of one or more selected from the group consisting of and derivatives thereof.

36. 36. The metal-phospholipid complex particle of claim 34 or 35, wherein the non-cationic or non-ionizable lipid described in (iii) comprises a combination of cholesterol and one or more selected from DSPC, DSPE, DSPA, or DSPG.

37. 37. The metal-phospholipid complex particle of claim 36, wherein the non-cationic or non-ionizable lipid described in (iii) comprises cholesterol (Formula 40) and DSPC (Formula 46).

38. The metal-phospholipid complex particles comprise: (i) a metal-phospholipid complex; (ii) conjugated lipids that inhibit particle aggregation, and (iii) The metal-phospholipid complex particles of any one of claims 34 to 37, which are composed of non-cationic or non-ionizable lipids, wherein the molar fraction of the metal-phospholipid complex in the feedstock is 10% to 40%, the molar fraction of the particle aggregation-inhibiting conjugated lipid in the feedstock is 2% to 10%, the molar fraction of the cholesterol in the feedstock is 35% to 75%, and the molar fraction of the non-cationic or non-ionizable lipid other than cholesterol in the feedstock is 0% to 40%.

39. the metal-phospholipid complex particles comprise (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic lipid or a non-ionizable lipid, wherein the molar fraction of the metal-phospholipid complex in the feedstock is 5% or more and less than 10%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the feedstock is 2% to 10%, the molar fraction of the cholesterol in the feedstock is 15% or more and less than 35%, 35% to 75%, or greater than 75% to 80%, and the molar fraction of the non-cationic lipid or non-ionizable lipid other than cholesterol in the feedstock is 0% to 40%, or greater than 40% to 51%; or the molar fraction of the metal-phospholipid complex in the feedstock is greater than 40% to 50%, the molar fraction of the particle aggregation-inhibiting conjugated lipid in the feedstock is between 2% and 10%, the molar fraction of the cholesterol in the feedstock is between 15% and less than 35%, between 35% and 75%, or between 75% and 80%, and the molar fraction of the non-cationic or non-ionizable lipid other than cholesterol in the feedstock is between 0% and 40%, or between 40% and 51%; or 38. The metal-phospholipid complex particles of any one of claims 34 to 37, wherein the molar fraction of the metal-phospholipid complex in the feedstock is 10% to 40%, the molar fraction of the particle aggregation inhibiting conjugated lipid in the feedstock is 2% to 10%, the molar fraction of the cholesterol in the feedstock is 15% to 35% or greater than 75% to 80%, and the molar fraction of the non-cationic or non-ionizable lipid other than cholesterol in the feedstock is 0% to 40%, or greater than 40% to 51%.

40. The metal-phospholipid complex particles according to claim 38 or 39, wherein the molar fraction of the metal-phospholipid complex in the raw material is 7% or more but less than 10%, 10% to 30%, or 20% to 30%, preferably 25%.

41. 41. The metal-phospholipid complex particles of any one of claims 38 to 40, wherein the molar fraction of lipids in the conjugation that inhibits particle aggregation in the raw material is 3% to 10% or 5% to 10%, preferably 10%.

42. 42. The metal-phospholipid complex particles according to any one of claims 38 to 41, wherein the molar fraction of cholesterol in the raw material is 15% or more but less than 35%, 35% to 56%, or 35% to 55%, preferably 40%.

43. 43. The metal-phospholipid complex particles of any one of claims 38 to 42, wherein the molar fraction of non-cationic 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%.

44. The molar fraction of the metal-phospholipid complex in the raw material is 15% to 25%, the molar fraction of the particle aggregation-inhibiting conjugation lipid in the raw material is 4% to 10%, the molar fraction of the cholesterol in the raw material is 40% to 46%, the molar fraction of the DSPC in the raw material is 25% to 35%, and the metal ion moiety in the metal-phospholipid complex is Fe 3+ 39. The metal-phospholipid complex particle according to claim 38, selected from the group consisting of:

45. The molar fraction of the metal-phospholipid complex in the raw material is 15%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 4%, the molar fraction of the cholesterol in the raw material is 46%, and the molar fraction of the DSPC in the raw material is 35%, or the molar fraction of the metal-phospholipid complex in the raw material is 25%, the molar fraction of the conjugated lipid that inhibits particle aggregation in the raw material is 10%, the molar fraction of the cholesterol in the raw material is 40%, and the molar fraction of the DSPC in the raw material is 25%, and the metal ion moiety in the metal-phospholipid complex is Fe 3+ 45. The metal-phospholipid complex particle of claim 44, selected from the group consisting of:

46. The molar fraction of the metal-phospholipid complex in the raw material is 10% to 30%, the molar fraction of the particle aggregation-inhibiting conjugation lipid in the raw material is 3% to 10%, the molar fraction of the cholesterol in the raw material is 35% to 56%, the molar fraction of the DSPC in the raw material is 34% to 40%, and the metal ion moiety in the metal-phospholipid complex is Al 3+ 39. The metal-phospholipid complex particle according to claim 38, selected from the group consisting of:

47. the molar fraction of the metal-phospholipid complex in the feedstock is between 10% and 30%, the molar fraction of the particle aggregation-inhibiting conjugated lipid in the feedstock is between 3% and 10%, the molar fraction of the cholesterol in the feedstock is between 35% and 56%, and the molar fraction of the DSPC in the feedstock is between 40% and 45%; or the molar fraction of the metal-phospholipid complex in the feedstock is between 10% and 30%, the molar fraction of the particle aggregation-inhibiting conjugated lipid in the feedstock is between 3% and 10%, the molar fraction of the cholesterol in the feedstock is between 15% and 35%, and the molar fraction of the DSPC in the feedstock is between 34% and 40% or greater than 40% and 45%; or The molar fraction of the metal-phospholipid complex in the raw material is 7% or more and less than 10%, the molar fraction of the particle aggregation-inhibiting conjugated lipid in the raw material is 3% to 10%, the molar fraction of the cholesterol in the raw material is 15% or more and less than 35% or 35% to 56%, the molar fraction of the DSPC in the raw material is 34% to 40%, or more than 40% to 45%, and the metal ion moiety in the metal-phospholipid complex is Al 3+ 40. The metal-phospholipid complex particle of claim 39, selected from the group consisting of:

48. The molar fraction of the metal-phospholipid complex in the raw material is 7%, the molar fraction of the particle aggregation-inhibiting conjugation lipid in the raw material is 3%, the molar fraction of the cholesterol in the raw material is 56%, the molar fraction of the DSPC in the raw material is 34%, and the metal ion moiety in the metal-phospholipid complex is Al 3+ 48. The metal-phospholipid complex particle of claim 47, selected from the group consisting of:

49. 49. The method for producing metal-phospholipid complex particles according to any one of claims 27 to 48, comprising mixing (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits particle aggregation, and (iii) a non-cationic or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation to obtain the metal-phospholipid complex particles.

50. 1. A drug-lipid particle comprising: (a) a drug, which is a negatively charged molecule; (b) a metal-phospholipid complex particle, the metal-phospholipid complex particle being a metal-phospholipid complex particle according to any one of claims 27 to 48; A drug-lipid particle comprising:

51. 51. The drug-lipid particle of claim 50, wherein the drug is encapsulated in the metal-phospholipid complex particle.

52. 52. The drug-lipid particle of claim 50 or 51, wherein the drug is one or a combination of more selected from nucleic acids, proteins, polypeptides, small molecules, nucleic acid analogs, protein analogs, and polypeptide analogs.

53. The drug-lipid particle of claim 52, wherein the nucleic acid is one or a combination of more than one selected from mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, and artificial nucleic acids.

54. The nucleic acids include an mRNA sequence encoding eGFP shown in SEQ ID No. 1, an mRNA sequence encoding the receptor-binding domain (RBD) of the S1 subunit of the novel coronavirus shown in SEQ ID No. 2, an mRNA sequence encoding NY-ESO-1 shown in SEQ ID No. 3, an siRNA sequence of the Bcl-2 gene shown in SEQ ID No. 4 as an antisense strand and SEQ ID No. 21 as a sense strand, an siRNA sequence of the PLK1 gene shown in SEQ ID No. 6 as an antisense strand and SEQ ID No. 23 as a sense strand, an siRNA sequence of the Gal-1 gene shown in SEQ ID No. 8, an ASO sequence of the STAT-3 gene shown in SEQ ID No. 10, an ASO sequence of the α-syn gene shown in SEQ ID No. 12, an ASO sequence of the Bcl-2 gene shown in SEQ ID No. 14, an mRNA sequence encoding the wild-type novel coronavirus S protein shown in SEQ ID No. 16, a double-stranded DNA sequence shown in SEQ ID No. 17 as an antisense strand and SEQ ID No. 25 as a sense strand, an siRNA sequence of the STAT-3 gene shown in SEQ ID No. 8, an ASO sequence of the α-syn gene shown in SEQ ID No. 12, an ASO sequence of the Bcl-2 gene shown in SEQ ID No. 14, an mRNA sequence encoding the wild-type novel coronavirus S protein shown in SEQ ID No. 16, and a double-stranded DNA sequence shown in SEQ ID No. 17 as an antisense strand and SEQ ID No. 25 as a sense strand. A drug-lipid particle described in claim 52 or 53, wherein the single-stranded DNA shown in SEQ ID No. 18, or the siRNA sequence of the B7-H4 gene whose sense strand is SEQ ID No. 19 and whose antisense strand is SEQ ID No. 26, is used.

55. 55. A method for producing the drug-lipid particle of any one of claims 50 to 54, comprising: A production method in which a drug is encapsulated in a metal-phospholipid complex particle to obtain the drug-lipid particle.

56. 56. The method of claim 55, wherein the metal-phospholipid complex, the lipid in the conjugation that inhibits particle aggregation, and a non-cationic or non-ionizable lipid other than the metal-phospholipid complex and the lipid in the conjugation that inhibits particle aggregation 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 uniformly mixed to obtain the drug-lipid particles.

57. 57. The method of claim 56, wherein the organic compound is ethanol.

58. 58. The method of claim 56 or 57, wherein the buffer is an enzyme-free PBS buffer.

59. 59. The method of any one of claims 56 to 58, wherein the mixing of the organic phase and the aqueous phase comprises a microfluidic chip or ultrasound.

60. 20. The use of the metal-phospholipid complex according to any one of claims 1 to 19 in a nucleic acid delivery system.

61. 61. The application of claim 60, wherein the nucleic acid delivery system is used to introduce a nucleic acid into a cell.

62. 62. The application of claim 61, wherein the nucleic acid is used to silence expression of a target sequence in a mammalian subject, to deliver a drug within a mammal, to deliver a drug from the body to mammalian cells, or to treat a disease or condition in a mammal.

63. 52. The use of a metal-phospholipid complex particle according to any one of claims 27 to 48 or a drug-lipid particle according to any one of claims 47 to 51 in a composition used for the delivery of a drug.

64. 64. The application of claim 63, wherein the composition is used to introduce a drug into a cell.

65. 65. The application of claim 63 or 64, wherein the composition is a drug.

66. 66. The application of claim 65, wherein the agent is used to silence expression of a target sequence in a mammalian subject, to deliver a drug within a mammalian body, to deliver a drug from the body to mammalian cells, or to treat a disease or condition in a mammal.

67. 67. The application of claim 62 or 66, wherein the mammal is a human.

68. 67. The application of claim 62 or 66, wherein the disease or condition is associated with the expression of a gene comprising a pharmacological target sequence.

69. 67. The application of claim 62 or 66, wherein the disease or condition comprises cancer, a viral infection, an autoimmune disease, diabetes or Alzheimer's disease.

70. 70. The application of claim 69, wherein the viral infection comprises Hepatitis A, Hepatitis B, Hepatitis C, SARS-Cov-2, HIV, HPV, influenza, smallpox or syphilis.

71. 70. The application of claim 69, wherein the cancer comprises liver cancer, glioma, melanoma, lung cancer, pancreatic cancer or breast cancer.

72. 66. The application of claim 65, wherein the drug is a vaccine.

73. 73. The use of claim 65 or 72, wherein the route of administration of the agent comprises intrathecal injection, intramuscular injection, intracranial injection, intravenous injection or intratumoral injection.

74. A pharmaceutical comprising a metal-phospholipid complex described in any one of claims 1 to 19, or a metal-phospholipid complex particle described in any one of claims 27 to 48, or a drug-lipid particle described in any one of claims 50 to 54.

75. 75. The agent of claim 74, wherein the agent is a vaccine.

76. The method of claim 75, wherein the vaccine is a novel coronavirus vaccine.

77. 19. Use of a metal-phospholipid complex according to any one of claims 1 to 19, or a metal-phospholipid complex particle according to any one of claims 27 to 48, or a drug-lipid particle according to any one of claims 50 to 54, or a medicament according to any one of claims 74 to 76 in the prevention / treatment of a disease or condition in a mammal.

78. 78. The application of claim 77, wherein the mammal is a human.

79. 78. The application of claim 77, wherein the disease or condition is associated with expression of a gene comprising a pharmacological target sequence.

80. 78. The application of claim 77, wherein the disease or condition comprises cancer, a viral infection, an autoimmune disease, diabetes, or Alzheimer's disease.

81. 81. The application of claim 80, wherein the viral infection comprises Hepatitis A, Hepatitis B, Hepatitis C, SARS-Cov-2, HIV, HPV, influenza, smallpox or syphilis.

82. 81. The application of claim 80, wherein the cancer comprises liver cancer, glioma, melanoma, lung cancer, pancreatic cancer or breast cancer.

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