CN112206326A - Amino acid self-assembly nano-carrier delivery system for targeting activation of CD44 molecule, preparation method and application thereof - Google Patents

Amino acid self-assembly nano-carrier delivery system for targeting activation of CD44 molecule, preparation method and application thereof Download PDF

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CN112206326A
CN112206326A CN202010662240.3A CN202010662240A CN112206326A CN 112206326 A CN112206326 A CN 112206326A CN 202010662240 A CN202010662240 A CN 202010662240A CN 112206326 A CN112206326 A CN 112206326A
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fmoc
vulnerable plaque
nanocarrier
delivery system
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CN112206326B (en
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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-assembly nano carrier delivery system for targeting and activating a CD44 molecule, wherein the nano carrier is an Fmoc modified amino acid or short peptide nano carrier, the nano carrier has a hollow spherical structure, the surface of the nano carrier is partially modified by a targeting ligand, and the targeting ligand is a ligand capable of being specifically combined with an activated CD44 molecule. Also provides a preparation method and application of the amino acid self-assembly nano carrier. The Fmoc-AC self-assembly 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-assembly nano-carrier delivery system for targeting activation of CD44 molecule, 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-assembly nano-carrier delivery system for targeted activation of CD44 molecules, and a preparation method and application thereof.
Background
At present, acute cardiovascular events mainly including acute myocardial infarction and sudden cardiac death become the first killers endangering human health. Statistically, about 2 million people die worldwide each year from acute cardiovascular events. In china, the situation is also not optimistic, and more than 70 million people die each year 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 last 70 s, the development process and mechanism of Acute Coronary Syndrome (ACS) and cerebral apoplexy caused by chronic atherosclerotic plaques have been explored.
In 1989, Muller and its team proposed the concept of "vulnerable plaques," which were thought to be the root cause of most acute cardiovascular events. Vulnerable plaque (also known as "unstable plaque") refers to atherosclerotic plaque that has a propensity to thrombus formation or is highly likely to rapidly progress into "plaque of offenders," and includes primarily ruptured plaque, erosive plaque, and partially calcified nodular lesions. A large number of studies indicate that most of acute myocardial infarction and cerebral apoplexy are caused by secondary thrombosis due to rupture of vulnerable plaques with mild and moderate stenosis. Naghavi and his team, etc. give histological definitions and criteria for vulnerable plaques. The main criteria include active inflammation, thin fibrous cap and large lipid core, endothelial denudation with surface platelet aggregation, fissuring or damaging plaque and severe stenosis. Secondary criteria included surface calcified plaques, yellow shiny plaques, intraplaque hemorrhage and positive remodeling. Therefore, early intervention is critical for vulnerable plaque. However, since the degree of stenosis of blood vessels due to vulnerable plaque is not high in general, many patients do not have precursor symptoms, and early diagnosis is difficult in clinic, so that the risk is extremely high. Therefore, how to identify and diagnose vulnerable plaque as early as possible and perform effective intervention becomes an urgent problem to be solved in preventing and treating acute myocardial infarction.
The techniques commonly used for vulnerable plaque diagnosis at present mainly include coronary angiography, intravascular ultrasound (IVUS), laser coherence tomography (OCT), etc., but these techniques are invasive examinations, and the diagnostic resolution and accuracy are not high, and these diagnostic techniques are expensive, and also limit the clinical popularization to some extent. Therefore, there is an urgent need for noninvasive diagnostic techniques and formulations for vulnerable plaques.
In addition, current methods of treating vulnerable plaques are primarily systemic administration, such as oral statins, aspirin, Matrix Metalloproteinase (MMPs) inhibitors, and/or fibrates, and the like. These drugs act to stabilize the plaque by regulating systemic blood lipids, fighting against inflammation, inhibiting protease and platelet production, etc., to reduce lipid in the plaque, improve vascular remodeling, etc. However, the therapeutic efficacy of current drugs used to treat vulnerable plaques is not ideal for clinical use. For example, the bioavailability of statins commonly used in clinical settings is low when administered orally, e.g., simvastatin is < 5%, atorvastatin is about 12%, and rosuvastatin is about 20%. Animal experiments also demonstrate that increasing the statin dose above 1mg/kg acts to increase the fibrous cap thickness and reduce plaque volume, which creates bottlenecks in the stability of oral statin administration and the effectiveness of reversing the plaque. Clinical trials have also confirmed that oral statin therapy for vulnerable plaques requires intensive high doses to have the effect of stabilizing vulnerable plaques, while systemic high dose statin therapy also risks an increased incidence of serious side effects (e.g. liver dysfunction, rhabdomyolysis, type II diabetes, etc.).
For the existing systemic administration, only a few effective components of the medicine can actually act on the pathological part after entering the body. This is the root cause of the drug effect restriction and the toxic and side effects. The targeted drug delivery system refers to a drug delivery system with targeted drug delivery capability. After being administered by a certain route, the drug contained in the targeting drug delivery system can be specifically enriched at the target site through the carrier with the targeting probe. The targeted drug delivery system can target the drug at a specific lesion site and release the effective ingredients at the target lesion site. Therefore, the targeted drug delivery system can enable the drug to form relatively high concentration at the target lesion site and reduce the dosage in blood circulation, thereby improving the drug effect, inhibiting toxic and side effects and reducing the damage to normal tissues and cells.
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 the improvement on the bioavailability of the drug is limited finally. In addition, the liposome has insufficient in vitro stability, phospholipid is easy to oxidize and hydrolyze during storage, and liposome vesicles are easy to aggregate and fuse with each other, so that the medicine encapsulated in the liposome vesicles is 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 by modifying nanocarriers with targeting ligands. However, a major problem with such targeting probes targeting vulnerable plaques in clinical practice is the insufficient specificity of the targeting site of these formulations. For example, the targeting site for such agents is mostly macrophage selective, but the targeting specificity of the probe is less than ideal since macrophages may be present throughout the body. Thus, a difficulty in the development of targeted formulations that target vulnerable plaques is finding a target in the cells within the vulnerable plaque with significant targeting specificity.
CD44 is a class of adhesion molecules that are widely distributed on the surface of lymphocytes, monocytes, endothelial cells, and the like. 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 (unable to bind HA), an induced activation state (able to bind HA after activation), and a structurally active state (able to bind HA without activation), whereas most normal cell surface CD44 is in a relatively quiescent state and thus unable to bind HA.
Numerous studies have followed to indicate that CD44 is not an ideal target with significant targeting specificity. This is because CD44 is widely distributed in the human body, especially on the surface of organs rich in reticuloendothelial mass. Therefore, the following problems are encountered in the development of a targeted drug delivery system targeting CD 44: such targeted drug delivery systems do not present specific targeting properties if the affinity of CD44 on the surface of the target cells to HA is insufficient to provide significant specificity.
Therefore, finding a specific target site existing at a vulnerable plaque part and a targeted drug delivery system suitable for targeting the vulnerable plaque, thereby developing a targeted drug delivery system capable of specifically targeting the vulnerable plaque and simultaneously realizing stable and sustained release of a drug, has become an urgent technical problem in the medical field.
To date, there HAs been no report on the expression state of CD44 on the surface of macrophages, monocytes, endothelial cells, lymphocytes and smooth muscle cells, which are mainly present within vulnerable plaque, and its affinity for HA, nor is there any prior art on designing a targeted drug delivery system capable of achieving stable sustained release of a drug for diagnosing or treating vulnerable plaque or a disease associated with vulnerable plaque using the interaction of HA and CD44 and the specific microenvironment of vulnerable plaque.
The amino acid or short peptide after chemical modification can be self-assembled into ordered spherical, vesicular and hollow tubular nano-structures through weak interaction between molecules under certain solution conditions, and the structures are maintained through pi-pi stacking effect generated by hydrogen bonds, benzene ring structures and amido bonds in molecules. The nano-drug carrier 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 clearance time in vivo, so that the continuity of the drug delivery time is hindered, and a scheme for solving the problems is a key for further exerting the drug delivery capacity of the amino acid or short peptide nano-carrier.
Disclosure of Invention
Therefore, the present invention aims to overcome the defects in the prior art and provide an amino acid self-assembly nano-carrier delivery system for targeting activation of CD44 molecule, a preparation method and application thereof.
Before setting forth the context of the present invention, the terms used herein are defined as follows:
the term "SEM" refers to: scanning electron microscopy.
The term "FT-IR" means: fourier transform infrared spectroscopy.
The term "TEM" refers to: transmission electron microscopy.
The term "Fmoc" means: fluorenylmethyloxycarbonyl.
The term "PEG" refers to: polyethylene glycol.
The term "edc.hcl" refers to: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
The term "sulfo-NHS" means: n-hydroxy thiosuccinimide.
The term "Col" means: collagen protein.
The term "SP" refers to: and (4) selecting the protein.
The term "OPN" refers to: osteopontin
The term "Col" means: collagen protein.
The term "Asp" means: aspirin.
The term "Clo" means: clopidogrel.
The term "At" means: atorvastatin.
The term "DXMS" refers to: dexamethasone.
The term "R" means: rosuvastatin.
The term "FDG" refers to: fluorodeoxyglucose.
The term "DPLA" refers to: iopromide.
The term "DKSC" refers to: iodixanol.
The term "DFC" refers to: an iodofluoroalcohol.
The term "GPA" refers to: gadoteric acid glucamine.
The term "GSA" refers to: gadolinium diamine.
The term "GPS" refers to: gadolinium pentaacetic acid.
"vulnerable plaque" also known as "unstable plaque" refers to atherosclerotic plaques having a propensity to thrombosis or a high probability of rapidly progressing to "offending plaque", and mainly includes ruptured plaques, erosive plaques, and partially calcified nodular lesions. A large number of studies indicate that most of acute myocardial infarction and cerebral apoplexy are caused by secondary thrombosis due to rupture of vulnerable plaques with mild and moderate stenosis. Histological manifestations of vulnerable plaque include active inflammation, thin fibrous caps and large lipid cores, endothelial denudation with surface platelet aggregation, fissuring or lesions of the plaque and severe stenosis, as well as surface calcified plaques, yellow shiny plaques, intra-plaque hemorrhage and positive remodeling.
"disease associated with vulnerable plaque" refers primarily to disease associated with "vulnerable plaque" during the development and progression of the disease, characterized by, caused by, or secondary to "vulnerable plaque". The disease related to vulnerable plaque mainly comprises diseases such as atherosclerosis, coronary atherosclerotic heart disease (including acute coronary syndrome, asymptomatic myocardial ischemia-occult coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, in-stent restenosis), cerebral atherosclerosis (including cerebral apoplexy), peripheral vascular atherosclerosis (including obliterative peripheral atherosclerosis, retinal atherosclerosis, carotid atherosclerosis, renal atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, atherosclerotic impotence), aortic dissection, hemangioma, thromboembolism, heart failure and cardiogenic shock.
"Targeted drug delivery system" refers to drug delivery systems having the ability to deliver drugs in a targeted manner. After administration by a certain route, the drug contained in the targeted drug delivery system is specifically enriched at the target site by the action of a special carrier or targeting warhead (e.g., targeting ligand). Currently known means for achieving targeted drug delivery include utilizing passive targeting properties of various particulate drug delivery systems, chemical modification of the surface of the particulate drug delivery system, 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. The ligand-mediated targeting drug delivery is characterized in that a specific receptor on certain organs and tissues can be specifically combined with a specific ligand, and a drug carrier is combined with the ligand, so that the drug is guided to a specific target tissue.
"hyaluronic acid (abbreviated as" HA ")" is a high molecular polymer of the formula: (C)14H21NO11) n is the same as the formula (I). It is a high-grade 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 exhibits various important physiological functions in the body due to its unique molecular structure and physicochemical properties, such as joint lubrication, regulation of permeability of vascular walls, regulation of proteins, and hydroelectric powerThe diffusion and the operation of the electrolyte, the healing of the wound and the like. More importantly, hyaluronic acid has a special water retention effect and is the best substance found in nature for retaining moisture.
By "derivative of hyaluronic acid" is meant herein any derivative of hyaluronic acid capable of retaining the hyaluronic acid's ability to specifically bind to the CD44 molecule 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 by hydrolysis or other means in vivo, and the like. It is within the skill of the art to determine whether a substance is a "derivative of hyaluronic acid" by determining the ability of the substance to specifically bind to the CD44 molecule on the cell surface at vulnerable plaque.
The "CD 44 molecule" is a kind of transmembrane proteoglycan adhesion molecule widely expressed in cell membrane of lymphocyte, monocyte, endothelial cell, etc. and consists of three sections, including extracellular section, transmembrane section and intracellular section. The CD44 molecule can mediate various cell-cell interactions and cell-extracellular matrix interactions, participate in the transduction of various signals in vivo, and change the biological functions 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, the CD44 molecule is also involved in the metabolism of hyaluronic acid.
"about" represents the set of all values within 5% of the value given thereafter.
The invention provides an amino acid self-assembly nano carrier delivery system for targeting an activated CD44 molecule, wherein the nano carrier is an Fmoc modified amino acid or short peptide nano carrier, the nano carrier has a hollow spherical structure, the surface of the nano carrier is partially modified by a targeting ligand, and the targeting ligand is a ligand capable of being specifically combined with the activated CD44 molecule.
The second aspect of the present invention provides an amino acid self-assembly nanocarrier delivery system for targeting vulnerable plaques, wherein the nanocarrier is an Fmoc-modified amino acid, or a 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 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 the following: 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 particle size of the nanocarrier is 50 to 1000nm, preferably 200 nm.
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 conditions are 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 derivatives of the monoclonal antibody HI44a, HI313, A3D8, H90, IM7, or hyaluronic acid capable of specifically binding to the CD44 molecule 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 antibody HI44a, IM 7.
The nanocarrier delivery system according to the first aspect or the second aspect of the invention, wherein the surface of the nanocarrier can be modified by modifying one or more of polyethylene glycol, a cell-penetrating peptide, a self-peptide on the surface of the nanocarrier, or by modifying both ligands.
The nanocarrier delivery system according to the first or second aspect of the invention, wherein the nanocarriers are loaded with a substance for diagnosing, preventing and/or treating a disease associated with the occurrence of a condition of activation of the CD44 molecule; and/or
The nanocarrier is loaded with a substance useful 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 the CD44 molecule 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, an optional CD44 activator and optionally hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to the CD44 molecule on the cell surface at vulnerable plaque.
The nanocarrier delivery system according to the first or second aspect of the invention, wherein the agent for diagnosing, preventing and/or treating a disease associated with the occurrence of a condition of activation of the CD44 molecule is a CD44 activator;
preferably, the activator of CD44 is the CD44 antibody mAb or IL5, IL12, IL18, TNF-a, 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, a polypeptide, a nucleic acid and a 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 a CT tracer, an MRI tracer and a nuclear tracer;
even more preferably:
the CT tracer is selected from an iodine nano contrast agent, a gold nano contrast agent, a tantalum oxide nano contrast agent, a bismuth nano contrast agent, a lanthanide series nano contrast agent or other tracers with similar structures; more preferably an iodinated contrast agent or nanogold, or other tracer of similar structure; further preferably iohexol, iocarmic acid, ioversol, iodixanol, iopromide, iobitrol, iomeprol, iopamidol, ioxilan, iozofenac acid, iodipamoic acid, iobenzamic acid, iodoglycanic acid, diatrizoic acid, sodium iothalamate, iodophenyl ester, iopanoic acid, ioxadifen acid, sodium ioioxadifen acid, propiolone, ioxolone, iotrolan, iopridol, meglumine cholate, iothalamic acid, diatrizoate, meglumine methopamoate, iodized oil or ethiodide, or other tracers of similar structure; preferably, the gold nanoparticles are gold nanoparticles;
the MRI tracer is selected from a longitudinal relaxation contrast agent and a transverse relaxation contrast agent; more preferably paramagnetic, ferromagnetic and supermagnetic contrast agents; further preferably Gd-DTPA and linear and cyclic polyamine polycarboxylic chelates thereof, porphyrin chelate of manganese, macromolecular gadolinium chelate, biomacromolecule modified gadolinium chelate, folic acid modified gadolinium chelate, dendrimer developer, liposome modified developer, gadolinium-containing fullerene, or other tracers with similar structures; and then preferably gadopentetate dimeglumine, gadoterate dimeglumine, gadobenate dimeglumine, ferric ammonium citrate effervescent granules and paramagnetic iron oxide, and preferably paramagnetic iron oxide or other tracers with similar structures; 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 a disease associated with vulnerable plaque is selected from one or more of statins, fibrates, antiplatelet drugs, PCSK9 inhibitors, anticoagulation drugs, angiotensin converting enzyme inhibitors, calcium antagonists, MMPs inhibitors, beta-blockers, glucocorticoids or other anti-inflammatory substances such as the IL-1 antibody canakinumab, and pharmaceutically acceptable salts thereof, including active preparations of these kinds 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 a disease associated with vulnerable plaque is selected from the group consisting of 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, antisense RNA-145 and nucleic acids such as aspirin strands thereof, aspirin, Acemetacin, troxerutin, dipyridamole, cilostazol, ticlopidine hydrochloride, ozagrel sodium, clopidogrel, prasugrel, cilostazol, beraprost sodium, ticagrelor, cagrelor, tirofiban, eptifibatide, abciximab, heparin, kexel, sapalin, flavamoeba sodium, warfarin, dabigatran, rivaroxaban, apixaban, idoxaban, bivalirudin, enoxaparin, titazaparin, aclarudilin, dicumarol, nitrocoumarin, lycium sodium, hirudin, argatroban, benazepril, captopril, enalapril, perindopril, fosinopril, lisinopril, moexipril, cilazapril, quinapril, ramipril, trandolapril, candesartan, losartan, valsartan, or, One or more of tasosartan, nifedipine, nicardipine, nitrendipine, amlodipine, nimodipine, nisoldipine, nilvadipine, isradipine, felodipine, lacidipine, diltiazem, verapamil, chlorhexidine, minocycline, MMI-166, metoprolol, atenolol, bisoprolol, carvedilol, batimastat, marimastat, prinomastat, BMS-279251, BAY 12-9566, TAA211, AAJ996A, nacetrapib, evacetapib, Torcetrapib, and Dalcetrapib, prednisone, methylprednisolone, betamethasone, beclometasone propionate, dipalmondone, prednisolone, hydrocortisone, dexamethasone, or other anti-inflammatory substances such as IL-1 antibody, and pharmaceutically active fragments or pharmaceutically acceptable salts thereof, and pharmaceutically active fragments of one or more of these species, including the pharmaceutically active species of these drugs, and endogenous anti-inflammatory cytokines such as interleukin 10.
A second aspect of the present invention provides a method of making the nanocarrier delivery system of the first aspect, the method comprising the step of strengthening the nanocarriers by adding dopamine.
A third aspect of the invention provides a pharmaceutical composition comprising a nanocarrier delivery system of the first or second aspect of the invention.
In a fourth aspect the invention provides a diagnostic formulation comprising a nanocarrier delivery system of the first or second aspect of the invention.
A fifth aspect of the invention provides the use of a nanocarrier delivery system of the first or second aspects of the invention in the manufacture of a product for the diagnosis, prevention and treatment of vulnerable plaque or a disease associated with vulnerable plaque.
Preferably, the vulnerable plaque is selected from one or more of a ruptured plaque, an erosive plaque, and a partially calcified nodular lesion;
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: acute coronary syndrome, asymptomatic myocardial ischemia-latent coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, and in-stent restenosis;
the cerebral atherosclerosis is stroke; and/or
The peripheral vascular atherosclerosis is selected from one or more of the following: atherosclerosis of the carotid, peri-occlusive atherosclerosis, retinal atherosclerosis, renal atherosclerosis, lower extremity atherosclerosis, upper extremity atherosclerosis, atherosclerotic impotence.
The patent provides a simple and convenient method for constructing a targeting nano-carrier by self-assembling Fmoc-protected amino acid or Fmoc-protected short peptide to form a nano-scale spherical structure, assembling and coupling a drug loading process with molecules, polymerizing the surface of dopamine to form a biological net structure, and reinforcing the nano-carrier, so that the stability and the biocompatibility of the self-assembled nano-carrier are greatly improved. The method is simple, green and controllable, has high drug loading, good compatibility with various drugs, good biocompatibility and high safety, and is easy to carry out targeted ligand modification. The self-assembly carrier is loaded with different drugs and coupled with different targeting ligands, and is used for diagnosis and treatment of vulnerable plaques.
Fmoc-protected amino acid molecules (Fmoc-amino acid, Fmoc-AC) are commonly used amino acid raw materials for peptide synthesis, and Fmoc protecting groups are used for protecting amino molecules in amino acids so that cross-linking can not occur in polypeptide solid phase synthesis. Research reports that the molecules can form a nano-scale fibrous structure or a nano-scale spherical structure through hydrophobic effect pi-pi accumulation, and the self-assembled nanosphere has the characteristics of good biocompatibility, easy 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-assembly nano-carriers reported in the follow-up research, the inventor selects Fmoc protection amino acid molecules, realizes simple assembly of the molecules by controlling the acidity of a solution, obtains a nano-scale hollow spherical structure for loading drugs, and forms a biocompatible shell-shaped structure on the surface of the Fmoc-AC self-assembly carrier through dopamine surface polymerization to reinforce the self-assembly nano-structure, thereby enhancing the stability of the carrier in serum. Finally, the hollow nano-carrier formed by self-assembly of the amino acid is used for loading various drugs to connect different target molecules for treating vulnerable plaques targeting CD44, and the drug/gene delivery capability and the treatment/gene interference effect of the Fmoc-AC nano-carrier are explored through a series of experimental processes.
In the nano-carrier, the Fmoc-AC nano-carrier can effectively improve the biocompatibility of the carrier on one hand through coupling polyethylene glycol, the in vivo circulation time, and the degradation of the carrier due to the combination of the carrier and serum albumin. In addition, the abundant functional group structure on the surface of the nano carrier is also very convenient for connecting a targeting carrier. The self-assembly structure has the advantages of good biocompatibility and high safety, and is a nano-drug carrier with important value.
The nano-carrier has the advantages of high drug loading, simple process, high yield, good applicability to various drugs, strong stability, good biocompatibility and convenience for coupling various targeting ligands. Amino acid self-assembling nanocarriers for targeting activated CD44 molecules, particularly vulnerable plaques. The invention also relates to the innovative preparation of said nanocarriers, in particular to the preparation and use of the self-assembled nano delivery system of amino acids, in particular in the diagnosis, prevention and treatment of vulnerable plaques or diseases associated with vulnerable plaques.
The amino acid self-assembly nanocarrier delivery system of the present invention may have the following beneficial effects, but is not limited thereto:
the nano particles and the nano composite material have great development potential in the fields of biomedical treatment and diagnosis. Compared with inorganic nano materials, the organic nano materials have better biocompatibility, safety and metabolizability, which also provides favorable conditions for clinical transformation of the organic nano materials. The Fmoc-AC self-assembly 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 invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows an SEM picture of the Fmoc-Lys assembly of example 1.
FIG. 2 shows FT _ IR spectra of Fmoc-Lys, Fmoc-Lys (R) -HA nanocarriers of example 1.
FIG. 3 shows an SEM picture of Fmoc-Lys (R) -PEG in example 2.
FIG. 4 shows FT _ IR spectra of Fmoc-Lys (R) -SP/PEG nanocarriers of example 2.
FIG. 5 shows FT _ IR spectra of Fmoc-Arg, Fmoc-Arg (R) -HA/PEG nanocarriers of example 3.
FIG. 6 shows SEM images of Fmoc-Arg (at) -PEG in example 4.
FIG. 7 shows the IR spectrum 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 SEM images of Fmoc-Lys (DPLA) -OPN/PEG in example 6.
FIG. 11 shows SEM results for Fmoc-Lys (DKSC) -OPN/PEG in example 6.
FIG. 12 shows SEM results for Fmoc-Lys (DFC) -OPN/PEG in example 6.
FIG. 13 shows Fmoc-Lys (Fe) in example 73O4DXMS) -TEM results of HI44 a/PEG.
FIG. 14 shows Fmoc-Lys (Fe) in example 73O4dXMS-PEG nano-carrier and Fmoc-Lys (Fe) added with targeting ligand3O4DXMS) -HI44a/PEG nanocarrier infrared results.
FIG. 15 shows SEM results for Fmoc-Lys (GPA) -HI44a/PEG in example 7.
FIG. 16 shows SEM results for Fmoc-Lys (GSA) -HI44a/PEG in example 7.
FIG. 17 shows SEM results for Fmoc-Lys (GPS) -HI44a/PEG in example 7.
FIG. 18 shows the infrared results of Fmoc-Lys (Asp/Clo) -PEG nanocarrier and Fmoc-Lys (Asp/Clo) -Col/PEG carrier after addition of targeting ligand in example 8.
Fig. 19 shows the effect of different holding times on hydrated particle size in test example 1.
FIG. 20 shows the effect of different storage times on the encapsulation efficiency in test example 1.
Fig. 21 shows the in vitro cumulative release rate (CRP%) for the nano-delivery system.
FIG. 22 is a graph showing 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 nano-delivery system of the present invention on carotid vulnerable plaque in model mice.
Figure 23 shows a graph of in vivo tracking effect of Fmoc-Lys (AuNP/At) -OPN/PEG and other CT tracer nano-delivery systems on carotid vulnerable plaque of model mice.
FIG. 24 is a graph showing the therapeutic effect of Fmoc-Lys (AuNP/At) -OPN/PEG nano-delivery system on carotid vulnerable plaque in model mice.
FIG. 25 shows Fmoc-Lys (Fe)3O4In vivo tracer efficacy profile of/DXMS) -HI44a/PEG and other MRI tracer nano-delivery systems on carotid vulnerable plaque in model mice.
FIG. 26 shows Fmoc-Lys(Fe3O4Efficacy of the/DXMS) -HI44a/PEG nano-delivery system for treatment of carotid vulnerable plaque in model mice.
FIG. 27 is a graph showing 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, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art, provided that they are not specifically illustrated.
The reagents and instrumentation used in the following examples are as follows:
reagent:
fmoc-protected lysine (Fmoc-Lys), Fmoc-protected arginine (Fmoc-Arg), Fmoc-protected arginine dipeptide (Fmoc-Arg) purchased from Shanghai Gill Biochemical, rosuvastatin purchased from Dalian Meiren Biotechnology, Hyaluronic Acid (HA) purchased from Zhejiang east Biotechnology, DMSO, ferric trichloride, Ammonia, ethanol, Chlorauric acid, NaBH4Lipoic acid was purchased from Beijing Chemicals, Inc., national drug group, dopamine 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl), N-hydroxythiosuccinimide (sulfo-NHS), collagen (Col), PEG activated by NHS ester with molecular weight of 1000 (PEG)1k-NHS), selectin SP, osteopontin, HI44a from Sigma-Aldrich, fluorodeoxyglucose, aspirin (Asp), Dexamethasone (DXSM) clopidogrel (Clo) from chinese food & drug testing institute.
The instrument comprises the following steps:
scanning electron microscope, purchased from zeiss, model number, germany: EVO18
Laser particle sizer, available from Marvin Intelligent laser particle sizer, model Zetasizer Nano ZS, UK
Transmission electron microscope, JEOL-2100 high resolution transmission electron microscope infrared spectrometer: saimer Feishale science and technology Fourier transform near infrared spectrometer, model Antaris II FT-NIR analyzer
Example 1 surface modification of Hyaluronic Acid (HA), rosuvastatin (R) -loaded lysine (Fmoc-Lys (R) -HA) Preparation of nanocarriers
1.1 preparation of blank Fmoc-Lys nanocarriers
10mg of Fmoc-Lys was weighed and dissolved in 10mL of ultrapure water to completely dissolve the Fmoc-Lys, and 0.1g L-1The pH value of the solution is adjusted to 11 by NaOH, and self-assembly is carried out by continuous ultrasonic treatment for 1h to obtain milky emulsion. A small amount of the solution was slowly dropped onto a silicon wafer, dried at room temperature, and observed using a scanning electron microscope, and FIG. 1 shows an SEM image of the Fmoc-Lys assembly in example 1.
1.2 preparation of Fmoc-Lys (R) nanocarrier loaded with rosuvastatin (R)
Weighing Fmoc-Lys 10mg, dissolving in 10mL ultrapure water to completely dissolve, and dropwise adding 1mg mL under ultrasonic condition-1Rosuvastatin in DMSO solution, 10mg mL-1The pH value of the solution is adjusted to 11 by NaOH, and self-assembly is carried out by continuous ultrasonic treatment for 1h to obtain milky emulsion. To this solution was added 1mmol L of final concentration-1Reacting dopamine hydrochloride at room temperature for 40 minutes, purifying by centrifugation after reaction, and freeze-drying and storing. A small amount of the solution was slowly dropped on a silicon wafer, air-dried at room temperature, and the structure was characterized by an infrared spectrum as shown in FIG. 2.
1.3 preparation of Fmoc-Lys (R) nanocarrier (Fmoc-Lys (R) -HA) to couple HA
1g of hyaluronic acid HA (molecular weight about 40kDa) was sufficiently dissolved in ultrapure water, and 0.1g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.12g of 0.12g N-hydroxythiosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. After stirring the reaction at room temperature for 1 hour, anhydrous ethanol was added to precipitate the activated HA.The precipitate was filtered, washed with ethanol and dried in vacuo to give activated HA. It was prepared to 0.1mg mL-10.2mL of the aqueous solution is sucked and dissolved in the purified Fmoc-Lys (R) solution to realize the coupling of HA on the nano-carrier, so as to obtain the target recognition nano-carrier Fmoc-Lys (R) -HA. Figure 2 shows FT _ IR spectra of targeting drug-loaded nanocarrier Fmoc-lys (r) -HA.
Example 2 Simultaneous coupling of polyethylene glycol, Selectin (SP), rosuvastatin (R) loaded Fmoc-Lys NanoS Preparation of Carrier (Fmoc-Lys (R) -SP/PEG)
2.1 preparation of polyethylene glycol coupled rosuvastatin loaded Fmoc-Lys nanocarriers (Fmoc-Lys (R) -PEG)
PEG functionalized with 100mg of NHS active ester with molecular weight of 10001kthe-NHS ester was dissolved in 1mM NaHCO3 solution (5mL, pH 8.6), 10mg Fmoc-Lys was added and stirred for 24 hours at room temperature, and the reacted Fmoc-Lys-PEG was precipitated with diethyl ether and lyophilized to obtain purified Fmoc-Lys-PEG.
Weighing Fmoc-Lys 50mg and 5mg Fmoc-Lys-PEG molecules, dissolving in 20mL ultrapure water to completely dissolve, and dropwise adding 1mg mL under ultrasonic condition-1Rosuvastatin in DMSO solution, 10mg mL-1The pH value of the solution is adjusted to 11 by NaOH, and self-assembly is carried out by continuous ultrasonic treatment for 1h to obtain milky emulsion. To this solution was added L of 1mmol in final concentration-1Reacting dopamine hydrochloride at room temperature for 40 minutes, purifying by centrifugation after reaction, and freeze-drying and storing. A small amount of the solution was slowly dropped on a silicon wafer, dried at room temperature, and observed using a scanning electron microscope, and FIG. 3 shows an SEM photograph 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 solution, and 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.5mg of N-hydroxythiosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. After stirring the reaction at room temperature for 1 hour. It was prepared to 1mg mL-11mL of the solution was pipetted into purified Fmoc-Lys (R) -PEG solutionCoupling SP on Fmoc-Lys (R) -PEG to obtain 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 Fmoc-protected sodium arginine simultaneously coupled with Hyaluronic Acid (HA) and PEG, loaded with rosuvastatin (R) Preparation of Rice Carrier Fmoc-Arg (R) -HA/PEG
3.1 preparation of polyethylene glycol coupled rosuvastatin (R) loaded Fmoc-Arg nanocarrier (Fmoc-Arg (R) -PEG)
Fmoc-Arg is a dipeptide with dimeric arginine, custom-synthesized and PEG functionalized by NHS active ester with molecular weight of 100mg and molecular weight of 1000 from Shanghai Gill Biochemical company1k-NHS ester dissolved in 1mM NaHCO3And adding 10mg of Fmoc-Arg into the solution (5mL, pH 8.6), stirring and reacting at room temperature for 24 hours, adding diethyl ether to precipitate Fmoc-Arg-PEG after reaction, and freeze-drying to obtain the purified Fmoc-Arg-PEG.
Weighing Fmoc-Arg 50mg and Fmoc-Arg-PEG molecules 5mg, dissolving in 20mL of ultrapure water to completely dissolve the molecules, and dropwise adding 1mg mL of the mixture under the ultrasonic condition-1Rosuvastatin (R) DMSO solution in 10mg mL-1The pH value of the solution is adjusted to 11 by NaOH, and self-assembly is carried out by continuous ultrasonic treatment for 1h to obtain milky emulsion. To this solution was added 1mmol L of final concentration-1Dopamine hydrochloride, reacting at room temperature for 40 minutes, purifying by centrifugation after the reaction, and freeze-drying to obtain Fmoc-Arg (R) -PEG, wherein the infrared spectrum of the product is shown in figure 5.
3.2 Fmoc-Arg (R) -PEG nanocarrier coupled to Hyaluronic Acid (HA)
Activated HA was obtained according to the method of example 1. It was prepared to 0.1mg mL-10.2mL of the solution is sucked and dissolved in a purified Fmoc-Arg (R) -PEG solution to realize the coupling of HA on the nano-carrier, so as to obtain a targeting recognition nano-carrier Fmoc-Arg (R) -HA/PEG figure 5 shows the FT _ IR spectrum of the targeting nano-carrier Fmoc-Arg (R) -HA/PEG.
Example 4 Simultaneous coupling of PEG, self-peptide (SEP) atorvastatin (At) loaded Fmoc-Arg nanocarriers Preparation of (Fmoc-Arg (at) -SEP/PEG) delivery System
4.1 preparation of atorvastatin (At) -loaded Fmoc-Arg nanocarriers (Fmoc-Arg (At) -PEG) coupled polyethylene glycol
Fmoc-Arg (at) -PEG was prepared according to the method of example 2. Weighing Fmoc-Arg 50mg and Fmoc-Arg-PEG molecules 5mg, dissolving in 20mL of ultrapure water to completely dissolve the molecules, and dropwise adding 1mg mL of the mixture under the ultrasonic condition-1Atorvastatin (At) DMSO solution, using 10mg mL-1The pH value of the solution is adjusted to 11 by NaOH, and self-assembly is carried out by continuous ultrasonic treatment for 1h to obtain milky emulsion. To this solution was added 1mmol L of final concentration-1Reacting dopamine hydrochloride at room temperature for 40 minutes, purifying by centrifugation after reaction, and freeze-drying and storing. A small amount of the solution was slowly dropped onto a silicon wafer, air-dried at room temperature, and 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 prepared to 0.1mg mL-10.2mL of the solution is sucked and dissolved in a purified Fmoc-Arg (at) -PEG solution to realize the coupling of HA on the nano-carrier, so as to obtain the targeting recognition nano-carrier Fmoc-Arg (at) -HA/PEG, and figure 4 shows the infrared spectrum of the targeting nano-carrier Fmoc-Arg (at) -HA/PEG
1mg of self-peptide (SEP) was dissolved well in 1ml of PBS buffer solution, and 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.5mg of N-hydroxythiosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. After reacting for 1h at room temperature, sucking 1mL of the SEP-NHS solution and dissolving in purified Fmoc-Arg (at) -PEG solution to realize coupling of SEP on Fmoc-Arg (at) -PEG, thereby obtaining the target recognition nano-carrier Fmoc-Arg (at) -SEP/PEG. The infrared spectrum is shown in FIG. 7.
Example 5 coupling monoclonal antibody (IM7) supporting atorvastatin (At) and miRNA-33 Fmoc-Arg NanoS Preparation of vector (Fmoc-Arg (At/miRNA-33) -IM7) delivery System
5.1 preparation of Fmoc-Arg nanocarrier (Fmoc-Arg (At/miRNA-33)) simultaneously loaded with miRNA-33 and atorvastatin (At)
Weighing Fmoc-Arg 10mg, dissolving in 10Dissolving the mixture completely in mL ultrapure water, and adding 1mg mL of the mixture dropwise under the ultrasonic condition-1Atorvastatin solution in DMSO, 10mg mL-1The pH value of the solution is adjusted to 11 by NaOH, and self-assembly is carried out by continuous ultrasonic treatment for 1h to obtain milky emulsion. To this solution was added 1mmol L of final concentration-1Reacting dopamine hydrochloride at room temperature for 40 minutes, purifying by centrifugation after reaction, and freeze-drying and storing. And continuously adding miRNA-33a (0.1mg) into the solution, continuously stirring 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 dissolved well in PBS and 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.5mg of N-hydroxythiosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. After stirring the reaction at room temperature for 1 hour, it was purified by ultrafiltration and prepared to 1mg mL-1An aqueous solution of (a). Sucking 1.0mL of activated IM7 solution, dissolving in purified Fmoc-Arg (At/miRNA-33) solution, realizing the coupling of IM7 on Fmoc-Arg (At/miRNA-33), and obtaining the targeting recognition nanocarrier Fmoc-Arg (At/miRNA-33) -IM 7. FIG. 8 shows Transmission Electron Microscopy (TEM) results of Fmoc-Arg (At/miRNA-33) -IM 7.
Example 6 Simultaneous coupling of polyethylene glycol (PEG), Osteopontin (OPN) loaded gold nanoparticles (AuNP) and atropic Preparation of Vavastatin (At) nanocarrier (Fmoc-Lys (AuNP/At) -OPN/PEG)
6.1 preparation of coupled PEG-loaded gold nanoparticles (AuNP) and atorvastatin (At) Fmoc-Lys nanocarrier Fmoc-Lys (AuNP/At) -OPN/PEG delivery System
Fmoc-Lys-PEG was prepared as in example 2. Weighing Fmoc-Lys 50mg and 5mg Fmoc-Lys-PEG molecules, dissolving in 20mL ultrapure water to completely dissolve the molecules, and dropwise adding 1mg mL-1Atorvastatin (At) DMSO solution using 10mg mL-1The pH value of the solution is adjusted to 11 by NaOH, and self-assembly is carried out by continuous ultrasonic treatment for 1h to obtain milky emulsion. To this solution was added 1mmol L of final concentration-1Dopamine hydrochloride reacts for 40 minutes at room temperatureThen purifying by centrifugation, and freeze-drying and storing.
1mL of 50mM HAuCl4The pH was adjusted to 11.0 with 1M NaOH, added dropwise to the above solution, stirred for 5 minutes and then reduced to HAuCl with 1mL of 0.1M sodium ascorbate4And obtaining the nano-carrier with the surface loaded with the gold nano-particles.
6.2 coupling of Osteopontin (OPN) to Fmoc-Lys (AuNP/At) -PEG surface
1mg of Osteopontin (OPN) was dissolved in PBS buffer solution sufficiently, and 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.5mg of N-hydroxythiosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. After stirring the reaction at room temperature for 1 hour. It was prepared to 1mg mL-1The solution is absorbed by 1mL and dissolved in the purified Fmoc-Lys (AuNP/At) -PEG solution to realize the coupling of OPN on the Fmoc-Lys (AuNP/At) -PEG, and the targeting recognition nano-carrier Fmoc-Lys (AuNP/At) -OPN/PEG is obtained. FIG. 9 is an infrared spectrum of Fmoc-Lys (AuNP/At) -OPN/PEG.
6.3 preparation of Fmoc-Lys nanocarrier delivery System with PEG-coupled, OPN loaded iodophor
Fmoc-Lys-PEG was prepared as in example 2. Weighing Fmoc-Lys 50mg and 5mg Fmoc-Lys-PEG molecules, dissolving in 20mL ultrapure water to completely dissolve, and dropwise adding 1mg mL under ultrasonic condition-1Ioprolide in water, 10mg mL-1The pH value of the solution is adjusted to 11 by NaOH, and self-assembly is carried out by continuous ultrasonic treatment for 1h to obtain milky emulsion. To this solution was added 1mmol L of final concentration-1Reacting dopamine hydrochloride at room temperature for 40 minutes, purifying by centrifugation after reaction, and freeze-drying and storing.
6.4 coupling of Osteopontin (OPN) to Fmoc-Lys (DPLA) -PEG surface
1mg of Osteopontin (OPN) was dissolved in PBS buffer solution sufficiently, and 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.5mg of N-hydroxythiosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. After stirring the reaction at room temperature for 1 hour. It was prepared to 1mg mL-11mL of the solution was pipetted and dissolved in purified Fmoc-Lys (DPLA) -PEG solution to achieve OPN in FmCoupling on the oc-Lys (DPLA) -PEG to obtain the target recognition nano-carrier Fmoc-Lys (DPLA) -OPN/PEG. FIG. 10 shows SEM images of Fmoc-Lys (DPLA) -OPN/PEG. The method can replace the iopromide to be iodixanol and iodofluoroalcohol to obtain Fmoc-Lys (DKSC) -OPN/PEG and Fmoc-Lys (DFC) -OPN/PEG nano-carriers. FIG. 11 shows SEM results for Fmoc-Lys (DKSC) -OPN/PEG. FIG. 12 shows SEM results for Fmoc-Lys (DFC) -OPN/PEG.
3 4Example 7 conjugation of monoclonal antibody HI44a, drug-loaded Dexamethasone (DXMS) paramagnetic Oxidation (FeONPs) 3 4Preparation of Fmoc-Lys nanocarrier (Fmoc-Lys (FeO/DXMS) -HI44 a/PEG)
7.1 coupling PEG Supported paramagnetic iron oxide (Fe)3O4NPs) and Dexamethasone (DXMS) Fmoc-Lys nanocarrier Fmoc-Lys (Fe)3O4Preparation of a/DXMS) -OPN/PEG delivery System
Fmoc-Lys-PEG was prepared as in example 2. Weighing Fmoc-Lys 50mg and 5mg Fmoc-Lys-PEG molecules, dissolving in 20mL ultrapure water to completely dissolve, and dropwise adding 1mg mL under ultrasonic condition-1Dexamethasone (DXMS) in DMF 10mg mL-1The pH value of the solution is adjusted to 11 by NaOH, and self-assembly is carried out by continuous ultrasonic treatment for 1h to obtain milky emulsion. To this solution was added 1mmol L of final concentration-1Reacting dopamine hydrochloride at room temperature for 40 minutes, purifying by centrifugation after reaction, and freeze-drying and storing.
To the above solution was added dropwise 1mL of 10mM ferric chloride (FeCl)3) Stirring the aqueous solution for 5 minutes to obtain surface-loaded paramagnetic iron oxide (Fe)3O4NPs) nanocarrier Fmoc-Lys (Fe)3O4/DXMS)-PEG。
7.2HI44a on nanocarriers (Fmoc-Lys (Fe)3O4Coupling on DXMS) -PEG
1mg of HI44a was thoroughly dissolved in ultrapure water, and 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.5mg of N-hydroxythiosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. After stirring and reacting for 1 hour at room temperature, ultrafiltration and centrifugal purification are carried out to obtain activationHI44 a. Pipette 1.0mL of HI44a solution into purified Fmoc-Lys (Fe)3O4In NP/DXMS) -PEG solution, realizing the coupling of HI44a to obtain the targeting recognition nano-carrier Fmoc-Lys (Fe)3O4NP/DXMS) -HI44 a/PEG. FIG. 13 shows Fmoc-Lys (Fe)3O4DXMS) -TEM results of HI44 a/PEG. FIG. 14 shows Fmoc-Lys (Fe)3O4dXMS-PEG nano-carrier and Fmoc-Lys (Fe) added with targeting ligand3O4DXMS) -HI44a/PEG nanocarrier infrared results.
7.3 preparation of coupled monoclonal antibody HI44a, Fmoc-Lys nanocarrier Fmoc-Lys (GPA) -HI44a/PEG for MRI tracer-loaded Fmoc-Lys nanocarrier
Fmoc-Lys-PEG was prepared as in example 2. Weighing Fmoc-Lys 50mg and 5mg Fmoc-Lys-PEG molecules, dissolving in 20mL ultrapure water to completely dissolve, and dropwise adding 1mg mL under ultrasonic condition-1Gadoterate Glucamine (GPA) DMF solution, using 10mg mL-1The pH value of the solution is adjusted to 11 by NaOH, and self-assembly is carried out by continuous ultrasonic treatment for 1h to obtain milky emulsion. Adding 1mmol L-1Reacting dopamine hydrochloride at room temperature for 40 minutes, purifying by centrifugation after reaction, and freeze-drying and storing.
1mg of HI44a was thoroughly dissolved in ultrapure water, and 0.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.5mg of N-hydroxythiosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. After stirring the reaction at room temperature for 1 hour, purification by ultrafiltration and centrifugation yielded activated HI44 a. Sucking 1.0mL of HI44a solution, dissolving in purified Fmoc-Lys (GPA) -PEG solution, realizing the coupling of HI44a, and obtaining the target recognition nano-carrier Fmoc-Lys (GPA) -HI44 a/PEG. FIG. 15 shows SEM results for Fmoc-Lys (GPA) -HI44 a/PEG.
The corresponding Fmoc-Lys (GSA) -HI44a/PEG, Fmoc-Lys (GPS) -HI44a/PEG nanophotograph agent can be obtained by replacing GPA with gadolinium diamine (GSA) and gadolinium pentanedioic acid (GPS). FIG. 16 shows SEM results for Fmoc-Lys (GSA) -HI44 a/PEG. FIG. 17 shows SEM results for Fmoc-Lys (GPS) -HI44 a/PEG.
Example 8 modification with polyethylene glycol, collagen (Col) while loading Aspirin (Asp), clopidogrel Preparation of (Clo) Fmoc-Lys nanocarriers (Fmoc-Lys (Asp/Clo) -Col/PEG)
Fmoc-Lys-PEG was prepared as in example 2. Weighing Fmoc-Lys 50mg and 5mg Fmoc-Lys-PEG molecules, dissolving in 20mL ultrapure water to completely dissolve, and dropwise adding 1mg mL under ultrasonic condition-1Aspirin (Asp, 1mL) and 1mg mL-1Clopidogrel (Clo, 1mL) DMF solution, 10mg mL-1The pH value of the solution is adjusted to 11, the solution is subjected to continuous ultrasonic treatment for 1 hour for self-assembly to obtain milky emulsion, and the milky emulsion with the final concentration of 1mmol L-1Reacting dopamine hydrochloride at room temperature for 40 minutes, purifying by centrifugation after reaction, and freeze-drying and storing. The Fmoc-Lys (Asp/Clo) -PEG can be obtained by centrifugal purification and freeze-drying. FIG. 18 shows the infrared results of Fmoc-Lys (Asp/Clo) -PEG nanocarriers.
10mg of collagen (col) was dissolved in ultrapure water sufficiently, and 3mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 3mg of N-hydroxythiosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. After stirring the reaction at room temperature for 1 hour, the activated Col was obtained by ultrafiltration and centrifugal purification. Respectively sucking 1.0mL of Col solution to be dissolved in purified Fmoc-Lys (Asp/Clo) -PEG solution to realize the coupling of Col, and obtaining the targeting recognition nano-carrier Fmoc-Lys (Asp/Clo) -Col/PEG. FIG. 18 shows the infrared results of Fmoc-Lys (Asp/Clo) -Col/PEG vector.
Test example 1 Property examination of NanoDelivery System
In this experimental example, the therapeutic agent-loaded nano delivery systems prepared in examples 1 to 8 were used as examples 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 strong ultraviolet absorption characteristics, and thus the contents thereof can be determined by using the ultraviolet absorption characteristics of rosuvastatin, atorvastatin, dexamethasone, aspirin, clopidogrel, fluorodeoxyglucose using HPLC-UV method (using Waters2487, Waters Corporation, usa). And (3) establishing a standard quantitative equation by using the concentrations (X) of different concentrations of rosuvastatin, atorvastatin, dexamethasone, aspirin, clopidogrel and fluorodeoxyglucose solution to the peak area (Y) of an HPLC chromatographic peak.
2. Determination of hydrated particle size:
the nanocarrier of the delivery system of the invention,
the hydrated particle sizes were determined by a laser particle sizer (BI-Zeta Plus/90Plus, Bruk Highen Corporation (Brookhaven Instruments Corporation), USA) with the specific results shown in Table 1.
3. Determination of encapsulation efficiency:
taking a certain amount of nano-carriers, 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)3O4dXMS) -HI44a/PEG, Fmoc-Lys (Asp/Clo) -Col/PEG was added to an excess of methanol/formic acid solution (100:1 volume ratio) at pH 2.0 and heated in a water bath for 2 hours to accelerate the release of the drug from the nanocarriers, and further sonication was used to accelerate the release of the drug from the dendrimers. The content of the drug in the resulting liquid was measured by HPLC (Waters2487, Watts Corporation, USA), and the encapsulation ratio was calculated by the following formula. The correlation results are shown in Table 1
Encapsulation ratio (%) ═ MAmount of drug to be packaged/MAmount of drug added) 100% … … … … … formula 1
TABLE 1 summary of various properties
Figure BDA0002579024850000211
Figure BDA0002579024850000221
Note: the data above are all expressed as "mean + standard deviation" of the results of 5 replicates.
4. Long term stability study
The nano-delivery system of the present invention was stored at 4 ℃, sampled at different time points, and tested for changes in hydrated particle size by a laser particle sizer (BI-Zeta Plus/90Plus, brueck sea Corporation (Brookhaven Instruments Corporation), usa). FIG. 19 is a graph showing the effect of different holding times on hydrated particle size.
5. Long term stability study
Nano-delivery system of the invention the nano-delivery system of the invention, stored at 4 ℃, sampled at different time points and examined for changes in encapsulation efficiency by ultrafiltration centrifugation to remove free drug. Figure 20 shows the effect of different holding times on the 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 at 37 ℃ for 120 h. At different time points, 2mL of the release solution was taken and supplemented with the same volume of PBS solution. The drug content in the release liquid was measured by HPLC (Waters2487, Watts Corporation, USA), and the cumulative release rate of the drug was calculated by equation 2.
Figure BDA0002579024850000231
The meaning of each parameter in formula 3 is as follows:
CRP: cumulative drug release rate
Ve: displaced volume of released liquid, here VeIs 2mL
V0: volume of release liquid in the delivery System, here V0Is 50mL
Ci: concentration of drug in release solution in unit of μ g/mL at the time of ith substitution sampling
M, drug: total mass of drug in delivery system, in μ g
n: number of times of replacement of released liquid
Cn: the concentration of drug in the delivery system was measured after the nth time of replacement of the delivery solution.
Fig. 21 shows the in vitro cumulative release rate (CRP%) for the nano-delivery system.
Experimental example 2 Fmoc-Lys (R) -HA, Fmoc-Lys (R) -SP/PEG of the present invention,
Fmoc-Arg(R)-HA/PEG，Fmoc-Arg(At)-SEP/PEG，
in vivo demonstration of the Effect of Fmoc-Arg (At/miRNA-33) -IM7 NanoTansmission System on arterial vulnerable plaque Test (experiment)
Hyaluronic Acid (HA) and Selectin (SP) are ligands of CD44 and 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 a medicament, cell-penetrating peptide (Tat) can increase local penetration and aggregation of the medicament, PEG is modified on the surface of a carrier, a long-circulating effect can be achieved, and the half-life period of the medicament is prolonged. miRNA-33a is capable of increasing 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 described in this invention on arterial vulnerable plaque.
The experimental method comprises the following steps:
(1) physiological saline solutions of free rosuvastatin and atorvastatin were prepared and therapeutic agent-loaded amino acid self-assembled nano delivery systems were prepared using the methods described in examples 1-5 above.
(2) Establishment of ApoE-/-mouse arterial vulnerable plaque model:
SPF-grade ApoE-/-mice (42, 5-6 weeks old, 20. + -.1 g body weight) were taken as experimental animals. After 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 being a common feed for mice) for 4 weeks, 1% sodium pentobarbital (formulated by adding 1mg sodium pentobarbital to 100ml of physiological saline) was intraperitoneally administered for anesthesia at a dose of 40 mg/kg. Then, the mice were fixed on a surgical plate in a supine position, sterilized with 75% (v/v) alcohol centering on the neck, the skin of the neck was cut off longitudinally, the anterior cervical gland was separated bluntly, and the pulsating left common carotid artery was observed on the left side of the trachea. Carefully separating the common carotid artery to the bifurcation, sleeving a silicone tube with the length of 2.5mm and the inner diameter of 0.3mm on the periphery of the left common carotid artery, and narrowing and fixing the proximal section and the distal section of the sleeve by thin silk threads. Local constriction causes proximal blood flow turbulence, increased shear forces, and resultant intimal damage. The carotid artery was repositioned and the anterior cervical skin was sutured intermittently. All manipulations were performed under a 10-fold visual microscope. And after the mouse wakes up after the operation, putting the mouse back into the cage, maintaining the ambient temperature at 20-25 ℃, and keeping the light on and off for 12 hours respectively. Intraperitoneal injections of Lipopolysaccharide (LPS) (1mg/kg in 0.2ml phosphate buffered saline, Sigma, USA) were initiated at week 4 post-surgery, 2 times per week for 10 weeks, inducing chronic inflammation. Mice were placed into 50ml syringes (with sufficient air holes reserved) for 8 weeks post-surgery to cause restrictive mental stress, 6 hours/day, 5 days per week, for 6 weeks. The mouse atherosclerotic vulnerable plaque model was modeled after 14 weeks postoperatively.
(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 group of animals did not undergo any therapeutic treatment;
rosuvastatin intravenous injection group: intravenous administration was carried out 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) -group HA: intravenous administration was carried out at a dose of 0.66mg rosuvastatin/kg body weight;
Fmoc-Lys (R) -SP/PEG set: intravenous administration was carried out at a dose of 0.66mg rosuvastatin/kg body weight;
Fmoc-Arg (R) -HA/PEG set: treatment was carried out by intravenous administration at a dose of 0.66mg rosuvastatin/kg body weight.
Fmoc-Arg (at) -SEP/PEG set: intravenous administration at a dose of 1.2mg atorvastatin/kg body weight;
Fmoc-Arg (At/miRNA) -IM7 set: the treatment was administered intravenously at a dose of 1.2mg atorvastatin/kg body weight.
Treatment of the treatment groups was performed 1 time every other day for a total of 5 times, except for 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 the percent plaque progression was calculated.
Percent plaque progression ═ (plaque area after treatment-plaque area before treatment)/luminal area.
The experimental results are as follows:
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 systems described herein on arterial vulnerable plaque. As shown, during the course of high-fat diet feeding (10 days), atherosclerosis progressed by 32.9% in the control group (not given any treatment); the treatment with rosuvastatin can delay the development of plaque, but the development is 30.1%; atorvastatin intravenous injection also delayed plaque progression, but also progressed by 31.7%; while targeted nano drug-loaded treatment significantly suppressed plaque progression and even showed 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%.
Taken together, for arterial vulnerable plaques in mice, neither free rosuvastatin nor atorvastatin exhibited the effect of reversing vulnerable plaques. However, when statin is loaded in the nano delivery system of the present invention, the therapeutic effect on vulnerable plaque is significantly improved, and the therapeutic effect on plaque reduction is achieved, and the nano system with functional modification has better effect.
Experimental example 3 Effect of Fmoc-Lys (AuNP/At) -OPN/PEG delivery System of the present invention on arterial vulnerable plaque In vivo experiment (CT tracing and treatment dual function)
Osteopontin (OPN) is a ligand of CD44 and can act to target vulnerable plaques, atorvastatin (At) has the effect of reversing plaques, and nanogold (AuNP) is a CT tracer. The purpose of this example is to verify the in vivo tracking and therapeutic effect of the nano delivery system loaded with CT tracer and atorvastatin on vulnerable plaque of artery.
(1) A physiological saline solution of free atorvastatin was prepared and a self-assembled nano delivery system of amino acids loaded with CT tracer and therapeutic agent was prepared using the method described in example 6 above.
(2)ApoE-/-The method for establishing the mouse artery vulnerable plaque model is the same as the experimental example 2.
(3) Tracing vulnerable plaques of experimental animals:
experimental animals were randomly divided into the following groups of 6 animals each:
free gold nanoparticle group: the dosage of the nano gold is 0.1mg/kg body weight;
Fmoc-Lys (AuNP/At) -OPN/PEG set: the dosage of the nano gold is 0.1mg/kg body weight;
Fmoc-Lys (DPLA) -OPN/PEG set: iopromide is administered in an amount of 0.1mg/kg body weight;
Fmoc-Lys (DKSC) -OPN/PEG set; the administration dose of iodixanol is 0.1mg/kg body weight;
Fmoc-Lys (DFC) -OPN/PEG set: the dosage of the iodofluoroalcohol to be administered is 0.1mg/kg body weight.
Injecting corresponding tracer agents into each experimental group through tail veins, carrying out CT imaging before administration and 2h after administration, and observing the identification condition of the atherosclerosis vulnerable plaques of 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 group of animals did not undergo any therapeutic treatment;
atorvastatin gavage group: performing gavage treatment at a dose of 20mg atorvastatin per kg body weight;
atorvastatin intravenous group: intravenous administration at a dose of 1.2mg atorvastatin/kg body weight;
Fmoc-Lys (AuNP/At) -OPN/PEG set: the treatment was administered intravenously at a dose of 1.2mg atorvastatin/kg body weight.
Treatment of the treatment groups was performed 1 time every other day for a total of 5 times, except for 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 the percent plaque progression was calculated.
Percent plaque progression ═ (plaque area after treatment-plaque area before treatment)/luminal area.
The experimental results are as follows:
figure 23 demonstrates the in vivo tracking effect of a tracer-loaded amino acid delivery system of the invention on arterial vulnerable plaque. As shown, the free gold nanoparticles showed some tracing effect for the vulnerable arterial plaque in mice. Compared with free nano gold particles, when nano gold, iopromide, iodixanol and iodofluoroalcohol are prepared in a targeted amino acid delivery system, the tracing effect of vulnerable plaques 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 be used for drug delivery, so that the recognition effect of nano-gold on vulnerable arterial plaque can be remarkably improved, and a better tracing effect can be generated.
FIG. 24 shows the in vivo therapeutic effect of Fmoc-Lys (AuNP/At) -OPN/PEG system of the present invention on vulnerable plaque of arteries. As shown, during the course of high-fat diet feeding (10 days), atherosclerosis of the control group (not given any treatment) progressed by 23%; by adopting atorvastatin for intragastric administration, the progress of the plaque can be delayed, but the progress is also 21%; atorvastatin intravenous injection also delayed plaque progression, but also progressed by 21.5%; while targeted nano drug-loaded therapy significantly suppressed plaque progression, even reversal and regression of plaque volume occurred, Fmoc-Lys (AuNP/At) -OPN/PEG caused plaque regression by 7.2%.
In conclusion, for the arterial vulnerable plaque in the mouse, whether the administration is intragastric or intravenous, the free atorvastatin has a certain treatment effect, but the free atorvastatin cannot reverse the vulnerable plaque. However, when atorvastatin and nanogold are formulated in the nano delivery system, the diagnosis and treatment effects on vulnerable plaques are remarkably improved, and the early warning of high-risk patients and the treatment effect on plaque reduction are achieved.
3 4Experimental example 4 Fmoc-Lys (FeO/DXMS) -HI44a/PEG delivery System of the present invention for arterial vulnerable plaque In vivo tracer assay (MRI tracer) and anti-inflammatory therapy
The monoclonal antibody (HI44a) is CD44 antibody and can be used for targeting vulnerable plaque, Dexamethasone (DXMS) has antiinflammatory and plaque progression inhibiting effects, and Fe3O4Is an MRI tracer. The purpose of this example is to verify the in vivo tracking and treatment effect of the MRI tracer and dexamethasone loaded amino acid self-assembled nano delivery system of the present invention on vulnerable arterial plaque. In addition, gadoterate meglumine, gadodiamide and gadopentetic acid can also be prepared into a nano preparation, and a targeted MRI (magnetic resonance imaging) tracing effect is displayed.
(1) An amino acid self-assembled nano-delivery system loaded with MRI tracer and therapeutic agents 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 the test example 2.
(3) Tracing vulnerable plaques of experimental animals:
experimental animals were randomly divided into the following groups of 6 animals each:
free Fe3O4Group (2): fe3O4The administration dose of (A) is 0.1mg/kg body weight
Fmoc-Lys(Fe3O4DXMS) -HI44a/PEG set: fe3O4The administration dose of (A) is 0.1mg/kg body weight;
Fmoc-Lys (GPA) -HI44a/PEG set: the administration dose of the meglumine gadotetate is 0.1mg/kg body weight;
Fmoc-Lys (GSA) -HI44a/PEG set: the administration dose of the gadodiamide is 0.1mg/kg body weight;
Fmoc-Lys (GPS) -HI44a/PEG set: the dose of gadopentetic acid administered was 0.1mg/kg body weight.
Injecting corresponding tracer agents into each experimental group through tail veins, carrying out MRI imaging before administration and 2h after administration, and observing the identification condition of atherosclerosis vulnerable plaques of 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 group of animals did not undergo any therapeutic treatment;
Fmoc-Lys(Fe3O4DXMS) -HI44a/PEG set: intravenous administration treatment was performed at a dose of 0.1mg dexamethasone/kg body weight;
treatment of the treatment groups was performed 1 time every other day for a total of 5 times, except for 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 the percent plaque progression was calculated.
Percent plaque progression ═ (plaque area after treatment-plaque area before treatment)/luminal area.
The experimental results are as follows:
figure 25 demonstrates the in vivo tracking effect of a tracer-loaded amino acid delivery system of the invention on arterial vulnerable plaque. Free Fe as shown in the figure3O4The particles exhibit some tracer effect on arterial vulnerable plaques in mice. With free Fe3O4Particle to particle ratio when Fe is compared3O4When the magnetic resonance imaging agent is prepared in a targeted amino acid delivery system, the tracing effect on vulnerable plaques is remarkably improved, and the tracing effect on vulnerable plaques is good when other MRI nano contrast agents are adopted. In conclusion, compared with a free MRI tracer agent, the amino acid delivery system with the surface modified with the targeting ligand is used for drug administration, so that the recognition effect of the MRI tracer agent on vulnerable arterial plaque can be obviously improved, and a better tracing effect can be generatedAnd (5) fruit.
FIG. 26 shows Fmoc-Lys (Fe) as described in the present invention3O4DXMS) -HI44a/PEG system for in vivo treatment of arterial vulnerable plaque. As shown, during the course of high fat diet feeding (10 days), atherosclerosis progressed 31% in the control group (not given any treatment); while the targeted nano drug-loaded therapy obviously inhibits the development of the plaque, even reverses and regresses the plaque volume, Fmoc-Lys (Fe)3O4DXMS) -HI44a/PEG resolved plaques by 8%.
In conclusion, for vulnerable arterial plaque in mice, dexamethasone and Fe are added3O4When the amino acid self-assembly nano delivery system is prepared, the diagnosis and treatment effects of vulnerable plaques are remarkably improved, and the early warning of high-risk patients and the treatment effect of reversing the growth of plaques (reducing plaques) are achieved.
Experimental example 5 Effect of Fmoc-Lys (Asp/Clo) -Col/PEG delivery System of the present invention on arterial vulnerable plaque In vivo experiments of
Aspirin (Asp) and clopidogrel (Clo) are antiplatelet drugs, which can act to reduce platelet aggregation and reduce the mortality rate of cardiovascular events. The purpose of this example is to demonstrate the in vivo therapeutic effect of Fmoc-Lys (Asp/Clo) -Col/PEG vector delivery system described in this invention on arterial vulnerable plaque.
The experimental method comprises the following steps:
(1) physiological saline solutions of free aspirin and clopidogrel were prepared and the 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: after feeding for 30 weeks with high fat diet, atherosclerotic plaque is formed in systemic artery of ApoE-/-mice, and vulnerable plaque is ruptured by snake venom induction to form 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 group of animals did not undergo any therapeutic treatment;
aspirin and clopidogrel gavage group: performing gavage administration treatment at a dosage of 100mg aspirin/kg body weight and 75mg clopidogrel/kg body weight;
Fmoc-Lys (Asp/Clo) -Col/PEG set: the treatment of intravenous administration was carried out at a dose of 10mg aspirin/kg body weight and 7.5mg clopidogrel/kg body weight;
treatment of the treatment groups was performed 1 time every other day for a total of 5 times, except for the vulnerable plaque model control group. For each group of animals, the mortality of the mice was observed for 1 month, and the Bleeding Time (BT) of the mice was measured by tail-cutting.
The experimental results are as follows:
FIG. 27 shows the in vivo therapeutic effect of Fmoc-Lys (Asp/Clo) -Col/PEG system described herein on arterial vulnerable plaques. As shown, mice in the control group (not given any treatment) had a mortality rate of 33%; by adopting aspirin and clopidogrel for intragastric administration, the death rate can be reduced to 29 percent; Fmoc-Lys (Asp/Clo) -Col/PEG treatment was able to reduce mortality to 16%. From the bleeding time, the Fmoc-Lys (Asp/Clo) -Col/PEG group was not significantly prolonged, while the bleeding time of mice orally administered with aspirin and clopidogrel was significantly prolonged.
In summary, oral dual antiplatelet therapy can reduce mortality but prolong bleeding time and increase bleeding risk for vulnerable plaque-ruptured animals. The anti-platelet drug is loaded to the nano delivery system, so that the anti-platelet drug has better curative effect than oral drugs, and the bleeding risk is not increased.
Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.

Claims (18)

1. An amino acid self-assembly nanocarrier delivery system for targeting an activated CD44 molecule, 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, the targeting ligand being a ligand capable of specifically binding to an activated CD44 molecule.
2. An amino acid self-assembled nanocarrier delivery system for targeting vulnerable plaques, 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, the targeting ligand being a ligand capable of specifically binding to an activated CD44 molecule.
3. The nanocarrier delivery system of claim 1 or 2, wherein said amino acid is selected from one of the following: 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 one or more 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.
4. The nanocarrier delivery system of any of claims 1 to 3, wherein the nanocarrier has a particle size of 50-1000 nm, preferably 200 nm.
5. The nanocarrier delivery system of any of claims 1 to 4, wherein the nanocarriers are prepared by self-assembly of starting materials in alkaline conditions.
6. The nanocarrier delivery system of claim 5, wherein the alkaline condition is a pH >8, preferably a pH >10, most preferably a pH of 11.
7. The nanocarrier delivery system of any of claims 1 to 6, wherein the targeting ligand is selected from the group consisting of GAGs, collagen, laminin, fibronectin, selectin, osteopontin, and monoclonal antibodies HI44a, HI313, A3D8, H90, IM7, or hyaluronic acid or derivatives of hyaluronic acid capable of binding specifically to CD44 molecules on the cell surface at vulnerable plaques.
Preferably, the targeting ligand is selected from the group consisting of self-peptide, collagen, hyaluronic acid, selectin, osteopontin or monoclonal antibody HI44a, IM 7.
8. The nanocarrier delivery system of any of claims 1 to 7, wherein the nanocarrier surface can be further modified, preferably by modifying the surface of the nanocarrier with one or more of polyethylene glycol, a cell-penetrating peptide, a self-peptide, or with dual ligands.
9. The nanocarrier delivery system of any of claims 1 to 8, wherein the nanocarriers are loaded with a substance for the diagnosis, prevention and/or treatment of a disease associated with the occurrence of a CD44 molecule activation status; and/or
The nanocarrier is loaded with a substance useful 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 the CD44 molecule 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, an optional CD44 activator and optionally hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to the CD44 molecule on the cell surface at vulnerable plaque.
10. The nanocarrier delivery system of claim 9, wherein said agent for diagnosing, preventing and/or treating a disease associated with the occurrence of a condition of activation of a CD44 molecule is a CD44 activator;
preferably, the activator of CD44 is the CD44 antibody mAb or IL5, IL12, IL18, TNF-a, LPS.
11. The nanocarrier delivery system of claim 9 or 10, wherein said 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, a polypeptide, a nucleic acid and a cytokine for diagnosing, preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque.
12. The nanocarrier delivery system of any of claims 9 to 11, wherein the substance for diagnosing, preventing and/or treating vulnerable plaque or a disease associated with vulnerable plaque is a substance for diagnosing vulnerable plaque or a disease associated with vulnerable plaque;
more preferably, the substance for diagnosing vulnerable plaque or a disease associated with vulnerable plaque is a tracer;
further preferably, the tracer is selected from the group consisting of a CT tracer, an MRI tracer and a nuclear tracer;
even more preferably:
the CT tracer is selected from an iodine nano contrast agent, a gold nano contrast agent, a tantalum oxide nano contrast agent, a bismuth nano contrast agent, a lanthanide series nano contrast agent or other tracers with similar structures; more preferably an iodinated contrast agent or nanogold, or other tracer of similar structure; further preferably iohexol, iocarmic acid, ioversol, iodixanol, iopromide, iobitrol, iomeprol, iopamidol, ioxilan, iozofenac acid, iodipamoic acid, iobenzamic acid, iodoglycanic acid, diatrizoic acid, sodium iothalamate, iodophenyl ester, iopanoic acid, ioxadifen acid, sodium ioioxadifen acid, propiolone, ioxolone, iotrolan, iopridol, meglumine cholate, iothalamic acid, diatrizoate, meglumine methopamoate, iodized oil or ethiodide, or other tracers of similar structure; preferably, the gold nanoparticles are gold nanoparticles;
the MRI tracer is selected from a longitudinal relaxation contrast agent and a transverse relaxation contrast agent; more preferably paramagnetic, ferromagnetic and supermagnetic contrast agents; further preferably Gd-DTPA and linear and cyclic polyamine polycarboxylic chelates thereof, porphyrin chelate of manganese, macromolecular gadolinium chelate, biomacromolecule modified gadolinium chelate, folic acid modified gadolinium chelate, dendrimer developer, liposome modified developer, gadolinium-containing fullerene, or other tracers with similar structures; and then preferably gadopentetate dimeglumine, gadoterate dimeglumine, gadobenate dimeglumine, ferric ammonium citrate effervescent granules and paramagnetic iron oxide, and preferably paramagnetic iron oxide or other tracers with similar structures; 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.
13. The nanocarrier delivery system of any of claims 9 to 12, 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 a disease associated with vulnerable plaque is selected from one or more of statins, fibrates, antiplatelet drugs, PCSK9 inhibitors, anticoagulation drugs, angiotensin converting enzyme inhibitors, calcium antagonists, MMPs inhibitors, beta-blockers, glucocorticoids or other anti-inflammatory substances such as the IL-1 antibody canakinumab, and pharmaceutically acceptable salts thereof, including active preparations of these kinds 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 a disease associated with vulnerable plaque is selected from the group consisting of 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, antisense RNA-145 and nucleic acids such as aspirin strands thereof, aspirin, Acemetacin, troxerutin, dipyridamole, cilostazol, ticlopidine hydrochloride, ozagrel sodium, clopidogrel, prasugrel, cilostazol, beraprost sodium, ticagrelor, cagrelor, tirofiban, eptifibatide, abciximab, heparin, kexel, sapalin, flavamoeba sodium, warfarin, dabigatran, rivaroxaban, apixaban, idoxaban, bivalirudin, enoxaparin, titazaparin, aclarudilin, dicumarol, nitrocoumarin, lycium sodium, hirudin, argatroban, benazepril, captopril, enalapril, perindopril, fosinopril, lisinopril, moexipril, cilazapril, quinapril, ramipril, trandolapril, candesartan, losartan, valsartan, or, One or more of tasosartan, nifedipine, nicardipine, nitrendipine, amlodipine, nimodipine, nisoldipine, nilvadipine, isradipine, felodipine, lacidipine, diltiazem, verapamil, chlorhexidine, minocycline, MMI-166, metoprolol, atenolol, bisoprolol, carvedilol, batimastat, marimastat, prinomastat, BMS-279251, BAY 12-9566, TAA211, AAJ996A, nacetrapib, evacetapib, Torcetrapib, and Dalcetrapib, prednisone, methylprednisolone, betamethasone, beclometasone propionate, dipalmondone, prednisolone, hydrocortisone, dexamethasone, or other anti-inflammatory substances such as IL-1 antibody, and pharmaceutically active fragments or pharmaceutically acceptable salts thereof, and pharmaceutically active fragments of one or more of these species, including the pharmaceutically active species of these drugs, and endogenous anti-inflammatory cytokines such as interleukin 10.
14. A method of manufacturing a nanocarrier delivery system according to any of claims 1 to 13, wherein the method comprises the step of strengthening the nanocarriers by adding dopamine.
15. A pharmaceutical composition comprising the nanocarrier delivery system of any of claims 1 to 13.
16. A diagnostic formulation comprising the nanocarrier delivery system of any of claims 1 to 13.
17. Use of a nanocarrier delivery system of any of claims 1-13 in the manufacture of a product for the diagnosis, prevention and treatment of vulnerable plaque or a disease associated with vulnerable plaque.
18. The use of claim 17, wherein the vulnerable plaque is selected from one or more of a ruptured plaque, an erosive plaque, and a partially calcified nodular lesion;
more preferably, the disease associated with vulnerable plaque is selected from one or more of the following: atherosclerosis, coronary atherosclerotic heart disease, cerebral atherosclerosis, peripheral vascular atherosclerosis, aortic dissection, hemangioma, thromboembolism, heart failure, and cardiogenic shock;
preferably, the coronary atherosclerotic heart disease is selected from one or more of: acute coronary syndrome, asymptomatic myocardial ischemia-latent coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, and in-stent restenosis;
the cerebral atherosclerosis is stroke; and/or
The peripheral vascular atherosclerosis is selected from one or more of the following: atherosclerosis of the carotid, peri-occlusive atherosclerosis, retinal atherosclerosis, renal atherosclerosis, lower extremity atherosclerosis, upper extremity atherosclerosis, atherosclerotic impotence.
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