CN117582416A - Medicine-lipid particle and preparation method and application thereof - Google Patents

Medicine-lipid particle and preparation method and application thereof Download PDF

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CN117582416A
CN117582416A CN202310975036.0A CN202310975036A CN117582416A CN 117582416 A CN117582416 A CN 117582416A CN 202310975036 A CN202310975036 A CN 202310975036A CN 117582416 A CN117582416 A CN 117582416A
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lipid
drug
formula
metal
polyphenol
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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 relates to the technical field of biological medicines, and particularly provides a medicine-lipid particle, a preparation method and application thereof. The drug-lipid particles provided herein include drug and metal-polyphenol complex particles, wherein the metal-polyphenol complex particles comprise a metal-polyphenol complex, a conjugated lipid that inhibits aggregation of the particles, and a non-cationic lipid or non-ionizable lipid other than the conjugated lipid that inhibits aggregation of the particles. The drug-lipid particles can realize high-efficiency systemic delivery of the drug, and simultaneously have significantly reduced toxicity compared with LNP containing cationic lipid or ionizable lipid, thereby realizing safe and effective treatment of diseases or symptoms.

Description

Medicine-lipid particle and preparation method and application thereof
Cross Reference to Related Applications
The present invention claims the priority of chinese patent application, filed at 2022, 08 and 09, under the application number 202210950179.1, entitled "drug-lipid particles, method for their preparation and use", the entire contents of which are incorporated herein by reference.
Technical Field
The invention relates to the technical field of biological medicines, in particular to a medicine-lipid particle, a preparation method and application thereof.
Background
Nucleic acid drugs refer to functional DNA or RNA that specifically edits a pathogenic gene or protein by binding, cleavage, knockout, insertion, etc. The discovery of the nucleic acid not only breaks through the traditional thought that the nucleic acid only carries genetic information, but also provides a powerful molecular tool for biomedicine and biosensing.
The nucleic acid medicine has the defects of easy rapid degradation of nuclease in a subject, weak transmembrane capability, short half-life in blood circulation and the like, and severely limits the clinical application of the nucleic acid medicine. Therefore, a safe and effective nucleic acid drug delivery system is one of the hot spots for nucleic acid drug development. Currently, vectors capable of delivering nucleic acid drugs can be largely classified into viral vectors and non-viral vectors. Viral vectors (including adenovirus, retrovirus and lentivirus vectors) can cause immune response after entering the human body, and are used less recently; 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 prolong 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.
Cationic liposomes are usually prepared by mixing cationic lipids with auxiliary lipids such as dioleoyl phosphatidylethanolamine (DOPE), cholesterol, etc. in a certain ratio. Cationic liposomes can be used to transport genes or drug molecules into target cells, however, cationic liposomes still contain some cytotoxicity during transfection and the like, and can produce some toxic effect on normal cells while carrying drugs to treat cancer cells, thus having some limitation on clinical application research. The cascade reaction caused by cationic liposome includes active oxygen generation, enzyme activation reaction, mitochondrial membrane potential change, apoptosis caused by cytochrome C and caspase release, etc.
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 (LipidNanoparticle, 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 component 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.
Disclosure of Invention
The present invention aims to provide drug-lipid particles and a method of preparation to at least alleviate one technical problem of the prior art.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the present invention provides a drug-lipid particle comprising:
(a) A drug, wherein the drug is a negatively charged molecule; and
(b) A metal-polyphenol composite particle comprising:
(i) A metal-polyphenol complex consisting of a polyphenol molecule portion and a metal ion portion, the polyphenol molecule portion and the metal ion portion being connected by a coordination bond;
(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) Non-cationic lipids or non-ionizable lipids other than conjugated lipids that inhibit particle aggregation.
In some embodiments, the polyphenol molecular moiety is selected from the group consisting of curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, taxol, fucoidan, polyhydrol, catechin, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, galloylglucose, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate, gallocatechin gallate, and combinations of one or more of its derivatives.
Wherein, "and derivatives thereof" means "curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, taxol, fucoidin, polyphenol of brown algae, polyphenol of flavanols, catechin, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, galloylglucose, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate or gallocatechin gallate". The polyphenol molecule moiety may be, for example, but not limited to, curcumin derivatives, hesperetin derivatives, catechins, catechin derivatives, curcumin and catechin, catechin and catechin derivatives, and the like. In the present invention, "and derivatives thereof" are all intended to have similar meanings.
Further, the polyphenol molecular moiety is selected from one or more of curcumin (formula 1), quercetin (formula 2), kaempferol (formula 3), rutin (formula 4), hesperetin (formula 5), naringenin (formula 6), eriodictyol (formula 7), luteolin (formula 8), apigenin (formula 9), taxol (formula 10), fucoidal polyphenol (formula 11), flavanolpolyphenol (formula 12), catechin (formula 13), ellagic acid (formula 14), gallic acid (formula 15), digallic acid (formula 16), propyl gallate (formula 17), epigallocatechin gallate (formula 18), galloylglucose (formula 19), hydroxyhydroquinone (formula 20), morin (formula 21), epicatechin gallate (formula 22), catechin gallate (formula 23), gallocatechin gallate (formula 24), and derivatives thereof.
Further, the polyphenol molecular moiety is selected from the group consisting of one or more of curcumin (formula 1), dihydrocurcumin (formula 25), hexahydrocurcumin (formula 26), curcumin sulfate (formula 27), bisdemethoxycurcumin (formula 28).
Further, the polyphenol molecular moiety is selected from one or more of curcumin (formula 1), hesperetin (formula 5) or catechin (formula 13), and derivatives thereof.
Further, the polyphenol molecular moiety is selected from curcumin (formula 1), hesperetin (formula 5) or catechin (formula 13).
In some embodiments, 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+ 。
In some embodiments, the conjugated lipid that inhibits aggregation of the particles in (ii) of the metal-polyphenol complex particles comprises polyethylene glycol (PEG) -lipid conjugate and/or PEG-Dialkoxypropyl (DAA).
Further, the PEG-lipid conjugate is selected from the group consisting of phosphatidylethanolamine-polyethylene glycol 2000 (formula 47), phosphatidylethanolamine-polyethylene glycol 700 (formula 48), phosphatidylethanolamine-polyethylene glycol 1000 (formula 49), phosphatidylethanolamine-polyethylene glycol 5000 (formula 50), and derivatives thereof, in combination of one or more thereof. 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 acylOr (b)
Phytantyl radical。
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 58), DSPE-PEG700 (formula 55), DSPE-PEG1000 (formula 56), or DSPE-PEG5000 (formula 57).
In some embodiments, the non-cationic lipid or non-ionizable lipid in (iii) of the metal-polyphenol complex particle 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-sphingosine phosphate (S1P), and derivatives thereof.
Further, the non-cationic lipid or non-ionizable lipid in (iii) is selected from one or more of lecithin (PC) (formula 29), phosphatidylethanolamine (PE) (formula 30), phosphatidylserine (PS) (formula 31), phosphatidic Acid (PA) (formula 32), phosphatidylglycerol (PG) (formula 33), 1-phosphoceramide (SP) (formula 34), phosphatidylinositol (PI) (formula 35), phosphatidylthreonine (PT) (formula 36), sphingomyelin (SM) (formula 37), lysolecithin (LPC) (formula 38), lysophosphatidylethanolamine (LPE) (formula 39), lysophosphatidylserine (LPS) (formula 40), lysophosphatidic acid (LPA) (formula 41), lysophosphatidylglycerol (LPG) (formula 42), lysophosphatidylinositol (LPI) (formula 43), lysophosphatidylthreonine (LPT) (formula 44), lysosphingomyelin (LSM) (formula 45), 1-phosphosphingosine (S1P) (formula 46), and derivatives thereof; wherein R1 and R2 are each independently a decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytantyl group.
It should be noted that, in the present invention, none of the cis-trans isomers of the components used has an effect on the technical effects to be achieved in the present invention.
Further, the non-cationic lipid or non-ionizable lipid of (iii) further comprises at least one of cholesterol and derivatives thereof.
In some embodiments, the non-cationic lipid or non-ionizable lipid in (iii) comprises cholesterol, and a combination of one or more selected from DSPC, DSPE, DSPA or DSPG.
Preferably, the cholesterol has the structural formula (formula 59)
In one embodiment, the non-cationic lipid or non-ionizable lipid of (iii) comprises cholesterol (formula 59), and a combination of one or more selected from DSPC (formula 51), DSPE (formula 52), DSPA (formula 53), or DSPG (formula 54);
in some embodiments, the non-cationic lipid or non-ionizable lipid in (iii) comprises cholesterol (formula 59) and DSPC (formula 51).
In some embodiments, the metal-polyphenol complexes of the invention are comprised of reacting a polyphenol molecular moiety selected from curcumin, hesperidin, or catechin with a metal ion moiety selected from Fe 3+ 、Ca 2+ Or Al 3+ 。
Further, the metal-polyphenol complex is composed of a polyphenol molecular moiety selected from curcumin (formula 1), hesperetin (formula 5) or catechin (formula 13) and a metal ion moiety selected from Fe 3+ 、Ca 2+ Or Al 3+ 。
Further, the molar ratio of the polyphenol molecular moiety to the metal ion moiety is 1 (0.5-2).
Further, the polyphenol molecular part is curcumin (formula 1), and the metal ion part is Fe 3+ 。
Further, curcumin (formula 1) and Fe 3+ The molar ratio of (2) is 1:1.
Further, the polyphenol molecule part is curcumin (formula 1), and the metal ion part is Al 3+ 。
Further, curcumin (formula 1) and Al 3+ The molar ratio of (2) is 1:1.
In some embodiments, the metal-polyphenol complex particles are made from (i) metal-polyphenol complexes in a molar ratio of 10% to 20% in the starting material, (ii) conjugated lipids that inhibit particle aggregation in a molar ratio of 2% to 10% in the starting material, cholesterol in a molar ratio of 0% to 48% in the starting material, and (iii) non-cationic lipids or non-ionizable lipids other than cholesterol in a molar ratio of 40% to 75% in the starting material.
In some embodiments, the metal-polyphenol complex particles are made from (i) metal-polyphenol 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 0% to 48% in the starting material, and (iii) non-cationic lipids other than cholesterol or non-ionizable lipids in a molar ratio of 30% to less than 40% or 40% to 75% in the starting material; or (b)
In some embodiments, the metal-polyphenol complex particles are made from (i) metal-polyphenol complexes in a molar ratio of 10% to 20% in the starting material, (ii) conjugated lipids that inhibit particle aggregation in a molar ratio of 2% to 10% in the starting material, cholesterol in a molar ratio of 0% to 48% in the starting material, and (iii) non-cationic lipids or non-ionizable lipids other than cholesterol in a molar ratio of 30% to less than 40% in the starting material.
Further, the metal-polyphenol compound accounts for 5 to less than 10%, 10 to 15% or 15 to 20%, preferably 5%, 10% or 15% of the raw material in mole ratio.
Further, the conjugated lipids that inhibit aggregation of the particles are present in the starting material at a molar ratio of 3% to 5% or 5% to 10%, preferably 3%, 5% or 10%.
Further, the cholesterol is present in the raw material in a molar ratio of 10% to 30%, 30% to 47% or 10% to 20%, preferably 10%, 30% or 47%.
Further, the molar ratio of the non-cationic lipid or non-ionizable lipid other than cholesterol in the raw material is 45% to 55%, 60% to 65% or 50% to 65%, preferably 45%, 55%, 60% or 65%.
In some embodiments, the metal-polyphenol complex (metal ion moiety is selected from Fe 3+ ) The molar ratio of the conjugated lipid for inhibiting particle aggregation in the raw material is 5-10%, the molar ratio of the cholesterol in the raw material is 10-30%, and the molar ratio of the non-cationic lipid or non-ionizable lipid except for cholesterol in the raw material is 60-65%. Preferably, the polyphenol molecular moiety is selected from curcumin (formula 1), the conjugated lipid that inhibits aggregation of particles is DSPE-PEG2000, and the non-cationic or non-ionizable lipid is cholesterol and DSPC.
In one embodiment, the metal-polyphenol complexThe metal ion part is selected from Fe 3+ ) The molar ratio of the conjugated lipid inhibiting particle aggregation was 15% in the starting material, the molar ratio of the conjugated lipid inhibiting particle aggregation was 10% in the starting material, the molar ratio of the cholesterol was 10% in the starting material, and the molar ratio of the non-cationic lipid or non-ionizable lipid other than cholesterol (e.g., DSPC, DSPA, DSPE or DSPG) was 65% in the starting material.
In one embodiment, the metal-polyphenol complex (metal ion moiety is selected from the group consisting of Fe 3+ ) The molar ratio of the conjugated lipid inhibiting aggregation of particles was 5% in the starting material, the molar ratio of cholesterol was 30% in the starting material, and the molar ratio of the non-cationic lipid other than cholesterol or the non-ionizable lipid was 60% in the starting material.
In some embodiments, the metal-polyphenol complex (metal ion moiety is selected from Al 3+ ) The conjugated lipid inhibiting aggregation of the particles is present in the starting material in a molar ratio of 5% to less than 10% or 10%, the conjugated lipid inhibiting aggregation of the particles is present in the starting material in a molar ratio of 3% to 5%, the cholesterol is present in the starting material in a molar ratio of 30% to 47%, and the non-cationic lipids or non-ionizable lipids other than cholesterol are present in the starting material in a molar ratio of 45% to 55%. Preferably, the polyphenol molecular moiety is selected from curcumin (formula 1), the conjugated lipid that inhibits aggregation of particles is DSPE-PEG2000, and the non-cationic or non-ionizable lipid is cholesterol and DSPC.
In one embodiment, the metal-polyphenol complex (metal ion moiety is selected from Al 3+ ) The molar ratio of the conjugated lipid inhibiting aggregation of particles was 5% in the starting material, the molar ratio of the conjugated lipid inhibiting aggregation of particles was 3% in the starting material, the molar ratio of cholesterol was 47% in the starting material, and the molar ratio of non-cationic lipids other than cholesterol or non-ionizable lipids was 45% in the starting material.
In one embodiment, the metal-polyphenol complex (metal ion moiety is selected from Al 3+ ) 10% by mole in the starting material, 5% by mole in the starting material of the conjugated lipid inhibiting aggregation of the particles, 30% by mole in the starting material of cholesterol, and 55% by mole in the starting material of non-cationic lipids or non-ionizable lipids other than cholesterol.
In some embodiments, the drug is encapsulated in the metal-polyphenol complex particles.
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.
In some embodiments, the nucleic acid is selected from the group consisting of mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, artificial nucleic acids, and combinations of one or more thereof.
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 carcinoma 1) 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 Kinase 1 ) shown in SEQ ID No.6 and a sense strand of PLK1 gene (polo-like Kinase 1) shown in SEQ ID No.23, an ASO sequence of the Gal-1 gene shown in SEQ ID No.8, an ASO sequence of STAT-3 gene shown in SEQ ID No.10, an alpha-synuclein gene shown in SEQ ID No.12 (alpha-synuclein/Leukemia-2 gene shown in SEQ ID No. 21), an antisense strand of siRNA sequence of SEQ ID No.6 and a single-stranded RNA sequence shown in SEQ ID No.23, an antisense strand of SEQ ID No.16, an antisense strand shown in SEQ ID No.18, or a single-stranded DNA sequence shown in SEQ ID No.14 and a sense strand shown in SEQ ID No. 18.
The present invention provides a method for producing the above-mentioned drug-lipid particle, wherein a drug is entrapped in the metal-polyphenol complex particles to obtain the drug-lipid particle.
In one embodiment, (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits aggregation of particles, and (iii) a non-cationic lipid or a non-ionizable lipid are mixed to obtain the metal-polyphenol complex particles.
In one embodiment, the drug-lipid particles are obtained by mixing (a) a drug, (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits aggregation of particles, and (iii) a non-cationic lipid or a non-ionizable lipid.
In some embodiments, the method of making comprises the steps of:
step one: reacting the polyphenol molecular moiety with the metal ion moiety through a coordination bond to form a metal-polyphenol complex;
step two: the metal-polyphenol complex particles prepared in the step one, conjugated lipid which inhibits aggregation of particles, non-cationic lipid or non-ionizable lipid, and drug are mixed to prepare the metal-polyphenol complex particles.
Further, polyphenol molecules are dissolved in ethanol, and then metal ions are added for reaction; the molar ratio of polyphenol molecules to metal ions is 1: (1-2); the reaction conditions included 1 hour at 60 ℃.
In one embodiment, the metal-polyphenol complex, the conjugated lipid that inhibits aggregation of the particles, and the non-cationic lipid or non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer to form an aqueous phase, and the organic phase is mixed with the aqueous phase to provide the drug-lipid particles. Preferably, the organic compound is ethanol; the buffer solution is enzyme-free Tris-HCl buffer solution; the mixing mode of the organic phase and the water phase comprises a micro-fluidic chip or ultrasound.
The invention provides the use of the drug-lipid particle in a composition for the delivery of a drug.
In some embodiments, the composition is used to introduce a drug into a cell.
In some embodiments, the composition is a pharmaceutical agent.
In some embodiments, the agent is used to silence expression of a target sequence in a mammalian subject.
In some embodiments, the agent is used to deliver a drug in a mammal.
In some embodiments, the agent is used to deliver a drug from within the body to a mammalian cell.
In some embodiments, the agent is used to treat a disease or disorder in a mammal.
In some embodiments, the mammal is a human.
In some embodiments, the disease or disorder is associated with expression of a gene comprising a target sequence for a drug.
In some embodiments, the disease or disorder includes cancer, viral infection, autoimmune disease, diabetes, or alzheimer's disease.
In some embodiments, the viral infection comprises hepatitis A, hepatitis B, hepatitis C, SARS-Cov-2 (2019 novel coronavirus), HIV (human immunodeficiency virus), HPV (human papilloma virus), influenza, smallpox, or syphilis.
In some embodiments, the cancer comprises liver cancer, glioma, melanoma, lung cancer, pancreatic cancer, or breast cancer.
In some embodiments, the agent is a vaccine.
In some embodiments, the route of administration of the agent comprises intrathecal injection, intramuscular administration, intracranial injection, intravenous injection, or intratumoral injection.
The present invention provides a pharmaceutical agent containing the drug-lipid particle.
In some embodiments, the agent is a vaccine.
In some embodiments, the vaccine is a novel coronavirus vaccine.
Compared with the prior art, the invention has the technical effects that:
in the prior art, a complex of metal ions and polyphenol molecules (such as curcumin) is mostly used as an active ingredient in the fields of antioxidation, anti-inflammation, antivirus and the like, however, the applicant finds that the metal-polyphenol complex provided by the invention can be used in a composition or a medicament, is mainly used as a carrier of the medicament for stability, transmission and transportation and the like of the medicament, and is combined with other carriers to realize effective administration of the medicament with negative charges.
The drug-lipid particle provided by the invention has small diameter suitable for systemic delivery, and under the condition of ensuring the effectiveness not lower than LNP, no cationic lipid or ionizable lipid is used, so that the drug-lipid particle has greatly reduced toxicity compared with LNP, obviously improved biological safety and is more beneficial to carrying negative charge drugs in organisms.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1-1 shows the eGFP-mRNA@MPNP (Fe) as provided in example 2.5.1 of the present invention 3+ ) Statistics of percent results of eGFP positive cells transfected with 293T;
FIGS. 1-2 show the RBD-mRNA@MPNP (Fe) of example 2.5.1 of the present invention 3+ ) Expression quantity statistics of RBD caused by transfection 293T;
FIGS. 1-3 show the RBD-mRNA@MPNP (Fe) of example 2.5.1 of the present invention 3+ ) Statistical graphs of the ability to induce humoral immunity;
FIGS. 1 to 4 show the result of the process of example 2.5.1 of the present invention, NY-ESO-1-mRNA@MPNP (Fe) 3+ ) Statistical graphs of the ability to induce humoral immunity;
FIGS. 1-5 show the RBD-mRNA@MPNP (Fe) of example 2.5.1 of the present invention 3+ ) Statistical graphs of the ability to induce cellular immunity;
FIGS. 1 to 6 show the result of the process of example 2.5.1 of the present invention, NY-ESO-1-mRNA@MPNP (Fe) 3+ ) Statistical graphs of the ability to induce cellular immunity;
FIGS. 1-7 show eGFP-mRNA@MPNP (Al) as provided in example 2.5.2 of the present invention 3+ ) Statistics of percent results of eGFP positive cells transfected with 293T;
FIGS. 1-8 show the RBD-mRNA@MPNP (Al) of example 2.5.2 of the present invention 3+ ) Expression quantity statistics of RBD caused by transfection 293T;
FIGS. 1-9In example 2.5.2 of the present invention, RBD-mRNA@MPNP (Al 3+ ) Statistical graphs of the ability to induce humoral immunity;
FIGS. 1-10 show the NY-ESO-1-mRNA@MPNP (Al) of example 2.5.2 of the present invention 3+ ) Statistical graphs of the ability to induce humoral immunity;
FIGS. 1-11 show the RBD-mRNA@MPNP (Al) of example 2.5.2 of the present invention 3+ ) Statistical graphs of the ability to induce cellular immunity;
FIGS. 1-12 show the result of the method of example 2.5.2 of the present invention for NY-ESO-1-mRNA@MPNP (Al) 3+ ) Statistical graphs of the ability to induce cellular immunity;
FIGS. 1-13 show the Bcl-2-siRNA@MPNP (Fe) of example 2.6.1 of the present invention 3+ ) The ability to silence a target gene;
FIGS. 1 to 14 show the PLK1-siRNA@MPNP (Fe) of example 2.6.1 of the present invention 3+ ) Statistical graphs of the ability to silence a target gene;
FIGS. 1 to 15 show the Gal-1-siRNA@MPNP (Fe) in example 2.6.1 of the present invention 3+ ) Statistical graphs of the ability to silence a target gene;
FIGS. 1-16 are the Bcl-2-siRNA@MPNP (Al) of example 2.6.2 of the present invention 3+ ) The ability to silence a target gene;
FIGS. 1-17 show the PLK1-siRNA@MPNP (Al) of example 2.6.2 of the present invention 3+ ) Statistical graphs of the ability to silence a target gene;
FIGS. 1-18 show Gal-1-siRNA@MPNP (Al) in example 2.6.2 of the present invention 3+ ) Statistical graphs of the ability to silence a target gene;
FIGS. 1 to 19 show STAT3-ASO@MPNP (Fe) in example 2.7.1 of the present invention 3+ ) Statistical graphs of the ability to silence a cellular target gene;
FIGS. 1 to 20 show the alpha-syn-ASO@MPNP (Fe) of example 2.7.1 of the present invention 3+ ) Statistical graphs of the ability to silence a cellular target gene;
FIGS. 1-21 show the Bcl-2-ASO@MPNP (Fe) of example 2.7.1 of the present invention 3+ ) Statistical graphs of the ability to silence a cellular target gene;
FIGS. 1-22 show STAT3-ASO@MPNP (Al) in example 2.7.2 of the present invention 3+ ) Statistical graphs of the ability to silence a cellular target gene;
FIGS. 1-23 show the α -syn-ASO@MPNP (Al) of example 2.7.2 of the present invention 3+ ) Statistical graphs of the ability to silence a cellular target gene;
FIGS. 1-24 show the Bcl-2-ASO@MPNP (Al) of example 2.7.2 of the present invention 3+ ) Statistical graphs of the ability to silence a cellular target gene;
FIGS. 1-25 show the S-mRNA@MPNP (Fe) of example 2.8.1 of the present invention 3+ ) Expression quantity statistics of S protein caused by transfection 293T;
FIGS. 1-26 show drug (dsDNA and ssDNA) -metal-polyphenol complex particles (Fe) according to example 2.8.1 of the present invention 3 + ) A functional detection result diagram of (2);
FIGS. 1-27 show the S-mRNA@MPNP (Al) of example 2.8.2 of the present invention 3+ ) Expression quantity statistics of S protein caused by transfection 293T;
FIGS. 1-28 are particles of drug (dsDNA and ssDNA) -metal-polyphenol complex (Al) in example 2.8.2 of the present invention 3 + ) A functional detection result diagram of (2);
FIG. 2-1 shows a metal-polyphenol compound (Fe) according to example 3.1 of the present invention 3+ ) Is a uv absorption diagram of (2);
FIG. 2-2 shows the metal-polyphenol complex (Al) of example 3.2 of the present invention 3+ ) Is a uv absorption diagram of (2);
FIGS. 2-3 show Fe at low pH (pH=5.0) in example 4 of the present invention 3+ A characterization picture of shedding from the metal-polyphenol complex;
FIGS. 2-4 are statistical graphs showing the efficiency of drug-lipid particle entrapped nucleic acid (mRNA and siRNA) in example 5 of the present invention;
FIGS. 2-5 are graphs showing the results and statistics of the nucleosome escape ability of siRNA/mRNA@MPNP and siRNA/mRNA@LNP of example 6 of the present invention;
FIGS. 2-6 are statistical graphs of the eGFP positive cell rates of MPNP and LNP according to example 7 of the present invention;
FIGS. 2-7 are statistical graphs of the mRNA expression promoting capacities of MPNP and LNP in example 8 of the present invention;
FIGS. 2-8 are statistical graphs of the humoral immunity-promoting capabilities of MPNP and LNP in example 8 of the present invention;
FIGS. 2-9 are statistics of the results of the ability of MPNP and LNP to boost cellular immunity in example 8 of the present invention;
FIG. 3-1 is a statistical chart showing the results of intratumoral injection of the drug-metal-polyphenol complex particles for treating liver cancer in example 11 of the present invention.
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. 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 of" 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.
All ranges provided herein are intended to include each specific range within the given range as well as combinations of sub-ranges between the given ranges. Moreover, unless otherwise indicated, all ranges provided herein include the endpoints of the ranges. Thus, ranges 1-5 specifically include 1, 2, 3, 4, and 5, as well as subranges such as 2-5, 3-5, 2-3, 2-4, 1-4, and the like.
All publications and patent applications cited in this specification are herein incorporated by reference and for any and all purposes, each individual publication or patent application is specifically and individually indicated to be incorporated by reference. In the event of a discrepancy between the present disclosure and any publication or patent application incorporated by reference herein, the present disclosure controls.
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", "complex lipids", "derived lipids". "simple lipids" which include glycerides, glycerides of fatty acids, waxes, long chain fatty acids and long chain alcohols or sterols; "Complex lipids" refers to molecules that contain non-lipid components, including phospholipids and glycolipids, in addition to fatty acids and alcohols; "derived lipids" are derived from simple lipids or complex lipids.
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-polyphenol complex particle (MPNP) is a "lipid vesicle" in which a drug, such as a nucleic acid mRNA, is encapsulated as an encapsulated ingredient in the MPNP, which may be fully encapsulated and/or partially encapsulated.
The phrase "polyphenol molecule moiety" herein refers to the structure of a polyphenol molecule that is originally in the possession of a polyphenol 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 "metal-polyphenol complex" herein is composed of the reaction of the above-mentioned polyphenol molecule moiety, the above-mentioned metal ion moiety, the above-mentioned polyphenol moiety and the above-mentioned metal ion moiety being linked by a coordination bond.
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-dimethyl ammonium 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 ', N' -dimethylaminoethane) carbamoyl) cholesterol (DC-Chol) and N- (1, 2 dimyristoxyprop-3-yl) -N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). 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-polyphenol complex particles, "non-cationic lipids or non-ionizable lipids other than conjugated lipids that inhibit particle aggregation" in (iii) refers to the non-cationic lipids or non-ionizable lipids in (iii) being the lipids remaining in the metal-polyphenol complex particles after the conjugated lipids that inhibit particle aggregation are excluded.
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-polyphenol complex particles, non-cationic lipids or non-ionizable lipids other than 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 which itself can spontaneously form bilayer vesicles in water, exemplified by most phospholipids.
In metal-polyphenol 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 hydrophilic 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 60:
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. patent application No. 10/136,707, published as U.S. p. a 2003/007829, and PCT patent application No. CA 02/00669, the entire contents of each of which are incorporated herein by reference.
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 61:
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 62:
in formula 62, R1 and R2 are independently selected and are long chain alkyl groups having from about 10 to about 22 carbon atoms. The long chain alkyl groups 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 62, 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 62, 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 63:
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 "small 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 "circular RNA" or "circular polyribonucleotides" or "circular 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 circular or ring-shaped structure by covalent or non-covalent bonds.
As used herein, the terms "microRNA" or "miRNA" or "microRNA" are used interchangeably and refer to non-coding single-stranded polynucleic nucleotides 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 ' -untranslated region,3' -UTR) of the mRNA of the target gene, thereby regulating a biological function of the cell.
As used herein, the term "ASO" or "antisense oligonucleotide" or "antisense oligonucleotide" is used interchangeably and refers to an artificially synthesized nucleic acid fragment 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 (deoxy) ribonucleotides of the gene, including antisense DNA and antisense RNA.
As used herein, the terms "siRNA" or "small interfering" or "short interfering" or "sizing RNA" or "small interfering RNA" or "short interfering RNA" or "silencing RNA" are used interchangeably and refer to a class of double stranded RNA molecules that are 20 to 25 nucleotides in length and 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 (substitution of DNA or RNA with PNA (peptide nucleic acids, peptide nucleic acid)) 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 (Batzer et al, nucleic acid acids Res.19:5081 (1991); ohtsuka et al, J.biol.chem.260:2605-2608 (1985); and Cassol et al (1992); rossolini et al, mol.cell.probes 8:91-98 (1994)). "nucleotide" 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.
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.
In the present invention, the polyphenol molecular moiety in the metal-polyphenol complex is mainly derived from natural plant extracts, such as curcumin, and has a wide range of biological effects including antibacterial, antiviral, antifungal, antioxidant and anti-inflammatory activities. 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.
In the present invention, the coordination bond between the polyphenol molecule part and the metal ion part in the metal-polyphenol complex is broken under the condition of low pH (ph=5.0) such as lysosomes, and the metal ion is detached from the metal-polyphenol complex.
In the invention, the proportion of each component in the metal-polyphenol compound can be adjusted according to the structure of the specific metal-polyphenol compound component. The basis for the adjustable proportion of the delivery is as follows: since the hydroxyl groups of the polyphenol molecules are linked to the metal ions by coordination bonds, the addition ratio of the polyphenol molecules to the metal ions can be adjusted according to the number of binding sites contained in the polyphenol molecules as long as the polyphenol molecules contain a plurality of binding sites.
Metal-polyphenol composite particles (Metal-chelated polyphenol complex nanoparticles, MPNP)
The principle of loading nucleic acid with metal-polyphenol complex particles assembled from metal-polyphenol complexes is: the polyphenol molecules are linked to the metal ions by coordination bonds, forming a metal-polyphenol complex, the metal ions of which are linked to the nucleic acid by coordination bonds, thereby ensuring that the metal-polyphenol complex self-assembles with other components into MPNP while loading the nucleic acid into the nanoparticle.
In this context, the term "non-cationic lipids or non-ionizable lipids other than conjugated lipids that inhibit aggregation of the particles" refers to component (iii) in the metal-polyphenol 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 to be achieved by the present invention.
In some embodiments, the molar ratio of metal-polyphenol complex in the feedstock is 5% to 30%, 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%, or 30%. Preferably 5% to less than 10%, 10% to 15% or 15% to 20%, more preferably 5%, 10% or 15%.
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%. Preferably 3% to 5% or 5% to 10%, more preferably 3%, 5% or 10%.
In some embodiments, the non-cationic lipid or non-ionizable lipid is optionally cholesterol in a molar ratio of 0% -48% in the starting material, e.g., 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47% or 48%. Preferably 10% to 30%, 30% to 47% or 10% to 20%, more preferably 10%, 30% or 47%.
In some embodiments, the metal-polyphenol complex particles contain, in addition to cholesterol, other non-cationic lipids or non-ionizable lipids in a molar ratio of 30% to 75%, e.g., 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75% in the starting material. Preferably 45% to 55%, 60% to 65% or 50% to 65%, further 45%, 55%, 60% or 65%.
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, sgRNA, DNA, ecDNA, an artificial nucleic acid), a metal-polyphenol complex, a non-cationic lipid or a non-ionizable lipid, and a bilayer stabilizing component, such as a conjugated lipid that inhibits 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-polyphenol complex particles to avoid degradation of the drug, enabling delivery of the drug into the cells.
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 RNA population is used, for example by transcription of cDNAs corresponding to the RNA population, or by use of RNA polymerase, RNA complements are also provided (e.g., formation of dsRNA, which is digested by e.coli (e.coli) RNAse III 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, cell 107:309 (2001)). The transcription unit is incorporated into a plasmid or DNA vector from which the interfering RNA is transcribed. Plasmids suitable for in vivo delivery of genetic material for therapeutic purposes are described in detail in U.S. patent nos. 5,962,428 and 5,910,488, both of which are incorporated herein by reference. The selected plasmid may provide transient or stable delivery of the target cell. It will be apparent to those skilled in the art that plasmids originally designed for expression of the desired gene sequences may be modified to include transcribed siRNAsTranscription unit cassette.
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-polyphenol complex, metal-polyphenol complex particles, and use of drug-lipid particles
In some embodiments, when the drug is a nucleic acid, the metal-polyphenol complex, metal-polyphenol complex particles can all be used in facilitating the escape of the drug lysosomes, as well as in facilitating the expression of the nucleic acid. The metal-polyphenol complex, metal-polyphenol complex particles can also be used to deliver drugs, introduce drugs into cells, and thereby achieve prevention and treatment of applicable diseases or conditions with drugs.
In some embodiments, the invention provides for the use of metal-polyphenol complexes, metal-polyphenol complex particles, drug-lipid particles, e.g., for compositions 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 prepared from the above metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles, having the above functions and uses of the metal-polyphenol complexes, metal-polyphenol complex particles, and drug-lipid particles, of a specific type such as, but not limited to, kits, medicaments, and the like, optionally containing other 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), stephenson JAMA 289.1494 (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., FIELDS VIROLOGY,2001, supra). Hepatitis C nucleic acid sequences that can be silenced include, but are not limited to: serine proteases (e.g., NS3/NS 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., forsman et al, cell 81:687 (1995); seol et al, mol.endocrinol.9:72 (1995), zaacki et al, PNAS USA 94:7909 (1997); sakai, et al, cell85:1037-1046 (1996); duncan, et al, J.biol. Chem.272:12778-12785 (1997); willy, et al, genes dev.9 (9): 1033-45 (1995); lehmann et al, J.biol.chem.272 (6): 3137-3140 (1997); janowski, et al, nature 383:728-731 (199; peet, et al, 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, blood 101:3157 (2003)); over-expressed sequences such as multi-drug resistance Genes (Nieth, et al, FEBS Lett.545:144 (2003); wu, et al, cancer Res.63:1515 (2003)), cyclin (Li, et al, cancer rRs.63:3593 (2003); zou, et al, genes Dev.16:2923 (2002)), beta-catenin (Verma, et al, clin Cancer Res.9:1291 (2003)), telomerase Genes (Kosciole, et al, mol Cancer Ther.2:209 (2003)), c-MYC, N-MYC, BCL-2, ERBB1 and ERBB2 (Nagy, et al exp.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, FEBS letters 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 genome of the native 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 can 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, kock et al, hepatolog 38 (6): 1410-8 (2003)), prenylation inhibitors (see, for example, border et al, J. Clin. Invert. 112 (3): 407-414 (2003)), ganciclovir (see, for example, yurdadin et al, J. Hepal. 37 (2): 266-71 (2002)), and saponins c and d (see, for example, chick et al, 60 (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 (such as vitamin A1, and extended release anti-cholesterol (such as vitamin c) and vitamin B1, such as, for example, vitamin c, 11, and stepstatin (steppe 1, 2003, 7). 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, tetrarhodimine isothiocynate (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, ajoint 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 & EN 36; theJournal OfNIH 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 similar results.
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 polyphenol complex nanoparticles (drug@mpnp).
Experimental example one preparation of drug-Metal-polyphenol Complex particles (mRNA-loaded Metal-chelated polyphenol complex nanoparticles, mRNA@MPNP)
EXAMPLE 1 preparation of Metal-polyphenol Complex (Metal-chelated polyphenol complex nanoparticles, MPNP)
Example 1.1 preparation of metal ion Fe 3+ Metal-polyphenol complexes
Dissolving curcumin (formula 1) in ethanol at a concentration of 1.5mg/ml, and adding anhydrous FeCl 3 Wherein curcumin (formula 1), anhydrous FeCl 3 The molar ratio of (2) is 1:1, reflux reaction at 60℃for 1 hour. After the reaction is finished, the solution is suspended, the product is dissolved by ultrapure water and filtered, and the product obtained after freeze-drying is the metal-polyphenol compound. The metal-polyphenol complex structure is shown below.
Analysis of results: curcumin (formula 1) and FeCl 3 Reacting at 60deg.C for 1 hr, with curcumin (formula 1) at concentration of 1.5mg/mL and curcumin (formula 1) and FeCl 3 The feed ratio of (2) is 1: the yield of the target product obtained at 1 was 95%.
EXAMPLE 1.2 preparation of metal ion Al 3+ Metal-polyphenol complexes
This example differs from example 1.1 in that FeCl 3 Replaced by Al (NO) 3 ) 3 ·9H 2 O。
The structure of the prepared metal-polyphenol compound is shown as follows.
Analysis of results: curcumin (formula 1) and Al (NO) 3 ) 3 ·9H 2 O was reacted at 60℃for 1 hour, with curcumin (formula 1) at a feed concentration of 1.5mg/mL and curcumin (formula 1) and Al (NO) 3 ) 3 ·9H 2 The feeding ratio of O is 1: the yield of the target product obtained at 1 was 98%.
Example 2 preparation of mRNA-Metal-polyphenol Complex particles (mRNA-loaded Metal-chelated polyphenol complex nanoparticles, mRNA@MPNP)
Preparation of metal ion Fe 3+ mRNA-metal-polyphenol complex particles
A metal-polyphenol complex was prepared according to the method in example 1.1, in which curcumin (formula 1), feCl 3 The metal-polyphenol complex was dosed at a 1:1 dosing ratio and dissolved in ethanol as an organic phase at different molar ratios with distearoyl phosphatidylcholine (DSPC, formula 51, as a non-cationic lipid or a non-ionizable lipid), cholesterol (CHOL, formula 59, as a non-cationic lipid or a non-ionizable lipid), and DSPE-PEG2000 (formula 58, as a conjugated lipid that inhibits particle aggregation). Wherein the metal-polyphenol complex, DSPC (formula 51), CHOL (formula 59) and DSPE-PEG2000 (formula 58) have a ratio of 5%, 60%, 30% and 5%, respectively. mRNA was dissolved in enzyme-free Tris-HCl buffer (molar concentration 0.1M) at pH5.0 at a concentration of 20. Mu.g/mL as an aqueous phase. Mixing the mass of the metal-polyphenol compound and the mass of mRNA in a mass ratio of 20:1 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). And preparing the eGFP-mRNA@MPNP. eGFP-mRNA@MPNP was incubated with 293T cells at a concentration of 2. Mu.g/mL (concentration of mRNA contained), the control group was incubated with MPNP without drug loading, and after 48h the cell suspension was harvested and the percentage of eGFP positive cells was detected by flow cytometry.
The prepared eGFP-mRNA@MPNP is subjected to detection on particle size, surface potential and stability, and the efficiency of encapsulating nucleic acid by the eGFP-mRNA@MPNP is 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 determined to be 10 mug/mL, the mass ratio of the metal-polyphenol compound to the nucleic acid is 20:1, the nucleic acid with the same concentration is dissolved in enzyme-free Tris-HCl buffer solution to be used as a positive control, and the negative control is enzyme-free Tris-HCl 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 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). 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 12-well plates at a density of 5X 10 5 Cells/well, when cell density reached 80%, cells were incubated with 1mL MPNP or eGFP-mrna@mpnp, where the eGFP-mrna@mpnp concentration was 2 μg/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.
Preparation of metal ions of Al 3+ mRNA-metal-polyphenol complex particles
A metal-polyphenol complex was prepared according to the method in example 1.2, in which curcumin (formula 1), al (NO) 3 ) 3 ·9H 2 O was dosed at a 1:1 dosing ratio and the metal-polyphenol complex was combined with distearoyl phosphatidylcholine (DSPC, formula 51, non-cationic lipid or non-ionizable lipid), cholesterol (CHOL, formula 59, non-cationic lipid or non-ionizable lipid) Ionizable lipids), and DSPE-PEG2000 (formula 58, conjugated lipids that inhibit particle aggregation) were dissolved in ethanol at different molar ratios as the organic phase. Wherein the metal-polyphenol complex, DSPC (formula 51), CHOL (formula 59) and DSPE-PEG2000 (formula 58) have a ratio of 5%, 45%, 47% and 3%, respectively. mRNA was dissolved in enzyme-free Tris-HCl buffer (molar concentration 0.1M) at pH5.0 at a concentration of 20. Mu.g/mL as an aqueous phase. Mixing the mass of the metal-polyphenol compound and the mass of mRNA in a mass ratio of 18:1 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). And preparing the eGFP-mRNA@MPNP. eGFP-mRNA@MPNP was incubated with 293T cells at a concentration of 2. Mu.g/mL (concentration of mRNA contained), the control group was incubated with MPNP, and after 48h the cell suspension was harvested and the percentage of eGFP positive cells was detected by flow cytometry.
The prepared eGFP-mRNA@MPNP is subjected to detection on particle size, surface potential and stability, and the efficiency of encapsulating nucleic acid by the eGFP-mRNA@MPNP is 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, the mass ratio of the metal-polyphenol complex to the nucleic acid is 18:1, the nucleic acid with the same concentration is dissolved in enzyme-free Tris-HCl buffer solution to be used as a positive control, and the negative control is enzyme-free Tris-HCl 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 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). 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 12-well plates at a density of 5X 10 5 Cells/well, when cell density reached 80%, cells were incubated with 1mL MPNP or eGFP-mrna@mpnp, where the eGFP-mrna@mpnp concentration was 2 μg/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.
The principle of loading nucleic acid from Metal-polyphenol complex particles (Metal-chelated polyphenol complex nanoparticles, MPNP) assembled from Metal-polyphenol complexes is: curcumin and Fe through coordination bond 3+ Or Al 3+ Are connected to form a metal-polyphenol complex, fe of the metal-polyphenol complex 3+ Or Al 3+ The coordination bond is connected with the nucleic acid, so that the metal-polyphenol complex and other components are self-assembled into MPNP, and the nucleic acid is loaded into the nanoparticle. The contribution of curcumin in MPNP-loaded nucleic acids has two possibilities: (1) curcumin interacts with nucleic acids to assist in loading the MPNP with nucleic acids, e.g., curcumin assists in loading the nucleic acids by inserting into the minor groove of the nucleic acids; (2) curcumin may not directly interact with nucleic acids.
EXAMPLE 2.1 ratio of the Components of the Metal-polyphenol Complex
Curcumin (formula 1) and FeCl in example 2 3 According to different throwing ratios (1:1, 3:2 and 2:1), other steps are the same as in example 2, different eGFP-mRNA@MPNP is prepared, and the nucleic acid entrapment rates of the eGFP-mRNA@MPNP are detected respectively.
Analysis of results: as shown in the results Table 1-1, when curcumin (formula 1), feCl 3 When the adding ratio of the metal-polyphenol compound particles is 1:1, the mRNA entrapment efficiency of the prepared metal-polyphenol compound particles is 85%; when curcumin (formula 1), feCl 3 When the adding ratio of the metal-polyphenol compound particles is 3:2, the mRNA entrapment efficiency of the prepared metal-polyphenol compound particles is 72%; when curcumin (formula 1), feCl 3 When the addition ratio of (2) to (1) was 2, the mRNA entrapment rate of the metal-polyphenol composite particles was 63%. Fe in drug-lipid particles 3+ Is to link curcumin with nucleic acid, each Fe 3+ At most three complexing sites, so curcumin, feCl 3 The dosing ratio in the drug-lipid particle should be 1:1, it is ensured that as much of the nucleic acid as possible is entrapped in the metal-polyphenol complex particles. Our results also demonstrate that when curcumin, feCl 3 When the addition ratio of the metal-polyphenol compound particles is 1:1, the mRNA entrapment rate of the prepared metal-polyphenol compound particles is highest. When curcumin, feCl 3 The ratio of the metal-polyphenol composite particles is in the range from 1:1 to 2:1, and the nucleic acid entrapment rate of the metal-polyphenol composite particles is above 60%.
TABLE 1-1 Metal ion is Fe 3+ Component addition ratio of metal-polyphenol composite and function of prepared metal-polyphenol composite particles
Curcumin (formula 1), feCl 3 Is added to the ratio of (2) mRNA entrapment Rate of Metal-polyphenol Complex particles
1:1 85％
3:2 72％
2:1 63％
Curcumin (formula 1), al (NO) in example 2 3 ) 3 ·9H 2 O is put according to different putting ratios (1:1, 3:2 and 2:1), other steps are the same as in example 2, different eGFP-mRNA@MPNP is prepared, and the nucleic acid entrapment rates of the eGFP-mRNA@MPNP are detected respectively.
Analysis of results: as shown in the results of tables 1-2, when curcumin (formula 1), al (NO 3 ) 3 ·9H 2 When the adding ratio of O is 1:1, the eGFP-mRNA entrapment efficiency of the prepared metal-polyphenol compound particles is 86%; when curcumin (formula 1), al (NO) 3 ) 3 ·9H 2 When the adding ratio of O is 3:2, the eGFP-mRNA entrapment efficiency of the prepared metal-polyphenol compound particles is 70%; when curcumin (formula 1), al (NO) 3 ) 3 ·9H 2 When the adding ratio of O is 2:1, the eGFP-mRNA entrapment efficiency of the prepared metal-polyphenol compound particles is 66%. Al in the metal-polyphenol composite particles 3+ The function of (2) is to link phospholipid complexes with nucleic acids, each Al 3+ At most three complexing sites, curcumin, al (NO) 3 ) 3 ·9H 2 The dosing ratio of O in the drug-lipid particles should be 1:1, it is ensured that the metal-polyphenol complex particles encapsulate as much nucleic acid as possible. The results of the experiment also demonstrate that when curcumin, al (NO 3 ) 3 ·9H 2 When the addition ratio of O is 1:1, the inclusion rate of eGFP-mRNA of the prepared metal-polyphenol compound particles is highest. When curcumin, al (NO) 3 ) 3 ·9H 2 The adding ratio of O is in the range from 1:1 to 2:1, and the nucleic acid entrapment rate of the metal-polyphenol compound particles is above 60%.
TABLE 1-2 Metal ion is Al 3+ Component addition ratio of metal-polyphenol composite and function of prepared metal-polyphenol composite particles
EXAMPLE 2.2 preparation of Metal-polyphenol Complex in drug-lipid particles, distearoyl phosphatidylcholine (DSPC), DSPE-PEG2000 and Cholesterol (CHOL) ratios
As compared with example 2, the ratios of the metal-polyphenol complex, distearoyl phosphatidylcholine (DSPC, formula 51), DSPE-PEG2000 (formula 58), cholesterol (CHOL, formula 59) are shown in tables 1 to 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-polyphenol complex (metal ion is Fe 3+ ) When the ratio is in the range of (5-20)%, DSPC is in the range of (40-75)%, CHOL is in the range of (0-48)%, DSPE-PEG2000 is in the range of (2-10)%, the particle diameter of the drug-lipid particles is in the range of 30-400 nm, the surface potential is in the range of-10 mV, the in vitro stability is more than or equal to 3 days, and the mRNA encapsulation rate is achieved>50%, the positive cell rate of eGFP protein is more than 65%. Wherein when the metal-polyphenol complex accounts for 5%, distearoyl phosphatidylcholine (DSPC) accounts for 60%, cholesterol (CHOL) accounts for 30% and DSPE-PEG2000 accounts for 5%, the performance of the drug-lipid particle is optimal, namely the particle size is in the range of 120nm, the surface potential is in the range of-1.99 mV, and the in vitro stability is improved>7 days, the mRNA entrapment rate was 85% and the positive cell rate of eGFP protein was 97%. Because the Metal-polyphenol complex particles (Metal-chelatedpolyphenol complex nanoparticles, MPNP) adsorb nucleic acids primarily by Metal-polyphenol complexes, the ratio of Metal-polyphenol complexes cannot be too low; the DSPC has the function of maintaining the stable structure of the nano particles, and the content of the DSPC is 40-75 percent, so that the performance is better; 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 percent; when the CHOL content is at 0%, From the experimental results, the stability of the drug-lipid particles is within an acceptable range, and when the CHOL content is more than 0% and less than 48%, the CHOL has the effect of enhancing the fluidity of the nanoparticles, thereby being beneficial to maintaining the stability of the nanoparticles.
The above results suggest that the metal-polyphenol complex (metal ion is Fe 3+ ) The drug-metal-polyphenol compound particles (mRNA@MPNP) have better drug carrying performance when the ratio of DSPE to PEG is in the range of (5-20)%, the ratio of DSPC to CHOL is in the range of (40-75)%, and the ratio of DSPE-PEG2000 is in the range of (2-10)%.
TABLE 1-3 Metal ion is Fe 3+ Ratio of the components in the drug-metal-polyphenol complex particles (mRNA@MPNP)
Analysis of results: as shown in the results tables 1 to 4, when the metal-polyphenol complex (metal ion is Al 3+ ) When the ratio is in the range of (5-20)%, the ratio of DSPC is in the range of (30-75)%, the ratio of CHOL is in the range of (0-48)%, the ratio of DSPE-PEG2000 is in the range of (2-10)%, the particle diameter of the metal-polyphenol compound particles is in the range of 30-400 nm, the surface potential is in the range of-10 mV, and the in vitro stability is not less than>3 days, mRNA entrapment rate>50%, the positive expression rate of eGFP protein is more than 65%. Wherein when the metal-polyphenol compound accounts for 5 percent, distearoyl phosphatidylcholine (DSPC) accounts for 45 percent, cholesterol (CHOL) accounts for 47 percent and DSPE-PEG2000 accounts for 3 percent, the metal-polyphenol compound particles have optimal performance, namely the particle diameter is in the range of 100nm, the surface potential is in the range of-2.74 mV, and the stability in vitro >7 days, the mRNA entrapment rate was 86% and the positive cell rate of eGFP protein was 97%. Because mrna@mpnp adsorbs nucleic acids primarily by metal-polyphenol complexes, the ratio of metal-polyphenol complexes must not be too low; when the DSPC content is within the range of (30-75)%, the stability of the nanoparticle is acceptableWithin the range; 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 is in the range of (2-10)% and has better performance; 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-polyphenol complex (metal ion is Al 3+ ) When the ratio is in the range of (5-20)%, the DSPC ratio is in the range of (30-75)%, the CHOL ratio is in the range of (0-48)%, the DSPE-PEG2000 ratio is in the range of (2-10)%, and the mRNA@MPNP has better drug carrying performance.
TABLE 1-4 Metal ion is Al 3+ Ratio of the components in the drug-metal-polyphenol complex particles (mRNA@MPNP)
Example 2.3 preparation of non-cationic lipids or non-ionizable lipid species in eGFP-mRNA@MPNP
In comparison with example 2, 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@mpnp can be replaced by other non-cationic lipids or non-ionizable lipids other than conjugated lipids that inhibit particle aggregation, DSPE, DSPA and DSPG were chosen to replace DSPC, respectively, and by examining particle size, surface potential, stability and mRNA entrapment rate, it was demonstrated that DSPC in eGFP-mrna@mpnp can be replaced by other non-cationic lipids or non-ionizable lipids, the function of which after replacement is equivalent to that of eGFP-mrna@mpnp containing DSPC (metal ions are Fe 3 And tables 1 to 6 (metal ion is Al) 3 +). Because the main role of the non-cationic lipid DSPC in eGFP-mrna@mpnp is to make the liposome membrane more fusogenic, more stable and less toxic, while other non-cationic lipids or non-ionizable lipids also have the function of making the liposome membrane more fusogenic, more stable and less toxic, the DSPC in the drug-lipid particles can be replaced by other non-cationic lipids or non-ionizable lipids and their efficacyIs not affected.
TABLE 1-5 Metal ion is Fe 3 Properties of non-cationic lipids other than conjugated lipids inhibiting particle aggregation in+ drug-metal-polyphenol complex particles (mRNA@MPNP)
Tables 1 to 6 Metal ions are Al 3 Properties of non-cationic lipids other than conjugated lipids inhibiting particle aggregation in+ drug-metal-polyphenol complex particles (mRNA@MPNP)
Example 2.4 preparation of conjugated lipid species for inhibiting aggregation of particles in eGFP-mRNA@MPNP
Compared with example 2, the substitution of DSPE-PEG2000 is shown in tables 1-7 (metal ion is Fe 3 And tables 1 to 8 (metal ion is Al) 3 Shown in +) and the rest of the conditions are the same.
Analysis of results: to explore that DSPE-PEG2000 in eGFP-mrna@mpnp could be replaced by other particle aggregation inhibiting conjugated lipids, three other particle aggregation inhibiting conjugated lipids, DSPE-PEG700, DSPE-PEG5000 and DSPE-PEG1000 were selected to replace DSPE-PEG2000, respectively, and by examining particle size, surface potential, stability and mRNA entrapment rate, it was demonstrated that DSPE-PEG2000 in eGFP-mrna@mpnp could be replaced by other particle aggregation inhibiting conjugated lipids, which after replacement were functionally equivalent to the efficacy of eGFP-mrna@mpnp comprising DSPE-PEG2000 (tables 1-7 and 1-8). Because the primary role of DSPE-PEG2000 in eGFP-mrna@mpnp is to inhibit aggregation, while other conjugated lipids that inhibit particle aggregation also have the function of inhibiting aggregation, DSPE-PEG2000 in eGFP-mrna@mpnp can be replaced by other conjugated lipids that inhibit particle aggregation, and its efficacy is not affected.
TABLE 1-7 Metal ion is Fe 3+ Inhibiting particle aggregation in drug-metal-polyphenol complex particles (mRNA@MPNP)Pooled conjugated lipid species
TABLE 1-8 Metal ion is Al 3+ Conjugated lipid species in drug-metal-polyphenol complex particles (mrna@mpnp) that inhibit particle aggregation
Example 2.5 preparation of mRNA@MPNP and characterization of the Effect
Example 2.5.1 the metal ion is Fe 3+ Preparation and effect characterization of mRNA@MPNP of (E)
Three kinds of mRNA@MPNP containing different target protein mRNA sequences were prepared by referring to the method of example 2 by replacing the mRNA in example 2 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 2, and eGFP-mRNA@MPNP, RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP are respectively obtained.
The eGFP-mRNA@MPNP was incubated with 293T cells at a concentration of 2. Mu.g/mL (concentration of the contained mRNA), the control group was incubated with MPNP, 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@MPNP 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 MPNP, after 24h, the supernatant was frozen at-20℃for later use after centrifugation, and the expression level of the fresh crown antigen RBD protein on the cells was detected using a commercially available novel crown antigen RBD ELISA detection kit, and the results are shown in FIGS. 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 3,3', 5' -tetramethyl benzidine (TMB) substrate solution into each well, 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.
The experimental animals were randomly divided into 2 groups (experimental and control groups) of 5 animals each. Wherein the RBD-mRNA@MPNP animal model is a BALB/c mouse, each mouse is subjected to first muscle administration on day 1 and second muscle administration on day 14, the experimental group is injected with RBD-mRNA@MPNP, and the control group is injected with metal-polyphenol complex particles (MPNPs) which are not loaded with mRNA. The dose per administration was 100. Mu.L, with the RBD-mRNA@MPNP 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@MPNP 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@MPNP and the control group was injected with metal-polyphenol complex particles (MPNPs) not loaded with mRNA. The dose per administration was 100 μl, wherein the mrna@mpnp formulation in the experimental group contained 30mg 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), adjusting pH to 9.6 with 1M NaOH solution after the solid is completely dissolved, and storing the prepared coating solution at 4deg.C.
2. Washing liquid: to 1L of 0.01M PBS solution, 0.5mL Tween-20 was added, and the mixture was left at room temperature after uniform mixing.
3. Sealing liquid: precisely weighing 20g BSA, adding into 1L 0.01M PBS 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 was weighed precisely and dissolved in 250mL 0.01M PBS solution, after the solid was completely dissolved, 1.25mL of LTwen-20 was added thereto, and after mixing well, it was stored at 4℃for further use.
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 0.15 mL。
6. Stop solution: 2M H 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@MPNP administration, single cell suspensions were prepared 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 kits, 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@MPBP, 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 93.7% for the eGFP-mrna@mpnp experimental group, while no eGFP signal was detected for the MPNP control group; as shown in FIGS. 1-2, the RBD protein encoded by the MPNP included RBD-mRNA was 166ng/mL in the 293T cell supernatant, and the RBD protein content of the 293T cell supernatant transfected with the empty vector MPNP was 0. The results suggest that the drug-metal-polyphenol complex particles (mRNA-MPNP) can encapsulate, deliver any mRNA and directly encode polypeptides in cells. As shown in FIGS. 1-3,1-4, RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP were both effective to induce humoral immunity in mice, producing high levels of antigen-specific binding antibodies. Wherein the titer of IgG antibody in the mice of the RBD-mRNA@MPNP treatment group reaches 84363.4; the titer of the IgG antibody of the mice treated with NY-ESO-1-mRNA@MPNP reaches 4283.56. As shown in FIGS. 1-5,1-6, RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP were both effective to induce cellular immunity in mice, i.e., to activate immune cells and produce a large amount of cytokines. Wherein RBD-mRNA@MPNP enables the expression amounts of cytokines IFN-gamma, IL-2 and IL-4 to reach 271.8pg/mL, 269.6pg/mL and 75.8pg/mL respectively; the expression levels of cytokines IFN-. Gamma., IL-2 and TNF-. Alpha.were 76.38pg/mL, 74.56pg/mL and 69.31pg/mL by the NY-ESO-1-mRNA@MPNP, respectively. The result shows that the medicine-metal-polyphenol compound particles (mRNA@MPNP) can encapsulate and deliver any mRNA, so that the expression of target proteins (antigens) is promoted, the humoral immunity and the cellular immunity of mice are effectively induced, high-level antigen-specific binding antibodies and cytokines are generated, and the functions of anti-novel coronavirus mRNA vaccines and anti-tumor mRNA vaccines are exerted.
Example 2.5.2 the metal ion is Al 3+ Preparation and effect characterization of mRNA@MPNP of (E)
This example differs from example 2.5.1 in that the metal ion Fe in example 2.5.1 3+ Replaced by Al 3 + 。
Analysis of results: as shown in fig. 1-7, the eGFP positive cell rate was 97.03% for the eGFP-mrna@mpnp experimental group, while no eGFP signal was detected for the MPNP control group; as shown in FIGS. 1-8, the RBD protein encoded by the MPNP included RBD-mRNA was 207ng/mL in the 293T cell supernatant, and the RBD protein content of the 293T cell supernatant transfected with the empty vector MPNP was 0. The results suggest that mRNA-MPNP can encapsulate, deliver any mRNA and directly encode the polypeptide in the cell. As shown in FIGS. 1-9,1-10, RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP were both effective to induce humoral immunity in mice, producing high levels of antigen-specific binding antibodies. Wherein the titer of IgG antibody in the mice of the RBD-mRNA@MPNP treatment group reaches 94828.6; the titer of the IgG antibody of the mice treated with NY-ESO-1-mRNA@MPNP reaches 5848.02. As shown in FIGS. 1-11,1-12, RBD-mRNA@MPNP and NY-ESO-1-mRNA@MPNP were both effective to induce cellular immunity in mice, i.e., to activate immune cells and produce a large amount of cytokines. Wherein RBD-mRNA@MPNP enables the expression amounts of cytokines IFN-gamma, IL-2 and IL-4 to respectively reach 306.2pg/mL, 289.6pg/mL and 88.2pg/mL; the expression levels of cytokines IFN-gamma, IL-2 and TNF-alpha reach 91.88pg/mL, 85.32pg/mL and 80.22pg/mL respectively by using NY-ESO-1-mRNA@MPNP. The result shows that the mRNA@MPNP can encapsulate and deliver any mRNA, so that the expression of target proteins (antigens) is promoted, the humoral immunity and the 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 2.6 preparation and Effect of siRNA-Metal-polyphenol Complex particles (siRNA@MPNP)
Example 2.6.1 the metal ion is Fe 3+ Preparation and Effect of siRNA-Metal-polyphenol Complex particles (siRNA-loaded Metal-chelated phospholipid complex nanoparticles, siRNA@MPNP)
Three kinds of siRNA@MPNP containing different siRNAs were prepared by referring to the method of example 2, replacing the mRNA in example 2 with the 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@MPNP was prepared in the same manner as in example 2.
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 (Bio Industries) 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@MPNP 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 RT-PCR technology, and the capacity of silencing the target genes of the cells by the siRNA@MPNP is counted.
RT-PCR specific procedure:
extraction of total RNA: the six-well plate was discarded, rinsed 3 times with PBS buffer, and cells were lysed by adding 1ml of LTrilzol to each well. 200. Mu.L of chloroform was added, the mixture was shaken well, allowed to stand at room temperature for 10min, and centrifuged at 13000rpm at 4℃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 13000rpm for 15min at 4℃to obtain 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 TaKa RaPrime 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 LPrime 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 LPCR 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). Repeating the experiment for three times, averaging to obtain Ct value of each group, and calculating tables of the experimental group and the control group And (5) reaching a difference multiple. The control gene was GAPDH. The RT-PCR primers were as follows: (1) bcl-2 primer: forward:5'-AGGATTGTGGCCTTCTTTGAG-3', reverse:5'-AGACAGCCAGGAGAAATC AAAC-3'; (2) PLK1 primer: forward:5'-ACCAGCACGTCGTAGGATTC-3', reverse:5'-CAAGCAATTTGCCGTAGG-3'; (3) gal-1 primer: forward:5'-CAATCAT GGCCTGTGGTCTG-3', reverse:5'-GTG TAGGCACAGGTTGTTGCTG-3'. (4) GAPDH primer: forward:5'-TCAGGGGTTTCACATTTGGCA-3', reverse:5'-GG AGCGGAAAACCA-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: as shown in fig. 1-13,1-14,1-15 (wherein scr siRNA is a random control sequence), three drug-metal-polyphenol complex particles (Bcl-2-sirna@mpnp, PLK1-sirna@mpnp, gal-1-sirna@mpnp) all significantly interfere with their corresponding target genes. The inhibition rate of Bcl-2-siRNA@MPNP to the target gene Bcl-2 reaches 67%; the inhibition rate of PLK1-siRNA@MPNP to a target gene PLK1 reaches 87%; the inhibition rate of Gal-1-siRNA@MPNP to the target gene Gal-1 reaches 64%. The result shows that the siRNA@MPNP can carry any siRNA to perform the intervention treatment of the target gene, and plays the role of carrying siRNA drugs, vaccines or other products.
Example 2.6.2 the metal ion is Al 3+ Preparation and Effect of siRNA-Metal-polyphenol Complex particles (siRNA-loaded Metal-chelated phospholipid complex nanoparticles, siRNA@MPNP)
This example differs from example 2.6.1 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@MPNPs significantly interfered with its corresponding target gene. The inhibition rate of Bcl-2-siRNA@MPNP to the target gene Bcl-2 reaches 72%; the inhibition rate of PLK1-siRNA@MPNP to a target gene PLK1 reaches 88.07%; the inhibition rate of Gal-1-siRNA@MPNP to the target gene Gal-1 reaches 70.11%. The result shows that the siRNA@MPNP can carry any siRNA to perform the intervention treatment of the target gene, and plays the role of carrying siRNA drugs, vaccines or other products.
EXAMPLE 2.7 preparation and Effect of ASO-Metal-polyphenol Complex particles (ASO@MPNP)
Example 2.7.1 the metal ion is Fe 3+ Preparation and Effect of ASO-metal-polyphenol Complex particles (ASO-loaded metal-chelated phospholipid complex nanoparticles, ASO@MPNP)
Three different ASO-containing drug-metal-polyphenol complex particles (aso@mpnp) were prepared separately by referring to the method of example 2, replacing the mRNA in example 2 with ASO. 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 drug (ASO) -metal-polyphenol complex particles were prepared in the same manner as in example 2. Incubation of different aso@mpnp with different cells: ASO@MPNP targeting STAT3 gene incubates U251 human brain glioblastoma cells; incubating SH-SY5Y human neuroblastoma cells with ASO@MPNP targeting the alpha-syn gene; ASO@MPNP targeting Bcl-2 gene was incubated with Daudi human lymphoma cells. At 1X 10 per well 6 After cell density inoculation in 6-well plates for about 24 hours, cells were collected after each well had been incubated with the above-mentioned ASO-containing drug-metal-polyphenol complex particles (ASO@MPNP) in which the concentration of ASO was 1. Mu.g/mL, respectively, and total RNA of the cells was extracted, and mRNA expression amounts of target genes (STAT 3, α -syn, bcl-2) were detected by RT-PCR technique, respectively, and the capacity of ASO@MPNP 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 (Bio Industries) 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 RPMI 1640+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.
RT-PCR specific procedure:
extraction of total RNA: the six-well plate was discarded, rinsed 3 times with PBS buffer, and cells were lysed by adding 1ml of LTrilzol to each well. 200. Mu.L of chloroform was added, the mixture was shaken well, allowed to stand at room temperature for 10min, and centrifuged at 13000rpm at 4℃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 13000rpm for 15min at 4℃to obtain 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 extracted RNA by using an ultra-micro ultraviolet visible spectrophotometer.
Reverse transcription of cDNA: using TaKa RaPrime 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 LPrime Script RT Enzyme Mix I, 1 mu LRT Primer Mix,4 mu L5X Prime Script Buffer 2,4 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 LPCR 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: 1-19,1-20,1-,21 (wherein scrASO is a random control sequence), all three ASO@MPNPs can obviously interfere with the corresponding target genes, wherein the inhibition rate of STAT 3-ASO@MPNPs to the target genes STAT3 reaches 72%; the inhibition rate of the alpha-syn-ASO@MPNP to the target gene alpha-syn reaches 78%; the inhibition rate of Bcl-2-ASO@MPNP to the target gene Bcl-2 reaches 62%. The results suggest that the drug-metal-polyphenol complex particles (aso@mpnp) can carry any ASO for targeted gene intervention therapy, and act as ASO-carrying drugs, vaccines or other products.
Example 2.7.2 the metal ion is Al 3+ Preparation and Effect of ASO-metal-polyphenol Complex particles (ASO-loaded metal-chelated phospholipid complex nanoparticles, ASO@MPNP)
This example differs from example 2.7.1 in that the metal ion Fe in example 2.7.2 3+ Replaced by Al 3 + 。
Analysis of results: as shown in fig. 1-22,1-23 and 1-24, all three aso@mpnp can obviously interfere with the corresponding target gene, wherein the inhibition rate of STAT3-aso@mpnp to the target gene STAT3 reaches 75.4%; the inhibition rate of the alpha-syn-ASO@MPNP to the target gene alpha-syn reaches 80.87%; the inhibition rate of Bcl-2-ASO@MPNP to the target gene Bcl-2 reaches 67.91%. The result shows that ASO@MPNP can carry any ASO to perform the intervention treatment of target genes, and plays a role in carrying ASO drugs, vaccines or other products.
EXAMPLE 2.8 preparation of drug (different kinds of nucleic acids) -Metal-polyphenol Complex particles and Effect thereof
Example 2.8.1 preparation of metal ion Fe 3+ Drug (different kinds of nucleic acid) -metal-polyphenol compound particles and effect thereof
The mRNA in example 2 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-polyphenol complex particles (Bcl-2-sirna@mpnp, STAT3-aso@mpnp, S-mrna@mpnp, dsdna@mpnp, ssdna@mpnp) encapsulating the above different kinds of nucleic acids were prepared separately by the method of example 2, and the remaining drug-lipid particles were prepared in the same manner as in example 2.
U251 cells at 1X 10 per well 6 After cell density inoculation in 6-well plates for about 24 hours, cells were collected after incubation with siRNA@MPNP (wherein the concentration of siRNA was 2. Mu.g/mL) or ASO@MPNP (wherein the concentration of ASO was 2. Mu.g/mL, respectively, for 72 hours, total RNA of the cells was extracted, mRNA expression amounts of target genes (Bcl-2, STAT 3) were detected, respectively, using RT-PCR technique, and the ability of siRNA@MPNP or ASO@MPNP to silence the target genes of the cells was calculated, and the results were as shown in FIGS. 1 to 13 in example 2.6 and 1 to 19 in example 2.7.
Incubating S-mRNA@MPNP with 293T cells at a concentration of 2 mug/mL (the concentration of the contained mRNA), incubating a control group with the MPNP, centrifuging after 24 hours, and freezing the supernatant at-20 ℃ for later use; the cell pellet was resuspended in 100. Mu.L PBS buffer, thawed 2 times and sonicated for 10min, and the supernatant was centrifuged, and the expression levels of S protein in both the cell supernatant and cell lysate were detected using a commercially available novel coronavirus S protein ELISA detection kit, 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@mpnp, the drug-lipid particles were removed, after washing the cells twice with PBS, after staining the nuclei with host 33342 dye for 3 minutes, the dye was removed, washing the cells 2 times with PBS, observing the cells with a high content imaging system, and calculating the efficiency of transfection of the drug-lipid particles, the results are shown in fig. 1-26.
ss-DNA@MPNP after incubation of HT22 mouse hippocampal neurons at 200nM concentration (concentration of DNA contained) for 2 hours, drug-lipid particles were removed, cells were washed twice with PBS, cells were observed with a high content imaging system, and the efficiency of transfection of DNA with drug-lipid particles 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 2.6.
The 293T cells were cultured in the same manner as in example 2.5.
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 2.6.
ELISA detects the expression level of S protein: the procedure of example 2.5 was repeated except that the "working solution for anti-RBD antibody" in the ELISA method for detecting RBD of example 2.5 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 2.6.
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 FIGS. 1-13 of example 2.6, the inhibition rate of the drug (double-stranded RNA) -metal-polyphenol complex particles (Bcl-2-siRNA@MPNP) to the target gene Bcl-2 reaches 67%; the inhibition rate of the drug (single-stranded DNA) -metal-polyphenol complex particles (STAT 3-aso@mpnp) of fig. 1-19 to the target gene STAT3 as in example 2.7 reaches 72%; as shown in FIGS. 1 to 25, the expression level of S protein in the supernatant of 293T cells transfected with drug (single-stranded RNA) -metal-polyphenol complex particles (S-mRNA@MPNP) was 134ng/mL, and the S protein content in the supernatant of 293T cells transfected with empty vector MPNP was 0; the efficiency of transfection of drug (double-stranded DNA) -metal-polyphenol complex particles (dsdna@mpnp) into cells was 100% (fig. 1-26); the efficiency of transfection of single-stranded DNA into cells with drug (single-stranded DNA) -metal-polyphenol complex particles (ssDNA@MPNP) was 100% (FIGS. 1-26). The results suggest that the drug-metal-polyphenol 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 2.8.2 preparation of metal ions to Al 3+ Drug (different kinds of nucleic acid) -metal-polyphenol compound particles and effect thereof
This example differs from example 2.8.1 in that the metal ion Fe in example 2.8.1 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-polyphenol complex particles (Bcl-2-sirna@mpnp) achieved an inhibition rate of 72% for the target gene Bcl-2; the inhibition ratio of the drug (single-stranded DNA) -metal-polyphenol complex particles (STAT 3-aso@mpnp) of fig. 1-22 to the target gene STAT3 as in example 3.7.2 reached 75.4%; as shown in FIGS. 1 to 27, the expression level of S protein in the supernatant of 293T cells transfected with drug (single-stranded RNA) -metal-polyphenol complex particles (S-mRNA@MPNP) was 157ng/mL, and the S protein content in the supernatant of 293T cells transfected with empty vector MPNP was 0; the efficiency of transfection of drug (double-stranded DNA) -metal-polyphenol complex particles (dsdna@mpnp) into cells was 100% (fig. 1-28); the efficiency of transfection of single-stranded DNA into cells with drug (single-stranded DNA) -metal-polyphenol complex particles (ssDNA@MPNP) was 100% (FIGS. 1-28). The results suggest that the drug-metal-polyphenol 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-polyphenol Complex particles
EXAMPLE 3 Synthesis characterization of Metal-polyphenol Complex
Example 3.1 the metal ion is Fe 3+ Synthesis characterization of metal-polyphenol complexes at the time
Curcumin and Fe 3+ The ligation was characterized by spectrophotometry: as shown in FIG. 2-1, curcumin and Fe 3+ After combination, the maximum absorption wavelength is shifted from 420nm to 372nm, and the conjugated structure of the metal-polyphenol compound is changed, thus proving that curcumin and Fe are successful 3+ Complexing.
Example 3.2 the metal ion is Al 3+ Synthesis characterization of metal-polyphenol complexes at the time
Curcumin and Al 3+ The ligation was characterized by spectrophotometry: as shown in FIG. 2-2, curcumin and Al 3+ After combination, the maximum absorption wavelength is shifted from 420nm to 433nm, and the conjugated structure of the metal-polyphenol compound is changed, thus proving that curcumin successfully reacts with Al 3+ Complexing.
Example 4 Fe at Low pH 3+ Characterization of exfoliation from metal-polyphenol complexes
Curcumin in metal-polyphenol complex binds Fe through coordination bond 3+ Curcumin and Fe under the condition of low pH value of lysosome 3+ The coordination bond between them is broken by the occurrence of a proton (hydrogen ion absorption). To demonstrate Fe in the metal-polyphenol complex 3+ Indeed, by the above mechanism, from lipid complexes, we devised the following experiments: the color of the metal-polyphenol complex was observed at physiological pH (ph=7.4) and lysosomal low pH (ph=5.0), respectively. As shown in fig. 2-3, the metal-polyphenol 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: under conditions of low pH of lysosomeLower Fe 3+ Can be detached from the metal-polyphenol complex.
Fe at low pH 3+ The principle of shedding from the metal-polyphenol 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-polyphenol complex (fig. 2-3).
Example 5 the metal ion is Fe 3+ Or is Al 3+ The efficiency of the drug-metal-polyphenol complex particles MPNP in encapsulating nucleic acids (siRNA and mRNA) and its comparison with LNP.
The preparation of nucleic acid-entrapped drug-metal-polyphenol complex particles siRNA@MPNP and mRNA@MPNP, respectively, was carried out by replacing the mRNA in example 2 with siRNA targeting the Bcl-2 gene (SEQ ID NO.4, 21 bp) and mRNA encoding the receptor binding domain (receptor binding domain, RBD) of the novel coronavirus S1 subunit (SEQ ID NO.2, 669 nt), respectively, and the preparation procedure of the remaining drug-metal-polyphenol complex particles was the same as in example 2.
The preparation of siRNA@LNP and mRNA@LNP according to the same drug loading amount of Bcl-2-siRNA@MPNP in example 2.6 and RBD-mRNA@MPNP in example 2.5 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@MPNP and the mRNA@MPNP, 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 Tris-HCl buffer solution with pH7.4, 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 Tris-HCl buffer solution to obtain siRNA@LNP and mRNA@LNP respectively.
The inclusion rates of the nucleic acids (siRNA and mRNA) with sirna@mpnp, mrna@mpnp, sirna@lnp and mrna@lnp were detected by agarose gel electrophoresis. The method for measuring the entrapment rate comprises the following steps: the dosage of nucleic acid (siRNA and mRNA) of each group of lipid nano particles is 10 mug/mL, the mass ratio of lipid to nucleic acid is that the metal in the metal-polyphenol compound is Fe 3+ When the mass ratio of the metal to the polyphenol compound is 20:1, the metal in the metal-polyphenol compound is Al 3+ When the nucleic acid is dissolved in a Tris-HCl buffer solution with the pH value of 5.0 according to the mass ratio of 18:1, the nucleic acid is used as a positive control group, and a negative control is a PBS buffer solution without the 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 FIGS. 2 to 4, MPNP (Fe 3+ ) The efficiency of the siRNA and mRNA are 87.78% and 83.17%; MPNP (Al) 3+ ) The efficiency of the siRNA and mRNA are 89.65% and 82.53%; the efficiency of LNP inclusion of siRNA and mRNA was 87.32% and 79.94%, respectively. The results suggest that there is no significant difference in the efficiency of MPNP and LNP-entrapped nucleic acids.
Example 6 the metal ion is Fe 3+ Or is Al 3+ Nucleic acid lysosomal escape ability of drug-metal-polyphenol complex particles MPNP and comparison with LNP
The Cy5-siRNA@MPNP (containing siRNA at a concentration of 100 nM) was prepared by substituting the Bcl-2-siRNA of example 2.6 (SEQ ID NO. 4) with Cy 5-labeled Bcl-2-siRNA; cy5-siRNA@LNP (100 nM concentration of siRNA contained) was prepared by substituting Bcl-2-siRNA (SEQ ID NO. 4) of example 5 with Cy 5-labeled Bcl-2-siRNA; the eGFP-mRNA (SEQ ID NO. 1) of example 2.5 was replaced with Cy 5-labeled mRNA (Cy 5-mRNA), and Cy5-mRNA@MPNP (the concentration of the contained mRNA was 2. Mu.g/mL) was prepared; the ability of the drug-lipid particle to facilitate lysosomal escape was examined by replacing RBD-mRNA (SEQ ID NO. 2) of example 5 with Cy 5-labeled mRNA (Cy 5-mRNA), preparing Cy5-mRNA@LNP (concentration of mRNA at 2. Mu.g/mL), incubating A549 cells with each of them separately with a lysosome probe Lysotracker Green for 3 hours, and observing the overlap of the fluorescent signal (red) of Cy5 with the fluorescent signal (green) of Lysotracker Green using a high content imaging system.
Determination criteria for the ability of drug-metal-polyphenol complex particles to promote nucleolytic escape: after incubating the cells for 3 hours with the drug-metal-polyphenol complex nanoparticles, the overlapping of the fluorescence signal of Cy5 (red) and the fluorescence signal of Lysotracker Green (green) was observed 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-polyphenol compound particles incubate cells for 3 hours, the overlapping rate of the red fluorescent signal and the green fluorescent signal is lower than 50%, which indicates that the nucleic acid can escape from the cell lysosome faster, and the drug-metal-polyphenol compound particles have better capability of promoting the escape of the nucleolytic lysosome.
Analysis of results: as shown in FIGS. 2-5, when Cy5-siRNA@MPNP (Fe 3+ ) And Cy5-mRNA@MPNP (Fe) 3+ ) After 3 hours of incubation of a549 cells, the overlapping rates of the red fluorescent signal and the green fluorescent signal were 39.20% ± 8.89% and 44.96% ± 3.85%, respectively, i.e., lysosome escape rates were 60.80% ± 8.89% and 55.04% ± 3.85%, respectively; when Cy5-siRNA@MPNP (Al 3+ ) And Cy5-mRNA@MPNP (Al) 3+ ) After 3 hours of incubation of a549 cells, the overlapping rates of the red fluorescent signal and the green fluorescent signal were 34.70% ± 4.98% and 39.10% ± 4.43%, respectively, i.e., lysosome escape rates were 65.30% ± 4.98% and 60.90% ± 4.43%, 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 76.02% + -7.90% and 85.33% + -4.87%, respectively, namely, the lysosome escape capacity is 23.98% + -7.90% and 14.67% + -4.87%, respectively. The medicine-lipid nanoparticle MPNP is provided with better ability of promoting the escape of nucleotidic lysosomes, and the capacity of promoting the escape of the lysosomes of the MPNP is obviously stronger than that of LNP.
Example 7 the metal ion is Fe 3+ Or is Al 3+ Drug-metal-polyphenol complex particles MPNP for promoting nucleic acid expression and comparison with LNP
The RBD-mRNA (SEQ ID NO. 2) of example 5 was replaced with mRNA encoding the fluorescent protein eGFP, and the other preparation methods were the same as in example 5, to obtain eGFP-mRNA@LNP.
The eGFP-mrna@mpnp prepared in example 2.5 and the above eGFP-mrna@lnp (with a concentration of 2 μg/mL of mRNA) were incubated with 293T cells, control groups incubated with MPNP or LNP, respectively, 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 2.
Analysis of results: analysis of results: as shown in FIGS. 2 to 6, MPNP (Fe 3+ )、MPNP(Al 3+ ) The percentage of eGFP positive cells after 293T cells treated with LNP was 93.47%, 97.06% and 63.09%, respectively. And (3) result prompting: MPNP functions better to promote nucleic acid expression than LNP. The possible reasons for this are: the MPNP is more capable of lysosomal escape than LNP in the case of the pro-nucleic acid of example 6, so that more nucleic acid loaded by the MPNP is efficiently released into the cytoplasm and translated into protein.
Example 8 drug-Metal-Polyphenol Complex particles MPNP Capacity to promote humoral and cellular immunity and comparison with LNP
Incubating RBD-mRNA@MPNP in example 2.5 and RBD-mRNA@LNP in example 5 with 293T cells at a concentration of 2 mug/mL (concentration of contained mRNA), incubating a control group with MPNP, centrifuging after 24 hours, and taking supernatant for freezing at-20 ℃ for later use; the cell pellet was resuspended in 100. Mu.L of PBS buffer, thawed 2 times and sonicated for 10min, and the supernatant was centrifuged, and the expression levels of RBD protein in both the cell supernatant and the cell lysate were detected using a commercially available novel coronavirus antigen RBD ELISA detection kit, as shown in FIGS. 2-7.
The ELISA method for detecting RBD expression levels was as described in example 2.5.
The experimental animals were randomly divided into 3 groups (experimental and control groups) of 5 animals each. Dynamic movementThe 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@MPNP (Fe 3+ )、RBD-mRNA@MPNP(Al 3+ ) Or RBD-mRNA@LNP, and the control group was injected with MPNP and LNP without mRNA. The dose per administration was 100. Mu.L, wherein RBD-mRNA@MPNP (Fe 3+ )、RBD-mRNA@MPNP(Al 3+ ) And the RBD-mRNA@LNP preparations each contained 30mg of mRNA. Mouse blood was collected 28 days after the first dose, serum was isolated and diluted in a gradient, and the titer of RBD total IgG antibodies against the novel coronavirus S1 subunit produced in mice was measured by a commercially available ELISA kit, and the results are shown in fig. 2-8.
ELISA the titres of RBD total IgG antibodies against the S1 subunit of the novel coronavirus were measured as described in example 2.5.
In the case of RBD-mRNA@MPNP (Fe) 3+ )、RBD-mRNA@MPNP(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 2-9.
ELISA detection of IFN-gamma, IL-2, IL-4 expression levels were performed as described in example 2.5.
Analysis of results: as shown in FIGS. 2-7, RBD-mRNA@MPNP (Fe 3+ )、RBD-mRNA@MPNP(Al 3+ ) And RBD-mRNA@LNP, which induce 293T cells to express a certain amount of RBD, but RBD-mRNA@MPNP (Al 3+ ) The capacity of inducing cells to express RBD is obviously higher than that of RBD-mRNA@MPNP (Fe) 3+ )，RBD-mRNA@MPNP(Fe 3+ ) The ability to induce cells to express RBD is significantly greater than RBD-mRNA@LNP: RBD-mRNA@MPNP (Fe) 3+ ) The expression level of RBD in the cell supernatant of the treatment group was 178ng/mL, and RBD-mRNA@MPNP (Al 3+ ) The expression level of RBD in the cell supernatant of the treatment group was 215ng/mL, and the expression level of RBD in the cell supernatant of the RBD-mRNA@LNP treatment group was 115.67ng/mL. As shown in the results of FIGS. 2-8, RBD-mRNA@MPNP was effective to induce humoral immunity in mice, to produce high levels of antigen-specific binding antibodies, and RBD-mRN A@MPNP(Al 3+ ) The ability to induce humoral immunity in mice was clearly better than that of RBD-mRNA@MPNP (Fe) 3+ )，RBD-mRNA@MPNP(Fe 3+ ) The ability to induce humoral immunity in mice was clearly better than RBD-mrna@lnp: RBD-mRNA@MPNP (Fe) 3+ ) IgG antibody titers in treated mice reached 84975; RBD-mRNA@MPNP (Al) 3+ ) IgG antibody titers in treated mice reached 96418; whereas the IgG antibody titer in mice of RBD-mrna@lnp treated groups was only 67476. As shown in FIGS. 2-9, RBD-mRNA@MPNP was effective in inducing cellular immunity in mice, i.e., activating immune cells and producing a large amount of cytokines, and mRNA@MPNP (Al 3+ ) The ability to induce cellular immunity in mice was clearly better than that of RBD-mRNA@MPNP (Fe) 3+ )，RBD-mRNA@MPNP(Fe 3+ ) The ability to induce cellular immunity in mice was clearly better than RBD-mrna@lnp: RBD-mRNA@MPNP (Fe) 3+ ) The expression amounts of cytokines IFN-gamma, IL-2 and IL-4 respectively reach 274.4pg/mL, 254.2pg/mL and 77.4pg/mL; RBD-mRNA@MPNP (Al) 3+ ) The expression amounts of cytokines IFN-gamma, IL-2 and IL-4 respectively reach 309pg/mL, 299pg/mL and 91.2pg/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@MPNP (Al 3+ ) The ability to deliver any mRNA and perform its function is significantly better than RBD-mRNA@MPNP (Fe 3+ )，RBD-mRNA@MPNP(Fe 3+ ) The ability to induce cellular immunity in mice was clearly better than RBD-mrna@lnp: the RBD-mRNA@MPNP can more effectively promote cells to express target proteins and can more effectively activate in vivo humoral immunity and cellular immunity, so that the medicine (mRNA) -lipid particles are obviously superior to LNP in the prior art in the aspect of the action of medicines, vaccines or other products carrying mRNA. The possible reasons for this are: 1) Compared with LNP, MPNP has stronger ability of promoting nucleolytic body escape; 2) Compared with LNP, MPNP has a stronger capacity for expressing a nucleic acid as a protein (antigen); 3) Compared with LNP, curcumin in MPNP is released and then used as an immune adjuvant (also called immune regulator), so that the humoral immunity and the cellular immunity can be activated to enhance the effect of MPNP in delivering mRNA vaccine, and the immune factor storm can be inhibited to inhibit excessive immune response harmful to organisms.
EXAMPLE 9 evaluation of in vivo safety of Metal-polyphenol Complex particles (MPNP)
SD rats were used as subjects for MPNP (Fe 3+ Or Al 3+ ) A sub-slow toxicity study was performed for 20 days and a recovery period of 20 days was established. 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 MPNP groups (25 mg/kg) (including 8 experimental groups and 6 recovery groups), and male and female halves; medium dose MPNP group (50 mg/kg) 14 (containing 8 experimental groups and 6 recovery groups), male and female halves; high dose group (100 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 MPNPs were dissolved in Tris-HCl buffer solution at pH7.4, and the control group was injected with the same amount of Tris-HCl buffer solution, and the low-dose MPNP group, the medium-dose MPNP group, and the high-dose MPNP group were respectively injected with 8mg/kg, 16mg/kg, and 32mg/kg of MPNPs.
Setting the basis of the MPNP administration dosage: when 200. Mu.g/kg of mRNA was entrapped (the actual amount required for the animal experiments with mRNA), the amount of empty vector MPNP required was 8mg/kg. To fully prove the safety of MPNP, 1, 2 and 4 times of the actual required dose of the MPNP animal experiment 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, rats in the low, medium and high dose MPNP groups survived well, had normal diet, appearance and behavioural activities at the end of the dosing period and at the end of the recovery period, compared with the control group, and no significant adverse reaction was seen after dosing; compared with the control group, the weight gain values of the male SD rats, the female SD rats in the low-dose, medium-dose and high-dose MPNP groups are not significantly different; there was no significant difference in the dirty body ratio between the low, medium and high dose MPNP groups compared to the control group.
At the end of the dosing period and at the end of the recovery period, no abnormality was seen in the blood normative index (white blood cell number, lymphocyte number, monocyte number, neutrophil number, lymphocyte percentage, monocyte percentage, neutrophil percentage, red blood cell number, hemoglobin, hematocrit, mean red blood cell volume, mean red blood cell hemoglobin content, mean red blood cell hemoglobin concentration, coefficient of variation in distribution width of red blood cells, platelet number, mean platelet volume, platelet distribution width, platelet volume) of the low, medium, high dose MPNP group compared to the control group; blood biochemical index of low, medium and high dose MPNP 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); immunologically relevant fingers of low, medium and high dose MPNP groups compared to control groupThe targets included thyroid function index (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), were not abnormal.
At the end of the administration period and the end of the recovery period, compared with a control group, the brain tissue structure of rats in the low, medium and high-dose MPNP groups is complete, the tissue staining is normal, the morphological structure of cells is 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; normal spleen structure and clear red and white marrow limitation; 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 MPNP (Fe) is injected into SD rats in a large amount for a long period of time 3+ Or Al 3+ ) No obvious chronic toxic reaction was found, suggesting that the safety of MPNP is higher.
TABLE 2-1 evaluation of in vivo safety of MPNP
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 10 in vivo safety comparison of Metal-polyphenol Complex particles (MPNPs) to LNPs
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 an important parameter for assessing the toxicity of LNP to the body. The drug-metal-polyphenol complex particles (MPNP) replace cationic lipids/ionizable lipids in LNP with metal-polyphenol complexes, therefore we have studied the median lethal dose (IC) of metal-polyphenol complexes and cationic lipids/ionizable lipids to biological cells 50 ) The differences in toxicity of LNP and MPNP were compared.
After incubating 293T cells with different concentrations of the metal-polyphenol complex (0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7. Mu.M), cationic lipid (DOTAP, 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7. Mu.M) and ionizable lipid (ALC 0315,0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7. Mu.M) respectively for 48 hours, cell activity was detected with a CCK8 activity detection kit, and the half-lethal IC of the metal-polyphenol complex, cationic lipid (DOTAP) and ionizable lipid (ALC 0315) to 293T cells was calculated, respectively 50 。
The detection method of CCK8 comprises the following steps:
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;
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;
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;
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;
survival (%) = [ a (dosing) -a (blank) ]/[ a (0 dosing) -a (blank) ]x100%.
IC 50 Is calculated by the following steps: 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 。
To compare the in vivo safety of MPNP and LNP, an in vivo experiment was performed to evaluate and compare the in vivo toxicity of MPNP (8 mg/kg) and LNP (3.24 mg/kg) loaded with an equivalent amount of nucleic acid (200. Mu.g/kg mRNA) according to the method of example 9.
Analysis of results: as shown in Table 2-2, IC of the metal-polyphenol complex 50 Significantly greater than the cationic lipid (DOTAP) and the ionizable lipid (ALC 0315). The metal-polyphenol complex has obviously lower toxicity than cationic lipid and ionizable lipid, and has obvious effect.
As shown in tables 2 to 3, at the end of the administration period and the end of the recovery period, MPNP (Fe 3+ ) Or MPNP (Al) 3+ ) The liver function indexes ALT, AST, ALP and cytokine IL-6, and the expression level of IL-1 beta in the group have no obvious abnormality. However, the liver function indexes ALT, AST, ALP and the cytokine IL-6, IL-1 beta expression levels of the LNP group are obviously increased compared with the control group. The results suggest that MPNP (Fe 3+ ) Or MPNP (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 MPNP (Fe) 3+ ) Or MPNP (Al) 3+ ) The core component of (a) is a metal-polyphenol compound which consists of natural micromolecular substance curcumin (food additive and pharmaceutical adjuvant approved by FDA) with high safety and safe metal ions, and the metal-polyphenol compound is formed after drug delivery is completed Is decomposed into natural molecules in vivo. In conclusion, since MPNP (Fe 3+ ) Or MPNP (Al) 3+ ) The composition contains no cationic lipid/ionizable lipid, and does not cause toxic side effects related to cationic lipid/ionizable lipid, so that MPNP (Fe) 3+ ) Or MPNP (Al) 3+ ) Is higher than LNP.
DOTAP structure
Structural formula of ALC0315
TABLE 2-2 Metal ion is Fe 3+ Or is Al 3+ IC of metal-polyphenol complex and cationic lipid (DOTAP) and ionizable lipid (ALC 0315) 50 Is a comparison of (2)
TABLE 2-3 Metal ion is Fe 3+ Or is Al 3+ Comparison of chronic toxicity test index of MPNP and LNP
Example III clinical application and route of administration of drug-Metal-polyphenol Complex particles MPNP
Example 11 the metal ion is Fe 3+ Or Al 3+ Clinical application and administration route of medicine-metal-polyphenol compound particle MPNP
The mRNA in example 2 was replaced with siRNA targeting the B7-H4 gene (B7-H4-siRNA) and its control (scr-siRNA), mRNA encoding the receptor binding domain (receptorbinding domain, RBD) of the novel coronavirus S1 subunit (RBD-mRNA), respectively.
The sequences of the different nucleic acids are as follows: (1) the sequence of the B7-H4-siRNA is SEQ IDNo.19 (sense strand) and SEQ ID No.26 (antisense strand) (25 bp), the random control sequences of which 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 (receptorbinding domain, RBD) of the novel coronavirus S1 subunit is SEQ ID No.2 (669 nt). The preparation of drug-metal-polyphenol complex particles (B7-H4-siRNA@MPNP (Fe) 3+ )、RBD-mRNA@MPNP(Fe 3+ )、B7-H4-siRNA@MPNP(Al 3+ )、RBD-mRNA@MPNP(Al 3+ ) The remaining drug-metal-polyphenol complex particles were prepared in the same manner as in example 2. The 2 different drug-metal-polyphenol complex particles (B7-H4-siRNA@MPNP and RBD-mRNA@MPNP) 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:
sense 5’-GGGAGACAC UCCAUC ACAGUCACUA-3’(SEQ ID No.19)。
antisense 5’-UAG UGACUG UGAUGGAGU GUC UCC C-3’(SEQ ID No.26)(25bp)。
the random control sequence of B7-H4-siRNA is as follows:
sense 5’-UUCUCCGAACGUGUCACGU-3’(SEQ ID No.20)。
antisense 5’-ACGUGACACGUUCGGAGAA-3’(SEQ ID No.27)(19bp)。
to evaluate B7-H4-siRNA @ MPNP (Fe 3+ ) And B7-H4-siRNA@MPNP (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): tris-HCl buffer control group, blank vector MPNP (Fe) 3+ ) Group and blank vector MPNP (Al) 3+ ) Group, scr-siRNA@MPNP (Fe) 3+ ) Control group, B7-H4-siRNA@MPNP (Fe) 3+ ) Treatment group, scr-siRNA@MPNP (Al 3+ ) Control group, B7-H4-siRNA@MPNP (Al) 3+ ) Treatment groups. Each group of mice was intratumorally injected with pH7.4 Tris-HCl buffer solution, MPNP (Fe) every 3 days 3+ )、MPNP(Al 3+ )、Scr-siRNA@MPNP(Fe 3+ )、B7-H4siRNA@MPNP(Fe 3+ )、Scr-siRNA@MPNP(Al 3+ )、B7-H4 siRNA@MPNP(Al 3+ ) 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@mpnp as an mRNA vaccine for the prevention of new coronaviruses, the experimental procedure and experimental method are as described in the previous example 2.5.
ELISA detection was as described in example 2.5.
Establishment of liver cancer mouse model: hepG2 cells were collected at 1X 10 7 The density of/mL 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@MPNP (Fe 3+ )、Scr-siRNA@MPNP(Al 3+ ) Has little inhibition effect on the growth of liver cancer HepG2 cells, while B7-H4-siRNA@MPNP (Fe) 3+ ) And B7-H4 siRNA@MPNP (Al) 3+ ) The composition shows high-efficiency treatment effect and can effectively inhibit the growth of liver cancer tumors. The results indicate that the drug-metal-polyphenol compound particles can encapsulate and deliver the B7-H4 siRNA, and inhibit the development of liver cancer by inhibiting the expression of target genes.
As shown in the previous examples 2.5, FIGS. 1-3, FIGS. 1-5, RBD-mRNA@MPNP (Fe 3+ ) The expression levels of the mouse IgG antibodies were set to 84363.4 (FIGS. 1-3), and the expression levels of the cytokines IFN-. Gamma.IL-2 and IL-4 were set to 271.8pg/mL, 269.6pg/mL and 75.8pg/mL, respectively (FIGS. 1-5). RBD-mRNA@MPNP (Al) 3+ ) The expression level of the mouse IgG antibody was set to 94828.6 (FIGS. 1-17), and the expression levels of the cytokines IFN-. Gamma., IL-2, and IL-4 were set to 306.2pg/mL, 289.6pg/mL, and 88.2pg/mL, respectively (FIGS. 1-19). The result shows that RBD-mRNA@MPNP 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@MPNP is effective in preventing infection by novel coronaviruses.
As shown in the figure 3-1, the B7-H4-siRNA@MPNP can effectively treat liver cancer by adopting an intratumoral injection administration route; as shown in examples 2.5, FIGS. 1-3, 1-5, 1-17 and 1-19, RBD-mRNA@MPNP was shown to be capable of activating humoral and cellular immunity by intramuscular administration, thereby exerting prophylaxis against novel coronavirus infections. And (3) result prompting: drug-metal-polyphenol complex particles can be administered by a variety of routes.
Embodiment four: curcumin, fe 3+ Function after being replaced by congener
Example 12 curcumin, fe 3+ Function after being replaced by congener
Reference example 1 with curcumin, fe 3+ The congener of (C) is respectively to curcumin and Fe 3+ Instead, 9 different drug-metal-polyphenol complex particles (eGFP-mrna@mpnp) were prepared by different combinations, each with a concentration of 2 μg/mL, respectively, with reference to example 2. Curcumin, fe 3+ The names and structures of the analogues are shown in Table 4-1, and curcumin and Fe in 9 mRNA@MPNP 3+ And the like are combined in the manner shown in Table 4-2. The reaction temperature in example 1 was 60℃and the reaction time was 2 hours, with the other conditions unchanged.
To compare the effects of the 9 different eGFP-mrna@mpnp with eGFP-mrna@lnp, we prepared LNP entrapping equal amounts of eGFP mRNA with reference to example 5, yielding eGFP-mrna@lnp.
The 9 different eGFP-mrna@mpnp and eGFP (both containing mRNA at a concentration of 2 μg/mL) were incubated with 293T cells, control groups incubated with MPNP 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 2.
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. Gold for the metal-polyphenol complex particles (MPNP)The generic-polyphenol complexes replace cationic lipids/ionizable lipids in LNP, therefore, we have examined the median lethal dose (IC) of 9 metal-polyphenol complexes and cationic lipid (DOTAP)/ionizable lipid (ALC 0315) to biological cells in table 4-2 50 ) The difference in toxicity of LNP was compared to 9 MPNPs.
The calculation method of IC50 is as described in example 10.
Analysis of results: as shown in tables 4-3, the percentage of eGFP positive cells after treatment of 293T cells with 9 different eGFP-mRNA@MPNPs was significantly higher than that of eGFP-mRNA@LNP, with the highest percentage of eGFP positive cells with mRNA@MPNP1. And (3) result prompting: curcumin, fe 3+ The function of the mRNA@MPNP formed after substitution by its congener is inferior to that of the mRNA@MPNP1, but slightly superior to that of the mRNA@LNP, probably because: the MPNP is more capable of lysosomal escape than LNP in the case of the pro-nucleic acid of example 5, so that more nucleic acid loaded by the MPNP is efficiently released into the cytoplasm and translated into protein.
The above results suggest that curcumin and Fe satisfy the following conditions 3+ The functions of the drug-metal-polyphenol composite particles formed after being replaced by the congeners are not affected: (1) the congener of curcumin is hydrophobic polyphenol capable of complexing with metal; (2) fe (Fe) 3+ Is a metal ion; (3) 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 Table 4-3, IC's for 9 metal-polyphenol 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-polyphenol complex is significantly less than that of the cationic lipid and the ionizable lipid, i.e. the complex is composed of curcumin and Fe 3+ And its congeners, the lipid particles (MPNP) 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; while the core component of MPNP is metal-polyphenol complex, which is natural small molecular substance with non-cationic lipid and high safety (wherein curcumin is approved by FDA) Food additives and pharmaceutical excipients) and safe metal ions, and which have been decomposed into natural molecules in vivo after completion of drug delivery. In conclusion, the composition is prepared from curcumin and Fe 3+ And the lipid particles (MPNP) composed of the same are free of cationic lipid/ionizable lipid and do not cause toxic or side effects related to the cationic lipid/ionizable lipid, so that the safety of the MPNP is higher than that of the LNP.
TABLE 4-1 curcumin, fe 3+ And the names and structures of the same
TABLE 4-2 curcumin, fe 3+ List of modes of combination and functions of metal-polyphenol complexes in drug-lipid nanoparticles prepared from the same
TABLE 4-3 curcumin, fe 3+ IC of metal-polyphenol compound prepared from same and analogue thereof 50
EXAMPLE 13 curcumin, fe of different Metal-Polyphenol complexes 3+ Component adding proportion and function of medicine-metal-polyphenol compound particles prepared by the same
Example 13.1 the metal ion is Fe 3+ Polyphenols, fe of different metal-polyphenol complexes 3+ Component adding proportion and function of medicine-metal-polyphenol compound particles prepared by the same
According to example 2.1, a metal-polyphenol complex was prepared, and curcumin was replaced with its congener hesperetin (1 molecule hesperetin contains 4 hydroxyl groups), and catechin (1 molecule catechin contains 5 hydroxyl groups) respectively, to prepare three metal-polyphenol complexes (mrna@mpnp1, mrna@mpnp4, mrna@mpnp7). Preparation of these three Metal-Polys Curcumin or its analogue and Fe in phenol compound 3+ The throwing proportion of (2) is respectively as follows: 1:1,1:1,1:2. And corresponding drug-metal-polyphenol complex particles prepared from the three metal-polyphenol complexes (mRNA@MPNP1, mRNA@MPNP4, mRNA@MPNP7). 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 three drug-lipid particles 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 2.5.
Analysis of results: as shown in tables 4 to 4, mRNA entrapment efficiency and the ability to promote expression of the target protein (i.e., positive cell rate) of the drug-metal-polyphenol complex particles prepared using different dosage ratios according to the chemical structures of the metal-polyphenol complex components were comparable. The results suggest that the ratio of metal-polyphenol complex components may be adjusted according to the structure of the particular metal-polyphenol complex component. The basis for the adjustable proportion of the delivery is as follows: 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+ 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.
TABLE 4-4 Metal ion is Fe 3+ Different component adding proportion of metal-polyphenol compound and function of medicine-lipid particle prepared by the same
Example 13.2 the metal ion is Al 3+ Polyphenols, al of different metal-polyphenol complexes 3+ Component adding proportion and function of medicine-metal-polyphenol compound particles prepared by the same
According to example 2.2, a metal-polyphenol complex was prepared, and curcumin was replaced with its congener hesperetin (1 molecule hesperetin contains 4 hydroxyl groups), catechin (1 molecule catechin contains 5 hydroxyl groups) to prepare three metal-polyphenol complexes (mRNA@MPNP3, mRNA@MPNP6mRNA@MPNP9). Curcumin or its analogue and Al in preparing these three metal-polyphenol compounds 3+ The throwing proportion of (2) is respectively as follows: 1:1,1:1,1:2. And corresponding drug-metal-polyphenol complex particles prepared from the three metal-polyphenol complexes (mRNA@MPNP3, mRNA@MPNP6, mRNA@MPNP9). 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 three drug-lipid particles 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 2.5.
Analysis of results: as shown in tables 4 to 5, mRNA entrapment efficiency and ability to promote expression of target protein of the drug-metal-polyphenol complex particles prepared using different dosage ratios according to chemical structures of the metal-polyphenol complex components were comparable. The results suggest that the ratio of metal-polyphenol complex components may be adjusted according to the structure of the particular metal-polyphenol complex component. The basis for the adjustable proportion of the delivery is as follows: 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.
TABLE 4-5 Metal ion is Al 3+ Different component adding proportion of metal-polyphenol compound and function of medicine-lipid particle prepared by the same
The preparation of mRNA@MPNP in example 2 above was completed as a group of subjects taught by the pharmaceutical engineering System Wang Shan of the university of Mitsnan chemical university.
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 (70)

1. A drug-lipid particle, wherein the drug-lipid particle comprises:
(a) A drug, wherein the drug is a negatively charged molecule; and
(b) A metal-polyphenol composite particle comprising:
(i) A metal-polyphenol complex consisting of a polyphenol molecular moiety and a metal ion moiety reacted, the polyphenol molecular moiety and the metal ion moiety being linked by a coordination bond;
(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) Non-cationic lipids or non-ionizable lipids other than conjugated lipids that inhibit particle aggregation.
2. The drug-lipid particle of claim 1, wherein the polyphenol molecular moiety is selected from the group consisting of curcumin, quercetin, kaempferol, rutin, hesperetin, naringenin, eriodictyol, luteolin, apigenin, taxol, fucoidan, polyphenol of a polyalkylene glycol, catechin, ellagic acid, gallic acid, digallic acid, propyl gallate, epigallocatechin gallate, galloylglucose, hydroxyhydroquinone, morin, epicatechin gallate, catechin gallate, gallocatechin gallate, and a combination of one or more of its derivatives.
3. The drug-lipid particle of claim 2, wherein the polyphenol molecular moiety is selected from one or more of curcumin (formula 1), quercetin (formula 2), kaempferol (formula 3), rutin (formula 4), hesperetin (formula 5), naringenin (formula 6), eriodictyol (formula 7), luteolin (formula 8), apigenin (formula 9), taxol (formula 10), fucoidan (formula 11), polyanthenol (formula 12), catechin (formula 13), ellagic acid (formula 14), gallic acid (formula 15), digallic acid (formula 16), propyl gallate (formula 17), epigallocatechin gallate (formula 18), galloylglucose (formula 19), hydroxyhydroquinone (formula 20), morin (formula 21), epigallocatechin gallate (formula 22), catechin gallate (formula 23), gallocatechin gallate (formula 24), and derivatives thereof;
4. A drug-lipid particle according to claim 3, wherein the polyphenol molecular moiety is selected from the group consisting of curcumin (formula 1), dihydrocurcumin (formula 25), hexahydrocurcumin (formula 26), curcumin sulphate (formula 27), bisdemethoxycurcumin (formula 28);
5. a drug-lipid particle according to claim 3, wherein the polyphenol molecular moiety is selected from one or more of curcumin (formula 1), hesperetin (formula 5) or catechin (formula 13), and derivatives thereof.
6. The drug-lipid particle of claim 5, wherein the polyphenol molecular moiety is selected from curcumin (formula 1), hesperetin (formula 5) or catechin (formula 13).
7. The drug-lipid particle of claim 1, 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.
8. The drug-lipid particle of claim 7, wherein the metal ion moiety is selected from Fe 3+ 、Ca 2+ 、Al 3+ A combination of one or more of the foregoing.
9. The drug-lipid particle of claim 8, wherein the metal ion moiety is selected from Fe 3+ 、Ca 2+ Or Al 3 + 。
10. The drug-lipid particle of claim 1, wherein the conjugated lipid that inhibits aggregation of particles comprises PEG-lipid conjugates and/or PEG-DAAs.
11. The drug-lipid particle of claim 10, wherein the PEG-lipid conjugate is selected from the group consisting of phosphatidylethanolamine-polyethylene glycol 2000 (formula 47), phosphatidylethanolamine-polyethylene glycol 700 (formula 48), phosphatidylethanolamine-polyethylene glycol 1000 (formula 49), phosphatidylethanolamine-polyethylene glycol 5000 (formula 50), and a combination 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 acylOr (b)
Phytantyl radical；
12. The drug-lipid particle of claim 11, wherein the PEG-lipid conjugate is selected from one or more of DSPE-PEG2000, DSPE-PEG700, DSPE-PEG1000, or DSPE-PEG 5000.
13. The drug-lipid particle of claim 12, wherein the PEG-lipid conjugate is selected from DSPE-PEG2000 (formula 58), DSPE-PEG700 (formula 55), DSPE-PEG1000 (formula 56), or DSPE-PEG5000 (formula 57);
14. the drug-lipid particle of claim 1, wherein the non-cationic lipid or non-ionizable lipid in (iii) 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.
15. The drug-lipid particle of claim 14, wherein the non-cationic lipid or non-ionizable lipid of (iii) is selected from the group consisting of lecithin (PC) (formula 29), phosphatidylethanolamine (PE) (formula 30), phosphatidylserine (PS) (formula 31), phosphatidic Acid (PA) (formula 32), phosphatidylglycerol (PG) (formula 33), 1-phosphoglyceride (SP) (formula 34), phosphatidylinositol (PI) (formula 35), phosphatidylthreonine (PT) (formula 36), sphingomyelin (SM) (formula 37), lysolecithin (LPC) (formula 38), lysophosphatidylethanolamine (LPE) (formula 39), lysophosphatidylserine (LPS) (formula 40), lysophosphatidic acid (LPA) (formula 41), lysophosphatidylglycerol (LPG) (formula 42), lysophosphatidylinositol (LPI) (formula 43), lysophosphatidylthreonine (LPT) (formula 44), lysosphingomyelin (LSM) (formula 45), 1-phosphosphingosine (S1P) (formula 46), and combinations of one or more of the derivatives thereof; wherein R1 and R2 are each independently a decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, erucyl, arachidoyl or phytantyl group;
16. the drug-lipid particle of claim 14, wherein the non-cationic lipid or non-ionizable lipid of (iii) further comprises at least one of cholesterol and derivatives thereof.
17. The drug-lipid particle of claim 16, wherein the non-cationic lipid or non-ionizable lipid of (iii) comprises cholesterol, and a combination of one or more selected from DSPC, DSPE, DSPA or DSPG.
18. The drug-lipid particle of claim 17, wherein the non-cationic lipid or non-ionizable lipid of (iii) comprises cholesterol (formula 59), and a combination of one or more selected from DSPC (formula 51), DSPE (formula 52), DSPA (formula 53), or DSPG (formula 54);
19. the drug-lipid particle of claim 18, wherein the non-cationic lipid or non-ionizable lipid of (iii) comprises cholesterol (formula 59) and DSPC (formula 51).
20. The drug-lipid particle of claim 2 or 7, wherein the metal-polyphenol complex is composed of a polyphenol molecular moiety selected from curcumin, hesperetin or catechin and a metal ion moiety selected from Fe 3+ 、Ca 2+ Or Al 3+ 。
21. The drug-lipid particle of claim 20, wherein the metal-polyphenol complex is comprised of a polyphenol molecular moiety selected from curcumin (formula 1), hesperetin (formula 5) or catechin (formula 13) and a metal ion moiety selected from Fe 3+ 、Ca 2+ Or Al 3+ 。
22. The drug-lipid particle of claim 21, wherein the molar ratio of the polyphenol molecule moiety to the metal ion moiety is 1 (0.5-2).
23. The drug-lipid particle of claim 22, wherein the polyphenol molecular moiety is curcumin (formula 1) and the metal ion moiety is Fe 3+ 。
24. The drug-lipid particle of claim 23, wherein curcumin (formula 1) and Fe 3+ The molar ratio of (2) is 1:1.
25. The drug-lipid particle of claim 22, wherein the polyphenol molecular moiety is curcumin (formula 1) and the metal ion moiety is Al 3+ 。
26. The drug-lipid particle of claim 25, wherein curcumin (formula 1) and Al 3+ The molar ratio of (2) is 1:1.
27. The drug-lipid particle of claim 16, wherein the metal-polyphenol complex particle is made of (i) a metal-polyphenol complex having a molar ratio of 10% to 20% in a starting material, (ii) a conjugated lipid that inhibits aggregation of particles, and (iii) a non-cationic lipid or a non-ionizable lipid, the conjugated lipid that inhibits aggregation of particles having a molar ratio of 2% to 10% in a starting material, the cholesterol having a molar ratio of 0% to 48% in a starting material, and the non-cationic lipid or non-ionizable lipid other than cholesterol having a molar ratio of 40% to 75% in a starting material.
28. The drug-lipid particle of claim 16, wherein the metal-polyphenol complex particle is made of (i) a metal-polyphenol complex having a molar ratio of 5% to less than 10% in a starting material, (ii) a conjugated lipid that inhibits aggregation of particles, and (iii) a non-cationic lipid or a non-ionizable lipid, the conjugated lipid that inhibits aggregation of particles having a molar ratio of 2% to 10% in a starting material, the cholesterol having a molar ratio of 0% to 48% in a starting material, and the non-cationic lipid or non-ionizable lipid other than cholesterol having a molar ratio of 30% to less than 40% or 40% to 75% in a starting material; or (b)
The metal-polyphenol complex particles are made of (i) metal-polyphenol complexes in a molar ratio of 10% to 20% in a raw material, (ii) conjugated lipids that inhibit aggregation of the particles in a molar ratio of 2% to 10% in a raw material, cholesterol in a molar ratio of 0% to 48% in a raw material, and (iii) non-cationic lipids or non-ionizable lipids other than cholesterol in a molar ratio of 30% to less than 40% in a raw material.
29. The drug-lipid particle of claim 27 or 28, wherein the metal-polyphenol complex is present in the starting material in a molar ratio of 5% to less than 10%, 10% to 15% or 15% to 20%.
30. The drug-lipid particle of claim 29, wherein the metal-polyphenol complex is present in the starting material at a molar ratio of 5%, 10% or 15%.
31. The drug-lipid particle of claim 27 or 28, wherein the conjugated lipid that inhibits aggregation of the particle is present in the starting material at a molar ratio of 3% to 5% or 5% to 10%.
32. The drug-lipid particle of claim 31, wherein the conjugated lipid that inhibits aggregation of the particle is present in the starting material at a molar ratio of 3%, 5% or 10%.
33. The drug-lipid particle of claim 27 or 28, wherein the cholesterol is present in the starting material in a molar ratio of 10-30%, 30-47% or 10-20%.
34. The drug-lipid particle of claim 33, wherein the cholesterol is present in the starting material at a molar ratio of 10%, 30% or 47%.
35. The drug-lipid particle of claim 27 or 28, wherein the non-cationic lipids or non-ionizable lipids other than cholesterol are present in the starting material in a molar ratio of 45-55%, 60-65% or 50-65%.
36. The drug-lipid particle of claim 35, wherein the non-cationic lipids or non-ionizable lipids other than cholesterol are present in the starting material in a molar ratio of 45%, 55%, 60% or 65%.
37. The drug-lipid particle of claim 27 or 28, wherein the metal-polyphenol complex comprises from 5% to less than 10% or from 10% to 15% by mole of the starting material, the conjugated lipid that inhibits aggregation of the particle comprises from 5% to 10% by mole of the starting material, the cholesterol comprises from 10% to 30% by mole of the starting material, the non-cationic lipid other than cholesterol or the non-ionizable lipid comprises from 60% to 65% by mole of the starting material, and the metal ion moiety of the metal-polyphenol complex is selected from Fe 3+ 。
38. The drug-lipid particle of claim 37, wherein the metal-polyphenol complex is present in the starting material at a molar ratio of 15%, the conjugated lipid inhibiting aggregation of the particleA molar ratio of 10% in the starting material, a molar ratio of 65% in the starting material of non-cationic lipids other than cholesterol or non-ionizable lipids, and a metal ion moiety selected from the group consisting of Fe 3+ 。
39. The drug-lipid particle of claim 37, wherein the metal-polyphenol complex comprises 5% by mole of the starting material, the conjugated lipid that inhibits aggregation of the particle comprises 5% by mole of the starting material, the cholesterol comprises 30% by mole of the starting material, the non-cationic lipid other than cholesterol comprises 60% by mole of the non-ionizable lipid, and the metal ion moiety of the metal-polyphenol complex is selected from Fe 3+ 。
40. The drug-lipid particle of claim 27 or 28, wherein the metal-polyphenol complex comprises from 5% to less than 10% or 10% by mole of the starting material, the conjugated lipid that inhibits aggregation of the particle comprises from 3% to 5% by mole of the starting material, the cholesterol comprises from 30% to 47% by mole of the starting material, the non-cationic lipid other than cholesterol or non-ionizable lipid comprises from 45% to 55% by mole of the starting material, and the metal ion moiety of the metal-polyphenol complex is selected from Al 3 + 。
41. The drug-lipid particle of claim 40, wherein the metal-polyphenol complex is present in a 5% molar ratio in the starting material, the conjugated lipid that inhibits aggregation of the particle is present in a 3% molar ratio in the starting material, the cholesterol is present in a 47% molar ratio in the starting material, and the non-cationic lipids other than cholesterol or non-ionizable lipids are present in a 45% molar ratio in the starting material; the metal ion part in the metal-polyphenol compound 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-polyphenol complex was present in a molar ratio of 10% in the starting material, the conjugated lipid inhibiting particle aggregation was present in a molar ratio of 5% in the starting material, the cholesterol was present in a molar ratio of 30% in the starting material, exceptThe mole ratio of the non-cationic lipid or the non-ionizable lipid other than cholesterol in the raw material is 55%; the metal ion part in the metal-polyphenol compound is selected from Al 3 + 。
42. The drug-lipid particle of claim 1, wherein the drug is encapsulated in the metal-polyphenol complex particle.
43. The drug-lipid particle of claim 42, wherein 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.
44. The drug-lipid particle of claim 43, wherein the nucleic acid is selected from the group consisting of mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, artificial nucleic acids, and combinations of one or more thereof.
45. The drug-lipid particle of claim 44, wherein the nucleic acid is the mRNA sequence encoding eGFP shown in SEQ ID No.1, the mRNA sequence encoding the receptor binding domain RBD of the novel coronavirus S1 subunit shown in SEQ ID No.2, the mRNA sequence encoding the NY-ESO-1 shown in SEQ ID No.3, the siRNA sequence encoding the Bcl-2 gene shown in SEQ ID No.4 and the sense strand shown in SEQ ID No.21, the siRNA sequence encoding the PLK1 gene shown in SEQ ID No.6 and the sense strand shown in SEQ ID No.23, the siRNA sequence encoding the Gal-1 gene shown in SEQ ID No.8, the ASO sequence encoding the STAT-3 gene shown in SEQ ID No.10, the ASO sequence encoding the alpha-syn gene shown in SEQ ID No.12, the ASO sequence encoding the Bcl-2 gene shown in SEQ ID No.14, the mRNA sequence encoding the wild-type novel coronavirus S protein shown in SEQ ID No.16, the siRNA sequence shown in SEQ ID No.6 and the sense strand shown in SEQ ID No.17 and the sense strand shown in SEQ ID No.26, the single strand shown in SEQ ID No. 7 and the sense strand shown in SEQ ID No.18 and the sense strand shown in SEQ ID No. 7.
46. The method for producing a drug-lipid particle as claimed in any one of claims 1 to 45, wherein the drug is entrapped in the metal-polyphenol complex particles to give the drug-lipid particle.
47. The process of claim 46, wherein (i) a metal-polyphenol complex, (ii) a conjugated lipid that inhibits aggregation of particles, and (iii) a non-cationic lipid or a non-ionizable lipid are mixed to provide the metal-polyphenol complex particles.
48. The process of claim 46, wherein (a) the drug, (i) the metal-polyphenol complex, (ii) the conjugated lipid that inhibits aggregation of particles, and (iii) the non-cationic lipid or the non-ionizable lipid are mixed to obtain the drug-lipid particle.
49. The method of manufacturing of claim 46, comprising the steps of:
step one: reacting the polyphenol molecular moiety with the metal ion moiety through a coordination bond to form a metal-polyphenol complex;
step two: the drug-lipid particles are prepared by mixing the metal-polyphenol complex prepared in the step one, the conjugated lipid which inhibits aggregation of particles, the non-cationic lipid or the non-ionizable lipid, and the drug.
50. The process of claim 49, wherein the polyphenol molecules are dissolved in ethanol and then added with metal ions to react to obtain the metal-polyphenol complex.
51. The process of claim 50, wherein the molar ratio of polyphenol molecules to metal ions is 1: (1-2).
52. The process of claim 50, wherein the reaction conditions comprise a reaction at 60℃for 1 hour.
53. The process of claim 49, wherein the metal-polyphenol complex, the conjugated lipid that inhibits aggregation of the particles, and the non-cationic lipid or the non-ionizable lipid are dissolved in an organic compound to form an organic phase, the drug is dissolved in a buffer to form an aqueous phase, and the organic phase and the aqueous phase are mixed to obtain the drug-lipid particles.
54. The process of claim 53 wherein the organic compound is ethanol.
55. The method of claim 53, wherein the buffer is an enzyme-free Tris-HCl buffer.
56. The process of claim 53 wherein the mixing of the organic phase and the aqueous phase comprises micro-fluidic chip or ultrasound.
57. Use of a drug-lipid particle of any one of claims 1 to 45 in a composition for delivery of a drug.
58. The use of claim 57, wherein the composition is for introducing a drug into a cell.
59. The use of claim 57, wherein the composition is a pharmaceutical agent.
60. The use of claim 59, 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.
61. The use of claim 60, wherein the mammal is a human.
62. The use of claim 60, wherein the disease or disorder is associated with expression of a gene comprising a target sequence for a drug.
63. The use of claim 60, wherein the disease or condition comprises cancer, a viral infection, an autoimmune disease, diabetes, or alzheimer's disease.
64. The use of claim 63, wherein the viral infection comprises hepatitis A, hepatitis B, hepatitis C, SARS-Cov-2, HIV, HPV, influenza, smallpox, or syphilis.
65. The use of claim 63, wherein the cancer comprises liver cancer, glioma, melanoma, lung cancer, pancreatic cancer, or breast cancer.
66. The use of claim 59, wherein the medicament is a vaccine.
67. The use of claim 59, wherein the route of administration of the agent comprises intrathecal injection, intramuscular administration, intracranial injection, intravenous injection, or intratumoral injection.
68. A medicament comprising the drug-lipid particle of any one of claims 1 to 45.
69. The agent of claim 68, wherein the agent is a vaccine.
70. The medicament of claim 69, wherein the vaccine is a novel coronavirus vaccine.
CN202310975036.0A 2022-08-09 2023-08-04 Medicine-lipid particle and preparation method and application thereof Pending CN117582416A (en)

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