Liposome nanocarrier delivery system for targeting active CD44 molecules, method for preparing same, and use thereof

Abstract

To provide a preparation method and the use of a nanocarrier especially a liposome delivery system, in the diagnosis, prevention and treatment of a vulnerable plaque or a disease associated with the vulnerable plaque.SOLUTION: The present invention provides a liposomal nanocarrier delivery system for targeting an active CD44 molecule, preparation method therefor, and uses thereof. The surface of the liposome is partially modified by a targeting ligand, wherein the targeting ligand is a ligand that can be specifically combined with the active CD44 molecule. The liposomal nanocarrier delivery system can be used for diagnosing, preventing, and treating vulnerable plaque or diseases related to vulnerable plaque.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of Chinese Patent Application No. 201810060265.9, filed on January 22, 2018, which is incorporated herein by reference in its entirety.

[0002] The present invention belongs to the technical field of targeted drug delivery, and particularly relates to nanocarriers for targeting activated CD44 molecules, particularly for targeting vulnerable plaques, and in particular to liposomal nanocarrier delivery systems. The present invention further relates to methods for preparing and using nanocarriers, in particular liposomal delivery systems, particularly in the diagnosis, prevention, and treatment of vulnerable plaques or diseases associated with vulnerable plaques. [Background technology]

[0003] Currently, acute cardiovascular events, mainly including acute myocardial infarction and sudden cardiac death, have become the number one threat to human health. Statistics show that approximately 20 million people die from acute cardiovascular events each year worldwide. The situation in China is also not optimistic. More than 700,000 people die from acute myocardial infarction and sudden cardiac death each year, making it one of the most prominent diseases seriously threatening the health of the Chinese people. Research has shown that most cases of acute myocardial infarction and sudden cardiac death are caused by atherosclerotic plaques. Since the 1970s, the processes and mechanisms linking chronic atherosclerotic plaques to acute coronary syndrome (ACS) and stroke have been continuously explored.

[0004] In 1989, Muller and colleagues (Circadian Variation and Triggers of Onset of Acute Cardiovascular Disease. Circulation. 1989; 79(4): 733-43) proposed the concept of "vulnerable plaque" and postulated that such plaques are the underlying cause of most acute cardiovascular and cerebrovascular events. Vulnerable plaque (also known as "unstable plaque") refers to atherosclerotic plaques that are prone to thrombus formation or that are likely to rapidly progress to "criminal plaques," including rupture-prone plaques, erosion-prone plaques, and partially calcified nodular lesions. Numerous studies have shown that most acute myocardial infarctions and strokes are caused by the rupture of unstable plaques with mild to moderate stenosis, followed by thrombosis. Naghavi and colleagues (New Developments in the Detection of Vulnerable Plaque. Curr Atheroscler Rep. 2001; 3(2): 125-35) provided a histological definition and diagnostic criteria for vulnerable plaque. The primary diagnostic criteria include active inflammation, a thin fibrous cap and a large lipid core, endothelial denudation with surface platelet aggregation, plaque fissures or lesions, and severe stenosis. Secondary diagnostic criteria include surface calcified plaque, yellow and shiny plaque, intraplaque hemorrhage, and positive remodeling. Therefore, early intervention is important for vulnerable plaque. However, because the degree of vascular stenosis caused by vulnerable plaque is usually not very high and many patients do not show precursory symptoms, early diagnosis of vulnerable plaque in clinical settings is very difficult, making it very dangerous. Therefore, an urgent problem to be solved in the prevention and treatment of acute myocardial infarction is how to accurately identify and diagnose vulnerable plaque as early as possible so that effective intervention can be performed.

[0005] Currently, commonly used techniques for diagnosing vulnerable plaque mainly include coronary angiography, intravascular ultrasound (IVUS), optical coherence tomography (OCT), etc. However, these techniques are all invasive tests with low diagnostic resolution and accuracy, and high costs, which limit their clinical use to some extent. Therefore, there is currently an urgent need for techniques and preparations for non-invasively diagnosing vulnerable plaque.

[0006] In addition, current methods for treating vulnerable plaques primarily rely on systemic administration, such as oral administration of statins (hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors), aspirin, matrix metalloproteinase (MMP) inhibitors, and / or fibrates. These drugs act to stabilize plaques by reducing lipids in plaques and improving vascular remodeling, for example, by regulating systemic blood lipid levels, combating inflammation, inhibiting proteases, and platelet production. However, in clinical applications, the therapeutic effects of current drugs for treating vulnerable plaques have proven unsatisfactory. For example, statins commonly used in clinical practice exhibit relatively low bioavailability when administered orally, such as less than 5% for simvastatin, approximately 12% for atorvastatin, and approximately 20% for rosuvastatin. Furthermore, animal experiments have confirmed that the fibrous cap thickness can be increased and the plaque volume can be reduced only when the statin dose is increased to a dose higher than 1 mg / kg, thereby posing a bottleneck in the stability of oral statins and their effect in reducing plaque. Furthermore, clinical trials have now confirmed that treatment of unstable plaque with oral statins requires very large doses to stabilize the unstable plaque, and that treatment with large systemic doses of statins poses a risk of increased incidence of severe side effects (such as liver dysfunction, rhabdomyolysis, and type II diabetes).

[0007] In the case of existing systemic administration, after a drug enters the body, usually only a very low percentage of the active ingredient can actually act on the lesion site. This is the fundamental reason for limiting the effectiveness of the drug and causing toxic side effects. A targeted drug delivery system refers to a drug delivery system capable of targeted drug delivery. After being administered via a certain route, the drug contained in the targeted drug delivery system is specifically concentrated at the target site by a carrier having a targeting probe. The targeted drug delivery system allows the drug to target a specific lesion site and release the active ingredient at the target lesion site. Therefore, the targeted drug delivery system can achieve a relatively high concentration of the drug at the target lesion site and a reduced dose of the drug in the blood circulation, thereby suppressing toxic side effects and reducing damage to normal tissues and cells while improving the drug effect.

[0008] In the field of vulnerable plaque diagnosis and treatment, there are several techniques for diagnosing vulnerable plaque by modifying nanocarriers with targeting ligands. However, a major problem with such targeting probes targeting vulnerable plaque in clinical practice is the insufficient specificity of these preparations to the target site. For example, most of such preparations select macrophages as the target site. However, because macrophages exist throughout the body, the targeting specificity of the probes is not satisfactory. Therefore, the difficulty in developing targeting preparations targeting vulnerable plaque lies in finding target sites with significant targeting specificity in cells within vulnerable plaque.

[0009] CD44 is a type of adhesion molecule that is widely distributed on the surface of lymphocytes, monocytes, endothelial cells, etc. The main ligand of the CD44 molecule is hyaluronan (abbreviated as "HA"). Based on the activation state of CD44-expressing cells, CD44 can exist in a relatively static state (unable to bind HA), an induced activated state (able to bind HA after activation), and a constitutively active state (able to bind HA without activation). CD44 on the surface of most normal cells is in a relatively static state and unable to bind HA.

[0010] Several previous studies have shown that CD44 is not an ideal targeting moiety with significant targeting specificity because it is widely distributed in the human body, especially on the surface of organs rich in the reticuloendothelial system. Therefore, the following problem will be encountered when developing targeted drug delivery systems using CD44 as a targeting moiety: if the CD44 on the surface of the target cell has insufficient affinity for HA to provide significant specificity, such targeted drug delivery systems will not exhibit specific targeting properties.

[0011] Therefore, finding a specific target site present in vulnerable plaque and a targeted drug delivery system suitable for targeting vulnerable plaque, and thereby developing a targeted drug delivery system that can specifically target vulnerable plaque and achieve stable and sustained release of a drug, has become an urgent technical problem to be solved in the medical field.

[0012] To date, there have been no reports on the expression status of CD44 on the surfaces of macrophages, monocytes, endothelial cells, lymphocytes, and smooth muscle cells that are primarily present within vulnerable plaques, or on their affinity for HA. Furthermore, there is no prior art for designing a targeted drug delivery system that achieves stable and sustained release of drugs by utilizing both the interaction between HA and CD44 and the specific microenvironment of vulnerable plaques for the diagnosis or treatment of vulnerable plaques or diseases associated with vulnerable plaques. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Muller et al, Circadian Variation and Triggers of Onset of Acute Cardiovascular Disease. Circulation. 1989; 79(4): 733-43 [Non-patent document 2] Naghavi et al, New Developments in the Detection of Vulnerable plaque. Curr Atheroscler Rep. 2001; 3(2): 125-35 Summary of the Invention [Means for solving the problem]

[0014] (1) Overview of the present invention The present inventors have found that, compared with normal cells, CD44 on the surface of cells in vulnerable plaques, such as endothelial cells, macrophages, and smooth muscle cells, is activated by the specific microenvironment of vulnerable plaques (such as inflammatory factors), and therefore their ability to bind to HA increases several orders of magnitude. This finding suggests that the large number of activated CD44 molecules present on the surface of cells in vulnerable plaques provides ideal targeting sites for a targeted drug delivery system that uses HA as a targeting ligand. To this end, the present invention provides a targeted drug delivery system for specifically targeting activated CD44 molecules, particularly for targeting vulnerable plaques.

[0015] The present inventors have also discovered that loading a CD44 activator can promote further activation of CD44 on the surface of diseased cells and rapidly amplify the targeting affinity of CD44 for HA, thereby significantly increasing the concentration of targeting compounds bound to the cell surface, which is of positive importance for tracer diagnosis and treatment of vulnerable plaques. To this end, the targeted drug delivery system of the present invention may be simultaneously loaded with a CD44 activator, thereby significantly increasing the concentration of tracer or therapeutic compounds in a short period of time, thereby improving diagnostic sensitivity or therapeutic efficacy.

[0016] The present inventors have also found that in vulnerable plaques, along with high levels of CD44 activation and overexpression, the endogenous macromolecule HA is produced in large quantities upon stimulation, which binds to CD44 on the cell surface and promotes the aggregation of cells such as macrophages and lymphocytes in vulnerable plaques. Such endogenous HA binds to CD44 on the cell surface, forming a barrier to drug entry and potentially reducing drug bioavailability. To this end, the targeted drug delivery system of the present invention may be loaded with low-molecular-weight hyaluronic acid or hyaluronic acid derivatives capable of specifically binding to CD44 molecules on the cell surface of vulnerable plaques. This eliminates the barrier formed by endogenous HA on the cell surface by competing with the binding of endogenous HA to the cell surface, facilitating successful intracellular drug release in diseased cells and providing significant therapeutic effects.

[0017] In summary, the present invention relates to the following aspects: The present invention provides a liposomal nanocarrier delivery system for targeting activated CD44 molecules.

[0018] The present invention provides a liposomal nanocarrier delivery system for targeting vulnerable plaque.

[0019] The present invention provides methods for preparing the liposomal nanocarrier delivery systems of the present invention for targeting vulnerable plaque.

[0020] The present invention further provides a medicament comprising the nanocarrier delivery system of the present invention for targeting vulnerable plaque and a pharmaceutically acceptable carrier.

[0021] The present invention further provides a diagnostic preparation comprising the nanocarrier delivery system of the present invention for targeting vulnerable plaque.

[0022] The present invention further provides use of the nanocarrier delivery system of the present invention for targeting vulnerable plaque in the preparation of a medicament for preventing and / or treating vulnerable plaque or a disease associated with vulnerable plaque.

[0023] The present invention further provides the use of the nanocarrier delivery system of the present invention for targeting vulnerable plaque in the preparation of a diagnostic preparation for diagnosing vulnerable plaque or a disease associated with vulnerable plaque.

[0024] The present invention further provides a method for preventing and / or treating vulnerable plaque or a disease associated with vulnerable plaque, comprising the step of administering to a subject in need thereof the nanocarrier delivery system of the present invention for targeting vulnerable plaque.

[0025] The present invention further provides a method for diagnosing vulnerable plaque or a disease associated with vulnerable plaque, comprising the step of administering to a subject in need thereof the nanocarrier delivery system of the present invention for targeting vulnerable plaque.

[0026] Specific embodiments of the technical solutions of the present invention and their implications will be described in detail below.

[0027] (2) Technical terms and their meanings The terms referred to in this specification have the following meanings: "Vulnerable plaque" (also known as "unstable plaque") refers to an atherosclerotic plaque that is prone to thrombus formation or likely to rapidly progress to "criminal plaque," including rupture-prone plaque, erosion-prone plaque, and partially calcified nodular lesion. Numerous studies have shown that most acute myocardial infarctions and strokes are caused by the rupture of unstable plaque with mild to moderate stenosis, followed by thrombosis. Histological signs of unstable plaque include active inflammation, a thin fibrous cap and large lipid core, endothelial denudation with surface platelet aggregation, plaque fissures or lesions, and severe stenosis, as well as surface calcified plaque, yellow and shiny plaque, intraplaque hemorrhage, and positive remodeling.

[0028] "Diseases associated with unstable plaque" primarily refer to diseases that are associated with, characterized by, or caused by unstable plaque, or that are secondary to unstable plaque during the onset and development of the disease. "Diseases associated with unstable plaque" primarily include atherosclerosis, coronary atherosclerotic heart disease (acute coronary syndrome, silent myocardial ischemia-occult coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, and in-stent restenosis), cerebral arteriosclerosis (including stroke), peripheral atherosclerosis (peripheral arterial occlusive disease, retinal arteriosclerosis, carotid artery atherosclerosis, renal atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, and atherosclerotic impotence), aortic dissection, hemangioma, thromboembolism, heart failure, cardiogenic shock, etc.

[0029] A "targeted drug delivery system" refers to a drug delivery system capable of targeted drug delivery. After administration via a certain route, the drug contained in the targeted drug delivery system is specifically concentrated at the target site through the action of a special carrier or targeting warhead (e.g., a targeting ligand). Current known methods for achieving targeted drug delivery include utilizing the passive targeting properties of various microparticle delivery systems, introducing chemical modifications to the surface of the microparticle delivery system, utilizing some special physical and chemical properties, utilizing antibody-mediated targeted drug delivery, utilizing ligand-mediated targeted drug delivery, and utilizing prodrug targeted drug delivery. Among these, ligand-mediated targeted drug delivery utilizes the characteristic that a drug carrier is combined with a ligand, and specific receptors in certain organs and tissues specifically bind to the specific ligand, thereby directing the drug to a specific target tissue.

[0030] A "liposome carrier" is a lipid-like bilayer drug carrier that encapsulates a drug in the lipid bilayer to form a microvesicle. It may also be a lipid bicelle structure, a disk-shaped vesicle typically formed by the self-assembly of long-chain and short-chain phospholipids or surfactants. The long-chain phospholipids form the flat surface of the disk, while the short-chain phospholipids surround the lateral edges of the disk. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) is often used as the long-chain phospholipid component. The surface charge of the bicelle can be varied, and versatility can be achieved by doping it with other phospholipid components with the same chain length but different head groups. Furthermore, the short-chain phospholipids form highly flexible regions, reducing the edge energy of the aggregates and stabilizing the bicelle.

[0031] "Hyaluronic acid" (abbreviated as "HA") is a macromolecular polymer (C 14 H 21 NO 11 ) nIt has the formula: It is a high-molecular polysaccharide composed of D-glucuronic acid and N-acetylglucosamine units. D-glucuronic acid and N-acetylglucosamine are linked by β-1,3-glycosidic bonds, and the disaccharide units are linked by β-1,4-glycosidic bonds. Due to its unique molecular structure and physical and chemical properties, hyaluronic acid exhibits various important physiological functions in living organisms, such as lubricating joints, regulating vascular wall permeability, regulating the diffusion and transport of proteins, water, and electrolytes, and promoting wound healing. It is particularly important that hyaluronic acid has a special water-retaining effect and is the substance with the best moisturizing properties found in nature.

[0032] "Derivatives of hyaluronic acid," as used herein, refers to any derivative of hyaluronic acid that maintains the ability of hyaluronic acid to specifically bind to CD44 molecules on the surface of cells in vulnerable plaque, including, but not limited to, pharmaceutically acceptable salts of hyaluronic acid, lower alkyl (alkyl containing 1 to 6 carbon atoms) esters, prodrugs capable of forming hyaluronic acid in vivo by hydrolysis or other means, etc. Determining whether a substance is a "derivative of hyaluronic acid" can be accomplished by measuring the ability of the substance to specifically bind to CD44 molecules on the surface of cells in vulnerable plaque, and is within the skill of one in the art.

[0033] The "CD44 molecule" is a type of transmembrane proteoglycan adhesion molecule that is widely expressed on the cell membrane of cells such as lymphocytes, monocytes, and endothelial cells and consists of three compartments: the extracellular compartment, the transmembrane compartment, and the intracellular compartment. The CD44 molecule mediates various interactions between cells and between cells and the extracellular matrix, and is involved in various signal transduction in the body, thereby altering the biological functions of cells. The main ligand of the CD44 molecule is hyaluronan, and receptor-ligand binding between the CD44 molecule and hyaluronan determines cell adhesion and / or migration in the extracellular matrix. In addition, the CD44 molecule is also involved in hyaluronan metabolism.

[0034] "About" refers to all values ​​in the series that fall within a range of ±5% of the numerical value given thereafter.

[0035] (3) Detailed description of the invention A first aspect of the present invention provides a liposomal nanocarrier delivery system for targeting activated CD44 molecules, wherein the surface of the nanocarrier is partially modified with a targeting ligand, and the targeting ligand is a ligand capable of specifically binding to activated CD44 molecules.

[0036] The second aspect of the present invention provides a liposomal nanocarrier delivery system for targeting vulnerable plaques, in which the surface of the nanocarrier is partially modified with a targeting ligand, which is capable of specifically binding to CD44 molecules on the cell surface of vulnerable plaques. Other modifications can be made to the surface of the nanocarrier to achieve better results. Modification of the surface of the carrier with PEG can achieve a long-circulation effect and extend the half-life of the drug. Modification of the surface of the carrier with a transmembrane peptide, a self-peptide SEP, or simultaneous modification with a biligand can all play a role in amplifying the drug effect.

[0037] According to the nanocarrier delivery system of the first or second embodiment, the liposome carrier is selected from bicelles, small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. A "liposome carrier" is a lipid bilayer drug carrier that encapsulates a drug in the lipid bilayer or hydrophilic lumen to form a microvesicle or disc-shaped structure.

[0038] The structure of a bicelle may generally be a disc-shaped vesicle formed by the self-assembly of long-chain phospholipids and short-chain phospholipids or surfactants. The long-chain phospholipids form the flat surface of the disc, while the short-chain phospholipids surround the lateral edges of the disc. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) is often used as the long-chain phospholipid component, allowing for the surface charge of the bicelle to be varied. This versatility can be achieved by doping with other phospholipid components with the same chain length but different head groups. Furthermore, 1,2-di-n-heptadecanoylphosphatidylcholine is often selected as the short-chain phospholipid, forming a highly flexible region, reducing the edge energy of the aggregates and stabilizing the bicelle.

[0039] According to the nanocarrier delivery system of the first or second aspect, the targeting ligand is selected from GAG, collagen, laminin, fibronectin, selectin, osteopontin (OPN), and monoclonal antibodies HI44a, HI313, A3D8, H90, and IM7, or a targeting ligand for vulnerable plaque. The hyaluronic acid or hyaluronic acid derivative is selected from hyaluronic acid or a hyaluronic acid derivative capable of specifically binding to a CD44 molecule on the cell surface of a subject.

[0040] According to the nanocarrier delivery system of the first or second aspect, the nanocarriers are loaded with a substance for diagnosing, preventing, and / or treating a disease associated with the presence of CD44 molecule activation.

[0041] According to the liposomal nanocarrier delivery system of the first or second aspect, the nanocarriers are loaded with an agent for diagnosing, preventing, and / or treating vulnerable plaque or a disease associated with vulnerable plaque.

[0042] In embodiments, the agent is for diagnosing vulnerable plaque or a disease associated with vulnerable plaque.

[0043] In an embodiment, the substance for diagnosing vulnerable plaque or a disease associated with vulnerable plaque is a tracer.

[0044] In embodiments, the tracer is selected from a CT tracer, an MRI tracer, and a radioisotope tracer.

[0045] In embodiments, the CT tracer is selected from an iodine-based nanoscale contrast agent, a gold-based nanoscale contrast agent, a tantalum oxide-based nanoscale contrast agent, a bismuth-based nanoscale contrast agent, a lanthanide-based nanoscale contrast agent, or other tracers with similar structure; more preferably, the CT tracer is selected from an iodinated contrast agent, or nanogold, or other tracers with similar structure; even more preferably, the CT tracer is selected from iohexol, iocarmic acid, ioversol, iodixan. ol, iopromide, iobitridol, iomeprol, iopamidol, ioxilan, acetolizoic acid, iodipamide, iobenzamic acid, ioglycamic acid, diatrizoic acid, sodium iothalamate, pantopac, iopanoic acid, iodoalfionic acid, sodium acetolizoate, sodium iodometamate, propriodone, geodon, iotrolan, iopidol, endografin, iothalamic acid, diatrizoic acid meglumine di, metrizoic acid, metrizamide, iodized oil, or ethiodized oil, or other tracers with a similar structure; preferably, the CT tracer is nanogold; and / or The MRI tracer is selected from longitudinal relaxation contrast agents and transverse relaxation contrast agents; more preferably, the MRI tracer is selected from paramagnetic contrast agents, ferromagnetic contrast agents, and superparamagnetic contrast agents; even more preferably, the MRI tracer is selected from Gd-DTPA and linear, cyclic polyamine polycarboxylate chelators and their manganese porphyrin chelators, macromolecular gadolinium chelators, biomacromolecule-modified gadolinium chelators, folate-modified gadolinium chelators, dendrimer contrast agents, liposome-modified contrast agents, and gadolinium-containing fullerenes, or other tracers with similar structures; and preferably, the MRI tracer is selected from gadopentetate dimeglumine, gadoterate meglumine, gadobenate dimeglumine, gadodiamide, ferric ammonium citrate effervescent granules, paramagnetic iron oxide (Fe3O4NP), or other tracers with similar structures; preferably, the MRI tracer is Fe3O4NP; The radioisotope tracer is selected from fludeoxyglucose labeled with carbon-14 (C), carbon-13 (C), phosphorus-32 (P), sulfur-35 (S), iodine-131 (I), hydrogen-3 (H), technetium-99 (Tc), and fluorine-18 (F); preferably, the radioisotope tracer is fluorine-18 labeled fludeoxyglucose.

[0046] In embodiments, the agent is 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.

[0047] In an embodiment, the agent is a CD44 activator.

[0048] In embodiments, the CD44 activator is a CD44 antibody mAb, IL5, IL12, IL18, TNF-α, or LPS.

[0049] In embodiments, the agent is a low molecular weight hyaluronic acid or hyaluronic acid derivative capable of specifically binding to CD44 molecules on the cell surface of vulnerable plaque; Preferably, the low molecular weight hyaluronic acid or hyaluronic acid derivative capable of specifically binding to CD44 molecules on the cell surface of vulnerable plaque has a molecular weight in the range of 1 to 500 KDa, preferably 1 to 20 KDa, more preferably 2 to 10 KDa.

[0050] In embodiments, the nanocarriers are co-loaded with an agent for diagnosing, preventing, and / or treating vulnerable plaque or a disease associated with vulnerable plaque, and a CD44 activator; Preferably, the nanocarriers are simultaneously loaded with a substance for preventing and / or treating vulnerable plaque or a disease associated with vulnerable plaque, and with low molecular weight hyaluronic acid or a hyaluronic acid derivative capable of specifically binding to CD44 molecules on the cell surface of vulnerable plaque; More preferably, the nanocarriers are simultaneously loaded with a substance for diagnosing vulnerable plaque or a disease associated with vulnerable plaque, a substance for preventing and / or treating vulnerable plaque or a disease associated with vulnerable plaque, optionally a CD44 activator, and optionally a low molecular weight hyaluronic acid or hyaluronic acid derivative capable of specifically binding to CD44 molecules on the cell surface of vulnerable plaque.

[0051] In embodiments, the agent is 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 one or more selected from the group consisting of statins, fibrates, antiplatelet drugs, PCSK9 inhibitors, anticoagulants, angiotensin converting enzyme inhibitors (ACEIs), calcium ion antagonists, MMP inhibitors, beta-receptor blockers, glucocorticoids, and other anti-inflammatory substances such as the IL-1 antibody canakinumab, and pharmaceutically acceptable salts thereof, including active preparations of the above drugs or substances, and endogenous anti-inflammatory cytokines such as interleukin 10 (IL-10); More preferably, the substance for preventing and / or treating vulnerable plaque or 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-PCSK9 antibodies such as evolocumab, alirocumab, bococizumab, RG7652, LY3015014, and LGT-209, and adnectins such as BMS-962476, ALN-PCSs, and the like. antisense RNAi oligonucleotides such as microRNA-33a, microRNA-27a / b, microRNA-106b, microRNA-302, microRNA-758, microRNA-10b, microRNA-19b, microRNA-26, microRNA-93, microRNA-128-2, microRNA-144, microRNA-145 antisense strands, and their nucleic acid analogs such as locked nucleic acids; aspirin, acemetacin, troxerutin, dipyridamole, cilostazol, ticlopidine hydrochloride, Ozagrel sodium, clopidogrel, prasugrel, cilostazol, beraprost sodium, ticagrelor, cangrelor, tirofiban, eptifibatide, abciximab, unfractionated heparin, clexane, fraxiparin, fondaparinux sodium, warfarin, dabigatran, rivaroxaban, apixaban, edoxaban, bivalirudin, enoxaparin, dalteparin, ardeparin, bishydroxycoumarin, coumarin nitrate, sodium citrate, hirudin, argatroban, benazepril, captopril, enalapril , perindopril, fosinopril, lisinopril, moexipril, cilazapril, perindopril, quinapril, ramipril, trandolapril, candesartan, eprosartan, irbesartan, losartan, telmisartan, valsartan, olmesartan, tasosartan, nifedipine, nicardipine, nitrendipine, amlodipine, nimodipine, nisoldipine, nilvadipine, isradipine, felodipine, lacidipine, diltiazem, verapamil, chlorhexidine, minocycline, MMI-166, metoprolol, atenolol,One or more of bisoprolol, propranolol, carvedilol, batimastat, marimastat, prinomastat, BMS-279251, BAY12-9566, TAA211, AAJ996A, nacetrapib, evacetrapib, torcetrapib, dalcetrapib, prednisone, methylprednisolone, betamethasone, beclomethasone dipropionate, diprospan, prednisolone, hydrocortisone, dexamethasone, and other anti-inflammatory substances such as the IL-1 antibody canakinumab, and active fragments or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable salts containing active structural fragments of the above substances, and one or more of endogenous anti-inflammatory cytokines such as interleukin-10 (IL-10).

[0052] A third aspect of the present invention provides a method for preparing a nano-delivery system for targeting vulnerable plaque according to the first or second aspect, comprising: (1) dissolving an appropriate amount of phospholipid molecules in a suitable organic solvent and preparing liposome nanocarriers by thin film hydration method, whereby drug molecules with lower polarity need to form a thin film together with the phospholipid molecules in this step; (2) an optional step of adding an aqueous medium, which may contain a water-soluble substance for diagnosing, preventing, and / or treating vulnerable plaque or a disease associated with vulnerable plaque, to the nanocarrier delivery system obtained in step (1) to form a coarse suspension; (3) dissolving the targeting ligand in a suitable buffer solution solvent, and adding the carrier molecule obtained in step (2) to the targeting ligand solution for reaction to obtain a nanocarrier delivery system; (4) an optional step of removing by dialysis any unloaded substance for diagnosing, preventing, and / or treating vulnerable plaque or a disease associated with vulnerable plaque contained in the crude suspension obtained in step (3) to obtain a loaded nanodelivery system. The present invention provides a method comprising:

[0053] A fourth aspect of the present invention provides a medicament comprising a nanocarrier delivery system according to the first or second aspect and a pharmaceutically acceptable carrier.

[0054] A fifth aspect of the present invention provides a diagnostic preparation comprising a nanocarrier delivery system according to the first or second aspect.

[0055] A sixth aspect of the present invention provides the use of a nanocarrier delivery system according to the first or second aspect, a medicament according to the fourth aspect, or a diagnostic preparation according to the fifth aspect in a preparation for preventing and / or treating a disease associated with the presence of CD44 molecule activation.

[0056] A seventh aspect of the present invention provides the use of a nanocarrier delivery system according to the first or second aspect, a medicament according to the fourth aspect, or a diagnostic preparation according to the fifth aspect in a preparation for preventing and / or treating vulnerable plaque or a disease associated with vulnerable plaque.

[0057] According to the use of the seventh aspect, the vulnerable plaque is one or more selected from the group consisting of rupture-prone plaque, erosion-prone plaque, and partially calcified nodular lesion; Preferably, the disease associated with vulnerable plaque is one or more selected from the group consisting of atherosclerosis, coronary artery sclerosis (acute coronary syndrome, silent myocardial ischemia-occult coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, and in-stent restenosis), cerebral arteriosclerosis (including stroke), peripheral vascular atherosclerosis (peripheral arterial occlusive disease, retinal arteriosclerosis, carotid artery atherosclerosis, renal atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, and atherosclerotic impotence), aortic dissection, hemangioma, thromboembolism, heart failure, and cardiogenic shock.

[0058] An eighth aspect of the present invention provides a method for preventing and / or treating a disease associated with the presence of CD44 molecule activation, the method comprising the step of administering to a subject in need thereof a nanocarrier delivery system according to the first or second aspect, a drug according to the fourth aspect, or a diagnostic preparation according to the fifth aspect.

[0059] A ninth aspect of the present invention provides a method for preventing and / or treating vulnerable plaque or a disease associated with vulnerable plaque, the method comprising administering a nanocarrier delivery system according to the first or second aspect, a drug according to the fourth aspect, or a diagnostic preparation according to the fifth aspect to a subject in need thereof; Preferably, the vulnerable plaque is one or more selected from the group consisting of rupture-prone plaque, erosion-prone plaque, and partially calcified nodular lesion; More preferably, the disease associated with unstable plaque is one or more selected from the group consisting of atherosclerosis, coronary artery sclerosis (acute coronary syndrome, silent myocardial ischemia-occult coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, and in-stent restenosis), cerebral arteriosclerosis (including stroke), peripheral vascular atherosclerosis (including peripheral arterial occlusive disease, retinal arteriosclerosis, carotid artery atherosclerosis, renal atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, and atherosclerotic impotence), aortic dissection, hemangioma, thromboembolism, heart failure, and cardiogenic shock. A method is provided.

[0060] A tenth aspect of the present invention provides a method for diagnosing a disease associated with the presence of CD44 molecule activation, the method comprising the step of administering to a subject in need thereof a nanocarrier delivery system according to the first or second aspect, a drug according to the fourth aspect, or a diagnostic preparation according to the fifth aspect.

[0061] In summary, the nanocarrier delivery system of the present invention has the following advantages for diseases associated with CD44 molecule activation: 1) The nanocarrier delivery system of the present invention can specifically bind to activated CD44 molecules and achieve stable and sustained drug release. 2) CD44 on the cell surface of vulnerable plaques is induced and activated by the extracellular matrix microenvironment, resulting in the overexpression of large amounts of CD44 and the significantly enhanced affinity of CD44-HA, so the interaction between CD44 and HA in vulnerable plaques has extremely significant affinity specificity. Therefore, CD44 in vulnerable plaques is an excellent target for nanocarrier delivery systems to target vulnerable plaques. 3) The nanocarrier delivery system for targeting vulnerable plaques can actively target vulnerable plaques and combine with lesion cells, thereby achieving sustained release of the loaded substance at the lesion, significantly increasing and continuously maintaining the substance concentration in the lesion area, thereby improving the diagnostic or therapeutic effect of the delivery system. 4) The nanocarrier delivery system for targeting vulnerable plaques according to the present invention may also be loaded with a CD44 activator, i.e., a CD44 activator such as IL5, IL12, IL18, TNF-α, or LPS. The loaded CD44 activator can promote further activation of CD44 on the surface of lesion cells, rapidly increasing the targeting affinity of CD44 to hyaluronic acid and significantly increasing the concentration of the targeted nanocarrier composition bound to the cell surface. This significantly increases the concentration of the tracer or therapeutic compound in a short period of time, thereby improving diagnostic resolution or therapeutic efficacy, and is therefore of positive importance for tracer diagnosis and treatment of vulnerable plaques. [Brief explanation of the drawings]

[0062] In order that the contents of the present invention may be fully understood, the present invention will now be described in more detail with reference to specific examples and the accompanying drawings. [Figure 1] 1 is an electron microscope photograph of LP1-(R)-HA of Example 1. [Figure 2]1 is an infrared spectrogram of LP1-(R)-HA of Example 1. [Figure 3] 1 is a characterization diagram of LP1-(R)-SP of Example 2. [Figure 4] 1 is a characterization diagram of LP1-(R)-HA / Tat in Example 3. [Figure 5] FIG. 1 is a characterization diagram of LP2-(At)-HA from Example 4. [Figure 6] 1 is a characterization diagram of LP2-(At)-SEP / IM7 of Example 5. [Figure 7] 1 is a characterization diagram of LP2-(At / miRNA-33a)-IM7 of Example 6. [Figure 8] 1 is a characterization diagram of LP2-(AuNP / R)-OPN in Example 7. [Figure 9] FIG. 1 is a characterization diagram of LP1-(Fe3O4 / DXMS)-HI44a of Example 8. [Figure 10] 10 is a characterization diagram of LP1-(Fe3O4 / IL-10)-HI44a of Example 9. [Figure 11] 1 is a characterization diagram of LP1-(Asp / Clo)-Col of Example 10. [Figure 12] 1 is a characterization diagram of LP1-(F-FDG)-OPN of Example 11. [Figure 13] FIG. 1 shows the effect of long-term storage on particle size stability in Experimental Example 1. [Figure 14] FIG. 1 shows the effect of long-term storage on the encapsulation rate in Experimental Example 1. [Figure 15] FIG. 1 shows the cumulative drug release rate in vitro of the liposome carrier of Experimental Example 1. [Figure 16] 1 shows a nuclear magnetic resonance imaging image of the mouse atherosclerotic vulnerable plaque model constructed in Experimental Example 2. [Figure 17]1 is a graph showing the results of determination of CD44 content (expressed as semiquantitative integrated values) on the surface of endothelial cells of normal arterial vessel walls and on the surface of endothelial cells of arterial vulnerable plaque in a mouse model. [Figure 18] Figure 1 shows the results of determining the avidity of CD44 for HA (expressed as avidity integrals) on the surface of endothelial cells from normal arterial vessel walls and on the surface of endothelial cells from vulnerable arterial plaque in a mouse model. [Figure 19] Graph showing the determination of the avidity of CD44 for HA on the surface of macrophages from outside and inside vulnerable arterial plaque in a mouse model (expressed as avidity integrals). [Figure 20] 1 is a graph showing the therapeutic effects of the LP1-(R)-HA, LP1-(R)-SP, and LP1-(R)-HA / Tat nanodelivery systems of the present invention on vulnerable carotid artery plaque in a mouse model. [Figure 21] 1 is a graph showing the therapeutic effects of the LP2-(At)-HA, LP2-(At)-SEP / IM7, and LP2-(At / miRNA-33a)-IM7 nanodelivery systems of the present invention on vulnerable carotid artery plaque in a mouse model. [Figure 22] 1 is a graph showing the in vivo tracing effect of LP2-(AuNP / R)-OPN of the present invention and other CT tracer nanodelivery systems on carotid artery vulnerable plaque in a mouse model. [Figure 23] 1 is a graph showing the therapeutic effect of the LP2-(AuNP / R)-OPN nanodelivery system of the present invention on vulnerable carotid artery plaque in a mouse model. [Figure 24] 1 is a graph showing the in vivo tracing effect of LP1-(Fe3O4 / DXMS)-HI44a, LP1-(Fe3O4 / IL-10)-HI44a, and other MRI tracer nanodelivery systems of the present invention on carotid artery vulnerable plaque in a mouse model. [Figure 25] 1 is a graph showing the therapeutic effect of the LP1-(Fe3O4 / DXMS)-HI44a and LP1-(Fe3O4 / IL-10)-HI44a nanodelivery systems of the present invention on vulnerable carotid artery plaque in a mouse model. [Figure 26] 1 is a graph showing the therapeutic effect of the LP1-(Asp / Clo)-Col nano-delivery system of the present invention on the rupture of vulnerable arterial plaque in a mouse model. [Figure 27] 1 is a graph showing the in vivo tracing effect of the radioisotope tracer LP1-(F-FDG)-OPN nanodelivery system of the present invention on carotid artery vulnerable plaque in a mouse model. DETAILED DESCRIPTION OF THE INVENTION

[0063] For a better understanding of the present invention, specific embodiments of the present invention are described in detail below with reference to examples. However, it should be understood that the description is intended only to further illustrate the features and advantages of the present invention, and is not intended in any way to limit the scope of the claims of the present invention.

[0064] The present invention will be further described below by means of specific examples, but it should be understood that these examples are for the purpose of further illustration only and should not be construed as limiting the invention in any way.

[0065] This section provides a basic description of the materials and experimental methods used to test the present invention. While many of the materials and methods for achieving the objectives of the present invention are known in the art, the present invention is further described herein in as much detail as possible. It will be apparent to one of ordinary skill in the art that the materials and methods of the present invention are well known in the art at the time of their application unless otherwise specified. [Example]

[0066] Preparation of rosuvastatin (R)-loaded liposomal nanovesicles (LP1-(R)-HA) decorated with hyaluronic acid (HA) In this example, a therapeutic agent-loaded liposomal nanovesicle, LP1-(R)-HA, was prepared by thin film dispersion. The surface of the nanovesicle in the liposomal delivery system was partially modified with the targeting ligand hyaluronic acid (abbreviated as "HA") and loaded with rosuvastatin (abbreviated as "R"), a substance for preventing and / or treating vulnerable plaque or vulnerable plaque-related diseases.

[0067] (1) Preparation of LP1-(R) liposome nanovesicle suspension: Four milligrams of distearoylphosphatidylcholine (DSPC), cholesterol, and dimyristoylphosphoethanolamine (DMPE) (molar ratio 4:1:1) were weighed and dissolved in 10 mL of chloroform. The drug rosuvastatin® (total drug-to-lipid molar ratio 1:10) was added. The organic solvent was removed by slow rotary evaporation (65°C water bath, 90 rpm, 30 minutes) to form a thin film on the container wall. The container was placed in a 50°C water bath kettle to allow the thin film to fully hydrate for 30 minutes to form a crude liposome nanovesicle suspension. The crude liposome nanovesicle suspension was sonicated in an ultrasonic bath, and then the suspension was further sonicated for 3 minutes with a probe-type ultrasonic sonicator (amplitude 20, interval 3 seconds). Unencapsulated drug in the micronized liposome nanovesicle suspension was removed by Sephadex column G-100.

[0068] (2) Hyaluronic Acid ("HA") Activation and Coupling: One gram of HA (having a molecular weight of approximately 100 kDa) was completely dissolved in ultrapure water, and 0.1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.12 g of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl groups. The solution was stirred at room temperature for 1 hour, and then absolute ethanol was added to precipitate the activated HA. The precipitate was filtered, washed with ethanol, and dried under reduced pressure to obtain activated HA. It was then dissolved in 0.1 mg mL of HA. -1 The activated HA was dissolved in water, and 0.2 mL of the solution was transferred to the liposomal nanovesicle suspension obtained in step (1) above. The activated carboxyl group of the activated HA was coupled to the amino group of the DSPE molecule incorporated into the lipid bilayer of the liposomal nanovesicle through the formation of an amide bond, yielding three liposomal delivery systems loaded with therapeutic agents: LP1-(R)-HA. Figure 1 shows an electron micrograph of LP1-(R)-HA. Figure 2 shows an infrared spectrogram of LP1-(R)-HA. [Example]

[0069] Preparation of rosuvastatin (R)-loaded and selectin (SP)-decorated liposomal nanovesicles (LP1-(R)-SP) In this example, a therapeutic agent-loaded liposomal nanovesicle, LP1-(R)-SP, was prepared by thin film dispersion. The surface of the nanovesicle in the liposomal delivery system was partially modified with a targeting ligand, selectin (abbreviated as "SP"), and loaded with rosuvastatin (abbreviated as "R"), a substance for preventing and / or treating vulnerable plaque or diseases associated with vulnerable plaque.

[0070] (1) Preparation of LP1-(R) liposome nanovesicles: LP1-(R) liposome nanovesicles were prepared by the method of Example 1.

[0071] (2) Activation and coupling of selectins (SP): 1 mg of SP was completely dissolved in ultrapure water, and 0.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.5 mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl groups. After stirring the solution at room temperature for 1 hour, the activated selectin was purified by ultrafiltration and dissolved in the liposome nanovesicle suspension obtained in step (1) above. The activated carboxyl groups of the activated SP were coupled to the amino groups of DMPE molecules incorporated into the lipid bilayer of the liposome nanovesicles via amide bond formation to obtain three therapeutic-loaded liposome delivery systems, LP1-(R)-SP. Figure 3 shows the characterization diagram of LP1-(R)-SP. [Example]

[0072] Preparation of liposomal nanovesicles (LP1-(R)-HA / Tat) loaded with rosuvastatin (R) and simultaneously modified with hyaluronic acid (HA) and transmembrane peptide (Tat) In this example, a therapeutic agent-loaded liposomal nanovesicle, LP1-(R)-HA / Tat, was prepared by thin film dispersion. The surface of the nanovesicle in the liposomal delivery system was partially modified with the targeting ligand hyaluronic acid (abbreviated as "HA") and the transmembrane peptide (Tat), and loaded with rosuvastatin (abbreviated as "R"), a substance for preventing and / or treating vulnerable plaque or diseases associated with vulnerable plaque.

[0073] 3.1 Preparation of LP1-(R) liposome nanovesicles: LP1-(R) liposome nanovesicles were prepared by the method of Example 1.

[0074] 3.2 Hyaluronic Acid ("HA") Activation and Coupling: 10 mg of HA (having a molecular weight of approximately 10 kDa) was completely dissolved in ultrapure water, and 5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 5 mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl groups. The solution was stirred at room temperature for 1 hour, and then absolute ethanol was added to precipitate the activated HA. The precipitate was filtered, washed with ethanol, and dried under reduced pressure to obtain activated HA. It was then dissolved in 0.1 mg mL -1 It was formulated in an aqueous solution.

[0075] 1 mg of Tat was completely dissolved in PBS buffer, and 0.1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.12 g of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl groups. The solution was stirred at room temperature for 1 h and then purified by ultrafiltration to remove unreacted small organic molecules. 0.1 mg mL of activated Tat was used. -1 It was formulated in an aqueous solution.

[0076] 1 mL of HA solution and 0.5 mL of Tat solution were transferred to the purified LP1-(R) liposome nanovesicle solution and dissolved. The activated carboxyl groups of the activated HA and Tat were coupled to the amino groups of the DMPE molecules incorporated into the lipid bilayer membrane of the liposome nanovesicle through the formation of amide bonds, thereby achieving double coupling of HA and Tat to LP1-(R), yielding the target-recognizing nanocarrier LP1-(R)-HA / Tat. Figure 4 shows the infrared characterization diagram of LP1-(R)-HA / Tat. [Example]

[0077] Preparation of atorvastatin (At)-loaded liposomal nanodiscs (LP2-(At)-HA) simultaneously modified with hyaluronic acid (HA) and PEG (1) Preparation of atorvastatin (At)-loaded liposomal nanodiscs LP2-(At): Four milligrams of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), short-chain 1,2-di-n-heptadecanoylphosphatidylcholine (DHPC), and dimyristoylphosphoethanolamine (DMPE) (molar ratio 7:2:1) were weighed and dissolved in 10 mL of chloroform. The organic solvent was removed by slow rotary evaporation (65°C water bath, 90 rpm, 30 min) to form a thin film on the wall of the container. 10 mL of water (concentration 1.0 mg / mL) was added to the round-bottom flask, and the flask was placed in a 50°C water bath kettle to completely hydrate the thin film, forming a crude liposome nanodisk suspension. The crude liposome nanodisc suspension was sonicated in an ultrasonic bath, and then the suspension was further sonicated for 3 minutes with a probe-type ultrasonic sonicator (amplitude 20, interval 3 seconds). Unencapsulated drug in the finely divided liposome nanodisc suspension was removed using a Sephadex G-100 column.

[0078] (2) Hyaluronic Acid ("HA") Activation and Coupling: 1 g of HA (having a molecular weight of approximately 100 kDa) was completely dissolved in ultrapure water, and 0.1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.12 g of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl groups. The solution was stirred at room temperature for 1 hour, and then absolute ethanol was added to precipitate the activated HA. The precipitate was filtered, washed with ethanol, and dried under reduced pressure to obtain activated HA. It was then dissolved in 0.1 mg mL -1The activated HA was dissolved in water, and 0.2 mL of the solution was transferred to the liposomal nanodisc suspension obtained in step (1) above. The activated carboxyl group of the activated HA was coupled to the amino group of PEG-NH2 (molecular weight 1000) and the amino group of the DMPE molecule incorporated into the lipid bilayer of the liposomal nanodisc via amide bond formation to obtain three liposomal delivery systems loaded with therapeutic agents, LP2-(At)-HA. Figure 5 shows the characterization diagram of LP2-(At)-HA. If PEG modification is not required, the above step of adding PEG-NH2 can be omitted to obtain LP2-(At)-HA without PEG modification. [Example]

[0079] Preparation of liposomal nanodiscs (LP2-(At)-SEP / IM7) loaded with atorvastatin (At) and decorated with self-peptide (SEP) and monoclonal antibody IM7 (1) Preparation of atorvastatin (At)-loaded liposome nanodiscs: Liposomal nanodiscs were prepared according to the method of Example 4.

[0080] (2) Activation and coupling of SEP or IM7: 1 mg of SEP was completely dissolved in ultrapure water, and 0.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.5 mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl groups. The solution was stirred at room temperature for 1 hour, and the activated SEP was purified by ultrafiltration and centrifugation. 1 mg of IM7 was completely dissolved in ultrapure water, and 0.1 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.1 mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl groups. The activated IM7 was purified by ultrafiltration and centrifugation.

[0081] Activated SEP or IM7 was dissolved in purified LP2-(At) solution to achieve double coupling of SEP / IM7 to LP2-(At), yielding the target-recognizing Nanodisc LP2-(At)-SEP / IM7. Figure 6 shows the characterization diagram of LP2-(At)-SEP / IM7. If SEP does not need to be modified, the above steps of activating and coupling SEP can be omitted to obtain LP2-(At)-IM7 without SEP modification. [Example]

[0082] Preparation of liposomal nanodiscs (LP2-(At / miRNA-33a)-IM7) loaded with atorvastatin (At) and microRNA (miRNA-33a) and modified with monoclonal antibody IM7 (1) Preparation of atorvastatin (At) and microRNA (miRNA-33a)-loaded liposomal nanodiscs LP2-(At): Four milligrams of DOTAP, short-chain 1,2-di-n-heptadecanoylphosphatidylcholine (DHPC), and dimyristoylphosphoethanolamine (DMPE) were weighed in a 7:2:1 molar ratio and dissolved in 10 mL of chloroform. The drug atorvastatin (At) (total drug-to-lipid molar ratio of 1:10) was added. The organic solvent was removed by slow rotary evaporation (65°C water bath, 90 rpm, 30 min) to form a thin film on the wall of the container. 10 mL of water (concentration: 1.0 mg / mL) was added to a round-bottom flask, and the flask was placed in a constant-temperature water bath kettle at 50°C. The thin film was allowed to fully hydrate, forming a coarse liposome nanodisk suspension. The crude liposomal nanodisc suspension was sonicated in an ultrasonic bath, and then further sonicated with a probe-type ultrasonic sonicator for 3 minutes (amplitude 20, interval 3 seconds) to obtain a finely divided liposomal nanodisc suspension. Unencapsulated atorvastatin in the finely divided liposomal nanodisc suspension was removed using a Sephadex G-100 column. After concentrating the filtrate, a certain amount of microRNA (miRNA-33a) was added and incubated for 2 hours to promote binding of the microRNA to the nanodisc surface. The product was stored at 4°C for later use.

[0083] 1 mg of IM7 was completely dissolved in ultrapure water, and 0.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.5 mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl groups. The activated sulfo-NHS-IM7 was purified by ultrafiltration and centrifugation. The activated sulfo-NHS-IM7 was dissolved in the purified LP2-(At / miRNA-33a) solution, allowing for dual coupling of IM7 to LP2-(At / miRNA-33a) to obtain the target-recognizing nanodisc LP2-(At / miRNA-33a)-IM7. Figure 7 shows the infrared characterization diagram of LP2-(At / miRNA-33a)-IM7. [Example]

[0084] Preparation of rosuvastatin(R) / gold nanoparticle (AuNP)-loaded liposome nanodiscs (LP2-(AuNP / R)-OPN) decorated with osteopontin (OPN) 7.1 Preparation of gold nanoparticles (AuNPs) 100 mL of 1 mM HAuCl4 solution was prepared, heated to boiling, and 1 mL of freshly prepared 0.1 M sodium borohydride was added with vigorous stirring to obtain AuNPs. The solution was purified by ultrafiltration, and the solution was concentrated to obtain 1 mM AuNPs.

[0085] 7.2 Preparation of Rosuvastatin (R) and Gold Nanoparticle (AuNP)-Loaded Liposomal Nanodiscs LP2-(AuNP / R) Four milligrams of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), short-chain 1,2-di-n-heptadecanoylphosphatidylcholine (DHPC), and dimyristoylphosphoethanolamine (DMPE) were weighed in a 7:2:1 molar ratio and the drug rosuvastatin (R) (total drug-to-lipid molar ratio was 1:10) was added. The organic solvent was then removed by slow rotary evaporation (65°C water bath, 90 rpm, 30 min) to form a thin film on the container wall. The flask was placed in a 50°C water bath kettle, and the thin film was allowed to fully hydrate for 30 min. Then, 1 mL of purified AuNP (1 mM) solution was added. The crude liposome nanodisc suspension was sonicated in an ultrasonic bath, followed by further sonication for 3 min with a probe-type ultrasonic sonicator (amplitude 20, interval 3 s). Unencapsulated rosuvastatin and unencapsulated AuNPs in the micronized liposome nanodisc suspension were removed by Sephadex column G-100.

[0086] 7.3 Preparation of liposomal nanodiscs loaded with AuNP / R and decorated with osteopontin (OPN) (LP2-(AuNP / R)-OPN) 1 mg of OPN was completely dissolved in ultrapure water, and 0.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.5 mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. The solution was stirred at room temperature for 1 hour, and then the activated OPN was purified by ultrafiltration and centrifugation. 1.0 mL of the OPN solution was dissolved in the purified LP2-(AuNP / R) solution to achieve coupling of OPN to LP2-(AuNP / R). Figure 8 shows the infrared characterization diagram of LP2-(AuNP / R)-OPN from Example 7.

[0087] Using alternative raw materials, we also successfully prepared the targeted CT tracers LP2-(iopromide)-OPN, LP2-(iodixanol)-OPN, and LP2-(iodofluoroalcohol)-OPN by similar preparation methods as described above. [Example]

[0088] Preparation of liposomal nanovesicles (LP1-(Fe3O4 / DXMS)-HI44a) loaded with dexamethasone (DXMS) and magnetic iron nanoparticles (Fe3O4NP) and modified with monoclonal antibody (HI44a) 8.1 Preparation of magnetic iron nanoparticles (Fe3O4NPs) Magnetic iron nanoparticles (Fe3O4NP) were obtained by preparing 100 mL of 10 mM ferric chloride (FeCl3) solution and adding 10 mL of freshly prepared 0.1 M ammonium hydroxide with vigorous stirring. This material was purified by an external magnetic field. The solution was concentrated and redispersed in pure water to prepare 10 mM Fe3O4NP.

[0089] 8.2 Preparation of dexamethasone (DXMS) and magnetic iron nanoparticle (Fe3O4NP) loaded liposomal nanovesicles LP1-(Fe3O4 / DXMS) Four milligrams of distearoylphosphatidylcholine (DSPC), cholesterol, and dimyristoylphosphoethanolamine (DMPE) (molar ratio 4:1:1) were weighed and dissolved in 10 mL of chloroform. The drug dexamethasone (DXMS) (total drug-to-lipid molar ratio 1:10) was added. The organic solvent was removed by slow rotary evaporation (65°C water bath, 90 rpm, 30 min) to form a thin film on the wall of the container. 10 mL of purified 1 mM Fe3O4NP solution was added, and the flask was placed in a 50°C water bath kettle to completely hydrate the thin film, forming a crude liposome nanovesicle suspension. The coarse liposome nanovesicle suspension was sonicated in an ultrasonic bath, and then further sonicated for 3 minutes with a probe-type ultrasonic sonicator (amplitude 20, interval 3 seconds) to completely disperse the liposome vesicles, forming a dispersion system, i.e., a finely divided liposome vesicle suspension. Unencapsulated drug and unencapsulated Fe3O4NPs in the finely divided liposome nanovesicle suspension were removed using a Sephadex G-100 column.

[0090] 8.3 Preparation of Fe3O4 / DXMS-loaded liposomal nanovesicles modified with HI44a (LP1-(Fe3O4 / DXMS)-HI44a) 1 mg of HI44a was completely dissolved in ultrapure water, and 0.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.5 mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. The solution was stirred at room temperature for 1 hour, and then the activated HI44a was purified by ultrafiltration and centrifugation. The HI44a solution was dissolved in the purified LP1-(Fe3O4 / DXMS) solution, and HI44a was coupled to LP1-(Fe3O4 / DXMS) to obtain the target-recognizing nanocarrier LP1-(Fe3O4 / DXMS)-HI44a. Figure 9 shows the characterization diagram of LP1-(Fe3O4 / DXMS)-HI44a from Example 8.

[0091] Using alternative raw materials, the inventors have also successfully prepared targeted MRI tracers LP1-(gadoterate meglumine)-HI44a, LP1-(gadodiamide)-HI44a, and LP1-(gadopentetic acid)-HI44a, using similar preparation methods as described above. [Example]

[0092] Preparation of liposomal nanovesicles (LP1-(Fe3O4 / IL-10)-HI44a) loaded with interleukin-10 (IL-10) and magnetic iron nanoparticles (Fe3O4NP) and modified with monoclonal antibody (HI44a) 9.1 Preparation of magnetic iron nanoparticles (Fe3O4NP) Magnetic iron nanoparticles (Fe3O4NP) were prepared according to the method in Example 8.

[0093] 9.2 Preparation of IL-10 and magnetic iron nanoparticle (Fe3O4NP) loaded liposomal nanovesicles LP1-(Fe3O4 / IL-10) Four milligrams of distearoylphosphatidylcholine (DSPC), cholesterol, and dimyristoylphosphoethanolamine (DMPE) (molar ratio 4:1:1) were weighed. The organic solvent was removed by slow rotary evaporation (65°C water bath, 90 rpm, 30 min) to form a thin film on the wall of the container. 10 mL of purified 1 mM Fe3O4NP solution was added, and the flask was placed in a 50°C water bath kettle to completely hydrate the thin film and form a coarse liposome nanovesicle suspension. The coarse liposome nanovesicle suspension was sonicated in an ultrasonic bath, and then further sonicated in a probe-type ultrasonic sonicator for 3 min (amplitude 20, interval 3 s) to completely disperse the liposome vesicles and form a dispersion, i.e., a finely divided liposome vesicle suspension. The solution was concentrated, 1 mg of IL-10 was added, and the mixture was incubated at room temperature for 10 hours to obtain LP1-(Fe3O4 / IL-10). Unencapsulated drug in the micronized liposome nanovesicle suspension was removed by Sephadex column G-100.

[0094] 9.3 Preparation of Fe3O4 / IL-10-loaded liposomal nanovesicles decorated with HI44a (LP1-(Fe3O4 / IL-10)-HI44a) 1 mg of HI44a was completely dissolved in ultrapure water, and 0.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.5 mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. The solution was stirred at room temperature for 1 hour, and then the activated HI44a was purified by ultrafiltration and centrifugation. The HI44a solution was dissolved in the purified LP1-(Fe3O4 / IL-10) solution, and HI44a was coupled to LP1-(Fe3O4 / IL-10) to obtain the target-recognizing nanocarrier LP1-(Fe3O4 / IL-10)-HI44a. Figure 10 shows the infrared characterization diagram of LP1-(Fe3O4 / IL-10)-HI44a from Example 9. [Example]

[0095] Preparation of aspirin (Asp) and clopidogrel (Clo)-loaded liposomal nanovesicles (LP1-(Asp / Clo)-Col) decorated with collagen (Col) 10.1 Preparation of aspirin (Asp) and clopidogrel (Clo) loaded liposomal nanovesicles LP1-(Asp / Clo) Four milligrams of distearoylphosphatidylcholine (DSPC), cholesterol, and dimyristoylphosphoethanolamine (DMPE) (molar ratio 4:1:1) were weighed and dissolved in 10 mL of chloroform. The drugs aspirin (Asp) and clopidogrel (Clo) (drug molar ratio 1:1, total drug-to-lipid molar ratio 1:10) were added. The organic solvent was removed by slow rotary evaporation (65°C water bath, 90 rpm, 30 minutes) to form a thin film on the wall of the container. The container was placed in a 50°C water bath kettle to fully hydrate the thin film, forming a crude liposome nanovesicle suspension. The crude liposome nanovesicle suspension was sonicated in an ultrasonic bath, and then further sonicated for 3 minutes with a probe-type ultrasonic sonicator (amplitude 20, interval 3 seconds). Unencapsulated drug in the micronized liposome nanovesicle suspension was removed by Sephadex column G-100.

[0096] 10.2 Preparation of loaded liposome nanovesicles (LP1-(Asp / Clo)-Col) modified with collagen (Col) Ten milligrams of Col was completely dissolved in ultrapure water, and 3 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 3 mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl groups. The solution was stirred at room temperature for 1 hour, and the activated Col was purified by ultrafiltration and centrifugation. 1.0 mL of activated Col was dissolved in the purified LP1-(Asp / Clo) solution, and the coupling of Col to LP1-(Asp / Clo) was achieved to obtain the target-recognizing liposomal vesicle LP1-(Asp / Clo)-Col. Figure 11 shows the infrared characterization diagram of LP1-(Asp / Clo)-Col from Example 10. [Example]

[0097] Preparation of liposomal nanovesicles (LP1-(F-FDG)-OPN) loaded with fluorine-18 (18F)-labeled fludeoxyglucose (F-FDG) and decorated with osteopontin (OPN) 11.1 Preparation of F-FDG-loaded liposome nanovesicles (LP1-(F-FDG)) 4 mg of distearoylphosphatidylcholine (DSPC), cholesterol, and dimyristoylphosphoethanolamine (DMPE) (molar ratio 4:1:1) were weighed and dissolved in 10 mL of chloroform. The organic solvent was removed by slow rotary evaporation (65°C water bath, 90 r / min, 30 min) to form a thin film on the wall of the container. 10 mL of 1 mg mL -1 F-FDG solution (total drug-to-lipid molar ratio 1:10) was added, and the container was placed in a hot water bath kettle at 50°C to completely hydrate the thin film to form a coarse liposome nanovesicle suspension. The coarse liposome nanovesicle suspension was sonicated in an ultrasonic bath, and then the suspension was further sonicated for 3 minutes with a probe-type ultrasonic sonicator (amplitude 20, interval 3 seconds). Unencapsulated drug in the finely divided liposome nanovesicle suspension was removed using a Sephadex G-100 column.

[0098] 11.2 Preparation of liposomal nanovesicles loaded with F-FDG and modified with osteopontin (OPN) (LP1-(F-FDG)) 1 mg of OPN was completely dissolved in ultrapure water, and 0.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.5 mg of N-hydroxysulfosuccinimide (sulfo-NHS) coupling agent were added to activate the carboxyl group. The solution was stirred at room temperature for 1 hour, and the activated OPN was purified by ultrafiltration and centrifugation. The activated OPN was dissolved in the purified LP1-(F-FDG) solution, and OPN was coupled to LP1-(F-FDG) to obtain the target-recognizing nanocarrier LP1-(F-FDG)-OPN. Figure 12 shows the infrared characterization diagram of LP1-(F-FDG)-OPN from Example 11.

[0099] Experimental Example 1: Investigation of the properties of the nanocarrier delivery system of the present invention In this example, the therapeutic agent-loaded nanocarrier delivery system prepared in Example 1 is taken as an example to demonstrate that the carrier delivery system of the present invention has stable and controllable properties and is therefore suitable for the diagnosis, prevention, and treatment of vulnerable plaque or diseases associated with vulnerable plaque.

[0100] 1. Method for determining drug concentration: The loaded drugs rosuvastatin, atorvastatin, dexamethasone, aspirin, clopidogrel, and fluorine-18 (18F)-labeled fludeoxyglucose have strong ultraviolet absorption properties, so their contents can be determined by HPLC-UV method (using a Waters 2487, Waters Corporation, USA) by utilizing the ultraviolet absorption properties of rosuvastatin, atorvastatin, dexamethasone, aspirin, clopidogrel, and fluorine-18 (18F)-labeled fludeoxyglucose. A standard quantification formula was established using the peak area (Y) of the HPLC chromatographic peak at different concentrations (X) of rosuvastatin, atorvastatin, dexamethasone, aspirin, clopidogrel, and fluorine-18 (18F)-labeled fludeoxyglucose solutions.

[0101] 2. Hydrodynamic size determination: The hydrodynamic sizes of the carrier delivery systems of the present invention, LP1-(R)-HA, LP1-(R)-SP, LP1-(R)-HA / Tat, LP2-(At)-HA, LP2-(At)-SEP / IM7, LP2-(At / miRNA-33a)-IM7, LP2-(AuNP / R)-OPN, LP1-(Fe3O4 / DXMS)-HI44a, LP1-(Fe3O4 / IL-10)-HI44a, LP1-(Asp / Clo)-Col, and LP1-(F-FDG)-OPN, were measured using a laser particle analyzer (BI-Zeta Plus / 90 Plus, Brookhaven Instruments Corporation, USA). The specific results are shown in Table 1.

[0102] 3. Determining the inclusion rate: A certain quality of drug suspension was taken, added to excess methanol and refluxed to extract the loaded drug, and ultrasonic extraction was further applied to accelerate the drug release from the carrier. The drug content in the resulting liquid was measured by HPLC (Waters 2487, Waters Corporation, USA), and the encapsulation rate was calculated according to Equation 1.

[0103]

number

[0104] 4. Determining Drug Loading Rate: The method for determining the drug loading rate was the same as that for determining the encapsulation rate, except for a slight difference in the calculation method. A certain quality of drug suspension was taken, added to excess methanol and refluxed to extract the loaded drug, and ultrasonic extraction was further applied to accelerate the drug release from the carrier. The drug content in the resulting liquid was measured by HPLC (Waters 2487, Waters Corporation, USA), and the encapsulation rate was calculated according to the following formula:

[0105]

number

[0106] The drug content in the resulting liquid was measured by HPLC (Waters 2487, Waters Corporation, USA), and the encapsulation rate was calculated according to Equation 2.

[0107] [Table 1] NOTE: The above data are expressed in the form of "mean + standard deviation" of the results of five determinations performed in parallel.

[0108] 5. Long-term stability study The nanocarrier delivery systems of the present invention, LP1-(R)-HA, LP1-(R)-SP, LP1-(R)-HA / Tat, LP2-(At)-HA, LP2-(At)-SEP / IM7, LP2-(At / miRNA-33a)-IM7, LP2-(AuNP / R)-OPN, LP1-(Fe3O4 / DXMS)-HI44a, LP1-(Fe3O4 / IL-10)-HI44a, LP1-(Asp / Clo)-Col, and LP1-(F-FDG)-OPN, were stored at 4°C and sampled at various time points. Changes in their hydrodynamic size were detected using a laser particle analyzer (BI-Zeta Plus / 90 Plus, Brookhaven Instruments Corporation, USA), and the results are shown in Figure 13. Figure 13 shows the effect of long-term storage on particle size stability.

[0109] 6. Investigation of long-term inclusion rate The nanocarrier delivery systems of the present invention, LP1-(R)-HA, LP1-(R)-SP, LP1-(R)-HA / Tat, LP2-(At)-HA, LP2-(At)-SEP / IM7, LP2-(At / miRNA-33a)-IM7, LP2-(AuNP / R)-OPN, LP1-(FeO / DXMS)-HI44a, LP1-(FeO / IL-10)-HI44a, LP1-(Asp / Clo)-Col, and LP1-(F-FDG)-OPN, were stored at 4°C and sampled at various time points. Free drug was removed by ultrafiltration and centrifugation to detect changes in their encapsulation rates. The results are shown in Figure 14. Figure 14 shows the effect of long-term storage on encapsulation rates.

[0110] 7. In vitro drug release performance study Two milliliters of the nanocarrier delivery systems of the present invention, LP1-(R)-HA, LP1-(R)-SP, LP1-(R)-HA / Tat, LP2-(At)-HA, LP2-(At)-SEP / IM7, LP2-(At / miRNA-33a)-IM7, LP2-(AuNP / R)-OPN, LP1-(Fe3O4 / DXMS)-HI44a, LP1-(Fe3O4 / IL-10)-HI44a, LP1-(Asp / Clo)-Col, and LP1-(F-FDG)-OPN, were placed in a dialysis bag and sealed. The dialysis bag was then placed in 50 mL of release medium (PBS solution, pH 7.4) and incubated at 37°C for 120 hours. 2 mL of the release solution was removed at various time points and replenished with the same volume of PBS solution. The drug content in the release solution was detected by HPLC (Waters 2487, Waters Corporation, USA), and the cumulative drug release rate was calculated according to Equation 3.

[0111]

number

[0112] The meaning of each parameter in Equation 3 is as follows: CRP: cumulative drug release rate Ve: Displacement volume of the discharge fluid, here Ve is 2 mL V0: Volume of the released fluid of the release system, here V0 is 50 mL Ci: Drug concentration in the released solution at the i-th replacement and sampling, unit is μg / mL M drug: total mass of drug in the cerasome or liposome delivery system, in μg n: Number of replacements of the released liquid Cn: Drug concentration in the release system measured after the nth replacement of the release solution

[0113] In vitro release is an important index for evaluating nanoparticle delivery systems. Figure 15 is a graph showing the change in cumulative drug release rate of the liposomal delivery system of the present invention.

[0114] Experimental Example 2: Study on targeting mechanism In this example, the density of CD44 on the surface of vulnerable plaque endothelial cells and its affinity for HA are studied, thereby providing experimental evidence for selecting CD44 within vulnerable plaque as the target for the delivery system of the present invention for targeting vulnerable plaque.

[0115] 1) Comparison of CD44 content on the surface of endothelial cells in vulnerable arterial plaques and on the surface of endothelial cells in normal arterial walls of mice Constructing a mouse model of atherosclerotic vulnerable plaque. SPF Grade ApoE - / -Mice (10 weeks old, weighing 20±1 g) were used as experimental animals. They were fed an adaptive high-fat diet (10% (w / w) fat, 2% (w / w) cholesterol, 0.5% (w / w) sodium cholate, and the remainder was normal mouse chow) for 4 weeks. They were then anesthetized by intraperitoneal injection of 1% sodium pentobarbital (prepared by adding 1 mg of sodium pentobarbital to 100 ml of saline) at a dose of 40 mg / kg. The mice were then fixed onto a surgical plate in the dorsal position, and the area around the neck was disinfected with 75% (v / v) alcohol. The cervical skin was then incised longitudinally, and the anterior cervical glands were roughly separated. The pulsating left common carotid artery could be observed to the left of the trachea. The common carotid artery was carefully isolated up to its bifurcation. A silicone cannula measuring 2.5 mm in length and 0.3 mm in inner diameter was placed around the outside of the left common carotid artery. The proximal and distal segments of the cannula were constricted and secured with fine sutures. Local constriction caused rapid blood flow at the proximal end, increasing shear forces and thus damaging the vascular intima. The carotid artery was repositioned, and the neck skin was sutured intermittently. All surgeries were performed under a 10x stereomicroscope. After recovery from surgery, mice were returned to their cages, and the ambient temperature was maintained at 20–25°C with a 12-h / 12-h light / dark cycle. Four weeks after surgery, lipopolysaccharide (LPS) (1 mg / kg in 0.2 ml of phosphate-buffered saline, Sigma, USA) was injected intraperitoneally twice weekly for 10 weeks to induce chronic inflammation. Eight weeks after surgery, the mice were placed in a 50 ml syringe (ensuring adequate ventilation) for 6 hours per day, 5 days per week, for a total of 6 weeks to induce psychological stress through restraint. The mouse model of vulnerable atherosclerotic plaque was completed 14 weeks after surgery. Figures 16(a) and (b) show magnetic resonance imaging images of the mouse model of vulnerable atherosclerotic plaque. The arrows indicate the formation of plaque in the left carotid artery, suggesting successful modeling. The right carotid artery can be used as a normal arterial wall for comparison.

[0116] Endothelial cells from normal arterial vessels and vulnerable plaques of the model mice were collected for immunohistochemical staining and image analysis to determine CD44 content. The specific experimental method was as follows:

[0117] Mouse carotid atherosclerotic plaque specimens were collected, fixed in 10 mL / L aqueous formaldehyde, embedded in paraffin, sectioned at 4 μm, and conventionally delipidated and hydrated. CD44 content was detected using the streptavidin-biotin-peroxidase complex method (SABC). The specimens were immersed in 30 mL / L H2O2 aqueous solution to block endogenous peroxidase activity, and then placed in citrate buffer for antigen microwave repair. A 50 g / L bovine serum albumin (BSA) blocking solution was then added dropwise, and the specimens were left at room temperature for 20 minutes. Mouse anti-CD44 polyclonal antibody (1:100) was then added dropwise, and the specimens were placed in a 4°C refrigerator overnight and then incubated at 37°C for 1 hour. The specimens were washed, then biotinylated goat anti-mouse IgG was added dropwise and incubated at 37°C for 30 minutes. Then, they were washed with phosphate-buffered saline (PBS), and horseradish peroxidase-labeled SABC complex was added dropwise and incubated at 37°C for 20 minutes. Each of the above steps was washed with PBS. Finally, development was performed with DAB (control of development under a microscope), and the specimens were restained with hematoxylin. The specimens were then dehydrated and sealed. The sections were analyzed using the immunohistochemistry analysis system of the BI-2000 Image Analysis System. Three sections were collected for each of the endothelial cells of normal arterial vessels and vulnerable plaques, and five representative fields were randomly selected. Positive CD44 expression was as follows: the cell membrane and cytoplasm were yellowish-brown / chocolate brown, with a clear background. The darker the color, the stronger the CD44 expression. Negative CD44 expression was as follows: no yellowish-brown particles were observed. The mean absorbance (A) values ​​of positive cells in the endothelial cells of normal arterial vessels and the endothelial cells of arterial vulnerable plaques were measured and compared, and the results are shown in Figure 17.

[0118] 17 shows the results of determining the CD44 content (semi-quantitative integrated value) on the surface of endothelial cells in normal arterial vessel walls and endothelial cells in vulnerable plaques in a mouse model. As shown in the figure, the CD44 content on the surface of endothelial cells in vulnerable plaques is approximately 2.3 times higher than that on the surface of endothelial cells in normal arterial vessels.

[0119] 2) Comparison of the affinity of CD44 on the surface of endothelial cells from vulnerable plaques and normal arterial walls in mice for ligands and antibodies Natural ligands for CD44 include HA, GAG, collagen, laminin, fibronectin, selectin, osteopontin (OPN), and monoclonal antibodies HI44a, HI313, A3D8, H90, and IM7.

[0120] Endothelial cells from normal arterial walls and vulnerable plaques in model mice were harvested and treated with 10 mg / ml aminofluorescein-labeled ligand / antibody. The samples were incubated in Dulberic modified Eagle's medium (DMEM) containing 10% calf serum, 100 U / ml penicillin, and 100 U / ml streptomycin at 37°C in a 5% CO2 incubator. After 30 minutes, mean fluorescence intensity (MFI) was measured using a flow cytometer (CytoFLEX, Beckman Coulter, USA), and the integrated binding affinity of FL-ligand / antibody to the surface of both cells was calculated (the binding affinity of CD44 on endothelial cells from normal arterial walls to the ligand / antibody is set to 1). The results are shown in Figure 18.

[0121] As shown in Figure 18, the integrated avidity value of CD44 on the surface of endothelial cells from vulnerable arterial plaques for HA is approximately 24-fold higher than that of endothelial cells from normal arterial walls. This indicates that most CD44 on the surface of endothelial cells from normal arterial walls is in a resting state unable to bind to the ligand HA, whereas CD44 on the surface of endothelial cells from vulnerable arterial plaques is activated by factors such as inflammatory factors in the internal environment, resulting in a significantly increased affinity for HA.

[0122] Other CD44 ligands show similar results to HA. The integrated avidity of CD44 on the surface of vulnerable plaque endothelial cells to GAG is 22-fold higher than that of normal cells. The integrated avidity of CD44 on the surface of vulnerable plaque endothelial cells to collagen is 21-fold higher than that of normal cells. The integrated avidity of CD44 on the surface of vulnerable plaque endothelial cells to laminin is 16-fold higher than that of normal cells. The integrated avidity of CD44 on the surface of vulnerable plaque endothelial cells to fibronectin is 18-fold higher than that of normal cells. The integrated avidity of CD44 on the surface of vulnerable plaque endothelial cells to selectin is 19-fold higher than that of normal cells. The integrated avidity of CD44 on the surface of vulnerable plaque endothelial cells to osteopontin is 17-fold higher than that of normal cells.

[0123] Similar results were observed with CD44 monoclonal antibodies. For HI44a, the integrated avidity of CD44 on the surface of vulnerable plaque endothelial cells was 15-fold higher than that of normal cells. For HI313, the integrated avidity of CD44 on the surface of vulnerable plaque endothelial cells was 21-fold higher than that of normal cells. For A3D8, the integrated avidity of CD44 on the surface of vulnerable plaque endothelial cells was 17-fold higher than that of normal cells. For H90, the integrated avidity of CD44 on the surface of vulnerable plaque endothelial cells was 9-fold higher than that of normal cells. For IM7, the integrated avidity of CD44 on the surface of vulnerable plaque endothelial cells was 8-fold higher than that of normal cells.

[0124] 3) Comparison of the affinity of CD44 on the surface of macrophages outside plaques with that of macrophages inside vulnerable arterial plaques for ligands / antibodies Peritoneal macrophages from model mice and macrophages inside vulnerable plaques were collected, and 10 mg / ml of aminofluorescein-labeled ligand / antibody was added. The samples were cultured in DMEM (containing 10% calf serum, 100 U / ml penicillin, and 100 U / ml streptomycin) at 37°C in a 5% CO2 incubator. After 30 minutes, mean fluorescence intensity (MFI) was determined by flow cytometry (CytoFLEX, Beckman Coulter, USA), and the integrated binding affinity of FL-HA to the surface of both cells was calculated (the affinity of CD44 on the surface of macrophages outside the plaque for the ligand / antibody was set to 1). The results are shown in Figure 19.

[0125] As shown in Figure 19, the binding affinity of CD44-HA on the surface of macrophages inside vulnerable plaques is approximately 40-fold higher than that on the surface of macrophages outside the plaques. This indicates that factors such as inflammatory factors in the internal environment also activate CD44 on the surface of macrophages inside vulnerable plaques, significantly increasing their affinity for HA.

[0126] Other CD44 ligands show similar results to those of HA. The integrated avidity of CD44 on the surface of macrophages from vulnerable plaques to GAGs is 33-fold higher than that of normal cells. The integrated avidity of CD44 on the surface of macrophages from vulnerable plaques to collagen is 38-fold higher than that of normal cells. The integrated avidity of CD44 on the surface of macrophages from vulnerable plaques to laminin is 37-fold higher than that of normal cells. The integrated avidity of CD44 on the surface of macrophages from vulnerable plaques to fibronectin is 35-fold higher than that of normal cells. The integrated avidity of CD44 on the surface of macrophages from vulnerable plaques to selectin is 33-fold higher than that of normal cells. The integrated avidity of CD44 on the surface of macrophages from vulnerable plaques to osteopontin is 33-fold higher than that of normal cells.

[0127] Similar results were observed with CD44 monoclonal antibodies. For HI44a, the integrated avidity of CD44 on the surface of macrophages from vulnerable plaques was 17-fold higher than that of normal cells. For HI313, the integrated avidity of CD44 on the surface of macrophages from vulnerable plaques was 20-fold higher than that of normal cells. For A3D8, the integrated avidity of CD44 on the surface of macrophages from vulnerable plaques was 16-fold higher than that of normal cells. For H90, the integrated avidity of CD44 on the surface of macrophages from vulnerable plaques was 9-fold higher than that of normal cells. For IM7, the integrated avidity of CD44 on the surface of macrophages from vulnerable plaques was 10-fold higher than that of normal cells.

[0128] Based on the results of the above experiments, the following conclusions can be drawn: compared with normal cells (endothelial cells in normal arterial blood vessel walls, macrophages outside plaques, etc.), the density of CD44 on the surface of cells in vulnerable plaques (including endothelial cells, macrophages, etc., which are important for the development of arterial vulnerable plaques) is significantly increased, and its affinity for ligands is significantly enhanced; therefore, the specific affinity of CD44 inside arterial vulnerable plaques for ligands is much higher than that of normal cells, which makes them highly advantageous as an excellent target for the cerasome delivery system of the present invention for targeting vulnerable plaques.

[0129] Experimental Example 3: In vivo experiment on the effects of the rosuvastatin delivery systems LP1-(R)-HA, LP1-(R)-SP, and LP1-(R)-HA / Tat of the present invention on vulnerable arterial plaque Hyaluronic acid (HA) and selectin (SP) are ligands for CD44 and can target vulnerable plaque. Rosuvastatin® can shrink plaque, and transmembrane peptide (Tat) can increase local drug penetration and aggregation. The purpose of this example is to verify the in vivo therapeutic effects of the LP1-(R)-HA, LP1-(R)-SP, and LP1-(R)-HA / Tat carrier delivery systems described in the present invention on vulnerable arterial plaque.

[0130] Experimental Method: (1) A physiological saline solution of free rosuvastatin was prepared, and a liposome nanocarrier delivery system loaded with a therapeutic agent was prepared by the methods described in Examples 1 to 3 above.

[0131] (2) ApoE in vulnerable arterial plaque - / - Establishment of the mouse model: SPF Grade ApoE - / -Mice (42 mice, 5-6 weeks old, weighing 20±1 g) were obtained as experimental animals. They were fed an adapted high-fat diet (10% (w / w) fat, 2% (w / w) cholesterol, 0.5% (w / w) sodium cholate, with the remainder consisting of normal mouse chow) for 4 weeks. They were then anesthetized by intraperitoneal injection of 1% sodium pentobarbital (prepared by adding 1 mg of sodium pentobarbital to 100 ml of saline) at a dose of 40 mg / kg. The mice were then fixed onto a surgical plate in the dorsal position, the area around the neck was disinfected with 75% (v / v) alcohol, the cervical skin was incised longitudinally, and the anterior cervical glands were roughly isolated. The pulsating left common carotid artery could be observed to the left of the trachea. The common carotid artery was carefully isolated up to its bifurcation. A silicone cannula measuring 2.5 mm in length and 0.3 mm in inner diameter was placed around the outside of the left common carotid artery. The proximal and distal segments of the cannula were constricted and secured with fine sutures. Local constriction caused rapid blood flow at the proximal end, increasing shear forces and thus damaging the vascular intima. The carotid artery was repositioned, and the neck skin was sutured intermittently. All surgeries were performed under a 10x stereomicroscope. After recovery from surgery, mice were returned to their cages, maintained at an ambient temperature of 20–25°C, and maintained on a 12-h / 12-h light / dark cycle. Four weeks after surgery, lipopolysaccharide (LPS) (1 mg / kg in 0.2 ml of phosphate-buffered saline, Sigma, USA) was injected intraperitoneally twice weekly for 10 weeks to induce chronic inflammation. Eight weeks after surgery, mice were placed in a 50 ml syringe (ensuring adequate ventilation) for 6 hours per day, 5 days per week, for a total of 6 weeks to induce psychological stress through restraint. The mouse model of vulnerable atherosclerotic plaque was completed 14 weeks after surgery.

[0132] (3) Grouping and treatment of experimental animals: The experimental animals were randomly divided into the following groups with 6 mice in each group: Control group of the vulnerable plaque model: animals in this group do not receive any therapeutic treatment. Intragastric administration of rosuvastatin: Treatment by intragastric administration of 10 mg of rosuvastatin per kg of body weight. Intravenous rosuvastatin group: Treatment with intravenous administration of rosuvastatin at a dose of 0.66 mg per kg of body weight. LP1-(R)-HA group: treatment with intravenous administration of rosuvastatin at a dose of 0.66 mg per kg of body weight. LP1-(R)-SP group: treatment with intravenous administration of rosuvastatin at a dose of 0.66 mg per kg of body weight. LP1-(R)-HA / Tat group: treatment with intravenous administration of rosuvastatin at a dose of 0.66 mg per kg of body weight.

[0133] Except for the control group in the vulnerable plaque model, the treatment groups were treated every other day for a total of five treatments. Carotid MRI scans were performed on animals in each group before and after treatment to detect plaque area and lumen area, and the percentage of plaque progression was calculated. Percentage of plaque progression = (post-treatment plaque area - pre-treatment plaque area) / luminal area.

[0134] Test Results: Figure 20 shows the in vivo therapeutic effects of the carrier delivery systems LP1-(R)-HA, LP1-(R)-SP, and LP1-(R)-HA / Tat of the present invention on vulnerable arterial plaque. As shown, during a 10-day high-fat diet, atherosclerosis in the control group (without any treatment) progressed by 36.23%. Intragastric administration of rosuvastatin can slow plaque progression, but it still progressed by 33.9%. Intravenous administration of rosuvastatin can also slow plaque progression, but it still progressed by 32.46%. However, targeted nanodrug delivery therapy significantly inhibited plaque progression and even reduced it, resulting in a reduction in plaque volume. The LP1-(R)-HA group eliminated 10.87% of plaques, the LP1-(R)-SP eliminated 8.74% of plaques, and the LP1-(R)-HA / Tat group eliminated 13.2% of plaques.

[0135] In summary, free rosuvastatin, whether administered intragastrically or intravenously, shows some therapeutic effect on vulnerable plaque in mice, but fails to prevent the continued growth of vulnerable plaque. However, when rosuvastatin is formulated into the nanodelivery system of the present invention, the therapeutic effect on vulnerable plaque is significantly improved, achieving a therapeutic effect of reducing plaque growth (stenotic plaque). Nanosystems with functional modifications show even better effects.

[0136] Experimental Example 4: In vivo experiment on the effects of the delivery systems LP2-(At)-HA, LP2-(At)-SEP / IM7, and LP2-(At / miRNA-33a)-IM7 of the present invention on vulnerable arterial plaque Hyaluronic acid (HA) and IM7 are ligands for CD44 and can target vulnerable plaques. Atorvastatin (At) can shrink plaques, self-peptide (SEP) can increase local drug penetration and aggregation, and miRNA-33a can increase cholesterol efflux. The purpose of this example is to verify the in vivo therapeutic effects of the LP2-(At)-HA, LP2-(At)-SEP / IM7, and LP2-(At / miRNA-33a)-IM7 carrier delivery systems described in this invention on vulnerable arterial plaques.

[0137] Experimental Method: (1) A saline solution of free atorvastatin was prepared, and a liposome nanocarrier delivery system loaded with a therapeutic agent was prepared by the methods described in Examples 4 to 6 above.

[0138] (2) ApoE in vulnerable arterial plaque - / - A mouse model was established according to Experimental Example 4.

[0139] (3) Grouping and treatment of experimental animals: The experimental animals were randomly divided into the following groups with 6 mice in each group: Control group of the vulnerable plaque model: animals in this group do not receive any therapeutic treatment. Intragastric administration of atorvastatin group: Treatment by intragastric administration of atorvastatin at a dose of 20 mg per kg of body weight. Intravenous atorvastatin group: Treatment with intravenous administration of atorvastatin at a dose of 1.2 mg per kg of body weight. PEG-free LP2-(At)-HA group: treatment with intravenous administration of atorvastatin at a dose of 1.2 mg per kg of body weight. LP2-(At)-HA group: treatment with intravenous administration of atorvastatin at a dose of 1.2 mg per kg of body weight. LP2-(At)-IM7 group: treatment with intravenous administration of atorvastatin at a dose of 1.2 mg per kg of body weight. LP2-(At)-SEP / IM7 group: treatment with intravenous administration of atorvastatin at a dose of 1.2 mg / kg body weight. LP2-(At / miRNA-33a)-IM7 group: treatment with intravenous administration of atorvastatin at a dose of 1.2 mg per kg of body weight.

[0140] Except for the control group in the vulnerable plaque model, the treatment groups were treated every other day for a total of five treatments. Carotid MRI scans were performed on animals in each group before and after treatment to detect plaque area and lumen area, and the percentage of plaque progression was calculated. Percentage of plaque progression = (post-treatment plaque area - pre-treatment plaque area) / luminal area.

[0141] Test Results: Figure 21 shows the in vivo therapeutic effects of the carrier delivery systems LP2-(At)-HA, LP2-(At)-SEP / IM7, and LP2-(At / miRNA-33a)-IM7 of the present invention on vulnerable arterial plaque. As shown, during a 10-day high-fat diet, atherosclerosis in the control group (without any treatment) progressed by 34.87%. Intragastric administration of atorvastatin slowed plaque progression, but still progressed by 33.21%. Intravenous administration of atorvastatin also slowed plaque progression, but still progressed by 32.98%. However, targeted nanodrug delivery therapy significantly inhibited plaque progression and even reduced plaque volume. The LP2-(At)-HA group without PEG eliminated 6.9% of plaques, the LP2-(At)-HA group eliminated 12.65% of plaques, the LP2-(At)-IM7 group eliminated 5.1% of plaques, the LP2-(At)-SEP / IM7 group eliminated 12.43% of plaques, and the LP2-(At / miRNA-33a)-IM7t group eliminated 14.22% of plaques.

[0142] In summary, free atorvastatin, whether administered intragastrically or intravenously, shows some therapeutic effect on vulnerable plaque in mice, but fails to prevent the continued growth of vulnerable plaque. However, when atorvastatin is formulated into the liposome nanocarrier delivery system of the present invention, the therapeutic effect on vulnerable plaque is significantly improved, achieving a therapeutic effect of reducing plaque growth (stenotic plaque). Nanosystems with PEG or SEP functional modifications show better efficacy, and nanocarriers loaded with statin and nucleic acid show significant efficacy.

[0143] Experimental Example 5: In vivo experiment on the effect of the delivery system LP2-(AuNP / R)-OPN of the present invention on vulnerable arterial plaque (dual functions of CT tracing and treatment) Osteopontin (OPN) is a ligand for CD44 and can target vulnerable plaque. Rosuvastatin® can shrink plaque, and nanogold (Au NP) is a CT tracer. The purpose of this example is to verify the in vivo tracing and therapeutic effects of the nanocarrier delivery system loaded with CT tracer and rosuvastatin described in this invention on vulnerable arterial plaque.

[0144] Experimental Method: (1) A saline solution of free rosuvastatin was prepared, and a liposome nanocarrier delivery system loaded with a CT tracer and a therapeutic agent was prepared by the method described in Example 7 above.

[0145] (2) ApoE in vulnerable arterial plaque - / - The mouse model was established according to Experimental Example 4.

[0146] (3) Tracing vulnerable plaques in experimental animals: The experimental animals were randomly divided into the following groups with 6 mice in each group: Free nanogold group: the dose of nanogold was 0.1 mg / kg body weight. LP2-(AuNP / R)-OPN group: the dose of nanogold was 0.1 mg / kg body weight. LP2-(iopromide)-OPN group: the dose of iopromide was 0.1 mg / kg body weight. LP2-(iodixanol)-OPN group: the dose of iodixanol was 0.1 mg / kg body weight. LP2-(iodofluoroalcohol)-OPN group: the dose of iodofluoroalcohol was 0.1 mg / kg body weight.

[0147] The corresponding tracer was injected into the tail vein of the animals in each experimental group. CT imaging was performed before and 2 hours after administration to observe and identify the atherosclerotic vulnerable plaques in each group.

[0148] (4) Grouping and treatment of experimental animals: The experimental animals were randomly divided into the following groups with 6 mice in each group: Control group of the vulnerable plaque model: animals in this group do not receive any therapeutic treatment. Intragastric administration of rosuvastatin: Treatment by intragastric administration of 10 mg of rosuvastatin per kg of body weight. Intravenous rosuvastatin group: Treatment with intravenous administration of rosuvastatin at a dose of 0.67 mg per kg of body weight. LP2-(AuNP / R)-OPN group: treatment with intravenous administration of rosuvastatin at a dose of 0.67 mg per kg of body weight.

[0149] Except for the control group in the vulnerable plaque model, the treatment groups were treated every other day for a total of five treatments. Carotid MRI scans were performed on animals in each group before and after treatment to detect plaque area and lumen area, and the percentage of plaque progression was calculated. Percentage of plaque progression = (post-treatment plaque area - pre-treatment plaque area) / luminal area.

[0150] Test Results: Figure 22 shows the in vivo tracing effect of the tracer-loaded liposomal delivery system of the present invention on vulnerable arterial plaque. As shown, free nanogold particles exhibit some tracing effect on vulnerable arterial plaque in mice. Compared with free nanogold particles, formulating nanogold, iopromide, iodixanol, and iodofluoroalcohol into a targeted liposomal delivery system significantly improves the tracing effect on vulnerable plaque. In summary, administration of the liposomal delivery system of the present invention, whose surface is modified with a targeting ligand, can improve the recognition effect of nanogold on vulnerable plaque and result in a better tracing effect compared with administration of free nanogold particles.

[0151] Figure 23 shows the in vivo therapeutic effect of the carrier delivery system LP2-(AuNP / R)-OPN of the present invention on vulnerable arterial plaque. As shown, during a 10-day high-fat diet, atherosclerosis in the control group (without any treatment) progressed by 31.23%. Intragastric administration of rosuvastatin slowed plaque progression, but still progressed by 30.9%. Intravenous administration of rosuvastatin also slowed plaque progression, but still progressed by 25.34%. However, targeted nanodrug delivery therapy significantly inhibited plaque progression and even reduced plaque volume. The LP2-(AuNP / R)-OPN group saw 8.32% plaque disappearance.

[0152] In summary, free rosuvastatin, whether administered intragastrically or intravenously, shows some therapeutic effect on arterial vulnerable plaque in mice, but fails to prevent the continued growth of vulnerable plaque. However, when rosuvastatin and nanogold are formulated into the nanodelivery system of the present invention, the diagnostic and therapeutic effects on vulnerable plaque are significantly improved, serving as an early warning for high-risk patients and achieving a therapeutic effect of reducing plaque growth (stenotic plaque).

[0153] Experimental Example 6: In vivo tracing experiments on the effects of the delivery systems of the present invention, LP1-(Fe3O4 / DXMS)-HI44a and LP1-(Fe3O4 / IL-10)-HI44a, on arterial vulnerable plaque (MRI tracing) and anti-inflammatory treatment The monoclonal antibody (HI44a) is a ligand for CD44 and can target vulnerable plaque. Dexamethasone (DXMS) has anti-inflammatory and plaque progression inhibitory effects, and Fe3O4 is an MRI tracer. The purpose of this example is to verify the in vivo tracing and therapeutic effects of the MRI tracer and dexamethasone-loaded nanocarrier delivery system described in this invention on vulnerable arterial plaque. In addition, gadoterate meglumine, gadodiamide, and gadopentetate can also be formulated into nanopreparations that exhibit targeted MRI tracing effects.

[0154] (1) A liposome nanocarrier delivery system loaded with an MRI tracer and a therapeutic agent was prepared by the methods described in Examples 8-9 above.

[0155] (2) ApoE in vulnerable arterial plaque - / - The mouse model was established according to Experimental Example 4.

[0156] (3) Tracing vulnerable plaques in experimental animals: The experimental animals were randomly divided into the following groups with 6 mice in each group: Free Fe3O4 group: the dose of Fe3O4 was 0.1 mg / kg body weight. LP1-(Fe3O4 / DXMS)-HI44a group: the dose of Fe3O4 was 0.1 mg / kg body weight. LP1-(Fe3O4 / IL-10)-HI44a group: the dose of Fe3O4 was 0.1 mg / kg body weight. LP1-(meglumine gadoterate)-HI44a group: the dose of meglumine gadoterate was 0.1 mg / kg body weight. LP1-(gadodiamide)-HI44a group: the dose of gadodiamide was 0.1 mg / kg body weight. LP1-(gadopentetate)-HI44a group: the dose of gadopentetate was 0.1 mg / kg body weight.

[0157] The animals in each experimental group were injected into the tail vein with the corresponding tracer, and MRI imaging was performed before and 2 hours after administration to identify the atherosclerotic vulnerable plaques in each group by observation.

[0158] (4) Grouping and treatment of experimental animals: The experimental animals were randomly divided into the following groups with 6 mice in each group: Control group of the vulnerable plaque model: animals in this group do not receive any therapeutic treatment. LP1-(Fe3O4 / DXMS)-HI44a group: treatment with intravenous administration of dexamethasone at a dose of 0.1 mg per kg of body weight. LP1-(Fe3O4 / IL-10)-HI44a group: treatment with intravenous administration of 0.1 μmol IL-10 per kg body weight.

[0159] Except for the control group in the vulnerable plaque model, the treatment groups were treated every other day for a total of five treatments. Carotid MRI scans were performed on animals in each group before and after treatment to detect plaque area and lumen area, and the percentage of plaque progression was calculated. Percentage of plaque progression = (post-treatment plaque area - pre-treatment plaque area) / luminal area.

[0160] Test Results: Figure 24 shows the in vivo tracing effect of the tracer-loaded liposomal delivery system of the present invention on vulnerable arterial plaque. As shown, free Fe3O4 particles exhibit some tracing effect on vulnerable arterial plaque in mice. Compared with free Fe3O4 particles, formulating Fe3O4 into a targeted liposomal delivery system significantly improves the tracing effect on vulnerable plaque. Furthermore, the use of other MRI nanocontrast agents results in a very good tracing effect on vulnerable plaque. In summary, administration of the liposomal delivery system of the present invention, whose surface is modified with a targeting ligand, can significantly improve the recognition effect of nanogold on vulnerable plaque, resulting in a better tracing effect, compared with administration of free MRI tracers.

[0161] Figure 25 shows the in vivo therapeutic effects of the carrier delivery systems LP1-(Fe3O4 / DXMS)-HI44a and LP1-(Fe3O4 / IL-10)-HI44a of the present invention on vulnerable arterial plaque. As shown, during a 10-day high-fat diet, atherosclerosis in the control group (without any treatment) progressed by 23.65%. However, targeted nanodrug delivery therapy significantly inhibited plaque progression and even reduced plaque volume. The LP1-(Fe3O4 / DXMS)-HI44a group achieved 9.54% plaque disappearance. The LP1-(Fe3O4 / IL-10)-HI44a group achieved 5.43% plaque disappearance.

[0162] In summary, in the case of unstable plaque in mice, the incorporation of dexamethasone or IL-10 into the nanodelivery system of the present invention significantly improved the diagnostic and therapeutic effects on vulnerable plaque, serving as an early warning for high-risk patients and achieving therapeutic effects in reducing plaque growth (stenotic plaque). Simultaneous loading of Fe3O4 can achieve MRI imaging and real-time monitoring of the disease.

[0163] Experimental Example 7: In vivo study of the effect of the delivery system LP1-(Asp / Clo)-Col of the present invention on vulnerable arterial plaque Aspirin (Asp) and clopidogrel (Clo) are antiplatelet drugs that can reduce platelet aggregation and mortality resulting from cardiovascular events. The purpose of this example is to verify the in vivo therapeutic effect of the LP1-(Asp / Clo)-Col carrier delivery system described in the present invention on vulnerable arterial plaque.

[0164] Experimental Method: (1) A saline solution of free aspirin and clopidogrel was prepared, and a therapeutic agent-loaded liposome nanocarrier delivery system was prepared by the method described in Example 10 above.

[0165] (2) ApoE in vulnerable arterial plaque - / - Establishing a mouse model: ApoE - / - Mice were fed a high-fat diet for 30 weeks to allow atherosclerotic plaques to form in their systemic arteries, and snake venom was administered to induce rupture of vulnerable plaques, resulting in acute coronary syndrome.

[0166] (3) Grouping and treatment of experimental animals: The experimental animals were randomly divided into the following groups with 10 mice in each group: Control group of the vulnerable plaque model: animals in this group do not receive any therapeutic treatment. Intragastric administration of aspirin and clopidogrel group: treatment by intragastric administration of 100 mg aspirin per kg body weight and 75 mg clopidogrel per kg body weight. LP1-(Asp / Clo)-Col group: treatment with intravenous administration of 10 mg aspirin per kg body weight and 7.5 mg clopidogrel per kg body weight.

[0167] Except for the control group in the vulnerable plaque model, the treatment groups were treated every other day for a total of five treatments. Animals in each group were observed for mouse mortality for one month. The bleeding time (BT) of the mice was detected by tail amputation.

[0168] Test Results: Figure 26 shows the in vivo therapeutic effect of the carrier delivery system LP1-(Asp / Clo)-Col of the present invention on vulnerable arterial plaque. As shown in the figure, the mortality rate of mice in the control group (without any treatment) was 50%. Intragastric administration of aspirin and clopidogrel can reduce the mortality rate to 30%. LP1-(Asp / Clo)-Col therapy can reduce the mortality rate to 10%. In terms of bleeding time, the LP1-(Asp / Clo)-Col group did not show a significant prolongation, but intragastric administration of aspirin and clopidogrel significantly prolonged the bleeding time of mice.

[0169] In summary, oral dual antiplatelet therapy can reduce mortality in animals with ruptured vulnerable plaques, but it can also prolong bleeding time and increase the risk of bleeding. However, loading antiplatelet drugs into a nanodelivery system shows better efficacy than oral drugs and does not increase the risk of bleeding.

[0170] Experimental Example 8: In vivo experiment on the effect of the delivery system LP1-(F-FDG)-OPN of the present invention on vulnerable arterial plaque Osteopontin (OPN) is a ligand for CD44 and can target vulnerable plaque. Rosuvastatin® can shrink plaque, and fluorine-18 (18F)-labeled fludeoxyglucose (F-FDG) is a radioisotope tracer. The purpose of this example is to verify the in vivo tracing and therapeutic effects of the radioisotope tracer-loaded liposome nanocarrier delivery system described in this invention on vulnerable arterial plaque.

[0171] Experimental Method: (1) A saline solution of free rosuvastatin was prepared, and a liposome nanocarrier delivery system loaded with a radioisotope tracer and a therapeutic agent was prepared by the method described in Example 11 above.

[0172] (2) ApoE in vulnerable arterial plaque - / - The mouse model was established according to Experimental Example 4.

[0173] (3) Tracing vulnerable plaques in experimental animals: The experimental animals were randomly divided into the following groups with 6 mice in each group: Free F-FDG group: The dose of F-FDG was 2 mSv / kg body weight. LP1-(F-FDG)-OPN group: The dose of F-FDG was 2 mSv / kg body weight. LP1-(99mTc)-OPN group: The dose of 99mTc was 2 mSv / kg body weight. LP1-(I-131)-OPN group: The dose of I-131 was 2 mSv / kg body weight.

[0174] The animals in each experimental group were injected into the tail vein with the corresponding tracer, and radioisotope imaging was performed before and 2 hours after administration to observe and identify the atherosclerotic vulnerable plaques in each group.

[0175] Test Results: Figure 27 shows the in vivo tracing effect of the liposomal delivery system of the present invention loaded with a radioisotope tracer on vulnerable arterial plaque. As shown, free F-FDG does not show any tracing effect on vulnerable arterial plaque in mice. Compared to free F-FDG, formulating technetium-99 (99mTc) and iodine-131 (I-131) into a targeted liposomal delivery system significantly improves the tracing effect on vulnerable plaque. In summary, administration of the liposomal delivery system of the present invention, whose surface is modified with a targeting ligand, can significantly improve the recognition effect of nanogold on vulnerable plaque, resulting in a better tracing effect, compared to free F-FDG.

[0176] Various aspects of the present invention have been illustrated by the above-described embodiments. It is clear that the above-described embodiments are merely illustrative and are not intended to limit the embodiments. Those skilled in the art may realize other variations or modifications in various forms in light of the above description. There is no need or method to cover all implementations. Obvious changes or variations arising therefrom still fall within the scope of the present invention.

[0177] [Claim 1] A liposome nanocarrier delivery system for targeting an activated CD44 molecule, wherein the surface of the nanocarrier is partially modified with a targeting ligand, the targeting ligand being capable of specifically binding to the activated CD44 molecule; The liposome nanocarrier delivery system is characterized in that, optionally, other modifications may be made to the surface of the nanocarrier, and the other modifications are preferably modifications on the surface of the carrier with one or more selected from the group consisting of PEG, a transmembrane peptide, and a self-peptide SEP, and a simultaneously modified biligand. [Claim 2] A liposome nanocarrier delivery system for targeting vulnerable plaque, wherein the surface of the nanocarrier is partially modified with a targeting ligand, and the targeting ligand is a ligand capable of specifically binding to CD44 molecules on the cell surface of the vulnerable plaque; The liposome nanocarrier delivery system is characterized in that, optionally, other modifications may be made to the surface of the nanocarrier, and the other modifications are preferably modifications on the surface of the carrier with one or more selected from the group consisting of PEG, a transmembrane peptide, and a self-peptide SEP, and a simultaneously modified biligand. [Claim 3] 3. The nanocarrier delivery system according to claim 1 or 2, wherein the liposome carrier is selected from the group consisting of small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. [Claim 4] the targeting ligand is selected from the group consisting of GAG, collagen, laminin, fibronectin, selectin, osteopontin (OPN), and monoclonal antibodies HI44a, HI313, A3D8, H90, and IM7, or is selected from hyaluronic acid or a hyaluronic acid derivative capable of specifically binding to CD44 molecules on the cell surface of vulnerable plaque; 4. The nanocarrier delivery system according to claim 1, wherein the targeting ligand is selected from collagen, hyaluronic acid, selectin, osteopontin, or monoclonal antibodies HI44a, IM7. [Claim 5] 5. The nanocarrier delivery system according to claim 1, wherein the nanocarrier is loaded with a substance for diagnosing, preventing, and / or treating a disease associated with the presence of CD44 molecule activation. [Claim 6] the nanocarriers are loaded with a substance for diagnosing, preventing, and / or treating vulnerable plaque or a disease associated with said vulnerable plaque; Preferably, the substance is a substance for diagnosing vulnerable plaque or a disease associated with the vulnerable plaque, More preferably, the substance for diagnosing vulnerable plaque or a disease associated with said vulnerable plaque is a tracer; More preferably, the tracer is selected from a CT tracer, an MRI tracer, and a radioisotope tracer; Even more preferably, The CT tracer is selected from an iodine-based nanoscale contrast agent, a gold-based nanoscale contrast agent, a tantalum oxide-based nanoscale contrast agent, a bismuth-based nanoscale contrast agent, a lanthanide-based nanoscale contrast agent, or other tracers with similar structures; more preferably, the CT tracer is selected from an iodinated contrast agent, or nanogold, or other tracers with similar structures; even more preferably, the CT tracer is selected from iohexol, iocarmic acid, ioversol, iodixanol, iopromide, iobitridol , iomeprol, iopamidol, ioxilan, acetolizoic acid, iodipamide, iobenzamic acid, ioglycamic acid, diatrizoic acid, sodium iothalamate, pantopac, iopanoic acid, iodoalfionic acid, sodium acetolizoate, sodium iodometamate, propriodone, geodon, iotrolan, iopidol, endografin, iothalamic acid, meglumine diatrizoate, metrizoic acid, metrizamide, iodized oil, or ethiodinated oil, or other tracers with similar structures; preferably, the CT tracer is nanogold; The MRI tracer is selected from longitudinal relaxation contrast agents and transverse relaxation contrast agents; more preferably, the MRI tracer is selected from paramagnetic contrast agents, ferromagnetic contrast agents, and superparamagnetic contrast agents; and even more preferably, the MRI tracer is selected from Gd-DTPA and linear and cyclic polyamine polycarboxylate chelators and their manganese porphyrin chelators, macromolecular gadolinium chelators, biomacromolecule-modified gadolinium chelators, folate-modified gadolinium chelators, dendrimers. a contrast agent, a liposome-modified contrast agent, and a gadolinium-containing fullerene, or other tracer with a similar structure; and preferably, the MRI tracer is selected from gadopentetate dimeglumine, gadoterate meglumine, gadobenate dimeglumine, gadodiamide, ferric ammonium citrate effervescent granules, paramagnetic iron oxide (Fe3O4NP), or other tracer with a similar structure; preferably, the MRI tracer is Fe3O4NP; and / or The radioisotope tracer is selected from fludeoxyglucose labeled with carbon-14 (C), carbon-13 (C), phosphorus-32 (P), sulfur-35 (S), iodine-131 (I), hydrogen-3 (H), technetium-99 (Tc), and fluorine-18 (F); preferably, the radioisotope tracer is fluorine-18 labeled fludeoxyglucose; 6. A nanocarrier delivery system according to any one of claims 1 to 5. [Claim 7] The nanocarrier delivery system of claim 6, wherein the substance is one or more selected from the group consisting of drugs, polypeptides, nucleic acids, and cytokines for diagnosing, preventing, and / or treating vulnerable plaque or diseases associated with the vulnerable plaque. [Claim 8] the substance is a CD44 activator, The nanocarrier delivery system according to any one of claims 5 to 7, wherein the CD44 activator is preferably CD44 antibody mAb, IL5, IL12, IL18, TNF-α, or LPS. [Claim 9] the substance is a low molecular weight hyaluronic acid or hyaluronic acid derivative capable of specifically binding to CD44 molecules on the cell surface of vulnerable plaque; The nanocarrier delivery system according to any one of claims 5 to 8, wherein the low molecular weight hyaluronic acid or the hyaluronic acid derivative capable of specifically binding to CD44 molecules on the cell surface of vulnerable plaque preferably has a molecular weight in the range of 1 to 500 KDa, preferably 1 to 20 KDa, more preferably 2 to 10 KDa. [Claim 10] the nanocarriers are simultaneously loaded with a substance for diagnosing, preventing, and / or treating vulnerable plaque or a disease associated with said vulnerable plaque and a CD44 activator; Preferably, the nanocarriers are simultaneously loaded with a substance for preventing and / or treating vulnerable plaque or a disease associated with the vulnerable plaque, and with low molecular weight hyaluronic acid or a hyaluronic acid derivative capable of specifically binding to CD44 molecules on the cell surface of vulnerable plaque; More preferably, the nanocarrier delivery system described in any one of claims 6 to 9 is characterized in that the nanocarrier is simultaneously loaded with a substance for diagnosing vulnerable plaque or a disease associated with vulnerable plaque, a substance for preventing and / or treating vulnerable plaque or a disease associated with vulnerable plaque, optionally a CD44 activator, and optionally low molecular weight hyaluronic acid or a hyaluronic acid derivative capable of specifically binding to CD44 molecules on the cell surface of vulnerable plaque. [Claim 11] the substance is for preventing and / or treating vulnerable plaque or a disease associated with said vulnerable plaque; Preferably, the substance for preventing and / or treating vulnerable plaque or a disease associated with said vulnerable plaque is one or more selected from the group consisting of statins, fibrates, antiplatelet drugs, PCSK9 inhibitors, anticoagulants, angiotensin-converting enzyme inhibitors (ACEI), calcium ion antagonists, MMP inhibitors, beta-receptor blockers, glucocorticoids, and other anti-inflammatory substances such as the IL-1 antibody canakinumab, and pharmaceutically acceptable salts thereof, including active preparations of the above drugs or substances, and endogenous anti-inflammatory cytokines such as interleukin-10 (IL-10); More preferably, the substance for preventing and / or treating vulnerable plaque or a disease associated with said vulnerable plaque is selected from the group consisting of lovastatin; atorvastatin; rosuvastatin; simvastatin; fluvastatin; pitavastatin; pravastatin; bezafibrate; ciprofibrate; clofibrate; gemfibrozil; fenofibrate; probucol; anti-PCSK9 antibodies such as evolocumab, alirocumab, bococizumab, RG7652, LY3015014, and LGT-209; and adnectins such as BMS-962476; ALN- Antisense RNAi oligonucleotides such as 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, and microRNA-145 antisense strands; and their nucleic acid analogs such as locked nucleic acids; aspirin; acemetacin; troxerutin; dipyridamole; cilostazol; and thiamin hydrochloride. lopidine; ozagrel sodium; clopidogrel; prasugrel; cilostazol; beraprost sodium; ticagrelor; cangrelor; tirofiban; eptifibatide; abciximab; unfractionated heparin; clexane; fraxiparin; fondaparinux sodium; warfarin; dabigatran; rivaroxaban; apixaban; edoxaban; bivalirudin; enoxaparin; dalteparin; ardeparin; bishydroxycoumarin; coumarin nitrate; sodium citrate; hirudin; argatroban; benazepril; captopril ;Enalapril;Perindopril;Fosinopril;Lisinopril;Moexipril;Cilazapril;Perindopril;Quinapril;Ramipril;Trandolapril;Candesartan;Eprosartan;Irbesartan;Losartan;Telmisartan;Valsartan;Olmesartan;Tasosartan;Nifedipine;Nicardipine;Nitrendipine;Amlodipine;Nimodipine;Nisoldipine;Nilvadipine;Isradipine;Felodipine;Lacidipine;Diltiazem;Verapamil;Chlorhexidine;Minocycline;MMI-166;Metoprolol;atenolol; bisoprolol; propranolol; carvedilol; batimastat; marimastat; prinomastat; BMS-279251; BAY 12-9566; TAA211; AAJ996A; nacetrapib; evacetrapib; torcetrapib; dalcetrapib; prednisone; methylprednisolone; betamethasone; beclomethasone dipropionate; diprospan; prednisolone; hydrocortisone; dexamethasone; and other anti-inflammatory substances such as the IL-1 antibody canakinumab; and active fragments or pharmaceutically acceptable salts thereof; and one or more pharmaceutically acceptable salts comprising active structural fragments of the above substances; and endogenous anti-inflammatory cytokines such as interleukin-10 (IL-10); 7. The nanocarrier delivery system of claim 6. [Claim 12] A method for preparing a nano-delivery system for targeting vulnerable plaque according to any one of claims 1 to 11, comprising: (1) dissolving an appropriate amount of phospholipid molecules in a suitable organic solvent and preparing liposome nanocarriers by thin film hydration method, whereby drug molecules with lower polarity need to form a thin film together with the phospholipid molecules in this step; (2) an optional step of adding an aqueous medium, which may contain a water-soluble substance for diagnosing, preventing, and / or treating vulnerable plaque or a disease associated with said vulnerable plaque, to the nanocarrier delivery system obtained in step (1) to form a coarse suspension; (3) dissolving the targeting ligand in a suitable buffer solution solvent, and adding the carrier molecule obtained in step (2) to the targeting ligand solution for reaction to obtain a nanocarrier delivery system; (4) an optional step of removing by dialysis any unloaded substance for diagnosing, preventing, and / or treating the vulnerable plaque or a disease associated with the vulnerable plaque contained in the crude suspension obtained in step (3) to obtain a loaded nanodelivery system. The method, characterized in that it comprises: [Claim 13] 12. A pharmaceutical comprising the nanocarrier delivery system according to claim 1 and a pharmaceutically acceptable carrier. [Claim 14] A diagnostic preparation comprising the nanocarrier delivery system according to any one of claims 1 to 11. [Claim 15] Use of a nanocarrier delivery system according to any one of claims 1 to 11, a pharmaceutical according to claim 13, or a diagnostic preparation according to claim 14 in a preparation for preventing and / or treating a disease associated with the presence of CD44 molecule activation. [Claim 16] Use of the nanocarrier delivery system according to any one of claims 1 to 11, the pharmaceutical according to claim 13, or the diagnostic preparation according to claim 14 in a preparation for preventing and / or treating vulnerable plaque or a disease associated with said vulnerable plaque. [Claim 17] the vulnerable plaque is selected from one or more of the group consisting of rupture-prone plaque, erosion-prone plaque, and partially calcified nodular lesion; 17. The use according to claim 16, wherein the disease associated with vulnerable plaque is selected from one or more of the group consisting of atherosclerosis, coronary artery disease (including acute coronary syndrome, silent myocardial ischemia-occult coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, and in-stent restenosis), cerebral arteriosclerosis (including stroke), peripheral vascular atherosclerosis (including peripheral arterial occlusive disease, retinal arteriosclerosis, carotid artery atherosclerosis, renal atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, and atherosclerotic impotence), aortic dissection, hemangioma, thromboembolism, heart failure, and cardiogenic shock. [Claim 18] A method for preventing and / or treating a disease associated with the presence of CD44 molecule activation, comprising the step of administering to a subject in need thereof a nanocarrier delivery system according to any one of claims 1 to 11, a pharmaceutical agent according to claim 13, or a diagnostic preparation according to claim 14. [Claim 19] A method for preventing, diagnosing, and / or treating vulnerable plaque or a disease associated with said vulnerable plaque, comprising the step of administering to a subject in need thereof the nanocarrier delivery system according to any one of claims 1 to 11, the medicament according to claim 13, or the diagnostic preparation according to claim 14; Preferably, the vulnerable plaque is selected from one or more of the group consisting of rupture-prone plaque, erosion-prone plaque, and partially calcified nodular lesion; More preferably, the method is characterized in that the disease associated with vulnerable plaque is selected from one or more of the group consisting of atherosclerosis, coronary artery sclerosis (including acute coronary syndrome, silent myocardial ischemia-occult coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, and in-stent restenosis), cerebral arteriosclerosis (including stroke), peripheral vascular atherosclerosis (including peripheral arterial occlusive disease, retinal arteriosclerosis, carotid artery atherosclerosis, renal atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, and atherosclerotic impotence), aortic dissection, hemangioma, thromboembolism, heart failure, and cardiogenic shock. [Claim 20] A method for diagnosing a disease associated with the presence of CD44 molecule activation, comprising the step of administering to a subject in need thereof a nanocarrier delivery system according to any one of claims 1 to 11, a pharmaceutical agent according to claim 13, or a diagnostic preparation according to claim 14.

Claims

1. A liposome nanocarrier delivery system, comprising: liposomal nanocarriers; a targeting ligand comprising hyaluronic acid or a hyaluronic acid derivative thereof having a molecular weight in the range of 2 KDa to 20 KDa or 2 KDa to 10 KDa; a pharmaceutical active ingredient comprising a statin selected from lovastatin, atorvastatin, rosuvastatin, fluvastatin, pitavastatin, and pravastatin; and PEG, transmembrane peptides, or self-peptides; Including, A liposomal nanocarrier delivery system that can compete with the binding of endogenous hyaluronic acid on the cell surface.

2. 2. The liposomal nanocarrier delivery system of claim 1, wherein the liposomal carrier is selected from the group consisting of small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.

3. The liposome nanocarrier delivery system described in claim 1, wherein the hyaluronic acid having a molecular weight in the range of 2KDa to 20KDa or 2KDa to 10KDa can specifically bind to CD44 molecules on the cell surface of vulnerable plaque.

4. The liposome nanocarrier delivery system of claim 1 , further comprising a CD44 activator.

5. The liposome nanocarrier delivery system of claim 4, wherein the CD44 activator is a CD44 antibody mAb, IL5, IL12, IL18, TNF-α, or LPS.

6. The liposome nanocarrier delivery system of claim 1, for use in the prevention and / or treatment of unstable plaque or diseases associated with unstable plaque, wherein the unstable plaque is selected from the group consisting of plaque prone to rupture, plaque prone to erosion, and partially calcified nodular lesion.

7. The liposome nanocarrier delivery system of claim 1, for use in the prevention and / or treatment of unstable plaque or a disease associated with unstable plaque, wherein the disease associated with unstable plaque is selected from atherosclerosis, coronary heart disease, cerebral arteriosclerosis, peripheral vascular atherosclerosis, aortic dissection, hemangioma, thromboembolism, heart failure, and cardiogenic shock.

8. The liposome nanocarrier delivery system according to claim 7, wherein the coronary arteriosclerotic heart disease is selected from acute coronary syndrome, silent myocardial ischemia-occult coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, and in-stent restenosis.

9. 8. The liposome nanocarrier delivery system of claim 7, wherein the peripheral atherosclerosis is selected from peripheral arterial occlusive disease, retinal arteriosclerosis, carotid atherosclerosis, renal atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, and atherosclerotic impotence.

10. The liposome nanocarrier delivery system according to claim 7, wherein the cerebral arteriosclerosis is stroke.

11. 8. The liposome nanocarrier delivery system according to claim 7, wherein the disease associated with vulnerable plaque is selected from atherosclerosis, coronary artery disease (including acute coronary syndrome, silent myocardial ischemia-occult coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, and in-stent restenosis), cerebral arteriosclerosis (including stroke), peripheral vascular atherosclerosis (including peripheral arterial occlusive disease, retinal arteriosclerosis, carotid artery atherosclerosis, renal atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, and atherosclerotic impotence), aortic dissection, hemangioma, thromboembolism, heart failure, and cardiogenic shock.

12. 10. A method for preparing the liposomal nanocarrier delivery system of claim 1, comprising: (1) Dissolving an appropriate amount of phospholipid molecules in a suitable organic solvent and preparing liposome nanocarriers by thin film hydration method; (2) adding an aqueous medium containing a pharmaceutically active ingredient, including a statin selected from lovastatin, atorvastatin, rosuvastatin, fluvastatin, pitavastatin, and pravastatin, to the nanocarrier delivery system obtained in step (1) to form a coarse suspension; (3) dissolving a targeting ligand comprising hyaluronic acid or its hyaluronic acid derivatives with a molecular weight ranging from 2KDa to 20KDa or 2KDa to 10KDa in a suitable buffer solution solvent, and adding the carrier molecules obtained in step (2) to the targeting ligand solution for reaction to obtain a nanocarrier delivery system; (4) an optional step of removing unloaded pharmaceutically active ingredients contained in the crude suspension obtained in step (3) by dialysis to obtain a loaded nanodelivery system. Including, method.

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