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CN117582511A - Metal-phospholipid composite particles, and preparation method and application thereof - Google Patents

Metal-phospholipid composite particles, and preparation method and application thereof Download PDF

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Publication number
CN117582511A
CN117582511A CN202310975085.4A CN202310975085A CN117582511A CN 117582511 A CN117582511 A CN 117582511A CN 202310975085 A CN202310975085 A CN 202310975085A CN 117582511 A CN117582511 A CN 117582511A
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Prior art keywords
metal
phospholipid complex
phospholipid
lipid
formula
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王珊
胡敦
孙毅毅
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Hunan Changxing Biomedical Co ltd
Hunan Jianrui Pharmaceutical Technology Co ltd
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Hunan Jianrui Pharmaceutical Technology Co ltd
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Abstract

The invention provides a metal-phospholipid composite particle, a preparation method and application thereof, and relates to the technical field of biology. The present invention provides metal phospholipid complex particles comprising a metal-phospholipid complex, conjugated lipids that inhibit aggregation of the particles, and non-cationic lipids or non-ionizable lipids. Under the condition that the effectiveness of the metal-phospholipid complex particles is not lower than that of LNP based on cationic lipid and/or ionizable lipid, the metal-phospholipid complex particles do not use the cationic lipid and the ionizable lipid, so that compared with LNP, the toxicity is greatly reduced, the biological safety is obviously improved, and the carrying of negative charge medicines in organisms is facilitated.

Description

Metal-phospholipid composite particles, and preparation method and application thereof
Cross Reference to Related Applications
The present disclosure claims 202210950392.2 to the chinese patent office at month 08 and 09 of 2022 entitled "metal-phospholipid complex particles, methods of making same, and uses thereof," priority of chinese patent application, the entire contents of which are incorporated herein by reference.
Technical Field
The invention relates to the technical field of biology, in particular to metal-phospholipid composite particles, a product and application thereof.
Background
With the continuous development of molecular biology technology, the relevance between genes and diseases is becoming more and more well known. Nucleic acid drugs refer to artificially synthesized DNA or RNA fragments with disease treatment function, and are attracting attention because they show great potential for use in disease diagnosis and treatment. Such drugs can act directly on pathogenic target genes or mRNAs, exerting therapeutic effects at the gene level. Compared with the traditional small molecule drugs and antibody drugs, the nucleic acid drugs are not limited by the drug property of target proteins, can treat more extensive diseases, and can radically regulate and control the expression of pathogenic genes. The nucleic acid medicine also has the obvious advantages of high efficiency, low toxicity, high specificity and the like, and is expected to become a third-large type medicine after the small molecular medicine and the antibody medicine.
However, nucleic acid drugs are susceptible to nuclease degradation in vivo and are difficult to act across cell membranes due to their large molecular weight and their negatively charged nature. Thus, the search for a safe, effective nucleic acid drug delivery system is a bottleneck in nucleic acid drug development. Currently, vectors capable of delivering nucleic acid drugs can be largely classified into viral vectors and non-viral vectors. The virus vector can cause immune response after entering the human body, and the current use is less; compared with small molecule conjugates directly conjugated with nucleic acid drugs, the nanoparticle can more effectively encapsulate the nucleic acid drugs, prevent the nucleic acid drugs from being rapidly degraded in vivo by nucleases, and further improve the in vivo circulation time of the nucleic acid drugs. The mechanism by which the nanoparticles encapsulate nucleic acids is by adsorption of negatively charged nucleic acids with positively charged cationic lipids. However, cationic lipids are relatively cytotoxic and their toxic mechanism of action is: (1) atrophy of cells, reduced number of mitoses and vacuolation of cytoplasm; (2) interact with biological proteins such as protein kinase C to disrupt their activity; (3) secretion of various pro-inflammatory cytokines and chemokines is triggered by activation of p38 mitogen-activated protein kinase and nuclear factor κb transcription factors. Further, an ionizable lipid is one containing positively charged ionizable amine groups that are uncharged under physiological conditions (ph=7.4), but are protonated and positively charged at lower pH values. Thus, ionizable lipids may be used to replace partially or completely cationic lipids as the main component of the nanoparticle responsible for adsorbing nucleic acids. When the nanoparticle containing the ionizable lipid enters the biological cell lysosome, the ionizable lipid becomes a positively charged lipid in a low pH (ph=4.0-6.5) environment within the lysosome. Although ionizable lipids reduce the cytotoxic and highly inflammatory effects of some permanently positively charged cationic lipids, their cytotoxicity and immunogenicity remain high. Lipid nanoparticles (Lipid Nanoparticle, LNP) based on cationic lipids and/or ionizable lipids are currently available for clinical nanoparticle nucleic acid drug delivery systems, wherein cationic lipids and/or ionizable lipids are the main components of LNP, responsible for adsorbing nucleic acids, while cationic lipids and/or ionizable lipids mediated cytotoxicity and immunogenicity remain one of the important reasons for greater LNP toxicity.
Thus, when negatively charged drugs (e.g., nucleic acid drugs, protein drugs, polypeptide drugs, small molecule drugs, etc.) are delivered with delivery systems, nanoparticle delivery systems developed that rely on cationic lipids and/or ionizable lipids are not fundamentally capable of solving the toxicity problems of nanoparticle delivery systems, and there is an urgent need for a liposome delivery system that does not use cationic lipids and/or ionizable lipids, but is low in toxicity.
In view of this, the present invention has been made.
Disclosure of Invention
A first object of the present invention is to provide a metal-phospholipid complex particle.
A second object of the present invention is to provide a method for producing the above metal-phospholipid complex particles.
A third object of the present invention is to provide the use of the above metal-phospholipid complex particles.
A fourth object of the present invention is to provide a pharmaceutical agent comprising the aforementioned metal-phospholipid complex particles.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the present invention provides Metal-phospholipid complex particles (Metal-chelatedphospholipidcomplex nanoparticles, MPP) comprising (i) a Metal-phospholipid complex, wherein the Metal-phospholipid complex is comprised of a phospholipid molecule moiety, a linker molecule moiety, and a Metal ion moiety, the phospholipid molecule moiety is linked to the linker molecule moiety, the linker molecule moiety is linked to the Metal ion moiety by a coordination bond, and the Metal-phospholipid complex is not a cationic lipid or an ionizable lipid; (ii) A conjugated lipid that inhibits aggregation of particles, wherein the conjugated lipid that inhibits aggregation of particles is not a cationic lipid or an ionizable lipid; and (iii) a non-cationic lipid or a non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits aggregation of particles. Wherein "(iii) a non-cationic lipid or a non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits aggregation of particles" may be referred to simply as a "non-cationic lipid or a non-ionizable lipid".
Further, the phospholipid molecule moiety is selected from the group consisting of one or more of lecithin (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic Acid (PA), phosphatidylglycerol (PG), 1-phosphoceramide (SP), phosphatidylinositol (PI), phosphatidylthreonine (PT), sphingomyelin (SM), lysolecithin (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysosphingomyelin (LSM), 1-phosphosphingosine (S1P), and derivatives thereof. Wherein, "and its derivatives" means "lecithin (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic Acid (PA), phosphatidylglycerol (PG), 1-phosphoceramide (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 1-sphingosine phosphate (S1P)". In the present invention, "and derivatives thereof" are all intended to have similar meanings. The phospholipid molecule moiety may be, for example, but 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, and the like.
Further, the phospholipid molecule moiety is selected from
Lecithin (PC) (1)
Phosphatidylethanolamine (PE) (2)
Phosphatidylserine (PS) (3)
Phosphatidic Acid (PA) (4)
Phosphatidylglycerol (PG) (5)
1-phosphoric acid ceramide (SP) (6)
Phosphatidylinositol (PI) (7)
Phosphatidylthreonine (PT) (8)
Sphingomyelin (SM) (9)
Lysolecithin (LPC) (10)
Lysophosphatidylethanolamine (LPE) (11)
Lysophosphatidylserine (LPS) (12)
Lysophosphatidic acid (LPA) (13)
Lysophosphatidylglycerol (LPG) (14)
Lysophosphatidylinositol (LPI) (formula 15)
Lysophosphatidylthreonine (LPT) (formula 16)
Lysosphingomyelin (LSM) (17)
Sphingosine-1-phosphate (S1P) (formula 18)And combinations of one or more of its derivatives;
wherein, R1 and R2 are respectively and independently:
sunflower acyl group
Lauroyl group
Myristoyl group
Palmitoyl group
Stearoyl group
Oleoyl radical
Linoleoyl group
Erucic acyl
Peanut acyl
Phytantyl radical
Further, the phospholipid molecule moiety is selected from one or more of lecithin (PC) (formula 1), phosphatidylethanolamine (PE) (formula 2), phosphatidic Acid (PA) (formula 4), phosphatidylglycerol (PG) (formula 5), and derivatives thereof.
Further, the phospholipid moiety is selected from the group consisting of distearyl phosphatidylcholine (DSPC), distearyl phosphatidylethanolamine (DSPE), distearyl phosphatidic acid (DSPA), distearyl phosphatidylglycerol (DSPG), and derivatives thereof.
Further, the phospholipid moiety is selected from distearoyl phosphatidylcholine (DSPC) (formula 46)
Distearoyl phosphatidylethanolamine (DSPE) (formula 47)
Distearyl phosphatidic acid (DSPA) (formula 48)
Distearoyl phosphatidylglycerol (DSPG) (formula 49)
And one of its derivativesOne or a combination of more.
Further, the phospholipid moiety is selected from the group consisting of DSPC (formula 46), DSPE (formula 47) or DSPA (formula 48) or DSPG (formula 49).
Further, the linker molecule moiety is selected from the group consisting of curcumin, chlorogenic acid, anthocyanin, quercetin, dihydromyricetin, hesperetin, naringenin, apigenin, catechin, tea polyphenol, epigallocatechin gallate, ellagic acid, morin, epicatechin gallate, catechin gallate, gallocatechin gallate or tubine C, and a combination of one or more of its derivatives.
Further, the linker molecule moiety is selected from
Curcumin (formula 19)
Chlorogenic acid (20)
Anthocyanin (21)R7 and R8 are H, OH or OCH 3 R3 is H or glycosyl, R4, R5 and R6 are OH or glycosyl;
quercetin (22)
Dihydromyricetin (formula 23)
Hesperetin (24)
Naringenin (25)
Apigenin (26)
Catechin (type 27)
Tea polyphenol (28)
Epigallocatechin gallate (29)
Ellagic acid (30)
Phellinus linteus (31)
Epicatechin gallate (32)
Catechin gallate (33)
Gallocatechin gallate (34)
Ping Beijian C (35)And combinations of one or more of its derivatives.
Further, the linker molecule moiety is selected from curcumin (formula 19),
DihydrogingerFlavin (36)
Hexahydrocurcumin (37)
Curcumin sulfate (38)
Bisdemethoxycurcumin (39)A combination of one or more of the foregoing.
Further, the linker molecule moiety is selected from the group consisting of curcumin (formula 19), hesperetin (formula 24), tea polyphenols (formula 28), and combinations of one or more of their derivatives.
Further, the linker molecule moiety is selected from curcumin (formula 19), hesperetin (formula 24) or tea polyphenols (formula 28).
Further, the metal ion moiety is selected from the group consisting of Fe 3+ 、Ag + 、Ba 2+ 、Ca 2+ 、Cd 2+ 、Cu 2+ 、Fe 2+ 、Mn 2+ 、Mg 2+ 、Mo 2 + 、Zn 2+ 、Pt 2+ 、Au 2+ 、Al 3+ 、Ce 3+ 、Co 3+ 、Cr 3+ 、Eu 3+ 、Gd 3+ 、Ni 3+ 、W 3+ 、V 3+ 、Zr 3+ A combination of one or more of the foregoing.
Further, the metal ion moiety is selected from the group consisting of Fe 3+ 、Ca 2+ 、Al 3+ A combination of one or more of the foregoing.
Further, the metal ion moiety is selected from the group consisting of Fe 3+ 、Ca 2+ Or Al 3+ 。
Further, the conjugated lipids that inhibit aggregation of the particles include polyethylene glycol (PEG) -lipid conjugates and/or PEG-Dialkoxypropyl (DAA).
Further, the PEG-lipid conjugate is selected from phosphatidylethanolamine-polyethylene glycol 2000 (PE-PEG 2000) (formula 42)
Phosphatidylethanolamine-polyethylene glycol 700 (PE-PEG 700) (formula 43)
Phosphatidylethanolamine-polyethylene glycol 1000 (PE-PEG 1000) (formula 44)
Phosphatidylethanolamine-polyethylene glycol 5000 (PE-PEG 5000) (formula 45)
And combinations of one or more of its derivatives;
r1, R2 are each independently: sunflower acyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytanoyl.
Further, the PEG-lipid conjugate is selected from the group consisting of one or more of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000, or DSPE-PEG 5000.
Further, the PEG-lipid conjugate is selected from DSPE-PEG2000 (formula 53)
DSPE-PEG700 (50)
DSPE-PEG1000 (51)
Or DSPE-PEG5000 (52)
A combination of one or more of the foregoing.
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).
Further, the non-cationic lipid or non-ionizable lipid other than the metal-phospholipid complex and the particle aggregation-inhibiting conjugated lipid in (iii) is a combination of one or more of cholesterol and derivatives thereof.
Further, the non-cationic lipid or non-ionizable lipid other than the metal-phospholipid complex and the particle aggregation-inhibiting conjugated lipid in (iii) is cholesterol (formula 40)And combinations of one or more of its derivatives.
Further, the non-cationic lipid or non-ionizable lipid in (iii) is cholesterol (formula 40).
Further, the non-cationic lipids or non-ionizable lipids of (iii) other than the metal-phospholipid complex and the particle aggregation inhibiting conjugated lipid further comprises, in addition to cholesterol, one or more selected from the group consisting of lecithin PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphoceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysolecithin LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, 1-phosphosphingosine S1P, cholesterol sulfate, and derivatives thereof.
Further, the non-cationic lipid or non-ionizable lipid of (iii) other than the metal-phospholipid complex and the particle aggregation-inhibiting conjugated lipid, further comprises, in addition to cholesterol, a lipid selected from the group consisting of: lecithin (PC) (1)
Phosphatidylethanolamine (PE) (2)
Phosphatidylserine (PS) (3)
Phosphatidic Acid (PA) (4)
Phosphatidylglycerol (PG) (5)
1-phosphoric acid ceramide (SP) (6)
Phosphatidylinositol (PI) (7)
Phosphatidylthreonine (PT) (8)
Sphingomyelin (SM) (9)
Lysolecithin (LPC) (10)
Lysophosphatidylethanolamine (LPE) (11)
Lysophosphatidylserine (LPS) (12)
Lysophosphatidic acid (LPA) (13)
Lysophosphatidylglycerol (LPG) (14)
Lysophosphatidylinositol (LPI) (formula 15)
Lysophosphatidylthreonine (LPT) (formula 16)
Lysosphingomyelin (LSM) (17)
Sphingosine-1-phosphate (S1P) (formula 18)
Cholesterol sulfate (formula 41)And combinations of one or more of its derivatives;
wherein, R1, R2 is: sunflower acyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytanoyl.
Further, the non-cationic or non-ionizable lipids of (iii) other than the metal-phospholipid complex and the particle aggregation inhibiting conjugated lipid comprise cholesterol, and one or more selected from DSPC, DSPE, DSPA or DSPG.
Further, the non-cationic or non-ionizable lipids other than the metal-phospholipid complex and the particle aggregation inhibiting conjugated lipid in (iii) include cholesterol (formula 40), and a combination of one or more selected from DSPC (formula 46), DSPE (formula 47), DSPA (formula 48), or DSPG (formula 49).
Further, the non-cationic lipids or non-ionizable lipids described in (iii) include cholesterol (formula 40) and DSPC (formula 46).
Further, the metal-phospholipid complex is made of a phospholipid molecule part selected from the group consisting of DSPC, DSPE or DSPA, a linker molecule part selected from the group consisting of curcumin, hesperetin or tea polyphenol, and a metal ion part selected from the group consisting of Fe 3+ 、Ca 2+ Or Al 3+ 。
Further, the metal-phospholipid complex is made of a phospholipid molecule part selected from the group consisting of DSPC (formula 46), DSPE (formula 47) and DSPA (formula 48), a linker molecule part selected from the group consisting of curcumin (formula 19), hesperetin (formula 24) and tea polyphenol (formula 28), and a metal ion part selected from the group consisting of Fe 3+ 、Ca 2+ Or Al 3+ 。
Further, the molar ratio of the phospholipid molecule moiety, the linker molecule moiety and the metal ion moiety is 1:1: (0.5-2).
Further, the phospholipid moiety is DSPC (formula 46), the linker moiety is selected from curcumin (formula 19), and the metal ion moiety is selected from Fe 3+ 。
Further, the molar ratio of the phospholipid molecule moiety, the linker molecule moiety, and the metal ion moiety is 1:1:1.
Further, DSPC, curcumin and Fe 3+ /Al 3+ The molar ratio of (2) is 1:1:1.
Further, DSPC, hesperetin and Fe 3+ /Al 3+ The molar ratio of (2) is 1:1:1.
Further, DSPC, tea polyphenols and Fe 3+ /Al 3+ The molar ratio of (2) is 1:1:2.
Further, fe 3+ Selected from FeCl 3 ，Al 3+ Selected from Al (NO) 3 ) 3 ·9H 2 O。
Further, the metal-phospholipid complex particles are made of (i) a metal-phospholipid complex in a molar ratio of 10% to 40% in the starting material, (ii) a conjugated lipid that inhibits aggregation of the particles in a molar ratio of 2% to 10% in the starting material, and (iii) a non-cationic lipid or non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits aggregation of the particles in a molar ratio of 0% to 40% in the starting material.
Further, wherein the metal-phospholipid complex particles are made of (i) metal-phospholipid complexes in a molar ratio of 5% to less than 10% in the starting material, (ii) conjugated lipids that inhibit aggregation of the particles in a molar ratio of 2% to 10% in the starting material, cholesterol in a molar ratio of 15% to less than 35%, 35% to 75% or greater than 75% to 80% in the starting material, and (iii) non-cationic lipids or non-ionizable lipids other than cholesterol in a molar ratio of 0% to 40% or greater than 40% to 51% in the starting material. Or (b)
The metal-phospholipid complex has a molar ratio of greater than 40% to 50% in the starting material, the conjugated lipid that inhibits aggregation of particles has a molar ratio of 2% to 10% in the starting material, the cholesterol has a molar ratio of 15% to less than 35%, 35% to 75% or greater than 75% to 80% in the starting material, and the non-cationic lipid or non-ionizable lipid other than cholesterol has a molar ratio of 0% to 40% or greater than 40% to 51% in the starting material. Or (b)
The metal-phospholipid complex has a molar ratio of 10% -40% in the raw material, the conjugated lipid inhibiting aggregation of particles has a molar ratio of 2% -10% in the raw material, the cholesterol has a molar ratio of 15% -less than 35% or more than 75% -80% in the raw material, and the non-cationic lipid or non-ionizable lipid other than cholesterol has a molar ratio of 0% -40% or more than 40% -51% in the raw material.
Further, the metal-phospholipid complex is present in the starting material in a molar ratio of 7% to less than 10%, 10% to 30% or 20% to 30%, preferably 25%.
Further, the conjugated lipids that inhibit aggregation of the particles are present in the starting material at a molar ratio of 3% to 10% or 5% to 10%, preferably 10%.
Further, the molar ratio of cholesterol in the raw material is 15% -less than 35%, 35% -56% or 35% -55%, preferably 40%.
Further, the mole ratio of the non-cationic lipid or the non-ionizable lipid except cholesterol in the raw material is 5% -30%, 25% -40%, more than 40% -45% or 20% -25%.
Further, the metal-phospholipid complex is 15 to 25% by mole in the starting material, the conjugated lipid that inhibits aggregation of particles is 4 to 10% by mole in the starting material, the cholesterol is 40 to 46% by mole in the starting material, the DSPC is 25 to 35% by mole in the starting material, and the metal ion portion of the metal-phospholipid complex is selected from the group consisting of Fe 3+ 。
Further, the metal-phospholipid complex (metal ion portion is Fe 3+ ) 15% mole in the feed, 4% mole in the feed of conjugated lipid inhibiting particle aggregation, 46% mole in the feed of cholesterol, and 35% mole in the feed of DSPC; or the metal-phospholipid complex (metal ion part is Fe 3+ ) 25% by mole of the starting material, of conjugated lipids inhibiting aggregation of the particlesThe molar ratio of the cholesterol in the raw material is 10%, the molar ratio of the cholesterol in the raw material is 40%, and the molar ratio of the DSPC in the raw material is 25%.
Further, the metal-phospholipid complex accounts for 10 to 30 percent of the mole of the raw material, the conjugated lipid for inhibiting aggregation of particles accounts for 3 to 10 percent of the mole of the raw material, the cholesterol accounts for 35 to 56 percent of the mole of the raw material, and the DSPC accounts for 34 to 40 percent of the mole of the raw material; the metal ion part in the metal-phospholipid complex is selected from Al 3+ 。
Further, the metal-phospholipid complex has a molar ratio of 10% to 30% in the starting material, the conjugated lipid that inhibits aggregation of particles has a molar ratio of 3% to 10% in the starting material, the cholesterol has a molar ratio of 35% to 56% in the starting material, the DSPC has a molar ratio of 40% to 45% in the starting material, and the metal ion portion of the metal-phospholipid complex is selected from the group consisting of Al 3+ The method comprises the steps of carrying out a first treatment on the surface of the Or (b)
The metal-phospholipid complex has a molar ratio of 10-30% in the raw material, the conjugated lipid that inhibits aggregation of particles has a molar ratio of 3-10% in the raw material, the cholesterol has a molar ratio of 15-35% in the raw material, the DSPC has a molar ratio of 34-40% or more than 40-45% in the raw material, and the metal ion portion of the metal-phospholipid complex is selected from Al 3+ The method comprises the steps of carrying out a first treatment on the surface of the Or (b)
The metal-phospholipid complex has a molar ratio of 7-10% in the raw material, the conjugated lipid inhibiting particle aggregation has a molar ratio of 3-10% in the raw material, the cholesterol has a molar ratio of 15-35% or 35-56% in the raw material, the DSPC has a molar ratio of 34-40% or more than 40-45% in the raw material, and the metal ion part of the metal-phospholipid complex is selected from Al 3+ 。
Further, the metal-phospholipid complex (metal ion portion is Al 3+ ) The molar ratio in the starting material was 7%, the conjugated lipid inhibiting aggregation of particles was 3%, the cholesterol was 56% and the DSPC was 34%.
Further, metal-phosphorusThe lipid complex is DSPC, curcumin and Fe 3+ The molar ratio of (1) to (1) was made such that the conjugated lipid inhibiting particle aggregation was DSPE-PEG2000, the non-cationic lipid or non-ionizable lipid was cholesterol and DSPC, the molar ratio of metal-phospholipid complex in the starting material was 15%, the molar ratio of DSPE-PEG2000 in the starting material was 4%, the molar ratio of cholesterol in the starting material was 46%, and the molar ratio of DSPC in the starting material was 35%.
Further, the metal-phospholipid complex is DSPC, curcumin and Fe 3+ The molar ratio of (1) to (1) was made such that the conjugated lipid inhibiting particle aggregation was DSPE-PEG2000, the non-cationic lipid or non-ionizable lipid was cholesterol and DSPC, the molar ratio of metal-phospholipid complex in the starting material was 25%, the molar ratio of DSPE-PEG2000 in the starting material was 10%, the molar ratio of cholesterol in the starting material was 40%, and the molar ratio of DSPC in the starting material was 25%.
Further, the metal-phospholipid complex is DSPC, curcumin and Al 3+ The molar ratio of (1:1:1) was made, the conjugated lipid that inhibited particle aggregation was DSPE-PEG2000, the non-cationic lipid or non-ionizable lipid was cholesterol and DSPC, the metal-phospholipid complex was 7% in the starting material, the DSPE-PEG2000 was 3% in the starting material, the cholesterol was 56% in the starting material, and the DSPC was 34% in the starting material.
The present invention provides a method for preparing the above metal-phospholipid complex particle comprising mixing (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits aggregation of particles, and (iii) a non-cationic lipid or a non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits aggregation of particles, to obtain the metal-phospholipid complex particle.
Further, the preparation method comprises the following steps:
step one: the phospholipid molecules are reacted with the linker molecules to form phospholipid complexes.
Step two: reacting the phospholipid complex prepared in the first step with a metal ion through a coordination bond to form a metal-phospholipid complex.
Step three: mixing the metal-phospholipid complex prepared in step two, (ii) conjugated lipid that inhibits aggregation of particles, (iii) non-cationic lipid or non-ionizable lipid to obtain the metal-phospholipid complex particles.
Further, in the first step, the phospholipid molecules and the linker molecules are dissolved in ethanol for reaction, and then n-hexane is added to precipitate to obtain the phospholipid complex. Further, the reaction conditions include a reaction time of 2 hours at 65 ℃.
Further, the molar ratio of the phospholipid molecules to the linker molecules is 1:1.
Further, in the second step, the metal-phospholipid complex is obtained after the phospholipid complex and the metal ions are dissolved in ethanol for reaction. Further, the reaction conditions include a reaction at 60℃for 2 hours.
Further, the molar ratio of the phospholipid complex to the metal ion is 1: (1-2).
The present invention provides the use of the above metal-phospholipid complex particles in drug-lipid particles.
Further, the drug-lipid particles contain a drug, which is a negatively charged molecule, and the metal-phospholipid complex particles, in which the drug is encapsulated.
Further, the drug is selected from the group consisting of a nucleic acid, a protein, a polypeptide, a small molecule, a nucleic acid analog, a protein analog, and a polypeptide analog.
Further, the nucleic acid is selected from one or more of mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA and artificial nucleic acid.
Further, the nucleic acid is an mRNA sequence of eGFP (enhanced green fluorescent protein ) shown in SEQ ID No.1, an mRNA sequence of RBD (receptor binding domain) shown in SEQ ID No.2, an mRNA sequence of NY-ESO-1 (New York esophageal squamous cell 1,New York esophageal squamous cell carcinoma1) shown in SEQ ID No.3, an antisense strand of Bcl-2 gene (B cell lymphoma/Leukemia-2 gene) shown in SEQ ID No.4 and a sense strand of SEQ ID No.21, an siRNA sequence of PLK1 gene (polo-like kinase1 ) shown in SEQ ID No.6 and a sense strand of PLK1 gene (polo-like kinase1 ) shown in SEQ ID No.23, an ASO sequence of the STAT-3 gene shown in SEQ ID No.8, an alpha-synuclein gene shown in SEQ ID No.12 (alpha-synuclein gene, alpha-syn-nucleotide) shown in SEQ ID No.21, an antisense strand of SEQ ID No.6 and a single-stranded RNA sequence shown in SEQ ID No.18, an antisense strand of SEQ ID No.14, an antisense strand of SEQ ID No.18, a single-stranded DNA sequence shown in SEQ ID No.19, and a sense strand of SEQ ID No. 18.
The present invention provides the use of the above metal-phospholipid complex particles in a composition for the delivery of a drug. Further, the composition is used for introducing a drug into a cell. The composition is a medicament. Further, the agent is useful for silencing expression of a target sequence in a mammalian subject, for delivering a drug in a mammal, for delivering a drug from within a body to a mammalian cell, or for treating a disease or condition of a mammal.
In the above technical solution, further, the mammal is a human. Further, the disease or disorder is associated with expression of a gene comprising a target sequence for a drug. Further, the disease or condition includes cancer, viral infection, autoimmune disease, diabetes, or Alzheimer's disease. Further, the viral infection includes hepatitis A, hepatitis B, hepatitis C, SARS-Cov-2 (2019 novel coronavirus), HIV (human immunodeficiency virus), HPV (human papilloma virus), influenza, smallpox or syphilis. Further, the cancer includes liver cancer, glioma, melanoma, lung cancer, pancreatic cancer or breast cancer.
Further, the agent is a vaccine. Further, the route of administration of the agent includes intrathecal injection, intramuscular administration, intracranial injection, intravenous injection or intratumoral injection.
A medicament comprising the metal-phospholipid complex particles of the present invention.
Further, the medicament is a vaccine.
Further, the vaccine is a novel coronavirus vaccine.
Compared with the prior art, the invention has the following beneficial effects:
under the condition that the effectiveness of the metal-phospholipid composite particles is not lower than that of LNP based on cationic lipid and/or ionizable lipid, the metal-phospholipid composite particles do not use the cationic lipid and the ionizable lipid, so that compared with LNP, the toxicity is greatly reduced, the biological safety is remarkably improved, and the carrying of negative charge medicines in organisms is facilitated.
Drawings
In order to more clearly illustrate the specific embodiments of the invention or the technical solutions in the prior art, the drawings that are necessary for the description of the specific embodiments or the prior art will be briefly described, it being obvious that the drawings in the following description are some embodiments of the invention and that other drawings may be obtained from these drawings without inventive effort for a person skilled in the art.
FIG. 1-1 shows the eGFP-mRNA@MPP (Fe) provided in example 3.5.1 of the present invention 3+ ) Percentage of eGFP positive cells due to transfection of 293T;
FIGS. 1-2 show RBD-mRNA@MPP (Fe) as provided in example 3.5.1 of the present invention 3+ ) Expression level of RBD resulting from transfection of 293T;
FIGS. 1-3 illustrate RBD-mRNA@MPP (Fe) provided in example 3.5.1 of the present invention 3+ ) The ability to induce humoral immunity;
FIGS. 1-4 show the NY-ESO-1-mRNA@MPP (Fe) provided in example 3.5.1 of the present invention 3+ ) The ability to induce humoral immunity;
FIGS. 1-5 illustrate RBD-mRNA@MPP (Fe) provided in example 3.5.1 of the present invention 3+ ) The ability to induce cellular immunity;
FIGS. 1-6 show the NY-ESO-1-mRNA@MPP (Fe) provided in example 3.5.1 of the present invention 3+ ) The ability to induce cellular immunity;
FIGS. 1-7 show eGFP-mRNA@MPP (Al) as provided in example 3.5.2 of the present invention 3+ ) Percentage of eGFP positive cells due to transfection of 293T;
FIGS. 1-8 illustrate RBD-mRNA@MPP (Al) provided in example 3.5.2 of the present invention 3+ ) Expression level of RBD resulting from transfection of 293T;
FIGS. 1-9 illustrate RBD-mRNA@MPP (Al) provided in example 3.5.2 of the present invention 3+ ) The ability to induce humoral immunity;
FIGS. 1-10 show the NY-ESO-1-mRNA@MPP (Al) provided in example 3.5.2 of the present invention 3+ ) The ability to induce humoral immunity;
FIGS. 1-11 illustrate RBD-mRNA@MPP (Al) provided in example 3.5.2 of the present invention 3+ ) The ability to induce cellular immunity;
FIGS. 1-12 show the NY-ESO-1-mRNA@MPP (Al) provided in example 3.5.2 of the present invention 3+ ) The ability to induce cellular immunity;
FIGS. 1-13 are the Bcl-2-siRNA@MPP (Fe) provided in example 3.6.1 of the present invention 3+ ) The ability to silence a target gene;
FIGS. 1-14 show PLK1-siRNA@MPP (Fe) as provided in example 3.6.1 of the present invention 3+ ) The ability to silence a target gene;
FIGS. 1-15 show Gal-1-siRNA@MPP (Fe) provided in example 3.6.1 of the present invention 3+ ) The ability to silence a target gene;
FIGS. 1-16 are the Bcl-2-siRNA@MPP (Al) provided in example 3.6.2 of the present invention 3+ ) The ability to silence a target gene;
FIGS. 1-17 show PLK1-siRNA@MPP (Al) as provided in example 3.6.2 of the present invention 3+ ) The ability to silence a target gene;
FIGS. 1-18 show Gal-1-siRNA@MPP (Al) provided in example 3.6.2 of the present invention 3+ ) The ability to silence a target gene;
FIGS. 1-19 show STAT3-ASO@MPP (Fe) provided in example 3.7.1 of the present invention 3+ ) The ability to silence a cellular target gene;
FIGS. 1-20 show a. Alpha. -syn-ASO@MPP (Fe) provided in example 3.7.1 of the present invention 3+ ) The ability to silence a cellular target gene;
FIGS. 1-21 show the Bcl-2-ASO@MPP (Fe) provided in example 3.7.1 of the present invention 3+ ) The ability to silence a cellular target gene;
FIGS. 1-22 show STAT3-ASO@MPP (Al) provided in example 3.7.2 of the present invention 3+ ) SilencingThe ability of the cell to target genes;
FIGS. 1-23 illustrate an α -syn-ASO@MPP (Al) provided in example 3.7.2 of the present invention 3+ ) The ability to silence a cellular target gene;
FIGS. 1-24 show the Bcl-2-ASO@MPP (Al) provided in example 3.7.2 of the present invention 3+ ) The ability to silence a cellular target gene;
FIGS. 1-25 show the S-mRNA@MPP (Fe) provided in example 3.8.1 of the present invention 3+ ) Expression level of S protein resulting from transfection of 293T;
FIGS. 1-26 show drug (dsDNA and ssDNA) -lipid particles (Fe) according to example 3.8.1 of the present invention 3+ ) Is a function of (2);
FIGS. 1-27 show the S-mRNA@MPP (Al) provided in example 3.8.2 of the present invention 3+ ) Expression level of S protein resulting from transfection of 293T;
FIGS. 1-28 show drug (dsDNA and ssDNA) -lipid particles (Al) according to example 3.8.1 of the present invention 3+ ) Is a function of (2);
FIG. 2-1 is a differential scanning calorimetry trace of the phospholipid complex provided in example 4.1 of the present invention;
FIG. 2-2 shows a metal-phospholipid complex (Fe) according to example 4.1 of the present invention 3+ ) Is a uv absorption diagram of (2);
FIGS. 2 to 3 show the metal-phospholipid complex (Al) according to example 4.2 of the present invention 3+ ) Is a uv absorption diagram of (2);
FIGS. 2-4 show the Fe at low pH (pH=5.0) provided in example 5 of the present invention 3+ Characterization of shedding from metal-phospholipid complexes;
FIGS. 2 to 5 show the drug-metal-phospholipid complex particles (Fe) according to example 6.1 of the present invention 3+ ) Elemental analysis of (a);
FIGS. 2-6 are diagrams showing drug-metal-phospholipid complex particles (Al) according to example 6.2 of the present invention 3+ ) Electron microscope analysis of the medium MPP;
FIGS. 2-7 show the efficiency of drug-lipid particle encapsulation of nucleic acids (mRNA and siRNA) provided in example 7 of the present invention;
FIGS. 2-8 are siRNA/mRNA@MPP and methods provided in example 8 of the present invention
Nucleic acid lysosomal escape ability of siRNA/mrna@lnp;
FIGS. 2-9 are graphs showing the eGFP positive cell rates for MPP and LNP provided in example 9 of the present invention;
FIGS. 2-10 are graphs comparing MPP provided in example 10 of the present invention with the mRNA expression enhancing capacity of LNP;
FIGS. 2-11 are graphs comparing the humoral immunity-enhancing capabilities of MPP and LNP provided in example 10 of the present invention;
FIGS. 2-12 are graphs comparing the MPP provided in example 10 of the present invention with the capability of LNP to promote cellular immunity;
FIG. 3-1 shows the effect of the drug-metal-phospholipid complex of example 13 of the present invention on intratumoral injection for treating liver cancer.
Detailed Description
Definition of the definition
For purposes of explanation, specific terms set forth in this specification, examples, and appended claims are generally described herein. Unless defined otherwise herein, the meanings of scientific and technical terms used herein are the same as commonly understood and used by one of ordinary skill in the art to which this invention belongs. In addition, unless the context requires otherwise, it should be understood that the singular terms shall include the same plural form and the plural terms shall include the singular. In particular, the terms "at least one" and "one" or "a plurality" as used herein and in the appended claims include one, two, three or more unless the context clearly dictates otherwise.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain standard deviations found in their respective testing measurements. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within an acceptable standard error of the average value, depending on the consideration of the person having ordinary skill in the art to which the present invention pertains. Except in the experimental examples, or where otherwise explicitly indicated, all ranges, amounts, values, and percentages used herein (e.g., to describe amounts of materials, lengths of time, temperatures, operating conditions, ratios of amounts, and the like) are to be understood to be modified by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that may vary depending upon the desired properties. At least these numerical parameters should be construed as the number of significant digits and by applying ordinary rounding techniques.
The term "lipid" refers to a group of organic compounds including, but not limited to, lipids of fatty acids. They are generally divided into three categories: "simple lipids", "compound lipids", "derivatized lipids". "simple lipids" which include fats and oils and waxes; "Compound lipids" which include phospholipids and glycolipids; "derivatized lipids" such as steroids.
The term "lipid vesicle" refers to any lipid composition useful for delivering a compound, including, but not limited to, liposomes, wherein a volume of water is enclosed by an amphiphilic lipid bilayer; or an interior wherein the lipid coating comprises a macromolecular component, such as an aqueous interior comprising mRNA, with concomitant reduction; or lipid aggregates or micelles, wherein the encapsulated ingredients are contained in a relatively chaotic lipid mixture. Herein, a metal-phospholipid complex particle (MPP) is a "lipid vesicle," in which a drug, such as a nucleic acid mRNA, is encapsulated as an encapsulated ingredient, which may be substantially encapsulated and/or partially encapsulated.
The term "phospholipid" refers to lipids containing phosphate groups, and belongs to complex lipids, also known as phospholipids, phospholipids. Phospholipids are the main components of the composition of biological membranes, and are divided into two main types, namely glycerophospholipids and sphingomyelins, and are respectively composed of glycerol and sphingosine. Phospholipids are amphiphilic molecules with a hydrophilic nitrogen or phosphorus-containing head at one end and a long hydrocarbon chain that is hydrophobic (oleophilic) at the other end. For this reason, the hydrophilic ends of phospholipid molecules are close to each other and the hydrophobic ends are close to each other, often forming a phospholipid bilayer, i.e. the structure of the cell membrane, together with other molecules such as proteins, glycolipids, cholesterol, etc.
The phrase "phospholipid molecule moiety" herein refers to a structure that is originally a phospholipid molecule after reaction with other substances.
The phrase "linker molecule moiety" herein refers to the structure of a linker molecule that is the original of the linker molecule after it has reacted with other substances.
The phrase "metal ion moiety" herein refers to a structure in which the metal ion moiety is a metal ion that is reacted with other species.
The phrase "phospholipid complexes" herein refers to complexes formed by the reactive attachment of the above-described phospholipid molecule moiety having a phosphate group to the above-described linker molecule moiety.
The phrase "metal-phospholipid complex" herein refers to a complex having a structure consisting of the above-mentioned phospholipid molecule part having a phosphate group, the above-mentioned linker molecule part, and the above-mentioned metal ion part, the above-mentioned phospholipid molecule part being linked to the above-mentioned linker molecule part, the above-mentioned linker part being linked to the above-mentioned metal ion part by a coordinate bond, and the metal-phospholipid complex being neither a cationic lipid nor an ionizable lipid.
The term "ionizable lipid" refers to a lipid containing positively charged ionizable amine groups that can be protonated at lower pH values and uncharged at physiological pH.
The term "neutral lipid" refers to any of a number of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol.
The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylglyceridephosphatidylserine, diacylglyceridephosphatidic acid, N-dodecanoyl phosphatidylethanolamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysyl phosphatidylglycerol, palmitoyl Oleoyl Phosphatidylglycerol (POPG), and other anionic groups attached to neutral lipids.
The term "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH. These lipids include, but are not limited to, N-dioleyl-N, N-dimethylammonium chloride ("DODAC"); n- (2, 3-dioleyloxy) propyl) -N, N-trimethylammonium chloride ("DOTMA"); n, N-distearyl-N, N-dimethyl ammonium bromide ("DDAB"); n- (2, 3-dioleoyloxy) propyl) -N, N-trimethylammonium chloride ("DOTAP"); 3- (N ', N' -dimethylaminoethane) carbamoyl) cholesterol ("DC-Chol") and N- (1, 2 dimyristoxyprop-3-yl) -N, N-dimethyl-N-hydroxyethyl ammonium bromide ("dmriie"). The following lipids are cationic and have a positive charge below physiological pH: DODAP, DODMA, DMDMA, etc.
The term "hydrophobic lipid" refers to compounds having non-polar groups including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and these groups optionally substituted with one or more aromatic, cycloaliphatic or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerols, dialkylglycerols, N-N-dialkylamino, 1, 2-diacyloxy-3-aminopropane and 1, 2-dialkyl-3-aminopropane.
The term "non-cationic lipid or non-ionizable lipid" refers to a lipid that is neither a cationic lipid nor a non-ionizable lipid, e.g., may be an anionic lipid, a neutral lipid.
Among the components of the metal-phospholipid complex particles, "non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation" in (iii) refers to the lipids remaining in the metal-phospholipid complex particles after the removal of the metal-phospholipid complex while the removal of the conjugated lipids that inhibit particle aggregation.
The term "fusogenic" refers to the ability of a liposome, drug-lipid particle, or other drug delivery system to fuse with a cell membrane. The membrane may be the plasma membrane or a membrane surrounding an organelle, such as an endosome, nucleus, etc.
In metal-phospholipid complex particles, non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation are primarily present as vesicle-forming lipids, the term "vesicle-forming lipids" is intended to include any amphiphilic lipid having a hydrophobic moiety and a polar head group and itself can spontaneously form bilayer vesicles in water, exemplified by most phospholipids.
In metal-phospholipid complex particles, the conjugated lipids that inhibit particle aggregation are primarily present as vesicle-employing lipids, and the term "vesicle-employing lipids" is intended to include any amphipathic lipid that stably binds to a lipid bilayer, as well as other amphipathic lipids, the hydrophobic portion of which is in contact with the inner, hydrophobic region of the bilayer membrane, and the polar head group portion of which is towards the outer, polar surface of the membrane. Lipids employing vesicles include lipids that can be independently adapted to employ a non-lamellar phase, and also to adopt a bilayer structure in the presence of bilayer stabilizing components. Conjugated lipids that inhibit aggregation of drug-lipid particles include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid derivatives), peptides, proteins, detergents, lipid derivatives, PEG-lipid derivatives such as PEG coupled to dialkoxypropyl, PEG coupled to diacylglycerol, PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramide (see, U.S. patent No. 5,885,613, incorporated herein by reference).
The term "amphiphilic lipid" refers to any suitable material in which the hydrophobic portion of the lipid material faces the hydrophobic phase and the hydrophilic portion faces the aqueous phase. Amphiphilic lipids are typically the major component of lipid vesicles. Hydrophilic properties come from the presence of polar or charged groups such as carbohydrates, phosphates, carboxyl groups, sulfato groups, amino groups, mercapto groups, nitro groups, hydroxyl groups, and other similar groups. Hydrophobicity may be imparted by the inclusion of non-polar groups including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphiphilic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, distearoyl phosphatidylcholine, or dioleoyl phosphatidylcholine. Other compounds lacking phosphorus, such as sphingomyelins, glycosphingolipids families, diacylglycerols and β -acyloxyacids are also in the group known as amphiphilic lipids. In addition, the amphipathic lipids described above may be mixed with other lipids, including triglycerides and sterols.
The term "diacylglycerol" refers to a compound having a 2-fatty acyl chain, wherein both R1 and R2 independently have 2-30 carbon atoms bonded to the 1-and 2-positions of glycerol through ester linkages. The acyl groups may be saturated or have varying degrees of unsaturation. Diacylglycerols have the following formula 54:
the term "diacylglycerol-coupled polyethylene glycol" in the present invention the conjugated lipid that inhibits aggregation of particles may be a diacylglycerol-coupled polyethylene glycol, i.e., a diacylglycerol-polyethylene glycol conjugate (DAG-PEG conjugate or PEG-DAG conjugate). In a preferred embodiment, the DAG-PEG conjugate is dilauryl glycerol (C12) -PEG conjugate, ditetradecylglycerol (C14) -PEG conjugate (DMG), dipalmitoyl glycerol (C16) -PEG conjugate or distearyl glycerol (C18) -PEG conjugate (DSG). Those skilled in the art will readily appreciate that other diacylglycerols may be used in the DAG-PEG conjugates of the present invention. Suitable DAG-PEG conjugates for use in the present invention and methods of making and using them are disclosed in U.S. application Ser. No. 10/136,707, published as U.S. P.A2003/0077829, and PCT patent application Ser. No. CA02/00669, each of which is incorporated herein by reference in its entirety.
The term "dialkoxypropyl" refers to a compound having a 2-alkyl chain, wherein R1 and R2 each independently have 2-30 carbons. The alkyl groups may be saturated or have varying degrees of unsaturation. Dialkoxypropyl has the following formula 55:
the term "dialkoxypropyl-coupled PEG", the conjugated lipid inhibiting particle aggregation in the present invention may be a dialkoxypropyl-coupled PEG, i.e., a dialkoxypropyl conjugate (PEG-DAA conjugate). In a preferred embodiment, the PEG-DAA conjugate has the following formula 56:
in formula 56, R1 and R2 are independently selected and are long chain alkyl groups having from about 10 to about 22 carbon atoms. The long chain alkyl groups may be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, lauryl (C12), tetradecyl (C14), hexadecyl (C16), octadecyl (C18), and icosyl (C20). In a preferred embodiment, R1 and R2 are the same, i.e., R1 and R2 are both tetradecyl (i.e., ditetradecyl), R1 and R2 are both octadecyl (i.e., dioctadecyl), and the like. In formula 56, the PEG is a polyethylene glycol having an average molecular weight of about 550 to about 10000 daltons and is optionally substituted at the terminal hydroxyl position with alkyl, alkoxy, acyl, or aryl groups. In a preferred embodiment, the PEG has an average molecular weight of about 1000 to about 5000 daltons, more preferably an average molecular weight of about 1,000 to about 3,000 daltons and even more preferably an average molecular weight of about 2000 daltons. The PEG may be optionally substituted with alkyl, alkoxy, acyl, or aryl groups. In formula 56, L is a linker moiety. Any linker moiety suitable for coupling PEG to the dialkoxypropyl backbone may be used. Suitable linker moieties include, but are not limited to, amido (-C (O) NH-), amino (-NR-), carbonyl (-C (O) -), carbonate (O-C (O) O-), carbamate (-NHC (O) O-), urea (-NHC (O) NH-), succinyl (- (O) CCH 2 CH 2 C (O) -), ethers, disulfides, and combinations thereof. Other suitable linkers are well known in the art.
Phosphatidylethanolamine having various acyl chain groups of varying chain length and saturation levels can be conjugated to polyethylene glycol to form a bilayer stabilizing component as a conjugated lipid that inhibits particle aggregation in the present invention. These phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. Phosphatidylethanolamine comprising saturated or unsaturated fatty acids is preferred, having a carbon chain length in the range of C10-C20. Phosphatidylethanolamine having mono-or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids may also be used. Suitable phosphatidylethanolamine include, but are not limited to, the following: dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dioleoyl phosphatidylethanolamine (DOPE), and distearoyl phosphatidylethanolamine (DSPE).
Such as phosphatidylethanolamine, a ceramide having a plurality of acyl chain groups of varying chain lengths and degrees of saturation may be coupled with polyethylene glycol to form a bilayer stabilizing component as a conjugated lipid that inhibits particle aggregation in the present invention. It will be clear to those skilled in the art that compared to phosphatidylethanolamine, ceramide has only one acyl group, which can be easily varied according to its chain length and degree of saturation. Ceramides suitable for use in accordance with the invention are commercially available. In addition, ceramide can be isolated, for example, from eggs and brain using well known isolation techniques, or synthesized using methods and techniques disclosed in U.S. patent No. 5,820,873, incorporated herein by reference in U.S. patent No. 5,820,873. Using the synthetic routes set forth in the foregoing applications, ceramides with saturated or unsaturated fatty acids having carbon chain lengths in the range of C2-C31 can be prepared.
The term "ATTA" or "polyamide" refers to, but is not limited to, the compounds disclosed in U.S. Pat. nos. 6,320,017 and 6,586,559, which are incorporated herein by reference. These compounds include compounds having the following formula 57:
wherein: r is a member selected from the group consisting of hydrogen, alkyl and acyl; r1 is a member selected from the group consisting of hydrogen and alkyl; or optionally R and R1 and the nitrogen atom to which they are attached form an azido moiety; r2 is a member selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, and amino acid side chains; r3 is a member selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, mercapto, hydrazino, amino, and NR4R5, wherein R4 and R5 are independently hydrogen or alkyl; n is 4-80; m is 2-6; p is 1-4; and q is 0 or 1. It will be clear to those skilled in the art that other polyamides may be used in the compounds of the present invention.
The term "congener" refers to an analog that performs the same or similar function, or a derivative of the same parent nucleus that performs the same or similar function.
As used herein, the terms "mRNA" or "messenger polyribonucleotide" or "messenger RNA" are used interchangeably and refer to a single-stranded polyribonucleotide transcribed from one strand of DNA as a template that carries genetic information that can direct protein synthesis.
As used herein, the terms "sgRNA" or "smallguide RNA" or "guide RNA" or "gRNA" are used interchangeably to guide insertion or deletion of uridine residues into the plastids (kinetoplastid) during RNA editing, a small non-coding RNA that can be paired with pre-mRNA. The gRNA edits the RNA molecule, approximately 60-80 nucleotides in length, transcribed from a single gene.
As used herein, the terms "circRNA" or "circularRNA" or "cyclic polyribonucleotides" or "cyclic RNA" are used interchangeably and refer to polyribonucleotide molecules having a free-end-free (i.e., no free 3 'and/or 5' end) structure, such as polyribonucleotides that form a cyclic or non-covalent structure.
As used herein, the terms "microRNA" or "miRNA" or "microRNA" are used interchangeably and refer to non-coding single-stranded polynucleic acids of about 22 nucleotides in length, having free 3 'and 5' ends, that are capable of inhibiting translation of a target gene protein by binding to the 3'-untranslated region (3' -UTR) of the mRNA of the target gene, thereby modulating a biological function of the cell.
As used herein, the terms "ASO" or "anti-sense oligonucleotide" are used interchangeably and refer to a nucleic acid fragment that is artificially synthesized, complementary to a segment of a target gene or mRNA, that can bind to the target gene/mRNA by the base complementarity principle, thereby blocking the expressed single-stranded poly (deoxyribonucleotides), including antisense DNA and antisense RNA, of the gene.
As used herein, the terms "siRNA" or "smallling" or "shortinging" or "silencingRNA" or "small interfering RNA" or "short interfering RNA" or "silencing RNA" are used interchangeably and refer to a class of double stranded RNA molecules that are 20 to 25 nucleotides in length and are capable of inducing the degradation of target gene mRNA.
As used herein, the term "ecDNA" or "extrachromosomal circular DNA" is used interchangeably and means DNA that breaks off from the chromosome, leaving the chromosome in a circular structure.
The term "nucleic acid derivative" refers to modifications or substitutions to a nucleic acid sequence, including but not limited to chemical modifications to residues, substitutions to nucleotides or deoxynucleotides, modifications to sequences that increase half-life or stability, labeling modifications. For example, chemical modifications include, but are not limited to, phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopicization of any one or more bases. Substitutions to nucleotides or deoxynucleotides include, but are not limited to, nucleic acid analogs (DNA or RNA replaced with PNA) that replace the sugar phosphate backbone with a polypeptide or other backbone. Modifications to the sequence that increase half-life or stability include, but are not limited to, modifications to PEG linkages, fluorine modifications. Label modifications include, but are not limited to, linking fluorophores, amino groups, biotin, digoxin, small peptides, and the like.
The term "artificial nucleic acid": artificially modified nucleic acid molecules including, but not limited to, base modification, ribose modification, PNA, etc.
The term "nucleic acid" refers to a polymer comprising at least two deoxynucleotides or nucleotides in single or double stranded form. Unless specifically limited, the term encompasses nucleic acids comprising 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 particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be obtained by generating sequences in which one or more (or all) of the selected codons are substituted at a third position with mixed base and/or deoxyinosine residues (Batzeret. Nucleic acids Res.19:5081 (1991); ohtsuka et al, J. Biol. Chem.260:2605-2608 (1985); and Cassolet al (1992); rossoliniet al, mol. Cell. Probes8:91-98 (1994)). "nucleotide" includes the sugar Deoxyribose (DNA) or Ribose (RNA), a base, and a phosphate group. Nucleotides are linked by phosphate groups. "bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications to replace new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and haloalkanes. The DNA may be present as antisense, plasmid DNA, parts of plasmid DNA, pre-compressed DNA, products of the Polymerase Chain Reaction (PCR), vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA or derivatives of these groups. The term nucleic acid is used interchangeably with gene, cDNA, mRNA encoded by a gene, and interfering RNA molecules.
The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that includes a partial length or full length coding sequence necessary to produce a polypeptide or polypeptide precursor (e.g., from a, B, C, D, E, G hepatitis virus; or a herpes simplex virus).
As used herein, "gene product" refers to a product of a gene such as a transcript including, for example, DNA, mRNA.
The phrase "expression silencing of a target gene" refers to the ability of the siRNA of the invention to initiate silencing of the target gene. To determine the extent of gene silencing, a sample or assay of cells in the target organism or culture that express a particular construct is compared to a control sample that does not express the construct. Control samples (lacking expression of construct) were set to a relative value of 100%. Inhibition of expression of the target gene is successfully obtained when the test value relative to the control is about 90%, preferably 50%, more preferably 25% -0%. Suitable assays include, for example, detection of protein or mRNA levels using techniques known to those skilled in the art such as spot blotting, northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzymatic action, and phenotypic assays known to those skilled in the art.
A "therapeutically effective amount" or "effective amount" of an siRNA is an amount sufficient to produce a desired effect, e.g., a reduction in expression of a target sequence compared to the normal expression level detected in the absence of the siRNA.
As used herein, the term "aqueous solution" refers to a composition that contains all or part of water.
As used herein, the term "organic lipid solution" refers to a composition that comprises, in whole or in part, an organic solvent having lipids.
As used herein, "systemic delivery" refers to the delivery of a compound that results in a broad biological distribution throughout an organism. Some techniques of administration may result in systemic delivery of certain compounds, but not others. Systemic delivery refers to the effective, preferably therapeutic, amount of a compound in contact with a substantial portion of the body. To achieve a broad biodistribution, blood survival is often required so that the compound is not rapidly degraded or cleared (such as by first passing through organs (liver, lung, etc.) or by rapid, non-specific cell binding) before reaching the disease site distal to the site of administration. Systemic delivery of the drug-lipid particles may be performed in any manner known in the art including, for example, intravenous, subcutaneous, intraperitoneal, and in a preferred embodiment, systemic delivery of the drug-lipid particles is by intravenous delivery.
As used herein, "local delivery" refers to the delivery of a compound directly to a target site in an organism. For example, the compound may be delivered locally by direct injection into a disease site such as a tumor or other target site such as an inflammation site or target organ such as liver, heart, pancreas, kidney, etc.
Metal-phospholipid complexes
In the present invention, the metal-phospholipid complex is composed of a phospholipid molecule portion, a linker molecule portion and a metal ion portion.
In the phospholipid molecule moiety, the cis-trans isomer of the phospholipid molecule of the present invention does not affect the effect of the present invention.
For the fraction of the linker molecule, which is mainly derived from natural plant extracts, such as curcumin, there is a wide range of biological effects including antibacterial, antiviral, antifungal, antioxidant and anti-inflammatory activity. In addition, it is also an effective immunomodulator, which can regulate the activity of various immune cells such as T cells, B cells, macrophages, neutrophils, natural killer cells, dendritic cells and the like, promote the balance of immunity and strengthen the immunity of organisms. Based on the potential immunopotentiation, anti-inflammation, anti-oxidation and anti-sars-cov-2 effects of curcumin molecules, the curcumin is expected to become a potential auxiliary treatment means for resisting COVID-19. In addition, the curcumin molecules have extremely high safety, are listed in the catalogues of food additives and pharmaceutic adjuvants, and the safety is favorable for the clinical drug registration of the drug-lipid whole body, so that the time length of the clinical drug registration is shortened.
For the metal ion moiety, the coordination bond between the linker molecule moiety and the metal ion moiety in the metal-phospholipid complex may be broken under conditions of low pH (ph=5.0) such as lysosomes, and the metal ion is detached from the metal-phospholipid complex.
The ratio of the components in the metal-phospholipid complex can be adjusted according to the structure of the specific metal-phospholipid complex component. The basis for the adjustable proportion of the delivery is as follows: because the phospholipid molecules are connected with the connector molecules by hydrogen bonds, as long as the phospholipid molecules contain a plurality of phosphate groups, the proportion of the phospholipid molecules to the connector molecules can be adjusted according to the number of the phosphate groups contained in the phospholipid molecules when the phospholipid compound is synthesized, namely, when the phospholipid molecules contain two phosphate groups, the proportion of the phospholipid molecules to the connector molecules can be adjusted to be 1:2; when the phospholipid molecule contains three phosphate groups, the ratio of the phospholipid molecule to the linker molecule can be adjusted to 1:3, a step of; because the hydroxyl groups of the linker molecule are linked to the metal ions by coordination bonds, the ratio of the linker molecule to the metal ions may be adjusted according to the number of binding sites contained in the linker, as long as the linker molecule contains a plurality of binding sites.
The phospholipid molecules with phosphate groups are connected with the connector molecules to obtain a phospholipid complex, and then the phospholipid complex is connected with metal ions to obtain the metal-phospholipid complex. Specifically, phospholipid molecules and connector molecules are dissolved in proper amount of ethanol according to a molar ratio, the mixture is reacted at about 65 ℃, and then n-hexane is added to precipitate to obtain a phospholipid complex; the phospholipid complex is then complexed with a metal ion (e.g., feCl 3 And the like) is dissolved in an appropriate amount of ethanol according to a molar ratio, and the reaction is carried out at about 60 ℃ to obtain a metal-phospholipid complex.
Metal-phospholipid complex particles (MPP)
The principle of loading nucleic acid with metal-phospholipid complex particles assembled from metal-phospholipid complexes is: the linker molecule and the phospholipid molecule are bound together by hydrogen bonds, while the linker molecule is linked to the metal ion by a coordination bond, forming a metal-phospholipid complex, the metal ion of which is linked to the negatively charged drug by a coordination bond, thereby ensuring self-assembly of the metal-phospholipid complex with other components (conjugated lipids that inhibit particle aggregation, and non-cationic lipids or non-ionizable lipids other than the metal-phospholipid complex and the conjugated lipids that inhibit particle aggregation) into MPP while loading the negatively charged drug into the nanoparticle MPP. In this context, the term "non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit aggregation of the particles" refers to component (iii) of the metal-phospholipid complex particles.
In some embodiments, conjugated lipids that inhibit aggregation of particles refer to conjugated lipids that inhibit aggregation of drug-lipid particles, the primary function being to prevent aggregation of drug-lipid particles, such as PEG coupled to dialkoxypropyl, PEG coupled to diacylglycerol, PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramide, preferably PEG-lipid conjugates. Wherein, the cis-trans isomer of the lipid does not affect the effect of the present invention.
In some embodiments, the metal-phospholipid complex is present in the feedstock at a molar ratio of 5% to less than 10%, 10% to 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%.
In a preferred embodiment, the metal-phospholipid complex is present in the starting material in a molar ratio of 7% to 40%, for example 10% to 40%, 7% to 30%, 15% to 25%, 20% to 30%, more preferably 15%, 25%, 7% or 30%.
In some embodiments, the conjugated lipid that inhibits aggregation of particles is present in the starting material at a molar ratio of 2% to 10%, e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. For example, the content may be 3% to 10%, 4% to 10%, 5% to 10%, and more preferably 3%, 4% or 10%.
In some embodiments, the non-cationic lipid or non-ionizable lipid is cholesterol in a molar ratio of 15% to less than 35%, 35% to 75%, or greater than 75% to 80%, such as 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%, or 80% in the starting material. For example, 35% to 75%, 15% to 56%, 40% to 46% and 35% to 55%, preferably 15%, 40%, 46% or 56%.
In some embodiments, the metal-phospholipid complex particles may optionally contain, in addition to cholesterol, other non-cationic lipids or non-ionizable lipids in a molar ratio of 0% -40% or greater than 40% -51%, such as 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or 51% in the feedstock. For example 5% to 30%, 25% to 35%, 34% to 45%, 20% to 25%, preferably 25%, 35%, 34% or 45%.
Drug-lipid particles
The drug-lipid particles described herein typically include a drug (which is a negatively charged molecule that may be selected from the group consisting of a nucleic acid, a protein, a polypeptide, a small molecule, a nucleic acid analog, a protein analog, a polypeptide analog, a member of the group consisting of one or more combinations of mRNA, siRNA, loop RNA, microRNA, DNA, ecDNA, an artificial nucleic acid), a metal-phospholipid complex, a non-cationic lipid or a non-ionizable lipid, and a bilayer stabilizing component, such as a conjugated lipid that inhibits aggregation of particles. Furthermore, the nucleic acids encapsulated in the drug-lipid particles of the invention are resistant to degradation with nucleases in aqueous solution.
In some embodiments, the drug is sufficiently encapsulated within the metal-phospholipid complex particles to avoid degradation of the drug, enabling delivery of the drug into the cell.
In some embodiments, the drug-lipid particles provided herein have a small diameter suitable for systemic delivery, with a particle size of 30-400 nm; the surface potential is-10 mV; the stability is at least 3 days, preferably more than 7 days; the cell delivery efficiency is 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%.
In some embodiments, the drug of the drug-lipid particle is preferably a nucleic acid, the nucleic acid component typically comprises mRNA, interfering RNA (i.e., siRNA) that can be provided in several forms including, for example, one or more isolated small interfering RNA (siRNA) duplex, longer double-stranded RNA (dsRNA), or siRNA or dsRNA translated from a transcription cassette in a DNA plasmid.
The RNA population can be used to provide long precursor RNAs, or long precursor RNAs that have substantial or complete identity to a selected target sequence that can be used to make the siRNA. The RNAs may be isolated, synthesized, and/or cloned from cells or tissues according to methods well known to those skilled in the art. The RNA may be a mixed population (obtained from cells or tissues, transcribed from cDNA, etc.), or may represent a single target sequence. RNA may be naturally occurring, e.g., isolated from tissue or cell samples, e.g., synthesized in vitro using T7 or SP6 polymerase and PCR products or cloned cDNA; or chemically synthesized.
For the formation of long dsrnas, the complements may also be transcribed and hybridized in vitro to form dsRNA for synthetic RNAs. If a naturally occurring population of RNAs is used, for example by transcription of cDNAs corresponding to the population of RNAs, or by use of RNA polymerase, RNA complements are also provided (e.g., formation of dsRNAs which are digested by E.coli (E.coli) RNAseIII or a nicking enzyme). The precursor RNAs are then hybridized to form double stranded RNAs for digestion. The dsRNAs may be directly encapsulated in SNALPs or may be digested in vitro prior to encapsulation.
Alternatively, one or more DNA plasmids encoding one or more siRNA templates may be encapsulated in a nucleic acid-lipid particle. For example, based on the naturally occurring transcription units of micronuclear RNAU6 or human RNase P RNA H1, siRNA can be transcribed from a DNA template in a plasmid with RNA polymerase III transcription units (see Brummelkamp, et al, science 296:550 (2002); donze, et al, nucleic acids Res.30:46 (2002); paddison, et al, genes Dev.16:948 (2002); yu, et al, proc. Natl. Acad. Sci.99:6047 (2002); lee, et al, nat. Biotech.20:500 (2002); yagishi, et al, nat. Biotech.20:497 (2002); paul, et al, nat. 20:505 (2002); and Sui, acad. Nat. 99:5515). Typically, the transcription unit or cassette will comprise an RNA transcription promoter sequence, such as an H1-RNA or U6 promoter, operably linked to a template for transcription of the desired siRNA sequence and a termination sequence comprising 2-3 uridine residues and a poly (T5) sequence (polyadenylation signal) (Brummelkamp, science, supra). The selected promoter may provide constitutive or inducible transcription. Methods of transcription of compositions and DNA-directed RNA interfering molecules are described in detail in U.S. patent No. 6,573,099, which is incorporated herein by reference. Preferably, the synthetic or transcribed siRNA has a 3 'overhang of about 1-4, preferably about 2-3 nucleotides and a 5' phosphate end (Elbashir, et al, genes Dev.15:188 (2001); et al, cell107:309 (2001)). The transcription unit is incorporated into a plasmid or DNA vector from which the interfering RNA is transcribed. Plasmids suitable for in vivo delivery of genetic material for therapeutic purposes are described in detail in U.S. patent nos. 5,962,428 and 5,910,488, both of which are incorporated herein by reference. The selected plasmid may provide transient or stable delivery of the target cell. Those of skill in the artIt will be clear to the skilled person that plasmids originally designed for expression of the desired gene sequences may be modified to contain a cassette of transcription units for transcription of the siRNA.
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, gene25:263-269 (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 GuidetoMethods and Applications (Innis et al, eds, 1990)). Expression libraries are also well known to those of skill in the art. Additional basic books disclosing the general methods used in the present invention include Sambrook et al Molecular Cloning, A Laboratory Manual (2 nd ed.1989); kriegler, gene Transfer and Expression: a Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994)).
Metal-phospholipid complexes, metal-phospholipid complex particles, use of drug-lipid particles
In some embodiments, when the drug is a nucleic acid, the metal-phospholipid complex, metal-phospholipid complex particles can be used to facilitate drug lysosomal escape, as well as to facilitate nucleic acid expression. The metal-phospholipid complex and metal-phospholipid complex particles can also be used for delivering drugs and introducing the drugs into cells, thereby realizing the prevention and treatment of applicable diseases or symptoms by the drugs.
In some embodiments, the invention provides for the use of the drug-lipid particle, e.g., in a composition that can effect delivery of a drug or introduction of a drug into a cell. The composition is, for example, a medicament which achieves: silencing expression of a target sequence in a mammalian subject, delivering a drug in a mammal (e.g., for treatment of a tumor, imaging agent, etc.), delivering a drug from within a body to a mammalian cell, or treating a disease or condition in a mammal, etc. In the medicament, the medicament-lipid particles are taken as main active ingredients, and can be prepared into different dosage forms, such as solid dosage forms (powder, granules, pills, tablets and gel), semisolid dosage forms (external paste and paste), liquid dosage forms (decoction, mixture, syrup, wine, injection), gas dosage forms (aerosol and smoke) and the like through different pharmaceutically acceptable auxiliary materials or preparation processes according to actual demands; such as those administered parenterally, rectally, parenterally, etc.
In some embodiments, the present invention provides products made of the above-described metal-phospholipid complexes, metal-phospholipid complex particles, and drug-lipid particles, having the above-described functions and uses of the metal-phospholipid complexes, metal-phospholipid complex particles, and drug-lipid particles, of a specific type such as, but not limited to, kits, medicaments, and the like, optionally containing additional excipients.
Target gene for drug-lipid particle action: in general, it is desirable to deliver drug-lipid particles such that translation (i.e., expression) of the target gene product is down-regulated or silenced. Suitable classifications of gene products include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., diseases and disorders in which liver is the target, and liver diseases and disorders), genes associated with tumorigenesis and cellular transformation, angiogenic genes, immunomodulator genes such as those associated with inflammation and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.
Genes associated with viral infection and survival include those that bind by viral expression, enter and replicate in cells. In particular viral sequences associated with chronic viral diseases. For example, viral sequences include sequences of hepatitis virus (Hamasaki, et al, FEBS Lett.543:51 (2003); yokota, et al, EMBO Rep.4:602 (2003), schloma, et al, hepatology 37:764 (2003), wilson, et al, proc.Natl. Acad.Sci.100:2783 (2003), kapad, et al, proc.Natl. Acad.Sci.100:2014 (2003), and FIELDSVIROLOGY (Knope et al eds.2001)), human Immunodeficiency Virus (HIV) (Banerjea, et al, mol Ther.8:62 (2003)), song, et al, J.Virol.77:7174 (2003), stephenson JAMA 289:1494 (2003), qin, et al, proc.Natl. Acad.Sci.100:183), herpes virus (Jia, et al, J.Virol.77:3301 (2003)), and Human Immunodeficiency Virus (HIV) (Banerjea, et al, mol. 8:62 (2003)), song, et al, J.Virol.77:7174 (2003), stephen, J.J.77:6021 (2003)). 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., FIELDSVIROLOGY,2001, supra). Hepatitis C nucleic acid sequences that can be silenced include, but are not limited to: serine proteases (e.g., NS3/NS 4), helicases (e.g., NS 3), polymerases (e.g., NS 5B), and envelope proteins (e.g., E1, E2, and p 7). Hepatitis A nucleic acid sequences are mentioned, for example, in Genbank accession NC-001489; hepatitis b nucleic acid sequences are mentioned, for example, in Genbank accession No. nc_ 003977; hepatitis C nucleic acid sequences are mentioned, for example, in Genbank accession NC-004102; hepatitis delta nucleic acid sequences are mentioned, for example, in Genbank accession number nc_ 001653; hepatitis E nucleic acid sequences are mentioned, for example, in Genbank accession NC-001434; and the hepatitis G nucleic acid sequence is mentioned, for example, in Genbank accession NC-001710. Silencing the sequence encoding the gene associated with viral infection and survival may conveniently be used in conjunction with the administration of conventional agents for the treatment of viral diseases.
Genes associated with metabolic diseases and disorders (e.g., disorders and liver diseases and disorders in which the liver is targeted) include, for example, genes expressed in dyslipidemia (e.g., liver X receptor (e.g., lxrα and lxrβ Genback accession number nm_ 007121)), farnesoid X Receptor (FXR) (Genbank accession number nm_ 005123), sterol Regulatory Element Binding Protein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl coenzyme-a reductase (HMG coenzyme-a reductase), apolipoprotein (ApoB), and apolipoprotein (ApoE)) and diabetes (e.g., glucose-6-phosphate) (see, e.g., for example, forsman et al, cell81:687 (1995); seol.endocrinol.9: 72 (1995), zaackietal, pnasesa 94:7909 (1997); sakai, et al, cell85:1037-1046 (1996); duncan, et al, J.biol. Chem.272:12778-12785 (1997); willy, et al, general dev.9 (9): 1033-45 (1995); lehmann, et al, j.biol.chem.272 (6): 3137-3140 (1997); janowski, et al, nature383:728-731 (199; peet, et al, cell93:693-704 (1998)). Those skilled in the art will appreciate that genes associated with metabolic diseases and disorders (e.g., diseases and disorders in which the liver is targeted and liver diseases and disorders) include genes expressed in the liver itself as well as genes expressed in other organs and tissues. Silencing a sequence encoding a gene associated with metabolic diseases and disorders can be conveniently used in conjunction with the administration of conventional agents for treating the diseases or disorders.
Examples of genes associated with tumorigenesis and cell transformation include translocation sequences such as MLL fusion genes, BCR-ABL (Wilda, et al, oncogene,21:5716 (2002); scherr, et al, blood 101:1566), TEL-AML1, FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO, and AML1-MTG8 (Heidenreich, et al, blood101:3157 (2003)); over-expressed sequences such as multi-drug resistance genes (Nieth, et al, FEBSLett.545:144 (2003); wu, et al, cancer rRs.63:1515 (2003)), cyclin (Li, et al, cancer rRs.63:3593 (2003); zou, et al, general Dev.16:2923 (2002)), beta-catenin (Verma, et al, clinCance rRs.9:1291 (2003)), telomerase genes (Kosciole, et al, molCancer Ther.2:209 (2003)), c-MYC, N-MYC, BCL-2, ERBB1 and ERBB2 (Nagy, et al exp. Cell Res.285:39 (2003)); and mutant sequences such as RAS (reviewed in Tuschl and Borkhardt, mol. Intermediates, 2:158 (2002)). Silencing of the sequence encoding the DNA repair enzyme was used in conjunction with administration of a chemotherapeutic agent (Collis, et al, cancer rRs.63:1550 (2003)). Genes encoding proteins associated with tumor migration, such as integrins, selectins and metalloproteinases, are also target sequences of interest. Any complete or partial gene sequence that favors or promotes tumorigenesis or cell transformation, tumor growth or tumor migration can be included as a template sequence.
Angiogenic genes can promote the formation of new blood vessels. Vascular Endothelial Growth Factor (VEGF) is the major research direction (Reich, et al, mol. Vis.9:210 (2003)).
An immunomodulator gene is a gene that modulates one or more immune responses. Examples of immunomodulator genes include cytokines such as growth factors (e.g., TGF- α, TGF- β, EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12 (Hill, et al, J. Immunol.171:691 (2003)), IL-15, IL-18, IL-20, etc.), interferons (e.g., IFN- α, IFN- β, IFN- γ, etc.), and TNF. Fas and Fas ligand genes are also target immunomodulator sequences of interest (Song, et al, nat. Med.9:347 (2003)). Genes encoding secondary signaling molecules in hematopoietic and lymphoid cells are also included in the present invention, for example, tec family kinases such as Bruton's tyrosine kinase (Btk) (heinone, et al, febslett.527:274 (2002)).
Cell receptor ligands include ligands that bind to cell surface receptors (e.g., insulin receptor, EPO receptor, G-protein coupled receptor, receptor with tyrosine kinase activity, cytokine receptor, growth factor receptor, etc.) to modulate (e.g., inhibit, activate, etc.) physiological pathways (e.g., glucose level regulation, blood cell development, mitogenesis, etc.) involved in the receptor. Examples of cellular receptor ligands include cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G-protein coupled receptor ligands, and the like. Templates encoding the expansion of trinucleotide repeats (e.g., CAG repeats) find use in silencing pathogenic sequences in neurodegenerative diseases caused by the expansion of trinucleotide repeats, such as spinal bulbar muscular atrophy and huntington's disease (replen, et al, hum. Mol. Genet.11:175 (2002)).
Injectable delivery: in some cases, such as in U.S. Pat. nos. 5,543,158; it is desirable to deliver the drug-lipid particles disclosed herein parenterally, intravenously, intramuscularly, subcutaneously, intradermally, or intraperitoneally as described in U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363. The drug-lipid particles can be injected locally to a target site (e.g., a disease site such as inflammation or tumor formation or to a target organ or tissue) or systemically for widespread distribution to organisms. Solutions of the drug-lipid particles may be prepared in water, suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Optionally, these formulations contain a preservative to prevent the growth of microorganisms. Typically, when administered intravenously, the drug-lipid particle formulation is formulated with a suitable pharmaceutical carrier. Typically, a common buffered saline solution (135-150 mM NaCl) will be used as a pharmaceutically acceptable carrier, but other suitable carriers will suffice. Additional suitable vectors are described, for example, in REMINGTON' S PHARMACEUTICAL SCIENCES, mack Publishing Company, philadelphia, pa., 17th ed. (1985). As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The formulation of an aqueous composition comprising a protein as an active ingredient is well understood in the art. Alternatively, these compositions are prepared as injectable solutions, liquid solutions or suspensions; solid forms suitable for solution or suspension in a liquid prior to injection may also be prepared. The formulation may also be emulsified.
The drug-lipid particles may be sterilized by conventional liposome sterilization techniques, such as filtration. The drug-lipid particles may contain pharmaceutically acceptable auxiliary substances which are suitable physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, etc. These compositions may be sterilized using the techniques referred to above, or alternatively, they may be produced under sterile conditions. The resulting aqueous solution may be packaged for use or filtered under sterile conditions and lyophilized, and the lyophilized formulation combined with the sterile aqueous solution prior to administration.
Prophylactic and therapeutic treatment: in some embodiments, the drug-lipid particles can be used for prophylactic or therapeutic treatment of a subject (e.g., a mammalian subject) having a disease or disorder associated with expression or overexpression of a target sequence. The drug-lipid particles are administered to a subject in an amount sufficient to elicit a therapeutic response in a patient. An amount sufficient to accomplish this is defined as a "therapeutically effective dose or amount" or "effective dose or amount". In determining an effective amount of drug-lipid particles to be administered in the treatment or prevention of a disease caused by expression or overexpression of a target gene, the physician evaluates circulating plasma levels of drug-lipid particles, drug-lipid particle toxicity and progression of the disease associated with expression or overexpression of the target gene. Administration may be accomplished in single or divided doses.
For example, the drug-lipid particles may be administered to a subject infected with or at risk of being infected with a pathogenic microorganism. The drug should preferably correspond to a sequence that has a critical role in the life cycle of the microorganism, and should also be unique to the microorganism (or at least absent from the natural genome of the patient undergoing treatment). The drug-lipid particles are introduced into the target cells, tissues or organs by ex vivo (ex vivo) or intravenous injection at a therapeutically effective dose. Silencing a sequence encoding a gene associated with a pathogenic infection may conveniently be used in combination with administration of conventional agents for the treatment of pathogenic diseases. The treatment may be administered prophylactically to a person at risk of being infected with a pathogenic microorganism or having been infected with a pathogenic microorganism.
In a preferred embodiment, the drug-lipid particles of the present invention may be conveniently used for the treatment of cancer, viral infections, autoimmune diseases, diabetes, alzheimer's disease. Viral infections include hepatitis A, hepatitis B, hepatitis C, SARS-Cov-2, HIV, HPV, influenza, smallpox, syphilis. For example, suitable sites for inhibiting hepatitis B virus include nucleic acid sequences encoding 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 associated with conventional treatments for hepatitis such as, for example, immunoglobulins, interferons (e.g., pegylated and unpegylated interferon a) (see, for example, medina et al, antiviralres.60 (2): 135-143 (2003), ribavirin (see, for example, hugle and Cerny, rev. Med. Virol.13 (6): 361-71 (2003)), adefovir and lamivudine (see, for example, kocketal, hepatolog 38 (6): 1410-8 (2003)), prenylation inhibitors (see, for example, bornietal, J. Clin. Et. 112 (3): 407-414 (2003)); acyclovir (see, for example, yurdaydin et al, jhepatil. 37 (2): 71 (2002)), and saponins c and d (see, for example, chicketal, 60. 69-2003).
In another embodiment, the drug-lipid particles of the invention may be conveniently used to treat diseases and conditions characterized by expression or overexpression of a gene or a gene cluster. In some aspects, the drug-lipid particles of the invention may be used to treat metabolic diseases and disorders (e.g., diseases and disorders in which the liver is the target and liver diseases and disorders) such as, for example, dyslipidemia and diabetes. Those skilled in the art will appreciate that silencing of genes associated with metabolic diseases and disorders may be combined with conventional treatment of such diseases. For example, the silencing of genes involved in dyslipidemia can be associated with the use of inhibin, bile acid sequestrants/resins and cholesterol absorption inhibitors such as ezetimibe, plant stanols/sterols, polyphenols, and nutraceuticals such as oat bran, flaxseed and soy protein, plant stanol (phytostanol) analogues, squalene synthetase inhibitors, bile acid transport inhibitors SREBP Cleavage Activated Protein (SCAP) activated ligands, niacin (niacin), acipimox, high dose fish oil, antioxidants and policosanol, microsomal triacylglycerol transporter (MTP) inhibitors, acyl-coa: cholesterol Acyltransferase (ACAT) inhibitors, gemcabene, li Feibei ro, pantothenate analogs, niacin-receptor agonists, anti-inflammatory agents (such as Lp-PLA (2) antagonists and AGI 1067) functional oils, PPAR-alpha, gamma, delta agonists, and dual PPAR-alpha,/gamma and 'pan' PPAR-alpha/gamma,/delta agonists, cholesteryl Ester Transfer Protein (CETP) inhibitors (such as torcetrapib), CETP vaccines, up-regulation of ATP-binding cassette transporter (ABC) A1, lecithin Cholesterol Acyltransferase (LCAT) and scavenger receptor type B1 (SRB 1), and synthetic apolipoprotein (Apo) E-related peptides, extended release niacin/lovastatin, atorvastatin/amlodipine, ezetimibe/simvastatin, atorvastatin/CETP inhibitors, tin/agonists, extended release niacin/simvastatin and pravastatin in development and extended release niacin/simvastatin and pravastatin (such as described below) and anti-stepstatin (stepstatin, such as sur-11, fig. 2003, fig. 11 and fig. 11, B1). Likewise, the silencing of genes involved in diabetes can be combined with treatment with insulin, dietary modification and exercise.
Similar methods are used to inhibit the expression of endogenous receptor cellular genes associated with tumorigenesis and cellular transformation, tumor growth and tumor migration; inhibiting expression of angiogenic genes; inhibiting expression of immunomodulator genes, such as those associated with inflammation and autoimmune responses; inhibiting expression of the ligand receptor gene; inhibiting expression of a gene associated with a neurodegenerative disorder; and inhibiting expression of additional genes associated with viral infection and survival. The target gene sequence of a specific target is as described above.
Detecting the particles: the drug-lipid particles herein are detected using any method known in the art. For example, the label may be coupled directly or indirectly to the component of the drug-lipid particle or other lipid-based carrier system using methods well known in the art. A wide variety of labels may be used, with the choice being made based on the sensitivity desired, the ease of conjugation to the drug-lipid particle component, stability requirements and the available tools and preparation for handling. Suitable labels include, but are not limited to, spectroscopic labels such as fluorescent dyes (e.g., fluorescein and derivatives such as Fluorescein Isothiocyanate (FITC) and oregon greentm; rhodamine and derivatives such as texas red, tetrarhodimineisothiocynate (TRITC), etc.), digoxigenin, biotin, phycoerythrin, AMCA, cydye, etc., radioactive labels such as 3h,125i,35s,14c,32p,33p, etc., enzymes such as horseradish peroxidase, alkaline phosphatase, etc., spectroscopic colorimetric labels such as colloidal gold or colored glass or plastic beads such as polystyrene, polypropylene, latex, etc.) the labels are detected using any means known in the art.
Detection of nucleic acid: the nucleic acids herein are detected and quantified by any of a number of means well known to those skilled in the art. Nucleic acid detection is performed by methods well known in the art such as southern blot analysis, northern blot analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Additional analytical biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high Performance Liquid Chromatography (HPLC), thin Layer Chromatography (TLC), hyperdiffusion chromatography may also be applied.
The sensitivity of hybridization assays can be increased by the use of nucleic acid amplification systems that double the target nucleic acid to be detected. In vitro amplification techniques suitable for amplifying sequences used as molecular probes or generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques sufficient to guide the skilled artisan through these in vitro amplification methods, including Polymerase Chain Reaction (PCR), ligase Chain Reaction (LCR), qβ -replicase amplification and other RNA polymerase mediated techniques (e.g., nasatm), are found in Sambrook, et al, molecular Cloning: a Laboratory Manual, coldSpring Harbor Laboratory Press,2000, and Ausubel et al, SHORT PROTOCOLSIN MOLECULAR BIOLOGY, eds, current Protocols, a joint venture between Greene Publishing Associates, inc.and John Wiley & Sons, inc., (2002), and Mullis et al (1987), U.S. Pat. nos. 4,683,202; PCR Protocols A Guide toMethods and Applications (Innis et al eds.) Academic Press Inc. san Diego, calif. (1990) (Innis); arnheim & Levinson (October 1, 1990), C & EN36; theJournal Of NIH Research,3:81 (1991); (Kwoh et al, proc.Natl.Acad.Sci.USA,86:1173 (1989); guatelli et al, proc.Natl.Acad.Sci.USA,87:1874 (1990); lomelal et al, J.Clin.chem.,35:1826 (1989); landieren et al, science,241:1077 (1988); van Brunt, biotechnology,8:291 (1990); wu and Wallace, gene,4:560 (1989); barringer et al, gene,89:117 (1990), and Sooknanan and Malek, biotechnology,13:563 (1995); improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al, U.S. Pat. No. 5,426,039. Nucleic acid sequence-based amplification (NAu. Misslight, sookara, SBQ and SBQ).
Nucleic Acids res., needham VanDevanter et al, 12:6159 (1984), typically as per Beaucage and Caruthers, tetrahedron letters, 22 (20): 18591862 The solid phase phosphoramidite triester method of (1981), wherein the oligonucleotide is chemically synthesized, for example, using an automated synthesizer, and is used as a probe, for example, in an in vitro amplification method, as a genetic probe, or as an inhibitor component. If necessary, e.g., pearson and Regnier, j.chrom.,255:137149 Purification of the oligonucleotides is typically performed by natural acrylamide gel electrophoresis or by anion exchange HPLC, as described in (1983). Academic Press, new York, methods in Enzymology,65, using Maxam and Gilbert (1980) in Grossman and Moldave (eds.): the chemical degradation method in 499 can confirm the sequence of the synthetic oligonucleotide.
The following examples provide illustration, but do not limit the claimed invention. Those skilled in the art will readily recognize various non-critical parameters that may produce substantially the same result.
The drug-lipid particles to be protected by the present invention refer to drug-lipid particles other than cation/ionizable lipids, i.e. drug-loaded metal-chelated phospholipid complex nanoparticles, drug@mpp.
Experimental example one, preparation of drug-Metal-phospholipid Complex particles
Example 1 preparation of phospholipid Complex
Linking a phospholipid molecule having a phosphate group to a linker molecule: distearoyl phosphatidylcholine (DSPC, formula 46) and curcumin (formula 19) were mixed in a molar ratio of 1:1 is added into a reaction bottle, a proper amount of ethanol is added for dissolution, and after reaction for 2 hours at 65 ℃, the mixture is concentrated and added with n-hexane. Precipitating to obtain phospholipid complex, filtering, and vacuum drying to obtain phospholipid complex. The phospholipid complex structure is shown below:
analysis of results: the yield of the target product obtained by reacting curcumin with DSPC at 65 ℃ for 2 hours was 94%.
Example 2 preparation of Metal-phospholipid complexes
EXAMPLE 2.1 preparation of the metal ion Fe 3+ Metal-phospholipid complexes when used
The phospholipid complexes prepared in example 1 are partially linked to metal ions: phospholipid complex and FeCl 3 According to the mole ratio of 1:1, adding the mixture into a reaction bottle, adding ethanol for dissolution, reacting for 2 hours at 60 ℃, suspending the reaction liquid, washing with ultrapure water, and vacuum drying to obtain the metal-phospholipid complex. The structure of the metal-phospholipid complex is shown below.
Analysis of results: phospholipid complexes and FeCl 3 Reacting for 2 hours at 60 ℃, wherein the feeding concentration of the phospholipid complex is 4.5mg/mL, and the phospholipid complex and FeCl are mixed 3 The feed ratio of (2) is 1: the yield of the target product obtained at 1 was 95%.
EXAMPLE 2.2 preparation of metal ion to Al 3+ Metal-phospholipid complexes when used
This example differs from example 2.1 in that FeCl 3 Replaced by Al (NO) 3 ) 3 ·9H 2 O. The structure of the prepared metal-phospholipid complex is shown below.
Analysis of results: phospholipid complexes with Al (NO) 3 ) 3 ·9H 2 O was reacted at 60℃for 2 hours at a phospholipid complex charge concentration of 4.5mg/mL and a phospholipid complex with Al (NO) 3 ) 3 ·9H 2 The feeding ratio of O is 1: the yield of the target product obtained at 1 was 95%.
Example 3 preparation of mRNA-Metal-phospholipid Complex particles (mRNA-loaded Metal-chelated phospholipidcomplexnanoparticles, mRNA@MPP)
Preparation of metal ion Fe 3+ When mRNA-metal-phospholipid complex particles
A metal-phospholipid complex was prepared according to the method in example 2.1, wherein DSPC, curcumin, feCl 3 The metal-phospholipid complex and distearoyl phosphatidylcholine (DSPC, formula 46, as a non-cationic lipid or a non-ionizable lipid), cholesterol (CHOL, formula 40, as a non-cationic lipid or a non-ionizable lipid), DSPE-PEG2000 (formula 53, as a conjugated lipid that inhibits particle aggregation) were dosed at a 1:1:1 ratio and dissolved in ethanol as an organic phase at different molar ratios. Wherein the metal-phospholipid complex, DSPC, CHOL and DSPE-PEG2000 account for 15%, 35%, 46% and 4%, respectively. mRNA was dissolved in an enzyme-free PBS buffer at a concentration of 20. Mu.g/mL as an aqueous phase (PBS composed of 0.137M sodium chloride, 0.0027M potassium chloride, 0.01M disodium hydrogen phosphate and 0.0018M potassium dihydrogen phosphate). The metal-phospholipid complex mass and the mRNA mass are mixed in a micro-fluidic chip according to the 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 phase and the aqueous phase in the microfluidic chip was 12ml/min. Wherein, the medicine mRNA is mRNA encoding fluorescent protein eGFP, and the sequence is SEQ ID NO.1 (720 nt). And preparing the eGFP-mRNA@MPP. eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2. Mu.g/mL (concentration of mRNA contained), control was incubated with MPP without drug loading, and after 48h the cell suspension was harvested and the percentage of eGFP positive cells was detected by flow cytometry.
Particle size, surface potential and stability were measured for the eGFP-mRNA@MPP prepared in example 3, and the efficiency of the eGFP-mRNA@MPP entrapment of nucleic acid was calculated.
Particle size detection method and result judgment standard: the nanoparticle size was measured using a malvern laser sizer Zetasizer and was considered acceptable in the 30-400 nm range.
Method for detecting surface potential and result judgment standard: the nanoparticles were tested for surface potential using a malvern laser sizer Zetasizer, with potentials in the range of-10 to 10mV considered acceptable.
Method for detecting stability and result judgment standard: the nanoparticles were placed at 4 ℃ for 7 days, and the particle size and surface potential of the nanoparticles were tested using a malvern laser sizer and were considered to be better in stability when there was no significant change in the particle size and surface potential over 3-7 days.
Method for calculating nucleic acid entrapment efficiency: in particular to an agarose gel electrophoresis method. Firstly, the nucleic acid feeding amount of each group of lipid nano particles is 10 mug/mL, and the metal ion is Fe 3+ The mass ratio of the metal-phospholipid complex to the mRNA is 40:1, the nucleic acid with the same concentration is dissolved in PBS buffer solution to be used as a positive control, and the negative control is PBS buffer solution. Agarose gel concentration is 1.5%, where the gel void allows only free nucleic acid to pass through but not lipid nanoparticles, and the electrophoresis is stopped when the free nucleic acid band is electrophoresed to a clear resolution. The grey scale value of the free nucleic acid in different groups is counted by using imageJ software, the positive control group is defined as 100%, the ratio of the free nucleic acid in each group relative to the positive control is the relative amount of the free nucleic acid, and the entrapment rate in each group is (100-relative amount of the free nucleic acid). Nucleic acid entrapment rates above 50% are considered acceptable ranges.
Cell culture method: human embryonic kidney cell line 293T was cultured with DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37℃with 5% CO 2 Is cultured under the condition of (2).
Flow cytometry method for analyzing the percentage of eGFP positive cells: 293T cells were seeded onto 24-well plates at a density of 5X 10 5 Cells/well, when cell density reached 80%, cells were incubated with 1mLMPP or eGFP-mRNA@MPP, where the eGFP-mRNA@MPP concentration was 2. Mu.g/mL. After 48h, the cell suspension was collected, 20000 cells were collected by flow cytometer FITC channel, and analyzed for eGThe percentage of FP positive cells was calculated as: eGFP positive cell rate calculation formula = number of cells expressing eGFP/total number of cells x 100%. The percentage of eGFP positive cells up to 40% or more was considered an acceptable range.
Preparation of metal ions of Al 3+ When mRNA-metal-phospholipid complex particles
A metal-phospholipid complex was prepared according to the method in example 2.2, wherein DSPC, curcumin, al (NO 3 ) 3 ·9H 2 O was dosed at a 1:1:1 dosing ratio and metal-phospholipid complex and distearoyl phosphatidylcholine (DSPC, formula 46, as a non-cationic lipid or a non-ionizable lipid), cholesterol (CHOL, formula 40, as a non-cationic lipid or a non-ionizable lipid), DSPE-PEG2000 (formula 53, as a conjugated lipid to inhibit particle aggregation) were dissolved in ethanol as an organic phase at different molar ratios. Wherein the metal-phospholipid complex, DSPC, CHOL and DSPE-PEG2000 account for 7%, 34%, 56% and 3% respectively. mRNA was dissolved in an enzyme-free PBS buffer at a concentration of 20. Mu.g/mL as an aqueous phase (PBS composed of 0.137M sodium chloride, 0.0027M potassium chloride, 0.01M disodium hydrogen phosphate and 0.0018M potassium dihydrogen phosphate). Mixing the metal phospholipid complex mass and the mRNA mass in a mass ratio of 13.3:1 in a microfluidic chip. The volume ratio of the aqueous phase to the organic phase was 3:1. The flow rate of the organic phase and the aqueous phase in the microfluidic chip was 12ml/min. Wherein, the medicine mRNA is mRNA encoding fluorescent protein eGFP, and the sequence is SEQ ID NO.1 (720 nt). The prepared eGFP-mRNA@MPP. eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2. Mu.g/mL (concentration of mRNA contained), control was incubated with MPP without drug loading, and after 48h the cell suspension was harvested and the percentage of eGFP positive cells was detected by flow cytometry.
Particle size, surface potential and stability were measured for the eGFP-mRNA@MPP prepared in example 3, and the efficiency of the eGFP-mRNA@MPP entrapment of nucleic acid was calculated.
Particle size detection method and result judgment standard: the nanoparticle size was measured using a malvern laser sizer Zetasizer and was considered acceptable in the 30-400 nm range.
Method for detecting surface potential and result judgment standard: the nanoparticles were tested for surface potential using a malvern laser sizer Zetasizer, with potentials in the range of-10 to 10mV considered acceptable.
Method for detecting stability and result judgment standard: the nanoparticles were placed at 4 ℃ for 7 days, and the particle size and surface potential of the nanoparticles were tested using a malvern laser sizer and were considered to be better in stability when there was no significant change in the particle size and surface potential over 3-7 days.
Method for calculating nucleic acid entrapment efficiency: in particular to an agarose gel electrophoresis method. Firstly, the nucleic acid feeding amount of each group of lipid nano particles is 10 mug/mL, and the metal ions are Al 3+ The mass ratio of the metal-phospholipid complex to the mRNA is 13.3:1, the nucleic acid with the same concentration is dissolved in PBS buffer solution to be used as a positive control, and the negative control is PBS buffer solution. Agarose gel concentration is 1.5%, where the gel void allows only free nucleic acid to pass through but not lipid nanoparticles, and the electrophoresis is stopped when the free nucleic acid band is electrophoresed to a clear resolution. The grey scale value of the free nucleic acid in different groups is counted by using imageJ software, the positive control group is defined as 100%, the ratio of the free nucleic acid in each group relative to the positive control is the relative amount of the free nucleic acid, and the entrapment rate in each group is (100-relative amount of the free nucleic acid). Nucleic acid entrapment rates above 50% are considered acceptable ranges.
Cell culture method: human embryonic kidney cell line 293T was cultured with DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37℃with 5% CO 2 Is cultured under the condition of (2).
Flow cytometry method for analyzing the percentage of eGFP positive cells: 293T cells were seeded onto 24-well plates at a density of 5X 10 5 Cells/well, when cell density reached 80%, cells were incubated with 1mLMPP or eGFP-mRNA@MPP, where the eGFP-mRNA@MPP concentration was 2. Mu.g/mL. After 48h, cell suspensions were collected, 20000 cells were collected using flow cytometer FITC channel and analyzed for the percentage of eGFP positive cells, the formula was: eGFP positive cell rate calculation formula = number of cells expressing eGFP/total number of cells x 100%. The percentage of eGFP positive cells up to 40% or more was considered an acceptable range.
From metalThe principle of loading the Metal-phospholipid complex particles (Metal-chelatedphospholipid complexnanoparticles, MPP) with nucleic acids of phospholipid complex assembly is: curcumin and DSPC are combined together through hydrogen bond, and simultaneously curcumin and Fe are combined through coordination bond 3+ Or Al 3+ Are connected to form a metal-phospholipid complex, fe of the metal-phospholipid complex 3+ Or Al 3+ Is linked to the nucleic acid by a coordination bond, thereby ensuring self-assembly of the metal-phospholipid complex with other lipid components into MPP while loading the nucleic acid into the nanoparticle. The contribution of curcumin in MMP loaded nucleic acids has two possibilities: (1) curcumin interacts with nucleic acids to assist in loading the MPP with nucleic acids, e.g., curcumin assists in loading the nucleic acids by inserting into the minor groove of the nucleic acid; (2) curcumin may not directly interact with nucleic acids.
EXAMPLE 3.1 ratio of the Components of the Metal-phospholipid Complex
DSPC, curcumin, feCl in example 3 3 According to different throwing ratios (1:1:1, 3:3:2 and 2:2:1), other steps are the same as in example 3, different eGFP-mRNA@MPP is prepared, and the nucleic acid entrapment rates are detected respectively.
Analysis of results: as shown in the results Table 1-1, when DSPC, curcumin, feCl 3 When the throwing ratio of the (2) to the (1:1:1), the eGFP-mRNA entrapment efficiency of the prepared medicine-lipid particles is 97%; when DSPC, curcumin and FeCl 3 When the throwing ratio of the prepared medicine-lipid particles is 3:3:2, the eGFP-mRNA entrapment efficiency of the prepared medicine-lipid particles is 70%; when DSPC, curcumin and FeCl 3 When the administration ratio of the prepared drug-lipid particles is 2:2:1, the eGFP-mRNA entrapment efficiency of the prepared drug-lipid particles is 60%. Fe in drug-lipid particles 3+ The function of (2) is to link phospholipid complexes with nucleic acids, each Fe 3+ At most three complexing sites are present, so DSPC, curcumin, feCl 3 The dosing ratio in the drug-lipid particle should be 1:1:1, it is ensured that as much of the nucleic acid as possible is entrapped in the drug-lipid particles. The results of the experiment also demonstrate that when DSPC, curcumin, feCl 3 When the administration ratio of the (2) to the (1:1:1) is 1, the inclusion rate of eGFP-mRNA of the prepared medicine-lipid particles is highest. When DSPC, curcumin and FeCl 3 The ratio of the drug to the lipid particles is within the range from 1:1:1 to 2:2:1, and the nucleic acid entrapment rate of the drug-lipid particles is above 60%.
TABLE 1-1 component addition ratio of Metal-phospholipid Complex and function of drug-lipid particles prepared therefrom
DSPC, curcumin and FeCl 3 Is added to the ratio of (2) mRNA entrapment Rate of drug-lipid particles
1:1:1 87％
3:3:2 70％
2:2:1 60％
DSPC, curcumin, al (NO) in example 3 3 ) 3 ·9H 2 O is put according to different putting ratios (1:1:1, 3:3:2 and 2:2:1), other steps are the same as in example 3, different eGFP-mRNA@MPP is prepared, and the nucleic acid entrapment rates are detected respectively.
Analysis of results: as shown in the results of tables 1-2, when DSPC, curcumin, al (NO 3 ) 3 ·9H 2 When the adding ratio of O is 1:1:1, the eGFP-mRNA entrapment efficiency of the prepared metal-phospholipid composite particles is 98%; when DSPC, curcumin, al (NO) 3 ) 3 ·9H 2 When the adding ratio of O is 3:3:2, the eGFP-mRNA entrapment efficiency of the prepared metal-phospholipid composite particles is 72%; when DSPC, curcumin, al (NO) 3 ) 3 ·9H 2 O (O)When the throwing ratio is 2:2:1, the eGFP-mRNA entrapment efficiency of the prepared metal-phospholipid complex particles is 58%. Al in the metal-phospholipid composite particles 3+ The function of (2) is to link phospholipid complexes with nucleic acids, each Al 3+ At most three complexing sites are present, so that DSPC, curcumin, al (NO 3 ) 3 ·9H 2 The dosing ratio of O in the drug-lipid particles should be 1:1:1, it is ensured that as much as possible of the nucleic acid is entrapped in the particles of the metal-phospholipid complex. The results of the experiment also demonstrate that when DSPC, curcumin, al (NO 3 ) 3 ·9H 2 When the adding ratio of O is 1:1:1, the inclusion rate of eGFP-mRNA of the metal-phospholipid complex particles prepared by the method is highest. When DSPC, curcumin, al (NO) 3 ) 3 ·9H 2 The adding ratio of O is in the range from 1:1:1 to 2:2:1, and the nucleic acid entrapment rate of the metal-phospholipid complex particles is above 58%.
TABLE 1-2 component addition ratio of Metal-phospholipid Complex and function of Metal-phospholipid Complex particles prepared thereby
EXAMPLE 3.2 preparation of Metal-phospholipid Complex, distearoyl phosphatidylcholine (DSPC), DSPE-PEG2000 and Cholesterol (CHOL) ratio in drug-lipid particles
In comparison with example 3, the ratios of metal-phospholipid complex, distearoyl phosphatidylcholine (DSPC), DSPE-PEG2000, cholesterol (CHOL) are shown in tables 1-3 (the metal ion is Fe 3+ ) And tables 1 to 4 (metal ion is Al 3+ ) The remaining conditions are the same as shown.
Analysis of results: as shown in the results tables 1 to 3, when the metal-phospholipid complex (the metal ion is Fe 3+ ) The particle size of the drug-lipid particles is 50-400 nm, the surface potential is-10 mV, the in vitro stability is more than 3 days, the mRNA entrapment rate is more than 50%, the positive expression of eGFP protein is realized when the ratio is in the range of (10-40)%, the DSPC ratio is in the range of (0-40)%, the CHOL ratio is in the range of (35-75)%, the DSPE-PEG2000 ratio is in the range of (2-10)%, the particle size is 50-400 nm The rate is more than 70 percent. Wherein the drug-lipid particles have optimal properties, i.e., particle size in the 110nm range, surface potential in the-2.04 mV range, in vitro stability, when the metal-phospholipid complex is 15%, distearoyl phosphatidylcholine (DSPC) is 35%, cholesterol (CHOL) is 46% and DSPE-PEG2000 is 4%>7 days, the mRNA entrapment rate was 87%, and the positive expression rate of eGFP protein was 97%. Because mrna@mpp adsorbs nucleic acids primarily by metal-phospholipid complexes, the metal-phospholipid complex ratio must not be too low; when the content of DSPC is in the range of 0-40%, the stability of the nano particles is in an acceptable range, and when the content of DSPC is 0%, the stability of the nano particles is maintained because the metal-phospholipid complex contains DSPC; the DSPE-PEG2000 has the functions of preventing nano particles from gathering and prolonging in vivo circulation time, and the content of the DSPE-PEG2000 can be better in the range of 2-10%; the CHOL acts to enhance the mobility of the nanoparticles, which maintains a certain level of stability of the nanoparticles.
The above results suggest that the metal-phospholipid complex (metal ion is Fe 3+ ) When the ratio 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)%, the ratio of DSPE-PEG2000 is in the range of (2-10)%, and the mRNA@MPP has better drug carrying performance.
Tables 1-3 eGFP-mRNA@MPP (Fe) 3+ ) Performance detection
Analysis of results: as shown in the results tables 1 to 4, when the metal-phospholipid complex (the metal ion is Al 3+ ) When the ratio is in the range of (5-50)%, when the ratio of DSPC is in the range of (0-51)%, when the ratio of CHOL is in the range of (15-80)%, when 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 mV, the in vitro stability is more than 3 days, the mRNA entrapment rate is more than 50%, and the positive expression rate of eGFP protein is more than 70%. Wherein when the metal-phospholipid complex is 7%, the distearoyl phosphatidylcholine (DSPC) is 34%, and the Cholesterol (CHOL) isAt 56% and DSPE-PEG2000 of 3%, the drug-lipid particles have optimal properties, i.e. particle size of 100nm, surface potential of-1.57 mV, in vitro stability>7 days, the mRNA entrapment rate was 92% and the positive expression rate of eGFP protein was 98%. Because mrna@mpp adsorbs nucleic acids primarily by metal-phospholipid complexes, the metal-phospholipid complex ratio must not be too low; when the content of DSPC is in the range of 0-51%, the stability of the nano particles is in an acceptable range, and when the content of DSPC is 0%, the stability of the nano particles is maintained because the metal-phospholipid complex contains DSPC; the DSPE-PEG2000 has the functions of preventing nano particles from gathering and prolonging in vivo circulation time, and the content of the DSPE-PEG2000 can be better in the range of 2-10%; the CHOL acts to enhance the mobility of the nanoparticles, which maintains a certain level of stability of the nanoparticles.
The above results suggest that the metal-phospholipid complex (metal ion is Al 3+ ) When the ratio 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)%, the ratio of DSPE-PEG2000 is in the range of (2-10)%, and the mRNA@MPP has better drug carrying performance.
Tables 1-4 eGFP-mRNA@MPP (Al) 3+ ) Performance detection
Example 3.3 preparation of non-cationic lipids or non-ionizable lipid species in eGFP-mRNA@MPP
In comparison with example 3, the substitution of distearoyl phosphatidylcholine (DSPC) is shown in tables 1-5 and tables 1-6, with the remaining conditions being the same.
Analysis of results: to explore that DSPC in eGFP-mRNA@MPP can be replaced by other non-cationic lipids or non-ionizable lipids, we select three other non-cationic lipids or non-ionizable lipids respectively, namely DSPE, DSPA and DSPG respectively replace DSPC, and by detecting particle size, surface potential, stability and mRNA encapsulation rate, it is proved that DSPC in eGFP-mRNA@MPP can be replaced by other non-cationic lipids or non-ionizable lipids, and the functions of the modified lipids are equivalent to that of the modified lipidsEfficacy of eGFP-mrna@mpp containing DSPC (tables 1-5 (metal ion is Fe 3+ ) And tables 1 to 6 (metal ion is Al 3+ )). Because the main role of the non-cationic lipid or non-ionizable lipid DSPC in eGFP-mrna@mpp is to make liposome membrane fusion better, more stable and less toxic, while other non-cationic lipids or non-ionizable lipids also have the function of making liposome membrane fusion better, more stable and less toxic, the DSPC in the drug-lipid particles can be replaced by other non-cationic lipids or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation, and their efficacy is not affected.
The structural formulas of the three non-cationic lipids or non-ionizable lipids (DSPE, DSPA, and DSPG) are shown below.
DSPE (47)
DSPA (48)
DSPG
Tables 1-5 contain eGFP-mRNA@MPP (Fe) of different classes of non-cationic or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation 3+ ) Performance of (3)
Tables 1-6 contain eGFP-mRNA@MPP (Al) of different classes of non-cationic or non-ionizable lipids other than metal-phospholipid complexes and conjugated lipids that inhibit particle aggregation 3+ ) Performance of (3)
Example 3.4 preparation of conjugated lipid species in eGFP-mRNA@MPP to inhibit particle aggregation
Compared with example 3, the substitution of DSPE-PEG2000 (formula 53) is shown in tables 1-7 (metal ion is Fe 3+ ) And tables 1 to 8 (metal ion is Al 3+ ) The remaining conditions are the same as shown. Three other conjugated lipids, DSPE-PEG700 (formula 50) and DSPE-PEG5000 (formula 52) and DSPE-PEG1000 (formula 51), inhibit particle aggregation.
Analysis of results: to explore that DSPE-PEG2000 in eGFP-mrna@mpp could be replaced by other particle aggregation inhibiting conjugated lipids, we selected three other particle aggregation inhibiting conjugated lipids, DSPE-PEG700, DSPE-PEG5000 and DSPE-PEG1000, respectively, to replace DSPE-PEG2000, and by examining particle size, surface potential, stability and mRNA entrapment rate, it was demonstrated that DSPE-PEG2000 in eGFP-mrna@mpp could be replaced by other particle aggregation inhibiting conjugated lipids, which after replacement were functionally equivalent to the efficacy of eGFP-mrna@mpp comprising DSPE-PEG2000 (tables 1-7 and 1-8). Because the primary role of DSPE-PEG2000 in eGFP-mrna@mpp is to inhibit aggregation, while other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation, DSPE-PEG2000 in eGFP-mrna@mpp can be replaced by other conjugated lipids that inhibit particle aggregation, and its efficacy is not affected.
Tables 1-7 eGFP-mRNA@MPP (Fe) containing conjugated lipids of different types inhibiting particle aggregation 3+ ) Performance of (3)
Tables 1-8 eGFP-mRNA@MPP (Al) containing various conjugated lipids inhibiting particle aggregation 3+ ) Performance of (3)
Example 3.5 preparation of mRNA@MPP and characterization of Effect
Example 3.5.1 the metal ion is Fe 3+ Preparation and effect characterization of mRNA@MPP of (C)
Three kinds of mRNA@MPPs containing different target protein mRNA sequences were prepared by the method of example 3 by replacing the mRNA of example 3 with two other kinds of mRNA. The three different mRNA sequences were: (1) the mRNA sequence of the coded fluorescent protein eGFP is SEQ ID NO.1 (720 nt); (2) the mRNA sequence encoding the receptor binding domain (receptor binding domain, RBD) of the S1 subunit of the novel coronavirus is SEQ ID NO.2 (669 nt); (3) the mRNA sequence encoding the tumor antigen NY-ESO-1 is SEQ ID NO.3 (543 nt). The preparation process of the rest of the drug (mRNA) -lipid particles is the same as that of example 3, and eGFP-mRNA@MPP, RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP are respectively obtained.
The eGFP-mRNA@MPP was incubated with 293T cells at a concentration of 2. Mu.g/mL (the concentration of the contained mRNA), the control group was incubated with MPP, the cell suspension was harvested after 48h, and the percentage of eGFP positive cells was detected by flow cytometry, as shown in FIGS. 1-1; the RBD-mRNA@MPP is incubated with 293T cells at a concentration of 2 mug/mL (the concentration of the contained mRNA), a control group is incubated with MPP, after 24 hours, supernatant is frozen at-20 ℃ for standby after centrifugation, and the expression level of fresh crown antigen RBD protein on the cells is detected by using a commercial novel crown antigen RBD ELISA detection kit, and the result is shown in figures 1-2.
Method for ELISA detection of RBD expression level:
1. sample collection: standing the cell supernatant at room temperature for 2 hours, centrifuging at 1000 Xg for 20min, and collecting the supernatant;
2. sample adding: and blank holes, standard substance holes and sample holes to be measured are respectively formed in the coating plate. Adding 100 mu L of sample diluent into blank holes, respectively adding sequentially gradient diluted standard substances into standard substance holes, adding 100 mu L of sample to be detected into sample holes to be detected, and incubating at 37 ℃ for 60min;
3. the liquid in the holes is discarded, the plate is washed for 3 times, and each time is soaked for 1-2min. 100 mu L of prepared biotin-labeled anti-RBD antibody working solution is added into each hole respectively, and the mixture is uniformly mixed and incubated for 60 minutes at 37 ℃;
4. discarding the liquid in the holes, washing the plate for 3 times, and soaking for 1-2min each time;
5. adding 100 mu L of prepared streptavidin HRP working solution into each hole, uniformly mixing, and incubating for 45min at 37 ℃;
6. discarding the liquid in the holes, washing the plate for 3 times, and soaking for 1-2min each time;
7. adding 100 mu L of TMB substrate solution (TMB) into each hole, and incubating for 15min at 37 ℃ in a dark place;
8. adding 100 mu L of stop solution into each hole to stop the reaction;
9. the optical density (OD value) of each well was measured at a wavelength of 450 nm.
Data analysis: and drawing a standard curve by taking the concentration of the standard substance as an abscissa and the OD value as an ordinate.
Experimental animals were randomly divided into 2 groups (experimental and control groups) of 5 animals each. Wherein, the RBD-mRNA@MPP animal model is BALB/c mice, each mouse is subjected to first muscle administration on the 1 st day and second muscle administration on the 14 th day, the experimental group is injected with RBD-mRNA@MPP, and the control group is injected with metal-phospholipid complex particles (MPP) which are not loaded with mRNA. The dose per administration was 100. Mu.L, with the RBD-mRNA@MPP preparation in the experimental group containing 30mg of mRNA. The mice were collected on day 28 from the first dose, serum was isolated and diluted in a gradient, and RBD total IgG antibodies raised against the novel coronavirus S1 subunit in the mice were detected by a commercially available ELISA kit, and the results are shown in fig. 1-3.
The animal model of NY-ESO-1-mRNA@MPP was C57BL/6 mice, each of which was given four intramuscular doses on days 1,7, 14, 21, the experimental group was injected with NY-ESO-1-mRNA@MPP and the control group was injected with metal-phospholipid complex particles (MPP) without mRNA. The dose per administration was 100. Mu.L, with the NY-ESO-1-mRNA@MPP preparation in the experimental group containing 30mg of mRNA. The mice were collected on day 28 from the first dose, serum was isolated and diluted in a gradient, and anti-NY-ESO-1 total IgG antibodies produced in the mice were detected by ELISA, and the results are shown in FIGS. 1-4.
A method for detecting an anti-NY-ESO-1 total IgG antibody in mice:
the ELISA method is prepared from the following reagents:
1. coating liquid: accurately weigh 8.4g NaHCO 3 Dissolving in 1L distilled water (DDW) until the solid is completely dissolvedThe pH of the whole solution was adjusted to 9.6 using 1M NaOH solution and the prepared coating solution was stored at 4℃for use.
2. Washing liquid: to 1L0.01MPBS solution was added 0.5ml of LTwen-20, and the mixture was left at room temperature after being mixed well.
3. Sealing liquid: precisely weighing 20g of BSA, adding into 1L0.01MPBS solution, performing ultrasonic treatment on undissolved BSA powder in the solution, and placing in a refrigerator at 4 ℃ for standby when the solid in the solution is completely dissolved and the solution is light yellow.
4. Antibody dilution: 2.5g BSA is precisely weighed and dissolved in 250ml0.01MPBS solution, after the solid is completely dissolved, 1.25ml LTwen-20 is added into the solution, and the solution is uniformly mixed and then stored at 4 ℃ for standby.
5. Color development liquid: 0.1M citric acid: 19.2g of citric acid was added to DDW water to 1000mL (A) 0.2M disodium hydrogen phosphate: 28.4g of anhydrous disodium hydrogen phosphate was added to 1000mL (B) of 0.1M citric acid solution (A) 24.3mL,0.2M phosphate buffer (B) 25.7mL, and 50mL of DDW water. 50mg of OPD (o-phenylenediamine) are added immediately before use, 30% H is added 2 O 2 0.15mL。
6. Stop solution: 2MH 2 SO 4 : concentrated sulfuric acid 55.5mL, DDW to 500mL.
ELISA method for determining antibody titer in mouse serum:
1. coating: NY-ESO-1 antigen was diluted to 1. Mu.g/mL with coating solution, added to 96-well plates, 50. Mu.L/well, and coated overnight at 4 ℃.
2. Closing: spin-drying the coating liquid in the pore plate, washing with the sealing liquid for 3 times, once for 5min, spin-drying, adding 150 mu L of sealing liquid into each pore, and incubating at 37 ℃ for 2h.
3. And (3) drying: and spin-drying the sealing liquid, and incubating for 1-2 h at 37 ℃ until the liquid at the bottom of the pore plate is completely dried.
4. Immunization: serum samples were initially diluted 1 with antibody dilutions: 1000, and then 1:2 sequentially carrying out serial dilution, adding diluted serum samples into a closed 96-well plate, and incubating at 37 ℃ for 2 hours at 100 mu L/well; spin-drying the liquid in the pore plate, adding washing liquid into the pore plate, adding 300 mu L/pore, slowly oscillating for 40s, and repeating the steps for three times; add 1 to the well plate: 1000 dilutions of biotinylated goat anti-mouse IgG antibody, 100. Mu.L/well, incubated at 37℃for 1h; spin-drying the liquid in the pore plate, adding washing liquid, and repeating the step of washing the plate; adding fresh prepared streptavidin marked horseradish peroxidase HRP working solution, 100 mu L/hole and incubating for 1h at 37 ℃; spin-drying the liquid in the pore plate, adding washing liquid, and repeating the step of washing the plate; adding a color development liquid under the dark condition, reacting for 5min at room temperature, and adding a stop solution to stop color development, wherein the color development is 50 mu L/hole; absorbance at 450nm was measured using a microplate reader.
Spleens of normal mice were collected on day 28 after RBD-mRNA@MPP was prepared into single cell suspensions under aseptic conditions, plated in cell well plates according to 100000 spleen cells/well, cultured for 48 hours with RBD protein at a final concentration of 10mg/mL, centrifuged to remove the supernatant, and the expression levels of IFN-gamma, IL-2, IL-4 were determined by ELISA kit, and the results are shown in FIGS. 1-5.
Spleens of normal mice were collected on day 28 after administration of NY-ESO-1-mRNA@MPP, single cell suspensions were prepared under aseptic conditions, plated in cell well plates according to 100000 spleen cells/well, cultured for 48 hours with addition of NY-ESO-1 protein at a final concentration of 10mg/mL, centrifuged to remove the supernatant, and the expression levels of IFN-gamma, IL-2, TNF-alpha were determined by ELISA kits, and the results are shown in FIGS. 1 to 6.
Analysis of results: as shown in fig. 1-1, the eGFP positive cell rate was 97% for the eGFP-mrna@mpp experimental group, while no eGFP signal was detected for the MPP control group; as shown in FIGS. 1-2, the RBD protein encoded by the MPP-entrapped RBD-mRNA was 193.3ng/mL in the supernatant of 293T cells, while the RBD protein content of the supernatant of 293T cells transfected with empty vector MPP was 0. The results suggest that mRNA-MPP can encapsulate, deliver any mRNA and directly encode polypeptides in cells. As shown in FIGS. 1-3,1-4, RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP were both effective to induce humoral immunity in mice, producing high levels of antigen-specific binding antibodies. Wherein the titer of IgG antibody in mice of the RBD-mRNA@MPP treatment group reaches 117268.8; the titer of the mouse IgG antibody of the NY-ESO-1-mRNA@MPP treatment group reaches 5319.52. As shown in FIGS. 1-5,1-6, RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP are both effective in inducing cellular immunity in mice, i.e., activating immune cells and producing substantial amounts of cytokines. Wherein RBD-mRNA@MPP enables the expression amounts of cytokines IFN-gamma, IL-2 and IL-4 to respectively reach 252.8pg/mL, 207.6pg/mL and 56.6pg/mL; the expression levels of cytokines IFN-gamma, IL-2 and TNF-alpha reach 70.79pg/mL, 75.29pg/mL and 75.27pg/mL respectively by using NY-ESO-1-mRNA@MPP. The result shows that mRNA@MPP can encapsulate and deliver any mRNA, so that the expression of target proteins (antigens) is promoted, the humoral immunity and cellular immunity of mice are effectively induced, high-level antigen specific binding antibodies and cytokines are generated, and the effects of anti-novel coronavirus mRNA vaccines and anti-tumor mRNA vaccines are exerted.
Example 3.5.2 the metal ion is Al 3+ Preparation and effect characterization of mRNA@MPP of (C)
This example differs from example 3.5.1 in that the metal ion Fe in example 3.5.1 3+ Replaced by Al 3+ 。
Analysis of results: as shown in fig. 1-7, the eGFP positive cell rate was 98.02% for the eGFP-mrna@mpp experimental group, while no eGFP signal was detected for the MPP control group; as shown in FIGS. 1-8, the RBD protein encoded by the MPP-entrapped RBD-mRNA was 212.6ng/mL in the 293T cell supernatant, while the RBD protein content of the 293T cell supernatant transfected with the empty vector MPP was 0. The results suggest that mRNA-MPP can encapsulate, deliver any mRNA and directly encode polypeptides in cells. As shown in FIGS. 1-9,1-10, RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP were both effective to induce humoral immunity in mice, producing high levels of antigen-specific binding antibodies. Wherein the titer of IgG antibody in mice of the RBD-mRNA@MPP treatment group reaches 129113; the titer of the mouse IgG antibody of the NY-ESO-1-mRNA@MPP treatment group reaches 6507.4. As shown in FIGS. 1-11,1-12, RBD-mRNA@MPP and NY-ESO-1-mRNA@MPP are both effective in inducing cellular immunity in mice, i.e., activating immune cells and producing substantial amounts of cytokines. Wherein RBD-mRNA@MPP enables the expression amounts of cytokines IFN-gamma, IL-2 and IL-4 to respectively reach 271.8pg/mL, 234.6pg/mL and 68.4pg/mL; the expression levels of cytokines IFN-gamma, IL-2 and TNF-alpha reach 83.8pg/mL, 98pg/mL and 97.8pg/mL respectively by using the NY-ESO-1-mRNA@MPP. The result shows that mRNA@MPP can encapsulate and deliver any mRNA, so that the expression of target proteins (antigens) is promoted, the humoral immunity and cellular immunity of mice are effectively induced, high-level antigen specific binding antibodies and cytokines are generated, and the effects of anti-novel coronavirus mRNA vaccines and anti-tumor mRNA vaccines are exerted.
EXAMPLE 3.6 preparation and Effect of siRNA-Metal-phospholipid Complex particles (siRNA-loadedmetal-chelated phospholipidcomplexnanoparticles, siRNA@MPP)
Example 3.6.1 the metal ion is Fe 3+ Preparation and Effect of siRNA-Metal-phospholipid Complex particles (siRNA @ MPP)
Three kinds of siRNA@MPPs containing different siRNAs were prepared by referring to the method of example 3, by replacing the mRNA in example 3 with an siRNA. The three different siRNA targeted genes, sequences and corresponding random control sequences are respectively: (1) the sequences of siRNA targeting Bcl-2 gene (Bcl-2-siRNA) are SEQ ID No.4 (antisense strand) and SEQ ID No.21 (sense strand) (19 bp), and the random control sequences thereof are SEQ ID No.5 (antisense strand) and SEQ ID No.22 (sense strand) (19 bp); (2) the sequences of siRNA targeting PLK1 gene (PLK 1-siRNA) are SEQ ID NO.6 (antisense strand) and SEQ ID NO.23 (sense strand) (21 bp), and the random control sequences thereof are SEQ ID NO.7 (antisense strand) and SEQ ID NO.24 (sense strand) (19 bp); (3) the sequence of siRNA targeting Gal-1 gene (Gal-1-siRNA) is SEQ ID NO.8 (19 bp); the random control sequence is SEQ ID NO.9 (19 bp). The remaining sirna@mpp was prepared in the same manner as in example 3.
The sequence of Bcl-2-siRNA is as follows:
Antisense：5′-CAGCUUAUAAUGGAUGUAC-3′(SEQ ID No.4)；
Sense：5′-GUACAUCCAUUAUAAGCUG-3′(SEQ ID No.21)(19bp)。
the random control sequence of Bcl-2-siRNA is as follows:
Antisense：5’-ACGUGACACGUUCGGAGAA-3’(SEQ ID No.5)；
Sense：5’-UUCUCCGAACGUGUCACGU-3’(SEQ ID No.22)(19bp)。
the sequence of PLK1-siRNA is as follows:
Antisense：5’-UAAGGAGGGUGAUCUUCUUCA-3’(SEQ ID No.6)；
Sense：5’-UGAAGAAGAUCACCCUCCUUA-3’(SEQ ID No.23)(21bp)。
the random control sequence of PLK1-siRNA is as follows:
Antisense：5’-CUUACGCUGAGUACUUCGA-3’(SEQ ID No.7)；
Sense：5’-UCGAAGUACUCAGCGUAAG-3’(SEQ ID No.24)(19bp)。
the sequence of Gal-1-siRNA is as follows:
5’-GCUGCCAGAUGGAUACGAA-3’(SEQ ID No.8)(19bp)。
the random control sequence of Gal-1-siRNA is as follows:
5’-GGAAAUCCCCCAACAGUGA-3’(SEQ ID No.9)(19bp)。
cell culture method: u251 human brain glioblastoma cells were grown as monolayers in high sugar (4.5 g/L) DMEM+10% Fetal Bovine Serum (FBS), 1% penicillin/streptomycin and 2 mml-glutamine (BioIndustries) medium, and at 37 ℃, 5% CO 2 Culturing under the condition, and passaging 2 times per week.
U251 cells at 1X 10 per well 6 After cell density inoculation in 6-well plates for about 24 hours, each well of cells is incubated with siRNA@MPP containing the siRNA (wherein the concentration of the siRNA is 2 mug/mL) for 72 hours respectively, the cells are collected, total RNA of the cells is extracted, mRNA expression amounts of target genes (Bcl-2, PLK1 and Gal-1) are detected respectively by using an RT-PCR technology, and the capacity of silencing the target genes of the cells by the siRNA@MPP is counted.
RT-PCR specific procedure:
extraction of total RNA: the six-well plate was discarded, the PBS buffer was rinsed 3 times, and 1mL Trizol was added to each well to lyse the cells. 200. Mu.L of chloroform was added, the mixture was shaken well, allowed to stand at room temperature for 10min, and centrifuged at 13000rpm4℃for 15min to obtain a layered three-phase liquid in which RNA was dissolved in the aqueous phase of the upper layer. The upper aqueous phase was aspirated and placed in a new enzyme-free 1.5ml centrifuge tube, 500. Mu.L of isopropanol was added, and the mixture was allowed to stand at room temperature for 10min and centrifuged at 13000rpm4℃for 15min to obtain an RNA pellet. Removing supernatant, adding 75% (v/v) ethanol newly prepared with RNase-free water into each tube, carefully blowing, blowing up white precipitate of RNA at the bottom of the tube, centrifuging at 7500rpm for 10min at 4 ℃, removing supernatant, and sucking the liquid at the bottom of the tube as much as possible. And (3) uncovering the cover, airing RNA sediment at the bottom of the pipe at room temperature, adding 50 mu L of enzyme-free water for dissolution, and detecting the purity and concentration of the RNA by using an ultra-micro ultraviolet visible spectrophotometer.
Reverse transcription of cDNA: using Ta Ka Ra Prime Script TM RT reagent Kit with g DNA Eraser kit reverse transcribes RNA into cDNA, and genomic DNA (gDNA) is removed before the reverse transcription step, so that the result is more accurate and reliable. Preparing a total RNA reverse transcription reaction system on ice: 1 mu L Prime Script RT Enzyme Mix I, 1 mu L RT Primer Mix,4 mu L5X Prime Script Buffer2,4 mu L RNase Free dH 2 O. After the reaction mixture was prepared, the mixture was allowed to react at 37℃for 15 minutes, then the reaction was terminated by 5sec at 85℃and then stored at 4℃for use.
RT-PCR procedure: the detection method is SYBR Green dye method, and no probe is needed. Specifically, real-time PCR was performed based on cDNA of different samples as a template. The reaction solution was prepared on ice: 5 mu L SYBR Premix Dimer Eraser (2X), 0.3 mu L of PCR Forward primer (10. Mu.M), 0.3 mu L of PCR Reverse primer (10. Mu.M), 0.2 mu L ROX Reference Dye II (50X), 1 mu L of cDNA template obtained in the previous step and 3.2 mu L of dH 2 O. The sample was applied to the well plate in an amount of 10. Mu.L per well, and after the completion of the sample application, the solution was centrifuged (1000 rpm,5 min) to remove the bubbles from the solution and the reaction solution. Real-time PCR detection was performed using an ABI ViiA7 Real-time fluorescent quantitative PCR apparatus, with a reaction program of 95℃for 30sec (1 cycle) & gt95℃for 5sec;55 ℃,30sec;72 ℃,30sec (40 cycles), 60 ℃ to 95 ℃ for 2min (1 cycle). The experiment is repeated three times, the Ct value of each group is obtained by taking the average value, and the expression difference multiple of the experimental group and the control group is calculated. The control gene was GAPDH. The RT-PCR primers were as follows: (1) bcl-2 primer: forward:5'-AGGATTGTGGCCTTCTTTGAG-3', reverse:5'-AGACAGCCAGGAGAAATCAAAC-3'; (2) PLK1 primer: forward:5'-ACCAGCACGTCGTAGGATTC-3', reverse:5'-CAAGCAATTTGCCGTAGG-3'; (3) gal-1 primer: forward:5'-CAATCATGGCCTGTGGTCTG-3', reverse:5'-GTGTAGGCACAGGTTGTTGCTG-3'. (4) GAPDH primer: forward:5'-TCAGGGGTTTCACATTTGGCA-3', reverse:5'-GGAGCGGAAAACCA-3'. RQ value for expression level of each target gene (2 -ΔΔCT ) To represent. The formula is as follows:
FoldChange＝2 –ΔΔCt
wherein, ΔΔΔΔ Ct is (Ct) =Δct Experimental group –ΔCt Control group ，ΔCt＝Ct Target gene -Ct Reference gene
The calculation method of the gene silencing efficiency comprises the following steps: the calculation method of the gene silencing efficiency comprises the following steps: 100% -experimental group gene expression level/control group gene expression level.
Analysis of results: as shown in FIGS. 1-13,1-14, and 1-15 (wherein scr siRNA is a random control sequence), each of the three siRNA@MPP significantly interfered with its corresponding target gene. The inhibition rate of Bcl-2-siRNA@MPP to the target gene Bcl-2 reaches 76%; the inhibition rate of PLK1-siRNA@MPP to a target gene PLK1 reaches 86%;
the inhibition rate of Gal-1-siRNA@MPP to the target gene Gal-1 reaches 73%. The result shows that the siRNA@MPP can carry any siRNA to perform the intervention treatment of the target gene, and plays the role of siRNA-carrying medicaments, vaccines or other products.
Example 3.6.2 the metal ion is Al 3+ Preparation and Effect of siRNA-Metal-phospholipid Complex particles (siRNA-loaded Metal-chelated phospholipid complex nanoparticles, siRNA@MPP)
This example differs from example 3.6.1 in that the metal ion Fe in example 3.6.1 3+ Replaced by Al 3 + 。
Analysis of results: as shown in FIGS. 1-16,1-17 and 1-18, each of the three siRNA@MPPs significantly interfered with its corresponding target gene. The inhibition rate of Bcl-2-siRNA@MPP to the target gene Bcl-2 reaches 81%; the inhibition rate of PLK1-siRNA@MPP to a target gene PLK1 reaches 90%; the inhibition rate of Gal-1-siRNA@MPP to the target gene Gal-1 reaches 79%. The result shows that the siRNA@MPP can carry any siRNA to perform the intervention treatment of the target gene, and plays the role of siRNA-carrying medicaments, vaccines or other products.
EXAMPLE 3.7 preparation and Effect of ASO-Metal-phospholipid Complex particles (ASO-loadedMetal-chelated phospholipidcomplexnanoparticles, ASO@MPP)
Example 3.7.1 the metal ion is Fe 3+ Preparation and Effect of ASO-metal-phospholipid Complex particles (ASO-loaded metal-chelatedphosepipipid complex nanoparticles)
Replacement of mRNA in example 3 with ASO, reference example3 to prepare three ASO@MPPs containing different ASOs respectively. The three different ASO targeted genes, sequences and corresponding random control sequences are respectively: (1) the ASO (STAT 3-ASO) sequence of the target STAT3 gene is SEQ ID No.10 (17 nt), and the random control sequence thereof is SEQ ID No.11 (18 nt); (2) the sequence of ASO (alpha-syn-ASO) of the targeting alpha-syn gene is SEQ ID NO.12 (16 nt), and the random control sequence thereof is SEQ ID NO.13 (16 nt); (3) the sequence of ASO (Bcl-2-ASO) targeting Bcl-2 gene is SEQ ID NO.14 (18 nt), and the random control sequence is SEQ ID NO.15 (20 nt). The remaining ASO-metal-phospholipid complex particles were prepared in the same manner as in example 3. Incubating different aso@mpp to different cells: ASO@MPP of the targeted STAT3 gene is used for incubating U251 human brain glioblastoma cells; incubating SH-SY5Y human neuroblastoma cells with ASO@MPP targeting the alpha-syn gene; ASO@MPP targeting Bcl-2 gene was used to incubate Daudi human lymphoma cells. At 1X 10 per well 6 After cell density inoculation in 6-well plates for about 24 hours, each well of cells was incubated with ASO@MPP containing ASO as described above (wherein the concentration of ASO is 1. Mu.g/mL) for 48 hours, the cells were collected, total RNA of the cells was extracted, mRNA expression amounts of target genes (STAT 3, α -syn, bcl-2) were detected by RT-PCR technique, and the ability of ASO@MPP to silence the target genes of the cells was calculated.
The sequence of SEQ ID No.10 (STAT 3-ASO sequence) is as follows:
5’-GCTCCAGCATCTGCTTC-3’(17nt)。
the sequence of SEQ ID No.11 (random control sequence of STAT 3-ASO) is as follows:
5’-GAAGCAGCAGATGCTGGA-3’(18nt)。
the sequence of SEQ ID No.12 (sequence of. Alpha. -syn-ASO) is as follows:
5’-GCTCCCTCCACTGTCT-3’(16nt)。
the sequence of SEQ ID No.13 (random control sequence of. Alpha. -syn-ASO) is as follows:
5’-ACTCCCGAACCTGTCT-3’(16nt)。
the sequence of SEQ ID No.14 (sequence of Bcl-2-ASO) is as follows:
5’-TCTCCCAGCGTGCGCCAT-3’(18nt)。
the sequence of SEQ ID No.15 (random control sequence of Bcl-2-ASO) is as follows:
5’-CAGCGTGCGCCATCCTTCCC-3’(20nt)。
cell culture: (1) u251 human brain glioblastoma cells were grown in monolayers in high sugar (4.5 g/L) DMEM+10% Fetal Bovine Serum (FBS), 1% penicillin/streptomycin and 2 mml-glutamine (BioIndustries) medium and at 37℃with 5% CO 2 Culturing under the condition, and passaging 2 times per week; (2) SH-SY5Y human neuroblastoma cells were grown in monolayers in high sugar (4.5 g/L) DMEM+10% Fetal Bovine Serum (FBS), 1% penicillin/streptomycin and 2 mml-glutamine (Bio Industries) medium and at 37℃with 5% CO 2 Culturing under the condition, and passaging 2 times per week; (3) daudi human lymphoma cells were grown in RPMI1640+10% Fetal Bovine Serum (FBS), 1% penicillin/streptomycin and 2 mml-glutamine (BioIndusties) medium at 37℃and 5% CO 2 Culturing under the condition, and passaging 2 times per week.
RT-PCR specific procedure:
extraction of total RNA: the six-well plate was discarded, the PBS buffer was rinsed 3 times, and 1mL Trizol was added to each well to lyse the cells. 200. Mu.L of chloroform was added, the mixture was shaken well, allowed to stand at room temperature for 10min, and centrifuged at 13000rpm4℃for 15min to obtain a layered three-phase liquid in which RNA was dissolved in the aqueous phase of the upper layer. The upper aqueous phase was aspirated and placed in a new enzyme-free 1.5ml centrifuge tube, 500. Mu.L of isopropanol was added, and the mixture was allowed to stand at room temperature for 10min and centrifuged at 13000rpm4℃for 15min to obtain an RNA pellet. Removing supernatant, adding 75% (v/v) ethanol newly prepared with RNase-free water into each tube, carefully blowing, blowing up white precipitate of RNA at the bottom of the tube, centrifuging at 7500rpm for 10min at 4 ℃, removing supernatant, and sucking the liquid at the bottom of the tube as much as possible. And (3) uncovering the cover, airing RNA sediment at the bottom of the pipe at room temperature, adding 50 mu L of enzyme-free water for dissolution, and detecting the purity and concentration of the RNA by using an ultra-micro ultraviolet visible spectrophotometer.
Reverse transcription of cDNA: using Ta Ka Ra Prime Script TM RT reagent Kit with g DNA Eraser kit reverse transcribes RNA into cDNA, and genomic DNA (gDNA) is removed before the reverse transcription step, so that the result is more accurate and reliable. Preparing a total RNA reverse transcription reaction system on ice: 1 mu L Prime Script RT Enzyme Mix I, 1 mu L RT Primer Mix,4 mu L5X Prime Script Buffer2,4 mu L RNase Free dH 2 O. Is prepared intoAfter the reaction mixture was allowed to react at 37℃for 15 minutes, then the reaction was terminated by allowing the reaction mixture to stand at 85℃for 5sec, and then the reaction mixture was stored at 4℃for use.
RT-PCR procedure: the detection method is SYBR Green dye method, and no probe is needed. Specifically, real-time PCR was performed based on cDNA of different samples as a template. The reaction solution was prepared on ice: 5 mu L SYBR Premix Dimer Eraser (2X), 0.3 mu L of PCR Forward primer (10. Mu.M), 0.3 mu L of PCR Reverse primer (10. Mu.M), 0.2 mu L ROX Reference Dye II (50X), 1 mu L of cDNA template obtained in the previous step and 3.2 mu L of dH 2 O. The sample was applied to the well plate in an amount of 10. Mu.L per well, and after the completion of the sample application, the solution was centrifuged (1000 rpm,5 min) to remove the bubbles from the solution and the reaction solution. Real-time PCR detection was performed using an ABI ViiA7 Real-time fluorescent quantitative PCR apparatus, with a reaction program of 95℃for 30sec (1 cycle) & gt95℃for 5sec;55 ℃,30sec;72 ℃,30sec (40 cycles), 60 ℃ to 95 ℃ for 2min (1 cycle). The experiment is repeated three times, the Ct value of each group is obtained by taking the average value, and the expression difference multiple of the experimental group and the control group is calculated. The control gene was GAPDH. The RT-PCR primer sequences were as follows: (1) STAT3 primer: forward:5'-TGATCACCTTTGAGACCGAGG-3', reverse:5'-GATCACCACAACTGG CAA GG-3'; (2) alpha-syn primer: forward 5'-TGACGGGTGTGACAGCAGTAG-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'-GGAGCGGAAAACCA-3'. RQ value for expression level of each target gene (2 -ΔΔCT ) To represent. The formula is as follows:
Fold Change＝2 –ΔΔCt
wherein, ΔΔΔΔ Ct is (Ct) =Δct Experimental group –ΔCt Control group ，ΔCt＝Ct Target gene -Ct Reference gene
The calculation method of the gene silencing efficiency comprises the following steps: 100% -experimental group gene expression level/control group gene expression level.
Analysis of results: 1-19,1-20 and 1-21 (wherein scrASO is a random control sequence), all three ASO@MPPs can obviously interfere with the corresponding target genes, wherein the inhibition rate of STAT3-ASO@MPP to the target genes STAT3 reaches 75%; the inhibition rate of the alpha-syn-ASO@MPP to the target gene alpha-syn reaches 71%; the inhibition rate of Bcl-2-ASO@MPP to the target gene Bcl-2 reaches 66%. The result suggests that ASO@MPP can carry any ASO to perform the intervention treatment of target genes, and plays a role of carrying ASO drugs, vaccines or other products.
Example 3.7.2 the metal ion is Al 3+ Preparation and Effect of ASO-metal-phospholipid Complex particles (ASO-loaded metal-chelated phospholipid complex nanoparticles, ASO@MPP)
This example differs from example 3.7.1 in that the metal ion Fe in example 3.7.2 3+ Replaced by Al 3+ 。
Analysis of results: 1-22,1-23 and 1-24, all three ASO@MPPs can obviously interfere with the corresponding target genes, wherein the inhibition rate of STAT3-ASO@MPP to the target genes STAT3 reaches 79%; the inhibition rate of the alpha-syn-ASO@MPP to the target gene alpha-syn reaches 80%; the inhibition rate of Bcl-2-ASO@MPP to the target gene Bcl-2 reaches 74%. The result suggests that ASO@MPP can carry any ASO to perform the intervention treatment of target genes, and plays a role of carrying ASO drugs, vaccines or other products.
EXAMPLE 3.8 preparation of drug (different kinds of nucleic acids) -Metal-phospholipid Complex particles and their Effect
Example 3.8.1 preparation of metal ions of Fe 3+ Drug (different kinds of nucleic acid) -metal-phospholipid complex particles and effect thereof
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 kinds of nucleic acid sequences are: (1) the sequences of double-stranded RNA (Bcl-2-siRNA) are SEQ ID NO.4 (antisense strand) and SEQ ID NO.21 (sense strand) (19 bp), and the random control sequences thereof are SEQ ID NO.5 (antisense strand) and SEQ ID NO.22 (sense strand) (19 bp); (2) the sequence of single-stranded DNA (STAT 3-ASO) is SEQ ID NO.10 (17 nt), and the random control sequence is SEQ ID NO.11 (18 nt); (3) the sequence of single-stranded RNA (mRNA encoding wild-type novel coronavirus S protein) is SEQ ID No.16 (3822 nt); (4) the sequences of double-stranded DNA (dsDNA) are SEQ ID NO.17 (antisense strand) and SEQ ID NO.25 (sense strand) (22 bp) (the 3' -end of which is labeled with a fluorescent probe Cy 3); (5) the sequence of the single-stranded DNA (ssDNA) is SEQ ID NO.18 (22 nt) (the 3' -end of which is labeled with a fluorescent probe Cy 3). Drug-metal-phospholipid complex particles (Bcl-2-sirna@mpp, STAT3-aso@mpp, S-mrna@mpp, dsdna@mpp, ssdna@mpp) encapsulating the above different kinds of nucleic acids were prepared separately by the method of example 3, and the remaining drug-lipid particles were prepared in the same manner as in example 3.
The sequence of the double stranded DNA is as follows:
Antisense：5’-TAGCTTATCAGACTGATGTTGA-3’(SEQ ID No.17)；
Sence:5’-TCAACATCAGTCTGATAAGCTA-3’(SEQ ID No.25)(22bp)。
the sequence of SEQ ID No.18 (sequence of single-stranded DNA) is as follows:
5’-TCAACATCAGTCTGATAAGCTA-3’(22nt)。
u251 cells at 1X 10 per well 5 After cell density inoculation in 12-well plates for about 24 hours, cells were collected after each well had been incubated with siRNA@MPP (wherein the concentration of siRNA was 2. Mu.g/mL) or ASO@MPP (wherein the concentration of ASO was 2. Mu.g/mL) for 72 hours, total RNA was extracted from the cells, mRNA expression levels of target genes (Bcl-2, STAT 3) were detected by RT-PCR technique, and the ability of siRNA@MPP or ASO@MPP to silence the target genes of the cells was calculated, as shown in FIGS. 1 to 13 in example 3.6 and 1 to 19 in example 3.7.
Incubating S-mRNA@MPP with 293T cells according to the concentration of 2 mug/mL (the concentration of the contained mRNA), incubating a control group with MPP, centrifuging after 24 hours, and freezing the supernatant at-20 ℃ for later use; the cell pellet was resuspended in 100 μLPBS buffer, thawed 2 times and sonicated for 10min, and the supernatant was centrifuged and assayed for protein expression levels of both cell supernatant and cell lysate using a commercially available novel coronavirus S protein ELISA assay kit, as shown in FIGS. 1-25.
After incubating a549 lung cancer cells for 2 hours at a concentration of 100nM (concentration of DNA contained) ds-dna@mpp was incubated for 2 hours, remaining drug-lipid particles were removed, after washing the cells twice with PBS, after staining nuclei with host 33342 dye for 3 minutes, after removing dye, washing the cells 2 times with PBS, observing the cells with a high content imaging system, and calculating the efficiency of drug-lipid particle transfection DNA, the results are shown in fig. 1-26.
ss-DNA@MPP after incubation of HT22 mouse hippocampal neurons at 200nM concentration (concentration of DNA contained) for 2 hours, remaining drug-lipid particles were removed, cells were washed twice with PBS, observed with a high content imaging system, and the efficiency of transfection of drug-lipid particles with DNA was calculated, and the results are shown in FIGS. 1-26.
The culture method of human brain glioblastoma U251 cells is the same as in example 3.6.
The 293T cells were cultured in the same manner as in example 3.
Method for culturing hippocampal neurons of HT22 mice: culturing with DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37deg.C, 5% CO 2 Is a member of the group (a) and (b).
The procedure of RT-PCR was as in example 3.6.
ELISA detects the expression level of S protein: the procedure of example 3.5 was repeated except that the "working solution for anti-RBD antibody" in the ELISA method for detecting RBD of example 3.5 was changed to the "working solution for anti-S protein antibody".
The calculation method of the gene silencing efficiency is the same as in example 3.6.
The transfection efficiency was calculated by: and randomly selecting 3-5 fields by using a high content imaging system to obtain cell morphology under a common light source, fluorescence signals when excitation/emission light is 550nm/570nm (excitation light of fluorescent dye Cy3 of marked DNA) under the same field, and fluorescence signals when excitation/emission light is 352nm/461nm (excitation light of fluorescent dye Hoechst33342 of marked cell nucleus), and calculating the proportion of the number of cells with Cy3 fluorescence signals in the randomly selected fields to the number of cells with Hochest33342 fluorescence signals in the cells in the same field, namely the transfection efficiency.
Analysis of results: as shown in fig. 1-13 of example 3.6.1, the drug (double-stranded RNA) -metal-phospholipid complex particles (Bcl-2-sirna@mpp) achieved 76% inhibition of the target gene Bcl-2; inhibition of the target Gene STAT3 by the drug (Single-stranded DNA) -Metal-phospholipid Complex particles (STAT 3-ASO@MPP) of FIGS. 1-19 as in example 3.7.1
The rate reaches 75 percent; as shown in fig. 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 161.3ng/mL, while the S protein content in the supernatant of 293T cells transfected with empty vector MPP was 0; the efficiency of transfection of drug (double-stranded DNA) -metal-phospholipid complex particles (dsdna@mpp) into double-stranded DNA into cells was 100% (fig. 1-26); the efficiency of transfection of single-stranded DNA into cells with drug (single-stranded DNA) -metal-phospholipid complex particles (ssDNA@MPP) was 100% (FIGS. 1-26). The results suggest that the drug-metal-phospholipid complex particles can encapsulate and perform any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA), where the length of the nucleic acid ranges from 16-3822nt.
Example 3.8.2 preparation of metal ions to Al 3+ Drug (different kinds of nucleic acid) -metal-phospholipid complex particles and effect thereof
This example differs from example 3.8.1 in that the metal ion Fe in example 3.8.1 3+ Replaced by Al 3+ 。
Analysis of results: as shown in fig. 1-16 of example 3.6.2, the drug (double-stranded RNA) -metal-phospholipid complex particles (Bcl-2-sirna@mpp) achieved an inhibition rate of 81% for the target gene Bcl-2; the inhibition of the target gene STAT3 by the drug (single-stranded DNA) -metal-phospholipid complex particles of fig. 1-22 (STAT 3-aso@mpp) as in example 3.7.2 reached 79%; as shown in FIGS. 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 178.7ng/mL, while the S protein content in the supernatant of 293T cells transfected with empty vector MPP was 0; the efficiency of transfection of drug (double-stranded DNA) -metal-phospholipid complex particles (dsdna@mpp) into double-stranded DNA into cells was 100% (fig. 1-28); the efficiency of transfection of single-stranded DNA into cells with drug (single-stranded DNA) -metal-phospholipid complex particles (ssDNA@MPP) was 100% (FIGS. 1-28). The results suggest that the drug-metal-phospholipid complex particles can encapsulate and perform any nucleic acid (double-stranded RNA, single-stranded RNA, double-stranded DNA, single-stranded DNA), where the length of the nucleic acid ranges from 16-3822nt.
Experimental example two characterization of drug-Metal-phospholipid Complex particles
EXAMPLE 4 Synthesis characterization of Metal-phospholipid complexes
Example 4.1 the metal ion is Fe 3+ Gold at the timeSynthetic characterization of genus-phospholipid complexes
The connection characterization method of DSPC and curcumin is differential scanning calorimetry, and the measurement conditions are as follows: 3-5 mg of the detection object is weighed, the heating rate is 10 ℃/min, the heating range is 30-300 ℃, curcumin, DSPC and phospholipid complex are respectively scanned, and a curve is drawn according to the obtained data, and the result is shown in the figure 2-1. As can be seen from the graph, curcumin has a lattice structure, and has obvious melting peaks at 185 ℃; DSPC is a mixture with a plurality of depressions in the curve, possibly with different components exhibiting thermal changes under different temperature conditions; no peak appears near the melting peak of curcumin in the phospholipid complex, which is basically a straight line, which indicates that curcumin and DSPC are combined and exist in an amorphous form, thus proving that the phospholipid complex is successfully prepared.
Phospholipid complex and Fe 3+ The ligation was characterized by spectrophotometry: as shown in FIG. 2-2, the phospholipid complex (CUR-HSPC) is combined with Fe 3+ After combination, the maximum absorption wavelength is shifted from 420nm to 375nm, and the conjugated structure of the phospholipid complex is changed, thus proving Fe 3+ Successful complexation with curcumin.
Example 4.2 the metal ion is Al 3+ Synthetic characterization of metal-phospholipid complexes at this time
This example differs from example 4.1 in that the phospholipid complex is combined with Al 3+ The ligation was characterized by spectrophotometry: as shown in FIGS. 2-3, the phospholipid complex (CUR-HSPC) was combined with Al 3+ After combination, the maximum absorption wavelength is shifted from 420nm to 433nm, and the conjugated structure of the phospholipid complex is changed, which proves that Al 3+ Successful complexation with curcumin.
Example 5 the metal ion is Fe 3+ Fe at low pH 3+ Characterization of shedding from metal-phospholipid complexes
The phospholipid complex in the metal-phospholipid complex binds Fe through coordination bond 3+ Under the condition of low pH value of lysosome, phospholipid complex and Fe 3+ The coordination bond between them is broken by the occurrence of a proton (hydrogen ion absorption). To demonstrate Fe in the metal-phospholipid complex 3+ Indeed by the above mechanism from fat complexThe following experiments were designed for exfoliation in the compound: the color of the metal-phospholipid complex was observed at physiological pH (ph=7.4) and lysosomal low pH (ph=5.0), respectively. As shown in fig. 2-4, the metal-phospholipid complex changed from brownish red to bright yellow under conditions of low lysosome pH (ph=5.0), suggesting Fe 3+ Has been detached from the complex. And (3) result prompting: fe at low pH of lysosome 3+ Can be detached from the metal-phospholipid complex.
Fe at low pH 3+ The principle of shedding from the metal-phospholipid complex is: curcumin and Fe 3+ The coordination bond between them is protonated at low pH (ph=5.0), i.e. curcumin combines a large number of protons from solution (H + ) Resulting in Fe 3+ Breaking the coordination bond with curcumin to make Fe 3+ Separation from curcumin ultimately results in Fe 3+ Separated from the metal-phospholipid complex (FIGS. 2-4).
Example 6 elemental analysis of MPP in drug-Metal-phospholipid Complex particles
Example 6.1 the metal ion is Fe 3+ Elemental analysis of MPP in drug-metal-phospholipid complex particles
The mRNA of example 3 was replaced with thiol-modified siRNA, and drug-metal-phospholipid complex particles sirna@mpp were prepared by the method of example 3. Elemental analysis was performed with a transmission electron microscopy instrument. The results are shown in FIGS. 2-5: C. n, O, P is a common element, and an Fe element analysis chart shows Fe 3+ Uniformly distributed on lipid nanoparticle, and S element analysis chart can specifically represent siRNA position due to modification of sulfhydryl group on siRNA, and can be known to complex with Fe 3+ Is demonstrated to successfully encapsulate siRNA.
Example 6.2 the metal ion is Al 3+ Electron microscope analysis of MPP in drug-metal-phospholipid complex particles
Drug-metal-phospholipid complex particles MPP were prepared according to the method of example 3. Morphological analysis was performed with a transmission electron microscopy instrument. The results are shown in FIGS. 2-6: the MPP in the drug-metal-phospholipid complex particles is in a standard sphere shape, has uniform particle size and is about 100 nm.
Example 7 the metal ion is Fe 3+ Or is Al 3+ The efficiency of the metal-phospholipid complex particles MPP in encapsulating nucleic acids (siRNA and mRNA) and its comparison with LNP.
The preparation of nucleic acid-entrapped drug-metal-phospholipid complex particles siRNA@MPP and mRNA@MPP, respectively, was performed by replacing the mRNA in example 3 with siRNA targeting the Bcl-2 gene (SEQ ID No.4, 19 bp) and mRNA encoding the Receptor Binding Domain (RBD) of the novel coronavirus S1 subunit (SEQ ID No.2, 669 nt), respectively, and the remaining drug-metal-phospholipid complex particles were prepared in the same manner as in example 3.
The preparation method of siRNA@LNP and mRNA@LNP according to the same drug loading rate of siRNA@MPP in example 3.6 and mRNA@MPP in example 3.5 comprises the following specific steps: the organic phase solution was formulated according to the onpatpro lipid nanoparticle formulation, i.e. ionizable lipids ALC0315, DSPE-PEG2000, DSPC and cholesterol in 50%:1.5%:10%:38.5% of the molar ratio was dissolved in ethanol. Bcl-siRNA or RBD-mRNA was added to the aqueous phase (0.1M, pH =4.0 acetate-sodium acetate buffer). Wherein the ratio of amino ester to nucleotide phosphate (N/P) is 6:1, simultaneously ensuring that the nucleic acid drug loading is the same as that of the siRNA@MPP and the mRNA@MPP, and rapidly mixing an aqueous phase and an organic phase at a volume ratio of 3:1 at a flow rate of 14 mL/min. After mixing, diluting ten times with enzyme-free PBS buffer solution, concentrating the mixed solution to one tenth by using a 100kDa ultrafiltration tube, repeatedly diluting and concentrating for 3 times, reducing the ethanol concentration in the mixed solution to below 0.0005%, and raising the pH value of the solution to the normal pH value (7.2-7.4) of the PBS buffer solution to obtain siRNA@LNP and mRNA@LNP respectively.
The inclusion rates of the nucleic acids (siRNA and mRNA) with sirna@mpp, mrna@mpp, sirna@lnp and mrna@lnp were detected by agarose gel electrophoresis. The method for measuring the entrapment rate comprises the following steps: the nucleic acid (siRNA and mRNA) dosage of each group of lipid nanoparticles is 10 mug/mL, the mass ratio of lipid to nucleic acid is 40:1, the nucleic acid is dissolved in PBS buffer solution, and the positive control group is the PBS buffer solution without nucleic acid. The concentration of agarose gel is 1.5%, at this time, the gap of gel only allows free nucleic acid to pass through, but not lipid nanoparticle, and when the free nucleic acid band electrophoresis is clear, electrophoresis is stopped, and the degradation of nucleic acid is prevented. The grey scale value of the free nucleic acid in different groups is counted by Image J software, the positive control group is defined as 100%, the ratio of the free nucleic acid in each group relative to the positive control is the relative amount of the free nucleic acid, and the entrapment rate in each group is (100-relative amount of the free nucleic acid).
Analysis of results: as shown in fig. 2-7, MPP (Fe 3+ ) The efficiency of the siRNA and mRNA are 85.86% and 87.11%; MPP (Al) 3+ ) The efficiency of the siRNA and mRNA are 89.73% and 92.23%; the efficiency of LNP inclusion of siRNA and mRNA was 84.98% and 79.12%, respectively. The results suggest that there is no significant difference in the efficiency of MPP and LNP nucleic acid entrapment.
Example 8 the metal ion is Fe 3+ Or is Al 3+ Nucleic acid lysosome escape ability of metal-phospholipid complex particle MPP and comparison with LNP
The Bcl-2-siRNA of example 3.6 (SEQ ID No. 4) was replaced with Cy 5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@MPP (the concentration of the contained siRNA was 100 nM), and the Bcl-2-siRNA of example 7 (SEQ ID No. 4) was replaced with Cy 5-labeled Bcl-2-siRNA to prepare Cy5-siRNA@LNP (the concentration of the contained siRNA was 100 nM); the ability of the drug-lipid particle to promote lysosome escape was examined by substituting eGFP-mRNA of example 3.5 (SEQ ID No. 1) with Cy 5-labeled eGFP-mRNA, preparing Cy5-mRNA@MPP (the concentration of the contained mRNA is 2. Mu.g/mL), substituting RBD-mRNA of example 7 with Cy 5-labeled RBD-mRNA, preparing Cy5-mRNA@LNP (the concentration of the contained mRNA is 2. Mu.g/mL), incubating A549 cells with the lysosome probe Lysotemore Green for 3 hours, and then observing the overlap of the fluorescent signal of Cy5 (red) with the fluorescent signal of Lysotemore Green using a high content imaging system.
Determination criteria for the ability of drug-metal-phospholipid complex particles to promote nucleolytic escape: after incubating the cells for 3 hours with the drug-lipid nanoparticle, the fluorescence signal of Cy5 (red) and the fluorescence signal of Lysotracker Green (green) were observed for overlap using a high content imaging system, and the overlapping rate of the red fluorescence signal and the green fluorescence signal was counted using imageJ software. When the drug-metal-phospholipid complex particles are incubated for 3 hours on cells, the overlapping rate of the red fluorescent signal and the green fluorescent signal is lower than 50%, so that the nucleic acid can escape from the cell lysosomes faster, and the lipid nano particles have better capability of promoting the escape of the nucleic acid lysosomes.
Analysis of results: as shown in FIGS. 2-8, when Cy5-siRNA@MPP (Fe 3+ ) And Cy5-mRNA@MPP (Fe) 3+ ) After incubating the A549 cells for 3 hours, the overlapping rate of the red fluorescent signal and the green fluorescent signal is 36.05% and 43.07%, respectively, that is, the escape rate of lysosomes is 63.95% and 56.93%, respectively; when Cy5-siRNA@MPP (Al 3+ ) And Cy5-mRNA@MPP (Al) 3+ ) After incubating the A549 cells for 3 hours, the overlapping rate of the red fluorescent signal and the green fluorescent signal is 32.39% and 40.17%, respectively, namely, the escape rate of lysosomes is 67.61% and 59.83%, respectively; and after the A549 cells are incubated for 3 hours by Cy5-siRNA@LNP and Cy5-mRNA@LNP, the overlapping rate of the red fluorescent signal and the green fluorescent signal is 76.89% and 86.87%, namely, the lysosome escaping capacity is 23.11% and 13.13%, respectively. The medicine-lipid nanoparticle MPP is suggested to have better capability of promoting the escape of nucleotidic lysosomes, and the capability of promoting the escape of the lysosomes of the MPP is obviously stronger than that of LNP.
Example 9 the metal ion is Fe 3+ Or is Al 3+ The ability of the metal-phospholipid complex particle MPP to promote nucleic acid expression and comparison with LNP
The RBD-mRNA of example 7 was replaced with mRNA encoding fluorescent protein eGFP, and the other preparation methods were the same as in example 7 to obtain eGFP-mRNA@LNP.
The eGFP-mrna@mpp prepared in example 3 and the above eGFP-mrna@lnp (with a concentration of 2 μg/mL of mRNA) were incubated with 293T cells, control groups were incubated with MPP or LNP, and after 48h, cell suspensions were harvested and the percentage of eGFP positive cells was detected by flow cytometry.
Flow cytometry methods the method of analyzing the eGFP positive cell rate was as described in example 3.
Analysis of results: as shown in fig. 2-9, MPP (Fe 3+ )、MPP(Al 3+ ) With LNP process 293The percentage of eGFP positive cells after T cells was 97.2%, 98.1% and 63.03%, respectively. And (3) result prompting: MPP has a better function of promoting nucleic acid expression than LNP. The possible reasons for this are: the MPP of example 8 has a greater capacity for lysosomal escape than LNP, so that more nucleic acid loaded by the MPP is efficiently released into the cytoplasm and translated into protein.
Example 10 the metal ion is Fe 3+ Or is Al 3+ The capacity of MPP of medicine-metal-phospholipid complex particles to promote humoral immunity and cellular immunity and comparison of MPP with LNP
Incubating the RBD-mRNA@MPP of example 3.5 and the RBD-mRNA@LNP of example 7 with 293T cells at a concentration of 2 mug/mL (the concentration of the contained mRNA), incubating a control group with MPP, centrifuging after 24 hours, and taking supernatant for freezing at-20 ℃ for later use; the cell pellet was resuspended in 100 μLPBS buffer, thawed 2 times and sonicated for 10min, and the supernatant was centrifuged, and the expression levels of RBD protein in both cell supernatant and cell lysate were detected using the commercially available novel coronavirus antigen RBDELISA detection kit, as shown in FIGS. 2-10.
The ELISA method for detecting RBD expression levels was as described in example 3.5.
The experimental animals were randomly divided into 3 groups (experimental and control groups) of 5 animals each. The animal model was BALB/c mice, each with a first intramuscular administration on day 1 and a second intramuscular administration on day 14, with the experimental groups injected with RBD-mRNA@MPP (Fe 3+ )、RBD-mRNA@MPP(Al 3+ ) Or RBD-mRNA@LNP, and the control group was injected with MPP and LNP which were not mRNA-loaded. The dose per administration was 100. Mu.L, wherein RBD-mRNA@MPP (Fe 3+ )、RBD-mRNA@MPP(Al 3+ ) And the RBD-mRNA@LNP preparations each contained 30mg of mRNA. Mouse blood was collected 28 days after the first administration, serum was separated and diluted in a gradient, and the titer of RBD total IgG antibodies against the novel coronavirus S1 subunit produced in the mice was measured by a commercially available ELISA kit, and the results are shown in fig. 2-11.
ELISA the titres of RBD total IgG antibodies against the S1 subunit of the novel coronavirus were measured as described in example 3.5.
In the case of RBD-mRNA@MPP (Fe 3+ )、RBD-mRNA@MPP(Al 3+ ) And the spleen of a normal mouse is collected on the 28 th day after RBD-mRNA@LNP, single cell suspension is prepared under the aseptic condition, the single cell suspension is plated in a cell pore plate according to 100000 spleen cells/hole, RBD protein with the final concentration of 10mg/mL is added for culturing for 48 hours, supernatant is removed by centrifugation, and the expression levels of IFN-gamma, IL-2 and IL-4 are measured by an ELISA kit, and the results are shown in figures 2-12.
ELISA detection of IFN-gamma, IL-2, IL-4 expression levels were performed as described in example 3.5.
Analysis of results: as shown in FIGS. 2-10, RBD-mRNA@MPP (Fe 3+ )、RBD-mRNA@MPP(Al 3+ ) And RBD-mRNA@LNP, which induce 293T cells to express a certain amount of RBD, but RBD-mRNA@MPP (Al 3+ ) The capacity of inducing cells to express RBD is obviously higher than that of RBD-mRNA@MPP (Fe 3+ )、RBD-mRNA@MPP(Fe 3+ ) The ability to induce cells to express RBD is significantly greater than RBD-mRNA@LNP: RBD-mRNA@MPP (Fe) 3+ ) The expression level of RBD in the cell supernatant of the treatment group is 205ng/mL, and RBD-mRNA@MPP (Al 3+ ) The expression level of RBD in the cell supernatant of the treatment group was 230ng/mL, and the expression level of RBD in the cell supernatant of the RBD-mRNA@LNP treatment group was 115.7ng/mL. As shown in the results of FIGS. 2-11, RBD-mRNA@MPP was effective to induce humoral immunity in mice, producing high levels of antigen-specific binding antibodies, and RBD-mRNA@MPP (Al 3+ ) The ability to induce humoral immunity in mice was clearly better than that of RBD-mRNA@MPP (Fe 3+ )，RBD-mRNA@MPP(Fe 3+ ) The ability to induce humoral immunity in mice was clearly better than RBD-mrna@lnp: RBD-mRNA@MPP (Fe) 3+ ) IgG antibody titers in treated mice reached 117233.8; RBD-mRNA@MPP (Al) 3+ ) IgG antibody titers in treated mice reached 133116; whereas the IgG antibody titer in mice of RBD-mrna@lnp treated groups was only 67476. As shown in FIGS. 2-12, RBD-mRNA@MPP (Al 3+ ) Can effectively induce cellular immunity of mice, namely activate immune cells and produce a large amount of cytokines, and mRNA@MPP (Al 3+ ) The ability to induce cellular immunity in mice was clearly better than that of RBD-mRNA@MPP (Fe 3+ )，RBD-mRNA@MPP(Fe 3+ ) The ability to induce cellular immunity in mice was clearly better than RBD-mrna@lnp: RBD-mRNA@MPP (Fe) 3+ ) The expression levels of cytokines IFN-gamma, IL-2 and IL-4 respectively reach 256.8pg/mL、207.6pg/mL、61.8pg/mL；
RBD-mRNA@MPP(Al 3+ ) The expression amounts of cytokines IFN-gamma, IL-2 and IL-4 respectively reach 298pg/mL, 249pg/mL and 74.6pg/mL; the RBD-mRNA@LNP allows the expression level of cytokines IFN-gamma, IL-2 and IL-4 to be only 104.2pg/mL, 79.2pg/mL and 27pg/mL. The results suggest that mRNA@MPP (Al 3+ ) The ability to deliver any mRNA and perform its function is significantly better than RBD-mRNA@MPP (Fe 3+ )，RBD-mRNA@MPP(Fe 3+ ) The ability to induce cellular immunity in mice was clearly better than RBD-mrna@lnp: the RBD-mRNA@MPP can more effectively promote cells to express target proteins and can more effectively activate humoral immunity and cellular immunity in vivo, so that the drug (mRNA) -lipid particles are obviously superior to LNP in the prior art in the action of drugs, vaccines or other products carrying mRNA. The possible reasons for this are: 1) Compared with LNP, MPP has stronger ability of promoting nucleolytic body escape; 2) Compared with LNP, MPP has stronger capability of promoting nucleic acid to express into protein (antigen); 3) Compared with LNP, curcumin in MPP is separated from DSPC in vivo and then used as an immune adjuvant (also called immune regulator), which can activate humoral immunity and cellular immunity to enhance the effect of MPP delivering mRNA vaccine, and can inhibit immune factor storm to inhibit excessive immune response harmful to organism.
Example 11 the metal ion is Fe 3+ Or is Al 3+ In vivo safety assessment of Metal-phospholipid Complex particles (MPP)
Submission toxicity studies were performed on MPP for 20 days and recovery periods were established for 20 days using SD rats as subjects. The specific experimental method is as follows:
SPF SD rats (220+ -20 g) 56, each half of which were kept in an environment with a temperature of 25℃and a humidity of 45% -55% and with light for 12 hours. After 3-5 days of adaptive feeding, the animals are randomly grouped according to gender: experimental group 32, recovery group 24. Blank Control group (Control) 14 (containing experimental group 8 and recovery group 6), male and female halves; 14 low-dose MPP groups (8 mg/kg) (including 8 experimental groups and 6 recovery groups), and male and female halves; medium dose MPP group (16 mg/kg) 14 (containing 8 experimental groups and 6 recovery groups), male and female halves; high dose group (32 mg/kg) 14 (containing 8 experimental groups and 6 recovery groups), male and female halves. The experimental group (32 total) dissected the materials after the end of the administration, and the recovery group (24 total) dissected the materials after the end of the administration, and the normal feeding was continued for 20 days.
The administration method comprises the following steps: the experimental animals were dosed by tail vein injection once every 2 days for 20 days, and SD rat body weights were recorded once a week. The prepared MPP was dissolved in DPBS, and the control group was injected with the same amount of DPBS, and the low-dose MPP group, the medium-dose MPP group, and the high-dose MPP group were respectively injected with 8mg/kg, 16mg/kg, and 32mg/kg of MPP.
Setting the basis of the MPP administration dosage: when 200. Mu.g/kg mRNA was entrapped (actual required amount for mRNA animal experiments), the required amount of empty vector MPP was 8mg/kg. To fully prove the safety of MPP, 1, 2 and 4 times of the actual required dose of MPP animal experiments are selected for administration, namely 8mg/kg, 16mg/kg and 32mg/kg.
The general index detection method comprises the following steps: the general status of each group of animals was observed after each administration, including survival, diet, appearance, behavioral activity, body weight, and whether there was a local response to the administration. Gross necropsy was performed during dissection, including timely weighing of major viscera wet weights, such as brain, heart, liver, spleen, lung, kidney, calculating the viscera-body ratio and recording pathological changes of each viscera. Where visceral volume ratio = wet rat visceral weight/body weight of rat x 100%.
Obtaining and preserving SD rat whole blood and serum: the rats were dissected after 20 days of administration and 20 days of recovery period, abdominal aorta was collected, i.e. isoflurane anesthetized SD rats and fixed on dissecting plates, the abdomen was sterilized with 75% ethanol, the abdomen of the rats was cut with sterile ophthalmology scissors, the abdominal aorta was exposed by gently poking the viscera with cotton balls, whole blood was collected with 500 μl negative pressure EDTAK2 anticoagulation blood collection tube, and stored at 4 ℃ for routine blood detection. Collecting whole blood by using a 5mL negative pressure common blood collecting tube, standing at normal temperature for 30min, centrifuging at 1500rpm at 4 ℃ for 15min, collecting supernatant, placing in a 1.5mL centrifuge tube, and preserving at-20 ℃ for detecting biochemical indexes and immunological related indexes of blood.
Blood routine detection method: blood routine index includes: white blood cell count, lymphocyte count, monocyte count, neutrophil count, lymphocyte percentage, monocyte percentage, neutrophil percentage, red blood cell count, hemoglobin, red blood cell backlog, 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, platelet volume. The whole blood sample is gently mixed upside down, a small amount of whole blood is taken, and the result is automatically analyzed by a full-automatic blood cell analyzer.
The method for detecting the biochemical indexes of the blood comprises the following steps: blood biochemical indicators include inorganic ions (Fe 2+ ，Na + ，K + ，Cl - ，Ca 2+ ) Liver function (ALT, AST, gamma-GT, T-BIL, D-BIL, ALP, ALB), kidney function (BUN, UA, CR), heart function (LDH, CK), sugar metabolism (GSP, GLU, INS), lipid metabolism (CHO, TG, LDL-C, HDL-C). Thawing serum sample, centrifuging at 3000rpm for 15min, collecting supernatant, packaging, setting corresponding parameters on full-automatic biochemical analyzer, adding prepared working solution, adding serum to be tested, and automatically measuring result.
The detection method of the immunological related index comprises the following steps: immunologically relevant indicators include thyroid function indicators (TT 3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN- γ, IFN- α, TNF- α), immunoglobulins (IgG, igA, igM), serum complement (C3, CH 50). The above index was detected by ELISA.
Major organ pathology examination method for SD rats: at the end of the dosing period and the recovery period, after the rats of each group are anesthetized, main organs of the rats, including whole brain, heart, liver, spleen, lung and kidney, are sheared off by the ophthalmology, gently rinsed with 0.9% physiological saline, fixed in 4% paraformaldehyde fixing solution, embedded in conventional paraffin, H & E stained, and observed by an optical microscope for histopathological changes of the organs of the rats of the control group and the experimental group.
Analysis of results: as shown in table 2-1, at the end of the dosing period and at the end of the recovery period, low, medium, and high doses of MPP (Fe 3 + ) Or MPP (Al) 3+ ) Group rats survived well, had normal diet, and were outsideNormal observation and behavior activities, and no obvious adverse reaction after administration; compared with the control group, the weight gain values of the male SD rats, the female SD rats and the low, medium and high dose MPP groups have no significant difference; there was no significant difference in the dirty body ratios of the low, medium, and high dose MPP groups compared to the control group.
At the end of the dosing and recovery period, low, medium, high doses of MPP (Fe 3+ ) Or MPP (Al) 3+ ) The blood routine index (white blood cell number, lymphocyte number, monocyte number, neutrophil number, lymphocyte percentage, monocyte percentage, neutrophil percentage, red blood cell number, hemoglobin, red blood cell backlog, average red blood cell volume, average red blood cell hemoglobin content, average red blood cell hemoglobin concentration, red blood cell distribution width variation coefficient, platelet number, average platelet volume, platelet distribution width, platelet pressure volume) of the group was not abnormal; blood biochemical index of low, medium, and high dose MPP group, including inorganic ion (Fe 2+ ，Na + ，K + ，Cl - ，Ca 2+ ) No abnormality was seen in liver function index (ALT, AST, gamma-GT, T-BIL, D-BIL, ALP, ALB), kidney function index (BUN, UA, CR), heart function index (LDH, CK), sugar metabolism index (GSP, GLU, INS), lipid metabolism index (CHO, TG, LDL-C, HDL-C); the immunological related indicators of the MPP groups at low, medium and high doses include thyroid function indicators (TT 3, TT4, TSH), cytokines (IL-1, IL-2, IL-4, IFN-gamma, IFN-alpha, TNF-alpha), immunoglobulins (IgG, igA, igM), serum complement (C3, CH 50) were not abnormal compared to the control group.
At the end of the dosing and recovery period, low, medium, high doses of MPP (Fe 3+ ) Or MPP (Al) 3+ ) The brain tissue structure of the group rat is complete, the tissue staining is normal, the cell morphology and structure are complete, and the nuclear shrinkage phenomenon and inflammatory cell infiltration are avoided; the myocardial tissue structure is complete, the myocardial cells are orderly, continuously and tightly arranged, the cell nuclei are clearly visible, and obvious cell congestion, edema or necrosis are not visible; liver cells are normal in morphology, and have no inflammatory cell aggregation and necrosis; spleen is normal in structure and red Bai Sui is clear in limitChu; the lung tissue structure is complete, the alveoli are consistent in size, and obvious inflammatory cell aggregation and infiltration are avoided; the kidney structure is normal.
The results suggest that MPP (Fe) is injected into SD rats in a large amount for a long period of time 3+ ) Or MPP (Al) 3+ ) No obvious chronic toxic reaction was found, suggesting that the safety of MPP is higher.
TABLE 2-1 evaluation of in vivo safety of MPP
Note that: ALT, glutamic-pyruvic transaminase; AST, glutamic-oxaloacetic transaminase; gamma-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, fructosamine; GLU, glucose; INS, insulin; CHO, cholesterol; TG, triglycerides; LDL-C, low density lipoprotein; HDL-C, high density lipoprotein; TT3, triiodothyronine; TT4, tetraiodothyronine; TSH, thyroid stimulating hormone; IL-1, interleukin 1; IL-2, interleukin 2; IL-4, interleukin 4; IFN-gamma, interferon gamma; IFN-alpha, interferon alpha; TNF- α, tumor necrosis factor α; igG, immunoglobulin G; igA, immunoglobulin a; igM, immunoglobulin M; c3, complement C3; CH50 Total complement CH50
Example 12 the metal ion is Fe 3+ Or is Al 3+ In vivo safety comparison of Metal-phospholipid Complex particles (MPP) with LNP
The main toxicity of LNP comes from its main components, cationic lipids and/or ionizable lipids. Free cationic lipids and/or ionizable lipids can cause significant toxicity to the body when LNP is metabolized in vivo. Semi-lethal dose (IC) of cationic lipids and/or ionizable lipids to biological cells 50 ) Is to evaluate LNImportant parameters of the toxicity of P to the organism. Metal-phospholipid complex particles (MPPs) replace cationic lipids/ionizable lipids in LNP with Metal-phospholipid complexes, therefore we have studied the median lethal dose (IC) of Metal-phospholipid complexes and cationic lipids/ionizable lipids to biological cells 50 ) Comparing LNP with MPP (Fe 3+ ) Or MPP (Al) 3+ ) Is a difference in toxicity.
After incubating 293T cells with different concentrations of the metal-phospholipid complexes (0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7. Mu.M), cationic lipids (DOTAP, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7. Mu.M) and ionizable lipids (ALC 0315,0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7. Mu.M, formula) respectively for 48 hours, the cell activity was detected with a CCK8 activity detection kit, and the median lethal IC of the metal-phospholipid complexes, cationic lipids (DOTAP) and ionizable lipids (ALC 0315) to 293T cells was calculated, respectively 50 。
DOTAP
ALC0315
The detection method of CCK8 comprises the following steps:
1. cell culture: culturing cells by using DMEM culture solution containing 10% FBS and 1% diabody until the cell density reaches 80% -90% of that of a culture bottle for later use;
2. washing the remaining medium in the flask with PBS, adding pancreatin, and rapidly transferring the flask to 37deg.C with 5% CO 2 Is provided. Note that after the cells were slightly rounded, digestion was stopped by adding culture medium. Transferring into a centrifuge tube, centrifuging at 1500RPM for 5min, and re-suspending cells with fresh culture medium;
3. counting: the cell suspension was diluted to 10,0000 cells per 1ml, 100 μl per well in 96-well plates, at least 5 multiplex wells per group, as desired. 37 ℃,5%CO 2 Adding medicine after culturing for 24 hours;
4. after the medicine is incubated for 48 hours, 10 percent of CCK8 is added, the medicine is incubated for 1 to 3 hours, and the absorbance is measured at 450nm by an enzyme-labeled instrument;
5. survival (%) = [ a (dosing) -a (blank) ]/[ a (0 dosing) -a (blank) ]x100%.
The calculation method of IC 50: the survival rate is taken as an ordinate, the drug concentration is taken as an abscissa, and the Graphpad is adopted by [ Inhibitor ]]Method of analysis of normalized response- -Variable slope to calculate IC 50 。
In order to compare the in vivo safety of MPP and LNP, MPP (8 mg/kg) and LNP (3.24 mg/kg) capable of carrying an equivalent amount of nucleic acid (200. Mu.g/kg mRNA) were taken and subjected to in vivo experiments in accordance with the method of example 11 to evaluate and compare the in vivo toxicity of MPP and LNP.
Analysis of results: as shown in Table 2-2, IC of metal-phospholipid complex 50 Significantly greater than the cationic lipid (DOTAP) and the ionizable lipid (ALC 0315). The results show that the metal-phospholipid complex is significantly less toxic than the cationic lipids and the ionizable lipids.
As shown in table results 2-3, at the end of the dosing period and at the end of the recovery period, MPP (Fe 3+ ) Or MPP (Al) 3+ ) No significant abnormalities were seen in liver function (ALT, AST, ALP) and cytokines (IL-6, IL-1β) of the group. However, liver function (ALT, AST, ALP) and cytokines (IL-6, IL-1β) were significantly elevated in the LNP group compared to the control group. The results suggest that MPP (Fe 3+ ) Or MPP (Al) 3+ ) Is higher than LNP due to the fact that: the LNP has the core component of artificial synthesized cationic lipid/ionizable lipid, high cytotoxicity and immunogenicity, stable structure and difficult catabolism in vivo; whereas MPP (Fe) 3+ ) Or MPP (Al) 3+ ) The core component of the drug is a metal-phospholipid complex, the metal-phospholipid complex is composed of phospholipid molecules, natural micromolecular substance curcumin with high safety (food additive and pharmaceutical excipients approved by FDA) and safe metal ions, and the metal-phospholipid complex is decomposed into natural molecules in vivo after drug delivery is completed. In conclusion, since MPP (Fe 3+ ) Or MPP (Al) 3 + ) Cationic-free/ionizable lipids in the compositionThe substance does not cause toxic and side reactions related to cationic lipids/ionizable lipids, so MPP (Fe 3+ ) Or MPP (Al) 3+ ) Is higher than LNP.
TABLE 2-2 Metal ion is Fe 3+ Or is Al 3+ IC of metal-phospholipid complex and cationic lipid (DOTAP) and ionizable lipid (ALC 0315) 50 Contrast to
TABLE 2-3 comparison of chronic toxicity test indicators for MPP and LNP
Example III clinical application of drug-Metal-phospholipid Complex particles and route of administration
Example 13 the metal ion is Fe 3+ Or Al 3+ Clinical application and administration route of medicine-metal-phospholipid complex particles
The mRNA in example 3 was replaced with siRNA targeting the B7-H4 gene (B7-H4-siRNA) and its control (scr-siRNA), mRNA encoding the receptor binding domain (receptor binding domain, RBD) of the novel coronavirus S1 subunit (RBD-mRNA), respectively.
The sequences of the different nucleic acids are as follows: (1) the sequences of the B7-H4-siRNA are SEQ ID No.19 (sense strand) and SEQ ID No.26 (antisense strand) (25 bp), and the random control sequences thereof are SEQ ID No.20 (sense strand) and SEQ ID No.27 (antisense strand) (19 bp); (2) the mRNA sequence encoding the receptor binding domain (receptor binding domain, RBD) of the novel coronavirus S1 subunit is SEQ ID No.2 (669 nt). The preparation of 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+ ) The procedure for the preparation of the remaining drug-metal-phospholipid complex particles was the same as in example 3. The 2 different drug-metal-phospholipid complex particles (B7-H4-siRNA@MPP and RBD-mRNA@MPP) are respectively used for treating liver cancer and are used as mRNA vaccines for preventing novel coronaviruses.
The sequence of B7-H4-siRNA is as follows:
sense5’-GGG AGA CAC UCC AUC ACA GUC ACUA-3’(SEQ ID No.19)。
antisense5’-UAG UGA CUG UGA UGG AGU GUC UCCC-3’(SEQ ID No.26)(25bp)。
the random control sequence of B7-H4-siRNA is as follows:
sense5’-UUCUCCGAACGUGUCACGU-3’(SEQ ID No.20)。
antisense5’-ACGUGACACGUUCGGAGAA-3’(SEQ ID No.27)(19bp)。
to evaluate B7-H4-siRNA @ MPP (Fe 3+ ) And B7-H4-siRNA@MPP (Al 3+ ) Treating liver cancer by preparing animal model of liver cancer with HepG2 cell until tumor size increases to about 100mm 3 Liver cancer mice were randomly divided into 7 groups (5 per group): PBS control, blank vector MPP (Fe 3+ ) Group, blank vector MPP (Al 3+ ) Group, scr-siRNA@MPP (Fe 3+ ) Control group, B7-H4-siRNA@MPP (Fe) 3+ ) Treatment group, scr-siRNA@MPP (Al 3+ ) Control group, B7-H4-siRNA@MPP (Al 3+ ) Treatment groups. Each group of mice was injected intratumorally with PBS, MPP (Fe) every 3 days 3+ )、MPP(Al 3+ )、Scr-siRNA@MPP(Fe 3+ )、B7-H4siRNA@MPP(Fe 3+ )、Scr-siRNA@MPP(Al 3+ )、B7-H4siRNA@MPP(Al 3+ ) One time, at a dose of 200. Mu.g siRNA/kg, 8 injections were performed. Tumor volumes were measured and recorded every 3 days. The results are shown in FIG. 3-1.
To evaluate the effect of RBD-mrna@mpp as an mRNA vaccine for the prevention of new coronaviruses, the experimental procedure and experimental method are as described in the previous example 3.5.
ELISA detection was as described in example 3.5.
Establishment of liver cancer mouse model: hepG2 cells were collected at 1X 10 7 /mL ofDensity was resuspended in PBS and kept on ice prior to inoculation. Then 100. Mu.L of the cell suspension was injected subcutaneously into the back region near the hind leg of female Balb/c nude mice to establish a liver cancer mouse model.
Analysis of results:
as shown in FIG. 3-1, scr-siRNA@MPP (Fe 3+ )、Scr-siRNA@MPP(Al 3+ ) Has little inhibition effect on the growth of liver cancer HepG2 cells, while B7-H4-siRNA@MPP (Fe) 3+ ) And B7-H4 siRNA@MPP (Al 3+ ) The composition shows high-efficiency treatment effect and can effectively inhibit the growth of liver cancer tumors. The result shows that the drug-metal-phospholipid complex particles can encapsulate and deliver the B7-H4 siRNA, and inhibit the development of liver cancer by inhibiting the expression of target genes.
Example 3.5 FIGS. 1-3, FIGS. 1-5 illustrate RBD-mRNA@MPP (Fe 3+ ) The expression levels of the mouse IgG antibodies were set to 117268.8 (FIGS. 1-3), and the expression levels of the cytokines IFN-. Gamma.IL-2 and IL-4 were set to 252.8pg/mL, 207.6pg/mL and 56.6pg/mL, respectively (FIGS. 1-5). RBD-mRNA@MPP (Al) 3+ ) The expression levels of the mouse IgG antibodies were set to 129113 (FIGS. 1-9), and the expression levels of the cytokines IFN-. Gamma.IL-2 and IL-4 were set to 271.8pg/mL, 234.6pg/mL and 68.4pg/mL, respectively (FIGS. 1-11). The result shows that RBD-mRNA@MPP can effectively induce humoral immunity of mice and generate high-level antigen-specific binding antibodies; meanwhile, the cell immunity of the mice can be effectively induced, namely immune cells are activated and a large amount of cytokines are produced. Therefore, RBD-mRNA@MPP is effective in preventing infection by novel coronaviruses.
As shown in the figure 3-1, B7-H4-siRNA@MPP can effectively treat liver cancer by adopting an intratumoral injection administration route; as shown in example 3.5 FIGS. 1-3, 1-5, 1-9 and 1-11, RBD-mRNA@MPP is capable of activating humoral and cellular immunity by intramuscular administration, thereby exerting a prophylactic effect on novel coronavirus infections. And (3) result prompting: drug-metal-phospholipid complex particles can be administered by a variety of routes.
Embodiment four: DSPC, curcumin, fe 3+ Or Al 3+ Function after being replaced by congener
Example 15 DSPC, curcumin, fe 3+ Or Al 3+ Function after being replaced by congener
Reference is made to examples 1, 2 and 3 using DSPC, curcumin, fe 3+ The congener of (a) is respectively used for DSPC, curcumin and Fe 3+ Instead, 28 different eGFP-mRNA@MPPs were prepared separately by different combinations, each containing mRNA at a concentration of 2. Mu.g/mL. DSPC, curcumin, fe 3+ The names and structures of the analogues are shown in Table 4-1, and DSPC, curcumin and Fe in 28 mRNA@MPP 3+ And the like are combined in the manner shown in Table 4-2. Wherein, the reaction temperature in example 1 was 65℃and the reaction time was 2 hours, the reaction temperature in example 2 was 60℃and the reaction time was 2 hours, and the other conditions were unchanged.
To compare the effects of the 28 different eGFP-mRNA@MPP with the eGFP-mRNA@LNP, we prepared LNPs entrapping equal amounts of eGFP-mRNA with reference to example 9, yielding eGFP-mRNA@LNP.
The 28 different eGFP-mRNA@MPP and eGFP-mRNA@LNP (both containing mRNA at a concentration of 2. Mu.g/mL) were incubated with 293T cells, control groups were incubated with MPP or LNP, and after 48h, cell suspensions were harvested and the percentage of eGFP positive cells was detected by flow cytometry.
Flow cytometry methods the method of analyzing the eGFP positive cell rate was as described in example 3.
The main toxicity of LNP comes from its main component, cationic lipids/ionizable lipids. Free cationic/ionizable lipids can cause significant toxicity to the body when LNP is metabolized in vivo. Semi-lethal dose (IC) of cationic/ionizable lipids to biological cells 50 ) Is an important parameter for assessing the toxicity of LNP to the body. Metal-phospholipid Complex particles (MPP) replace cationic lipids/ionizable lipids in LNP with Metal-phospholipid complexes, therefore we have examined the half-lethal amount (IC) of 28 Metal-phospholipid complexes and cationic lipids (DOTAP)/ionizable lipids (ALC 0315) in Table 4-2 on biological cells 50 ) The differences in toxicity of LNP and 28 MPPs were compared.
The calculation method of IC50 is as described in example 12.
Analysis of results: as shown in tables 4-3, the percentage of eGFP positive cells after 293T cells were treated with 28 different eGFP-mRNA@MPP was significantly higher than that of eGFP-mRNA@LNP, consisting of DSPC, curcumin, fe 3+ The percentage of eGFP positive cells composed of mRNA@MPP was highest. And (3) result prompting: DSPC, curcumin, fe 3+ mRNA@MPP formed by substitution of the same species has inferior functions to those formed by DSPC, curcumin and Fe 3+ The combined mrna@mpp, but superior to the mrna@lnp function, may be due to: the MPP of example 8 has a greater capacity for lysosomal escape than LNP, so that more nucleic acid loaded by the MPP is efficiently released into the cytoplasm and translated into protein.
The above results suggest that DSPC, curcumin, fe should satisfy the following conditions 3+ The function of the drug-metal-phospholipid complex particles formed by replacing the drug-metal-phospholipid complex particles with the analogues is not affected: (1) the congener of DSPC is an amphipathic phospholipid molecule; (2) fe (Fe) 3+ Is a metal ion; (3) the congener of curcumin and the congener of DSPC form a phospholipid complex and can be complexed with metal ions; (4) curcumin and Fe 3+ The coordination bond between them is capable of breaking in response to the low pH environment of the lysosome.
As shown in tables 4-3, IC's for 28 metal-phospholipid complexes 50 Are all significantly larger than the cationic lipid (DOTAP) and the ionizable lipid (ALC 0315). It is suggested that the toxicity of the metal-phospholipid complex is clearly less than that of the cationic lipids and ionizable lipids, i.e. by DSPC, curcumin, fe 3+ And its congeners, the lipid nanoparticles (MPP) are safer than LNP because: the LNP has the core component of artificial synthesized cationic lipid/ionizable lipid, high cytotoxicity and immunogenicity, stable structure and difficult catabolism in vivo; the core component of MPP is metal-phospholipid complex, which is composed of phospholipid molecules, natural small molecular substances with high safety (wherein curcumin is food additive and pharmaceutical adjuvant approved by FDA) and safe metal ions, and the metal-phospholipid complex is decomposed into natural molecules in vivo after drug delivery is completed. In conclusion, the composition is prepared from DSPC, curcumin and Fe 3+ And the lipid particles (MPP) composed of the same substances are free of cationic lipid/ionizable lipid and cannot cause toxic and side effects related to the cationic lipid/ionizable lipid, so that the safety of the MPP is higher than that of the LNP.
TABLE 4-1 DSPC, curcumin, fe 3+ And the names and structures of the same
TABLE 4-2DSPC, curcumin, fe 3+ List of modes of combination and functions of metal-phospholipid complexes in drug-lipid particles prepared from the same
TABLE 4-3DSPC, curcumin, fe 3+ IC of metal-phospholipid complex prepared from same and analogue thereof 50
EXAMPLE 16 DSPC, curcumin, and analogues thereof, fe of different Metal-phospholipid complexes 3+ Component adding proportion and function of medicine-metal-phospholipid composite particles prepared by same
According to example 3, three metal-phospholipid complexes (mrna@mpp1, mrna@mpp4, mrna@mpp29) were prepared by preparing metal-phospholipid complexes and replacing curcumin with its congener hesperetin (1 molecule hesperetin contains 4 hydroxyl groups), tea polyphenols (1 molecule tea polyphenols contains 8 hydroxyl groups), respectively. Preparation of the three metal-phospholipid complexes of DSPC, curcumin or its analogues, feCl 3 The throwing proportion of (2) is respectively as follows: 1:1:1,1:1:1,1:1:2. And corresponding drug-metal-phospholipid complex nanoparticles prepared with the three metal-phospholipid complexes (mrna@mpp1, mrna@mpp4, mrna@mpp29). Wherein mRNA is mRNA encoding eGFP fluorescent protein, and the sequence of the mRNA is SEQ ID No.1 (720 nt). The mRNA entrapment rate of these 4 drug-lipid nanoparticles and their ability to promote eGFP fluorescent protein expression after treatment of 293T cells were tested according to the experimental procedure and experimental methods described in example 3.5.
Analysis of results: as shown in tables 4 to 4, mRNA entrapment efficiency and ability to promote expression of a target protein of the drug-metal-phospholipid complex particles prepared using different dosage ratios according to the chemical structure of the metal-phospholipid complex components were comparable. The results suggest that the ratio of the metal-phospholipid complex components may be adjusted according to the structure of the specific metal-phospholipid complex components. The basis for the adjustable proportion of the delivery is as follows: since the congener of DSPC is hydrogen-bonded to the congener of curcumin, the ratio of the congener of DSPC to the congener of curcumin can be adjusted according to the number of the phosphoric acid groups contained in the congener of DSPC when the phospholipid complex is synthesized, i.e., when the congener of DSPC contains two phosphoric acid groups, the ratio of the congener of DSPC to the congener of curcumin can be adjusted to 1:2; when the congener of DSPC contains three phosphate groups, the dosing ratio of the congener of DSPC and the congener of curcumin can be adjusted to 1:3, a step of; because of hydroxy groups and Fe of the curcumine congener 3+ Is linked by coordination bonds, the congener of curcumin and Fe as long as the congener of curcumin contains a plurality of binding sites 3+ Ratio of the substances of the same species to be administeredThe number of binding sites included in the curcumin congener can be adjusted, for example.
Tables 4-4 different ratios of the components of the metal-phospholipid complexes and the function of the drug-lipid particles prepared therefrom
EXAMPLE 17 DSPC, curcumin, and analogues, al of different Metal-phospholipid complexes 3+ Component adding proportion and function of medicine-metal-phospholipid composite particles prepared by same
According to example 3, three metal-phospholipid complexes (mrna@mpp2, mrna@mpp5, mrna@mpp30) were prepared by preparing metal-phospholipid complexes and replacing curcumin with its congener hesperetin (1 molecule hesperetin contains 4 hydroxyl groups), tea polyphenols (1 molecule tea polyphenols contains 8 hydroxyl groups), respectively. Preparation of these three metal-phospholipid complexes DSPC, curcumin or its analogues, al (NO) 3 ) 3 ·9H 2 The throwing proportion of O is respectively as follows: 1:1:1,1:1:1,1:1:2. And corresponding drug-metal-phospholipid complex nanoparticles prepared with the three metal-phospholipid complexes (mrna@mpp1, mrna@mpp4, mrna@mpp29). Wherein mRNA is mRNA encoding eGFP fluorescent protein, and the sequence of the mRNA is SEQ ID No.1 (720 nt). The mRNA entrapment rate of these 4 drug-lipid nanoparticles and their ability to promote eGFP fluorescent protein expression after treatment of 293T cells were tested according to the experimental procedure and experimental methods described in example 3.5.
Analysis of results: as shown in tables 4 to 5, mRNA entrapment efficiency and ability to promote expression of a target protein of the drug-metal-phospholipid complex particles prepared using different dosage ratios according to the chemical structure of the metal-phospholipid complex components were comparable. The results suggest that the ratio of the metal-phospholipid complex components may be adjusted according to the structure of the specific metal-phospholipid complex components. The basis for the adjustable proportion of the delivery is as follows: because the congener of DSPC is hydrogen-bonded with the congener of curcumin, the congener of DSPC and the congener of curcumin are synthesized when the phospholipid complex is synthesized as long as the congener of DSPC contains a plurality of phosphate groupsThe proportion of the substance can be adjusted according to the number of the phosphoric acid groups contained in the congener of the DSPC, namely, when the congener of the DSPC contains two phosphoric acid groups, the proportion of the congener of the DSPC and the congener of the curcumin can be adjusted to be 1:2; when the congener of DSPC contains three phosphate groups, the dosing ratio of the congener of DSPC and the congener of curcumin can be adjusted to 1:3, a step of; because of the hydroxy group and Al of the curcumine congener 3+ Is linked by coordination bonds, the congener of curcumin and Al as long as the congener of curcumin contains a plurality of binding sites 3+ The ratio of administration of the congeners of curcumin can be adjusted according to the number of binding sites contained in the congeners of curcumin.
Tables 4-5 component addition ratios of different Metal-phospholipid complexes and Functions of drug-lipid particles prepared therefrom
mRNA@MPP in example 3 was prepared as taught by the university of south China chemical engineering System Wang Shan Programming group.
Unless defined otherwise, all technical and scientific terms used throughout this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of inconsistency, the meaning described throughout the present invention or the meaning derived from what is described throughout the present invention. In addition, the terminology used in the description of the embodiments of the invention presented herein is for the purpose of describing the embodiments of the invention only and is not intended to be limiting.
Note that the above is only a preferred embodiment of the present invention and the technical principle applied. It will be understood by those skilled in the art that the present invention is not limited to the particular embodiments described herein, but is capable of various obvious changes, rearrangements and substitutions as will now become apparent to those skilled in the art without departing from the scope of the invention. Therefore, although the present invention has been described in detail by way of the above embodiments, the present invention is not limited to the above embodiments, and may include many other equivalent embodiments without departing from the technical spirit of the present invention, which fall within the scope of the present invention.

Claims (63)

1. A metal-phospholipid complex particle, wherein the metal-phospholipid complex particle comprises:
(i) A metal-phospholipid complex, wherein the metal-phospholipid complex is comprised of a phospholipid molecule moiety, a linker molecule moiety, and a metal ion moiety, the phospholipid molecule moiety is linked to the linker molecule moiety, the linker molecule moiety is linked to the metal ion moiety by a coordination bond, and the metal-phospholipid complex is not a cationic lipid or an ionizable lipid;
(ii) A conjugated lipid that inhibits aggregation of particles, wherein the conjugated lipid that inhibits aggregation of particles is not a cationic lipid or an ionizable lipid; and
(iii) A non-cationic lipid or a non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits particle aggregation.
2. The metal-phospholipid complex particle of claim 1, wherein the phospholipid molecule moiety is selected from one or more of lecithin (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic Acid (PA), phosphatidylglycerol (PG), 1-phosphoceramide (SP), phosphatidylinositol (PI), phosphatidylthreonine (PT), sphingomyelin (SM), lysolecithin (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylthreonine (LPT), lysosphingomyelin (LSM), 1-phosphosphingosine (S1P), and derivatives thereof.
3. The metal-phospholipid complex of claim 2, wherein the phospholipid molecule moiety is selected from the group consisting of
Lecithin (PC)
PhospholipidAcylethanolamine (PE)
Phosphatidylserine (PS)
Phosphatidic Acid (PA)
Phosphatidylglycerol (PG)
1-phosphoric acid ceramide (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 combinations of one or more of its derivatives;
wherein, R1 and R2 are respectively and independently:
sunflower acyl group
Lauroyl group
Myristoyl group
Palmitoyl group
Stearoyl group
Oleoyl radical
Linoleoyl group
Erucic acyl
Peanut acyl
Phytantyl radical
4. A metal-phospholipid complex particle according to claim 3 wherein the phospholipid molecule moiety is selected from the group consisting of lecithin (PC, formula 1), phosphatidylethanolamine (PE, formula 2), phosphatidic acid (PA, formula 4), phosphatidylglycerol (PG, formula 5), and a combination of one or more of its derivatives.
5. The metal-phospholipid complex particle of claim 4, wherein the phospholipid molecule moiety is selected from the group consisting of DSPC, DSPE, DSPA, DSPG, and combinations of one or more of its derivatives.
6. The metal-phospholipid complex particle of claim 5, wherein the phospholipid molecule moiety is selected from the group consisting of DSPC (formula 46)
DSPE (47)
DSPA (48)
DSPG (49)
And combinations of one or more of its derivatives.
7. The metal-phospholipid complex particle of claim 6, wherein the phospholipid molecule moiety is selected from DSPC (formula 46), DSPE (formula 47) or DSPA (formula 48).
8. The metal-phospholipid complex particle of claim 1, wherein the linker molecule moiety is selected from the group consisting of curcumin, chlorogenic acid, anthocyanin, quercetin, dihydromyricetin, hesperetin, naringenin, apigenin, catechin, tea polyphenol, epigallocatechin gallate, ellagic acid, morin, epicatechin gallate, catechin gallate, gallocatechin gallate or tubingene C, and a combination of one or more of its derivatives.
9. The metal-phospholipid complex particle of claim 8, wherein the linker molecule moiety is selected from curcumin
Chlorogenic acid
Anthocyanin
Wherein R7 and R8 are H, OH or OCH 3 R3 is H or glycosyl, R4, R5 and R6 are OH or glycosyl,
Quercetin
Dihydromyricetin
Hesperetin
Naringenin
Apigenin
Catechin
Tea polyphenols
Epigallocatechin gallate
Ellagic acid
Mulberry pigment
Epicatechin gallate
Catechin gallate
Gallocatechin gallate
Ping Beijian CAnd combinations of one or more of its derivatives.
10. The metal-phospholipid complex particle of claim 9, wherein the linker molecule moiety is selected from curcumin (formula 19), dihydrocurcumin (formula 36)Hexahydrocurcumin (formula 37)>Sulfuric acidCurcumin (formula 38)>Didemethoxycurcumin (formula 39)/(I)>A combination of one or more of the foregoing.
11. The metal-phospholipid complex particle of claim 9, wherein the linker molecule moiety is selected from the group consisting of curcumin (formula 19), hesperetin (formula 24), tea polyphenols (formula 28), and combinations of one or more of its derivatives.
12. The metal-phospholipid complex particle of claim 11, wherein the linker molecule moiety is selected from curcumin (formula 19), hesperetin (formula 24) or tea polyphenols (formula 28).
13. The metal-phospholipid complex particle of any one of claims 1 to 12, wherein the metal ion moiety is selected from Fe 3+ 、Ag + 、Ba 2+ 、Ca 2+ 、Cd 2+ 、Cu 2+ 、Fe 2+ 、Mn 2+ 、Mg 2+ 、Mo 2+ 、Zn 2+ 、Pt 2+ 、Au 2+ 、Al 3+ 、Ce 3+ 、Co 3+ 、Cr 3+ 、Eu 3+ 、Gd 3+ 、Ni 3+ 、W 3+ 、V 3+ 、Zr 3+ A combination of one or more of the foregoing.
14. The metal-phospholipid complex particle of claim 13, wherein the metal ion moiety is selected from Fe 3+ 、Ca 2+ 、Al 3+ A combination of one or more of the foregoing.
15. The metal-phospholipid complex particle of claim 14, wherein the metal ion moiety is selected from Fe 3+ 、Ca 2+ Or Al 3+ 。
16. The metal-phospholipid complex particle of claim 1, wherein the conjugated lipid that inhibits aggregation of the particle comprises a PEG-lipid conjugate and/or PEG-DAA.
17. The metal-phospholipid complex particle of claim 16, wherein the PEG-lipid conjugate is selected from phosphatidylethanolamine-polyethylene glycol 2000 (formula 42)
Phosphatidylethanolamine-polyethylene glycol 700 (43)
Phosphatidylethanolamine-polyethylene glycol 1000 (44)
Phosphatidylethanolamine-polyethylene glycol 5000 (45)
And derivatives thereof, and combinations of one or more thereof;
r1, R2 are each independently: sunflower acyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytanoyl.
18. The metal-phospholipid complex particle of claim 17, wherein the PEG-lipid conjugate is selected from one or more of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000, or DSPE-PEG 5000.
19. The metal-phospholipid complex particle of claim 18, wherein the PEG-lipid conjugate is selected from DSPE-PEG2000 (formula 53)
DSPE-PEG700 (50)
DSPE-PEG1000 (51)
Or DSPE-PEG5000 (52)
20. The metal-phospholipid complex particle of claim 1, wherein the non-cationic lipid or non-ionizable lipid of (iii) is a combination of one or more of cholesterol and derivatives thereof.
21. The metal-phospholipid complex particle of claim 20, wherein the non-cationic lipid or non-ionizable lipid of (iii) is cholesterol (formula 40)
22. The metal-phospholipid complex particle of claim 20, wherein the non-cationic lipid or non-ionizable lipid of (iii) further comprises one or more selected from the group consisting of lecithin PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidic acid PA, phosphatidylglycerol PG, 1-phosphoceramide SP, phosphatidylinositol PI, phosphatidylthreonine PT, sphingomyelin SM, lysolecithin LPC, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS, lysophosphatidic acid LPA, lysophosphatidylglycerol LPG, lysophosphatidylinositol LPI, lysophosphatidylthreonine LPT, lysosphingomyelin LSM, 1-phosphosphingosine S1P, cholesterol sulfate, and derivatives thereof.
23. The metal-phospholipid complex particle of claim 22, wherein the non-cationic lipid or non-ionizable lipid of (iii) further comprises a lipid selected from the group consisting of lecithin (PC, formula 1), phosphatidylethanolamine (PE, formula 2), phosphatidylserine (PS, formula 3), phosphatidic acid (PA, formula 4), phosphatidylglycerol (PG, formula 5), 1-phosphoceramide (SP, formula 6), phosphatidylinositol (PI, formula 7), phosphatidylthreonine (PT, formula 8), sphingomyelin (SM, formula 9), lysolecithin (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), 1-phosphosphingosine (S1P, formula 18), cholesterol sulfate
And combinations of one or more of its derivatives.
24. The metal-phospholipid complex particle of claim 23, wherein the non-cationic lipid or non-ionizable lipid of (iii) comprises cholesterol, and one or more combinations selected from DSPC, DSPE, DSPA or DSPG.
25. The metal-phospholipid complex particle of claim 24, wherein the non-cationic lipid or non-ionizable lipid of (iii) comprises cholesterol (formula 40) and DSPC (formula 46).
26. The metal-phospholipid complex particle according to any one of claims 2, 8 or 13, wherein the metal-phospholipid complex is made of a phospholipid molecule moiety selected from DSPC, DSPE or DSPA, a linker molecule moiety selected from curcumin, hesperetin or tea polyphenols, and a metal ion moiety selected from Fe 3+ 、Ca 2+ Or Al 3+ 。
27. The metal-phospholipid complex particle of claim 26, wherein the metal-phospholipid complex is made of a phospholipid molecular moiety selected from DSPC (formula 46), DSPE (formula 47) or DSPA (formula 48), a linker molecular moiety selected from curcumin (formula 19), hesperetin (formula 24) or tea polyphenol (formula 28), and a metal ion moiety selected from Fe 3+ 、Ca 2+ Or Al 3+ 。
28. The metal-phospholipid complex particle of claim 26, wherein the molar ratio of the phospholipid molecule moiety, the linker molecule moiety and the metal ion moiety is 1:1 (0.5-2).
29. The metal-phospholipid complex particle of claim 28, wherein the molar ratio of the phospholipid molecule moiety, the linker molecule moiety, and the metal ion moiety is 1:1:1.
30. The metal-phospholipid complex particle of claim 22, wherein the metal-phospholipid complex particle is made of (i) metal-phospholipid complex, (ii) conjugated lipid that inhibits aggregation of particles, and (iii) non-cationic lipid or non-ionizable lipid, the metal-phospholipid complex being present in the starting material in a molar ratio of 10% to 40%, the conjugated lipid that inhibits aggregation of particles being present in the starting material in a molar ratio of 2% to 10%, the cholesterol being present in the starting material in a molar ratio of 35% to 75%, and the non-cationic lipid or non-ionizable lipid other than cholesterol being present in the starting material in a molar ratio of 0% to 40%.
31. The metal-phospholipid complex particle of claim 22, wherein the metal-phospholipid complex particle is made of (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits aggregation of particles, and (iii) a non-cationic lipid or a non-ionizable lipid, the metal-phospholipid complex being present in the starting material in a molar ratio of 5% to less than 10%, the conjugated lipid that inhibits aggregation of particles being present in the starting material in a molar ratio of 2% to 10%, the cholesterol being present in the starting material in a molar ratio of 15% to less than 35%, 35% to 75% or greater than 75% to 80%, the non-cationic lipid or non-ionizable lipid other than cholesterol being present in the starting material in a molar ratio of 0% to 40% or greater than 40% to 51%; or (b)
The metal-phospholipid complex accounts for more than 40-50% of the raw materials in mole, the conjugated lipid for inhibiting aggregation of particles accounts for 2-10% of the raw materials in mole, the cholesterol accounts for 15-35%, 35-75% or more than 75-80% of the raw materials in mole, and the non-cationic lipid or non-ionizable lipid except the cholesterol accounts for 0-40% or more than 40-51% of the raw materials in mole; or (b)
The metal-phospholipid complex accounts for 10% -40% of the raw materials in mole, the conjugated lipid for inhibiting particle aggregation accounts for 2% -10% of the raw materials in mole, the cholesterol accounts for 15% -less than 35% or more than 75% -80% of the raw materials in mole, and the non-cationic lipid or non-ionizable lipid except for the cholesterol accounts for 0% -40% or more than 40% -51% of the raw materials in mole.
32. Metal-phospholipid complex particles according to claim 30 or 31, wherein the metal-phospholipid complex is present in the feedstock in a molar ratio of 7% to less than 10%, 10% to 30% or 20% to 30%, preferably 25%.
33. A metal-phospholipid complex particle according to claim 30 or 31, wherein the conjugated lipid that inhibits aggregation of the particle is present in the starting material in a molar ratio of 3% to 10% or 5% to 10%, preferably 10%.
34. Metal-phospholipid complex particles according to claim 30 or 31, wherein the cholesterol is present in the starting material in a molar ratio of 15% to less than 35%, 35% to 56% or 35% to 55%, preferably 40%.
35. A metal-phospholipid complex particle according to claim 30 or 31, wherein the molar ratio of non-cationic lipids other than cholesterol or non-ionisable lipids in the feedstock is 5-30%, 25-40%, greater than 40-45% or 20-25%.
36. The metal-phospholipid complex particles of claim 30, wherein the metal-phospholipid complex is present in the starting material in a molar ratio of 15-25%, the conjugated lipid that inhibits aggregation of the particles is present in the starting material in a molar ratio of 4-10%, the cholesterol is present in the starting material in a molar ratio of 40-46%, the DSPC is present in the starting material in a molar ratio of 25-35%, and the metal ion moiety in the metal-phospholipid complex is selected from the group consisting of Fe 3+ 。
37. The metal-phospholipid complex particle of claim 36, wherein the metal-phospholipid complex is present in the feedstock at a molar ratio of 15%, the conjugated lipid that inhibits aggregation of the particle is present in the feedstock at a molar ratio of 4%, the cholesterol is present in the feedstock at a molar ratio of 46%, and the DSPC is present in the feedstock at a molar ratio of 35%; or the metal-phospholipid complex is present in the feedstock at a molar ratio of 25%, the conjugated lipid that inhibits aggregation of particles is present in the feedstock at a molar ratio of 10%, the cholesterol is present in the feedstock at a molar ratio of 40%, and the DSPC is present in the feedstock at a molar ratio of 25%; the metal-phospholipid The metal ion part in the complex is selected from Fe 3+ 。
38. The metal-phospholipid complex particle of claim 30, wherein the metal-phospholipid complex is present in the feedstock in a molar ratio of 10% to 30%, the conjugated lipid that inhibits aggregation of the particle is present in the feedstock in a molar ratio of 3% to 10%, the cholesterol is present in the feedstock in a molar ratio of 35% to 56%, and the DSPC is present in the feedstock in a molar ratio of 34% to 40%; the metal ion part in the metal-phospholipid complex is selected from Al 3+ 。
39. The metal-phospholipid complex particle of claim 31, wherein the metal-phospholipid complex is present in the feedstock in a molar ratio of 10% to 30%, the conjugated lipid that inhibits aggregation of the particle is present in the feedstock in a molar ratio of 3% to 10%, the cholesterol is present in the feedstock in a molar ratio of 35% to 56%, and the DSPC is present in the feedstock in a molar ratio of 40% to 45%; or (b)
The metal-phospholipid complex accounts for 10-30% of the raw material in mole, the conjugated lipid for inhibiting particle aggregation accounts for 3-10% of the raw material in mole, the cholesterol accounts for 15-35% of the raw material in mole, and the DSPC accounts for 34-40% or more than 40-45% of the raw material in mole; or (b)
The metal-phospholipid complex accounts for 7-10% of the raw material in mole, the conjugated lipid for inhibiting particle aggregation accounts for 3-10% of the raw material in mole, the cholesterol accounts for 15-35% or 35-56% of the raw material in mole, and the DSPC accounts for 34-40% or more than 40-45% of the raw material in mole; the metal ion part in the metal-phospholipid complex is selected from Al 3+ 。
40. A metal-phospholipid complex particle according to claim 39 wherein the metal-phospholipid complex is present in the feedstock at a molar ratio of 7%, the conjugated lipid that inhibits aggregation of the particle is present in the feedstock at a molar ratio of 3%, and the cholesterol is present in the feedstock56% by mole, 34% by mole of DSPC in the feedstock; the metal ion part in the metal-phospholipid complex is selected from Al 3+ 。
41. The method of any one of claims 1 to 40, wherein the method of preparing comprises mixing (i) a metal-phospholipid complex, (ii) a conjugated lipid that inhibits aggregation of particles, and (iii) a non-cationic lipid or a non-ionizable lipid other than the metal-phospholipid complex and the conjugated lipid that inhibits aggregation of particles, to obtain the metal-phospholipid complex particle.
42. The preparation method according to claim 41, wherein the preparation method comprises the steps of:
step one: reacting and linking phospholipid molecules with linker molecules to form phospholipid complexes;
step two: reacting the phospholipid complex prepared in the first step with a metal ion through a coordination bond to form a metal-phospholipid complex;
step three: mixing the metal-phospholipid complex prepared in step two, (ii) conjugated lipid that inhibits aggregation of particles, (iii) non-cationic lipid or non-ionizable lipid to obtain the metal-phospholipid complex particles.
43. A method according to claim 42, wherein in step one, the phospholipid molecules and the linker molecules are dissolved in ethanol to react, and then n-hexane is added to precipitate the phospholipid complex.
44. The method of claim 43, wherein the molar ratio of the phospholipid molecules to the linker molecules is 1:1.
45. The process of claim 43 or 44, wherein the reaction conditions comprise a reaction at 65℃for 2 hours.
46. A method according to claim 42, wherein in step two, the metal-phospholipid complex is obtained after the reaction of the phospholipid complex with a metal ion in ethanol.
47. The method according to claim 46, wherein the molar ratio of the phospholipid complex to the metal ion is 1: (1-2).
48. The method according to claim 46 or 47, wherein the reaction conditions include a reaction at 60℃for 2 hours.
49. Use of metal-phospholipid complex particles according to any one of claims 1 to 40 in drug-lipid particles.
50. Use of metal-phospholipid complex particles according to any one of claims 1 to 40 in a composition for the delivery of a drug.
51. The use of claim 50, wherein the composition is for introducing a drug into a cell.
52. The use of claim 51, wherein the composition is a medicament.
53. The use of claim 52, wherein the agent is for silencing expression of a target sequence in a mammalian subject, for delivering a drug in a mammal, for delivering a drug from within a body to a mammalian cell, or for treating a disease or condition in a mammal.
54. The use of claim 53, wherein the mammal is a human.
55. The use according to claim 53, wherein the disease or condition is associated with expression of a gene comprising a target sequence for a drug.
56. The use of claim 53, wherein the disease or condition comprises cancer, a viral infection, an autoimmune disease, diabetes, or Alzheimer's disease.
57. The use according to claim 56, wherein said viral infection comprises hepatitis A, hepatitis B, hepatitis C, SARS-Cov-2, HIV, HPV, influenza, smallpox or syphilis.
58. The use of claim 56, wherein said cancer comprises liver cancer, glioma, melanoma, lung cancer, pancreatic cancer or breast cancer.
59. The use of claim 52, wherein the medicament is a vaccine.
60. The use of any one of claims 52 to 59, wherein the route of administration of the medicament comprises intrathecal injection, intramuscular administration, intracranial injection, intravenous injection or intratumoral injection.
61. A pharmaceutical agent comprising the metal-phospholipid complex particle of any one of claims 1 to 40.
62. The agent of claim 61, wherein the agent is a vaccine.
63. The agent of claim 62, wherein the vaccine is a novel coronavirus vaccine.
CN202310975085.4A 2022-08-09 2023-08-04 Metal-phospholipid composite particles, and preparation method and application thereof Pending CN117582511A (en)

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