CN112274646B - Amphiphilic protein-macromolecule conjugate delivery system for targeted activation of CD44 molecules, preparation method and application thereof - Google Patents

Amphiphilic protein-macromolecule conjugate delivery system for targeted activation of CD44 molecules, preparation method and application thereof Download PDF

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CN112274646B
CN112274646B CN202010663991.7A CN202010663991A CN112274646B CN 112274646 B CN112274646 B CN 112274646B CN 202010663991 A CN202010663991 A CN 202010663991A CN 112274646 B CN112274646 B CN 112274646B
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delivery system
hsa
acid
protein
plaque
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CN112274646A (en
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马茜
孙洁芳
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Beijing Inno Medicine Co ltd
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
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    • A61K51/04—Organic compounds
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    • A61K51/081—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the protein being an albumin, e.g. human serum albumin [HSA], bovine serum albumin [BSA], ovalbumin
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    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A61K9/5107—Excipients; Inactive ingredients
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Abstract

The invention provides an amphiphilic protein-macromolecule conjugate nano-carrier delivery system for targeted activation of CD44 molecules, wherein the amphiphilic protein-macromolecule conjugate is a nano-structure formed by self-assembly after in-situ grafting coupling of macromolecule fragments on protein molecules, and the surface of the amphiphilic protein-macromolecule conjugate is partially modified by a targeting ligand which can be specifically bound with the activated CD44 molecules. Methods of making and using the delivery system are also provided. The ProS self-assembled structure designed by the invention has the advantages of good biocompatibility, high drug loading capacity, good safety and strong stability, and is a nano drug carrier with important application value.

Description

Amphiphilic protein-macromolecule conjugate delivery system for targeted activation of CD44 molecules, preparation method and application thereof
Technical Field
The invention belongs to the technical field of targeted drug delivery, and particularly relates to a nano-carrier, in particular to an amphiphilic protein-polymer conjugate, for targeted activation of CD44 molecules, in particular to targeting vulnerable plaques. The invention also relates to a preparation method, performance improvement and application expansion of the nanocarrier, in particular to application in diagnosis, prevention and treatment of vulnerable plaque or diseases related to vulnerable plaque of an amphiphilic protein-polymer conjugate drug delivery system.
Background
At present, acute cardiovascular events, which are mainly acute myocardial infarction and sudden cardiac death, have become the first killer for endangering human health. It is counted that about 2 tens of millions of people die each year worldwide from an acute cardiovascular event. In China, too, the situation is optimistic, and more than 70 tens of thousands of people die annually from acute myocardial infarction and sudden cardiac death, which has become one of the most important diseases seriously threatening the health of residents in China. Studies have shown that most acute myocardial infarction and sudden cardiac death are caused by atherosclerotic plaques. Since the 70 s of the last century, the course and mechanism of the occurrence of Acute Coronary Syndrome (ACS) and stroke due to chronic atherosclerotic plaques has been explored.
In 1989, muller and his team proposed the concept of "vulnerable plaque", which is thought to be the root cause of most acute cardiovascular events. Vulnerable plaque (vulnerable plaque), also known as "unstable plaque", refers to atherosclerotic plaque that has a tendency to form thrombus or is highly likely to progress rapidly to "criminal plaque", primarily including ruptured plaque, erosive plaque, and partially calcified nodular lesions. Numerous studies have shown that most acute myocardial infarction and stroke are due to rupture of vulnerable plaques with light and moderate stenosis, and secondary thrombosis. Naghavi and its team et al give histological definition and criteria for vulnerable plaque. The main criteria include active inflammation, thin fibrous caps and large lipid cores, endothelial denudation with surface platelet aggregation, plaque fissures or lesions, and severe stenosis. Secondary criteria include surface calcification spots, yellow shiny plaques, intra-plaque bleeding and positive reconstitution. Thus, early intervention is critical for vulnerable plaque. However, since the degree of vascular stenosis caused by vulnerable plaque is not high in general, many patients have no pre-symptoms, and early diagnosis is difficult to be clinically performed, so that the risk is extremely high. Therefore, how to identify and diagnose vulnerable plaque as early as possible and to perform effective intervention becomes a problem to be solved in preventing and treating acute myocardial infarction.
The techniques commonly used for vulnerable plaque diagnosis at present mainly comprise coronary angiography, intravascular ultrasound (IVUS), laser coherence tomography (OCT) and other techniques, but the techniques belong to invasive examination, the diagnostic resolution and accuracy are not high, and meanwhile, the diagnostic techniques are expensive, so that the clinical popularization is limited to a certain extent. Thus, there is an urgent need for noninvasive diagnostic techniques and formulations for vulnerable plaque.
In addition, current methods of treating vulnerable plaque are primarily systemic administration, such as oral statin, aspirin, inhibitors of Matrix Metalloproteinases (MMPs), and/or fibrates, among others. These drugs have the effect of stabilizing plaque by reducing lipid in plaque, improving vascular remodeling, etc. by regulating systemic blood lipid, anti-inflammation, inhibiting protease and platelet production, etc. However, the therapeutic effect of the drugs currently used for treating vulnerable plaques is found to be not ideal in clinical applications. For example, the oral administration of the statin commonly used in clinic has a relatively low bioavailability, e.g., < 5% simvastatin, about 12% atorvastatin, and about 20% rosuvastatin. Animal experiments also prove that the effect of increasing the thickness of the fibrous cap and reducing the plaque volume can be achieved when the dosage of the statin is increased to more than 1mg/kg, which causes the stability of oral administration of the statin and the effect of reversing the plaque to encounter a bottleneck. Clinical trials have also demonstrated that oral statin therapy requires a high dose of reinforcement to stabilize vulnerable plaque, while systemic large dose statin therapy also presents a risk of increased incidence of serious side effects (e.g., liver dysfunction, rhabdomyolysis, type II diabetes, etc.).
For existing systemic administration, only a very small fraction of the active ingredient usually acts on the lesion actually after entering the body. This is the root cause of the adverse side effects of drugs and restricts the therapeutic effects of drugs. Targeted drug delivery system refers to drug delivery systems having targeted drug delivery capability. After administration via a certain route, the drug contained in the targeted delivery system will be specifically enriched at the target site by the carrier with the targeting probe. The targeted drug delivery system is capable of targeting the drug to a specific lesion and releasing the active ingredient at the targeted lesion. Therefore, the targeted drug delivery system can enable the drug to form relatively high concentration at the target lesion site and reduce the drug dosage in blood circulation, thereby inhibiting toxic and side effects and reducing the damage to normal tissues and cells while improving the drug efficacy.
Currently, the nanocarriers commonly used in targeted drug delivery systems are liposomes. Although the liposome has the advantages of improving the drug effect and reducing the toxic and side effects of the drug, the liposome has poor in vivo stability, so that the circulation time is insufficient, and finally the bioavailability of the drug is improved to a limited extent. In addition, the liposome has insufficient in vitro stability, phospholipid is easy to oxidize and hydrolyze during storage, liposome vesicles are easy to mutually aggregate and fuse, and medicines wrapped in the liposome vesicles are easy to leak. This has limited the development of targeted drug delivery systems to some extent.
In addition, in the field of diagnosis and treatment of vulnerable plaque, there are also some techniques for diagnosing vulnerable plaque using targeting ligand-modified nanocarriers. However, a major problem in clinical practice of such targeting probes targeting vulnerable plaques is the lack of specificity of the targeting sites of these formulations. For example, the targeting sites of such formulations are mostly macrophages, but since macrophages can be present throughout the body, the targeting specificity of the probe is not ideal. Thus, a difficulty in the development of targeting agents that target vulnerable plaques is the discovery of targets with significant targeting specificity in cells within vulnerable plaques.
CD44 is a class of adhesion molecules that are widely distributed on the surface of lymphocytes, monocytes, endothelial cells, etc. The primary ligand of the CD44 molecule is hyaluronic acid (abbreviated as "HA"). Based on the activation state of the expressing cells, CD44 can be classified into a relatively quiescent state (incapable of binding HA), an induced activation state (capable of binding HA after activation) and a structurally active state (capable of binding HA without activation), whereas most normal cell surface CD44 is in a relatively quiescent state and thus incapable of binding HA.
Extensive research has been continued to demonstrate that CD44 is not an ideal target with significant targeting specificity. This is because CD44 is widely distributed in the human body, and particularly exists in a large amount on the surface of organs rich in reticuloendothelial. Thus, the following problems are encountered in the development of targeted drug delivery systems targeting CD 44: such targeted drug delivery systems do not have specific targeting properties if the affinity of CD44 to HA on the target cell surface is insufficient to provide significant specificity.
Therefore, finding a specific target site existing at a vulnerable plaque site and a targeting drug delivery system suitable for targeting the vulnerable plaque, thereby developing a targeting drug delivery system capable of specifically targeting the vulnerable plaque and simultaneously realizing stable and sustained release of a drug, has become a technical problem to be solved in the medical field.
To date, there HAs been no report on the expression status of CD44 on the surface of macrophages, monocytes, endothelial cells, lymphocytes and smooth muscle cells that are mainly present within vulnerable plaques and their affinity to HA, nor is there any prior art regarding the use of the interaction of HA and CD44 and the design of targeted drug delivery systems for diagnosing or treating vulnerable plaques or diseases associated with vulnerable plaques that are capable of achieving stable sustained release of drugs using the specific microenvironment of the vulnerable plaques.
The protein with good biocompatibility can be self-assembled into a nano carrier under physiological conditions by coupling with hydrophobic polymer fragments, the protein forms a hydrophilic segment of a giant amphiphilic polymer, and the hydrophobic polymer forms a hydrophobic end, so that the target drug delivery can be realized by loading chemical drugs and microRNAs. The protein-polymer conjugate integrates the properties and advantages of the protein and the linked polymer, and also shows some new properties. The existence of the protein endows the conjugate with excellent biocompatibility and biological function, while the introduction of the polymer chain can increase the stability of the protein and the drug loading capacity, and can endow the conjugate with amphiphilicity and the property of the polymer chain, for example, the introduction of the temperature-sensitive polymer chain can endow the conjugate with temperature sensitivity. The research shows that the novel amphiphilic protein-macromolecule combination body has similar assembly performance with the traditional amphiphilic small molecules and block copolymers in aqueous solution, and can be self-assembled to form assemblies with various morphologies. The carrier is derived from biological molecules, so that the carrier has small cytotoxicity and good biocompatibility, thus showing good application prospect, providing safety guarantee for further application of protein assembly carrier and providing favorable conditions for clinical transformation.
In order to further enhance the biocompatibility of the nano-drug delivery vehicle, protein assemblies provide a new direction of investigation for this field. The biological source of the human serum albumin ensures the biological safety of the drug carrier, and the biocompatible polymer hydrophobic fragments (polylactic acid, polycaprolactone and the like) are gently derived on the surface of the drug carrier, so that the molecules have good amphipathic property, can form vesicle structures through self-assembly, further encapsulate various molecules, and further have the greatest advantages of no toxicity, good biocompatibility, biodegradability, easy preparation and modification, flexible carrier design, high biocompatibility and strong structural stability.
In a continuing study, protein-polymer conjugates were used to construct cell membrane-like structures, mimic biological processes, and verify the potential for use in drug carriers. The prior art also prepares the bovine serum albumin modified polyacrylamide fragments to form nano vesicles through self-assembly, the stability of the vesicles is improved through forming tannic acid-iron ion coordination complexes on the surfaces of the vesicle structures, the vesicles are used for simulating the physiological process of cells, enzyme loading is used for catalytic reaction, and the permeability of the vesicles to small molecules is verified. However, the carrier is not modified on the surface, and has the biggest problem of poor in vivo stability, and is particularly easy to be rapidly degraded and cleared by protease widely existing in vivo. It was reported that proteome loading would be 50. Mu.g mL -1 Proteases degrade within 10 minutes. Undoubtedly, such fragile structures greatly limit the potential applications of protein assemblies for in vivo drug delivery. Therefore, a key point for improving the application properties of the vector is to propose improvement in the stability of the protein vector under physiological conditions.
The elicitation given by the multi-layered cell surface structure in simple organisms, such as in gram-negative bacteria, that the surface of the cell has an outer membrane composed of lipopolysaccharides with the function of protecting the bacteria from damage, suggests that the inventors further coat outside the cell a biocompatible protective layer similar to the cell wall or plasma membrane structure for stabilizing the protein-polymer conjugate structure, which would greatly improve the applicability of such vectors.
Disclosure of Invention
The hollow protein assembly is used for constructing nano drug carriers, and has good application prospect, but still faces the problems of insufficient in vivo stability and easy clearance by protease. In view of this problem, the present application proposes a strategy for forming several biocompatible protective layers on the surface of protein assemblies, for stabilizing the self-assembled nano-carrier structure of proteins and for ensuring the biocompatibility of the carrier. And the feasibility of the method is verified by taking targeted treatment of vulnerable plaque as an embodiment.
Before setting forth the present disclosure, the terms used herein are defined as follows:
"protein-polymer conjugate" means: the protein molecule is grafted and coupled with a biosafety macromolecule fragment in situ to prepare a protein-macromolecule grafted compound, and the amphiphilic protein compound molecule forms a giant protein nanostructure by self-assembly under physiological conditions.
"physiological condition" means: the biochemical reactions required to maintain the normal performance of vital activities, temperature, ionic strength, pH, protein, enzyme concentration levels, and to maintain vital operations.
"vulnerable plaque" is also known as "unstable plaque" and refers to an atherosclerotic plaque that has a tendency to form thrombosis or is highly likely to rapidly progress to "criminal plaque," and includes primarily ruptured plaque, erosive plaque, and partially calcified nodular lesions. Numerous studies have shown that most acute myocardial infarction and stroke are due to rupture of vulnerable plaques with light and moderate stenosis, and secondary thrombosis. Histological manifestations of vulnerable plaque include active inflammation, thin fibrous caps and large lipid cores, endothelial denudation with surface platelet aggregation, plaque fissures or lesions, and severe stenosis, as well as surface calcification plaque, yellow shiny plaque, intra-plaque hemorrhage, and positive remodeling.
"vulnerable plaque-associated disease" refers primarily to diseases associated with, characterized by, caused by, or secondary to "vulnerable plaque" during the occurrence and progression of the disease. The "diseases associated with vulnerable plaque" mainly include diseases such as atherosclerosis, coronary heart disease (including acute coronary syndrome, asymptomatic myocardial ischemia-occult coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, and in-stent restenosis), cerebral atherosclerosis (including cerebral apoplexy), peripheral vascular atherosclerosis (including peripheral occlusive atherosclerosis, retinal atherosclerosis, carotid atherosclerosis, renal atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, atherosclerosis impotence), aortic dissection, hemangioma, thromboembolism, heart failure, and cardiogenic shock.
"targeted drug delivery system" refers to a drug delivery system having targeted drug delivery capability. After administration via a route, the drug contained in the targeted delivery system will be specifically enriched at the target site by the action of a particular carrier or targeting bullet (e.g., targeting ligand). Means for achieving targeted drug delivery are currently known including utilizing the passive targeting properties of various microparticle drug delivery systems, chemical modification at the surface of microparticle drug delivery systems, utilizing some specific physicochemical properties, utilizing antibody-mediated targeted drug delivery, utilizing ligand-mediated targeted drug delivery, utilizing prodrug targeted drug delivery, and the like. Wherein, the ligand-mediated targeted drug delivery is to use the characteristic that specific receptors on certain organs and tissues can specifically bind with the specific ligands, and the drug carrier is bound with the ligands, so that the drug is guided to specific target tissues.
"hyaluronic acid (abbreviated" HA ")" is a polymer of high molecular weight, molecular formula: (C) 14 H 21 NO 11 ) n. It is a higher polysaccharide composed of units of D-glucuronic acid and N-acetylglucosamine. D-glucuronic acid and N-acetylglucosamine are connected by beta-1, 3-glycosidic bond, and disaccharide units are connected by beta-1, 4-glycosidic bond. Hyaluronic acid shows various important physiological functions in the body by virtue of unique molecular structure and physicochemical properties, such as lubricating joints, regulating permeability of vascular wall, regulating protein, water electrolyte diffusion and operation, and promoting wound healingEtc. Particularly, hyaluronic acid has a special water-retaining effect, and is the substance with the best water retention in the nature which is found at present.
By "derivative of hyaluronic acid" is meant herein any derivative of hyaluronic acid capable of retaining the specific binding capacity of hyaluronic acid to CD44 molecules on the cell surface at vulnerable plaques, including but not limited to pharmaceutically acceptable salts of hyaluronic acid, lower alkyl (alkyl containing 1-6 carbon atoms) esters, prodrugs capable of forming hyaluronic acid in vivo by hydrolysis or other means, and the like. Determining whether a substance is a "derivative of hyaluronic acid" can be accomplished by measuring the specific binding capacity of the substance to CD44 molecules on the cell surface at vulnerable plaques, which is within the skill of the person skilled in the art.
The "CD44 molecule" is one kind of transmembrane proteoglycan adhesion molecule expressed widely on lymphocyte, monocyte, endothelial cell, etc. cell membrane and consists of three sections including extracellular section, transmembrane section and intracellular section. CD44 molecules can mediate interactions between a variety of cells and cells, and between cells and extracellular matrix, and are involved in the transduction of a variety of signals in the body, thereby altering the biological function of cells. The primary ligand of the CD44 molecule is hyaluronic acid, and receptor-ligand binding between it and hyaluronic acid determines cell adhesion and/or migration in the extracellular matrix. In addition, CD44 molecules are involved in the metabolism of hyaluronic acid.
"about" represents the set of all values within + -5% of the values given thereafter.
The term "HSA" refers to: human serum albumin.
The term "PCL" refers to: polycaprolactone.
The term "PLLA" refers to: poly-L-lactic acid.
The term "edc.hcl" means: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
The term "sulfo-NHS" refers to: n-hydroxysulfosuccinimide.
The term "Col" refers to: collagen.
The term "PEG" refers to: polyethylene glycol.
The term "SP" refers to: proteins are selected.
The term "OPN" refers to: osteopontin
The term "Col" refers to: collagen.
The term "Asp" refers to: aspirin.
The term "Clo" means: clopidogrel.
The term "At" refers to: atorvastatin.
The term "DXMS" refers to: dexamethasone.
The term "R" refers to: rosuvastatin.
The term "FDG" refers to: fluorodeoxyglucose.
The term "DPLA" refers to: iopromide.
The term "DKSC" refers to: iodixanol.
The term "DFC" refers to: iofluor alcohol.
The term "GPA" refers to: gadoteric acid meglumine.
The term "GSA" refers to: gadolinium diamine.
The term "GPS" refers to: gadolinium spray acid.
In a first aspect, the present invention provides an amphiphilic protein-polymer conjugate nanocarrier delivery system for targeted activation of CD44 molecules, wherein the amphiphilic protein-polymer conjugate is formed by coupling a protein and a hydrophobic polymer fragment through a chemical bond, and the surface of the amphiphilic protein-polymer conjugate is partially modified by a targeting ligand, wherein the targeting ligand is a ligand capable of specifically binding to an activated CD44 molecule.
In a second aspect, the present invention provides an amphiphilic protein-macromolecule conjugate nanocarrier delivery system for targeting vulnerable plaques, wherein the amphiphilic protein-macromolecule conjugate is formed by coupling a protein and a hydrophobic macromolecule fragment through a chemical bond, and the surface of the amphiphilic protein-macromolecule conjugate is partially modified by a targeting ligand, wherein the targeting ligand is a ligand capable of specifically binding to an activated CD44 molecule.
The delivery system according to the first or second aspect, wherein the protein is selected from one or more of the following: immunoglobulin, bovine serum albumin, human serum albumin, gene recombinant human serum albumin, ovalbumin, gelatin, collagen, milk protein, silk protein, elastin; preferably human serum albumin.
The delivery system provided in the first aspect or the second aspect, wherein the polymer fragment is a hydrophobic polymer fragment with biocompatibility, biodegradability and good safety, and the polymer fragment can be polymerized by one monomer or several different monomers, and the polymer fragment is not modified or modified; preferably, the polymeric fragment is selected from one or more of the following: polystyrene (PS), polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), polyglycolic acid (PGA), polylactic acid (including both left and right handed) (PLA), poly (N-isopropylacrylamide) (PNIAAm), polyhydroxyethyl acrylate (PHEA), polymethacrylic acid (PMMA), polyvinyl alcohol (PVA) and polyethylene glycol dimethacrylate (PEGDMA), polytetrafluoroethylene; more preferred are Polycaprolactone (PCL), poly L-lactic acid (PLLA), poly lactic acid-co-glycolic acid (PLGA).
The delivery system according to the first or second aspect, wherein the protein molecule is self-assembled to form an amphiphilic protein-polymer conjugate after in situ grafting of the coupled hydrophobic polymer fragment onto the protein molecule;
preferably, the self-assembly condition is ultrasound induced two-phase assembly.
The delivery system according to the first or second aspect, wherein the amphiphilic protein-polymer conjugate is surface-modified with a natural polymer, the natural compound being an organic layer that can undergo self-aggregation or form a complex network with metal ions;
preferably, the natural compound is selected from one or more of the following: dopamine, phytic acid, tannic acid, chitosan, trehalose and plant polyphenol; more preferably, the natural compound dopamine self-polymerizes or tanninsacid-Fe 3+ A complex.
The delivery system according to the first or second aspect, wherein the targeting ligand is selected from GAGs, collagen, laminin, fibronectin, selectin, osteopontin and monoclonal antibodies HI44a, HI313, A3D8, H90, IM7, or hyaluronic acid or derivatives of hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at vulnerable plaques;
Preferably, the targeting ligand is selected from collagen, hyaluronic acid, selectin, osteopontin or monoclonal antibodies HI44a, IM7.
The delivery system according to the first or second aspect, wherein the nanocarrier surface may be further modified, preferably by modifying one or more of polyethylene glycol, a transmembrane peptide, a self-peptide, or a dual ligand simultaneous modification on the carrier surface.
The delivery system according to the first or second aspect, wherein the nanocarrier is loaded with a substance for diagnosing, preventing and/or treating a disease associated with the occurrence of a CD44 molecule activation condition; and/or
The nanocarriers are loaded with a substance for diagnosing, preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque;
preferably, the nanocarrier is loaded with both a substance for preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque and hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at the vulnerable plaque;
more preferably, the nanocarrier is simultaneously loaded with a substance for diagnosing vulnerable plaque or a disease associated with vulnerable plaque, a substance for preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque, optionally a CD44 activator and optionally hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at vulnerable plaque.
The delivery system according to the first or second aspect, wherein the substance for diagnosing, preventing and/or treating a disease associated with the occurrence of a CD44 molecule activation condition is a CD44 activator;
preferably, the CD44 activator is a CD44 antibody mAb or IL5, IL12, IL18, TNF- α, LPS.
The delivery system according to the first or second aspect, wherein the substance for diagnosing, preventing and/or treating vulnerable plaque or vulnerable plaque-related diseases is selected from one or more of a drug, polypeptide, nucleic acid and cytokine for diagnosing, preventing and/or treating vulnerable plaque or vulnerable plaque-related diseases.
The delivery system according to the first or second aspect, wherein the substance for diagnosing, preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque is a substance for diagnosing vulnerable plaque or a disease associated with vulnerable plaque;
more preferably, the substance for diagnosing vulnerable plaque or a disease associated with vulnerable plaque is a tracer;
further preferably, the tracer is selected from the group consisting of CT tracers, MRI tracers and nuclide tracers;
still further preferably:
The CT tracer is selected from iodine nano contrast agent, gold nano contrast agent, tantalum oxide nano contrast agent, bismuth nano contrast agent, lanthanide nano contrast agent or other tracer with similar structure; more preferably iodinated contrast agent or nanogold, or other similarly structured tracers; further preferred are iohexol, iocaic acid, ioversol, iodixanol, iopromide, iobitol, iomeprol, iopamidol, ioxilan, aceiobenzoic acid, cholic acid, iobenzamic acid, iogancaic acid, diatrizoic acid, sodium iotazinate, iophenyl ester, iopanoic acid, ioafoic acid, sodium acetate iobenzoate, propidone, ioaodone, iotrolan, iopidol, meglumine of cholic acid, iotaloic acid, diatrizoic amine, mezoic acid, meglumine, iodized oil or ethidium iodide, or other similarly structured tracers; preferably nano gold;
the MRI tracer is selected from the group consisting of longitudinal relaxation contrast agents and transverse relaxation contrast agents; more preferably paramagnetic, ferromagnetic and super-magnetic contrast agents; further preferred are Gd-DTPA and porphyrin chelates of linear, cyclic polyamine polycarboxylic chelates and manganese, macromolecular gadolinium chelates, biomacromolecule modified gadolinium chelates, folic acid modified gadolinium chelates, dendrimer developers, liposome modified developers and gadolinium-containing fullerenes, or other similarly structured tracers; and preferably gadoferamic acid meglumine, gadoferamine, ferric ammonium citrate effervescent granules, paramagnetic iron oxide, preferably paramagnetic iron oxide or other tracers of similar structure; and/or
The nuclide tracer is selected from fluorodeoxyglucose labeled with carbon 14, carbon 13, phosphorus 32, sulfur 35, iodine 131, hydrogen 3, technetium 99, and fluorine 18.
The delivery system according to the first or second aspect, wherein the substance for diagnosing, preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque is a substance for preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque;
preferably, the substance for preventing and/or treating vulnerable plaque or vulnerable plaque-related diseases is selected from one or more of statin drugs, fibrates, antiplatelet drugs, PCSK9 inhibitors, anticoagulants, angiotensin converting enzyme inhibitors, calcium antagonists, MMPs inhibitors, beta blockers, glucocorticoids or other anti-inflammatory substances such as IL-1 antibody canakinumab, and pharmaceutically acceptable salts thereof, including active formulations of these classes of drugs or substances, and endogenous anti-inflammatory cytokines such as interleukin 10;
more preferably, the substance for preventing and/or treating vulnerable plaque or vulnerable plaque-related diseases is selected from lovastatin, atorvastatin, rosuvastatin, simvastatin, fluvastatin, pitavastatin, pravastatin, bezafibrate, ciprofibrate, clofibrate, gemfibrozil, fenofibrate, probucol, anti-PCSK 9 antibodies such as evolocumab, alirocumab, bococizumab, RG7652, LY3015014 and LGT-209, or adnectins such as BMS-962476, antisense RNAi oligonucleotides such as ALN-PCSsc, nucleic acids such as microRNA-33a, microRNA-27a/b, microRNA-106b, microRNA-302, microRNA-758, microRNA-10b, microRNA-19b, microRNA-26, microRNA-93, microRNA-128-2, microRNA-144, microRNA-145 antisense strands and nucleic acid analogues thereof such as locked nucleic acids, aspirin, acimetacin, troxerutin, dipyridamole, cilostazol, ticlopidine hydrochloride, ozagrel sodium, clopidogrel, prasugrel, cilostazol, belipratropium sodium, ticagrelor, canceririol, tirofiban, etiquetin, acimumab, common heparin, kesai, fast-green, huang Dagan sunflower sodium, warfarin, dabigatran, rivaroxaban, apixaban, edoxaban, bivalirudin, enoxaparin, tetaran, adequan, biscoumarin, coumarin nitrate, sodium matrisulfonate, hirudin, argatroban, benazepril, captopril, enalapril, perindopril, fosinopril, lisinopril, moexipril, cilazapril, perindopril, quinapril, ramipril, trandolapril, candesartan, eprosartan, irbesartan, losartan, telmisartan, valsartan, olmesartan, tasosartan, nifedipine, nicardipine, nitrendipine, nimodipine, nilodipine, nilvadipine, isradipine, felodipine, lacidipine, diltiazem, verapamil, chlorhexidine, minocycline, MMI-166, metoprolol, atenolol, bisoprolol, propranolol, carvedilol, bamomastat, marimastat, pranlostat, BMS-279251, BAY 12-9566, TAA211, AAJ996A, nacetrapib, evacetrapib, torcetrapib and dalretrapib, prednisone, methylprednisone, betamethasone, beclomethasone propionate, prednisolone, hydrocortisone, dexamethasone or other anti-inflammatory substances such as one or more of the IL-1 antibodies canakinumab, and pharmaceutically acceptable salts thereof, including endogenous cytokines such as endogenous cytokines of these structures and endogenous cytokines 10.
A third aspect of the present invention provides a method of preparing the delivery system of the first or second aspect, the method comprising the steps of:
(1) Coupling the protein and the hydrophobic polymer fragment to prepare a protein-hydrophobic polymer complex;
(2) The protein-hydrophobic polymer compound prepared in the step (1) is self-assembled to prepare an amphiphilic protein-polymer conjugate;
(3) And (3) coupling the target ligand to the amphiphilic protein-polymer conjugate prepared in the step (2).
The method according to the third aspect of the present invention, wherein in the step (2), the self-assembly step further includes a drug entrapping process.
In a fourth aspect the invention provides a pharmaceutical composition comprising the nanocarrier delivery system of the first or second aspect.
In a fifth aspect the present invention provides a diagnostic formulation comprising the nanocarrier delivery system of the first or second aspect.
A sixth aspect of the invention provides the use of the nanocarrier delivery system of the first or second aspect in the manufacture of a product for the diagnosis, prevention and treatment of vulnerable plaques or diseases associated with vulnerable plaques.
The use according to the sixth aspect of the invention, wherein the vulnerable plaque is selected from one or more of a ruptured plaque, an erosive plaque and a partially calcified nodular lesion;
More preferably, the disease associated with vulnerable plaque is selected from one or more of the following: atherosclerosis, coronary atherosclerotic heart disease, cerebral atherosclerosis, peripheral vascular atherosclerosis, aortic dissection, hemangioma, thromboembolism, heart failure and cardiogenic shock;
preferably, the coronary atherosclerotic heart disease is selected from one or more of the following: acute coronary syndrome, asymptomatic myocardial ischemia-occult coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, and in-stent restenosis;
the cerebral atherosclerosis is cerebral apoplexy; and/or
The peripheral vascular atherosclerosis is selected from one or more of the following: carotid atherosclerosis, peripheral occlusive atherosclerosis, retinal atherosclerosis, renal atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, and atherosclerosis impotence.
In recent years, human serum albumin is used as a novel biological material, and has good biocompatibility, accurate geometric structure, easy functionalization and very high combination property on medicines, so that the formed serum albumin nano-particles can load medicines with high efficiency. Functional groups such as amine groups and carboxyl groups on the surface of serum albumin nanoparticles can be used for coupling drugs and targeting ligands. These advantages have led to an increasing interest in the field of nanomedicine. The application selects Human Serum Albumin (HSA) as a base material, and prepares a plurality of novel protein-polymer grafted complexes by grafting and coupling biosafety polymer fragments (polycaprolactone and polylactic acid) on protein molecules in situ through surface initiated free radical polymerization reaction from the molecular design perspective, wherein the protein-polymer conjugates can form self-assembled protein nanostructures (ProS) for loading medicines under physiological conditions. The application is based on improving the in-vivo stability of the assembled protein nano-carrier, and proposes to further form a biocompatible protective layer (polydopamine, phytic acid/rare earth ion compound) on the surface of the assembled protein nano-carrier, so that the stability of the self-assembled nano-carrier is improved, and the self-assembled protein nano-carrier has the advantages of good biocompatibility, structural diversity, easiness in functionalization and the like. The application of the drug carrier in the field of vulnerable plaque treatment is realized by coupling the carrier with different target molecules. Through a series of experimental processes, the drug/gene delivery capacity and the therapeutic and developing effects of the ProS nano-carrier are verified.
According to the invention, a protein self-assembly structure is constructed by coupling hydrophobic polymer fragments on the surface of human serum albumin and is used for loading medicines and biomolecules, a vesicle structure is reinforced by utilizing dopamine to polymerize on the surface of the protein self-assembly structure or by forming a coordination metal complex structure, a single ligand or multiple ligands are further coupled on the surface of the protein self-assembly structure, and targeted identification is carried out on activated CD44 so as to diagnose and treat vulnerable plaque.
In the PorS nano carrier, the protein-polymer conjugate can be assembled in an aqueous phase by coupling with a hydrophobic polymer fragment, and a biocompatible composite interface is further modified on the surface of the protein-polymer conjugate, so that on one hand, the in vivo stability of the carrier can be effectively improved, the in vivo circulation time is prolonged, and the protein carrier is prevented from being rapidly degraded by in vivo protease. In addition, the functional group structure with rich surfaces of the nano-carrier is also very convenient for connecting the targeting carrier. The ProS structure has the advantages of strong structural stability, good biocompatibility and high safety, and is a nano drug carrier with important application value.
In summary, the present invention relates to the following aspects:
the present invention provides a protein-macromolecule conjugate nanocarrier delivery system for targeting activated CD44 molecules.
The invention provides a protein-macromolecule conjugate nano-carrier delivery system for targeting vulnerable plaques.
The present invention provides a variety of surface modification strategies that improve the in vivo stability of protein-polymer conjugate nanocarrier delivery systems.
The invention also provides a method for preparing the protein-polymer conjugate delivery system for targeting vulnerable plaques.
The invention also provides a medicament comprising the protein-polymer conjugate delivery system for targeting vulnerable plaques and a pharmaceutically acceptable carrier.
The invention also provides a diagnostic formulation comprising the protein-polymer conjugate delivery system of the invention that targets vulnerable plaques.
The invention also provides application of the protein-macromolecule conjugate targeting vulnerable plaque in preparation of medicines for preventing and/or treating vulnerable plaque or diseases related to vulnerable plaque.
The invention also provides application of the protein-macromolecule conjugate targeting vulnerable plaque in preparation of diagnostic preparations for diagnosing vulnerable plaque or diseases related to vulnerable plaque.
The present invention also provides a method for preventing and/or treating vulnerable plaque or disease associated with vulnerable plaque, comprising administering to a subject in need thereof the vulnerable plaque-targeting protein-polymer conjugate of the present invention.
The invention also provides a method for diagnosing vulnerable plaque or a disease associated with vulnerable plaque, the method comprising administering to a subject in need thereof a vulnerable plaque-targeting protein-polymer conjugate of the invention.
The specific embodiments of the technical scheme of the present invention and the meanings thereof will be described in detail hereinafter.
The present invention provides a protein-macromolecule conjugate delivery system for targeting activated CD44 molecules, the surface of the protein-macromolecule conjugate being partially modified with a targeting ligand, which is a ligand capable of specifically binding to the activated CD44 molecule.
The present invention provides a protein-polymer conjugate delivery system for targeting vulnerable plaques, the surface of which is partially modified by a targeting ligand, which is a ligand capable of specifically binding to CD44 molecules on the cell surface at the vulnerable plaque.
Other modifications can be made to the surface of the protein-polymer conjugate to provide better results. PEG is modified on the surface of the carrier, so that the long circulation effect can be achieved, and the half life of the drug can be prolonged; PEG is modified on the surface of the carrier, and the penetrating peptide Tat, self peptide SEP or double ligands are modified simultaneously, so that the effect of amplifying the drug effect can be achieved.
The delivery system according to the present invention, wherein the protein-polymer conjugate is selected from a series of hydrophobic polymer fragments having good biocompatibility such as human serum albumin, poly-L-lactic acid (PLLA), polycaprolactone (PCL), poly-lactic-co-glycolic acid (PLGA), and the like, and is coupled to form an amphiphilic protein-polymer conjugate (ProS) by chemical bonds.
The protein-polymer conjugate according to the first or second aspect of the present invention, wherein the protein-polymer conjugate is surface-modified with a natural polymer selected from the group consisting of dopamine, phytic acid, tannic acid, and the like, which can undergo self-polymerization or form a complex network with metal ions.
The protein-polymer conjugate delivery system according to the present invention, wherein the targeting ligand is selected from GAGs, collagen, laminin, fibronectin, selectin, osteopontin (OPN) and monoclonal antibodies HI44a, HI313, A3D8, H90, IM7, or hyaluronic acid or derivatives of hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at vulnerable plaques;
Preferably, the targeting ligand is selected from collagen, hyaluronic acid, selectin, osteopontin or monoclonal antibodies HI44a, IM7.
The nanocarrier delivery system according to the present invention, wherein the nanocarrier is loaded with a substance for diagnosing, preventing and/or treating a disease associated with the occurrence of a CD44 molecule activation condition.
The protein-polymer conjugate according to the present invention, wherein the nanocarrier is loaded with a substance for diagnosing, preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque;
in one embodiment, the substance is a substance for diagnosing vulnerable plaque or a disease associated with vulnerable plaque;
in one embodiment, the substance for diagnosing vulnerable plaque or a disease associated with vulnerable plaque is a tracer;
in one embodiment, the tracer is selected from the group consisting of CT tracers, MRI tracers and nuclide tracers;
in one embodiment, the CT tracer is selected from the group consisting of iodine nanocontrast agents, gold nanocontrast agents, tantalum oxide nanocontrast agents, bismuth nanocontrast agents, lanthanide nanocontrast agents, or other similarly structured tracers; more preferably iodinated contrast agent or nanogold, or other similarly structured tracers; further preferred are iohexol, iocaic acid, ioversol, iodixanol, iopromide, iobitol, iomeprol, iopamidol, ioxilan, aceiobenzoic acid, cholic acid, iobenzamic acid, iogancaic acid, diatrizoic acid, sodium iotazinate, iophenyl ester, iopanoic acid, ioafoic acid, sodium acetate iobenzoate, propidone, ioaodone, iotrolan, iopidol, meglumine of cholic acid, iotaloic acid, diatrizoic amine, mezoic acid, meglumine, iodized oil or ethidium iodide, or other similarly structured tracers, preferably nanogold; and/or
The MRI tracer is selected from the group consisting of longitudinal relaxation contrast agents and transverse relaxation contrast agents; more preferably paramagnetic, ferromagnetic and super-magnetic contrast agents; further preferred are Gd-DTPA and porphyrin chelates of linear, cyclic polyamine polycarboxylic chelates and manganese, macromolecular gadolinium chelates, biomacromolecule modified gadolinium chelates, folic acid modified gadolinium chelates, dendrimer developers, liposome modified developers and gadolinium-containing fullerenes, or other similarly structured tracers; still more preferably, gadoferate meglumine, gadoferamine, ferric ammonium citrate effervescent granule, paramagnetic iron oxide (Fe) 3 O 4 NPs), or other similarly structured tracers, preferably Fe 3 O 4 NPs；
The nuclide tracer is selected from the group consisting of carbon 14% 14 C) 13% of carbon 13 C) Phosphorus 32% 32 P, sulfur 35% 35 S, iodine 131% 131 I) Hydrogen 3% 3 H) Technetium 99% 99 Tc, fluorine 18% 18 F) Labeled fluorodeoxyglucose.
In one embodiment, the substance is one or more of a drug, polypeptide, nucleic acid, and cytokine for diagnosing, preventing, and/or treating vulnerable plaque or a disease associated with vulnerable plaque.
In one embodiment, the substance is a CD44 activator;
In one embodiment, the CD44 activator is a CD44 antibody mAb or IL5, IL12, IL18, TNF- α, LPS.
In one embodiment, the substance is hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at vulnerable plaques;
preferably, the molecular weight of the small molecule hyaluronic acid or derivative of hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at vulnerable plaques is in the range of 1-500KDa, preferably 1-20KDa, more preferably 2-10KDa.
In one embodiment, the nanocarrier is loaded with both a substance for diagnosing, preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque and a CD44 activator;
preferably, the nanocarrier is loaded with both a substance for preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque and hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at the vulnerable plaque;
more preferably, the nanocarrier is simultaneously loaded with a substance for diagnosing vulnerable plaque or a disease associated with vulnerable plaque, a substance for preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque, optionally a CD44 activator and optionally hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at vulnerable plaque.
In one embodiment, the substance is a substance for preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque;
preferably, the substance for preventing and/or treating vulnerable plaque or vulnerable plaque-related diseases is selected from one or more of statin drugs, fibrates, antiplatelet drugs, PCSK9 inhibitors, anticoagulants, angiotensin Converting Enzyme Inhibitors (ACEI), calcium antagonists, MMPs inhibitors, beta blockers, glucocorticoids or other anti-inflammatory substances such as IL-1 antibody canakinumab, and pharmaceutically acceptable salts thereof, including active formulations of these classes of drugs or substances, and endogenous anti-inflammatory cytokines such as interleukin 10 (IL-10);
more preferably, the substance for preventing and/or treating vulnerable plaque or vulnerable plaque-related diseases is selected from lovastatin, atorvastatin, rosuvastatin, simvastatin, fluvastatin, pitavastatin, pravastatin, bezafibrate, ciprofibrate, clofibrate, gemfibrozil, fenofibrate, probucol, anti-PCSK 9 antibodies such as evolocumab, alirocumab, bococizumab, RG7652, LY3015014 and LGT-209, or adnectins such as BMS-962476, antisense RNAi oligonucleotides such as ALN-PCSsc, nucleic acids such as microRNA-33a, microRNA-27a/b, microRNA-106b, microRNA-302, microRNA-758, microRNA-10b, microRNA-19b, microRNA-26, microRNA-93, microRNA-128-2, microRNA-144, microRNA-145 antisense strands and nucleic acid analogues thereof such as locked nucleic acids, aspirin, acimetacin, troxerutin, dipyridamole, cilostazol, ticlopidine hydrochloride, ozagrel sodium, clopidogrel, prasugrel, cilostazol, belipratropium sodium, ticagrelor, canceririol, tirofiban, etiquetin, acimumab, common heparin, kesai, fast-green, huang Dagan sunflower sodium, warfarin, dabigatran, rivaroxaban, apixaban, edoxaban, bivalirudin, enoxaparin, tetaran, adequan, biscoumarin, coumarin nitrate, sodium matrisulfonate, hirudin, argatroban, benazepril, captopril, enalapril, perindopril, fosinopril, lisinopril, moexipril, cilazapril, perindopril, quinapril, ramipril, trandolapril, candesartan, eprosartan, irbesartan, losartan, telmisartan, valsartan, olmesartan, tasosartan, nifedipine, nicardipine, nitrendipine, nimodipine, nisoldipine, nilvadipine, isradipine, felodipine, lacidipine, diltiazem, verapamil, chlorhexidine, minocycline, MMI-166, metoprolol, atenolol, bisoprolol, propranolol, carvedilol, bamomastat, marimastat, pranlostat, BMS-279251, BAY 12-9566, TAA211, AAJ996A, nacetrapib, evacetrapib, torcetrapib and dalretrapib, prednisone, methylprednisone, betamethasone, beclomethasone propionate, prednisolone, hydrocortisone, dexamethasone or other anti-inflammatory substances such as the IL-1 antibody canakinumab), and one or more of their pharmaceutically acceptable salts, including these endogenous cytokines and endogenous cytokines such as the endogenous cytokine 10-10.
The present invention also provides a method for preparing a delivery system for targeting vulnerable plaque, the method comprising the steps of:
the specific study contents are as follows:
(1) Protein-polymer conjugate: synthesis and structural performance characterization of HSA-Polycaprolactone (PCL), HSA-poly-l-lactic acid (PLLA), HSA-poly-lactic-co-glycolic acid (PLGA) conjugates. The end group maleimido PCL, PLLA, PLGA was synthesized and the amphipathic HSA-PCL, HSA-PLLA was prepared by a coupling reaction between maleimide and the free thiol group of HSA. The structure of the HSA-PCL and HSA-PLLA compound is verified by infrared spectroscopy (FT-IR), and the results show that the HSA-PCL, the HSA-PLLA and the HSA-PLGA can be assembled in water to obtain nanometer assemblies with different morphologies, and the assembly performance of the HSA-PCL, the HSA-PLLA and the HSA-PLGA is further characterized by using a Transmission Electron Microscope (TEM), dynamic Light Scattering (DLS) and the like.
(2) Construction of nanotargeting drug carriers (ProS) based on HSA-PCL, HSA-PLLA, HSA-PLGA conjugates. The self-assembly performance of the prepared amphipathic HSA-PCL and HSA-PLA conjugate is utilized to construct the ProS nano-carrier for encapsulating various medicines. In order to improve the stability of the nano-carrier, different interface modification strategies are adopted: (1) the in-situ polymerization of polydopamine on the surface of the nano-carrier wraps ProS, so that the in-vivo stability of the nano-carrier is greatly improved, the polydopamine has high biosafety, and abundant functional groups are provided for facilitating the coupling of the targeting ligand in the next step, and the in-vitro performance of the nano-carrier is verified by coupling various ligands. Endocytosis and cytotoxicity of the constructed ProS nanocarriers were evaluated using cell and animal models. (2) The surface of the carrier is coated with ProS by forming the coordination complex of phytic acid and rare earth ions in situ, so that the in-vivo stability of the carrier is greatly improved, and the surface of the carrier is coated with the coordination complex of phytic acid-rare earth ions (PA-Re) n+ ) Not only has high biological safety, but also is beneficial to the next step of target couplingThe in vitro stability and the drug release performance of the nano-carrier are verified by coupling various ligands to the ligands. Endocytosis and cytotoxicity of the constructed targeted ProS nanocarriers were evaluated using cell and animal models. The results show that dopamine-coated and ligand-modified ProS has a better therapeutic effect than unmodified ProS. MTT results show that the prepared ProS nano-carrier has very low cytotoxicity and better biocompatibility. After the medicine is entrapped, the targeting modified ProS nano-carrier has obvious inhibition effect on the growth of vulnerable plaque.
The invention provides a medicament comprising the protein-polymer conjugate delivery system and a pharmaceutically acceptable carrier.
The present invention provides a diagnostic formulation comprising the protein-polymer conjugate delivery system.
The invention provides application of the protein-macromolecule combination delivery system, the medicine and the diagnostic preparation in preparing medicines for preventing and/or treating diseases related to the occurrence of CD44 molecule activation conditions.
The invention provides application of the protein-macromolecule combination delivery system, the medicine and the diagnostic preparation in preparing the medicine and/or the diagnostic preparation for preventing and/or treating vulnerable plaque or diseases related to vulnerable plaque.
Preferably, the vulnerable plaque is selected from one or more of a ruptured plaque, an erosive plaque, and a partially calcified nodular lesion;
preferably, the vulnerable plaque-associated disease is selected from one or more of atherosclerosis, coronary heart disease (including acute coronary syndrome, asymptomatic myocardial ischemia-occult coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, in-stent restenosis), cerebral atherosclerosis (including cerebral stroke), peripheral vascular atherosclerosis (including peripheral occlusive atherosclerosis, retinal atherosclerosis, carotid atherosclerosis, renal atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, atherosclerosis impotence), aortic dissection, hemangioma, thromboembolism, heart failure, and cardiogenic shock.
The present invention provides a method for preventing and/or treating a disease associated with the occurrence of a CD44 molecule activation condition, the method comprising: the protein-polymer conjugate delivery system, the drug, and the diagnostic agent described above are administered to a subject in need thereof.
The present invention also provides a method for preventing, diagnosing and/or treating vulnerable plaque or diseases associated with vulnerable plaque, the method comprising: the protein-polymer conjugate delivery system, the drug, and the diagnostic agent described above are administered to a subject in need thereof.
Preferably, the vulnerable plaque is selected from one or more of a ruptured plaque, an erosive plaque, and a partially calcified nodular lesion.
More preferably, the disease associated with vulnerable plaque is selected from one or more of atherosclerosis, coronary atherosclerotic heart disease (including acute coronary syndrome, asymptomatic myocardial ischemia-occult coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, in-stent restenosis), cerebral atherosclerosis (including cerebral stroke), peripheral vascular atherosclerosis (including peripheral occlusive atherosclerosis, retinal atherosclerosis, carotid atherosclerosis, renal atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, atherosclerosis impotence), aortic dissection, hemangioma, thromboembolism, heart failure, and cardiogenic shock.
The present invention also provides a method for diagnosing a disease associated with the occurrence of a CD44 molecule activation condition, the method comprising: administering to a subject in need thereof the nanocarrier delivery system of the first or second aspect, the drug of the fourth aspect, or the diagnostic formulation of the fifth aspect.
The protein-polymer conjugate nanocarrier delivery system of the present invention may have, but is not limited to, the following beneficial effects:
nanoparticles and nanocomposite materials have great potential for development in biomedical therapy and diagnostics. The giant amphoteric molecule formed by taking protein as a polar end group and a hydrophobic polymer as a tail has similarity with the traditional surfactant and amphiphilic block copolymer in terms of structure and performance, can realize self-assembly in solution, and has potential application prospect in a drug carrier and a nano-reactor. Compared with inorganic nanomaterials, the nanomaterials carrier based on the biological molecules has better biocompatibility, safety and metabolizability, and provides favorable conditions for the clinical transformation of the organic nanomaterials. The ProS self-assembled structure designed by the invention has the advantages of good biocompatibility, high drug loading capacity, good safety and strong stability, and is a nano drug carrier with important application value.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the IR spectrum of the human serum albumin and polymer complex in example 1.
FIG. 2 shows the transmission electron microscopy results of the HSA-PCL conjugate of example 2.
FIG. 3 shows the infrared spectra of blank HSA-PCL, HSA-PCL (R) and HSA-PCL (R) @ HA targeted nanomedicines in example 2.
FIG. 4 shows the transmission electron microscopy results of the HSA-PLLA conjugate of example 3.
FIG. 5 shows the IR spectra of HSA-PLLA, non-HA-linked nano-carrier HSA-PLLA (At) and targeting nano-carrier HSA-PLLA (At) @ SP/PEG in example 3.
FIG. 6 shows the transmission electron microscopy results of the HSA-PLLA conjugate of example 4.
FIG. 7 shows the IR spectra of HSA-PLGA (R) and HSA-PLGA (R) @ HA/Tat, which are nano-carriers without targeting ligand, in example 4.
FIG. 8 shows the IR spectra of the nano-carrier HSA-PCL (R) and the targeting nano-carrier HSA-PCL (R) @ HA/PEG without targeting ligand in example 5.
FIG. 9 shows the IR spectra of the nano-carriers HSA-PLLA (At) and the targeting nano-carriers HSA-PLLA (At) @ IM7 without the targeting ligand attached in example 6.
Fig. 10 shows the infrared spectra of the targeted drug-loaded nanocarriers HSA-PCL (At/miRNA) @ SEP in example 7.
FIG. 11 shows TEM results of HSA-PLLA (AuNP/R) @ OPN/PEG in example 8.
FIG. 12 shows HSA-PLGA (Fe 3 O 4 DXMS) @ infrared spectrum of HI44 a.
FIG. 13 shows the IR spectra of HSA-PCL (Asp/Clo) and HSA-PCL (Asp/Clo) @ Col/PEG in example 10.
FIG. 14 shows the effect of different time periods on hydrated particle size in test example 1.
Fig. 15 shows the effect of different shelf-life on encapsulation efficiency in test example 1.
Fig. 16 shows the in vitro cumulative release rate (CRP%) of the nano-delivery system in test example 1.
FIG. 17 shows the therapeutic effect of the HSA-PCL (R) @ HA, HSA-PLLA (At) @ SP/PEG, HSA-PLGA (R) @ HA/Tat, HSA-PCL (At) @ HA/PEG, HSA-PLLA (At) @ IM7, HSA-PCL (At/miRNA) @ SEP nanodelivery system of test example 2 of the present invention on carotid vulnerable plaque in model mice.
FIG. 18 shows the in vivo tracer effect of HSA-PLLA (AuNP/R) @ OPN/PEG and other CT tracer nano-delivery systems in experimental example 3 of the invention on carotid vulnerable plaque in model mice.
FIG. 19 shows a graph of the therapeutic effect of the HSA-PLLA (AuNP/R) @ OPN/PEG nano-delivery system in test example 3 of the present invention on carotid vulnerable plaque in model mice.
FIG. 20 shows the in vivo tracer effect of HSA-PLGA (Fe 3O 4/DXMS) @ HI44a and other MRI tracer nano-delivery systems in accordance with test example 4 of the invention on vulnerable carotid plaques in model mice.
FIG. 21 shows a graph of the therapeutic effect of the HSA-PLGA (Fe 3O 4/DAMS) @ HI44a nanodelivery system in test example 4 on carotid vulnerable plaque in model mice.
FIG. 22 shows a graph of the therapeutic effect of the HSA-PCL (Asp/Clo) @ Col/PEG nanodelivery system on arterial vulnerable plaque rupture in model mice in test example 5.
Detailed Description
The invention is further illustrated by the following specific examples, which are, however, to be understood only for the purpose of more detailed description and are not to be construed as limiting the invention in any way.
This section generally describes the materials used in the test of the present invention and the test method. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein. It will be apparent to those skilled in the art that in this context, the materials and methods of operation used in the present invention are well known in the art, if not specifically described.
The reagents and instrumentation used in the following examples were as follows:
reagent: HA was purchased from Zhejiang east biotechnology limited, HSA,4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (aminomercapto coupling agent, SMCC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC.HCl), and N-hydroxysulfosuccinimide was purchased from Solebao biotechnology limited. Aminated polyethylene glycol (PEG-NH) 2 Molecular weight 1000) aminated PLGA (PLGA-NH 2 Molecular weight 1000), aminated PCL (PCL-NH) 2 Molecular weight 1000), aminated PLLA (PLLA-NH) 2 Molecular weight 1000) available from Siamiliaxi Biotechnology Co., ltd., feCl 3 6-mercaptohexanol, DMSO, tannic acid, chloroform, chloroauric acid, naBH 4 Lipoic acid, dopamine hydrochloride was purchased from carbofuran reagent company. Rosuvastatin (R) atorvastatin (At), aspirin (Asp), clopidogrel (Clo), dexamethasone (DXMS) were purchased from the chinese pharmaceutical biologicals institute.
Selectin SP, transmembrane peptide (Tat), self peptide (SEP), monoclonal antibody IM7, osteopontin (OPN), PBS, ferric trichloride (FeCl 3), HI44a, collagen (Col) were purchased from Sigma-Aldrich, carbosulfan and the national pharmaceutical community beijing chemical reagent company. miRNA-33 is custom synthesized by Shanghai Biotechnology company.
Instrument:
scanning electron microscope, available from zeiss, model germany: EVO18
Laser particle sizer, commercially available from Markov intelligent laser particle sizer, model Zetasizer Nano ZS transmission electron microscope, JEOL-2100 high resolution transmission electron microscope
Infrared spectrometer: simer-Feishi technology Fourier transform near infrared spectrometer, model Antaris II FT-NIR analyzer
EXAMPLE 1 preparation of 3 human serum albumin-Polymer complexes
1.1 preparation of reduced human serum albumin (rHSA)
Into a three-necked flask, a phosphate buffer (PBS buffer (pH=8.3, 0.1M Na) having a concentration of 1mg/mL of HSA was added 2 HPO 4 /NaH 2 PO 4 1mM ethylenediamine tetraacetic acid (EDTA)), the system was deoxygenated by three cycles of vacuum-nitrogen-charging under magnetic stirring, and KBH 5-fold excess over HSA was added under nitrogen protection 4 The powder was reacted for four hours, purified by ultrafiltration tube, lyophilized to give rHSA, and stored at-20 ℃.
1.2 preparation of human serum albumin-polycaprolactone (HSA-PCL) Complex
PCL-NH realization by using dual-function coupling agent SMCC 2 Coupling with rHSA, the amino group of PCL is coupled with the mercapto group on rHSA, and the specific method is as follows: into a nitrogen-filled three-necked flask, 1mg/mL rHSA in PBS buffer (pH=6.8, 0.1M Na) 2 HPO 4 /NaH 2 PO 4 1mM EDTA), magnetic stirring, PCL-NH was added dropwise 2 And DMF solution of SMCC (PCL-NH) 2 And a molar ratio of SMCC of 1:10). The reaction was allowed to react overnight, and the reaction system was dialyzed against 1L of DMF, with the molecular weight cut-off of the dialysis bag being mw=35 000. After 24 hours, the dialysate was changed to 1 liter of deoxygenated deionized water and dialysis was continued for 24 hours. Freeze-drying to obtain HSA-PCL conjugate, freeze-drying at-20deg.C and storing.
1.3 preparation of human serum albumin-Poly L-lactic acid (HSA-PLLA) Complex
PLLA-NH Using Dual-function coupling agent SMCC 2 Couple with rHSATogether, PLLA-NH 2 The amino group on rHSA and the sulfhydryl group on rHSA are coupled by the following specific method: into a nitrogen-filled three-necked flask, 1mg/mL rHSA in PBS buffer (pH=6.8, 0.1M Na) 2 HPO 4 /NaH 2 PO 4 1mM EDTA), magnetic stirring, PLLA-NH was added dropwise 2 And DMF solution of SMCC (PLLA-NH 2 And a molar ratio of SMCC of 1:10). The reaction was allowed to react overnight, and the reaction system was dialyzed against 1L of DMF, with the molecular weight cut-off of the dialysis bag being mw=35 000. After 24 hours, the dialysate was changed to 1 liter of deoxygenated deionized water and dialysis was continued for 24 hours. Freeze-drying to obtain HSA-PLLA compound, and freeze-drying at-20deg.C for storage.
1.4 preparation of human serum albumin-polylactic acid-glycolic acid Complex (HSA-PLGA)
PLGA-NH realization by using bifunctional coupling agent SMCC 2 Coupling with rHSA, PLGA-NH 2 The amino group on rHSA and the sulfhydryl group on rHSA are coupled by the following specific method: into a nitrogen-filled three-necked flask, 1mg/mL rHSA in PBS buffer (pH=6.8, 0.1M Na) 2 HPO 4 /NaH 2 PO 4 1mM EDTA), magnetic stirring, PLGA-NH was added dropwise 2 And DMF solution of SMCC (PLGA-NH) 2 And a molar ratio of SMCC of 1:10). The reaction was allowed to react overnight, and the reaction system was dialyzed against 1L of DMF, with the molecular weight cut-off of the dialysis bag being mw=35 000. After 24 hours, the dialysate was changed to 1 liter of deoxygenated deionized water and dialysis was continued for 24 hours. Freeze-drying to obtain HSA-PLGA, and freeze-drying at-20deg.C for storage. FIG. 1 shows an infrared spectrum of a human serum albumin and polymer complex.
EXAMPLE 2 Targeted attachment of Hyaluronic Acid (HA), rosuvastatin (R) -loaded HSA-PCL conjugate (HSA-PCL) Preparation of (R) @ HA
2.1 preparation of non-drug carrying HSA-PCL conjugate
The blank HSA-PCL self-assembly body is prepared by an emulsion-solvent evaporation method, and the specific operation is as follows: 20 mg of HSA-PCL conjugate was weighed out and dissolved in 8 ml of deionized water, and the solution was placed in an ice bath. Under the action of a probe type ultrasonic generator, 4 ml of dichloromethane is injected at a constant speed by using a syringe, the ultrasonic time is 5 minutes, and the ultrasonic power is 150W. At the end of the sonication, the resulting emulsion was spin evaporated at 30℃for 30 minutes by means of a rotary evaporator, removing the organic phase at a speed of 80 rpm. And (3) after the dichloromethane is completely volatilized, obtaining an assembly solution of the HSA-PCL conjugate, and characterizing the appearance of the assembly through a transmission electron microscope. FIG. 2 shows the transmission electron microscopy results of HSA-PCL conjugates.
2.2 preparation of rosuvastatin (R) -loaded HSA-PCL conjugate HSA-PCL (HSA-PCL (R))
20 mg of HSA-PCL conjugate was weighed out and dissolved in 8 ml of deionized water, and the solution was placed in an ice bath. 5mg of rosuvastatin (R) was dissolved in 5mL of DMSO. Under the action of a probe type ultrasonic generator, the DMSO solution is injected into the probe type ultrasonic generator at a constant speed by using an injector, the ultrasonic time is 5 minutes, and the ultrasonic power is 150W. And (5) ending the ultrasonic treatment to obtain a milky white solution.
2.3 preparation of HSA-PCL conjugate (HSA-PCL (R) @ HA) to which Hyaluronic Acid (HA) is targeted and which is to be attached to rosuvastatin (R)
The HSA-PCL (R) emulsion thus obtained was added with 1mL of 10mM tannic acid solution and 1mL of 0.1M Tris-HCl buffer solution (pH 8.0), stirred for 30 minutes, followed by dropwise addition of 0.5mL of 1mM FeCl3 solution, to form a complex network structure (TA-Fe) 3+ ) Thereby reinforcing the carrier, and after 2 hours of reaction, unreacted PDA was removed by centrifugation to obtain purified HSA-PCL (R).
1g of hyaluronic acid HA (molecular weight about 40 kDa) was dissolved in ultrapure water and carboxyl groups were activated by the addition of 0.1g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.12. 0.12g N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent. After stirring the reaction at room temperature for 1 hour, the activated HA was precipitated by adding absolute ethanol. The precipitate was filtered, washed with ethanol and dried in vacuo to give activated HA. It was formulated as 0.1mg mL -1 Is dissolved in purified HSA-PCL (R) solution to effect the coupling of HA to HSA-PCL (R). Ultrafiltering, concentrating, freeze drying to remove organic phase to obtain the target recognition nano carrier HSA-PCL (R) @ HA. FIG. 3 is an infrared spectrum of blank HSA-PCL, HSA-PCL (R) and HSA-PCL (R) @ HA targeted nanomedicine.
EXAMPLE 3 HSA-doped atorvastatin (At) with both Selectin (SP) and polyethylene glycol (PEG) Preparation of PLLA conjugate (HSA-PLLA (At) @ SP/PEG)
3.1 preparation of non-drug loaded HSA-PLLA conjugate
The blank HSA-PLLA self-assembly body is prepared by an emulsion-solvent evaporation method and comprises the following specific operations: 20 mg of HSA-PCL conjugate was weighed out and dissolved in 8 ml of deionized water, and the solution was placed in an ice bath. Under the action of a probe type ultrasonic generator, 4 ml of dichloromethane is injected at a constant speed by using a syringe, the ultrasonic time is 5 minutes, and the ultrasonic power is 150W. At the end of the sonication, the resulting emulsion was spin evaporated at 30℃for 30 minutes by means of a rotary evaporator, removing the organic phase at a speed of 80 rpm. And after the dichloromethane is completely volatilized, obtaining an assembly solution of the HSA-PLLA conjugate, and characterizing the appearance of the assembly through a transmission electron microscope. FIG. 4 shows the transmission electron microscopy results of the HSA-PLLA conjugate.
3.2 preparation of atorvastatin (At) -loaded HSA-PLLA conjugate HSA-PLLA (HSA-PLLA (At))
20 mg of HSA-PLLA conjugate was weighed out and dissolved in 8 ml of deionized water, and the solution was placed in an ice bath. 5mg of atorvastatin (At) was dissolved in 5mL of DMSO. Under the action of a probe type ultrasonic generator, the DMSO solution is injected into the probe type ultrasonic generator at a constant speed by using an injector, the ultrasonic time is 5 minutes, and the ultrasonic power is 150W. And (5) ending the ultrasonic treatment to obtain a milky white solution.
3.3 preparation of HSA-PLLA conjugate (HSA-PLLA (At) @ SP/PEG) with simultaneous attachment of Selectin (SP) and polyethylene glycol (PEG), atorvastatin (At) supported
Adding 10mg of dopamine hydrochloride into the HSA-PLLA (At) emulsion, adjusting the pH to 9.5, forming a Polydopamine (PDA) layer on the surface of the HSA-PLLA (At) through dopamine self-polymerization reaction, reinforcing the carrier, reacting for 1 hour, and adding 2mg of PEG-NH 2 The reaction was continued for 1 hour. Centrifugation to remove unreacted PDA and PEG-NH 2 Purified HSA-PLLA (At) @ PEG was obtained.
1mg of selectin SP was dissolved well in PBS buffer, and 0.5mg ED was addedHCl and 0.5mg of sulfo-NHS coupling agent activate carboxyl. After stirring the reaction at room temperature for 1 hour, the mixture was prepared to 1mg mL -1 SP aqueous solution of (c). And adding the purified HSA-PLLA (At) @ PEG solution to realize coupling of SP on the HSA-PLLA (At) @ PEG, performing ultrafiltration concentration, and freeze-drying to remove an organic phase to obtain the targeted recognition nano carrier HSA-PLLA (At) @ SP/PEG. FIG. 5 is an infrared spectrum of the non-HA-linked nanocarriers HSA-PLLA (At) and the targeting nanocarriers HSA-PLLA (At) @ SP/PEG.
EXAMPLE 4 Simultaneous modification of the transmembrane peptide (Tat) and Hyaluronic Acid (HA), rosuvastatin (R) -loaded HSA-PLGA Preparation of conjugate (HSA-PLGA (R) @ HA/Tat)
4.1 preparation of non-drug loaded HSA-PLGA conjugate
The blank HSA-PLGA self-assembly body is prepared by an emulsion-solvent evaporation method, and the specific operation is as follows: 20 mg of HSA-PCL conjugate was weighed out and dissolved in 8 ml of deionized water, and the solution was placed in an ice bath. Under the action of a probe type ultrasonic generator, 4 ml of dichloromethane is injected at a constant speed by using a syringe, the ultrasonic time is 5 minutes, and the ultrasonic power is 150W. At the end of the sonication, the resulting emulsion was spin evaporated at 30℃for 30 minutes by means of a rotary evaporator, removing the organic phase at a speed of 80 rpm. And after the dichloromethane is completely volatilized, obtaining an assembly solution of the HSA-PLGA conjugate, and characterizing the appearance of the assembly through a transmission electron microscope. FIG. 6 shows the transmission electron microscopy results of HSA-PLLA conjugate.
4.2 preparation of rosuvastatin (R) -loaded HSA-PLGA conjugate HSA-PLGA (HSA-PLGA (R))
20 mg of HSA-PLGA conjugate was weighed out and dissolved in 8 ml of deionized water, and the solution was placed in an ice bath. 5mg of rosuvastatin (R) was dissolved in 5mL of DMSO. Under the action of a probe type ultrasonic generator, the DMSO solution is injected into the probe type ultrasonic generator at a constant speed by using an injector, the ultrasonic time is 5 minutes, and the ultrasonic power is 150W. And (5) ending the ultrasonic treatment to obtain a milky white solution.
4.3 preparation of HSA-PLGA conjugate (HSA-PLGA (R) @ HA/Tat) with simultaneous modification of the transmembrane peptide (Tat) and Hyaluronic Acid (HA), and rosuvastatin (R) loading
The HSA-PLGA (R) emulsion obtained above was added with 10mg of dopamine hydrochloride, the pH was adjusted to 9.5, and a Polydopamine (PDA) layer was formed on the surface of HSA-PLGA (R) by dopamine self-polymerization, thereby reinforcing the carrier, and after 1 hour of reaction, 2mg of Tat was added and the reaction was continued for 1 hour. Unreacted PDA and Tat were removed by centrifugation to give purified HSA-PLGA (R) @ Tat.
1g of hyaluronic acid HA (molecular weight about 10 kDa) was dissolved in ultrapure water and carboxyl groups were activated by adding 0.1g of EDC. HCl and 0.12g of sulfo-NHS coupling agent. After stirring the reaction at room temperature for 1 hour, the activated HA was precipitated by adding absolute ethanol. The precipitate was filtered, washed with ethanol and dried in vacuo to give activated HA. It was configured to 1mg mL -1 Is a solution of (a) and (b). Absorbing 1mL of activated HA solution, dissolving in purified HSA-PLGA (R) @ Tat solution to realize the coupling of HA on HSA-PLGA (R) @ Tat, ultrafiltering, concentrating, freeze-drying to remove organic phase, and obtaining the targeted recognition nano carrier HSA-PLGA (R) @ HA/Tat. FIG. 7 is an infrared spectrum of HSA-PLGA (R) and HSA-PLGA (R) @ HA/Tat, a nanocarrier, to which no targeting ligand is attached.
EXAMPLE 5 Simultaneous modification of Hyaluronic Acid (HA) and PEG, atorvastatin (At) -loaded HSA-PCL conjugate Preparation of (HSA-PCL (At) @ HA/PEG)
5.1 preparation of atorvastatin (At) -loaded HSA-PCL conjugate HSA-PCL (HSA-PCL (At))
20 mg of HSA-PCL conjugate was weighed out and dissolved in 8 ml of deionized water, and the solution was placed in an ice bath. 5mg of atorvastatin (At) was dissolved in 5mL of DMSO. Under the action of a probe type ultrasonic generator, the DMSO solution is injected into the probe type ultrasonic generator at a constant speed by using an injector, the ultrasonic time is 5 minutes, and the ultrasonic power is 150W. And (5) ending the ultrasonic treatment to obtain a milky white solution.
Adding 10mg of dopamine hydrochloride into the HSA-PCL (At) emulsion obtained in the above, regulating the pH to 9.5, forming a Polydopamine (PDA) layer on the surface of the HSA-PCL (At) through dopamine self-polymerization reaction, reinforcing the carrier, reacting for 1 hour, and adding 5mg of PEG-NH 2 And the reaction was continued for 2 hours, and unreacted PDA and PEG-NH were removed by centrifugation 2 Purified HSA-PCL (At) @ PEG was obtained.
1g of hyaluronic acid HA(molecular weight of about 40 kDa) was dissolved in ultrapure water, and carboxyl groups were activated by adding 0.1g of EDC.HCl and 0.12g of sulfo-NHS coupling agent. After stirring the reaction at room temperature for 1 hour, the activated HA was precipitated by adding absolute ethanol. The precipitate was filtered, washed with ethanol and dried in vacuo to give activated HA. It was formulated as 0.1mg mL -1 0.2mL of the solution was taken up in the purified HSA-PCL (At) solution to effect the coupling of HA to HSA-PCL (At) @ PEG. Ultrafiltering, concentrating, freeze drying to remove organic phase to obtain target recognition nano carrier HSA-PCL (At) @ HA/PEG.
FIG. 8 is an infrared spectrum of the nano-carrier HSA-PCL (At) and the targeting nano-carrier HSA-PCL (At) @ HA/PEG without the targeting ligand attached.
EXAMPLE 6 modification of the HSA-PLLA conjugate of IM7 mab loaded with atorvastatin (At) (HSA-PLLA (At) @) Preparation of IM 7)
6.1 preparation of atorvastatin (At) -loaded HSA-PLLA conjugate HSA-PLLA (HSA-PLLA (At))
20 mg of HSA-PLGA conjugate was weighed out and dissolved in 8 ml of deionized water, and the solution was placed in an ice bath. Under the action of a probe type ultrasonic generator, 4mL 1mg mL is injected at a constant speed by a syringe -1 The ultrasound time was 5 minutes and the ultrasound power was 150W. And (5) ending the ultrasonic treatment to obtain a milky white solution.
The HSA-PLLA (At) emulsion thus obtained was added with 1mL of 10mM tannic acid solution and 1mL of 0.1M Tris-HCl buffer solution (pH 8.0), stirred for 30 minutes, and then 0.5mL of 1mM FeCl was added dropwise 3 Solution by forming a complex network structure (TA-Fe) on the surface of HSA-PLLA (At) 3+ ) Thereby reinforcing the carrier, and after 2 hours of reaction, unreacted molecules were removed by centrifugation to obtain purified HSA-PCL (R).
1mg of IM7 was dissolved well in PBS buffer and carboxyl groups were activated by the addition of 0.1g EDC.HCl and 0.12g sulfo-NHS coupling agent. After stirring at room temperature for 1 hour, purification by ultrafiltration was carried out to prepare 1mg mL -1 Is added into purified HSA-PLLA (At) solution drop by drop to realize the coupling of IM7 on the HSA-PLLA (At) to obtain the targeted recognition nano carrier HSA-PLLA (At) @ IM7. FIG. 9 shows the IR spectrum of the targeting nanocarrier HSA-PLLA (At) @ IM7. Ultrafiltering, concentrating, freeze drying to remove organic phase to obtain target recognition nano carrier HSA-PLLA (At) @ IM7. FIG. 9 is an infrared spectrum of the nano-carrier HSA-PLLA (At) and the targeting nano-carrier HSA-PLLA (At) @ IM7 without the targeting ligand attached.
EXAMPLE 7 modification of self-peptide (SEP) with simultaneous loading of microRNA (miRNA-33 a) and atorvastatin (At) HSA Preparation of PCL complex HSA-PCL (At/miRNA) @ SEP
7.1 preparation of atorvastatin (At) -loaded HSA-PCL conjugate HSA-PCL (HSA-PCL (At))
20 mg of HSA-PCL conjugate was weighed out and dissolved in 8 ml of deionized water, and the solution was placed in an ice bath. Under the action of a probe type ultrasonic generator, 4mL 1mg mL is injected at a constant speed by a syringe -1 The ultrasonic time of DMSO solution of atorvastatin was 5 minutes and the ultrasonic power was 150W. And (5) ending the ultrasonic treatment to obtain a milky white solution.
Adding 10mg of dopamine hydrochloride and 1mg of miRNA-33a into the HSA-PCL (At) emulsion, adjusting the pH to 9.5, simultaneously adding a poly-dopamine (PDA) layer which is formed by carrying miRNA-33a on the surface of HSA-PLLA (At) through dopamine self-polymerization reaction, thereby achieving the purpose of reinforcing a carrier to load miRNA-33a, centrifuging to remove unreacted PDA and miRNA-33a after 2 hours of reaction, and obtaining purified HSA-PCL (At/miRNA).
7.2 preparation of HSA-PCL (At/miRNA) Complex conjugated to self peptide (SEP)
1mg of self peptide (SEP) was dissolved well in 1ml of buffer solution of LPBS, and carboxyl groups were activated by adding 0.5mg of EDC.HCl and 0.5mg of sulfo-NHS coupling agent. After reacting for 1h At room temperature, 1mL of the SEP-NHS solution is dissolved in purified HSA-PCL (At/miRNA) solution to realize the coupling of SEP on the HSA-PCL (At/miRNA), ultrafiltration purification is carried out, and the purified target recognition nano carrier HSA-PCL (At/miRNA) @ SEP can be obtained after freeze drying. FIG. 10 is an infrared spectrum of a targeted drug-loaded nanocarrier HSA-PCL (At/miRNA) @ SEP.
Example 8 simultaneous coupling of polyethylene glycol (PEG), osteopontin (OPN) and gold nanoparticle (AuNP) Reshu Preparation of HSA-PLLA Complex of vastatin (R) (HSA-PLLA (AuNP/R) @ OPN/PEG)
8.1 Synthesis of gold nanoparticles:
100mL of 1mM aqueous chloroauric acid solution was prepared, and 1mL of 0.1M NaBH was added with stirring at room temperature 4 Thus obtaining gold nanoparticles. The pH of the solution was adjusted to alkaline, 5mL of 1mM 6-mercaptohexanol solution was added, stirred at room temperature for 24 hours, and MHO-AuNPs were purified using a 3K ultrafiltration tube to obtain hydrophobic gold nanoparticles (MHO-AuNPs).
8.2 preparation of gold nanoparticle (AuNP) rosuvastatin (R) -Supported HSA-PLLA Complex nanocarriers
20 mg of HSA-PLLA conjugate was weighed out and dissolved in 8 ml of deionized water, and the solution was placed in an ice bath. Under the action of a probe type ultrasonic generator, 4mL 1mg mL is injected at a constant speed by a syringe -1 Rosuvastatin and MHO-AuNPs in DMSO, the sonication time was 5 minutes and the sonication power was 150W. And (5) ending the ultrasonic treatment to obtain a milky white solution.
The HSA-PLLA (AuNP/R) emulsion obtained above was added with 10mg of dopamine hydrochloride and the pH was adjusted to 9.5, and after 1 hour of reaction, 4mg of PEG-NH was further added 2 After continuing the reaction for 1 hour, unreacted PDA and PEG-NH were removed by centrifugation 2 Purified HSA-PLLA (AuNP/R) @ PEG was obtained.
8.3 preparation of a delivery System for simultaneously coupling Osteopontin (OPN) and PEG, gold nanoparticle-loaded (AuNP) rosuvastatin (R) HSA-PLLA Complex HSA-PLLA (AuNP/R) @ OPN/PEG
1mg of Osteopontin (OPN) was well dissolved in PBS buffer, and carboxyl groups were activated by the addition of 0.5mg EDC.HCl and 0.5mg sulfo-NHS coupling agent. After stirring the reaction at room temperature for 1 hour. It was configured to 1mg mL -1 And (3) absorbing 1mL of the solution to be dissolved in purified HSA-PLLA (AuNP/R) @ PEG solution to realize the coupling of OPN on the surface of the solution, thereby obtaining the targeted recognition nano carrier HSA-PLLA (AuNP/R) @ OPN/PEG. FIG. 11 shows TEM results of HSA-PLLA (AuNP/R) @ OPN/PEG.
8.4 preparation method of HSA-PLLA@OPN/PEG
20 mg of HSA-PLLA conjugate was weighed and dissolved in 8 ml of deionized water, and the solution was setIn an ice bath. Under the action of a probe type ultrasonic generator, 4mL 1mg mL is injected at a constant speed by a syringe -1 The chloroform solution was sonicated for 5 minutes with an ultrasonic power of 150W. And (5) ending the ultrasonic treatment to obtain a milky white solution.
Adding 10mg of dopamine hydrochloride into the HSA-PLLA emulsion obtained in the above way, regulating the pH value to 9.5, reacting for 1h, and continuously adding 4mg of PEG-NH 2 After the reaction was continued for 1 hour. Centrifugation to remove unreacted PDA and PEG-NH 2 Purified HSA-PLLA@PEG was obtained.
1mg of Osteopontin (OPN) was well dissolved in PBS buffer, and carboxyl groups were activated by the addition of 0.5mg EDC.HCl and 0.5mg sulfo-NHS coupling agent. After stirring the reaction at room temperature for 1 hour. It was configured to 1mg mL -1 Absorbing 1mL of the solution and dissolving in the purified HSA-PLLA@PEG solution to realize the coupling of OPN on the surface of the solution, thereby obtaining the targeted recognition nano carrier HSA-PLLA@OPN/PEG.
Example 9 coupling of monoclonal antibody HI44a, drug-loaded Dexamethasone (DXMS) paramagnetic iron oxide 3 4 3 4 Preparation of HSA-PLGA complexes (HSA-PLGA (FeO/DXMS) @ HI44 a) of (FeONPs)
9.1 paramagnetic iron oxide (Fe 3 O 4 NPs) are prepared
100mL of 10mM ferric trichloride (FeCl) was prepared 3 ) Adding 10mL of newly prepared 0.1M ammonia water into the aqueous solution under the condition of intense stirring to prepare magnetic iron nano particles (Fe 3 O 4 NPs), adding 10mg mL-1 oleic acid ethanol solution, reacting for 4 hours, purifying the material by external magnetic field, and dispersing the concentrated solution in pure water to obtain 10mM hydrophobic Fe 3 O 4 NPs。
9.2 monoclonal antibody HI44a modified drug-loaded Dexamethasone (DXMS) paramagnetic iron oxide (Fe) 3 O 4 NPs) HSA-PLGA complexes
20 mg of HSA-PLGA conjugate was weighed out and dissolved in 8 ml of deionized water, and the solution was placed in an ice bath. Under the action of a probe type ultrasonic generator, 4mL 1mg mL is injected at a constant speed by a syringe -1 Dexamethasone (DAMS) andparamagnetic iron oxide (Fe) 3 O 4 NPs) was sonicated for 5 minutes at an ultrasonic power of 150W. And (5) ending the ultrasonic treatment to obtain a milky white solution.
The HSA-PLGA (Fe) 3 O 4 DXMS) emulsion, adding 10mg dopamine hydrochloride and adjusting pH to 9.5, reacting for 2 hours, centrifuging to remove unreacted PDA, and lyophilizing the sample to obtain purified HSA-PLGA (Fe) 3 O 4 /DXMS)。
FIG. 12 shows HSA-PLGA (Fe 3 O 4 /DXMS). 1mg of HI44a was dissolved in ultrapure water, and carboxyl groups were activated by adding 1.0mg of EDC.HCl and 0.5mg of sulfo-NHS coupling agent. After stirring the reaction at room temperature for 1 hour, activated HI44a was obtained by ultrafiltration and centrifugation. 1.0mL of HI44a solution was pipetted into purified HSA-PLGA (Fe 3 O 4 DXMS) solution to achieve HI44a in HSA-PLGA (Fe 3 O 4 Coupling on/DXMS) to obtain the target recognition nano carrier HSA-PLGA (Fe) 3 O 4 The IR spectrum of @ DAMS) @ HI44a is shown in FIG. 12.
9.3 preparation of HSA-PLGA coupled HI44a (HSA-PLGA@HI44a)
20 mg of HSA-PLGA conjugate was weighed out and dissolved in 8 ml of deionized water, and the solution was placed in an ice bath. Under the action of a probe type ultrasonic generator, 4mL of chloroform solution is injected at a constant speed by using a syringe, the ultrasonic time is 5 minutes, and the ultrasonic power is 150W. And (5) ending the ultrasonic treatment to obtain a milky white solution. The HSA-PLGA emulsion obtained above was added with 10mg of dopamine hydrochloride and the pH was adjusted to 9.5, and after 2 hours of reaction, unreacted PDA was removed by centrifugation, and the sample was lyophilized to obtain purified HSA-PLGANP.
1mg of HI44a was dissolved in ultrapure water, and carboxyl groups were activated by adding 1.0mg of EDC.HCl and 0.5mg of sulfo-NHS coupling agent. After stirring the reaction at room temperature for 1 hour, activated HI44a was obtained by ultrafiltration and centrifugation. 1.0mL of HI44a solution is absorbed and dissolved in the purified HSA-PLGA solution, and the coupling of HI44a on the HSA-PLGA is realized, so that the targeting recognition nano carrier HSA-PLGA@HI44a is obtained.
EXAMPLE 10 modification of collagen (Col) with Simultaneous Asp-Supported Asp, clopidogrel (Clo) HSA-PCL Preparation of Complex (HSA-PCL (Asp/Clo) @ Col/PEG)
20mg of HSA-PCL conjugate was weighed out and dissolved in 8mL of deionized water, and the solution was placed in an ice bath. Under the action of a probe type ultrasonic generator, 4mL 1mg mL is injected at a constant speed by a syringe -1 Asp (Asp) and clopidogrel (Clo) in chloroform, the ultrasonic power was 150W and the ultrasonic time was 5 minutes. And (5) ending the ultrasonic treatment to obtain a milky white solution. 10mg of dopamine hydrochloride was added to the HSA-PCL (Asp/Clo) emulsion obtained above and the pH was adjusted to 9.5, and after 1 hour of reaction, an amino-modified polyethylene glycol (PEG-NH) having a molecular weight of 1kD was added dropwise 2 ) Aqueous solution (1 mL,2mg mL) -1 ) And the reaction was continued for 1 hour, the unreacted drug and PDA were removed by centrifugation, and the sample was lyophilized to give purified HSA-PCL (Asp/Clo) @ PEG.
10mg of col was fully dissolved in ultrapure water and carboxyl groups were activated by adding 3mg of EDC. HCl and 3mg of sulfo-NHS coupling agent. After stirring the reaction at room temperature for 1 hour, activated Col was obtained by ultrafiltration and centrifugation. 1.0mL of Col solution is absorbed and dissolved in purified HSA-PCL (Asp/Clo) @ PEG solution, and the coupling of Col on HSA-PCL (Asp/Clo) @ PEG is realized, so that the targeting recognition nano carrier HSA-PCL (Asp/Clo) @ Col/PEG is obtained. FIG. 13 is an infrared spectrum of HSA-PCL (Asp/Clo) @ PEG and HSA-PCL (Asp/Clo) @ Col/PEG.
Experimental example 1 investigation of the properties of the nano-delivery system of the present invention
In this example, taking the therapeutic agent-loaded nano-delivery systems prepared in examples 2 to 10 as an example, the delivery system of the present invention has stable and controllable properties, thereby being suitable for diagnosis, prevention and treatment of vulnerable plaque or diseases associated with vulnerable plaque.
1. Drug concentration assay:
the carrier drugs rosuvastatin, atorvastatin, dexamethasone, aspirin, clopidogrel, fluorodeoxyglucose have very strong ultraviolet absorption properties, so that the content thereof can be determined by using the ultraviolet absorption properties of rosuvastatin, atorvastatin, dexamethasone, aspirin, clopidogrel, fluorodeoxyglucose by using HPLC-UV method (using Waters2487, wate company (Waters Corporation), usa). Standard quantitative equations were established for the peak area (Y) of HPLC chromatographic peaks with different concentrations of rosuvastatin, atorvastatin, dexamethasone, aspirin, clopidogrel, concentration of fluorodeoxyglucose solution (X).
2. Determination of the particle size of the hydrate:
the nano-carrier of the delivery system of the present invention, HSA-PCL (R) @ HA, HSA-PLLA (At) @ SP/PEG, HSA-PLGA (R) @ HA/Tat, HSA-PCL (At) @ HA/PEG, HSA-PLLA (At) @ IM7, HSA-PCL (At/miRNA) @ SEP, HSA-PLLA (AuNP/R) @ OPN/PEG, HSA-PLGA (Fe 3O 4/DAMS) @ HI44a, HSA-PCL (Asp/Clo) @ Col/PEG hydrated particle sizes were all determined by a laser particle sizer (BI-Zeta Plus/90Plus, bricke Hai company (Brookhaven Instruments Corporation), U.S.S.), the specific results are shown in Table 1.
3. Determination of drug loading:
taking a certain amount of nano-carrier, HSA-PCL (R) @ HA, HSA-PLLA (At) @ SP/PEG, HSA-PLGA (R) @ HA/Tat, HSA-PCL (At) @ HA/PEG, HSA-PLLA (At) @ IM7, HSA-PCL (At/miRNA) @ SEP, HSA-PLLA (AuNP/R) @ OPN/PEG, HSA-PLGA (Fe 3O 4/DXMS) @ HI44a, HSA-PCL (Asp/Clo) @ Col/PEG, adding excessive methanol/formic acid solution with pH of 2.0, heating in a water bath for 60 degrees for 2 hours to accelerate the release of the hydrogel nano-carrier, and further adopting an ultrasonic method to accelerate the release of the drug from the dendritic nano-carrier. The drug content in the obtained liquid was measured by HPLC (Waters 2487, watter company (Waters Corporation), usa) and the encapsulation efficiency was calculated by the following formula. The correlation results are shown in Table 1
Encapsulation efficiency (%) = (M-coated drug amount/M-added drug amount) ×100% … … … … … table 1 list of various properties
Figure SMS_1
And (3) injection: the above data are all expressed as "mean + standard deviation" of the results of 5 replicates.
4. Long-term stability investigation
The nano-delivery system of the present invention, HSA-PCL (R) @ HA, HSA-PLLA (At) @ SP/PEG, HSA-PLGA (R) @ HA/Tat, HSA-PCL (At) @ HA/PEG, HSA-PLLA (At) @ IM7, HSA-PCL (At/miRNA) @ SEP, HSA-PLLA (AuNP/R) @ OPN/PEG, HSA-PLGA (Fe 3O 4/DXMS) @ HI44a, HSA-PCL (Asp/Clo) @ Col/PEG, was stored At 4℃and sampled At various time points and the variation in hydrated particle size was detected by a laser particle sizer. FIG. 14 is a graph showing the effect of different time periods on hydrated particle size.
5. Long-term stability investigation
The nano delivery system of the present invention was prepared by storing HSA-PCL (R) @ HA, HSA-PLLA (At) @ SP/PEG, HSA-PLGA (R) @ HA/Tat, HSA-PCL (At) @ HA/PEG, HSA-PLLA (At) @ IM7, HSA-PCL (At/miRNA) @ SEP, HSA-PLLA (AuNP/R) @ OPN/PEG, HSA-PLGA (Fe 3O 4/DXMS) @ 44a, HSA-PCL (Asp/Clo) @ Col/PEG At 4℃at various time points, and removing the free drug by ultrafiltration to examine the change in drug loading.
Fig. 15 is a graph showing the effect of different shelf life on encapsulation efficiency.
6. In vitro drug release performance study
2mL of the nano delivery system HSA-PCL (R) @ HA, HSA-PLLA (At) @ SP/PEG, HSA-PLGA (R) @ HA/Tat, HSA-PCL (At) @ HA/PEG, HSA-PLLA (At) @ IM7, HSA-PCL (At/miRNA) @ SEP, HSA-PLLA (AuNP/R) @ OPN/PEG, HSA-PLGA (Fe 3O 4/DAMS) @ HI44a, HSA-PCL (Asp/Clo) @ Col/PEG of the present invention was placed in a dialysis bag for sealing. The dialysis bag was then placed in 50mL of release medium (PBS solution, ph=7.4) and incubated for 120h at 37 ℃. At various time points 2mL of release solution was taken and the same volume of PBS solution was replenished. The drug content in the release liquid was measured by HPLC (Waters 2487, waters (Waters Corporation), usa) and the cumulative release rate of the drug was calculated by equation 2.
Figure SMS_2
The meaning of each parameter in formula 3 is as follows:
CRP: cumulative drug release rate
V e : displacement volume of release liquid, here V e Is 2mL
V 0 : the volume of release liquid in the release system, here V 0 50mL of
C i : drug concentration in release solution in μg/mL at the ith displacement sampling
M Medicament : total mass of drug in delivery system in μg
n: number of times of displacing the release liquid
Cn: drug concentration in the delivery system measured after the n-th displacement of the delivery fluid.
Fig. 16 is an in vitro cumulative release rate (CRP%) of the nano-delivery system.
Experimental example 2 HSA-PCL (R) @ HA, HSA-PLLA (At) @ SP/PEG, HSA-PLGA (R) @ HA/Tat of the present invention, HSA-PCL (At) @ HA/PEG, HSA-PLLA (At) @ IM7, HSA-PCL (At/miRNA) @ SEP nano drug delivery system In vivo experiments on the effects of vulnerable plaques in the pulse
Hyaluronic Acid (HA), selectin (SP) and IM7 are ligands of CD44, can play a role in targeting vulnerable plaques, rosuvastatin (R) and atorvastatin (At) have a plaque reversing effect, self peptide (SEP) can increase local penetration and aggregation of drugs, and transmembrane peptide (Tat) can increase local penetration and aggregation of drugs, PEG is modified on the surface of a carrier, so that the effects of long circulation can be achieved, and the half life of the drugs can be prolonged. miRNA-33a can increase cholesterol efflux. The purpose of this example is to demonstrate the in vivo therapeutic effect of the HSA-PCL (R) @ HA, HSA-PLLA (At) @ SP/PEG, HSA-PLGA (R) @ HA/Tat, HSA-PCL (At) @ HA/PEG, HSA-PLLA (At) @ IM7, HSA-PCL (At/miRNA) @ SEP vector delivery system of the present invention on arterial vulnerable plaque.
The experimental method comprises the following steps:
(1) Physiological saline solutions of free rosuvastatin and atorvastatin were formulated and the therapeutic agent-loaded amphiphilic protein-polymer conjugate delivery systems were prepared using the methods described in examples 1-7 above.
(2) Establishment of ApoE-/-mouse arterial vulnerable plaque model:
SPF-grade ApoE-/-mice (42, 5-6 weeks old, body weight 20.+ -. 1 g) were used as experimental animals. After 4 weeks of feeding mice with an adaptive high fat diet (10% fat (w/w), 2% cholesterol (w/w), 0.5% sodium cholate (w/w) and the remainder of the normal feed for the mice), the mice were anesthetized by intraperitoneal injection with 1% sodium pentobarbital (preparation method comprising adding 1mg sodium pentobarbital to 100ml of physiological saline) at a dose of 40 mg/kg. The mice were then fixed to the surgical plate in a supine position, sterilized with 75% (v/v) alcohol centered on the neck, and the neck skin was cut longitudinally, the cervical prostate was blunt-separated, and the pulsatile left common carotid artery was visible on the left side of the trachea. The common carotid artery was carefully separated to the bifurcation, a silicone tube sleeve of 2.5mm length and 0.3mm inside diameter was placed around the left common carotid artery, and both the proximal and distal sections of the sleeve were secured with a thin wire narrowing. Local constriction causes turbulence in the proximal blood flow, increased shear forces, and resulting damage to the intima of the vessel. The carotid artery was repositioned and the anterior cervical skin was sutured intermittently. All manipulations were performed under a 10-fold stereoscopic microscope. After the operation, the mice are recovered to the cage after the recovery, the ambient temperature is maintained at 20-25 ℃, and the lamplight is kept on and off for 12 hours. Lipopolysaccharide (LPS) (1 mg/kg, sigma, USA in 0.2ml phosphate buffered saline) was injected intraperitoneally, 2 times per week, for 10 weeks, to induce chronic inflammation. Mice were placed in 50ml syringes (reserved for adequate ventilation holes) 8 weeks post-surgery to create restrictive mental stress, 6 hours/day, 5 days per week for a total of 6 weeks. The mouse atherosclerosis vulnerable plaque model is molded after 14 weeks of operation.
(3) Grouping and treating experimental animals:
experimental animals were randomly divided into the following groups of 6 animals each:
vulnerable plaque model control group: the animals of this group were not subjected to any therapeutic treatment;
rosuvastatin intravenous group: intravenous administration at a dose of 0.66mg rosuvastatin/kg body weight;
atorvastatin intravenous group: intravenous administration at a dose of 1.2mg atorvastatin/kg body weight;
HSA-PCL (R) @ HA group: intravenous administration at a dose of 0.66mg rosuvastatin/kg body weight;
HSA-PLLA (At) @ SP/PEG group: intravenous administration at a dose of 1.2mg atorvastatin/kg body weight;
HSA-PLGA (R) @ HA/Tat group: intravenous administration at a dose of 0.66mg rosuvastatin/kg body weight;
HSA-PCL (At) @ HA/PEG group: intravenous administration at a dose of 1.2mg atorvastatin/kg body weight;
HSA-PLLA (At) @ IM7 group: intravenous administration at a dose of 1.2mg atorvastatin/kg body weight;
HSA-PCL (At/miRNA) @ SEP group: intravenous administration was performed at a dose of 1.2mg atorvastatin/kg body weight.
Treatment in the treatment group was performed 1 time every other day for 5 times in addition to the vulnerable plaque model control group. For each group of animals, carotid MRI scans were performed before and after treatment to detect plaque and luminal area and calculate percent plaque progression.
Percent plaque progression = (plaque area after treatment-plaque area before treatment)/lumen area.
Experimental results:
FIG. 17 shows the in vivo therapeutic effect of the HSA-PCL (R) @ HA, HSA-PLLA (At) @ SP/PEG, HSA-PLGA (R) @ HA/Tat, HSA-PCL (At) @ HA/PEG, HSA-PLLA (At) @ IM7, HSA-PCL (At/miRNA) @ SEP vector delivery system of the present invention on arterial vulnerable plaque. As shown, during the course of high fat diet feeding (10 days), atherosclerosis in the control group (without any treatment) progressed by 26.9%; treatment with rosuvastatin can delay plaque progression, but also 25.8%; intravenous atorvastatin also delayed plaque progression but also progressed by 26.7%; the targeted nano drug-loaded treatment obviously suppresses plaque progression, even the plaque volume is reversed and resolved, the HSA-PCL (R) @ HA group eliminates plaque by 10.7%, the HSA-PLLA (At) @ SP/PEG group eliminates plaque by 11.3%, the HSA-PLGA (R) @ HA/Tat group eliminates plaque by 12.5%, the HSA-PCL (At) @ HA/PEG group eliminates plaque by 10.2%, the HSA-PLLA (At) @ IM7 group eliminates plaque by 7.1%, and the HSA-PCL (At/miRNA) @ SEP group eliminates plaque by 9.3%.
In summary, neither free rosuvastatin nor atorvastatin exhibited an effect of reversing vulnerable plaque in mice. However, when statin is loaded in the nano-delivery system disclosed by the invention, the treatment effect of the statin on vulnerable plaque is remarkably improved, the treatment effect of plaque reduction is achieved, and the nano-system effect with functional modification is better.
Test example 3 Effect of the HSA-PLLA (AuNP/R) @ OPN/PEG delivery System of the invention on vulnerable plaque in arteries In vivo experiment (CT tracing and therapeutic double-function)
Osteopontin (OPN) is a ligand of CD44, and can play a role in targeting vulnerable plaques, rosuvastatin (R) has a plaque reversing effect, and nanogold (AuNP) is a CT tracer. The aim of this example is to verify the in vivo tracer and therapeutic effect of the CT tracer and rosuvastatin loaded nano delivery system of the present invention on vulnerable arterial plaque.
(1) A physiological saline solution of free atorvastatin was formulated and an amphiphilic protein-polymer conjugate nano-delivery system loaded with a CT tracer and therapeutic agent was prepared using the method described in example 8 above.
(2) The method for establishing the ApoE-/-mouse arterial vulnerable plaque model is the same as that of test example 2.
(3) Vulnerable plaque tracking in experimental animals:
experimental animals were randomly divided into the following groups of 6 animals each:
free group of nano gold particles: the dosage of the nano gold is 0.1mg/kg body weight;
HSA-PLLA (AuNP/R) @ OPN/PEG group: the dosage of the nano gold is 0.1mg/kg body weight;
HSA-PLLA@OPN/PEG group: the HSA content was determined for the HSA-PLLA (AuNP/R) @ OPN/PEG group drug, and the same concentration of HSA-PLLA @ OPN/PEG was used with the HSA concentration as the standard.
The experimental groups were injected with the corresponding tracer through the tail vein, and CT imaging was performed before and 2 hours after administration, and the identification of atherosclerosis vulnerable plaque was observed for each group of animals.
(4) Grouping and treating experimental animals:
experimental animals were randomly divided into the following groups of 6 animals each:
vulnerable plaque model control group: the animals of this group were not subjected to any therapeutic treatment;
rosuvastatin gavage group: gastric lavage treatment was performed at a dose of 10mg rosuvastatin/kg body weight;
rosuvastatin intravenous group: intravenous administration at a dose of 0.67mg rosuvastatin/kg body weight;
HSA-PLLA (AuNP/R) @ OPN/PEG group: intravenous administration was performed at a dose of 0.67mg rosuvastatin/kg body weight.
Treatment in the treatment group was performed 1 time every other day for 5 times in addition to the vulnerable plaque model control group. For each group of animals, carotid MRI scans were performed before and after treatment to detect plaque and luminal area and calculate percent plaque progression.
Percent plaque progression = (plaque area after treatment-plaque area before treatment)/lumen area.
Experimental results:
figure 18 demonstrates the in vivo tracer effect of the tracer-loaded amphiphilic protein-polymer conjugate delivery system of the invention on vulnerable arterial plaque. As shown, the free nano gold particles exhibit a certain tracing effect on vulnerable arterial plaque in mice. When the nanogold is formulated in a targeted amphiphilic protein-polymer conjugate delivery system, the tracer effect on vulnerable plaques is very significantly improved compared to free nanogold particles. In conclusion, compared with free nano gold particles, the amphiphilic protein-polymer conjugate delivery system with the surface modified with the targeting ligand can remarkably improve the recognition effect of nano gold on vulnerable arterial plaque and generate better tracing effect.
FIG. 19 shows the in vivo therapeutic effect of the HSA-PLLA (AuNP/R) @ OPN/PEG system of the present invention on arterial vulnerable plaque. As shown, during the course of high fat diet feeding (10 days), atherosclerosis in the control group (without any treatment) progressed by 28%; rosuvastatin gastric lavage treatment can delay plaque progression, but also progress by 27.1%; rosuvastatin intravenous injection also delayed plaque progression, but also progressed by 26.4%; whereas targeted nanodrug delivery treatment significantly suppressed plaque progression, even with reversal and regression of plaque volume, HSA-PLLA (AuNP/R) @ OPN/PEG regressed plaque by 12.4%. In summary, free rosuvastatin shows a therapeutic effect on arterial vulnerable plaque in mice, whether by intragastric administration or intravenous administration, but it cannot reverse vulnerable plaque. However, when rosuvastatin and nanogold are formulated in the nano delivery system of the present invention, the diagnosis and treatment effects on vulnerable plaque are significantly improved, and the early warning of high-risk patients and the treatment effects on plaque reduction are achieved.
Test example 4 in vivo administration of the HSA-PLGA (Fe 3O 4/DXMS) @ HI44a delivery System of the invention to arterial vulnerable plaque Trace experiments (MRI Trace) and anti-inflammatory treatments
Monoclonal antibody (HI 44 a) is CD44 antibody, and can target vulnerable plaque, dexamethasone (DXSS) has antiinflammatory and plaque progression inhibiting effects, and Fe 3 O 4 Is an MRI tracer. The aim of this example is to verify the in vivo tracer and therapeutic effect of the delivery system of the amphiphilic protein-polymer conjugate loaded with MRI tracer and dexamethasone on vulnerable arterial plaque.
(1) An amphiphilic protein-polymer conjugate nanodelivery system loaded with an MRI tracer and a therapeutic agent was prepared using the method described in example 9 above. (2) The method for establishing the ApoE-/-mouse arterial vulnerable plaque model is the same as that of test example 2.
(3) Vulnerable plaque tracking in experimental animals:
experimental animals were randomly divided into the following groups of 6 animals each:
free Fe 3 O 4 Group: fe (Fe) 3 O 4 Is administered at a dose of 0.1mg/kg body weight
HSA-PLGA(Fe 3 O 4 DXMS) @ HI44a group: fe (Fe) 3 O 4 Is administered at a dose of 0.1mg/kg body weight;
the experimental groups were injected with the corresponding tracer through the tail vein, and MRI imaging was performed before and 2 hours after the administration, and the recognition of atherosclerosis vulnerable plaque was observed for each group of animals.
HSA-plga@hid44a group: for HSA-PLGA (Fe) 3 O 4 The HSA content was determined for the drug of group/DAMS) @ HI44a using the same concentration of HSA-PLGA @ HI44a as standard for HSA concentration.
(4) Grouping and treating experimental animals:
experimental animals were randomly divided into the following groups of 6 animals each:
vulnerable plaque model control group: the animals of this group were not subjected to any therapeutic treatment;
HSA-PLGA (Fe 3O 4/DXMS) @ HI44a group: intravenous administration at a dose of 0.1mg dexamethasone/kg body weight;
treatment in the treatment group was performed 1 time every other day for 5 times in addition to the vulnerable plaque model control group. For each group of animals, carotid MRI scans were performed before and after treatment to detect plaque and luminal area and calculate percent plaque progression.
Percent plaque progression = (plaque area after treatment-plaque area before treatment)/lumen area.
Experimental results:
figure 20 demonstrates the in vivo tracer effect of the tracer-loaded amphiphilic protein-polymer conjugate delivery system of the invention on vulnerable arterial plaque. As shown in the figure, free Fe 3 O 4 The particles exhibit a certain tracing effect on vulnerable arterial plaque in mice. With free Fe 3 O 4 When Fe is compared with the particles 3 O 4 When formulated in a targeted amphiphilic protein-polymer conjugate delivery system, the tracer effect on vulnerable plaques is greatly improved. In conclusion, compared with the free MRI tracer, the application of the amphiphilic protein-polymer conjugate delivery system with the surface modified with the targeting ligand in the invention can obviously improve the identification effect of the MRI tracer on vulnerable arterial plaque, and generate better indication The trace effect.
FIG. 21 shows the in vivo therapeutic effect of the HSA-PLGA (Fe 3O 4/DAMS) @ HI44a system of the present invention on arterial vulnerable plaque. As shown, during the course of high fat diet feeding (10 days), atherosclerosis in the control group (without any treatment) progressed by 28.3%; whereas targeted nanodrug delivery treatment significantly suppressed plaque progression, even with reversal and regression of plaque volume, HSA-PLGA (fe3o4/DXMS) @ HI44a resolved plaque by 12.4%. Taken together, when dexamethasone and Fe are used for treating arterial vulnerable plaque in mice 3 O 4 When the amphiphilic protein-polymer conjugate nano delivery system is prepared, the diagnosis and treatment effects on vulnerable plaque are remarkably improved, and the early warning of high-risk patients and the treatment effects of reversing plaque growth (shrinking plaque) are achieved.
Test example 5 Effect of the HSA-PCL (Asp/Clo) @ Col/PEG delivery System of the invention on vulnerable plaque in arteries In vivo experiments
Asp (Asp) and clopidogrel (Clo) are antiplatelet agents that act to reduce platelet aggregation and reduce mortality from cardiovascular events. The purpose of this example was to demonstrate the in vivo therapeutic effect of the HSA-PCL (Asp/Clo) @ Col/PEG carrier delivery system of the present invention on arterial vulnerable plaques.
The experimental method comprises the following steps:
(1) Physiological saline solutions of free aspirin and clopidogrel were formulated and an amphiphilic protein-polymer conjugate nano-delivery system loaded with a therapeutic agent was prepared using the method described in example 10 above.
(2) Establishment of ApoE-/-mouse arterial vulnerable plaque rupture model: the ApoE-/-mice were given a high fat diet for 30 weeks to form atherosclerotic plaques throughout the arteries, and the venom was given to induce rupture of vulnerable plaques, forming acute coronary syndrome.
(3) Grouping and treating experimental animals:
experimental animals were randomly divided into the following groups of 10 animals each:
plaque rupture model control group: the animals of this group were not subjected to any therapeutic treatment;
aspirin and clopidogrel gavage group: gastric administration at a dose of 100mg aspirin/kg body weight and 75mg clopidogrel/kg body weight;
HSA-PCL (Asp/Clo) @ Col/PEG group: intravenous administration treatment was performed at a dose of 10mg aspirin/kg body weight and 7.5mg clopidogrel/kg body weight;
treatment in the treatment group was performed 1 time every other day for 5 times in addition to the vulnerable plaque model control group. For each group of animals, mortality was observed for mice for 1 month, and mice Bleeding Time (BT) was detected by tail-off.
Experimental results:
FIG. 22 shows the in vivo therapeutic effect of the HSA-PCL (Asp/Clo) @ Col/PEG system of the present invention on arterial vulnerable plaque. As shown, mice in the control group (not given any treatment) had a mortality of 40%; the aspirin and clopidogrel are adopted for gastric lavage treatment, so that the death rate can be reduced to 28%; HSA-PCL (Asp/Clo) @ Col/PEG treatment reduced mortality to 12%. From the bleeding time, the HSA-PCL (Asp/Clo) @ Col/PEG group was not significantly prolonged, whereas the bleeding time of mice orally administered with aspirin and clopidogrel was significantly prolonged.
In summary, for animals with vulnerable plaque rupture, oral dual anti-platelet therapy can reduce mortality, but extend bleeding time and increase bleeding risk. The antiplatelet drug is loaded to the nano delivery system, so that better curative effect than oral drug is achieved, and the bleeding risk is not increased.
Although the present invention has been described to a certain extent, it is apparent that appropriate changes may be made in the individual conditions without departing from the spirit and scope of the invention. It is to be understood that the invention is not to be limited to the described embodiments, but is to be given the full breadth of the claims, including equivalents of each of the elements described.

Claims (41)

1. An amphiphilic protein-polymer conjugate nanocarrier delivery system for targeted activation of CD44 molecules, characterized in that the amphiphilic protein-polymer conjugate is formed by coupling a protein and a hydrophobic polymer fragment through a chemical bond, and the surface of the amphiphilic protein-polymer conjugate is partially modified by a targeting ligand, wherein the protein is human serum albumin; the hydrophobic macromolecule fragment is selected from polycaprolactone, poly-L-lactic acid and poly-lactic acid-glycolic acid copolymer; the targeting ligand is a ligand capable of specifically binding to an activated CD44 molecule, the targeting ligand being selected from GAGs, collagens, laminin, fibronectin, selectins, osteopontin, monoclonal antibodies HI44a, HI313, A3D8, H90, IM7, or hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at vulnerable plaques; the derivatives of hyaluronic acid are pharmaceutically acceptable salts of hyaluronic acid, alkyl esters containing 1-6 carbon atoms, prodrugs capable of forming hyaluronic acid in vivo by hydrolysis or other means;
wherein the nanocarrier is loaded with a substance for diagnosing, preventing and/or treating vulnerable plaque disease.
2. The delivery system of claim 1, wherein the protein molecule self-assembles to form an amphiphilic protein-polymer conjugate after in situ grafting of the coupled hydrophobic polymer fragment onto the protein molecule.
3. The delivery system of claim 2, wherein the self-assembly condition is ultrasound-induced two-phase assembly.
4. The delivery system of claim 1, wherein the amphiphilic protein-polymer conjugate is surface modified with a natural polymer that is an organic layer that can self-polymerize or form a complex network with metal ions.
5. The delivery system of claim 4, wherein the natural high molecular polymer is selected from one or more of the following: dopamine, phytic acid, tannic acid, chitosan, trehalose and plant polyphenols.
6. The delivery system of claim 5, wherein the natural high molecular polymer is dopamine autopolymerization or tannic acid-Fe 3+ A complex.
7. The delivery system of claim 1, wherein the nanocarrier surface is further modified.
8. The delivery system of claim 7, wherein the modification is one or more of polyethylene glycol, a transmembrane peptide, a self peptide, or a dual ligand simultaneous modification on the surface of the carrier.
9. An amphiphilic protein-polymer conjugate nanocarrier delivery system for targeting vulnerable plaques, characterized in that the amphiphilic protein-polymer conjugate is formed by coupling a protein and a hydrophobic polymer fragment through a chemical bond, and the surface of the amphiphilic protein-polymer conjugate is partially modified by a targeting ligand, wherein the protein is human serum albumin; the high molecular fragment is selected from polycaprolactone, poly-L-lactic acid and poly-lactic acid-glycolic acid copolymer; the targeting ligand is a ligand capable of specifically binding to an activated CD44 molecule, the targeting ligand is selected from GAG, collagen, laminin, fibronectin, selectin, osteopontin, monoclonal antibodies HI44a, HI313, A3D8, H90, IM7, or derivatives of hyaluronic acid or hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at vulnerable plaques, the derivatives of hyaluronic acid being pharmaceutically acceptable salts of hyaluronic acid, alkyl esters containing 1-6 carbon atoms, prodrugs capable of forming hyaluronic acid in vivo via hydrolysis or other means,
wherein the nanocarrier is loaded with a substance for diagnosing, preventing and/or treating vulnerable plaque.
10. The delivery system of claim 9, wherein the protein molecule self-assembles to form an amphiphilic protein-polymer conjugate after in situ grafting of the coupled hydrophobic polymer fragment onto the protein molecule.
11. The delivery system of claim 10, wherein the self-assembly condition is ultrasound-induced two-phase assembly.
12. The delivery system of claim 9, wherein the amphiphilic protein-polymer conjugate is surface modified with a natural polymer that is an organic layer that can self-polymerize or form a complex network with metal ions.
13. The delivery system of claim 12, wherein the natural high molecular polymer is selected from one or more of the following: dopamine, phytic acid, tannic acid, chitosan, trehalose and plant polyphenols.
14. The delivery system of claim 13, wherein the natural high molecular polymer is dopamine self-polymerization or tannic acid-Fe 3+ A complex.
15. The delivery system of claim 9, wherein the nanocarrier surface is further modified.
16. The delivery system of claim 15, wherein the modification is one or more of polyethylene glycol, a transmembrane peptide, a self peptide, or a dual ligand simultaneous modification on the surface of the carrier.
17. The delivery system of any one of claims 1 to 16, wherein the targeting ligand is selected from collagen, hyaluronic acid, selectin, osteopontin or monoclonal antibody HI44a, IM7.
18. The delivery system of any one of claims 1 to 16, wherein the nanocarrier is loaded with a substance for diagnosing, preventing and/or treating a disease associated with the occurrence of a CD44 molecule activation condition.
19. The delivery system according to any one of claims 1 to 16, wherein the nanocarrier is loaded with both a substance for preventing and/or treating vulnerable plaques and hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at vulnerable plaques.
20. The delivery system of claim 19, wherein the nanocarrier is simultaneously loaded with a substance for diagnosing vulnerable plaques, a substance for preventing and/or treating vulnerable plaques, an optional CD44 activator and an optional hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at vulnerable plaques.
21. The delivery system of claim 18, wherein the substance for diagnosing, preventing and/or treating a disease associated with the occurrence of a CD44 molecule activation condition is a CD44 activator.
22. The delivery system of claim 21, wherein the CD44 activator is a CD44 antibody mAb or IL5, IL12, IL18, TNF- α, LPS.
23. The delivery system according to any one of claims 1 to 16, wherein the substance for diagnosing, preventing and/or treating vulnerable plaque is selected from one or more of a drug, a polypeptide, a nucleic acid and a cytokine for diagnosing, preventing and/or treating vulnerable plaque.
24. The delivery system according to any one of claims 1 to 16, wherein the substance for diagnosing, preventing and/or treating vulnerable plaque is a substance for diagnosing vulnerable plaque.
25. The delivery system of claim 24, wherein the substance for diagnosing vulnerable plaque is a tracer.
26. The delivery system of claim 25, wherein the tracer is selected from the group consisting of a CT tracer, an MRI tracer, and a nuclide tracer.
27. The delivery system of claim 26, wherein the CT tracer is selected from the group consisting of iodine nanocontrast agents, gold nanocontrast agents, tantalum oxide nanocontrast agents, bismuth nanocontrast agents, lanthanide nanocontrast agents;
The MRI tracer is selected from the group consisting of longitudinal relaxation contrast agents and transverse relaxation contrast agents; and/or
The nuclide tracer is selected from fluorodeoxyglucose labeled with carbon 14, carbon 13, phosphorus 32, sulfur 35, iodine 131, hydrogen 3, technetium 99, and fluorine 18.
28. The delivery system of claim 27, wherein the CT tracer is an iodinated contrast agent or nanogold;
the MRI tracer is paramagnetic contrast agent, ferromagnetic contrast agent and super-magnetic contrast agent.
29. The delivery system of claim 28, wherein the CT tracer is selected from the group consisting of iohexol, iocaic acid, ioversol, iodixanol, iopromide, iobitol, iomeprol, iopamidol, ioxilan, aceiobenzoic acid, cholanic acid, iobenzamic acid, iogancaic acid, diatrizoic acid, sodium iotazinate, iophenyl ester, iopanoic acid, ioafoic acid, sodium aceiobenzoate, propidone, ioaone, iotrolan, iopidol, meglumine of cholate, iotala acid, diatrizosamine, mezoic acid, methoglucammonium, iodized oil, and ethiodized oil;
the MRI tracer is selected from Gd-DTPA and porphyrin chelate of linear, cyclic polyamine polycarboxylic chelate and manganese, macromolecular gadolinium chelate, biomacromolecule modified gadolinium chelate, folic acid modified gadolinium chelate, dendrimer developer, liposome modified developer and gadolinium-containing fullerene.
30. The delivery system of claim 29, wherein the CT tracer is nanogold;
the MRI tracer is selected from gadofoshan, ferric ammonium citrate effervescent granule, and paramagnetic ferric oxide.
31. The delivery system of claim 30, wherein the MRI tracer is paramagnetic iron oxide.
32. The delivery system according to any one of claims 1 to 16, wherein the substance for diagnosing, preventing and/or treating vulnerable plaque is a substance for preventing and/or treating vulnerable plaque.
33. The delivery system of claim 32, wherein the substance for preventing and/or treating vulnerable plaque is selected from one or more of a statin, a fibrate, an antiplatelet, a PCSK9 inhibitor, an anticoagulant, an angiotensin converting enzyme inhibitor, a calcium ion antagonist, an MMPs inhibitor, a beta blocker, a glucocorticoid, an IL-1 antibody canakinumab, or a pharmaceutically acceptable salt thereof, or an endogenous anti-inflammatory cytokine.
34. The delivery system of claim 33, wherein the delivery system comprises, the substance for preventing and/or treating vulnerable plaque is selected from lovastatin, atorvastatin, rosuvastatin, simvastatin, fluvastatin, pitavastatin, pravastatin, bezafibrate, ciprofibrate, clofibrate, gemfibrozil, fenofibrate, probucol, anti-PCSK 9 antibody, adnectin, antisense RNAi oligonucleotide, microRNA-33a, microRNA-27a/b, microRNA-106b, microRNA-302, microRNA-758, microRNA-10b, microRNA-19b, microRNA-26, microRNA-93, microRNA-128-2, microRNA-144, microRNA-145 antisense strand, locked nucleic acid, aspirin, acemetacin, troxegrel, dipyridamole, cilostazol, ticlopidine hydrochloride, sodium, clopyralid, moxazole, prasugrel, cilostazol, sodium, beraprost, sodium, fluxol, and microRNA-19b cangrelor, tirofiban, eptifibatide, acipimab, plain heparin, kesai, fast-b lin, huang Dagan sodium, warfarin, dabigatran, rivaroxaban, apixaban, idexaban, bivalirudin, enoxaparin, tetanus heparin, aclarubin, coumarin nitrate, sodium matrisulfonate, hirudin, argatroban, benazepril, captopril, enalapril, perindopril, fosinopril, lisinopril, moexipril, cilazapril, perindopril, quinapril, ramipril, tranilartan, eprosartan, irbesartan, losartan, telmisartan, valsartan, olmesartan, tamoxirtan, nifedipine, nicardipine, nitrendipine, amlodipine, nimodipine, nisoldipine, nisole, fosapril, valsartan, nilvadipine, isradipine, felodipine, lacidipine, diltiazem, verapamil, chlorhexidine, minocycline, MMI-166, metoprolol, atenolol, bisoprolol, propranolol, carvedilol, pamphlestat, marimastat, praline stat, BMS-279251, BAY 12-9566, TAA211, AAJ996A, nacetrapib, evacetrapib, torcetrapib, dalcetrapib, prednisone, methylprednisone, betamethasone, beclomethasone propionate, deborosone, prednisolone, hydrocortisone, dexamethasone, the IL-1 antibody canakinumab, or one or more of their pharmaceutically acceptable salts, or endogenous anti-inflammatory cytokines.
35. The delivery system of claim 34, wherein the substance for preventing and/or treating vulnerable plaque is selected from evolocumab, alirocumab, bococizumab, RG7652, LY3015014, LGT-209, BMS-962476, ALN-PCSsc, interleukin 10.
36. A method of preparing a delivery system according to any one of claims 1 to 35, comprising the steps of:
(1) Coupling the protein and the hydrophobic polymer fragment to prepare a protein-hydrophobic polymer complex;
(2) The protein-hydrophobic polymer compound prepared in the step (1) is self-assembled to prepare an amphiphilic protein-polymer conjugate;
(3) And (3) coupling the target ligand to the amphiphilic protein-polymer conjugate prepared in the step (2).
37. The method of claim 36, wherein in step (2), the self-assembling step further comprises a drug entrapment process.
38. A pharmaceutical composition comprising the nanocarrier delivery system of any of claims 1 to 35.
39. A diagnostic formulation comprising the nanocarrier delivery system of any of claims 1 to 35.
40. Use of the nanocarrier delivery system of any of claims 1 to 35 in the manufacture of a product for the diagnosis, prevention and treatment of vulnerable plaques.
41. The use of claim 40, wherein the vulnerable plaque is selected from one or more of a ruptured plaque, an erosive plaque, and a partially calcified nodular lesion.
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