CN112206326B - Amino acid self-assembled nanocarrier delivery system for targeted activation of CD44 molecules, preparation method and application thereof - Google Patents

Amino acid self-assembled nanocarrier delivery system for targeted activation of CD44 molecules, preparation method and application thereof Download PDF

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CN112206326B
CN112206326B CN202010662240.3A CN202010662240A CN112206326B CN 112206326 B CN112206326 B CN 112206326B CN 202010662240 A CN202010662240 A CN 202010662240A CN 112206326 B CN112206326 B CN 112206326B
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马茜
孙洁芳
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Beijing Inno Medicine Co ltd
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Abstract

The invention provides an amino acid self-assembled nanocarrier delivery system for targeted activation of CD44 molecules, wherein the nanocarrier is an Fmoc modified amino acid or short peptide nanocarrier, the nanocarrier has a hollow spherical structure, and the surface of the nanocarrier is partially modified by a targeting ligand, and the targeting ligand is a ligand capable of specifically binding with the activated CD44 molecules. Also provides a preparation method and application of the amino acid self-assembled nano-carrier. The Fmoc-AC 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

Amino acid self-assembled nanocarrier 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 an amino acid self-assembled nano-carrier delivery system for targeted activation of CD44 molecules, a preparation method and application thereof.
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 chemically modified amino acid or short peptide can self-assemble into ordered spherical, vesicle and hollow tubular nanometer structure through weak intermolecular interaction under certain solution condition, and the structure is maintained through pi-pi stacking effect produced by hydrogen bond, benzene ring structure and amide bond. The preparation method has the characteristics of simple preparation, obvious effect, low cytotoxicity and the like, can be used as a nano-drug carrier, but has the defects of poor stability, easy degradation and excessively fast in vivo clearance time, and can prevent the sustainability of drug delivery time, so that the scheme for solving the problems is the key for further playing the drug delivery capacity of the amino acid or short peptide nano-carrier.
Disclosure of Invention
It is therefore an object of the present invention to overcome the drawbacks of the prior art and to provide an amino acid self-assembled nanocarrier delivery system for targeted activation of CD44 molecules, a method of preparation and use thereof.
Before setting forth the present disclosure, the terms used herein are defined as follows:
the term "SEM" refers to: scanning electron microscope.
The term "FT-IR" refers to: fourier transform infrared spectra.
The term "TEM" refers to: transmission electron microscope.
The term "Fmoc" refers to: fluorenylmethoxycarbonyl.
The term "PEG" refers to: polyethylene glycol.
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 "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.
"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 walls, regulating protein, water electrolyte diffusion and operation, promoting wound healing and the like. 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.
In a first aspect the present invention provides an amino acid self-assembled nanocarrier delivery system for targeted activation of CD44 molecules, the nanocarrier being an Fmoc-modified amino acid or a short peptide nanocarrier, the nanocarrier having a hollow spherical structure and the surface of the nanocarrier being partially modified by a targeting ligand, the targeting ligand being a ligand capable of specifically binding to an activated CD44 molecule.
In a second aspect, the present invention provides an amino acid self-assembled nanocarrier delivery system for targeting vulnerable plaques, the nanocarrier being an Fmoc-modified amino acid, or a short peptide nanocarrier, the nanocarrier having a hollow spherical structure, and the surface of the nanocarrier being partially modified by a targeting ligand, the targeting ligand being a ligand capable of specifically binding to an activated CD44 molecule.
The nanocarrier delivery system according to the first or second aspect of the invention, wherein the amino acid is selected from one of: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine; preferably lysine and/or arginine; and/or
The short peptide consists of the following amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.
The nanocarrier delivery system according to the first or second aspect of the invention, wherein the nanocarriers have a particle size of 50 to 1000nm, preferably 200nm.
The nanocarrier delivery system according to the first or second aspect of the invention, wherein the nanocarrier is prepared by self-assembling a starting material in alkaline conditions.
Preferably, the alkaline condition is pH >8, preferably pH >10, most preferably ph=11.
The nanocarrier delivery system according to the first or second aspect of the invention, wherein the targeting ligand is selected from the group consisting of self-peptide, GAG, collagen, laminin, fibronectin, selectin, osteopontin and monoclonal antibodies HI44a, HI313, A3D8, H90, IM7, or derivatization of hyaluronic acid or hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at vulnerable plaques, preferably the targeting ligand is selected from the group consisting of collagen, hyaluronic acid, selectin, self-peptide, osteopontin or monoclonal antibodies HI44a, IM7.
The nanocarrier delivery system according to the first or second aspect of the invention, 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 nanocarrier delivery system according to the first or second aspect of the 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; 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 nanocarrier delivery system according to the first or second aspect of the invention, 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 nanocarrier delivery system according to the first or second aspect of the invention, wherein the substance for diagnosing, preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque is selected from 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.
The nanocarrier delivery system according to the first or second aspect of the invention, wherein the substance 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 nanocarrier delivery system according to the first or second aspect of the invention, 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 second aspect of the present invention provides a method of preparing a nanocarrier delivery system according to the first aspect, the method comprising the step of adding dopamine to consolidate the nanocarriers.
A third aspect of the invention provides a pharmaceutical composition comprising the nanocarrier delivery system of the first or second aspect of the invention.
In a fourth aspect the present invention provides a diagnostic formulation comprising the nanocarrier delivery system of the first or second aspect of the invention.
In a fifth aspect, the present invention provides the use of a nanocarrier delivery system of the first or second aspect of the invention in the manufacture of a product for the diagnosis, prophylaxis and treatment of vulnerable plaques or diseases associated with vulnerable plaques.
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 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.
The patent provides a spherical structure of nanometer scale is formed through Fmoc-protected amino acid or Fmoc-protected short peptide self-assembly, the drug carrying process is coupled with molecular assembly, a biological reticular structure is formed through dopamine surface polymerization, the nano-carrier is reinforced, a simple method for constructing the targeting nano-carrier is obtained, and the stability and biocompatibility of the self-assembled nano-carrier are greatly improved. The method is simple, green, controllable, high in drug loading, good in compatibility with various drugs, good in biocompatibility and high in safety, and is easy to modify the targeting ligand. The self-assembled carrier is loaded with different medicines and coupled with different targeting ligands, and is used for diagnosing and treating vulnerable plaque.
Fmoc-protected amino acid molecules (Fmoc-amino acids, fmoc-ACs) are commonly used amino acid starting materials for synthetic peptides, and Fmoc protecting groups are used to protect amino molecules in amino acids from cross-linking during solid phase synthesis of polypeptides. Researches report that the molecules can form a nano-scale fibrous structure or a nano-scale spherical structure through the hydrophobic effect pi-pi stacking effect, and the self-assembled nanosphere has the characteristics of good biocompatibility, easiness in functionalization and the like, and is particularly suitable for being applied to nano drug carriers. In the patent, aiming at the problems of dipeptide self-assembled nano-carriers reported in the past, the inventor selects Fmoc protective amino acid molecules, realizes simple and convenient assembly of the molecules by controlling the acidity of a solution, obtains a hollow spherical structure with nano scale for loading drugs, and reinforces the self-assembled nano-structure by polymerizing the surface of dopamine on the surface of the Fmoc-AC self-assembled carrier, so that the stability of the carrier in serum is enhanced. Finally, various drugs are loaded on the hollow nano-carrier formed by the self-assembly of the amino acid and are connected with different target molecules for targeting the vulnerable plaque treatment of CD44, and the drug/gene delivery capacity and the treatment/gene interference effect of the Fmoc-AC nano-carrier are explored through a series of experimental processes.
In the nano-carrier, fmoc-AC nano-carrier can effectively improve the biocompatibility of the carrier on one hand and the circulation time in the body by coupling polyethylene glycol, and the carrier is combined with serum albumin so as to be degraded. In addition, the functional group structure with rich surfaces of the nano-carrier is also very convenient for connecting the targeting carrier. The self-assembled structure has the advantages of good biocompatibility and high safety, and is a nano-drug carrier with important value.
The nano-carrier has high drug loading capacity, simple process, high yield, good applicability to various drugs, strong stability and good biocompatibility, and is convenient for coupling various targeting ligands. Amino acid self-assembled nanocarriers for targeted activation of CD44 molecules, particularly for targeting vulnerable plaques. The invention also relates to the innovative preparation of said nanocarriers, in particular to the preparation method and the use of the amino acid self-assembled nanocarrier delivery system, in particular in the diagnosis, prevention and treatment of vulnerable plaques or diseases associated with vulnerable plaques.
The amino acid self-assembled 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. Compared with inorganic nano materials, the organic nano materials have better biocompatibility, safety and metabolizability, which also provides favorable conditions for the clinical transformation of the organic nano materials. The Fmoc-AC 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 an SEM image of Fmoc-Lys assembly in example 1.
FIG. 2 shows the FT_IR spectral results of Fmoc-Lys, fmoc-Lys (R), fmoc-Lys (R) -HA nanocarriers in example 1.
FIG. 3 shows an SEM image of Fmoc-Lys (R) -PEG in example 2.
FIG. 4 shows the FT_IR spectral results of Fmoc-Lys (R) -SP/PEG nanocarriers in example 2.
FIG. 5 shows the FT_IR spectrum of Fmoc-Arg, fmoc-Arg (R), fmoc-Arg (R) -HA/PEG nanocarrier in example 3.
FIG. 6 shows an SEM image of Fmoc-Arg (At) -PEG of example 4.
FIG. 7 shows the results of IR spectra of Fmoc-Arg (At) -SEP/PEG nanocarriers in example 4.
Fig. 8 shows TEM results of Fmoc-Arg (At/miRNA-33) -IM7 in example 5.
FIG. 9 shows the IR spectra of Fmoc-Lys (AuNP/At) -PEG and Fmoc-Lys (AuNP/At) -OPN/PEG nanocarriers after modification of targeting ligands in example 6.
FIG. 10 shows an SEM image of Fmoc-Lys (DPLA) -OPN/PEG in example 6.
FIG. 11 shows SEM results of Fmoc-Lys (DKSC) -OPN/PEG in example 6.
FIG. 12 shows SEM results of Fmoc-Lys (DFC) -OPN/PEG in example 6.
FIG. 13 shows Fmoc-Lys (Fe 3 O 4 TEM results of/DXMS) -HI44 a/PEG.
FIG. 14 shows Fmoc-Lys (Fe 3 O 4 Fmoc-Lys (Fe) after adding targeting ligand to/DXMS) -PEG nano-carrier 3 O 4 Infrared results of/DXMS) -HI44a/PEG nanocarriers.
FIG. 15 shows SEM results of Fmoc-Lys (GPA) -HI44a/PEG of example 7.
FIG. 16 shows SEM results of Fmoc-Lys (GSA) -HI44a/PEG of example 7.
FIG. 17 shows SEM results of Fmoc-Lys (GPS) -HI44a/PEG of example 7.
FIG. 18 shows the infrared results of Fmoc-Lys (Asp/Clo) -PEG nanocarriers and Fmoc-Lys (Asp/Clo) -Col/PEG carriers after addition of targeting ligand in example 8.
FIG. 19 shows the effect of different shelf-life on hydrated particle size in test example 1.
Fig. 20 shows the effect of different shelf-life on encapsulation efficiency in test example 1.
Figure 21 shows the in vitro cumulative release rate (CRP%) of the nano-delivery system.
FIG. 22 shows a graph of the therapeutic effect of Fmoc-Lys (R) -HA, fmoc-Lys (R) -SP/PEG, fmoc-Arg (R) -HA/PEG, fmoc-Arg (At) -SEP/PEG, fmoc-Arg (At/miRNA-33) -IM7/PEG nanodelivery system of the present invention on carotid vulnerable plaque in model mice.
Figure 23 shows an in vivo tracer effect of Fmoc-Lys (AuNP/At) -OPN/PEG and other CT tracer nano delivery systems on carotid vulnerable plaques in model mice.
FIG. 24 shows a graph of the therapeutic effect of Fmoc-Lys (AuNP/At) -OPN/PEG nanodelivery systems on carotid vulnerable plaque in model mice.
FIG. 25 shows Fmoc-Lys (Fe 3 O 4 Graph of in vivo tracer effect of/DXMS) -HI44a/PEG and other MRI tracer nanodelivery systems on carotid vulnerable plaque in model mice.
FIG. 26 shows Fmoc-Lys (Fe 3 O 4 Graph of therapeutic effect of the/DXMS) -HI44a/PEG nanodelivery system on carotid vulnerable plaque in model mice.
FIG. 27 shows a graph of the therapeutic effect of Fmoc-Lys (Asp/Clo) -Col/PEG nano delivery system on arterial vulnerable plaque rupture in model mice.
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:
fmoc-protected lysine (Fmoc-Lys), fmoc-protected arginine (Fmoc-Arg), fmoc-protected arginine dipeptide (Fmoc-Arg) from Shanghai Jier Biochemical, rosuvastatin from Dalian Mei Lun Biotechnology, hyaluronic Acid (HA) from Zhejiang Dong Biotechnology, DMSO, ferric trichloride, ammonia, ethanol, chloroauric acid, naBH 4 Lipoic acid is purchased from Beijing chemical reagent Co., ltd., dopamine 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl), N-hydroxythiosuccinimide (sulfo-NHS), collagen (Col), NHS ester activated PEG (PEG) with molecular weight of 1000 1k -NHS), selectin SP, osteopontin, HI44a was purchased from Sigma-Aldrich, fluorodeoxyglucose, aspirin (Asp), dexamethasone (DXSM) clopidogrel (Clo) was purchased from chinese food and drug assay institute.
Instrument:
scanning electron microscope, available from zeiss, model germany: EVO18
Laser particle sizer, commercially available from malvern 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 surface modification of Hyaluronic Acid (HA), lysine loaded with rosuvastatin (R) (Fmoc-Lys (R) -HA) Preparation of nanocarriers
1.1 blank Fmoc-Lys nanocarrier preparation
Fmoc-Lys (10 mg) was weighed and dissolved in 10mL of ultrapure water to completely dissolve the Fmoc-Lys, and 0.1. 0.1g L was used -1 The pH of the solution is adjusted to 11, and the solution is subjected to self-assembly after being subjected to continuous ultrasonic treatment for 1h, so that the milky emulsion is obtained. A small amount of the solution was slowly dropped onto a silicon wafer, dried at room temperature, and observed by a scanning electron microscope, and FIG. 1 shows an SEM image of the Fmoc-Lys assembly in example 1.
1.2 preparation of rosuvastatin (R) -loaded Fmoc-Lys (R) nanocarriers
Fmoc-Lys (10 mg) was weighed and dissolved in 10mL of ultrapure water to completely dissolve the Fmoc-Lys, and 1mg mL was added dropwise under ultrasonic conditions -1 Rosuvastatin DMSO solution, 10mg mL was used -1 The pH of the solution is adjusted to 11, and the solution is subjected to self-assembly after being subjected to continuous ultrasonic treatment for 1h, so that the milky emulsion is obtained. Adding 1mmolL of final concentration to the solution -1 The dopamine hydrochloride is reacted for 40 minutes at room temperature, and after the reaction, the dopamine hydrochloride is purified by centrifugation and is freeze-dried for storage. A small amount of the solution was slowly dropped onto a silicon wafer, dried at room temperature, and the structure was characterized by using an infrared spectrum, which is shown in fig. 2.
1.3 preparation of Fmoc-Lys (R) -HA-coupled nanocarriers (Fmoc-Lys (R) -HA)
1g of hyaluronic acid HA (molecular weight about 40 kDa) was dissolved in ultrapure water and 0.1g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.12. 0.12g N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl groups. 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 And (3) absorbing 0.2mL of the solution to dissolve in the purified Fmoc-Lys (R) solution to realize the coupling of HA on the nano-carrier, thereby obtaining the targeting recognition nano-carrier Fmoc-Lys (R) -HA. Fig. 2 shows ft_ir spectra of targeting drug-loaded nanocarriers Fmoc-Lys (R) -HA.
Example 2 Simultaneous coupling of polyethylene glycol, selectin (SP), rosuvastatin (R) -loaded Fmoc-Lys nanos Preparation of the Carrier (Fmoc-Lys (R) -SP/PEG)
2.1 preparation of rosuvastatin-loaded Fmoc-Lys nanocarrier (Fmoc-Lys (R) -PEG) coupled with polyethylene glycol
100mg of NHS active ester functionalized PEG with molecular weight 1000 1k The NHS ester was dissolved in 1mM NaHCO3 solution (5 mL, pH 8.6), 10mg of Fmoc-Lys was added and the reaction was stirred at room temperature for 24 hours, fmoc-Lys-PEG after ether precipitation was added and the purified Fmoc-Lys-PEG was obtained after lyophilization.
Fmoc-Lys 50mg and 5mg Fmoc-Lys-PEG molecules were weighed, dissolved in 20mL of ultra pure water to completely dissolve them, and 1mg mL was added dropwise under ultrasonic conditions -1 Rosuvastatin DMSO solution, 10mg mL was used -1 The pH of the solution is adjusted to 11, and the solution is subjected to self-assembly after being subjected to continuous ultrasonic treatment for 1h, so that the milky emulsion is obtained. To this solution was added 1mmol L as a final concentration -1 The dopamine hydrochloride is reacted for 40 minutes at room temperature, and after the reaction, the dopamine hydrochloride is purified by centrifugation and is freeze-dried for storage. A small amount of the solution was slowly dropped onto a silicon wafer, dried at room temperature, and observed by a scanning electron microscope, and FIG. 3 shows an SEM image of Fmoc-Lys (R) -PEG in example 2.
2.2 Fmoc-Lys (R) -SP/PEG nanocarriers coupled to Selectin (SP)
1mg of selectin SP was dissolved well in PBS buffer, and 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.5mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. 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 dissolve in the purified Fmoc-Lys (R) -PEG solution to realize the coupling of the SP on the Fmoc-Lys (R) -PEG, thereby obtaining the targeting recognition nano carrier Fmoc-Lys (R) -SP/PEG. FIG. 4 is FT-IR spectrum of targeting nanocarrier Fmoc-Lys (R) -SP/PEG.
Example 3 simultaneous coupling of Hyaluronic Acid (HA) and PEG, fmoc-protected arginine sodium loaded with rosuvastatin (R) Preparation of Fmoc-Arg (R) -HA/PEG as rice carrier
3.1 preparation of rosuvastatin (R) -loaded Fmoc-Arg nanocarriers (Fmoc-Arg (R) -PEG) coupled with polyethylene glycol
Fmoc-Arg is dipeptide with dimeric arginine, and is custom-made and synthesized by Shanghai Jier biochemical company,
100mg of NHS active ester functionalized PEG with molecular weight 1000 1k -NHS ester in 1mM NaHCO 3 To the solution (5 mL, pH 8.6), 10mg of Fmoc-Arg was added and the reaction was stirred at room temperature for 24 hours, fmoc-Arg-PEG after the ether precipitation reaction was added, and the purified Fmoc-Arg-PEG was obtained after lyophilization.
Fmoc-Arg 50mg and 5mg of Fmoc-Arg-PEG molecule were weighed, dissolved in 20mL of ultrapure water to completely dissolve the Fmoc-Arg-PEG molecule, and 1mg of the Fmoc-Arg-PEG molecule was added dropwise under ultrasonic conditions -1 Rosuvastatin (R) DMSO solution, 10mg mL was used -1 The pH of the solution is adjusted to 11, and the solution is subjected to self-assembly after being subjected to continuous ultrasonic treatment for 1h, so that the milky emulsion is obtained. Adding 1mmolL of final concentration to the solution -1 After reaction at room temperature for 40 minutes, purification by centrifugation and lyophilization gave Fmoc-Arg (R) -PEG, the infrared spectrum of which is shown in FIG. 5.
3.2 Fmoc-Arg (R) -PEG nanocarriers coupled to Hyaluronic Acid (HA)
Activated HA was obtained as in example 1. It was formulated as 0.1mg mL -1 And 0.2mL of the solution is absorbed and dissolved in the purified Fmoc-Arg (R) -PEG solution to realize the coupling of HA on the nanocarrier, thereby obtaining the targeting recognition nanocarrier Fmoc-Arg (R) -HA/PEG fig. 5 shows the ft_ir spectrum of the targeting nanocarrier Fmoc-Arg (R) -HA/PEG.
Example 4 Fmoc-Arg nanocarriers of self-peptide (SEP) -supported atorvastatin (At) coupled simultaneously with PEG Preparation of (Fmoc-Arg (At) -SEP/PEG) delivery System
4.1 preparation of atorvastatin (At) -supported Fmoc-Arg nanocarriers of polyethylene glycol (Fmoc-Arg (At) -PEG)
Fmoc-Arg (At) -PEG was prepared according to the method of example 2. Fmoc-Arg 50mg and 5mg of Fmoc-Arg-PEG molecule were weighed, dissolved in 20mL of ultrapure water to completely dissolve the Fmoc-Arg-PEG molecule, and 1mg of the Fmoc-Arg-PEG molecule was added dropwise under ultrasonic conditions -1 Atorvastatin (At) DMSO solution using 10mg mL -1 The pH of the solution is adjusted to 11, and the solution is subjected to self-assembly after being subjected to continuous ultrasonic treatment for 1h, so that the milky emulsion is obtained. Adding 1mmolL of final concentration to the solution -1 The dopamine hydrochloride is reacted for 40 minutes at room temperature, and after the reaction, the dopamine hydrochloride is purified by centrifugation and is freeze-dried for storage. Slowly dripping a small amount of solution on a silicon waferAir-dried At room temperature, observed using a scanning electron microscope, and an SEM image of Fmoc-Arg (At) -PEG is shown in FIG. 6.
4.2 Fmoc-Arg (At) -PEG nanocarriers coupled to self-peptide (SEP)
It was formulated as 0.1mg mL -1 Absorbing 0.2mL of the solution to be dissolved in the purified Fmoc-Arg (At) -PEG solution to realize the coupling of HA on the nano-carrier, thus obtaining the targeting recognition nano-carrier Fmoc-Arg (At) -HA/PEG. FIG. 7 is the infrared spectrum of the targeting nano-carrier Fmoc-Arg (At) -HA/PEG
1mg of self peptide (SEP) was dissolved in 1ml of buffer solution and 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.5mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. After reacting for 1h At room temperature, 1mL of the SEP-NHS solution is dissolved in purified Fmoc-Arg (At) -PEG solution to realize coupling of SEP on Fmoc-Arg (At) -PEG, and the targeting recognition nano-carrier Fmoc-Arg (At) -SEP/PEG is obtained. The infrared spectrum is shown in fig. 7.
Example 5 Fmoc-Arg nanobody coupled monoclonal antibody (IM 7) Supported atorvastatin (At) and miRNA-33 Preparation of vector (Fmoc-Arg (At/miRNA-33) -IM 7) delivery System
5.1 preparation of Fmoc-Arg nanocarriers (Fmoc-Arg (At/miRNA-33)) simultaneously carrying miRNA-33 and atorvastatin (At)
Fmoc-Arg 10mg was weighed and dissolved in 10mL of ultrapure water to completely dissolve the Arg, and 1mg of Arg was added dropwise under ultrasonic conditions -1 A DMSO solution of atorvastatin was used with 10mg mL -1 The pH of the solution is adjusted to 11, and the solution is subjected to self-assembly after being subjected to continuous ultrasonic treatment for 1h, so that the milky emulsion is obtained. Adding 1mmolL of final concentration to the solution -1 The dopamine hydrochloride is reacted for 40 minutes at room temperature, and after the reaction, the dopamine hydrochloride is purified by centrifugation and is freeze-dried for storage. And continuing to add miRNA-33a (0.1 mg) into the solution, continuing to stir for 30 minutes, centrifuging and purifying the sample, and freeze-drying to obtain Fmoc-Arg (At/miRNA).
5.2 coupling of monoclonal antibody IM7 to Fmoc-Arg (At/miRNA-33) surface
1mg of monoclonal antibody IM7 was well dissolved in PBS,carboxyl groups were activated by the addition of 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.5mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent. After stirring the reaction at room temperature for 1 hour, it was purified by ultrafiltration and was prepared as 1mg mL -1 Is a solution of (a) and (b). 1.0mL of activated IM7 solution is absorbed and dissolved in purified Fmoc-Arg (At/miRNA-33) solution, so that the coupling of IM7 on Fmoc-Arg (At/miRNA-33) is realized, and the targeted recognition nano-carrier Fmoc-Arg (At/miRNA-33) -IM7 is obtained. FIG. 8 shows Transmission Electron Microscopy (TEM) results of Fmoc-Arg (At/miRNA-33) -IM7.
Example 6 simultaneous coupling of polyethylene glycol (PEG), osteopontin (OPN) supported gold nanoparticle (AuNP) and atto Preparation of a Vastatin (At) nanocarrier (Fmoc-Lys (AuNP/At) -OPN/PEG)
6.1 preparation of coupled PEG-Supported gold nanoparticles (AuNP) and atorvastatin (At) Fmoc-Lys nanocarriers Fmoc-Lys (AuNP/At) -OPN/PEG delivery System
Fmoc-Lys-PEG was prepared as in example 2. Fmoc-Lys 50mg and 5mg Fmoc-Lys-PEG molecules were weighed, dissolved in 20mL of ultra pure water to completely dissolve them, and 1mg mL was added dropwise under ultrasonic conditions -1 Atorvastatin (At) DMSO solution using 10mg mL -1 The pH of the solution is adjusted to 11, and the solution is subjected to self-assembly after being subjected to continuous ultrasonic treatment for 1h, so that the milky emulsion is obtained. Adding 1mmolL of final concentration to the solution -1 The dopamine hydrochloride is reacted for 40 minutes at room temperature, and after the reaction, the dopamine hydrochloride is purified by centrifugation and is freeze-dried for storage.
1mL of 50mM HAuCl 4 The pH was adjusted to 11.0 with 1M NaOH, added dropwise to the above solution, stirred for 5 minutes, and 1mL of 0.1M sodium ascorbate was added to reduce HAuCl 4 Obtaining the nano-carrier with gold nano-particles loaded on the surface.
6.2 coupling of Osteopontin (OPN) to Fmoc-Lys (AuNP/At) -PEG surface
1mg of Osteopontin (OPN) was well dissolved in PBS buffer, and 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.5mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. After stirring the reaction at room temperature for 1 hour. Compounding it with Is set to 1mg mL -1 And (3) absorbing 1mL of the solution to be dissolved in purified Fmoc-Lys (AuNP/At) -PEG solution, and realizing the coupling of OPN on the Fmoc-Lys (AuNP/At) -PEG to obtain the targeted recognition nano carrier Fmoc-Lys (AuNP/At) -OPN/PEG. FIG. 9 is an infrared spectrum of Fmoc-Lys (AuNP/At) -OPN/PEG.
6.3 preparation of Fmoc-Lys nanocarrier delivery System coupled with PEG and OPN loaded with iodine imaging agent
Fmoc-Lys-PEG was prepared as in example 2. Fmoc-Lys 50mg and 5mg Fmoc-Lys-PEG molecules were weighed, dissolved in 20mL of ultra pure water to completely dissolve them, and 1mg mL was added dropwise under ultrasonic conditions -1 Aqueous solution of iopromide was used with 10mg mL -1 The pH of the solution is adjusted to 11, and the solution is subjected to self-assembly after being subjected to continuous ultrasonic treatment for 1h, so that the milky emulsion is obtained. Adding 1mmolL of final concentration to the solution -1 The dopamine hydrochloride is reacted for 40 minutes at room temperature, and after the reaction, the dopamine hydrochloride is purified by centrifugation and is freeze-dried for storage.
6.4 coupling of Osteopontin (OPN) to Fmoc-Lys (DPLA) -PEG surface
1mg of Osteopontin (OPN) was well dissolved in PBS buffer, and 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.5mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. 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 the purified Fmoc-Lys (DPLA) -PEG solution, so as to realize the coupling of OPN on the Fmoc-Lys (DPLA) -PEG, and obtain the targeted recognition nano carrier Fmoc-Lys (DPLA) -OPN/PEG. FIG. 10 shows SEM images of Fmoc-Lys (DPLA) -OPN/PEG. According to the method, the Fmoc-Lys (DKSC) -OPN/PEG can be obtained by replacing iopromide with iodixanol and iofluor alcohol. FIG. 11 shows SEM results of Fmoc-Lys (DKSC) -OPN/PEG. FIG. 12 shows SEM results of Fmoc-Lys (DFC) -OPN/PEG.
3 4 Example 7 coupling monoclonal antibody HI44a, drug-loaded Dexamethasone (DXMS) paramagnetic Oxidation (FeONPs) 3 4 Fmoc-Lys nanocarriers (Fmoc-Lys (FeO/DXMS) -HI44a/PEG preparation
7.1 coupling PEG Supported paramagnetic iron oxide (Fe) 3 O 4 NPs) and Dexamethasone (DXMS) Fmoc-Lys nanocarriers Fmoc-Lys (Fe) 3 O 4 Preparation of a/DXMS) -OPN/PEG delivery System
Fmoc-Lys-PEG was prepared as in example 2. Fmoc-Lys 50mg and 5mg Fmoc-Lys-PEG molecules were weighed, dissolved in 20mL of ultra pure water to completely dissolve them, and 1mg mL was added dropwise under ultrasonic conditions -1 Dexamethasone (DXMS) in DMF, 10mg mL was used -1 The pH of the solution is adjusted to 11, and the solution is subjected to self-assembly after being subjected to continuous ultrasonic treatment for 1h, so that the milky emulsion is obtained. Adding 1mmolL of final concentration to the solution -1 The dopamine hydrochloride is reacted for 40 minutes at room temperature, and after the reaction, the dopamine hydrochloride is purified by centrifugation and is freeze-dried for storage.
1mL of 10mM ferric chloride (FeCl) was added dropwise to the above solution 3 ) The aqueous solution is stirred for 5 minutes to obtain the surface-loaded paramagnetic iron oxide (Fe) 3 O 4 NPs) nanocarrier Fmoc-Lys (Fe) 3 O 4 /DXMS)-PEG。
7.2HI44a on nanocarriers (Fmoc-Lys (Fe) 3 O 4 Coupling to/DXMS) -PEG
1mg of HI44a was sufficiently dissolved in ultrapure water, and 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.5mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. 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 Fmoc-Lys (Fe 3 O 4 Coupling HI44a is realized in NP/DXMS) -PEG solution, and the targeting recognition nano carrier Fmoc-Lys (Fe) is obtained 3 O 4 NP/DXMS) -HI44a/PEG. FIG. 13 shows Fmoc-Lys (Fe 3 O 4 TEM results of/DXMS) -HI44a/PEG. FIG. 14 shows Fmoc-Lys (Fe 3 O 4 Fmoc-Lys (Fe) after adding targeting ligand to/DXMS) -PEG nano-carrier 3 O 4 Infrared results of/DXMS) -HI44a/PEG nanocarriers.
7.3 preparation of conjugated monoclonal antibody HI44a, fmoc-Lys nanocarrier Fmoc-Lys (GPA) -HI44a/PEG loaded with MRI tracer
According to example 2Fmoc-Lys-PEG was prepared. Fmoc-Lys 50mg and 5mg Fmoc-Lys-PEG molecules were weighed, dissolved in 20mL of ultra pure water to completely dissolve them, and 1mg mL was added dropwise under ultrasonic conditions -1 Meglumine Gadoterate (GPA) in DMF using 10mg mL -1 The pH of the solution is adjusted to 11, and the solution is subjected to self-assembly after being subjected to continuous ultrasonic treatment for 1h, so that the milky emulsion is obtained. Adding 1mmolL of final concentration to the solution -1 The dopamine hydrochloride is reacted for 40 minutes at room temperature, and after the reaction, the dopamine hydrochloride is purified by centrifugation and is freeze-dried for storage.
1mg of HI44a was sufficiently dissolved in ultrapure water, and 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.5mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. 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 the purified Fmoc-Lys (GPA) -PEG solution to effect HI44a coupling to give the targeted recognition nanocarrier Fmoc-Lys (GPA) -HI44a/PEG. FIG. 15 shows SEM results of Fmoc-Lys (GPA) -HI44a/PEG.
Substitution of GPA with Gadodiamine (GSA), gadofoshan (GPS) gives the corresponding Fmoc-Lys (GSA) -HI44a/PEG, fmoc-Lys (GPS) -HI44a/PEG nanophotographic agents. FIG. 16 shows SEM results of Fmoc-Lys (GSA) -HI44a/PEG. FIG. 17 shows SEM results of Fmoc-Lys (GPS) -HI44a/PEG.
Example 8 Simultaneous Asp-loading of clopidogrel modified with polyethylene glycol, collagen (Col) Preparation of (Clo) Fmoc-Lys nanocarriers (Fmoc-Lys (Asp/Clo) -Col/PEG)
Fmoc-Lys-PEG was prepared as in example 2. Fmoc-Lys 50mg and 5mg Fmoc-Lys-PEG molecules were weighed, dissolved in 20mL of ultra pure water to completely dissolve them, and 1mg mL was added dropwise under ultrasonic conditions -1 Asp (Asp, 1 mL) and 1mg mL -1 Clopidogrel (Clo, 1 mL) in DMF was used 10mg mL -1 The pH of the solution was adjusted to 11 by means of NaOH and self-assembly was carried out by means of a continuous ultrasonic treatment for 1h, a milky emulsion was obtained, to which solution a final concentration of 1mmolL L was added -1 The dopamine hydrochloride is reacted for 40 minutes at room temperature, and after the reaction, the dopamine hydrochloride is purified by centrifugation and is freeze-dried for storage. Centrifugal purification and freeze-drying to obtain Fmoc-Lys (A)sp/Clo) -PEG. FIG. 18 shows the IR results of Fmoc-Lys (Asp/Clo) -PEG nanocarriers.
10mg of collagen (col) was fully dissolved in ultrapure water, and 3mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 3mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. After stirring the reaction at room temperature for 1 hour, activated Col was obtained by ultrafiltration and centrifugation. 1.0mL of Col solution is respectively absorbed and dissolved in purified Fmoc-Lys (Asp/Clo) -PEG solution, and the coupling of Col is realized, so that the targeting recognition nano-carrier Fmoc-Lys (Asp/Clo) -Col/PEG is obtained. FIG. 18 shows the IR results for Fmoc-Lys (Asp/Clo) -Col/PEG carriers.
Test example 1 Property investigation of Nano delivery System
In this test example, the therapeutic agent-loaded nano-delivery systems prepared in examples 1 to 8 were exemplified to demonstrate that the delivery system of the present invention has stable and controllable properties, and thus is 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 nanocarriers of the delivery system of the present invention,
the hydrated particle sizes were measured by a laser particle sizer (BI-Zeta Plus/90Plus, bruce sea (Brookhaven Instruments Corporation), U.S.), and the specific results are shown in Table 1.
3. Determination of encapsulation efficiency:
taking a certain amount of nano-carrier, fmoc-Lys (R) -HA, fmoc-Lys (R) -SP/PEG, fmoc-Arg (R) -HA/PEG, fmoc-Arg (At) -SEP/PEG, fmoc-Arg (At/miRNA-33) -IM7, fmoc-Lys (AuNP/At) -OPN/PEG, fmoc-Lys (Fe) 3 O 4 The drug release from the dendritic nanocarriers was accelerated by adding an excess of methanol/formic acid solution (100:1 volume ratio) at pH 2.0 to/DXMS) -HI44a/PEG, fmoc-Lys (Asp/Clo) -Col/PEG, and heating in a water bath for 2 hours at 60℃and further using an ultrasonic method. 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% … … … … … equation 1
Table 1 list of various properties
Figure GDA0004196567460000211
Figure GDA0004196567460000221
Note that: 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 was stored at 4℃and sampled at various time points and tested for changes in hydrated particle size by a laser particle sizer (BI-Zeta Plus/90Plus, bruce Haievin (Brookhaven Instruments Corporation), USA). FIG. 19 is a graph showing the effect of different shelf life on hydrated particle size.
5. Long-term stability investigation
The nano-delivery system of the present invention was stored at 4 ℃ and sampled at various time points, and the change in encapsulation efficiency was examined by removing free drug by ultrafiltration centrifugation. Fig. 20 shows the effect of different shelf-life on encapsulation efficiency.
6. In vitro drug release performance study
2mL of the nano-delivery system of the present invention was placed in a dialysis bag and sealed. 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 GDA0004196567460000231
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 medicine: 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.
Figure 21 shows the in vitro cumulative release rate (CRP%) of the nano-delivery system.
Experimental example 2 Fmoc-Lys (R) -HA, fmoc-Lys (R) -SP/PEG, fmoc-Arg (R) -HA/PEG of the present invention, Fmoc-Arg (At) -SEP/PEG, fmoc-Arg (At/miRNA-33) -IM7 nano-drug delivery system for vulnerable plaque of artery In vivo experiments of the influence of (a)
Hyaluronic Acid (HA) and Selectin (SP) 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 Fmoc-Lys (R) -HA, fmoc-Lys (R) -SP/PEG, fmoc-Arg (R) -HA/PEG, fmoc-Arg (At) -SEP/PEG, fmoc-Arg (At/miRNA-33) -IM7 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 methods described in examples 1-5 above were used to prepare therapeutic agent-loaded amino acid self-assembled nano-delivery systems.
(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;
Fmoc-Lys (R) -HA group: intravenous administration at a dose of 0.66mg rosuvastatin/kg body weight;
Fmoc-Lys (R) -SP/PEG group: intravenous administration at a dose of 0.66mg rosuvastatin/kg body weight;
Fmoc-Arg (R) -HA/PEG group: intravenous administration was performed at a dose of 0.66mg rosuvastatin/kg body weight.
Fmoc-Arg (At) -SEP/PEG group: intravenous administration at a dose of 1.2mg atorvastatin/kg body weight;
Fmoc-Arg (At/miRNA) -IM7 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. 22 shows the in vivo therapeutic effect of Fmoc-Lys (R) -HA, fmoc-Lys (R) -SP/PEG, fmoc-Arg (R) -HA/PEG, fmoc-Arg (At) -SEP/PEG, fmoc-Arg (At/miRNA-33) -IM7 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 32.9%; rosuvastatin treatment can delay plaque progression, but also 30.1%; intravenous atorvastatin also delayed plaque progression but also progressed by 31.7%; whereas targeted nanodrug delivery treatment significantly suppressed plaque progression even with reversal and regression of plaque volume, the Fmoc-Lys (R) -HA group eliminated plaque by 8.27%, the Fmoc-Lys (R) -SP/PEG group eliminated plaque by 12.21%, the Fmoc-Arg (R) -HA/PEG group eliminated plaque by 11%, the Fmoc-Arg (At) -SEP/PEG group eliminated plaque by 6.23%, and the Fmoc-Arg (At/miRNA-33) -IM7 group eliminated plaque by 12.49%.
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.
Experimental example 3 Effect of Fmoc-Lys (AuNP/At) -OPN/PEG delivery System of the invention on vulnerable plaque in arteries In vivo experiments (CT tracing and therapeutic dual-function)
Osteopontin (OPN) is a ligand of CD44, and can play a role in targeting vulnerable plaques, atorvastatin (At) 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 atorvastatin-loaded nano-delivery system of the present invention on vulnerable arterial plaque.
(1) A physiological saline solution of free atorvastatin was formulated and the CT tracer and therapeutic agent loaded amino acid self-assembled nano-delivery system was prepared using the method described in example 6 above.
(2)ApoE -/- The method for establishing the 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;
Fmoc-Lys (AuNP/At) -OPN/PEG group: the dosage of the nano gold is 0.1mg/kg body weight;
Fmoc-Lys (DPLA) -OPN/PEG group: the dose of iopromide is 0.1mg/kg body weight; fmoc-Lys (DKSC) -OPN/PEG group; iodixanol is administered at a dose of 0.1mg/kg body weight; fmoc-Lys (DFC) -OPN/PEG group: the dose of iofluor was 0.1mg/kg body weight.
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;
atorvastatin lavage group: gastric lavage treatment was performed at a dose of 20mg atorvastatin/kg body weight;
atorvastatin intravenous group: intravenous administration at a dose of 1.2mg atorvastatin/kg body weight;
Fmoc-Lys (AuNP/At) -OPN/PEG 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:
figure 23 demonstrates the in vivo tracer effect of the tracer-loaded amino acid delivery systems 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. Compared with free nano gold particles, when nano gold, iopromide, iodixanol and iofluool are formulated in a targeted amino acid delivery system, the tracing effect on vulnerable plaque is remarkably improved. In conclusion, compared with free nano gold particles, the amino acid delivery system with the surface modified with the targeting ligand can remarkably improve the identification effect of nano gold on vulnerable arterial plaque and generate better tracing effect.
FIG. 24 shows the in vivo therapeutic effect of Fmoc-Lys (AuNP/At) -OPN/PEG system of the invention on vulnerable arterial plaque. As shown, during the course of high fat diet feeding (10 days), atherosclerosis in the control group (without any treatment) progressed by 23%; the atorvastatin gastric lavage treatment can delay plaque progression, but also progress by 21%; intravenous atorvastatin also delayed plaque progression but also progressed by 21.5%; whereas targeted nanodrug delivery treatment significantly suppressed plaque progression, even with reversal and regression of plaque volume, fmoc-Lys (AuNP/At) -OPN/PEG resolved plaque 7.2%.
In summary, free atorvastatin exhibits a therapeutic effect on arterial vulnerable plaque in mice, whether administered by gavage or by intravenous injection, but it is unable to reverse vulnerable plaque. However, when atorvastatin and nanogold are formulated in the nano-delivery system disclosed by the invention, the diagnosis and treatment effects on vulnerable plaques are remarkably improved, and the early warning of high-risk patients and the treatment effects on plaque reduction are achieved.
Experimental example 4 Fmoc-Lys (Fe 3 O 4 Delivery System for arterial vulnerable plaque In vivo tracer assay (MRI tracer) and anti-inflammatory treatment
The monoclonal antibody (HI 44 a) is an antibody of CD44 and can play a role in targeting vulnerable plaqueDexamethasone (DXMS) has anti-inflammatory and plaque progression inhibiting effects, fe 3 O 4 Is an MRI tracer. The aim of the example is to verify the in vivo tracing and therapeutic effect of the MRI tracer and dexamethasone-loaded amino acid self-assembled nano-delivery system of the invention on vulnerable arterial plaque. In addition, gadoteric acid meglumine, gadodiamide and gadopentetic acid can also be prepared into nano preparations, and the targeting MRI tracing effect is shown.
(1) An amino acid self-assembled nano-delivery system loaded with an MRI tracer and a therapeutic agent was prepared using the method described in example 7 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
Fmoc-Lys(Fe 3 O 4 /DXMS) -HI44a/PEG group: fe (Fe) 3 O 4 Is administered at a dose of 0.1mg/kg body weight;
Fmoc-Lys (GPA) -HI44a/PEG group: the administration dose of gadoteridol is 0.1mg/kg body weight;
Fmoc-Lys (GSA) -HI44a/PEG group: the dosage of gadolinium diamine is 0.1mg/kg body weight;
Fmoc-Lys (GPS) -HI44a/PEG group: the dose of gadofosprate is 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.
(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;
Fmoc-Lys(Fe 3 O 4 /DXMS) -HI44a/PEG group: intravenous administration at a dose of 0.1mg dexamethasone/kg body weightProcessing;
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 25 demonstrates the in vivo tracer effect of the tracer-loaded amino acid delivery systems 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 the compound is prepared in a targeted amino acid delivery system, the trace effect of vulnerable plaque is remarkably improved, and other MRI nanometer contrast agents are adopted, so that the trace effect of vulnerable plaque is good. In conclusion, compared with the free MRI tracer, the amino acid delivery system with the surface modified with the targeting ligand can remarkably improve the identification effect of the MRI tracer on vulnerable arterial plaque and generate better tracing effect.
FIG. 26 shows Fmoc-Lys (Fe 3 O 4 In vivo therapeutic effects of the/DXMS) -HI44a/PEG system on vulnerable arterial plaque. As shown, during the course of high fat diet feeding (10 days), atherosclerosis in the control group (without any treatment) progressed by 31%; while targeted nanodrug delivery treatment significantly inhibited plaque progression, even with reversal and regression of plaque volume, fmoc-Lys (Fe 3 O 4 The plaque was resolved by 8% by/DXMS) -HI44 a/PEG.
Taken together, when dexamethasone and Fe are used for treating arterial vulnerable plaque in mice 3 O 4 When the amino acid self-assembled nano delivery system is prepared in the invention, the diagnostic and therapeutic effects of vulnerable plaque are obviously improved, and the early warning of high-risk patients and the reversion of plaque are realizedTherapeutic effects of block growth (plaque reduction).
Test example 5 Effect of Fmoc-Lys (Asp/Clo) -Col/PEG delivery System of the invention on vulnerable plaque in arteries In vivo experiments in (2)
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 Fmoc-Lys (Asp/Clo) -Col/PEG carrier delivery systems of the present invention on vulnerable arterial plaque.
The experimental method comprises the following steps:
(1) Physiological saline solutions of free aspirin and clopidogrel were formulated and a therapeutic agent-loaded amino acid self-assembled nano-delivery system was prepared using the method described in example 8 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;
Fmoc-Lys (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. 27 shows the in vivo therapeutic effect of Fmoc-Lys (Asp/Clo) -Col/PEG system of the present invention on vulnerable arterial plaque. As shown, mice in the control group (not given any treatment) had a mortality of 33%; the aspirin and clopidogrel are adopted for gastric lavage treatment, so that the death rate can be reduced to 29%; fmoc-Lys (Asp/Clo) -Col/PEG treatment enabled a reduction in mortality to 16%. From the bleeding time, fmoc-Lys (Asp/Clo) -Col/PEG groups were not significantly prolonged, whereas mice orally administered aspirin and clopidogrel had significantly prolonged bleeding times.
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 (42)

1. An amino acid self-assembled nanocarrier delivery system for targeted activation of CD44 molecules, characterized in that the nanocarrier is Fmoc-modified lysine or arginine, the nanocarrier has a hollow spherical structure, and the surface of the nanocarrier is partially modified by a targeting ligand, which is a ligand capable of specifically binding to an activated CD44 molecule, selected from the group consisting of GAG, collagen, laminin, fibronectin, selectin, osteopontin, and 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, which is a pharmaceutically acceptable salt of hyaluronic acid, an alkyl ester containing 1-6 carbon atoms, a prodrug 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.
2. The nanocarrier delivery system of claim 1, wherein the nanocarrier surface is further amenable to other modifications.
3. The nanocarrier delivery system of claim 2, 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 support.
4. The nanocarrier delivery system of claim 1, wherein the nanocarriers have a particle size of 50-1000 nm.
5. The nanocarrier delivery system of claim 4, wherein the nanocarriers have a particle size of 200 nm.
6. The nanocarrier delivery system of claim 1, wherein the nanocarriers are prepared by self-assembly of raw materials in alkaline conditions.
7. The nanocarrier delivery system of claim 6, wherein the alkaline condition is a pH >8.
8. The nanocarrier delivery system of claim 7, wherein the alkaline condition is a pH >10.
9. The nanocarrier delivery system of claim 8, wherein the alkaline condition is ph=11.
10. An amino acid self-assembled nanocarrier delivery system for targeting vulnerable plaques, characterized in that the nanocarrier is Fmoc-modified lysine or arginine, the nanocarrier has a hollow spherical structure, and the surface of the nanocarrier is partially modified by a targeting ligand, which is a ligand capable of specifically binding to an activated CD44 molecule, selected from the group consisting of GAG, collagen, laminin, fibronectin, selectin, osteopontin, and 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, which is a pharmaceutically acceptable salt of hyaluronic acid, an alkyl ester containing 1-6 carbon atoms, a prodrug 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.
11. The nanocarrier delivery system of claim 10, wherein the nanocarrier surface is further amenable to other modifications.
12. The nanocarrier delivery system of claim 11, 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 support.
13. The nanocarrier delivery system of claim 10, wherein the nanocarriers have a particle size of 50-1000 nm.
14. The nanocarrier delivery system of claim 13, wherein the nanocarriers have a particle size of 200 nm.
15. The nanocarrier delivery system of claim 10, wherein the nanocarriers are prepared by self-assembling raw materials in alkaline conditions.
16. The nanocarrier delivery system of claim 15, wherein the alkaline condition is a pH >8.
17. The nanocarrier delivery system of claim 16, wherein the alkaline condition is a pH >10.
18. The nanocarrier delivery system of claim 17, wherein the alkaline condition is ph=11.
19. The nanocarrier delivery system of any of claims 1 to 18, wherein the targeting ligand is selected from the group consisting of self peptide, collagen, hyaluronic acid, selectin, osteopontin, or monoclonal antibody HI44a, IM7.
20. The nanocarrier delivery system of any of claims 1 to 18, 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.
21. The nanocarrier delivery system of any of claims 1 to 18, wherein the nanocarrier is loaded with both a substance for preventing and/or treating 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, which derivative of hyaluronic acid is a pharmaceutically acceptable salt of hyaluronic acid, an alkyl ester containing 1-6 carbon atoms, a prodrug capable of forming hyaluronic acid in vivo via hydrolysis or other means.
22. The nanocarrier delivery system of claim 21, which is simultaneously loaded with a substance for diagnosing vulnerable plaques, a substance for preventing and/or treating vulnerable plaques, 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 plaques, which is a pharmaceutically acceptable salt of hyaluronic acid, an alkyl ester containing 1-6 carbon atoms, a prodrug capable of forming hyaluronic acid in vivo via hydrolysis or other means.
23. The nanocarrier delivery system of claim 20, 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.
24. The nanocarrier delivery system of claim 23, wherein the CD44 activator is a CD44 antibody mAb or IL5, IL12, IL18, TNF-a, LPS.
25. The nanocarrier delivery system of any of claims 1 to 18, wherein the substance for diagnosing, preventing and/or treating vulnerable plaque is selected from one or more of a drug, polypeptide, nucleic acid, and cytokine for diagnosing, preventing and/or treating vulnerable plaque.
26. The nanocarrier delivery system of any of claims 1 to 18, wherein the substance for diagnosing, preventing and/or treating vulnerable plaque is a substance for diagnosing vulnerable plaque.
27. The nanocarrier delivery system of claim 26, wherein the substance for diagnosing vulnerable plaque is a tracer.
28. The nanocarrier delivery system of claim 27, wherein the tracer is selected from the group consisting of a CT tracer, an MRI tracer, and a nuclide tracer.
29. The nanocarrier delivery system of claim 28, wherein the CT tracer is selected from the group consisting of an iodine nanocontrast agent, a gold nanocontrast agent, a tantalum oxide nanocontrast agent, a bismuth nanocontrast agent, a lanthanide nanocontrast agent;
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.
30. The nanocarrier delivery system of claim 29, 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.
31. The nanocarrier delivery system of claim 30, 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 iobenzoate, propidone, ioaone, iotrolan, iopidol, meglumine of iotazinate, diatrizamine, mezoic acid, meglumine of iodate, or 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.
32. The nanocarrier delivery system of claim 31, wherein the CT tracer is nanogold;
the MRI tracer is selected from gadofoshan, ferric ammonium citrate effervescent granule, and paramagnetic ferric oxide.
33. The nanocarrier delivery system of claim 32, wherein the MRI tracer is paramagnetic iron oxide.
34. The nanocarrier delivery system of any of claims 1 to 18, wherein the substance for diagnosing, preventing and/or treating vulnerable plaque is a substance for preventing and/or treating vulnerable plaque.
35. The nanocarrier delivery system of claim 34, wherein the substance for preventing and/or treating vulnerable plaque is selected from one or more of a statin, a fibrate, an antiplatelet drug, a PCSK9 inhibitor, an anticoagulant drug, 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.
36. The nanocarrier delivery system of claim 35, wherein, 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, troxerutin, dipyridamole, cilostazol, ticlopidine hydrochloride, ozagrel sodium, clopidogrel, prasugrel, cilostazol, beraprost sodium, ticagrelor, cangrelor, tirofiban, eptifibatide, acipimab, common heparin, kesai, fast-Bilin, huang Dagan sodium, warfarin, dabigatran, rivaroxaban, apixaban, irishaban, bivalirudin, enoxaparin, tetalaheparin, aclidinium, biscoumarin, coumarin nitrate, sodium medlar, hirudin, argatroban, benazepril, captopril, enalapril, perindopril, fosinopril, lisinopril, moexipril, cilazapril, perindopril, quinapril, ramipril, trandolapril, candesartan, eprosartan, irbesartan, losartan, telmisartan, valsartan, olmesartan, tartan, nifedipine, nicardipine, nimodipine, enopril, telmisartan, valsartan, one or more of nisoldipine, nilvadipine, isradipine, felodipine, lacidipine, diltiazem, verapamil, chlorhexidine, minocycline, MMI-166, metoprolol, atenolol, bisoprolol, propranolol, carvedilol, pamamastat, marimastat, praline stat, BMS-279251, BAY 12-9566, TAA211, AAJ996A, nacetrapib, evacetrapib, torcetrapib, dalcetrapib, prednisone, methylprednisone, betamethasone, beclomethasone propionate, deborosone, prednisolone, hydrocortisone, dexamethasone, IL-1 antibody canakinumab, or a pharmaceutically acceptable salt thereof, or an endogenous anti-inflammatory cytokine.
37. The nanocarrier delivery system of claim 36, 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.
38. The method of any one of claims 1 to 37, wherein the method comprises the step of adding dopamine to consolidate the nanocarriers.
39. A pharmaceutical composition comprising the nanocarrier delivery system of any of claims 1 to 37.
40. A diagnostic formulation comprising the nanocarrier delivery system of any of claims 1 to 37.
41. Use of the nanocarrier delivery system of any of claims 1 to 37 in the manufacture of a product for the diagnosis, prevention and treatment of vulnerable plaques.
42. The use of claim 41, 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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