WO2018188635A1 - 一种用于靶向活化cd44分子的硅质体递送系统、其制备方法和用途 - Google Patents

一种用于靶向活化cd44分子的硅质体递送系统、其制备方法和用途 Download PDF

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WO2018188635A1
WO2018188635A1 PCT/CN2018/082850 CN2018082850W WO2018188635A1 WO 2018188635 A1 WO2018188635 A1 WO 2018188635A1 CN 2018082850 W CN2018082850 W CN 2018082850W WO 2018188635 A1 WO2018188635 A1 WO 2018188635A1
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siliceous
delivery system
vulnerable plaque
group
molecule
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PCT/CN2018/082850
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English (en)
French (fr)
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马茜
孙洁芳
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北京茵诺医药科技有限公司
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Priority to JP2020505962A priority Critical patent/JP7485366B2/ja
Priority to US16/605,120 priority patent/US11737977B2/en
Priority to CN201880015119.2A priority patent/CN110402137B/zh
Priority to EP18785183.7A priority patent/EP3610859A4/en
Publication of WO2018188635A1 publication Critical patent/WO2018188635A1/zh
Priority to JP2022161864A priority patent/JP2022188222A/ja

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase

Definitions

  • the invention belongs to the field of targeted drug delivery technology, and in particular relates to a silicon body delivery system for targeting activated CD44 molecules, in particular to vulnerable fragile plaques.
  • the invention also relates to methods of preparation and use of the siliceous delivery system, particularly in the diagnosis, prevention and treatment of vulnerable plaque or diseases associated with vulnerable plaque.
  • Vulnerable plaque refers to atherosclerotic plaques that have a tendency to thrombosis or are likely to progress rapidly into "criminal plaques", including Rupture plaques, aggressive plaques, and partially calcified nodular lesions.
  • the techniques currently used for the diagnosis of vulnerable plaque mainly include coronary angiography, intravascular ultrasound (IVUS), and laser coherence tomography (OCT), but these techniques are all invasive, and the diagnostic resolution and The accuracy is not high, and these diagnostic techniques are expensive, which also limits the clinical popularity to a certain extent. Therefore, there is an urgent need for non-invasive diagnostic techniques and formulations for vulnerable plaque.
  • the current treatment of vulnerable plaques is mainly systemic administration, such as oral statins (hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors), aspirin, matrix metalloproteinases (MMPs) inhibition Agents and/or fibrates, etc.
  • HMG-CoA hydroxymethylglutaryl coenzyme A
  • MMPs matrix metalloproteinases
  • statins commonly used in clinical practice are relatively low, such as ⁇ 5% for simvastatin, about 12% for atorvastatin, and about 20% for rosuvastatin.
  • Animal experiments have also confirmed that when the dose of statin is increased to more than 1 mg/kg, it can increase the thickness of the fibrous cap and reduce the volume of plaque, which makes the stability and reversal of oral administration of statins. The effect of the block has encountered a bottleneck.
  • a targeted drug delivery system refers to a drug delivery system that has the ability to target administration. After administration via a route, the drug contained in the targeted drug delivery system is specifically enriched in the target site by a vector with a targeting probe.
  • the targeted drug delivery system is capable of targeting the drug to a particular lesion site and releasing the active ingredient at the target lesion site. Therefore, the targeted drug delivery system can make the drug form a relatively high concentration in the target lesion site, and reduce the dose in the blood circulation, thereby improving the drug effect while suppressing toxic side effects and reducing damage to normal tissues and cells.
  • nanocarriers commonly used in targeted drug delivery systems are liposomes.
  • liposomes have the advantage of improving the drug efficacy and reducing the toxic side effects of the drug, due to poor stability in vivo, the cycle time is insufficient, and finally the bioavailability of the drug is limited.
  • the in vitro stability of the liposome is also insufficient, and the phospholipid is easily oxidatively hydrolyzed during storage, and the liposome vesicles are easily aggregated and fused to each other, and the drug enclosed therein is prone to leakage. This all limits the development of targeted drug delivery systems to some extent.
  • CD44 is a type of adhesion molecule that is widely distributed on the surface of lymphocytes, monocytes, endothelial cells, and the like.
  • the main ligand for the CD44 molecule is hyaluronic acid (abbreviated as "HA").
  • HA hyaluronic acid
  • CD44 Based on the activation state of the expressed cells, CD44 can be classified into a relatively static state (which cannot bind to HA), an induced activation state (which can bind to HA after activation), and a structurally active state (which can bind to HA without activation), while most normal cells
  • the CD44 of the surface is in a relatively static state and thus cannot be combined with HA.
  • CD44 is not an ideal target with significant targeting specificity. This is because CD44 is widely distributed in the human body, especially on the surface of organs rich in reticuloendothelial. Therefore, the development of a targeted drug delivery system targeting CD44 encounters the problem that if the affinity of CD44 on the surface of the target cell to HA is insufficient to provide significant specificity, then such a targeted drug delivery system will There is no specific targeting performance.
  • the inventors have found that CD44 on the surface of vulnerable plaque cells such as endothelial cells, macrophages, and smooth muscle cells is exposed to the microenvironment of vulnerable plaques (such as under the influence of inflammatory factors) compared to normal cells.
  • the induced activation results in a sudden increase in the ability to bind to HA by several tens of times.
  • This finding suggests that the presence of a large number of activated CD44 molecules on the cell surface at vulnerable plaques provides an ideal target for targeted drug delivery systems with HA as a targeting ligand.
  • the present invention provides a targeted delivery system capable of specifically targeting activated CD44 molecules, particularly targeting vulnerable plaques.
  • the present invention also provides a targeted drug delivery system capable of specifically targeting vulnerable plaque while at the same time enabling stable sustained release of the drug.
  • loading CD44 activator can promote the further activation of CD44 on the surface of the lesion cells, can amplify the targeting affinity of CD44 for HA in a short time, and significantly increase the concentration of the targeted siliceous composition bound to the cell surface, which It has positive significance for the trace diagnosis and treatment of vulnerable plaque.
  • the targeted drug delivery system of the present invention can be loaded with a CD44 activator which can significantly increase the concentration of the tracer or therapeutic compound in a short period of time to improve diagnostic resolution or therapeutic efficacy.
  • the inventors have also found that in vulnerable plaques, with the high activation and overexpression of CD44, the endogenous macromolecular HA is also stimulated to generate a large amount, and binds to CD44 on the cell surface, promoting macrophages and lymphocytes. Aggregation within vulnerable plaques.
  • Such endogenous HA which binds to CD44 on the cell surface, forms a barrier to drug entry and reduces the bioavailability of the drug.
  • the targeted drug delivery system of the present invention can be loaded with a small molecule of hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at the vulnerable plaque, which The competitive binding of endogenous HA on the surface relieves the barrier formed by endogenous HA on the cell surface, which facilitates the smooth entry of the drug into the lesion cells and provides a significant therapeutic effect.
  • the present invention relates to the following aspects:
  • the present invention provides a siliceous delivery system for targeting activated CD44 molecules.
  • the present invention provides a siliceous delivery system for targeting vulnerable plaque.
  • the present invention also provides a method for preparing a siliceous delivery system for targeting vulnerable plaques of the present invention.
  • the invention also provides a medicament comprising a siliceous delivery system for targeting vulnerable plaques of the invention and a pharmaceutically acceptable carrier.
  • the invention also provides a diagnostic formulation comprising a silicon body delivery system for targeting vulnerable plaques according to the invention.
  • the present invention also provides the use of the present invention for a vulnerable plaque-targeting siliceous delivery system for the manufacture of a medicament for the prevention and/or treatment of vulnerable plaque or diseases associated with vulnerable plaque.
  • the invention also provides the use of a sacrificial plaque-targeting siliceous delivery system of the invention for the preparation of a diagnostic preparation for the diagnosis of a vulnerable plaque or a disease associated with a vulnerable plaque.
  • the invention also provides a method for preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque, the method comprising administering to a subject in need thereof a targeted vulnerable plaque according to the invention Block of siliceous delivery system.
  • the present invention also provides a method for diagnosing a vulnerable plaque or a disease associated with a vulnerable plaque, the method comprising administering to a subject in need thereof the silicon-targeted vulnerable plaque of the present invention Body delivery system.
  • Vulnerable plaque also known as “unstable plaque” refers to atherosclerotic plaques that have a tendency to thrombosis or are likely to progress rapidly into "criminal plaques”, mainly including ruptured plaques and erosive plaques. Block and partial calcified nodular lesions. A large number of studies have shown that most of the acute myocardial infarction and stroke are caused by the rupture of vulnerable plaques with mild to moderate stenosis and secondary thrombosis.
  • Histological manifestations of vulnerable plaque include active inflammation, thin fibrous cap and large lipid core, endothelial exfoliation with surface platelet aggregation, plaque fissures or damage, and severe stenosis, as well as surface calcification, yellow luster Plaque, intraplaque hemorrhage and positive remodeling.
  • “Disease associated with vulnerable plaque” mainly refers to the “vulnerable plaque” associated with the occurrence and development of the disease, which is characterized by “vulnerable plaque”, caused by “vulnerable plaque” or secondary to “Vulnerable plaque” disease.
  • "Severe diseases associated with vulnerable plaque” mainly include atherosclerosis, coronary atherosclerotic heart disease (including acute coronary syndrome, asymptomatic myocardial ischemia - occult coronary heart disease, angina pectoris, myocardial infarction, Ischemic heart disease, sudden death, in-stent restenosis), cerebral atherosclerosis (including stroke), peripheral vascular atherosclerosis (including carotid atherosclerosis, renal atherosclerosis, lower extremity) Atherosclerosis, upper extremity atherosclerosis, aortic dissection, hemangioma, thromboembolism, heart failure, and cardiogenic shock.
  • Targeteted drug delivery system refers to a drug delivery system that has the ability to target administration. After administration via a route, the drug contained in the targeted drug delivery system is specifically enriched in the target site by the action of a specific carrier or targeting warhead (e.g., a targeting ligand).
  • a specific carrier or targeting warhead e.g., a targeting ligand.
  • Means currently known for achieving targeted administration include the use of passive targeting properties of various microparticle delivery systems, chemical modification on the surface of microparticle delivery systems, utilization of specific physicochemical properties, and utilization of antibody-mediated targets.
  • the siliceous body of the present invention is an active substance delivery system which is morphologically formed by a lipid bilayer having closed vesicles having a hydrophilic cavity therein.
  • the lipid bilayer is formed from a lipid component including a siliceous monomer molecule.
  • the siliceous monomer molecule is an inorganic-organic composite lipid molecule capable of forming a siliceous body, which is composed of a head having a siloxane structure and a hydrophobic tail, wherein the hydrophobic tail is organic hydrophobic Double molecular chain.
  • the siliceous monomer molecule is typically a trialkoxysilylated lipid which forms a siloxane bond (Si-O-Si) via an in situ sol-gel process, thereby An inorganic polysiloxane network having a rigidity itself is formed on the surface of the siliceous body.
  • the siliceous delivery system used herein to target vulnerable plaques is based on the discovery that "the specific binding between a large number of activated CD44 molecules present on the cell surface at vulnerable plaques and HA" A ligand-mediated targeted drug delivery system is designed.
  • Hyaluronic acid (hyaluronic acid, abbreviated as” the HA )" is a polymer of the polymer, molecular formula: (C 14 H 21 NO 11 ) n. It is a higher polysaccharide consisting of the units D-glucuronic acid and N-acetylglucosamine. D-glucuronic acid and N-acetylglucosamine are linked by a ⁇ -1,3-glycosidic bond, and the disaccharide units are linked by a ⁇ -1,4-glycosidic bond.
  • Hyaluronic acid displays various important physiological functions in the body with its unique molecular structure and physicochemical properties, such as lubricating joints, regulating the permeability of blood vessel walls, regulating protein, water and electrolyte diffusion and operation, and promoting wound healing. It is especially important that hyaluronic acid has a special water retention effect and is the best moisturizing substance found in nature.
  • Derivative of hyaluronic acid refers to any derivative of hyaluronic acid capable of retaining the specific binding ability of hyaluronic acid to CD44 molecules on the surface of cells at vulnerable plaques, including but not limited to transparent
  • Judging whether a substance is a "derivative of hyaluronic acid” can be achieved by measuring the specific binding ability of the substance to the CD44 molecule on the cell surface at the vulnerable plaque, which is within the skill of those skilled in the art. Inside.
  • CD44 molecule is a type of transmembrane proteoglycan adhesion molecule widely expressed on the cell membrane of lymphocytes, monocytes, endothelial cells, etc., from the extracellular segment, the transmembrane segment and the intracellular segment.
  • the composition of the sections. CD44 molecules can mediate the interaction between a variety of cells and cells, cells and extracellular matrix, participate in the transmission of various signals in the body, and thus change the biological function of cells.
  • the primary ligand for the CD44 molecule is hyaluronic acid, and its receptor-ligand binding to hyaluronic acid determines the adhesion and/or migration of cells in the extracellular matrix.
  • CD44 molecules are also involved in the metabolism of hyaluronic acid.
  • Alkyl means a saturated aliphatic hydrocarbon group of both branched and straight chain having the specified number of carbon atoms.
  • C 1-6 alkyl means an alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, iso Propyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and the like.
  • C 10-24 alkyl refers to an alkyl group having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbon atoms.
  • Alkenyl means having the specified number of carbon atoms and one or more, preferably one to six, for example one, two, three, four, five or six carbon-carbon double bonds (which may be along the chain) An unsaturated aliphatic hydrocarbon group in a linear or branched configuration present at any stable point).
  • C 10-24 alkenyl group refers to a 10,11,12,13,14,15,16,17,18,19,20,21,22,23 or 24 carbon atoms and one or more Preferably, one to six, for example one, two, three, four, five or six carbon-carbon double bond alkenyl groups.
  • a silicon plastid delivery system for targeting activated CD44 molecules the surface of which is partially modified by a targeting ligand, said targeting ligand It is a ligand that specifically binds to an activated CD44 molecule.
  • the present invention provides a silt delivery system for targeting vulnerable plaque, the surface of which is partially modified by a targeting ligand, said targeting ligand It is a ligand that specifically binds to CD44 molecules on the cell surface at vulnerable plaques.
  • the present invention provides a silicon body delivery system for targeting vulnerable plaque, characterized in that the silicon body delivery system comprises a silicon body vesicle, wherein the silicon The surface of the plastid vesicles is partially modified by the targeting ligand.
  • the siliceous vesicle is a closed vesicle formed of a lipid bilayer having a hydrophilic cavity therein, wherein the surface of the vesicle has an inorganic polysiloxane network And coupled targeting ligands.
  • the lipid bilayer is a distearoylphosphatidylethanolamine (DSPE) molecule comprising a siliceous monomer molecule coupled to a targeting ligand by a covalent bond and The components selected from other lipid molecules present are formed.
  • DSPE distearoylphosphatidylethanolamine
  • the siliceous monomer molecule a distearoylphosphatidylethanolamine (DSPE) molecule coupled to a targeting ligand by a covalent bond, and optionally other lipid molecules
  • DSPE distearoylphosphatidylethanolamine
  • the weight ratio between the two is 1-10:0.2-1:1-9.
  • the siliceous monomer molecule a distearoylphosphatidylethanolamine (DSPE) molecule coupled to a targeting ligand by a covalent bond, and optionally other lipid molecules
  • DSPE distearoylphosphatidylethanolamine
  • the weight ratio between the two is 2-10:1-3:0-3.
  • the siliceous monomer molecule the weight ratio between the distearoylphosphatidylethanolamine (DSPE) molecule coupled to the targeting ligand via a covalent bond, and other lipid molecules The ratio is 3-7:1.5-2.5:1.5-2.5.
  • DSPE distearoylphosphatidylethanolamine
  • the siliceous monomer molecule the weight ratio between the distearoylphosphatidylethanolamine (DSPE) molecule coupled to the targeting ligand via a covalent bond, and other lipid molecules The ratio is 3-7:0.5-1:1.5-2.5.
  • DSPE distearoylphosphatidylethanolamine
  • the siliceous monomer molecule the weight ratio between the distearoylphosphatidylethanolamine (DSPE) molecule coupled to the targeting ligand via a covalent bond, and other lipid molecules The ratio is 4-6:0.5:2.
  • DSPE distearoylphosphatidylethanolamine
  • the siliceous monomer molecule the weight ratio between the distearoylphosphatidylethanolamine (DSPE) molecule coupled to the targeting ligand via a covalent bond, and other lipid molecules The ratio is 4-6:2:2.
  • DSPE distearoylphosphatidylethanolamine
  • the siliceous monomer molecule is an inorganic-organic complex lipid molecule capable of forming a silicon body, the inorganic-organic complex lipid molecule being composed of a head having a siloxane structure and hydrophobicity
  • the tail is constructed wherein the hydrophobic tail is an organic hydrophobic bimolecular chain.
  • the siliceous monomer molecule is a monomeric molecule having the following structural formula:
  • R 1 represents a C 16 alkyl group
  • L is a linking group composed of 4 to 12 carbon atoms (preferably 4 to 10 carbon atoms) and 1 to 2 nitrogen atoms, wherein 0-1 carbon atoms in the linking group are substituted by an oxo group, that is, Forming a carbonyl group, provided that (1) if a carbonyl group is present in the linking group, the carbonyl group is adjacent to a nitrogen atom; (2) one nitrogen atom in the linking group may be quaternized, and the quaternization a nitrogen atom forms a salt with a suitable counter ion;
  • R 2 and R 3 independently of each other represent a C 10-24 alkyl group or a C 10-24 alkenyl group.
  • the siliceous monomer molecule is selected from one or more of the following compounds:
  • the components of the lipid bilayer comprise other lipid molecules.
  • the additional lipid molecule is selected from one or more of a neutral phospholipid, a negatively charged phospholipid, and a positively charged lipid.
  • the lipid is selected from one or more of the group consisting of choline phospholipids, glycerophospholipids, ethanolamine phospholipids, serine phospholipids, and phosphatidic acid.
  • the other lipid molecule is a choline phospholipid.
  • the other lipid molecule is a positively charged lipid.
  • the positively charged lipid is selected from the group consisting of 3 ⁇ -[N-(N',N'-dimethylaminoethyl)aminoformyl]cholesterol (DC-chol), N-[1- (2,3-Dioleyl)propyl-]-N,N,N-triethylamine chloride (DOTMA), 2,3-dioleyloxy N-[2(arginyl amide)ethyl] -N,N-dimethyl-1-propyl-trioxoacetate (DOSPA) and 1,2-dioleyloxypropyl-N,N,N-trimethylammonium bromide (DOTAP) One or more.
  • DC-chol 3 ⁇ -[N-(N',N'-dimethylaminoethyl)aminoformyl]cholesterol
  • DOTMA 2,3-dioleyloxy N-[2(arginyl amide)ethyl] -N,N-d
  • the lipid is 1,2-dioleyloxypropyl-N,N,N-trimethylammonium bromide (DOTAP).
  • DOTAP 1,2-dioleyloxypropyl-N,N,N-trimethylammonium bromide
  • the targeting ligand has a molecular weight of 50,000 to 400,000 Da.
  • the targeting ligand has a molecular weight of 80,000 to 150,000 Da.
  • the targeting ligand has a molecular weight of about 100,000 Da.
  • the siliceous vesicles have a particle size ranging from 50 nm to 400 nm.
  • the siliceous vesicles have a particle size ranging from 50 nm to 300 nm.
  • the siliceous vesicles have a particle size ranging from 150 nm to 250 nm.
  • the siliceous vesicles have a particle size ranging from 180 nm to 220 nm.
  • the targeting ligand in the delivery system is selected from the group consisting of GAG, collagen, laminin, fibronectin, selectin, osteopontin (OPN), and monoclonal antibodies HI44a, HI313, A3D8, H90, IM7, or hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to the CD44 molecule on the cell surface at the vulnerable plaque.
  • siliceous delivery system of the present invention it is loaded with a substance for diagnosing, preventing and/or treating a disease associated with the presence of a CD44 molecule activation condition.
  • the siliceous body is loaded with a substance for diagnosing, preventing, and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque.
  • the substance is one or more of a drug, polypeptide, nucleic acid, and cytokine for use in diagnosing, preventing, and/or treating a disease associated with a vulnerable plaque or a vulnerable plaque.
  • the substance is a substance for diagnosing a vulnerable plaque or a disease associated with a vulnerable plaque.
  • the substance for diagnosing a vulnerable plaque or a disease associated with a vulnerable plaque is a tracer.
  • the tracer is selected from the group consisting of a CT tracer and an MRI tracer.
  • the CT tracer is selected from the group consisting of an iodine nanocontrast agent, a gold nano contrast agent, a cerium oxide nano contrast agent, a cerium nano contrast agent, a lanthanide nano contrast agent, or other similarly structured tracer.
  • the CT tracer is selected from the group consisting of iodinated contrast agents or nanogold, or other similarly structured tracers.
  • the CT tracer is selected from the group consisting of iohexol, iodine acid, ioversol, iodixanol, iopromide, iodipropanol, iomeprol, iopamidol, iodine , vinegar iodobenzoic acid, biliary acid, iodobenzene acid, iodomate, diatrizoic acid, sodium iodate, iodophenyl ester, iopanoic acid, iodophenolic acid, sodium iodine acetate, iodine Polysaccharides, acetophenone, iodooxone, iodine, iodopyrylene, meglumine, iodine, diatrizoate, guanine, mepium glucosamine, iodized oil or Ethyl iodide
  • the MRI tracer is selected from the group consisting of a longitudinal relaxation contrast agent and a transverse relaxation contrast agent.
  • the MRI tracer is selected from the group consisting of a paramagnetic contrast agent, a ferromagnetic contrast agent, and a supermagnetic contrast agent.
  • the MRI tracer is selected from the group consisting of Gd-DTPA and its linear, cyclic polyamine polycarboxylate chelate and manganese porphyrin chelate, macromolecular europium chelate, biolarge Molecularly modified ruthenium chelates, folic acid modified ruthenium chelates, dendrimer developers, liposome modified developers, and fluorene-containing fullerenes, or other similar structures of tracers.
  • the MRI tracer is selected from the group consisting of gadopentetate gadolinium, gadolinium mesylate, mussel glucamine, guanidine diamine, ferric ammonium citrate effervescent granules, paramagnetic iron oxide, or Other similar structures of tracers.
  • the substance loaded is a CD44 activator.
  • the CD44 activator is a CD44 antibody mAb or IL5, IL12, IL18, TNF-[alpha], LPS.
  • the substance to be loaded is a small molecule hyaluronic acid having a molecular weight ranging from 2000 to 5000 Da or a hyaluronic substance capable of specifically binding to a CD44 molecule on the cell surface at a vulnerable plaque.
  • Acid derivative is a small molecule hyaluronic acid having a molecular weight ranging from 2000 to 5000 Da or a hyaluronic substance capable of specifically binding to a CD44 molecule on the cell surface at a vulnerable plaque.
  • the small molecule hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to a CD44 molecule on the cell surface at the vulnerable plaque has a molecular weight ranging from 2500 to 4500 D.
  • the small molecule hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to a CD44 molecule on the cell surface at the vulnerable plaque has a molecular weight ranging from 3000 to 4000 Da.
  • the molecular weight of the small molecule hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to a CD44 molecule on the cell surface at the vulnerable plaque is about 3411 Da.
  • the siliceous body is simultaneously loaded with a substance and a CD44 activator for diagnosing, preventing and/or treating a disease of a vulnerable plaque or associated with a vulnerable plaque.
  • the siliceous body is simultaneously loaded with a substance for preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque, and a small molecule having a molecular weight ranging from 2000 to 5000 Da.
  • Hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to CD44 molecules on the surface of cells at vulnerable plaques.
  • the siliceous body is simultaneously loaded with a substance for diagnosing a vulnerable plaque or a disease associated with a vulnerable plaque, for preventing and/or treating a vulnerable plaque or Substances of vulnerable plaque-associated diseases, optionally CD44 activators and optionally small molecule hyaluronic acid having a molecular weight range of 2000 to 5000 Da or capable of specifically binding to CD44 molecules on the cell surface at vulnerable plaque a derivative of hyaluronic acid.
  • the substance to be loaded is a substance for preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque.
  • the substance for preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque is selected from the group consisting of a statin, a fibrate, an antiplatelet drug, a PCSK9 inhibitor, an antibiotic One or more of a clotting drug, an angiotensin converting enzyme inhibitor (ACEI), a calcium ion antagonist, a MMPs inhibitor, a beta blocker, and a pharmaceutically acceptable salt thereof, including these species Active structural fragment of the drug.
  • ACEI angiotensin converting enzyme inhibitor
  • MMPs inhibitor a beta blocker
  • a pharmaceutically acceptable salt thereof including these species Active structural fragment of the drug.
  • the substance for preventing and/or treating a vulnerable plaque or a disease associated with a 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, antisense RNAi oligonucleotides such as ALN-PCSsc, nucleic acids such as microRNA-33a, microRNA-27a/b, microRNA-106b, microRNA-302, microRNA-758, microRNA-10b, microRNA- 19b, microRNA-26, microRNA-93, microRNA-128-2, microRNA-144, microRNA
  • the siliceous delivery system comprises a siliceous vesicle, wherein a surface of the siliceous vesicle is partially modified by a targeting ligand, the targeting ligand It is a derivative of hyaluronic acid or hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at the vulnerable plaque.
  • the siliceous delivery system for targeting vulnerable plaques of the present invention can be prepared according to any of the methods known in the art.
  • a silicon wafer delivery system for targeting vulnerable plaques of the present invention is prepared using a film dispersion process.
  • the present invention provides a method for preparing a siliconulosic delivery system for targeting vulnerable plaques according to the present invention, the method comprising the steps of:
  • a siliceous monomer molecule a distearoylphosphatidylethanolamine (DSPE) molecule, optionally other lipid molecules, and optionally a lipid-soluble one for diagnosis, prevention, and/or treatment a substance that damages plaque or disease associated with vulnerable plaques is dissolved in a suitable organic solvent;
  • DSPE distearoylphosphatidylethanolamine
  • aqueous medium optionally containing a water-soluble substance for the diagnosis, prevention and/or treatment of vulnerable plaque or diseases associated with vulnerable plaque, and in a film at a constant temperature of 40-60 ° C
  • the components are sufficiently hydrated to form a crude siliceous vesicle suspension
  • step 4) treating the crude siliceous vesicle suspension obtained in step 3) by ultrasonication, shaking, homogenization, extrusion or other suitable method to obtain a purified siliceous vesicle suspension;
  • step 5) optionally removing the unloaded plaque contained in the purified siliceous vesicle suspension obtained in step 4) by dialysis for the diagnosis, prevention and/or treatment of vulnerable plaque or associated with vulnerable plaque Substance of disease;
  • step 6) placing the purified siliceous vesicle suspension obtained in step 4) or 5) for at least 24 hours to promote hydrolysis and condensation of the siloxane to form an inorganic polysiloxane network;
  • step 8) adding the activated targeting ligand to the siliceous vesicle suspension obtained in step 6) to form an amide between the activated targeting ligand and the distearoylphosphatidylethanolamine (DSPE) molecule The bond is coupled to obtain a silt delivery system for targeting vulnerable plaque.
  • DSPE distearoylphosphatidylethanolamine
  • the invention provides a medicament comprising the siliceous delivery system of the invention, and a pharmaceutically acceptable carrier.
  • the medicament comprises a siliceous delivery system for targeting vulnerable plaques of the invention and a pharmaceutically acceptable carrier.
  • the invention provides a diagnostic formulation comprising a silicon body delivery system for targeting vulnerable plaques according to the invention.
  • the diagnostic formulation comprises a siliceous delivery system that targets vulnerable plaques of the invention.
  • a siliconulosic delivery system for the preparation of a medicament for the diagnosis, prevention and/or treatment of a disease associated with the presence of a CD44 molecule activation condition.
  • the plastid delivery system is for use in the manufacture of a medicament for the diagnosis, prevention, and/or treatment of a vulnerable plaque or a disease associated with a vulnerable plaque.
  • the invention provides a method for diagnosing, preventing and/or treating a disease associated with the presence of a CD44 molecule activation condition, the method comprising administering a silicon body delivery system according to the invention.
  • the invention provides a method for diagnosing, preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque, the method comprising administering a silicon body of the invention Delivery system.
  • the vulnerable plaque is selected from one or more of a ruptured plaque, an aggressive plaque, and a partially calcified nodular lesion.
  • the disease associated with vulnerable plaque is selected from the group consisting of coronary atherosclerotic heart disease, atherosclerosis, hemangioma, thromboembolism, angina pectoris, myocardial infarction, sudden cardiac death, heart rate One or more of failure, cardiogenic shock, ischemic cardiomyopathy, and stroke.
  • the invention includes any combination of any of the above embodiments, including various preferred embodiments.
  • the invention includes endpoint values of the ranges, any specific values within the range, and sub-ranges that are comprised of any two specific values within the range.
  • the siliceous delivery system of the present invention has the following advantages for diseases in which the CD44 molecule is activated:
  • the siliceous delivery system of the present invention is capable of specifically binding to activated CD44 molecules and is capable of achieving stable sustained release of the drug.
  • CD44 in vulnerable plaques is activated by the extracellular matrix microenvironment, overexpressed in a large amount, and the affinity of CD44-HA is significantly increased, making the interaction between CD44 and HA in vulnerable plaques extremely significant. Affinity specificity.
  • CD44 within the vulnerable plaque constitutes an excellent target for the presently described vulnerable plaque-targeting siliceous delivery system.
  • the siliceous delivery system targeting the vulnerable plaque of the present invention can actively target into the vulnerable plaque and bind to the focal cells.
  • the siliceous delivery system can achieve sustained release of the loaded material at the lesion, significantly increasing and sustaining the concentration of the substance in the lesion area, thereby improving the diagnostic or therapeutic effect of the delivery system.
  • the siliceous delivery system for vulnerable plaques can encapsulate small molecule hyaluronic acid having a molecular weight ranging from 2000 to 5000 Da or can be specific to CD44 molecules on the cell surface at vulnerable plaques.
  • Sexually bound derivatives of hyaluronic acid The small molecule hyaluronic acid or its derivative binds to the endogenous hyaluronic acid on the cell surface by competitive binding with the endogenous hyaluronic acid on the cell surface, thereby facilitating the diagnosis or treatment of the active substance to enter smoothly.
  • the lesion is inside the cell.
  • lipid pool in the vulnerable plaque contains a large amount of oxidized low density lipoprotein (ox-LDL).
  • ox-LDL oxidized low density lipoprotein
  • liposomes are unstable in such an internal environment, and are highly disintegrable to achieve a controlled release function; whereas the silicon-based delivery system targeting vulnerable plaques of the present invention is vulnerable to plaques.
  • the lipid pool in the block is relatively stable and can release the drug continuously, thereby maintaining the drug concentration at the lesion.
  • the siliceous delivery system targeting the vulnerable plaque of the present invention may also be loaded with a CD44 activating substance, namely a CD44 activator such as IL5, IL12, IL18, TNF- ⁇ , LPS.
  • a CD44 activator such as IL5, IL12, IL18, TNF- ⁇ , LPS.
  • the CD44 activator can promote the further activation of CD44 on the surface of the lesion cells, and can amplify the targeting affinity of CD44 for hyaluronic acid in a short time, significantly increasing the concentration of the targeted siliceous composition bound to the cell surface, which is vulnerable to Tracing diagnosis and treatment of plaques is of positive significance because it can significantly increase the concentration of tracer or therapeutic compound in a short period of time to improve diagnostic resolution or therapeutic efficacy.
  • the present invention relates to a vulnerable plaque-targeting siliceous delivery system which has good mechanical stability, thermal stability and stability in a vulnerable plaque microenvironment, and has good storage stability;
  • the siliceous monomer molecule contains Si-C and Si-O bonds, and the chemical composition makes it biodegradable;
  • the siliceous vesicles can be loaded with fat-soluble substances, amphoteric compounds or water-soluble substances.
  • materials that can be loaded in the present invention's siliceous delivery system include, but are not limited to, those listed in the present invention, as long as they can be incorporated into the siliceous body and can be used for diagnosis, prevention, and/or treatment.
  • the purpose can be.
  • the substance can be easily incorporated into the siliceous body as long as it is not a substance which is insoluble in the aqueous phase and the organic solvent or a substance which is very soluble in both the aqueous phase and the organic solvent.
  • the substance is a fat-soluble substance, an amphoteric compound or a water-soluble substance.
  • Figure 1 is a schematic illustration of the construction of a silt delivery system for targeting vulnerable plaques of the present invention.
  • Figure 2 is an infrared spectrum of HA-CL1 in which the IR spectrum of rosuvastatin-loaded CL1 is linked before HA (black, underline) and after attachment (red, upper line).
  • Figure 3 is a graph showing the change in particle size of the siliceous delivery system of the present invention and the liposome delivery system as a control stored at 4 ° C for 90 days.
  • Figure 4 is a graph showing the change in drug encapsulation efficiency of a siliceous delivery system of the present invention and a liposome delivery system as a control stored at 4 ° C for 90 days.
  • Figure 5 is a graph showing changes in drug cumulative release rate for a siliceous delivery system of the present invention and a liposome delivery system as a control.
  • Figure 6 is a graph showing changes in drug cumulative release rate for three of the present invention.
  • Figure 7 is a magnetic resonance imaging image of a mouse atherosclerotic vulnerable plaque model constructed in Example 4.
  • Figure 8 is a graph showing the percentage of vehicle carotid plaque drug exposure after administration of the siliceous delivery system of the present invention and the liposome delivery system as a control.
  • Figure 9 is a graph showing the results of CD44 content (in semi-quantitative integration) on the surface of endothelial cells at normal arterial wall endothelial cells and arterial vulnerable plaques of model mice.
  • Fig. 10 is a graph showing the results of binding of CD44 to HA on the surface of endothelial cells of normal arterial wall and arterial vulnerable plaques of model mice (indicated by binding force integral).
  • Fig. 11 is a graph showing the results of measurement of binding ability (integrated with binding force) of CD44 on the surface of endothelial cells of normal arterial blood vessel wall endothelial cells and arterial vulnerable plaques of model mice.
  • Fig. 12 is a graph showing the results of binding of CD44 to HA on the surface of macrophages in model mice and on the surface of macrophages in arterial vulnerable plaques (indicated by binding force integral).
  • Fig. 13 is a graph showing the results of binding of CD44 to various ligands/antibodies on the surface of macrophage of macroscopic phagocytes and arterial vulnerable plaques of model mice (indicated by binding force integral).
  • Figure 14 is a graph showing the in vivo therapeutic effect (expressed as percentage of plaque progression) of a saxoplasmic delivery system of the present invention on carotid vulnerable plaques in model mice.
  • Figure 15 is a graph showing the in vivo tracing effect (indicated by CT values) of a saxoplasmic delivery system of the present invention on carotid vulnerable plaques of model mice.
  • Figure 16 is an in vivo CT trace of the sarcoplasmic-HA-ioxolol delivery system for arterial vulnerable plaque.
  • Figure 17 is an in vivo MRI tracing of arterial vulnerable plaques by a siliceous-HA-demetphate delivery system.
  • Figure 18 is an in vivo MRI tracing of arterial vulnerable plaques by CD44 polyclonal antibody-siliceous-gluconate delivery system.
  • Figure 19 is an in vivo MRI tracing of arterial vulnerable plaques by CD44 multiple ligand-siliceous-guanidine bisamine delivery systems.
  • Example 1 Three siliceous monomers used in the present invention
  • siliceous monomers C1, C2 and C3 used in the siliceous delivery system of the present invention are known, which can be obtained according to the preparation methods described herein.
  • siliceous monomer C1 N, N-dihexadecyl-N ⁇ -(6-((3-triethoxysilyl)propyldimethylammonio)hexanoyl)propylamine
  • a siliceous delivery system HA-CL1@R, HA CL2@R, and HA-CL3@R loaded with a therapeutic agent were prepared by a film dispersion method.
  • the surfaces of the siliceous vesicles of the above three siliceous delivery systems are partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA") and are both loaded for the prevention and/or treatment of vulnerable plaques.
  • HA targeting ligand hyaluronic acid
  • R the substance rosuvastatin (represented by the abbreviation "R") of the disease associated with vulnerable plaque, except that the siliceous monomer molecules used in the preparation of the three siliceous delivery systems are The siliceous monomers C1, C2 and C3 described in Example 1.
  • HA-CL1@R The specific preparation methods of HA-CL1@R, HA-CL2@R and HA-CL3@R are:
  • rosuvastatin aqueous solution concentration 2 mg/mL
  • the crude siliceous vesicle suspension was sonicated for 10 min, and then ultrasonically probed for 5 min (amplitude 20, interval 3 s) to obtain a stable system formed by the dispersion of siliceous vesicles, ie, refined siliceous body.
  • Vesicle suspension Unencapsulated rosuvastatin in the purified siliceous vesicle suspension was removed in a dialysis bag. The siliceous vesicle suspension is then placed for at least 24 hours to promote the formation of a rigid inorganic polysiloxane network on the surface of the siliceous body of the siliceous monomer molecules.
  • HA molecular weight of about 100 KDa
  • EDC.HCl 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • the g-N-hydroxythiosuccinimide (sulfo-NHS) coupling agent activates the carboxyl group.
  • acetone was added to precipitate activated HA.
  • the precipitate was filtered, washed with ethanol and dried in vacuo to give activated HA.
  • the coupling of HA to CL was confirmed by infrared characterization.
  • the sample for infrared characterization was a sample of rosuvastatin HA-CL1@R loaded by weighing 6 mg of C1 and 1 mg of DSPE in a round bottom flask and dissolving with 10 mL of chloroform. The organic solvent was completely removed by rotary evaporation (55 ° C water bath, 90 r/min, 30 min) to form a film on the walls of the vessel.
  • siliceous vesicles are obtained and allowed to stand for at least 24 hours to promote the formation of a rigid inorganic polysiloxane network on the surface of the siliceous body.
  • HA molecular weight of about 100 KDa
  • carboxyl group was activated by adding 10 mg of EDC.HCl and 12 mg of sulfo-NHS coupling agent. After the reaction was stirred at room temperature for 1 hour, acetone was added to precipitate activated HA. The precipitate was filtered, washed with ethanol and dried in vacuo to give activated HA. It was configured to be an aqueous solution of 0.1 mg mL-1, and 0.2 mL of the solution was dissolved in the suspension of the siliceous vesicle obtained in the above step (1) to activate the activated carboxyl group and the siliceous sac in the activated HA.
  • the amino group of the doped DSPE molecule in the bubbled lipid bilayer has a coupling by forming an amide bond, thereby obtaining a targeted siliceous body HA-CL1@R1.
  • HA-CL1@R was separated by high speed centrifugation at 12,000 rpm. After vacuum drying, it was used for infrared spectroscopy.
  • the 1100 nm absorption peak demonstrates the presence of siliceous bodies, and the absorption peaks at 1700 nm and 2910 nm indicate successful coupling of siliceous bodies and HA.
  • a siliceous delivery system HACL1@LMHA, HA-CL2@LMHA, and HA-CL3@LMHA loaded with small molecule hyaluronic acid were prepared by a film dispersion method.
  • the surfaces of the siliceous vesicles of the above three siliceous delivery systems are partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA") and each load a small molecule hyaluronic acid having a molecular weight of about 3411 Da (
  • the molecules are the siliceous monomers C1, C2 and C3 described in Example 1, respectively.
  • the preparation method is the same as that of HA-CL3@R, except that in step (1), 10 mL of rosuvastatin aqueous solution (concentration: 2 mg/mL) is replaced with 10 mL of an aqueous solution of small molecular weight hyaluronic acid having a molecular weight of 3411 Da (concentration). 0.5 mg/mL), and the unencapsulated small molecule hyaluronic acid in the purified siliceous vesicle suspension was removed with a dialysis bag.
  • a silt delivery system HA-CL1@R+LMHA, HA-CL2@R+LMHA and HA-CL3, which simultaneously load the therapeutic agent rosuvastatin and small molecule hyaluronic acid, is prepared by a film dispersion method. @R+LMHA.
  • the surface of the siliceous vesicles of the above three siliceous delivery systems are partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA"), and both are loaded with the therapeutic agent rosuvastatin (abbreviated as “abbreviation” R” represents) and a small molecule hyaluronic acid having a molecular weight of about 3411 Da, except that the silicon monomer molecules used in the preparation of the three siliceous delivery systems are respectively the silicon described in Example 1.
  • HA-CL1@R+LMHA Specific preparation method of HA-CL1@R+LMHA, HA-CL2@R+LMHA and HA-CL3@R+LMHA and the siliceous delivery system HA-CL1@R of the therapeutic agent described in the above point 1.
  • the preparation methods of HA-CL2@R and HACL3@R are basically the same, except that 10 mL of rosuvastatin aqueous solution (concentration: 2 mg/mL) and 10 mL of small molecular weight molecular weight of 3411 Da are simultaneously used in the step (1).
  • aqueous acid solution concentration of 0.5 mg/mL
  • unencapsulated rosuvastatin and small molecule hyaluronic acid in the purified siliceous vesicle suspension were removed with a dialysis bag.
  • a CD44 activator-loaded siliceous delivery system HA-CL1@S, HACL2@S, and HA-CL3@S were prepared by a film dispersion method.
  • the surface of the siliceous vesicles of the above three siliceous delivery systems are partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA"), and both are loaded with CD44 activator--CD44 antibody mAb (using abbreviations) "S" indicates) the only difference is that the siliceous monomer molecules used in the preparation of the three siliceous delivery systems are the siliceous monomers C1, C2 and C3 described in Example 1, respectively.
  • the preparation method is the same as that of HA-CL3@R, except that in step (1), 10 mL of rosuvastatin aqueous solution (concentration: 2 mg/mL) is replaced with an aqueous solution of 10 m LCD44 antibody mAb (concentration: 0.7 mg/mL). And the unencapsulated CD44 antibody mAb in the purified siliceous vesicle suspension was separated and removed through a Sephadex column G-100.
  • CD44 activators can also be prepared using LPS, with similar results.
  • a silt delivery system HACL1@R+S, HA-CL2@R+S, and HA-CL3@R+S loaded with a therapeutic agent and a CD44 activator were prepared by a film dispersion method.
  • the surface of the siliceous vesicles of the above three siliceous delivery systems are partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA”), and both are loaded with the therapeutic agent rosuvastatin (abbreviated as “abbreviation” R” represents) and CD44 activator - CD44 antibody mAb (represented by the abbreviation "S”), except that the siliceous monomer molecules used in the preparation of the three siliceous delivery systems are respectively examples The siliceous monomers C1, C2 and C3 described in 1.
  • HA targeting ligand hyaluronic acid
  • R therapeutic agent rosuvastatin
  • S CD44 activator - CD44 antibody mAb
  • HA-CL1@R+S Specific preparation method of HA-CL1@R+S, HA-CL2@R+S and HA-CL3@R+S and the siliceous delivery system HA-CL1@R of the therapeutic agent described in the above point 1.
  • the preparation methods of HA-CL2@R and HA-CL3@R are basically the same, except that in step (1), 10 mL of aqueous solution of rosuvastatin (concentration: 2 mg/mL) and 10 mL of CD44 antibody mAb are simultaneously used ( The concentration was 0.7 mg/mL), and the unencapsulated rosuvastatin and CD44 antibody mAb in the purified siliceous vesicle suspension were removed with a Sephadex column G-100.
  • CD44 activators can also be prepared using LPS, with similar results.
  • a load tracer-based siliceous delivery system HA-CL1@T, HACL2@T, and HA-CL3@T were prepared by a film dispersion method.
  • the surfaces of the siliceous vesicles of the above three siliceous delivery systems are partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA"), and both are loaded with the MRI tracer guanidinic acid (abbreviated as "abbreviation” T”), except that the siliceous monomer molecules used in the preparation of the three siliceous delivery systems are the siliceous monomers C1, C2 and C3 described in Example 1, respectively.
  • HA targeting ligand hyaluronic acid
  • T MRI tracer guanidinic acid
  • the preparation method is the same as that of HA-CL3@R, except that in step (1), 10 mL of rosuvastatin aqueous solution (concentration: 2 mg/mL) is replaced with 10 mL of hydrazine acid aqueous solution (concentration: 3 mg/mL). And removing the unencapsulated sputum spray acid from the purified siliceous vesicle suspension with a dialysis bag.
  • the tracer can also be prepared using dextran or guanidine diamine, and similar results are obtained.
  • a silicon trace delivery system HA-CL1@AuNPs, HACL2@AuNPs, and HA-CL3@AuNPs loaded with a tracer were prepared by a thin film dispersion method.
  • the surface of the siliceous vesicles of the above three siliceous delivery systems are partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA") and both loaded with the CT tracer nanogold (using the abbreviation "AuNPs" "Expression” differs only in that the siliceous monomer molecules used in the preparation of the three siliceous delivery systems are the siliceous monomers C1, C2 and C3 described in Example 1, respectively.
  • HA-CL1@AuNPs HA-CL2@AuNPs
  • HA-CL3@AuNPs siliceous delivery system
  • the preparation method is the same as that of HA-CL3@R, except that in step (1), 10 mL of rosuvastatin aqueous solution (concentration: 2 mg/mL) is replaced with 10 mL of nano gold solution (concentration: 1 mg/mL), and Unencapsulated gold nanoparticles in the purified siliceous vesicle suspension were removed using a Sephadex column G-100.
  • a load tracer-based siliceous delivery system HA-CL1@I, HA-CL2@I, and HA-CL3@I were prepared by a film dispersion method.
  • the surface of the siliceous vesicles of the above three siliceous delivery systems are partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA") and both loaded with the CT tracer iodixanol or iodopro Amine (represented by the abbreviation "I”), except that the siliceous monomer molecules used in the preparation of the three siliceous delivery systems are respectively the siliceous monomer C1 described in Example 1. C2 and C3.
  • HA-CL1@I HA-CL2@I and HA-CL3@I and the siliceous delivery system HA-CL1@R, HA-CL2@R of the therapeutic agent described in the above point 1.
  • the preparation method is the same as that of HA-CL3@R, except that in step (1), 10 mL of rosuvastatin aqueous solution (concentration: 2 mg/mL) is replaced with 10 mL of iodixanol or iopromide solution (concentration: 1 ⁇ g). /mL), and the unencapsulated iodixanol or iopromide in the purified siliceous vesicle suspension was removed using a Sephadex column.
  • a silt delivery system HACL1@AuNPs+S, HA-CL2@AuNPs+S, and HA-CL3@AuNPs+S which simultaneously loaded a tracer and a CD44 activator, were prepared by a thin film dispersion method.
  • the surfaces of the siliceous vesicles of the above three siliceous delivery systems are partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA"), and both are loaded with CT tracer nanogold (using the abbreviation " AuNPs "represented” and CD44 activator - CD44 antibody mAb (represented by the abbreviation "S”), except that the siliceous monomer molecules used in the preparation of the three siliceous delivery systems are respectively examples The siliceous monomers C1, C2 and C3 described in 1.
  • HA-CL1@AuNPS+S Specific preparation method of HA-CL1@AuNPS+S, HA-CL2@AuNPS+S and HA-CL3@AuNPS+S and the siliceous delivery system HA-CL1@R of the therapeutic agent described in the above point 1.
  • the preparation methods of HA-CL2@R and HACL3@R are basically the same, except that in step (1), 10 mL of nano gold solution (concentration of 1 mg/mL) and 10 mL of CD44 antibody mAb aqueous solution (concentration of 0.7 mg) are simultaneously used. /mL), and the unencapsulated nanogold and CD44 antibody mAb in the purified siliceous vesicle suspension was removed with a Sephadex column G-100.
  • a silt delivery system HA-CL1@I+S, HA-CL2@I+S, and HA-CL3@I+S which simultaneously loaded a tracer and a CD44 activator, were prepared by a thin film dispersion method.
  • the surface of the siliceous vesicles of the above three siliceous delivery systems are partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA") and both are loaded with the CT tracer iodixanol or iodine.
  • HA targeting ligand hyaluronic acid
  • Proline represented by the abbreviation "I”
  • CD44 activator - LPS represented by the abbreviation "S”
  • siliceous monomers C1, C2 and C3 described in Example 1 are respectively.
  • HA-CL1@I+S Specific preparation method of HA-CL1@I+S, HA-CL2@I+S and HA-CL3@I+S and the siliceous delivery system HA-CL1@R of the therapeutic agent described in the above point 1.
  • the preparation methods of HA-CL2@R and HA-CL3@R are basically the same, except that in step (1), 10 ml of an aqueous solution of iodixanol or iopromide solution (1 ⁇ g/mL) and 10 ml of LPS are simultaneously used ( The concentration was 0.7 mg/mL), and the unencapsulated iodixanol or iopromide and LPS in the purified siliceous vesicle suspension were removed with a Sephadex column G-200.
  • a silicon-based delivery system HA-CL1@R+LMHA+ is prepared by film dispersion method while loading the therapeutic agent rosuvastatin, small molecule hyaluronic acid, tracer guanidinoic acid and CD44 activator.
  • the surface of the siliceous vesicles of the above three siliceous delivery systems are partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA”), and both are loaded with the therapeutic agent rosuvastatin (abbreviated as “abbreviation” R” represents), a small molecule hyaluronic acid having a molecular weight of about 3411 Da, an MRI tracer guanidinoic acid (represented by the abbreviation "T”), and a CD44 activator--CD44 antibody mAb (represented by the abbreviation "S”), Only the siliceous monomer molecules used in the preparation of the three siliceous delivery systems are the siliceous monomers C1, C2 and C3 described in Example 1, respectively.
  • HA targeting ligand hyaluronic acid
  • R therapeutic agent rosuvastatin
  • T an MRI tracer guanidinoic acid
  • S CD44 activator--CD44 antibody mAb
  • HA-CL1@R+LMHA+T+S HA-CL2@R+LMHA+T+S
  • HA-CL3@R+LMHA+T+S The preparation methods of the siliceous delivery systems HA-CL1@R, HACL2@R and HA-CL3@R are basically the same, except that 10 mL of rosuvastatin aqueous solution is simultaneously used in step (1) (concentration is 2 mg/ mL), 10 mL aqueous solution of small molecule hyaluronic acid having a molecular weight of 3411 Da (concentration: 0.5 mg/mL), 10 mL aqueous solution of hydrazine acid (concentration: 3.0 mg/mL), and aqueous solution of 10 m LCD44 antibody mAb (concentration: 0.7 mg/mL) (mL), and the unencapsulated rosuvastatin, small molecule hyaluronic acid,
  • LPS can also be used for the CD44 activator, and the tracer can also be similarly prepared using gadolinium citrate to obtain similar results.
  • siliceous delivery systems HA-CL1, HA-CL2, HA-CL3 were prepared by thin film dispersion.
  • the surfaces of the siliceous vesicles of the above three siliceous delivery systems are partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA") and are not loaded for any diagnosis, prevention and/or treatment.
  • HA targeting ligand hyaluronic acid
  • a substance that is vulnerable to plaque or disease associated with vulnerable plaque differs only in that the siliceous monomer molecules used in the preparation of the three siliceous delivery systems are respectively the silicon described in Example 1.
  • the preparation method was basically the same except that in the step (1), 10 mL of the aqueous rosuvastatin solution (concentration: 2 mg/mL) was replaced with pure water, and the step of removing the unencapsulated material by dialysis was eliminated.
  • a non-targeted therapeutic agent-loaded siliceous delivery system CL1@R, CL2@R, and CL3@R was prepared using a thin film dispersion process.
  • the surfaces of the siliceous vesicles of the above three siliceous delivery systems are not modified by the targeting ligand hyaluronic acid (abbreviated as "HA") and are both loaded for the prevention and/or treatment of vulnerable plaque or
  • the substance rosuvastatin (represented by the abbreviation "R") of the disease associated with vulnerable plaques differs only in that the siliceous monomer molecules used in the preparation of the three siliceous delivery systems are implemented separately
  • a non-targeted load tracer-based silicon delivery system CL1@AuNPS, CL2@AuNPS, and CL3@AuNPS were prepared by thin film dispersion.
  • the surface of the siliceous vesicles of the above three siliceous delivery systems were not modified by the targeting ligand hyaluronic acid (abbreviated as "HA"), and both were loaded with CT tracer nanogold (using the abbreviation "AuNPs"
  • HA targeting ligand hyaluronic acid
  • CT tracer nanogold using the abbreviation "AuNPs”
  • the siliceous monomer molecules used in the preparation of the three siliceous delivery systems are the siliceous monomers C1, C2 and C3 described in Example 1, respectively.
  • the specific preparation methods of CL1@AuNPS, CL2@AuNPS and CL3@AuNPS are the same as the load tracer-based siliceous delivery systems HA-CL1@AuNPs, HA-CL2@AuNPs and HA-CL3@ described in point 7 above.
  • the preparation method of AuNPs is basically the same, except that step (2) is omitted.
  • a non-targeted load tracer-based silicon delivery system CL1@I, CL2@I, and CL3@I were prepared by thin film dispersion.
  • the surface of the siliceous vesicles of the above three siliceous delivery systems were not modified by the targeting ligand hyaluronic acid (abbreviated as "HA"), and both were loaded with the CT tracer iodixanol (abbreviated as "abbreviation” I”), except that the siliceous monomer molecules used in the preparation of the three siliceous delivery systems are the siliceous monomers C1, C2 and C3 described in Example 1, respectively.
  • a liposome delivery system HA-PL@R loaded with a therapeutic agent was prepared by a film dispersion method.
  • the surface of the liposome vesicles of the liposome delivery system HA-PL@R are partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA”) and are both loaded for prevention and/or treatment.
  • HA targeting ligand hyaluronic acid
  • R rosuvastatin
  • HA-PL@R The specific preparation method of HA-PL@R is:
  • DSPC distearoylphosphatidylcholine
  • DSPE distearoylphosphatidylethanolamine
  • the crude liposome vesicle suspension was sonicated in a water bath, and finally ultrasonically treated with a probe sonicator for 3 min (amplitude 20, interval 3 s) to obtain a dispersion system in which liposome vesicles were sufficiently dispersed, that is, a purified liposome sac.
  • Bubble suspension Unencapsulated rosuvastatin in the purified liposome vesicle suspension was removed in a dialysis bag.
  • HA molecular weight of about 100 KDa
  • EDC.HCl 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • the g-N-hydroxythiosuccinimide (sulfo-NHS) coupling agent activates the carboxyl group.
  • the activated HA was precipitated by adding absolute ethanol. The precipitate was filtered, washed with ethanol and dried in vacuo to give activated HA.
  • the amino acid of the doped DSPE molecule in the bubbled lipid bilayer has a coupling by forming an amide bond, thereby obtaining a therapeutic agent-loaded liposome delivery system HA-PL@R.
  • a load tracer-loaded liposome delivery system HA-PL@T was prepared by a thin film dispersion method.
  • the surface of the liposome vesicles of the liposome delivery system HA-PL@T is partially modified by the targeting ligand hyaluronic acid (abbreviated as "HA”) and both are loaded with the MRI tracer guanidinic acid. (Expressed by the abbreviation "T").
  • the specific preparation method of HA-PL@T is basically the same as the preparation method of the liposome delivery system HA-PL@R of the therapeutic agent described in the above 13th point, except that 10 mL is used in the step (1).
  • An aqueous solution of rosuvastatin (concentration 2.0 mg/mL) was replaced with 10 mL of a tracer aqueous solution of guanidinoic acid (concentration of 3.0 mg/mL), and an unencapsulated tracer of the purified liposomal vesicle suspension
  • the sputum spray acid is removed with a dialysis bag.
  • the tracer can also be similarly prepared using gadolinium citrate to give similar results.
  • the siliceous delivery systems HA-CL1@R, HACL2@R and HA-CL3@R of the therapeutic agent prepared in Example 2 were taken as an example to prove that the silicon plastid delivery system of the present invention has Stable and controllable properties, suitable for the diagnosis, prevention and treatment of vulnerable plaque or diseases associated with vulnerable plaque. Meanwhile, for convenience of comparison, the liposome delivery system HA-PL@R (as a comparative example) of the therapeutic agent prepared in Example 2 was also used in the present example.
  • Rosuvastatin has a strong ultraviolet absorption property, and thus its content can be determined by using the UV-absorbing property of rosuvastatin by HPLC-UV method (using Waters 2487, Waters Corporation, USA). A standard quantitative equation was established for the peak area (Y) of the HPLC chromatographic peaks using different concentrations of rosuvastatin solution (X).
  • silicate particle delivery systems HA-CL1@R, HA-CL2@R and HA-CL3@R of the present invention and the hydrated particle size of the liposome delivery system HA-PL@R as a comparative example were all laser size analyzers ( BI-Zeta Plus/90 Plus, Brookhaven Instruments Corporation, USA), the specific results are shown in Table 1.
  • the method for determining the drug loading is similar to the method for determining the encapsulation rate, except that the calculation method is slightly different.
  • the siliceous vesicle suspension is taken, an excess of HCl is added to form a strong acidic environment, and the ultrasonic method is further used to accelerate the release of the drug from the siliceous vesicles.
  • the drug content in the resulting liquid was measured by HPLC (Waters 2487, Waters Corporation, USA), and the drug loading amount was calculated by Formula 2.
  • the siliceous delivery systems HA-CL1@R, HA-CL2@R and HA-CL3@R of the present invention and the liposome delivery system HA-PL@R as a control were stored at 4 ° C at different time points. Samples were taken and their hydrated particle size changes were measured by a laser particle size analyzer (BI-Zeta Plus/90 Plus, Brookhaven Instruments Corporation, USA). The results are shown in FIG. It can be seen that the particle size of the liposome delivery system HA-PL@R is significantly increased as the storage time is extended. This is most likely due to instability of the liposome vesicles, prone to aggregation or fusion. Moreover, due to poor stability, liposome vesicles are easily removed by the reticuloendothelial system in the body, resulting in a short half-life and limited when applied to humans.
  • the siliceous delivery systems HA-CL1@R, HA-CL2@R and HA-CL3@R of the present invention have better storage stability than the liposome delivery system HA-PL@R.
  • HA-PL@R liposome delivery system
  • siliceous delivery systems HA-CL1@R, HA-CL2@R and HA-CL3@R of the present invention and the liposome delivery system HA-PL@R as a control were stored at 4 ° C and sampled at different time points. The free drug was removed by ultrafiltration to examine the change in encapsulation efficiency. The results are shown in Fig. 4.
  • Equation 3 The meaning of each parameter in Equation 3 is as follows:
  • Ve displacement volume of the release solution, where Ve is 2 mL
  • V0 volume of the release liquid in the release system, where V0 is 50mL
  • Ci concentration of drug in the release solution at the time of the i-th replacement sampling, in ⁇ g/mL
  • M drug total mass of drug in a siliceous or liposome delivery system, in ⁇ g
  • Cn drug concentration in the release system measured after the nth replacement of the release solution.
  • Figure 5 is a graph showing the change in drug cumulative release rate of the siliceous delivery system HA-CL1@R of the present invention and the liposome delivery system HA-PL@R as a control.
  • the liposome delivery system HA-PL@R almost released 100% of the drug within 30 hours.
  • the siliceous delivery system HA-CL1@R released faster in the first 3 hours and released about 15% in 3 hours. After that, the drug release rate gradually slowed down, and only 59.5% of the drug was released after 120 hours.
  • the faster release rate in the early stage may be caused by the release behavior of the drug which is partially adsorbed or precipitated on the surface of the siliceous vesicle and can be rapidly dissolved and diffused into the release medium, and the late drug release is mainly encapsulated in Drug release within the siliceous vesicles manifests as a sustained, slow release behavior.
  • the in vitro release test results show that the release of the drug from the siliceous vesicles can be effectively retarded because the surface of the siliceous vesicles is covered by the inorganic polysiloxane network, so between the lipid bilayers The voids are reduced, increasing the density of the lipid bilayer.
  • In vitro release experiments indicate that siliceous vesicles have slow and sustained release characteristics as drug carriers.
  • Example 4 In vivo release stability study of the siliceous delivery system of the present invention
  • the sussapstatin-loaded siliceous delivery systems HA-CL1@R and HACL2@R prepared in Example 2 were used as an example to demonstrate the silicon of the present invention compared to the liposome delivery system.
  • the plastid delivery system is able to remain relatively stable at vulnerable plaques, thereby achieving long-lasting sustained release of the drug.
  • the rosuvastatin-loaded liposome delivery system HA-PL@R prepared in Example 2 (as a comparative example) was also used in the present example.
  • mice SPF-grade ApoE-/- mice (18, 10 weeks old, body weight 20 ⁇ 1 g) were taken as experimental animals.
  • the mice were given an adaptive high-fat diet (fat 10% (w/w), cholesterol 2% (w/w), sodium cholate 0.5% (w/w), and the rest were normal feed for mice) after 4 weeks of feeding.
  • Anesthesia was intraperitoneally injected with 1% sodium pentobarbital (prepared by adding 1 mg of sodium pentobarbital to 100 ml of physiological saline) at a dose of 40 mg/kg.
  • the mouse was fixed on the surgical plate in the supine position, disinfected with the neck centered with 75% (v/v) alcohol, the neck skin was cut longitudinally, and the anterior cervical gland was bluntly separated, on the left side of the trachea.
  • the left common carotid artery can be seen on the side. Carefully separate the common carotid artery to the bifurcation.
  • the silicone tube sleeve with a length of 2.5 mm and an inner diameter of 0.3 mm was placed on the outer circumference of the left common carotid artery.
  • the proximal and distal centripet segments of the cannula were narrowed and fixed by filaments. .
  • LPS lipopolysaccharide
  • mice were placed in a 50 ml syringe (sufficient venting reserved) to cause restrictive mental stress, 6 hours/day, 5 days per week for a total of 6 weeks.
  • the mouse atherosclerotic vulnerable plaque model was completed at 14 weeks postoperatively.
  • Figure 7 (a) and (b) show the MRI image of the mouse atherosclerotic vulnerable plaque model. It can be seen from the arrow pointing part that the left carotid plaque has formed. Successful modeling, right arterial artery can be compared as normal arterial wall.
  • mice were randomly divided into three groups according to the targeted delivery system used, namely the siliceous delivery system group 1 (using the sussapatin-loaded siliceous delivery system HA-CL1@ prepared in Example 2) R), a group of siliceous delivery systems 2 (using the sussapstatin-loaded siliceous delivery system HA-CL2@R prepared in Example 2) and a liposome delivery system set (using the preparation prepared in Example 2)
  • the rosuvastatin-loaded liposome delivery system HA-PL@R was 6 per group.
  • HA-CL1@R, HA-CL2@R and HA-PL@R were administered to a single dose of 5 mg of rosuvastatin/kg body weight for the above three groups of mice, respectively.
  • the percentage of drug exposure at the vulnerable plaque of the artery was determined by liquid chromatography-mass spectrometry (which reflects the change in the concentration of rosuvastatin at the vulnerable plaque after injection of the test drug):
  • rosuvastatin 0.0141g placed in a 25mL volumetric flask, dissolved in methanol and diluted to the mark, shaken, prepared into a concentration of 56.4 ⁇ g / mL of rosuvastatin reference stock; rosulva
  • the statin reference stock solution was diluted with methanol to a series of standard solutions of 10, 1, 0.5, 0.125, 0.05, 0.025, 0.01, 0.002, 0.0004 ⁇ g/mL, and stored at 4 ° C for use.
  • the animals were sacrificed (one mouse at each time point) before and 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 168 h (seven days) before administration, and the carotid plaques were quickly removed for physiological placement.
  • the surface water was dried by a filter paper, and each was cut 1 cm, and the wet weight was weighed, and 1 ml of physiological saline was added to homogenize to prepare a homogenate.
  • 1 ml of the homogenate was taken, and 20 ⁇ L of methanol, 100 ⁇ L of an internal standard solution having a concentration of 15.2 ng/mL, 100 ⁇ L of a 10% (v/v) formic acid aqueous solution, and 5 mL of ethyl acetate were added thereto, and the mixture was mixed and centrifuged at 14,000 rpm for 10 minutes. 4 ml of the organic layer solution was taken and dried with nitrogen.
  • Rosuvastatin simulates drug-containing plasma samples. Operate according to plasma treatment (add 50 ⁇ L of 15.2 ng/mL internal standard solution, 50 ⁇ L of 10% (v/v) formic acid aqueous solution, 2.5 mL of ethyl acetate, mix, centrifuge at 14000 rpm for 10 min, take 2 ml of organic layer solution, and blow with nitrogen.
  • the liquid phase separation was carried out using a Shimadzu modula RLC system (Tokyo, Japan) system including: 1 DGU-20A3R vacuum degasser, 2 LC-20 ADXR solvent delivery modules, 1 SIL-20ACXR autosampler, 1 One SPD-M20A PDA system and one CBM-20A controller.
  • the liquid phase system was connected in-line with an ABSciex 5500 Qtrap mass spectrometer (FosteRCity, CA, USA) equipped with an ESI interface. Analyst software (Version 1.6.2, ABSciex) is used for data acquisition and processing.
  • Chromatographic analysis was performed using a CortecsTM UPLC C18 column (150 mm x 2.1 mm internal diameter (i.d.), 1.6 ⁇ m particle size) (Waters Corporation, USA), and the column temperature and sample chamber temperatures were set to 40 ° C and 4 ° C, respectively.
  • the mobile phase was a 0.1% (v/v) aqueous solution of formic acid and acetonitrile (40:60, v/v) in an amount of 2 ⁇ l.
  • the flow rate was 0.2 mL/min and the analysis time for a single sample was 4 min.
  • the ion source used for mass spectrometry is the ESI source, positive ion scan mode.
  • the spray voltage was set to 4500V and the source temperature was set to 500 °C.
  • Each compound was detected by multiple reaction monitoring (MRM).
  • the ion channels of each component were: rosuvastatin calcium m/z 482.2 ⁇ 258.2, acetaminophen m/z 152.2 ⁇ 110.
  • the collision energy and cone voltage of each compound were optimized: rosuvastatin 43V and 100V, acetaminophen 23V and 100V.
  • the retention times of rosuvastatin calcium and acetaminophen were 2.07 min and 1.49 min, respectively.
  • the linear range, correlation coefficient (r), linear equation and LLOQ of rosuvastatin are shown in Table 2. As can be seen from the table, the R value of rosuvastatin is greater than 0.999, which satisfies the requirements of quantitative analysis.
  • Percentage of drug exposure weight of drug / weight of tissue.
  • the density of CD44 on the surface of vulnerable plaque endothelial cells and the affinity with HA are studied to select CD44 in the vulnerable plaque as the targeted vulnerable plaque according to the present invention.
  • the target of the block's siliceous delivery system provides an experimental basis.
  • a mouse atherosclerotic vulnerable plaque model was constructed according to the method described in Example 4 above.
  • the endothelial cells of the normal arterial vascular endothelial cells and arterial vulnerable plaques of the model mice were taken for CD44 determination by immunohistochemical staining and image analysis.
  • the specific experimental methods are as follows:
  • the mouse carotid atherosclerotic vulnerable plaque specimens were fixed with 10 mL/L formaldehyde solution, paraffin-embedded, 4 ⁇ m sections, conventional dewaxing, hydration treatment, and avidin-biotin-enzyme complex method. (SABC) detects CD44 content.
  • the specimen was immersed in a 30 mL/L H 2 O 2 aqueous solution to block the activity of endogenous peroxidase, and placed in a citrate buffer for antigen microwave repair. Then 50 g/L bovine serum albumin (BSA) blocking solution was added dropwise and allowed to stand at room temperature for 20 min.
  • BSA bovine serum albumin
  • a murine anti-CD44 polyclonal antibody (1:100) was added dropwise, placed in a refrigerator at 4 ° C overnight, and incubated at 37 ° C for 1 h. After washing, biotinylated goat anti-mouse IgG was added dropwise and reacted at 37 ° C for 30 min. Then, it was washed with phosphate buffered saline (PBS), and horseradish peroxidase-labeled SABC complex was added dropwise, and incubated at 37 ° C for 20 min; each step was washed with PBS.
  • PBS phosphate buffered saline
  • Figure 9 shows the results of surface CD44 content determination (in semi-quantitative integration) of endothelial cells at normal arterial wall endothelial cells and arterial vulnerable plaques of model mice. As shown, the surface CD44 content of endothelial cells at arterial vulnerable plaques was approximately 2.3 times that of normal arterial endothelial cells.
  • hyaluronic acid labeled with aminofluorescein represented by "FL-HA" was added at a concentration of 10 mg/ml, and used in Dürr.
  • DMEM Borco-modified Eagle's medium
  • the binding force integral of FL-HA on the surface of endothelial cells at arterial vulnerable plaques was approximately 24 times that of FL-HA on the surface of endothelial cells of normal arterial walls. This indicates that most of the CD44 on the surface of endothelial cells of normal arterial wall is in a quiescent state that cannot bind to ligand HA, and CD44 on the surface of endothelial cells at the vulnerable plaque of arteries is activated by factors such as inflammatory factors in the internal environment. The affinity with HA has increased significantly.
  • Natural ligands for CD44 include: HA, GAG, collagen, laminin, fibronectin, selectin, osteopontin (OPN), and monoclonal antibodies HI44a, HI313, A3D8, H90, IM7, and the like.
  • the normal arterial wall endothelial cells of the model mice and the endothelial cells at the vulnerable plaques of the arteries were added with a ligand/antibody labeled with aminofluorescein at a concentration of 10 mg/ml, and the improved Iggar medium was used in Durbreco. (DMEM) medium (containing 10% by volume of calf serum, 100 U/ml penicillin, 100 U/ml streptomycin) was cultured in a 37 ° C, 5% CO 2 incubator. After 30 minutes, the mean fluorescence intensity (MFI) was determined by flow cytometry (CytoFLEX, Beckman Coulter, USA), and the binding integral of FL-ligand/antibody on both cell surfaces was calculated (to normal arterial vessels). The binding of CD44 to the ligand/antibody of the wall endothelial cells is 1). The result is shown in FIG.
  • the binding force of CD44 on the surface of endothelial cells at the arterial vulnerable plaque was 24 times greater than that on the surface of endothelial cells on the normal arterial wall. This indicates that most of the CD44 on the surface of endothelial cells of normal arterial wall is in a quiescent state that cannot bind to ligand HA, and CD44 on the surface of endothelial cells at the vulnerable plaque of arteries is activated by factors such as inflammatory factors in the internal environment. The affinity with HA has increased significantly.
  • CD44 other ligands similar to HA, the binding capacity of CD44 and GAG on the surface of vulnerable plaque endothelial cells is 22 times that of normal cells, and the binding capacity of CD44 and collagen in vulnerable plaque endothelial cells is 21 times that of normal cells.
  • the binding capacity of CD44 and laminin in vulnerable plaque endothelial cells is 16 times that of normal cells.
  • the binding force of CD44 and fibronectin in vulnerable plaque endothelial cells is 18 times that of normal cells, and vulnerable spots are vulnerable.
  • the binding capacity of CD44 and selectin in block endothelial cells was 19 times that of normal cells, and the binding capacity of CD44 and osteopontin in vulnerable plaque endothelial cells was 17 times that of normal cells.
  • CD44 monoclonal antibodies Similar results were observed for CD44 monoclonal antibodies: the binding of CD44 to H144a on the surface of vulnerable plaque endothelial cells was 15 times that of normal cells, and the binding capacity of CD44 and H1313 in vulnerable plaque endothelial cells was 21 times that of normal cells. The binding capacity of CD44 and A3D8 in vulnerable plaque endothelial cells is 17 times that of normal cells. The binding capacity of CD44 and H90 in vulnerable plaque endothelial cells is 9 times that of normal cells, and vulnerable plaque endothelial cells CD44 and IM7. The binding force integral is 8 times that of normal cells.
  • the macrophages in the peritoneal cavity of the model mice and the macrophages in the vulnerable plaques of the arteries were added to the hyaluronic acid labeled with aminofluorescein at a concentration of 10 mg/ml (represented by "FL-HA").
  • DMEM medium containing 10% by volume of calf serum, 100 U/ml penicillin, 100 U/ml streptomycin was cultured in a 37 ° C, 5% CO 2 incubator.
  • the binding force of FL-HA on the surface of macrophages in arterial vulnerable plaques was about 40 times that of FL-HA on the surface of macrophage outside the plaque. This indicates that CD44 on the surface of macrophages in arterial vulnerable plaques is also activated by factors such as inflammatory factors in the internal environment, and the affinity with HA is significantly increased.
  • the macrophages in the peritoneal cavity of the model mice and the macrophages in the vulnerable plaques of the arteries were added to the ligand/antibody labeled with aminofluorescein at a concentration of 10 mg/ml, and the volume fraction was 10% calf serum, 100 U/ml penicillin, 100 U/ml streptomycin) were cultured in a 37 ° C, 5% CO 2 incubator. After 30 minutes, the mean fluorescence intensity (MFI) was determined using a flow cytometer (CytoFLEX, Beckman Coulter, USA) and the binding integral of FL-HA on both cell surfaces was calculated (with extra-plaque macrophages) The CD44 on the cell surface has a ligand/antibody affinity of 1). The result is shown in FIG.
  • the binding force of CD44-HA on the surface of macrophages in arterial vulnerable plaques was about 40 times that of CD44-HA on the surface of extraplaque macrophages. This indicates that CD44 on the surface of macrophages in arterial vulnerable plaques is also activated by factors such as inflammatory factors in the internal environment, and the affinity with HA is significantly increased.
  • CD44 other ligands similar to HA, the binding capacity of CD44 and GAG on the surface of vulnerable plaque macrophages is 33 times that of normal cells, and the binding integral of CD44 and collagen in vulnerable plaque macrophages is normal cells. 38 times, the binding capacity of CD44 and laminin in vulnerable plaque macrophages is 37 times that of normal cells, and the binding force of CD44 and fibronectin in vulnerable plaque macrophages is 35 times that of normal cells.
  • the binding capacity of vulnerable plaque macrophage CD44 to selectin is 33 times that of normal cells, and the binding capacity of vulnerable plaque macrophage CD44 to osteopontin is 33 times that of normal cells.
  • CD44 monoclonal antibodies Similar results were observed for CD44 monoclonal antibodies: the binding of CD44 to H144a on the surface of vulnerable plaque macrophages was 17 times that of normal cells, and the binding integral of vulnerable plaque macrophages CD44 and H1313 was normal cells. 20 times, the binding strength of CD44 and A3D8 in vulnerable plaque macrophages is 16 times that of normal cells. The binding force of CD44 and H90 in vulnerable plaque macrophages is 9 times that of normal cells, and vulnerable plaques are giant. The binding capacity of phagocytic CD44 to IM7 is 10 times that of normal cells.
  • Example 6 In vivo experiment of the effect of the siliceous delivery system of the present invention on vulnerable plaque of arteries
  • the purpose of this example was to verify the in vivo therapeutic effect of the loaded therapeutic agent-loaded siliceous delivery system of the present invention on arterial vulnerable plaque.
  • hyaluronic acid nanomicelle system (PDLLA/Chol-HA@R): 1 g of cholesterol was dissolved in 30 mL of acetone, and 1 g of succinic anhydride was added. The reaction solution was stirred at 70 ° C for 3 h. The solvent was distilled off under reduced pressure, and the crude product was dissolved in water/anhydroethanol (1:10) to give crystals. 500 mg of cholesterol-succinate was weighed and dissolved in 20 mL of anhydrous chloroform, and 6 mL of chloroform containing 1 mL of thionyl chloride was added dropwise.
  • mice SPF-grade ApoE-/- mice (30, 5-6 weeks old, body weight 20 ⁇ 1 g) were taken as experimental animals. The mice were given an adaptive high-fat diet (fat 10% (w/w), cholesterol 2% (w/w), sodium cholate 0.5% (w/w), and the rest were normal feed for mice) after 4 weeks of feeding. Anesthesia was intraperitoneally injected with 1% sodium pentobarbital (prepared by adding 1 mg of sodium pentobarbital to 100 ml of physiological saline) at a dose of 40 mg/kg.
  • anesthesia was intraperitoneally injected with 1% sodium pentobarbital (prepared by adding 1 mg of sodium pentobarbital to 100 ml of physiological saline) at a dose of 40 mg/kg.
  • the mouse was fixed on the surgical plate in the supine position, disinfected with the neck centered with 75% (v/v) alcohol, the neck skin was cut longitudinally, and the anterior cervical gland was bluntly separated, on the left side of the trachea.
  • the left common carotid artery can be seen on the side. Carefully separate the common carotid artery to the bifurcation.
  • the silicone tube sleeve with a length of 2.5 mm and an inner diameter of 0.3 mm was placed on the outer circumference of the left common carotid artery.
  • the proximal and distal centripet segments of the cannula were narrowed and fixed by filaments. .
  • LPS lipopolysaccharide
  • mice were placed in a 50 ml syringe (sufficient venting reserved) to cause restrictive mental stress, 6 hours/day, 5 days per week for a total of 6 weeks.
  • the mouse atherosclerotic vulnerable plaque model was completed at 14 weeks postoperatively.
  • the experimental animals were randomly divided into the following groups, 6 in each group:
  • Vulnerable plaque model control group this group of animals did not undergo any therapeutic treatment
  • Rosuvastatin intragastric administration intragastric administration at a dose of 5 mg rosuvastatin / kg body weight;
  • Rosuvastatin intravenous group intravenous administration of 5 mg rosuvastatin / kg body weight;
  • HA-PL@R group intravenous administration at a dose of 5 mg rosuvastatin / kg body weight;
  • PDLLA/Chol-HA@R group intravenous administration at a dose of 5 mg rosuvastatin/kg body weight;
  • HA-CL1@R group intravenous administration at a dose of 5 mg rosuvastatin / kg body weight;
  • HA-CL1@R+LMHA group intravenous administration of 5 mg rosuvastatin and 1.25 mg of small molecule hyaluronic acid/kg body weight of 3411 Da;
  • HA-CL1@R+S group intravenous administration was carried out at a dose of 5 mg rosuvastatin and 1.75 mg CD44 antibody mAb/kg body weight.
  • the treatment group was treated once every other day for a total of 5 treatments.
  • carotid MRI scans were performed before and after treatment to detect plaque and lumen area, and the percentage of plaque progression was calculated.
  • Percentage of plaque progression (plaque area after treatment - plaque area before treatment) / lumen area.
  • Figure 14 is a graph showing the in vivo therapeutic effect of the therapeutic agent-loaded siliceous delivery system of the present invention on arterial vulnerable plaque.
  • free rosuvastatin has a certain therapeutic effect, whether it is administered by intragastric administration or intravenous administration, but it cannot prevent the damage.
  • the plaque continues to grow.
  • free rosuvastatin when rosuvastatin is formulated in a liposome delivery system or a hyaluronic acid nanomicelle delivery system, it has a certain degree of therapeutic effect on vulnerable plaque. The improvement, but still can not prevent the continued growth of vulnerable plaque.
  • rosuvastatin when rosuvastatin was formulated in the siliceous delivery system of the present invention, its therapeutic effect on vulnerable plaques was significantly improved and reversed plaque growth (ie, reduced plaques) The therapeutic effect of the block).
  • the inventors have unexpectedly discovered that when rosuvastatin is formulated in combination with small molecule hyaluronic acid or CD44 activator-CD44 antibody mAb in a siliceous delivery system, arterial vulnerability is observed in mice. Plaques produce a very significant therapeutic effect.
  • the administration of the therapeutic agent using the siliceous delivery system of the present invention significantly reverses the growth of arterial vulnerable plaque and has a better therapeutic effect than the free drug and liposome delivery system.
  • Example 7 In vivo tracer experiment of arterial vulnerable plaque by the siliceous delivery system of the present invention
  • the purpose of this example was to verify the in vivo tracer effect of the load tracer-based siliceous delivery system of the present invention on arterial vulnerable plaque.
  • Example 2 (1) Using a commercially available nanogold solution, the method described in Example 2 above was used to prepare a silicon trace delivery system HA-CL1@AuNPs, a load tracer and a CD44 activator loaded with a CT tracer nanogold.
  • a mouse atherosclerotic vulnerable plaque model was constructed according to the method described in Example 4 above.
  • Model mice were fed a high-fat diet (same as in Example 4) for 16 weeks. Twenty-four model mice were randomly divided into free nanogold particles (6, given a commercially available nanogold solution, nanogold was dosed at 0.1 mg/kg body weight), and CL1@AuNPS group (6, given CL1).
  • nanogold was administered at a dose of 0.1 mg/kg body weight
  • HA-CL1@AuNPs group (6, given HA-CL1@AuNPs, nanogold was administered at a dose of 0.1 mg/kg body weight) and HA- In the CL1@AuNPs+S group (6, given HA-CL1@AuNPs+S, the dose of nanogold was 0.1 mg/kg body weight, and the dose of CD44 activator-CD44 antibody mAb was about 0.07 mg/kg. body weight).
  • Each experimental group was injected with the corresponding tracer through the tail vein, and CT imaging was performed before administration and 4 hours after administration to observe the identification of atherosclerotic vulnerable plaque in each group.
  • Figure 15 illustrates the in vivo tracer effect of the load tracer-based silicon delivery system of the present invention on arterial vulnerable plaque.
  • the free nanogold particles showed a certain trace effect on arterial vulnerable plaques in mice.
  • its traceability to vulnerable plaques is somewhat improved.
  • nanogold is formulated in the siliceous delivery system of the present invention having a surface-modified ligand hyaluronic acid, its traceability to vulnerable plaques is significantly improved.
  • the inventors have unexpectedly discovered that when nanogold is formulated in combination with a CD44 activator-CD44 antibody mAb in a siliceous delivery system, a very significant indication is given to arterial vulnerable plaque in mice. Trace effect.
  • the administration of the siliceous delivery system with the surface-modified ligand hyaluronic acid described in the present invention can be significantly improved compared to the free nanogold particles and the non-targeted siliceous delivery system.
  • the recognition of nano-gold on arterial vulnerable plaques produces better tracer effects.
  • Example 8 In vivo tracer experiment of arterial vulnerable plaques by the siliceous-HA-ioxolol delivery system of the present invention (CT tracer)
  • the purpose of this example was to verify the in vivo tracer effect of the CT tracer-loaded silicon plastid delivery system of the present invention on arterial vulnerable plaque.
  • a silicon trace delivery system HA-CL1@I, a load tracer and a CD44 activator loaded with a CT tracer were prepared by the method described in Example 1 above.
  • the siliceous delivery system HA-CL1@I+S and the non-targeted load tracer-based siliceous delivery system CL1@I (as a comparative example).
  • a mouse atherosclerotic vulnerable plaque model was constructed according to the method described in Example 6 above.
  • Figure 16 illustrates the in vivo tracer effect of the load tracer-based silicon delivery system of the present invention on arterial vulnerable plaque.
  • intravenous iodixanol did not exhibit any tracer effect on arterial vulnerable plaque in mice.
  • intravenous iodixanol when it is loaded into a non-targeted siliceous delivery system, its traceability to vulnerable plaques is somewhat improved.
  • iodixanol is formulated in a siliceous delivery system of the present invention having a surface-modified ligand hyaluronic acid, its tracer effect on vulnerable plaques is significantly improved.
  • the inventors have unexpectedly discovered that when iodixanol is formulated in combination with a CD44 activator-LPS in a siliceous delivery system, a very significant indication is given to arterial vulnerable plaque in mice. Trace effect.
  • the administration of the siliceous delivery system with the surface-modified ligand hyaluronic acid described in the present invention can be significantly improved compared to the free iodixanol and the non-targeted siliceous delivery system.
  • the recognition of arterial vulnerable plaque by iodixanol produces a better tracer effect.
  • Example 9 In vivo tracer experiment (MRI tracing) of arterial vulnerable plaques by the siliceous-HA-demetrate glucamine delivery system of the present invention
  • the purpose of this example was to verify the in vivo tracer effect of the MRI tracer-loaded siliceous delivery system of the present invention on arterial vulnerable plaque.
  • a MRI tracer-loaded siliceous delivery system and a blank siliceous delivery system were prepared by the method described in the above Example 1 using a commercially available bulk drug, glucuronide.
  • a mouse atherosclerotic vulnerable plaque model was constructed according to the method described in Example 6 above.
  • Figure 17 illustrates the in vivo tracer effect of the load tracer-based silicon delivery system of the present invention on arterial vulnerable plaque.
  • the blank siliceous body did not exhibit any tracer effect on arterial vulnerable plaque in mice.
  • the gadolinium glutamate was loaded in a targeted siliceous delivery system, its traceability to vulnerable plaques was significantly improved in a dose-dependent manner. improve.
  • the administration of the siliceous-HA-demetrate glucamine delivery system of the present invention can exhibit the recognition of arterial vulnerable plaques compared to a blank siliceous delivery system. Good MRI tracer effect.
  • Example 10 In vivo tracer experiment (MRI tracing) of arterial vulnerable plaques by CD44 multiple monoclonal antibody-siliceous-gluconate delivery system of the present invention
  • the purpose of this example is to verify the in vivo tracer effect of the nano-delivery system composed of a plurality of different CD44 monoclonal antibodies on the vulnerable plaque of arteries on the surface of the silicon body loaded with the MRI tracer of the present invention.
  • a mouse atherosclerotic vulnerable plaque model was constructed according to the method described in Example 6 above.
  • Figure 18 is a graph showing the in vivo tracer effect of the nano-delivery system of the various CD44s of the present invention as probes on arterial vulnerable plaques.
  • a siliceous nanosystem using a CD44 multi-monoclonal antibody of the present invention as a targeting probe (including gadolinium-silicoid-H144a) is used as compared with a blank siliceous group.
  • Nanosystems, gadolinium-silica-A3D8 nanosystems, glucuronide-siliceous-H90 nanosystems can all show the recognition of arterial vulnerable plaques, resulting in better MRI Trace effect.
  • Example 11 In vivo tracer experiment (MRI tracing) of arterial vulnerable plaques of CD44 multiple ligand-siliceous-guanidine bisamine delivery system of the present invention
  • the purpose of this example was to verify the in vivo tracer effect of the nano-delivery system composed of a plurality of different CD44 ligands on the surface of the silicon body loaded with the MRI tracer of the present invention on arterial vulnerable plaque.
  • a silicon-based nano-delivery system loaded with an MRI tracer using a commercially available bulk drug, bisamine, using the method described in Example 1 above to prepare a different CD44 ligand as a targeting probe.
  • a mouse atherosclerotic vulnerable plaque model was constructed according to the method described in Example 6 above.
  • Model mice were fed a high-fat diet (same as in Example 5) for 16 weeks. 42 model mice were randomly divided into blank siliceous group (6), HA-silic body group (6), and collagen- ⁇ bis-silicon group (6, given commercially available sputum double).
  • Amine bulk drug, bismuth bisamine is administered at a concentration of 0.5 mg/ml, administered in an amount of 10 ml/kg), laminin-oxime bis-silicon group (6, given commercially available guanidine diamine)
  • the drug substance, the concentration of bismuth diamine is 0.5 mg/ml, the dosage is 10 ml/kg, and the fibronectin-oxime bis-silicon group (6, which is commercially available as bismuth bisamine)
  • the drug, bismuth diamine is administered at a concentration of 0.5 mg/ml, and the dose is 10 ml/kg), and the protein-guanidine bisamine-silica group (6, which is commercially available as a bismuth diamine drug substance, is selected.
  • the bisamine was administered at a concentration of 0.5 mg/ml, and the dose was 10 ml/kg.
  • the osteopontin-oxime bis-silicon group (6, given a commercially available bismuth diamine bulk drug, bismuth bisamine) The administration concentration was 0.5 mg/ml, and the administration amount was 10 ml/kg).
  • Each experimental group was injected with the corresponding tracer through the tail vein, and MRI imaging was performed before and after the administration, and the identification of atherosclerotic vulnerable plaques in each group was observed.
  • Figure 19 is a graph showing the in vivo tracer effect of the nano-delivery system of the CD44 of the present invention as a probe on arterial vulnerable plaque.
  • the various CD44 ligands including HA, collagen, laminin, fibronectin, selectin, osteopontin
  • the siliceous nanosystems to the probes can all show the recognition of vulnerable plaques of arteries, resulting in better MRI tracer effects.
  • Example 12 Tissue distribution of the HA-multiple particle size siliceous-iodoxacin delivery system of the present invention
  • the purpose of this example was to verify the tissue distribution of a different size particle size silicon nano-delivery system of the loaded CT tracer of the present invention.
  • a mouse atherosclerotic vulnerable plaque model was constructed according to the method described in Example 6 above.
  • Model mice were fed a high-fat diet (same as in Example 5) for 16 weeks. Thirty-six model mice were randomly divided into a 280 nm siliceous group (12), a 200 nm siliceous group (12), and a 160 nm siliceous group (12).
  • the carrier was commercially available.
  • the iodixanol bulk drug, iodixanol is administered at a concentration of 0.5 mg/ml, and the dose is 10 ml/kg.
  • Each experimental group is injected with a corresponding tracer through the tail vein, and before administration, Tissue collection was performed at 4 time points after administration (3 animals per time point), and the iodixanol tissue distribution of each group of animals was observed.
  • Table 3 shows the tissue distribution of different particle size silicimetric nano-delivery systems loaded with CT tracers of the present invention. As shown in the table, the 200 nm siliceous body described in the present invention can be better enriched in arterial vulnerable plaques compared to the other particle size siliceous groups.
  • Example 13 Tissue Distribution of Various Molecular Weight HA-Siliposome-Iodixanol Delivery Systems of the Invention
  • the purpose of this example was to verify the tissue distribution of the different molecular weight HA of the CT tracer of the present invention as a recognition ligand for the siliceous nano delivery system.
  • a siliceous nano-delivery system (silica particle size: 200 nm) was prepared by the following method: 1 g of sodium hyaluronate HA1, HA2, HA3 (molecular weight: 200,000 Da, respectively) , 100,000 Da, 30,000 Da) fully dissolved in ultrapure water, adding 0.1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 0.12
  • EDC.HCl 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • the g-N-hydroxythiosuccinimide (sulfo-NHS) coupling agent activates the carboxyl group.
  • activated HA was configured to be an aqueous solution of 0.1 mg mL-1, and 0.2 mL of the solution was dissolved in the suspension of the siliceous vesicle obtained in the above step to activate the activated carboxyl group and the lipid of the silicate vesicle in the activated HA.
  • the amino group of the DSPE molecule doped in the bilayer is coupled by forming an amide bond, thereby obtaining three kinds of therapeutic agent-loaded siliceous delivery systems HA1-CL1@R, HA2-CL1@R and HA3-CL1@. R.
  • a mouse atherosclerotic vulnerable plaque model was constructed according to the method described in Example 6 above.
  • Model mice were fed a high-fat diet (same as in Example 5) for 16 weeks. Thirty-six model mice were randomly divided into HA1-CL1@R group (12), HA2-CL1@R group (12), and HA3-CL1@R group (12). The carrier was loaded with commercially available iodide. The saffin raw material drug, iodixanol was administered at a concentration of 0.5 mg/ml, and the dose was 10 ml/kg. Each experimental group was injected with a corresponding tracer through the tail vein, and before and after administration. Tissue collection was performed at 4 time points (3 animals per time point), and the distribution of iodixanol tissue in each group of animals was observed.
  • Table 4 shows the tissue distribution of different molecular weight HA-siliceous nano-delivery systems loaded with CT tracers of the present invention. As shown in the table, the 100,000 DaHA described in the present invention can be better enriched in arterial vulnerable plaques compared to other molecular weight HA.

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Abstract

一种用于靶向活化的CD44分子的硅质体递送系统、制备方法及其用途，所述硅质体的表面部分地被靶向配体修饰，所述靶向配体是能与活化的CD44分子特异性结合的配体。该硅质体递送系统可用于易损斑块或与易损斑块相关的疾病的诊断、预防和治疗。

Description

一种用于靶向活化CD44分子的硅质体递送系统、其制备方法和用途 技术领域
本发明属于靶向给药技术领域，具体涉及一种用于靶向活化CD44分子，尤其是靶向易损斑块的硅质体递送系统。本发明也涉及所述硅质体递送系统的制备方法和用途，特别是在易损斑块或与易损斑块相关的疾病的诊断、预防和治疗中的用途。
背景技术
目前，以急性心肌梗死和心源性猝死为主的急性心血管事件已成为危害人类健康的头号杀手。据统计，全世界每年约有2千万人死于急性心血管事件。在中国，情况同样不容乐观，每年有超过70万人死于急性心肌梗死和心源性猝死，这已经成为严重威胁我国居民健康的最重要疾病之一。研究表明，大部分的急性心肌梗死和心源性猝死均是由动脉粥样硬化斑块引起的。自上世纪70年代以来，人们一直在探索慢性动脉粥样硬化斑块导致急性冠脉综合征(ACS)及脑卒中的发生过程及机制。
1989年，Muller及其团队(Circadian variation and triggers of onset of acute cardiovascular disease.Circulation.1989；79(4)：733-43)提出了“易损斑块”的概念，认为此类斑块是导致大多数急性心脑血管事件的根本原因。易损斑块(vulnerable plaque，又称为“不稳定斑块(unstable plaque)”)是指具有血栓形成倾向或极有可能快速进展成为“罪犯斑块”的动脉粥样硬化斑块，主要包括破裂斑块、侵蚀性斑块和部分钙化结节性病变。大量的研究表明，大部分的急性心肌梗死及脑卒中是由于轻、中度狭窄的易损斑块破裂，继发血栓形成所致。Naghavi及其团队(New developments in the detection of vulnerable plaque.Curr Atheroscler Rep.2001；3(2)：125-35)等给出了易损斑块的组织学定义和标准。主要的标准包括活动性炎症、薄的纤维帽和大的脂质核心、内皮剥脱伴表面血小板聚集、斑块有裂隙或损伤以及严重的狭窄。次要的标准包括表面钙化斑、黄色有光泽的斑块、斑块内出血和正性重构。因此，对于易损斑块而言，早期干预至关重要。但是由于一般情况下易损斑块所导致的血管狭窄程度并不高，很多患者没有前驱症状，导致临床上很难进行早期诊断，使得其危险性极高。因此，如何尽早准确的识别和诊断易损斑块，进行有效的干预成为预防及治疗急性心肌梗死中亟待解决的问题。
目前常用于易损斑块诊断的技术主要包括冠脉造影、血管内超声(IVUS)、激光相干断层显像(OCT)等技术，但这些技术均属于有创性的检查，并且诊断分辨率和准确性不高，同时这些诊断技术费用昂贵，也在一定程度上限制了临床上的普及。因此，目前急需针对易损斑块的无创诊断技术和制剂。
另外，目前治疗易损斑块的方法主要是全身给药，例如口服他汀类药物(羟甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂)、阿司匹林、基质金属蛋白酶(MMPs)抑制剂和/或贝特类药物等。这些药物通过调节全身血脂、对抗炎症、抑制蛋白酶和血小板生成等来减少斑块内的脂质，改善血管重构等，从而起到稳定斑块的作用。然而，临床应用中发现 目前用于治疗易损斑块的药物的治疗效果并不理想。例如，临床常用的他汀类药物的口服给药生物利用度比较低，如辛伐他汀为＜5％，阿托伐他汀为约12％，瑞舒伐他汀为约20％。动物实验也证实，当他汀类药物的剂量增加到1mg/kg以上时才可以起到增加纤维帽厚度和减少斑块体积的作用，这就使得他汀类药物的口服给药的稳定性及逆转斑块的效果遭遇了瓶颈。目前临床试验也已经证实，口服他汀类药物治疗易损斑块需要采用强化大剂量才能具有稳定易损斑块的作用，而全身大剂量使用他汀类药物治疗也存在严重副作用(例如肝功能异常、横纹肌溶解、II型糖尿病等)发生率升高的风险。
对于现有的全身性给药而言，药物在进入体内后通常仅有极少一部分有效成分能够真正作用于病变部位。这是制约药物疗效，并导致药物毒副作用的根本原因。靶向给药系统是指具有靶向给药能力的给药系统。在经某种途径给药以后，靶向给药系统所包含的药物会通过带有靶向探针的载体特异性地富集于靶部位。靶向给药系统能够使药物瞄准特定的病变部位，并在目标病变部位释放有效成分。因此，靶向给药系统可以使药物在目标病变部位形成相对较高的浓度，并减少血液循环中的药量，从而在提高药效的同时抑制毒副作用，减少对正常组织和细胞的伤害。
目前，靶向给药系统通常所使用的纳米载体是脂质体。虽然脂质体具有提高药效、降低药物毒副作用的优势，但是由于其体内稳定性差，导致循环时间不足，最终对药物的生物利用度提升有限。另外，脂质体的体外稳定性同样不足，存储期间磷脂易氧化水解，而且脂质体囊泡之间容易相互聚集融合、包裹在其中的药物容易发生渗漏的问题。这均在一定程度上限制了靶向给药系统的发展。
另外，在易损斑块的诊断和治疗领域中，也存在一些利用靶向配体修饰纳米载体来诊断易损斑块的技术。然而，此类靶向易损斑块的靶向探针在临床实际应用中的主要问题在于这些制剂的靶向位点的特异性不足。例如，此类制剂的靶向位点大多选择巨噬细胞，但由于巨噬细胞可存在于身体各处，所以所述探针的靶向特异性不够理想。因此，靶向易损斑块的靶向制剂的研制中存在的难点在于发现易损斑块内的细胞中的具有显著靶向特异性的靶位。
CD44是一类黏附分子，广泛分布于淋巴细胞、单核细胞、内皮细胞等的表面。CD44分子的主要配体是透明质酸(hyaluronic acid，缩写为“HA”)。基于表达细胞的活化状态，可以将CD44分为相对静止状态(不能结合HA)、诱导活化状态(激活后可结合HA)和结构活跃状态(无需激活即可与HA结合)，而大多数正常细胞表面的CD44处于相对静止状态，从而不能与HA相结合。
继往大量研究表明CD44并不是具有显著靶向特异性的理想靶位。这是因为CD44在人体内广泛分布，尤其是大量存在于网状内皮丰富的器官表面上。因此，以CD44为靶位的靶向给药系统的研发中会遇到如下问题：如果靶位细胞表面的CD44与HA的亲和力不足以提供显著的特异性，那么此类靶向给药系统就不会存在特异性靶向性能。
因此，寻找易损斑块部位存在的特异性靶位以及适合于靶向易损斑块的靶向给药系统，由此开发能够特异性地靶向易损斑块，并同时能够实现药物的稳定持续释放的靶向 给药系统，已经成为医学领域中的一个亟待解决的技术问题。
迄今为止，对于易损斑块内主要存在的巨噬细胞、单核细胞、内皮细胞、淋巴细胞和平滑肌细胞的表面上的CD44的表达状态及其与HA的亲和力尚无任何报道，也不存在任何关于利用HA和CD44的相互作用以及易损斑块的特定微环境设计用于诊断或治疗易损斑块或与易损斑块相关的疾病的能够实现药物的稳定持续释放的靶向给药系统的现有技术。
发明内容
(1)发明概述
发明人发现，与正常细胞相比，易损斑块中的细胞诸如内皮细胞、巨噬细胞和平滑肌细胞等的表面的CD44会被易损斑块的微环境(诸如在炎症因子的影响下)所诱导活化，导致与HA的结合能力会骤然增强数十倍。这一发现提示，易损斑块处的细胞表面存在的大量活化的CD44分子为以HA为靶向配体的靶向给药系统提供了理想的靶位。为此，本发明提供了一种能够特异性地靶向活化的CD44分子，尤其是靶向易损斑块的靶向给药系统。
发明人还发现，在易损斑块处存在大量的脂质如胆固醇等，这些脂质能够起到乳化剂的作用，会严重影响常规的靶向给药系统脂质体的稳定性，从而导致脂质体外层结构被快速地破坏或侵蚀(萃取)而崩解，这使得脂质体内包封的药物提前释放，无法起到药物持续释放的作用。然而，如果采用新型的药物递送系统纳米载体来代替脂质体，则会显著地改善包封药物在易损斑块处的释放特性，不会受到脂质侵蚀的影响，在易损斑块处的微环境中可以维持较好的稳定性，从而能够实现药物的持续释放。为此，本发明还提供一种能够特异性地靶向易损斑块，并同时能够实现药物的稳定持续释放的靶向给药系统。
发明人还发现，负载CD44活化剂可以促使病灶细胞表面的CD44进一步活化，可以在短时间内放大CD44对HA的靶向亲和力，显著增加结合在细胞表面的靶向硅质体组合物浓度，这对于易损斑块的示踪诊断和治疗具有积极意义。为此，本发明所述的靶向给药系统可以负载CD44活化剂，其可以在短时间内显著增加示踪剂或治疗剂化合物的浓度以提高诊断分辨率或治疗效果。
发明人还发现，在易损斑块内，伴随CD44的高度活化与过表达，同时内源性大分子HA也受激大量生成，并与细胞表面CD44结合，促进巨噬细胞和淋巴细胞等在易损斑块内的聚集。此种在细胞表面结合CD44的内源性HA会形成药物进入的屏障，会降低药物的生物利用度。为此，本发明所述的靶向给药系统可以负载小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物，其通过与细胞表面的内源性HA的竞争性结合，解除内源性HA在细胞表面形成的屏障，有利于药物顺利进入病灶细胞内，显著提供治疗效果。
总而言之，本发明涉及如下方面：
本发明提供了一种用于靶向活化的CD44分子的硅质体递送系统。
本发明提供了一种用于靶向易损斑块的硅质体递送系统。
本发明还提供了一种用于制备本发明所述的靶向易损斑块的硅质体递送系统的方法。
本发明还提供了一种药物，其包含本发明所述的靶向易损斑块的硅质体递送系统和药学上可接受的载体。
本发明还提供了一种诊断制剂，其包含本发明所述的靶向易损斑块的硅质体递送系统。
本发明还提供了本发明所述的靶向易损斑块的硅质体递送系统在制备用于预防和/或治疗易损斑块或与易损斑块相关的疾病的药物中的用途。
本发明还提供了本发明所述的靶向易损斑块的硅质体递送系统在制备用于诊断易损斑块或与易损斑块相关的疾病的诊断制剂中的用途。
本发明还提供了一种用于预防和/或治疗易损斑块或与易损斑块相关的疾病的方法，所述方法包括向需要其的对象施用本发明所述的靶向易损斑块的硅质体递送系统。
本发明还提供了一种用于诊断易损斑块或与易损斑块相关的疾病的方法，所述方法包括向需要其的对象施用本发明所述的靶向易损斑块的硅质体递送系统。
本发明技术方案的具体实施方式及其含义将在下文中进行详细说明。
(2)技术术语及其含义
本文中所提及的术语具有以下含义：
“易损斑块”又称“不稳定斑块”，是指具有血栓形成倾向或极有可能快速进展成为“罪犯斑块”的动脉粥样硬化斑块，主要包括破裂斑块、侵蚀性斑块和部分钙化结节性病变。大量的研究表明，大部分的急性心肌梗死及脑卒中是由于轻、中度狭窄的易损斑块破裂，继发血栓形成所致。易损斑块的组织学表现包括活动性炎症、薄的纤维帽和大的脂质核心、内皮剥脱伴表面血小板聚集、斑块有裂隙或损伤以及严重的狭窄，以及表面钙化斑、黄色有光泽的斑块、斑块内出血和正性重构。
“与易损斑块相关的疾病”主要是指疾病的发生和发展过程中与“易损斑块”相关、以为“易损斑块”特征、由“易损斑块”引起或继发于“易损斑块”的疾病。“与易损斑块相关的疾病”主要包括动脉粥样硬化症、冠状动脉粥样硬化性心脏病(包括急性冠脉综合征、无症状心肌缺血-隐匿性冠心病、心绞痛、心肌梗死、缺血性心脏病、猝死、支架内再狭窄)、脑动脉粥样硬化症(包括脑卒中)、外周血管动脉粥样硬化症(包括颈动脉粥样硬化症、肾动脉粥样硬化症、下肢动脉粥样硬化症、上肢动脉粥样硬化症)、主动脉夹层、血管瘤、血栓栓塞、心力衰竭和心源性休克等疾病。
“靶向给药系统”是指具有靶向给药能力的给药系统。在经某种途径给药以后，靶向给药系统所包含的药物会通过特殊载体或靶向弹头(例如，靶向配体)的作用特异性地富 集于靶部位。目前已知的用于实现靶向给药的手段包括利用各种微粒给药系统的被动靶向性能、在微粒给药系统的表面进行化学修饰、利用一些特殊的理化性能、利用抗体介导靶向给药、利用配体介导靶向给药、利用前体药物靶向给药等。其中，利用配体介导靶向给药是利用某些器官和组织上的特定的受体可与其特异性的配体发生专一性结合的特点，将药物载体与配体结合，从而将药物导向特定的靶组织。
“硅质体(cerasome)”是20世纪九十年代末期开发出来的一种新型的形态学上稳定的脂质载体。本发明所述的硅质体是一种活性物质的递送系统，从形态上来说，它是由脂质双分子层形成的，内部具有亲水性空腔的闭合囊泡。所述脂质双分子层是由包括硅质体单体分子在内的脂质组分形成的。所述硅质体单体分子是能够形成硅质体的无机-有机复合脂质分子，其由具有硅氧烷结构的头部和疏水性的尾部构成，其中所述疏水性的尾部是有机疏水双分子链。所述硅质体单体分子一般是三烷氧基甲硅烷基化的脂质，后者经由原位发生的溶胶(sol-gel)过程形成硅氧烷键(Si-O-Si)，从而在硅质体的表面形成自身具有刚性的无机聚硅氧烷网状结构。本文中所用的用于靶向易损斑块的硅质体递送系统就是基于“易损斑块处的细胞表面存在的大量活化的CD44分子与HA之间的专一性结合”这一发现而设计的配体介导的靶向给药系统。
“透明质酸(hyaluronic acid，缩写为“HA”)”是一种高分子的聚合物，分子式：(C 14H 21NO 11)n。它是由单位D-葡萄糖醛酸及N-乙酰葡糖胺组成的高级多糖。D-葡萄糖醛酸及N-乙酰葡糖胺之间由β-1，3-配糖键相连，双糖单位之间由β-1，4-配糖键相连。透明质酸以其独特的分子结构和理化性质在机体内显示出多种重要的生理功能，如润滑关节，调节血管壁的通透性，调节蛋白质，水电解质扩散及运转，促进创伤愈合等。尤为重要的是，透明质酸具有特殊的保水作用，是目前发现的自然界中保湿性最好的物质。
“透明质酸的衍生物”在本文中是指任何能够保留透明质酸与易损斑块处的细胞表面上的CD44分子的特异性结合能力的透明质酸的衍生物，包括但不限于透明质酸的药学上可接受的盐、低级烷基(含有1-6个碳原子的烷基)酯、在体内能够经水解或其它方式形成透明质酸的前体药物等。判断某种物质是否是“透明质酸的衍生物”可以通过测定该物质与易损斑块处的细胞表面上的CD44分子的特异性结合能力来实现，这属于本领域技术人员的技能范围之内。
“CD44分子”是一类广泛地表达于淋巴细胞、单核细胞、内皮细胞等细胞的细胞膜上的跨膜蛋白多糖黏附分子，由胞外区段、跨膜区段和胞内区段等三个区段构成。CD44分子可介导多种细胞与细胞、细胞与细胞外基质之间的相互作用，参与体内的多种信号的传导，从而改变细胞的生物学功能。CD44分子的主要配体是透明质酸，它与透明质酸之间的受体-配体结合决定了细胞在细胞外基质中的黏附和/或迁移。此外，CD44分子还参与透明质酸的代谢。
“烷基”是指具有规定数量的碳原子的支链和直链二者的饱和脂肪族烃基团。例如，“C 1-6烷基”是指具有1、2、3、4、5或6个碳原子的烷基，例如甲基、乙基、丙基、丁基、戊基、己基、异丙基、异丁基、仲丁基、叔丁基、异戊基、新戊基等。类似地，“C 10-24 烷基”是指具有10、11、12、13、14、15、16、17、18、19、20、21、22、23或24个碳原子的烷基。
“烯基”是指具有规定数量的碳原子以及一个或多个、优选一至六个、例如一个、两个、三个、四个、五个或六个碳-碳双键(其可沿链存在于任何稳定点)的直链或支链构型的不饱和脂肪族烃基团。例如，“C 10-24烯基”是指具有10、11、12、13、14、15、16、17、18、19、20、21、22、23或24个碳原子以及一个或多个、优选一至六个、例如一个、两个、三个、四个、五个或六个碳-碳双键的烯基。
“约”代表在其后面给出的数值的±5％的范围内的所有值构成的集合。
(3)发明详述
根据本发明的一个方面，本发明提供了一种用于靶向活化的CD44分子的硅质体递送系统，所述硅质体的表面部分地被靶向配体修饰，所述靶向配体是能与活化的CD44分子特异性结合的配体。
根据本发明的一个方面，本发明提供了一种用于靶向易损斑块的硅质体递送系统，所述硅质体的表面部分地被靶向配体修饰，所述靶向配体是能与易损斑块处的细胞表面上的CD44分子特异性结合的配体。
根据本发明的一个方面，本发明提供了一种用于靶向易损斑块的硅质体递送系统，其特征在于，所述硅质体递送系统包括硅质体囊泡，其中所述硅质体囊泡的表面部分地被靶向配体修饰。
在一个实施方案中，所述硅质体囊泡是由脂质双分子层形成的，内部具有亲水性空腔的闭合囊泡，其中该囊泡的表面具有无机聚硅氧烷网状结构和偶联的靶向配体。
在一个实施方案中，所述脂质双分子层是由包括硅质体单体分子、与靶向配体通过共价键偶联在一起的二硬脂酰磷脂酰乙醇胺(DSPE)分子和任选存在的其它脂质分子在内的组分形成。
在一个实施方案中，所述硅质体单体分子、与靶向配体通过共价键偶联在一起的二硬脂酰磷脂酰乙醇胺(DSPE)分子和任选存在的其它脂质分子之间的重量配比为1-10∶0.2-1∶1-9。
在一个实施方案中，所述硅质体单体分子、与靶向配体通过共价键偶联在一起的二硬脂酰磷脂酰乙醇胺(DSPE)分子和任选存在的其它脂质分子之间的重量配比为2-10∶1-3∶0-3。
在一个实施方案中，所述硅质体单体分子、与靶向配体通过共价键偶联在一起的二硬脂酰磷脂酰乙醇胺(DSPE)分子和其它脂质分子之间的重量配比为3-7∶1.5-2.5∶1.5-2.5。
在一个实施方案中，所述硅质体单体分子、与靶向配体通过共价键偶联在一起的二硬脂酰磷脂酰乙醇胺(DSPE)分子和其它脂质分子之间的重量配比为3-7∶0.5-1∶1.5-2.5。
在一个实施方案中，所述硅质体单体分子、与靶向配体通过共价键偶联在一起的二硬脂酰磷脂酰乙醇胺(DSPE)分子和其它脂质分子之间的重量配比为4-6∶0.5∶2。
在一个实施方案中，所述硅质体单体分子、与靶向配体通过共价键偶联在一起的二硬脂酰磷脂酰乙醇胺(DSPE)分子和其它脂质分子之间的重量配比为4-6∶2∶2。
在一个实施方案中，所述硅质体单体分子是能够形成硅质体的无机-有机复合脂质分子，所述无机-有机复合脂质分子由具有硅氧烷结构的头部和疏水性的尾部构成，其中所述疏水性的尾部是有机疏水双分子链。
在一个实施方案中，所述硅质体单体分子为具有如下结构通式的单体分子：
(R 1O) 3Si-L-C(O)-N(R 2)(R 3)
其中：
R 1代表C 16烷基；
L是由4-12个碳原子(优选4-10个碳原子)和1-2个氮原子构成的连接基，其中所述连接基中有0-1个碳原子被氧代基取代，即形成羰基，条件是(1)如果所述连接基中存在羰基，则所述羰基与氮原子毗邻；(2)所述连接基中的1个氮原子可以被季铵化，并且该季铵化的氮原子与适当的抗衡离子形成盐；
R 2和R 3彼此独立地代表C 10-24烷基或C 10-24烯基。
在一个实施方案中，所述硅质体单体分子选自以下化合物中的一种或多种：
Figure PCTCN2018082850-appb-000001
在一个实施方案中，所述脂质双分子层的组分包括其它脂质分子。
在一个实施方案中，所述其它脂质分子选自中性磷脂、负电荷磷脂和正电荷脂质中的一种或多种。
在一个实施方案中，所述脂质选自胆碱磷脂、甘油磷脂、乙醇胺磷脂、丝氨酸磷脂、磷脂酸中的一种或多种。
在一个实施方案中，所述其它脂质分子为胆碱磷脂。
在一个实施方案中，所述其它脂质分子为正电荷脂质。
在一个实施方案中，所述正电荷脂质选自3β-[N-(N′，N′-二甲基胺乙基)胺基甲酰基]胆固醇(DC-chol)、N-[1-(2，3-二油酰基)丙基-]-N，N，N-三乙胺氯(DOTMA)、2，3-二油酰氧 N-[2(精氨酸基酰胺)乙基]-N，N-二甲基-1-丙基-三氧乙酸胺(DOSPA)和1，2-二油酰氧丙基-N，N，N-三甲基溴化铵(DOTAP)中的一种或多种。
在一个实施方案中，所述脂质为1，2-二油酰氧丙基-N，N，N-三甲基溴化铵(DOTAP)。
在一个实施方案中，所述靶向配体具有5万～40万Da的分子量。
在一个实施方案中，所述靶向配体具有8万～15万Da的分子量。
在一个实施方案中，所述靶向配体具有约10万Da的分子量。
在一个实施方案中，所述硅质体囊泡的粒径范围为50nm～400nm。
在一个实施方案中，所述硅质体囊泡的粒径范围为50nm～300nm。
在一个实施方案中，所述硅质体囊泡的粒径范围为150nm～250nm。
在一个实施方案中，所述硅质体囊泡的粒径范围为180nm～220nm。
在本发明的硅质体递送系统中，所述递送系统中的靶向配体选自GAG、胶原、层黏连蛋白、纤黏连蛋白、选择蛋白、骨桥蛋白(OPN)以及单克隆抗体HI44a，HI313，A3D8，H90，IM7，或透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物。
在本发明的硅质体递送系统中，其负载有用于诊断、预防和/或治疗与出现CD44分子活化状况相关的疾病的物质。
在一个实施方案中，所述硅质体负载有用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质。
在一个实施方案中，所述物质是用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的药物、多肽、核酸和细胞因子中的一种或多种。
在一个实施方案中，所述物质是用于诊断易损斑块或与易损斑块相关的疾病的物质。
在一个实施方案中，所述用于诊断易损斑块或与易损斑块相关的疾病的物质是示踪剂。
在一个实施方案中，所述示踪剂选自CT示踪剂和MRI示踪剂。
在一个实施方案中，所述CT示踪剂选自碘纳米造影剂、金纳米造影剂、氧化钽纳米造影剂、铋纳米造影剂、镧系纳米造影剂，或其他类似结构的示踪剂。
在一个实施方案中，所述CT示踪剂选自碘化造影剂或纳米金，或其他类似结构的示踪剂。
在一个实施方案中，所述CT示踪剂选自碘海醇、碘卡酸、碘佛醇、碘克沙醇、碘普罗胺、碘比醇、碘美普尔、碘帕醇、碘昔兰、醋碘苯酸、胆影酸、碘苯扎酸、碘甘卡酸、泛影酸、碘他拉酸钠、碘苯酯、碘番酸、碘阿芬酸、醋碘苯酸钠、碘多啥、丙碘酮、碘奥酮、碘曲仑、碘吡多、胆影酸葡甲胺、碘他拉酸、泛影葡胺、甲泛影酸、甲泛葡铵、碘化油或乙碘油，或其他类似结构的示踪剂。
在一个实施方案中，所述MRI示踪剂选自纵向弛豫造影剂和横向弛豫造影剂。
在一个实施方案中，所述MRI示踪剂选自顺磁性造影剂、铁磁性造影剂和超磁性造影剂。
在一个实施方案中，所述MRI示踪剂选自Gd-DTPA及其线型、环型多胺多羧类螯合物和锰的卟啉螯合物，大分子钆螯合物、生物大分子修饰的钆螯合物、叶酸修饰的钆螯合物、树状大分子显影剂、脂质体修饰的显影剂和含钆富勒烯，或其他类似结构的示踪剂。
在一个实施方案中，所述MRI示踪剂选自钆喷酸葡胺、钆特酸葡胺、钆贝葡胺、钆双胺、枸橼酸铁铵泡腾颗粒、顺磁性氧化铁，或其他类似结构的示踪剂。
在本发明的硅质体递送系统中，其负载的物质是CD44活化剂。在一个实施方案中，所述CD44活化剂是CD44抗体mAb或IL5、IL12、IL18、TNF-α、LPS。
在本发明的硅质体递送系统中，其负载的物质是是分子量范围为2000～5000Da的小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物。
在一个实施方案中，所述小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物的分子量范围为2500～4500Da。
在一个实施方案中，所述小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物的分子量范围为3000～4000Da。
在一个实施方案中，所述小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物的分子量是约3411Da。
在本发明的硅质体递送系统中，所述硅质体同时负载有用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质和CD44活化剂。
在本发明的硅质体递送系统中，所述硅质体同时负载有用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质和分子量范围为2000～5000Da的小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物。
在本发明的硅质体递送系统中，所述硅质体同时负载有用于诊断易损斑块或与易损斑块相关的疾病的物质、用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质、任选的CD44活化剂和任选的分子量范围为2000～5000Da的小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物。
在本发明的硅质体递送系统中，其负载的物质是用于预防和/或治疗易损斑块或与易 损斑块相关的疾病的物质。
在一个实施方案中，所述用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质选自他汀类药物、贝特类药物、抗血小板药物、PCSK9抑制剂、抗凝药物、血管紧张素转换酶抑制剂(ACEI)、钙离子拮抗剂、MMPs抑制剂、β受体阻滞剂，以及它们的药学上可接受的盐中的一种或多种，包括这些种类药物的活性结构片段。
在一个实施方案中，所述用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质选自洛伐他汀、阿托伐他汀、瑞舒伐他汀、辛伐他汀、氟伐他汀、匹伐他汀、普伐他汀，苯扎贝特、环丙贝特、氯贝特、吉非贝齐、非诺贝特、普罗布考，抗PCSK9抗体如evolocumab、alirocumab、bococizumab、RG7652、LY3015014和LGT-209，反义RNAi寡核苷酸如ALN-PCSsc，核酸如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反义链以及它们的核酸类似物如锁核酸，或adnectin如BMS-962476，阿司匹林、阿西美辛、曲克芦丁、双嘧达莫、西洛他唑、盐酸噻氯匹定、奥扎格雷钠、氯吡格雷、普拉格雷、西洛他唑、替罗非班、贝列前素钠、替格瑞洛、坎格瑞洛、替罗非班、依替巴肽、阿昔单抗、普通肝素、克赛、速碧林、黄达肝葵钠、华法林、达比加群、利伐沙班、阿哌沙班、依度沙班、比伐卢定、依诺肝素、替他肝素、阿地肝素、双香豆素、硝酸香豆素、枸杞酸钠、水蛭素、阿加曲班，贝那普利、卡托普利、依那普利、培多普利、福辛普利、赖诺普利、莫昔普利、西拉普利、培哚普利、喹那普利、雷米普利、群多普利、坎地沙坦，依普罗沙坦、厄贝沙坦、氯沙坦、替米沙坦、缬沙坦、奥美沙坦或他索沙坦、硝苯地平、尼卡地平、尼群地平、氨氯地平、尼莫地平、尼索地平、尼伐地平、伊拉地平、非洛地平、拉西地平、地尔硫卓、维拉帕米、氯己定、米诺环素、MMI-166、美托洛尔、阿替洛尔、比索洛尔、普萘洛尔、卡维地络、巴马司他、马立马司他、普啉司他、BMS-279251、BAY 12-9566、TAA211、AAJ996A、nacetrapib、evacetrapib、Torcetrapib和Dalcetrapib以及它们的药效片段或药学上可接受的盐中的一种或多种，以及它们的药学上可接受的盐中的一种或多种，包括这些种类药物的活性结构片段。
在本发明的硅质体递送系统中，所述硅质体递送系统包括硅质体囊泡，其中所述硅质体囊泡的表面部分地被靶向配体修饰，所述靶向配体是透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物。
本发明所述的用于靶向易损斑块的硅质体递送系统可以根据本领域已知的任何一种方法来制备。例如，采用薄膜分散法制备本发明所述的用于靶向易损斑块的硅质体递送系统。
因此，根据本发明的一个方面，本发明提供了一种用于制备本发明所述的用于靶向易损斑块的硅质体递送系统的方法，所述方法包括以下步骤：
1)将适量的硅质体单体分子、二硬脂酰磷脂酰乙醇胺(DSPE)分子、任选存在的其它脂质分子以及任选存在的脂溶性的用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质溶解于合适的有机溶剂中；
2)通过在旋转蒸发或其它适宜的条件下除去有机溶剂，使步骤1)中的组分在容器壁上形成薄膜；
3)加入任选含有水溶性的用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质的水性介质，并在40-60℃恒温条件下将薄膜中的组分充分水化，形成粗制硅质体囊泡悬浮液；
4)通过超声、振荡、匀浆化、挤压或其它适当的方法处理步骤3)中得到的粗制硅质体囊泡悬浮液，得到精制硅质体囊泡悬浮液；
5)任选地通过透析法除去步骤4)中得到的精制硅质体囊泡悬浮液中所含有的未负载的用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质；
6)将步骤4)或5)中得到的精制硅质体囊泡悬浮液放置至少24小时以促进硅氧烷水解缩合形成无机聚硅氧烷网状结构；
7)在水性条件下，通过加入适量的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC.HCl)和N-羟基硫代琥珀酰亚胺(sulfo-NHS)偶联剂活化靶向配体中的羧基，得到活化的靶向配体；
8)将活化的靶向配体加入到步骤6)中得到的硅质体囊泡悬浮液中，使活化的靶向配体与二硬脂酰磷脂酰乙醇胺(DSPE)分子之间通过形成酰胺键实现偶联，得到用于靶向易损斑块的硅质体递送系统。
根据本发明的一个方面，本发明提供了一种药物，其包含本发明所述的硅质体递送系统，以及药学上可接受的载体。
在一个实施方案中，所述药物包含本发明所述的靶向易损斑块的硅质体递送系统和药学上可接受的载体。
根据本发明的一个方面，本发明提供了一种诊断制剂，其包含本发明所述的靶向易损斑块的硅质体递送系统。
在一个实施方案中，所述诊断制剂包含本发明所述的靶向易损斑块的硅质体递送系统。
根据本发明的一个方面，本发明提供了本发明所述的硅质体递送系统在制备用于诊断、预防和/或治疗与出现CD44分子活化状况相关的疾病的药物中的用途。
在一个实施方案中，所述的质体递送系统在制备用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的药物中的用途。
根据本发明的一个方面，本发明提供了一种用于诊断、预防和/或治疗与出现CD44分子活化状况相关的疾病的方法，所述方法包括给予本发明所述的硅质体递送系统。
在一个实施方案中，本发明提供了一种用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的方法，所述方法包括给予本发明所述的硅质体递送系统。
在一个实施方案中，所述易损斑块选自破裂斑块、侵蚀性斑块和部分钙化结节性病变中的一种或多种。
在一个实施方案中，所述与易损斑块相关的疾病选自冠状动脉粥样硬化性心脏病、动脉粥样硬化症、血管瘤、血栓栓塞、心绞痛、心肌梗死、心源性猝死、心力衰竭、心源性休克、缺血性心肌病、脑卒中中的一种或多种。
本发明包括以上任意实施方案(包括各种优选实施方案)的任意组合。另外，对于任意给定的范围，本发明包括所述范围的端点值、在该范围内的任意具体值以及由在该范围内的任意两个具体值构成的子范围。
总而言之，本发明所述的硅质体递送系统，对于出现CD44分子活化状况的疾病而言，具有如下优点：
1)本发明的硅质体递送系统能够特异性结合至活化的CD44分子，并能够实现药物的稳定持续释放。
2)易损斑块中的细胞表面CD44被细胞外基质微环境所诱导活化，大量过表达，并且CD44-HA的亲和力显著提高，使得易损斑块内的CD44与HA的相互作用具有极为显著的亲和特异性。由此，易损斑块内的CD44构成了本发明所述的靶向易损斑块的硅质体递送系统的优良靶点。
3)本发明所述的靶向易损斑块的硅质体递送系统能够主动靶向进入易损斑块内并与病灶细胞结合。因此，该硅质体递送系统可以实现所负载的物质在病灶处的持续释放，显著增加并持续保持病灶区域的物质浓度，从而提高该递送系统的诊断或治疗效果。
4)本发明所述的靶向易损斑块的硅质体递送系统可包封分子量范围为2000～5000Da的小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物。上述小分子透明质酸或其衍生物通过与细胞表面的内源性透明质酸的竞争性结合，解除内源性透明质酸在细胞表面形成的屏障，从而有利于诊断或治疗活性物质顺利进入病灶细胞内。
5)易损斑块内存在巨大的脂质池，其含有大量的氧化低密度脂蛋白(ox-LDL)。而脂质体在这样的内环境中是不稳定的，极易崩解从而不能实现可控释放的功能；而本发明所述的靶向易损斑块的硅质体递送系统在易损斑块的脂质池中是相对稳定的，可以持续释放药物，从而保持病灶处的药物浓度。
6)本发明所述的靶向易损斑块的硅质体递送系统还可以负载促CD44活化物质即CD44活化剂例如IL5、IL12、IL18、TNF-α、LPS。负载CD44活化剂可以促使病灶细胞表面的CD44进一步活化，可以在短时间内放大CD44对透明质酸的靶向亲和力，显著 增加结合在细胞表面的靶向硅质体组合物浓度，这对于易损斑块的示踪诊断和治疗具有积极意义，因为其可以在短时间内显著增加示踪剂或治疗剂化合物的浓度以提高诊断分辨率或治疗效果。
6)本发明所述的靶向易损斑块的硅质体递送系统具有良好的力学稳定性、热稳定性以及在易损斑块微环境中的稳定性，并且具有良好的储存稳定性；硅质体单体分子中含有Si-C、Si-O键，这样的化学组成使其可以被生物降解；硅质体囊泡中可以负载脂溶性物质、两性化合物或水溶性物质。通过调整硅质体囊泡表面的无机聚硅氧烷网状结构的缩合度和空隙，可以控制活性物质的体内外释放，同时不破坏硅质体囊泡的形态学稳定性。
特别需要指出的是，本领域技术人员在现有技术水平的基础上，可以通过借鉴本文公开的内容而实施本发明。并且，在不脱离本发明的精神和实质的前提下，本领域技术人员可以对本发明进行类似的改进和等同替换，而这些类似的改进和等同替换对于本领域技术人员来说是显而易见的，它们都被视为包括在本发明中。例如，对于本发明硅质体递送系统中可以负载的物质，包括但不限于本发明中所列的物质，其只要能够被掺入至硅质体中并且可以用于诊断、预防和/或治疗目的即可。一般而言，只要所述物质不是在水相和有机溶剂中都不溶解的物质或者在水相和有机溶剂中溶解性都非常好的物质，都能够被容易地掺入至硅质体中。优选地，所述物质是脂溶性物质、两性化合物或水溶性物质。
附图说明
为了使本发明的内容得到更充分的理解，下面通过本发明的具体实施例并结合附图，对本发明作进一步详细地说明，其中：
图1为本发明所述的用于靶向易损斑块的硅质体递送系统的构建示意图。
图2为HA-CL1的红外图谱，其中负载瑞舒伐他汀的CL1连接HA前(黑色，下面的线)及连接后(红色，上面的线)的红外光谱。
图3为本发明的硅质体递送系统和作为对照的脂质体递送系统在4℃储存90天的粒径变化图。
图4为本发明的硅质体递送系统和作为对照的脂质体递送系统在4℃储存90天的药物包封率变化图。
图5为本发明的硅质体递送系统和作为对照的脂质体递送系统的药物累积释放率变化图。
图6为三种本发明的硅质体递送系统的药物累积释放率变化图。
图7为实施例4中构建的小鼠动脉粥样硬化易损斑块模型的核磁共振成像图。
图8为给予本发明的硅质体递送系统和作为对照的脂质体递送系统后小鼠颈动脉斑块药物暴露百分比。
图9为模型小鼠的正常动脉血管壁内皮细胞和动脉易损斑块处内皮细胞表面的CD44含量测定结果(以半定量积分表示)图。
图10为模型小鼠的正常动脉血管壁内皮细胞和动脉易损斑块处内皮细胞表面的CD44与HA的结合力测定结果(以结合力积分表示)图。
图11为模型小鼠的正常动脉血管壁内皮细胞和动脉易损斑块处内皮细胞表面的CD44与与各种配体/抗体的结合力测定结果(以结合力积分表示)图。
图12为模型小鼠的斑块外巨噬细胞和动脉易损斑块内巨噬细胞表面的CD44与HA的结合力测定结果(以结合力积分表示)图。
图13为模型小鼠的斑块外巨噬细胞和动脉易损斑块内巨噬细胞表面的CD44与各种配体/抗体的结合力测定结果(以结合力积分表示)图。
图14为本发明的硅质体递送系统对模型小鼠的颈动脉易损斑块的体内治疗效果(以斑块进展百分比表示)图。
图15为本发明的硅质体递送系统对模型小鼠的颈动脉易损斑块的体内示踪效果(以CT值表示)图。
图16为硅质体-HA-碘克沙醇递送系统对动脉易损斑块的体内CT示踪。
图17为硅质体-HA-钆特酸葡胺递送系统对动脉易损斑块的体内MRI示踪。
图18为CD44多种单克隆抗体-硅质体-钆特酸葡胺递送系统对动脉易损斑块的体内MRI示踪。
图19为CD44多种配体-硅质体-钆双胺递送系统对动脉易损斑块的体内MRI示踪。
具体实施方式
为了进一步理解本发明，下面结合实施例对本发明的具体实施方案进行详细描述。但是应当理解，这些描述的目的只是为进一步说明本发明的特征和优点，而不构成对本发明的权利要求的任何限制。
实施例1：本发明使用的三种硅质体单体
本发明硅质体递送系统中使用到的硅质体单体C1、C2和C3是已知的，其可以按照中所述的制备方法获得。
其中，硅质体单体C1：N，N-双十六烷基-N α-(6-((3-三乙氧基甲硅烷基)丙基二甲基铵基)己酰基)丙氨酰胺溴化物的制备参见Nature Protocols，2006，1(3)，1227-1234
Figure PCTCN2018082850-appb-000002
硅质体单体C1的分子结构
硅质体单体C2：N，N-双十六烷基-N′-(3-三乙氧基甲硅烷基丙基)琥珀酰胺的制备参见 J.Am，Chem.Soc.，2002，124，7892-7893
Figure PCTCN2018082850-appb-000003
硅质体单体C2的分子结构
硅质体单体C3：N，N-双十六烷基-N′-[(3-三乙氧基甲硅烷基)丙基]脲的制备参见Thin Solid Films，2003，438-439，20-26
Figure PCTCN2018082850-appb-000004
硅质体单体C3的分子结构
实施例2：递送系统的制备
1.负载治疗剂的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R的制备
在本实施例中，采用薄膜分散法制备负载治疗剂的硅质体递送系统HA-CL1@R、HA CL2@R和HA-CL3@R。上述三种硅质体递送系统的硅质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均负载用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质瑞舒伐他汀(用缩写“R”表示)，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
HA-CL1@R、HA-CL2@R和HA-CL3@R的具体制备方法为：
(1)硅质体囊泡悬浮液的制备：
称取6mg C1(5mg C2或4mg C3)、2mg 1，2-二油酰氧丙基-N，N，N-三甲基溴化铵(DOTAP)和2mg二硬脂酰磷脂酰乙醇胺(DSPE)置于圆底烧瓶中，并加入10mL氯仿溶解。通过旋转蒸发(55℃水浴，90r/min，30min)完全除去有机溶剂，从而在容器壁上形成薄膜。加入10mL瑞舒伐他汀水溶液(浓度为2mg/mL)，并将烧瓶放在50℃的恒温水浴锅中使薄膜充分水化30min，形成粗制硅质体囊泡悬浮液。将粗制硅质体囊泡悬浮液水浴超声10min，然后采用探头式超声仪超声5min(振幅20，间隔为3s)，得到硅质体囊泡充分分散而形成的稳定体系，即精制硅质体囊泡悬浮液。将精制硅质体囊泡悬浮液中未包封的瑞舒伐他汀用透析袋除去。然后将硅质体囊泡悬浮液放置至少24小时以促进硅质体单体分子在硅质体的表面形成自身具有刚性的无机聚硅氧烷网状结构。
(2)透明质酸(“HA”)的活化和偶联：
将1g HA(分子量为约100KDa)充分溶解于超纯水中，加入0.1g 1-(3-二甲氨基丙基)-3- 乙基碳二亚胺盐酸盐(EDC.HCl)和0.12g N-羟基硫代琥珀酰亚胺(sulfo-NHS)偶联剂活化羧基。在室温搅拌反应1小时后，加入丙酮沉淀活化的HA。将沉淀过滤、用乙醇洗涤并真空干燥，得到活化的HA。将其配置成为0.1mg mL -1的水溶液，吸取0.2mL溶液溶于上面的步骤(1)中得到的硅质体囊泡悬浮液中，使活化的HA中的活化的羧基和硅质体囊泡的脂质双分子层中掺杂的DSPE分子具有的氨基通过形成酰胺键实现偶联，从而得到三种负载治疗剂的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R。
HA与CL的偶联通过红外表征来确证。用于红外表征的样品为负载瑞舒伐他汀HA-CL1@R样品，其制备方法为：称取6mg C1和1mg DSPE置于圆底烧瓶中，并加入10mL氯仿溶解。通过旋转蒸发(55℃水浴，90r/min，30min)完全除去有机溶剂，从而在容器壁上形成薄膜。加入10mL瑞舒伐他汀水溶液(浓度为2mg/mL)，并将烧瓶放在50℃的恒温水浴锅中使薄膜充分水化30min，通过水浴超声10min和探头式超声仪超声5min(振幅20，间隔为3s)，得到硅质体囊泡，放置至少24小时以促进硅质体单体分子在硅质体的表面形成自身具有刚性的无机聚硅氧烷网状结构。将0.1g HA(分子量为约100KDa)充分溶解于超纯水中，加入10mg EDC.HCl和12mg sulfo-NHS偶联剂活化羧基。在室温搅拌反应1小时后，加入丙酮沉淀活化的HA。将沉淀过滤、用乙醇洗涤并真空干燥，得到活化的HA。将其配置成为0.1mg mL-1的水溶液，吸取0.2mL溶液溶于上面的步骤(1)中得到的硅质体囊泡悬浮液中，使活化的HA中的活化的羧基和硅质体囊泡的脂质双分子层中掺杂的DSPE分子具有的氨基通过形成酰胺键实现偶联，从而得到靶向硅质体HA-CL1@R1。自偶联反应开始24h后，通过以12000rpm的转速高速离心分离HA-CL1@R。真空干燥后，将其用于红外光谱表征。如图2所示，1100nm吸收峰证明了硅质体的存在，1700nm和2910nm处吸收峰表明了硅质体和HA的成功偶联。
2.负载小分子透明质酸的硅质体递送系统HA-CL1@LMHA、HA-CL2@LMHA和HA-CL3@LMHA的制备
在本实施例中，采用薄膜分散法制备负载小分子透明质酸的硅质体递送系统HACL1@LMHA、HA-CL2@LMHA和HA-CL3@LMHA。上述三种硅质体递送系统的硅质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均负载分子量为约3411Da的小分子透明质酸(分子式为(C 14H 21NO 11) n，n＝9，用缩写“LMHA”表示，下同)，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
HA-CL1@LMHA、HA-CL2@LMHA和HA-CL3@LMHA的具体制备方法与上述第1点中所述的负载治疗剂的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R的制备方法相同，区别仅在于在步骤(1)中将10mL瑞舒伐他汀水溶液(浓度为2mg/mL)替换成10mL分子量为3411Da的小分子透明质酸的水溶液(浓度为0.5mg/mL)，以及将精制硅质体囊泡悬浮液中未包封的小分子透明质酸用透析袋除去。
3.负载治疗剂和小分子透明质酸的硅质体递送系统HA-CL1@R+LMHA、HACL2@R+LMHA和HA-CL3@R+LMHA的制备
在本实施例中，采用薄膜分散法制备同时负载治疗剂瑞舒伐他汀和小分子透明质酸的硅质体递送系统HA-CL1@R+LMHA、HA-CL2@R+LMHA和HA-CL3@R+LMHA。上述三种硅质体递送系统的硅质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均同时负载治疗剂瑞舒伐他汀(用缩写“R”表示)和分子量为约3411Da的小分子透明质酸，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
HA-CL1@R+LMHA、HA-CL2@R+LMHA和HA-CL3@R+LMHA的具体制备方法与上述第1点中所述的负载治疗剂的硅质体递送系统HA-CL1@R、HA-CL2@R和HACL3@R的制备方法基本相同，区别仅在于在步骤(1)中同时使用10mL瑞舒伐他汀水溶液(浓度为2mg/mL)和10mL分子量为3411Da的小分子透明质酸的水溶液(浓度为0.5mg/mL)，以及将精制硅质体囊泡悬浮液中未包封的瑞舒伐他汀和小分子透明质酸用透析袋除去。
4.负载CD44活化剂的硅质体递送系统HA-CL1@S、HA-CL2@S和HA-CL3@S的制备
在本实施例中，采用薄膜分散法制备负载CD44活化剂的硅质体递送系统HA-CL1@S、HACL2@S和HA-CL3@S。上述三种硅质体递送系统的硅质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均负载CD44活化剂--CD44抗体mAb(用缩写“S”表示)，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
HA-CL1@S、HA-CL2@S和HA-CL3@S的具体制备方法与上述第1点中所述的负载治疗剂的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R的制备方法相同，区别仅在于在步骤(1)中将10mL瑞舒伐他汀水溶液(浓度为2mg/mL)替换成10mLCD44抗体mAb的水溶液(浓度为0.7mg/mL)，以及将精制硅质体囊泡悬浮液中未包封的CD44抗体mAb通过葡聚糖凝胶柱G-100分离去除。
同理，CD44活化剂同样可以使用LPS进行制备，得到了类似的结果。
5.负载治疗剂和CD44活化剂的硅质体递送系统HA-CL1@R+S、HA-CL2@R+S和HA-CL3@R+S的制备
在本实施例中，采用薄膜分散法制备同时负载治疗剂和CD44活化剂的硅质体递送系统HACL1@R+S、HA-CL2@R+S和HA-CL3@R+S。上述三种硅质体递送系统的硅质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均同时负载治疗剂瑞舒伐他汀(用缩写“R”表示)和CD44活化剂--CD44抗体mAb(用缩写“S”表示)，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述 的硅质体单体C1、C2和C3。
HA-CL1@R+S、HA-CL2@R+S和HA-CL3@R+S的具体制备方法与上述第1点中所述的负载治疗剂的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R的制备方法基本相同，区别仅在于在步骤(1)中同时使用10mL瑞舒伐他汀水溶液(浓度为2mg/mL)和10mL CD44抗体mAb的水溶液(浓度为0.7mg/mL)，以及将精制硅质体囊泡悬浮液中未包封的瑞舒伐他汀和CD44抗体mAb用葡聚糖凝胶柱G-100除去。
同理，CD44活化剂同样可以使用LPS进行制备，得到了类似的结果。
6.负载示踪剂的硅质体递送系统HA-CL1@T、HA-CL2@T和HA-CL3@T的制备
在本实施例中，采用薄膜分散法制备负载示踪剂的硅质体递送系统HA-CL1@T、HACL2@T和HA-CL3@T。上述三种硅质体递送系统的硅质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均负载MRI示踪剂钆喷酸(用缩写“T”表示)，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
HA-CL1@T、HA-CL2@T和HA-CL3@T的具体制备方法与上述第1点中所述的负载治疗剂的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R的制备方法相同，区别仅在于在步骤(1)中将10mL瑞舒伐他汀水溶液(浓度为2mg/mL)替换成10mL钆喷酸的水溶液(浓度为3mg/mL)，以及将精制硅质体囊泡悬浮液中未包封的钆喷酸用透析袋除去。
同理，示踪剂同样可以使用钆特酸葡胺或钆双胺进行制备，得到了类似的结果。
7a.负载示踪剂的硅质体递送系统HA-CL1@AuNPs、HA-CL2@AuNPs和HACL3@AuNPs的制备
在本实施例中，采用薄膜分散法制备负载示踪剂的硅质体递送系统HA-CL1@AuNPs、HACL2@AuNPs和HA-CL3@AuNPs。上述三种硅质体递送系统的硅质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均负载CT示踪剂纳米金(用缩写“AuNPs”表示)，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
HA-CL1@AuNPs、HA-CL2@AuNPs和HA-CL3@AuNPs的具体制备方法与上述第1点中所述的负载治疗剂的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R的制备方法相同，区别仅在于在步骤(1)中将10mL瑞舒伐他汀水溶液(浓度为2mg/mL)替换成10mL纳米金溶液(浓度为1mg/mL)，以及将精制硅质体囊泡悬浮液中未包封的纳米金用葡聚糖凝胶柱G-100除去。
7b.负载示踪剂的硅质体递送系统HA-CL1@碘克沙醇、HA-CL2@碘克沙醇和HA-CL3@碘克沙醇的制备
在本实施例中，采用薄膜分散法制备负载示踪剂的硅质体递送系统HA-CL1@I、 HA-CL2@I和HA-CL3@I。上述三种硅质体递送系统的硅质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均负载CT示踪剂碘克沙醇或碘普罗胺(用缩写“I”表示)，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
HA-CL1@I、HA-CL2@I和HA-CL3@I的具体制备方法与上述第1点中所述的负载治疗剂的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R的制备方法相同，区别仅在于在步骤(1)中将10mL瑞舒伐他汀水溶液(浓度为2mg/mL)替换成10mL碘克沙醇或碘普罗胺溶液(浓度为1μg/mL)，以及将精制硅质体囊泡悬浮液中未包封的碘克沙醇或碘普罗胺用葡聚糖凝胶柱除去。
8a.负载示踪剂和CD44活化剂的硅质体递送系统HA-CL1@AuNPs+S、HACL2@AuNPs+S和HA-CL3@AuNPs+S的制备
在本实施例中，采用薄膜分散法制备同时负载示踪剂和CD44活化剂的硅质体递送系统HACL1@AuNPs+S、HA-CL2@AuNPs+S和HA-CL3@AuNPs+S。上述三种硅质体递送系统的硅质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均同时负载CT示踪剂纳米金(用缩写“AuNPs”表示)和CD44活化剂--CD44抗体mAb(用缩写“S”表示)，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
HA-CL1@AuNPS+S、HA-CL2@AuNPS+S和HA-CL3@AuNPS+S的具体制备方法与上述第1点中所述的负载治疗剂的硅质体递送系统HA-CL1@R、HA-CL2@R和HACL3@R的制备方法基本相同，区别仅在于在步骤(1)中同时使用10mL纳米金溶液(浓度为1mg/mL)和10mL CD44抗体mAb的水溶液(浓度为0.7mg/mL)，以及将精制硅质体囊泡悬浮液中未包封的纳米金和CD44抗体mAb用葡聚糖凝胶柱G-100除去。
8b.负载示踪剂和CD44活化剂的硅质体递送系统HA-CL1@I+S、HA-CL2@I+S和HA-CL3@I+S的制备
在本实施例中，采用薄膜分散法制备同时负载示踪剂和CD44活化剂的硅质体递送系统HA-CL1@I+S、HA-CL2@I+S和HA-CL3@I+S。上述三种硅质体递送系统的硅质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均同时负载CT示踪剂碘克沙醇或碘普罗胺(用缩写“I”表示)和CD44活化剂--LPS(用缩写“S”表示)，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
HA-CL1@I+S、HA-CL2@I+S和HA-CL3@I+S的具体制备方法与上述第1点中所述的负载治疗剂的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R的制备方法基本相同，区别仅在于在步骤(1)中同时使用10ml碘克沙醇或碘普罗胺水溶液(1μg/mL)和10ml LPS的水溶液(浓度为0.7mg/mL)，以及将精制硅质体囊泡悬浮液中未包封的碘克沙醇或 碘普罗胺和LPS用葡聚糖凝胶柱G-200除去。
9.负载治疗剂、小分子透明质酸、示踪剂和CD44活化剂的硅质体递送系统HACL1@R+LMHA+T+S、HA-CL2@R+LMHA+T+S和HA-CL3@R+LMHA+T+S的制备
在本实施例中，采用薄膜分散法制备同时负载治疗剂瑞舒伐他汀、小分子透明质酸、示踪剂钆喷酸和CD44活化剂的硅质体递送系统HA-CL1@R+LMHA+T+S、HACL2@R+LMHA+T+S和HA-CL3@R+LMHA+T+S。上述三种硅质体递送系统的硅质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均同时负载治疗剂瑞舒伐他汀(用缩写“R”表示)、分子量为约3411Da的小分子透明质酸、MRI示踪剂钆喷酸(用缩写“T”表示)和CD44活化剂--CD44抗体mAb(用缩写“S”表示)，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
HA-CL1@R+LMHA+T+S、HA-CL2@R+LMHA+T+S和HA-CL3@R+LMHA+T+S的具体制备方法与上述第1点中所述的负载治疗剂的硅质体递送系统HA-CL1@R、HACL2@R和HA-CL3@R的制备方法基本相同，区别仅在于在步骤(1)中同时使用10mL瑞舒伐他汀水溶液(浓度为2mg/mL)、10mL分子量为3411Da的小分子透明质酸的水溶液(浓度为0.5mg/mL)、10mL钆喷酸的水溶液(浓度为3.0mg/mL)和10mLCD44抗体mAb的水溶液(浓度为0.7mg/mL)，以及将精制硅质体囊泡悬浮液中未包封的瑞舒伐他汀、小分子透明质酸、钆喷酸和CD44抗体mAb用葡聚糖凝胶柱G-100除去。
同理，CD44活化剂也可以使用LPS，示踪剂也可以使用钆特酸葡胺进行类似制备，得到类似的结果。
10.空白硅质体递送系统HA-CL1、HA-CL2、HA-CL3(作为对比例)的制备
在本实施例中，采用薄膜分散法制备空白硅质体递送系统HA-CL1、HA-CL2、HA-CL3。上述三种硅质体递送系统的硅质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均不负载任何用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
HA-CL1、HA-CL2、HA-CL3的具体制备方法与上述第1点中所述的负载治疗剂的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R的制备方法基本相同，区别仅在于在步骤(1)中将10mL瑞舒伐他汀水溶液(浓度为2mg/mL)替换成纯水，以及免去透析除去未包封物质的步骤。
11.非靶向的负载治疗剂的硅质体递送系统CL1@R、CL2@R和CL3@R(作为对比 例)的制备
在本实施例中，采用薄膜分散法制备非靶向的负载治疗剂的硅质体递送系统CL1@R、CL2@R和CL3@R。上述三种硅质体递送系统的硅质体囊泡的表面均未被靶向配体透明质酸(缩写为“HA”)修饰，并且均负载用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质瑞舒伐他汀(用缩写“R”表示)，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
CL1@R、CL2@R和CL3@R的具体制备方法与上述第1点中所述的负载治疗剂的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R的制备方法基本相同，区别仅在于免去了步骤(2)。
12a.非靶向的负载示踪剂的硅质体递送系统CL1@AuNPs、CL2@AuNPs和CL3@AuNPs(作为对比例)的制备
在本实施例中，采用薄膜分散法制备非靶向的负载示踪剂的硅质体递送系统CL1@AuNPS、CL2@AuNPS和CL3@AuNPS。上述三种硅质体递送系统的硅质体囊泡的表面均未被靶向配体透明质酸(缩写为“HA”)修饰，并且均负载CT示踪剂纳米金(用缩写“AuNPs”表示)，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
CL1@AuNPS、CL2@AuNPS和CL3@AuNPS的具体制备方法与上述第7点中所述的负载示踪剂的硅质体递送系统HA-CL1@AuNPs、HA-CL2@AuNPs和HA-CL3@AuNPs的制备方法基本相同，区别仅在于免去了步骤(2)。
12b.非靶向的负载示踪剂的硅质体递送系统CL1@I、CL2@I和CL3@I(作为对比例)的制备
在本实施例中，采用薄膜分散法制备非靶向的负载示踪剂的硅质体递送系统CL1@I、CL2@I和CL3@I。上述三种硅质体递送系统的硅质体囊泡的表面均未被靶向配体透明质酸(缩写为“HA”)修饰，并且均负载CT示踪剂碘克沙醇(用缩写“I”表示)，区别仅在于所述三种硅质体递送系统的制备中所使用的硅质体单体分子分别为实施例1中所述的硅质体单体C1、C2和C3。
CL1@I、CL2@I和CL3@I的具体制备方法与上述第7点中所述的负载示踪剂的硅质体递送系统HA-CL1@I、HA-CL2@I和HA-CL3@I的制备方法基本相同，区别仅在于免去了步骤(2)。
13.负载治疗剂的脂质体递送系统HA-PL@R(作为对比例)的制备
在本实施例中，采用薄膜分散法制备负载治疗剂的脂质体递送系统HA-PL@R。所述脂质体递送系统HA-PL@R的脂质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均负载用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质 瑞舒伐他汀(用缩写“R”表示)。
HA-PL@R的具体制备方法为：
(1)脂质体囊泡悬浮液的制备：
称取4mg二硬脂酰磷脂酰胆碱(DSPC)、1mg胆固醇、1mg二硬脂酰磷脂酰乙醇胺(DSPE)(其质量比为4∶1∶1)，并加入10mL氯仿溶解。通过缓慢旋转蒸发(65℃水浴，90r/min，30min)除去有机溶剂，从而在容器壁上形成薄膜。向圆底烧瓶中加入10mL的瑞舒伐他汀水溶液(浓度为2.0mg/mL)，将烧瓶放在50℃的恒温水浴锅中使薄膜充分水化，形成粗制脂质体囊泡悬浮液。将粗制脂质体囊泡悬浮液水浴超声，最后用探头超声仪超声处理3min(振幅20，间隔为3s)，得到脂质体囊泡充分分散而形成的分散体系，即精制脂质体囊泡悬浮液。将精制脂质体囊泡悬浮液中未包封的瑞舒伐他汀用透析袋除去。
(2)透明质酸(“HA”)的活化和偶联：
将1g HA(分子量为约100KDa)充分溶解于超纯水中，加入0.1g 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC.HCl)和0.12g N-羟基硫代琥珀酰亚胺(sulfo-NHS)偶联剂活化羧基。在室温搅拌反应1小时后，加入无水乙醇沉淀活化的HA。将沉淀过滤、用乙醇洗涤并真空干燥，得到活化的HA。将其配置成为0.1mg mL-1的水溶液，吸取0.2mL溶液溶于上面的步骤(1)中得到的脂质体囊泡悬浮液中，使活化的HA中的活化的羧基和脂质体囊泡的脂质双分子层中掺杂的DSPE分子具有的氨基通过形成酰胺键实现偶联，从而得到负载治疗剂的脂质体递送系统HA-PL@R。
14a.负载示踪剂的脂质体递送系统HA-PL@T(作为对比例)的制备
在本实施例中，采用薄膜分散法制备负载示踪剂的脂质体递送系统HA-PL@T。所述脂质体递送系统HA-PL@T的脂质体囊泡的表面均部分地被靶向配体透明质酸(缩写为“HA”)修饰，并且均负载MRI示踪剂钆喷酸(用缩写“T”表示)。
HA-PL@T的具体制备方法与上述第13点中所述的负载治疗剂的脂质体递送系统HA-PL@R的制备方法基本相同，区别仅在于在步骤(1)中将10mL的瑞舒伐他汀水溶液(浓度为2.0mg/mL)替换成10mL示踪剂钆喷酸的水溶液(浓度为3.0mg/mL)，以及将精制脂质体囊泡悬浮液中未包封的示踪剂钆喷酸用透析袋除去。
同理，示踪剂也可以使用钆特酸葡胺进行类似制备，得到类似的结果。
实施例3：本发明的硅质体递送系统的性质考察
在本实施例中，以实施例2中制备的负载治疗剂的硅质体递送系统HA-CL1@R、HACL2@R和HA-CL3@R为例来证明本发明的硅质体递送系统具有稳定可控的性质，从而适合于易损斑块或与易损斑块相关的疾病的诊断、预防和治疗。同时，为了便于比较，本实施例中还使用了实施例2中制备的负载治疗剂的脂质体递送系统HA-PL@R(作为对比例)。
1.药物浓度测定法：
瑞舒伐他汀具有很强的紫外吸收特性，因此可以通过采用HPLC-UV法(使用Waters2487，沃特世公司(Waters Corporation)，美国)利用瑞舒伐他汀的紫外吸收特性测定其含量。用不同浓度的瑞舒伐他汀溶液的浓度(X)对HPLC色谱峰的峰面积(Y)建立标准定量方程。
2.水合粒径的测定：
本发明的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R和作为对比例的脂质体递送系统HA-PL@R的水合粒径均由激光粒度仪(BI-Zeta Plus/90 Plus，布鲁克海文公司(Brookhaven Instruments Corporation)，美国)测定，具体结果如表1所示。
3.包封率的测定：
取1.0mL硅质体囊泡悬浮液，通过加入过量的HCl使悬浮液形成强酸性环境，并进一步采用超声法，使药物加速从硅质体囊泡中释放出来。用HPLC(Waters 2487，沃特世公司(Waters Corporation)，美国)测定所得液体中的药物含量，并通过公式1计算包封率。
Figure PCTCN2018082850-appb-000005
4.载药量的测定：
载药量的测定方法类似于包封率的测定方法，只是计算方法略有不同。取硅质体囊泡悬浮液，加入过量的HCl使悬浮液形成强酸性环境，并进一步采用超声法，使药物加速从硅质体囊泡中释放出来。用HPLC(Waters 2487，沃特世公司(Waters Corporation)，美国)测定所得液体中的药物含量，并通过公式2计算载药量。
Figure PCTCN2018082850-appb-000006
表1：各种性质一览表
Figure PCTCN2018082850-appb-000007
注：以上数据均以平行测定5次的结果的“平均值+标准差”的形式表示。
5.长期稳定性考察
将本发明的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R和作为对照的脂质体递送系统HA-PL@R在4℃储存，于不同的时间点取样，并通过激光粒度仪(BI-Zeta Plus/90Plus，布鲁克海文公司(Brookhaven Instruments Corporation)，美国)检测其水合粒径的变化，结果见图3所示。可见，随着储存时间的延长，脂质体递送系统HA-PL@R的粒径明显增大。这很可能是由于脂质体囊泡不稳定、容易发生聚集或融合而导致的。而且，由于稳定性较差，脂质体囊泡很容易被体内的网状内皮系统清除，致使其半衰期很短，在应用于人体时受到限制。
与之不同的是，在放置90天后，HA-CL1@R、HA-CL2@R和HA-CL3@R的平均水合粒径几乎保持不变，而且在整个试验期间未见分层、絮凝等现象。而HA-PL@R的平均水合粒径从183nm变为2μm左右，并且从外观上看，放置10天后就已经出现明显沉淀，在放置90天后，HA-PL@R已经呈絮状沉淀，无法重新分散。由此可见，与脂质体递送系统HA-PL@R相比，本发明的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R具有更好的储存稳定性，从而具有作为长循环靶向药物递送系统的应用潜力。
6.长期包封率考察
将本发明的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R和作为对照的脂质体递送系统HA-PL@R在4℃储存，于不同时间点取样，通过超滤离心除去游离药物考察其包封率的变化，结果见图4。
从图中可以看出，在放置90天后，硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R中的药物含量未见明显变化。相比而言，脂质体递送系统HA-PL@R中的药物含量陡然下降到30％左右。这说明包封的药物的释放速率和脂质双分子层的性质有很大关系。硅质体囊泡表面的无机聚硅氧烷网状结构有效地保护了内部的脂质双分子层结构，使脂质双分子层的通透性变差，所以药物不易泄漏。相比而言，脂质体囊泡由于无表面的无机聚硅氧烷网状结构的保护作用而稳定性差，容易发生药物的泄露。
从以上数据可以充分地说明本发明的硅质体递送系统的长期储存稳定性很好，在4℃储存三个月后粒径变化不大且药物的泄漏率很低。
7.体外释药性能研究
取2mL本发明的硅质体递送系统HA-CL1@R和2mL作为对照的脂质体递送系统HAPL@R置于透析袋内密封。然后将透析袋置于50mL释放介质(PBS溶液，pH＝7.4)中，于37℃孵育120h。在不同时间点取2mL释放液并补充相同体积的PBS溶液。用HPLC(Waters2487，沃特世公司(Waters Corporation)，美国)检测释放液中的药物含量，并通过公式3计算出药物的累积释放率。
Figure PCTCN2018082850-appb-000008
公式3中各参数意义如下：
CRP：药物累积释放率
Ve：释放液的置换体积，此处Ve为2mL
V0：释放体系中释放液的体积，此处V0为50mL
Ci：第i次置换取样时释放液中药物的浓度，单位μg/mL
M药物：硅质体或脂质体递送系统中的药物的总质量，单位μg
n：置换释放液的次数
Cn：第n次置换释放液后测定的释放体系中的药物浓度。
体外释放是评价纳米粒子递送系统的一项重要指标。图5为本发明的硅质体递送系统HA-CL1@R和作为对照的脂质体递送系统HA-PL@R的的药物累积释放率变化图。由图中可以看出，脂质体递送系统HA-PL@R在30h内几乎释放掉100％的药物。硅质体递送系统HA-CL1@R在起始的3小时内释放速度较快，3小时共释放了15％左右。之后，药物释放速率逐渐变缓，经过120小时仅有59.5％的药物被释放出来。前期较快的释药速率可能是由于部分吸附或沉淀在硅质体囊泡表面的可迅速溶解和扩散到释放介质中的药物的释放行为所导致的，而后期的药物释放主要是包封于硅质体囊泡内的药物释放，表现为持续、缓慢的释放行为。体外释放实验结果显示药物从硅质体囊泡中的释放能够被有效地延缓，这是由于硅质体囊泡的表面被无机聚硅氧烷网状结构覆盖，所以脂质双分子层之间的空隙减少，提高了脂质双分子层的致密度。体外释放实验结果说明硅质体囊泡作为药物载体具有缓慢和持续的释放特性。
另外，通过类似的研究发现本发明的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R的药物体外释放性能相近，其中HA-CL3@R的药物释放速度最快(参见图6)。这指示了本发明的三种硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R具有类似的药物释放机理和特性。
实施例4：本发明的硅质体递送系统的体内释放稳定性研究
在本实施例中，以实施例2中制备的负载瑞舒伐他汀的硅质体递送系统HA-CL1@R和HACL2@R为例来证明与脂质体递送系统相比，本发明的硅质体递送系统能够在易损斑块处保持相对稳定，从而实现长时间持续释放药物的效果。同时，为了便于比较，本实施例中还使用了实施例2中制备的负载瑞舒伐他汀的脂质体递送系统HA-PL@R(作为对比例)。
实验方法：
取SPF级ApoE-/-小鼠(18只，10周龄，体重20±1g)作为实验动物。给予小鼠适应性 高脂饮食(脂肪10％(w/w)，胆固醇2％(w/w)，胆酸钠0.5％(w/w)，其余部分为小鼠普通饲料)喂养4周后，用1％的戊巴比妥钠(配制方法为将1mg戊巴比妥钠加入至100ml的生理盐水中)以40mg/kg的剂量腹腔注射麻醉。然后，将小鼠以仰卧位固定于手术板上，用75％(v/v)酒精以颈部为中心进行消毒，纵向剪开颈部皮肤，钝性分离颈前腺体，在气管的左侧可见搏动的左颈总动脉。小心分离颈总动脉至分叉处，将长度为2.5mm、内径为0.3mm的硅胶管套置于左颈总动脉的外周，套管的近心段和远心段均以细丝线缩窄固定。局部紧缩造成近端血流湍流，剪切力增加，造成血管内膜损伤。将颈动脉复位，间断缝合颈前皮肤。所有操作均在10倍体视显微镜下进行。术后待小鼠苏醒后将其放回笼中，维持环境温度在20～25℃，灯光保持开闭各12h。术后第4周开始腹腔注射脂多糖(LPS)(1mg/kg，在0.2ml磷酸盐缓冲盐水中，Sigma，USA)，每周2次，持续10周，诱导慢性炎症。术后8周将小鼠置入50ml注射器(预留充足通气孔)内造成限制性精神应激，6小时/天，每周5天，共持续6周。小鼠动脉粥样硬化易损斑块模型于术后14周造模完毕。图7中的(a)和(b)给出了所述小鼠动脉粥样硬化易损斑块模型的核磁共振成像图，由箭头指向部分可以看出左侧颈动脉斑块已经形成，提示造模成功，右侧劲动脉可作为正常动脉血管壁进行对比。
根据所使用的靶向递送系统的不同，将小鼠随机分成三组，即硅质体递送系统1组(使用实施例2中制备的负载瑞舒伐他汀的硅质体递送系统HA-CL1@R)、硅质体递送系统2组(使用实施例2中制备的负载瑞舒伐他汀的硅质体递送系统HA-CL2@R)和脂质体递送系统组(使用实施例2中制备的负载瑞舒伐他汀的脂质体递送系统HA-PL@R，作为对比例)，每组6只。
在实验当天，对于上述三组小鼠，分别以5mg瑞舒伐他汀/kg体重的剂量单次静脉注射给予HA-CL1@R、HA-CL2@R和HA-PL@R。采用液相色谱-质谱法检测动脉易损斑块处的药物暴露百分比(其反映了注射实验药物后易损斑块处的瑞舒伐他汀的浓度随时问的变化)：
(1)标准溶液配制
精密称取瑞舒伐他汀0.0141g，置于25mL容量瓶中，用甲醇溶解并稀释至刻度，摇匀，配制成浓度为56.4μg/mL的瑞舒伐他汀对照品储备液；将瑞舒伐他汀对照品储备液用甲醇稀释成10，1，0.5，0.125，0.05，0.025，0.01，0.002，0.0004μg/mL的系列标准溶液，4℃冷藏备用。
(2)内标溶液配制
精密称取对乙酰氨基酚0.0038g，置于25mL容量瓶中，用甲醇溶解并稀释至刻度，摇匀，配制成浓度为0.152mg/mL的对乙酰氨基酚储备液；将乙酰氨基酚储备液用甲醇稀释成15.2ng/mL的内标溶液，4℃冷藏备用。
(3)颈动脉样品前处理
分别于给药前和给药后2h，4h，8h，12h，24h，48h，72h，168h(七天)将动物处死(每个时间点一只小鼠)，迅速取出颈动脉斑块置于生理盐水中，用滤纸吸干表面水分，各剪取1cm，称量湿重，加1ml生理盐水匀浆，制成匀浆液。
取匀浆液1ml，加入甲醇20μL、浓度为15.2ng/mL的内标溶液100μL、10％(v/v)甲酸水溶液100μL、乙酸乙酯5mL，混匀，以14000rpm离心10min。取有机层溶液4ml，用氮气吹干。然后，用200μL流动相(0.1％(v/v)甲酸水溶液和乙腈(40：60，v/v))溶解，以14000rpm离心10min，取上清液，移入进样瓶，待测。
(4)标准曲线样品制备
取系列浓度的瑞舒伐他汀溶液10μL，加入500μL空白血浆，涡旋使其充分混匀，制备成浓度分别为200，20，10，2.5，1，0.5，0.2，0.04，0.008ng/mL的瑞舒伐他汀模拟含药血浆样品。按照血浆处理进行操作(加入15.2ng/mL内标溶液50μL，10％(v/v)甲酸水溶液50μL，乙酸乙酯2.5mL，混匀，以14000rpm离心10min，取有机层溶液2ml，用氮气吹干，然后用100μL流动相溶解，以14000rpm离心10min，取上清液，移入进样瓶，待测)，建立标准曲线。以瑞舒伐他汀峰面积和内标峰面积比(y)为纵坐标，以血药浓度(x)为横坐标，用加权最小二乘法进行线性回归。
(5)液相色谱-质谱分析
液相分离采用Shimadzu modulaRLC system(东京，日本)系统进行，所述系统包括：1个DGU-20A3R真空脱气器，2个LC-20ADXR溶剂输送模块，1个SIL-20ACXR自动进样器，1个SPD-M20A PDA系统和1个CBM-20A控制器。该液相系统与装有ESI接口的ABSciex5500Qtrap质谱仪(FosteRCity，CA，USA)在线连接。Analyst软件(Version 1.6.2，ABSciex)用于数据采集与处理。
色谱分析采用CortecsTM UPLC C18柱(150mm×2.1mm内径(i.d.)，1.6μm粒度)(Waters公司，美国)，柱温和样品室温度分别设为40℃和4℃。流动相为0.1％(v/v)甲酸水溶液和乙腈(40∶60，v/v)，进样量为2μl。流速为0.2mL/min，单个样品分析时间为4min。
质谱检测采用的离子源为ESI源，正离子扫描模式。喷雾电压设为4500V，源温度设为500℃。通过多反应监测(MRM)方式检测各化合物，各成分离子通道分别为：瑞舒伐他汀钙m/z 482.2→258.2，对乙酰氨基酚m/z 152.2→110。优化各化合物碰撞能量和锥孔电压分别为：瑞舒伐他汀43V和100V，对乙酰氨基酚23V和100V。瑞舒伐他汀钙和对乙酰氨基酚的保留时间分别为2.07min和1.49min。
(6)标准曲线
瑞舒伐他汀的线性范围、相关系数(r)、线性方程及LLOQ如表2所示。从表中可以看出，瑞舒伐他汀的R值大于0.999，满足定量分析的要求。
表2 瑞舒伐他汀的线性方程及LLOQ
Figure PCTCN2018082850-appb-000009
药物暴露百分比＝药物的重量/组织的重量。
结果如图8中显示。如图所示，在注射HA-PL@R之后，模型小鼠的易损斑块中的瑞舒伐他汀的浓度在很快达到峰值后迅速消减，这表明脂质体囊泡在易损斑块处不稳定，容易发生崩解而快速泄露药物。相反，在注射HA-CL1@R和HA-CL2@R之后，模型小鼠的易损斑块中的瑞舒伐他汀浓度较快达到峰值，并且在相当长的时间内浓度平缓下降，这表明本发明的硅质体递送系统能够在易损斑块中保持稳定，从而实现在长时间内持续地释放药物的效果。
实施例5：靶向机理的研究
在本实施例中，对易损斑块内皮细胞表面上的CD44的密度以及与HA之间的亲和力进行研究，从而为选择易损斑块内的CD44作为本发明所述的靶向易损斑块的硅质体递送系统的靶点提供了实验依据。
1)小鼠动脉易损斑块处内皮细胞表面与正常动脉血管壁内皮细胞表面的CD44含量比较
按照上述实施例4中所述的方法，构建小鼠动脉粥样硬化易损斑块模型。取模型小鼠的正常动脉血管内皮细胞和动脉易损斑块处内皮细胞，采用免疫组织化学染色和图像分析方法进行CD44含量测定，具体实验方法如下：
取小鼠颈动脉粥样硬化易损斑块标本，经10mL/L甲醛水溶液固定、石蜡包埋、4μm切片、常规脱蜡、水化处理后，采用亲和素-生物素-酶复合物法(SABC)检测CD44含量。将标本浸入30mL/L H 2O 2水溶液以阻断内源性过氧化物酶的活性，并置入柠檬酸盐缓冲液中行抗原微波修复。然后滴加50g/L牛血清白蛋白(BSA)封闭液，于室温放置20min。然后，滴加鼠抗CD44多克隆抗体(1∶100)，于4℃冰箱放置过夜，再在37℃孵育1h。洗涤后，滴加生物素化的山羊抗鼠IgG，于37℃反应30min。然后用磷酸盐缓冲盐水(PBS)洗涤，并滴加辣根过氧化物酶标记的SABC复合物，于37℃孵育20min；以上每步均用PBS冲洗。
最后用DAB显色(显色时在显微镜下控制)，随后用苏木素复染、脱水和封片。采用BI-2000图像分析系统的免疫组织化学分析系统分析切片，其中针对正常动脉血管内皮细胞和动脉易损斑块处内皮细胞组各采集3张切片，随机取5个具有代表性的视野。CD44表达阳性为：细胞膜、细胞质呈棕黄色/棕褐色且背景清晰，并且颜色越深说明CD44表达越强。
未出现棕黄颗粒为CD44表达阴性。测量正常动脉血管内皮细胞和动脉易损斑块处内皮细胞组的阳性细胞平均吸光度(A)值并进行对比。结果如图9所示。
图9示出模型小鼠的正常动脉血管壁内皮细胞和动脉易损斑块处内皮细胞的表面CD44含量测定结果(以半定量积分表示)。如图所示，动脉易损斑块处内皮细胞的表面CD44含量约为正常动脉血管内皮细胞表面CD44含量的2.3倍。
2a)小鼠动脉易损斑块处内皮细胞表面与正常动脉血管壁内皮细胞表面的CD44与HA的亲和力比较
取模型小鼠的正常动脉血管壁内皮细胞和动脉易损斑块处内皮细胞，加入浓度为10mg/ml的标记有氨基荧光素的透明质酸(用“FL-HA”表示)，并用杜尔伯科改良伊格尔培养基(DMEM)培养基(含体积分数为10％的小牛血清、100U/ml青霉素、100U/ml链霉素)于37℃、5％CO 2培养箱中培养。30分钟后，利用流式细胞仪(CytoFLEX，贝克曼库尔特，美国)测定平均荧光强度(MFI)，并计算两种细胞表面的FL-HA的结合力积分(以正常动脉血管壁内皮细胞的结合力为1)。结果如图10所示。
如图10所示，动脉易损斑块处内皮细胞表面的FL-HA的结合力积分约为正常动脉血管壁内皮细胞表面的FL-HA的结合力积分的24倍。这表明正常动脉血管壁内皮细胞表面的CD44大多数处于不能与配体HA结合的静止状态，而动脉易损斑块处内皮细胞表面的CD44受到内环境中的诸如炎症因子等因素的影响而激活，与HA的亲和力显著增加。
2b)小鼠动脉易损斑块处内皮细胞表面与正常动脉血管壁内皮细胞表面的CD44与配体及抗体的亲和力比较
CD44的天然配体包括：HA、GAG、胶原、层黏连蛋白、纤黏连蛋白、选择蛋白、骨桥蛋白(OPN)以及单克隆抗体HI44a，HI313，A3D8，H90，IM7等。
取模型小鼠的正常动脉血管壁内皮细胞和动脉易损斑块处内皮细胞，加入浓度为10mg/ml的标记有氨基荧光素的配体/抗体，并用杜尔伯科改良伊格尔培养基(DMEM)培养基(含体积分数为10％的小牛血清、100U/ml青霉素、100U/ml链霉素)于37℃、5％CO 2培养箱中培养。30分钟后，利用流式细胞仪(CytoFLEX，贝克曼库尔特，美国)测定平均荧光强度(MFI)，并计算两种细胞表面的FL-配体/抗体的结合力积分(以正常动脉血管壁内皮细胞的CD44与配体/抗体的结合力为1)。结果如图11所示。
如图11所示，动脉易损斑块处内皮细胞表面的CD44与HA的结合力积分约为正常动脉血管壁内皮细胞表面的结合力积分的24倍。这表明正常动脉血管壁内皮细胞表面的CD44大多数处于不能与配体HA结合的静止状态，而动脉易损斑块处内皮细胞表面的CD44受到内环境中的诸如炎症因子等因素的影响而激活，与HA的亲和力显著增加。
CD44其他配体，与HA相似，易损斑块内皮细胞表面的CD44与GAG的结合力积分是正常细胞的22倍，易损斑块内皮细胞CD44与胶原的结合力积分是正常细胞的21倍，易损斑块内皮细胞CD44与层黏连蛋白的结合力积分是正常细胞的16倍，易损斑块内皮细胞CD44与纤黏连蛋白的结合力积分是正常细胞的18倍，易损斑块内皮细胞CD44与选择蛋白的结合力积分是正常细胞的19倍，易损斑块内皮细胞CD44与骨桥蛋白的结合力积分是正常细胞的17倍。
CD44单克隆抗体也出现类似结果：易损斑块内皮细胞表面的CD44与H144a的结合力积分是正常细胞的15倍，易损斑块内皮细胞CD44与H1313的结合力积分是正常细胞的21 倍，易损斑块内皮细胞CD44与A3D8的结合力积分是正常细胞的17倍，易损斑块内皮细胞CD44与H90的结合力积分是正常细胞的9倍，易损斑块内皮细胞CD44与IM7的结合力积分是正常细胞的8倍。
3a)斑块外巨噬细胞和动脉易损斑块内巨噬细胞表面的CD44与HA的亲和力比较
取模型小鼠的腹腔内的巨噬细胞和动脉易损斑块内的巨噬细胞，加入浓度为10mg/ml的标记有氨基荧光素的透明质酸(用“FL-HA”表示)，用DMEM培养基(含体积分数为10％的小牛血清、100U/ml青霉素、100U/ml链霉素)于37℃、5％CO 2培养箱中培养。30分钟后，利用流式细胞计(CytoFLEX，贝克曼库尔特，美国)测定平均荧光强度(MFI)，并计算两种细胞表面上的FL-HA的结合力积分(以斑块外巨噬细胞表面的CD44与HA的亲和力为1)。结果如图12所示。
如图12所示，动脉易损斑块内巨噬细胞表面的FL-HA的结合力约为斑块外巨噬细胞表面的FL-HA的结合力的40倍。这表明动脉易损斑块内的巨噬细胞表面的CD44同样受到内环境中的诸如炎症因子等因素的影响而激活，与HA的亲和力显著增加。
综合上述实验的结果，可以得出如下结论：与正常细胞(诸如正常动脉血管壁内皮细胞、斑块外巨噬细胞)相比，易损斑块内的细胞(包括内皮细胞、巨噬细胞等，其对于动脉易损斑块的发展具有重要影响)表面上的CD44的密度显著提高，并且与HA的亲和力显著增强，从而导致动脉易损斑块内的CD44与HA配体的特异性亲和能力远远高于正常细胞，使其非常有利于作为本发明所述的靶向易损斑块的硅质体递送系统的优良靶点。
3b)斑块外巨噬细胞和动脉易损斑块内巨噬细胞表面的CD44与配体/抗体的亲和力比较
取模型小鼠的腹腔内的巨噬细胞和动脉易损斑块内的巨噬细胞，加入浓度为10mg/ml的标记有氨基荧光素的配体/抗体，用DMEM培养基(含体积分数为10％的小牛血清、100U/ml青霉素、100U/ml链霉素)于37℃、5％CO 2培养箱中培养。30分钟后，利用流式细胞计(CytoFLEX，贝克曼库尔特，美国)测定平均荧光强度(MFI)，并计算两种细胞表面上的FL-HA的结合力积分(以斑块外巨噬细胞表面的CD44与配体/抗体亲和力为1)。结果如图13所示。
如图13所示，动脉易损斑块内巨噬细胞表面的CD44-HA的结合力约为斑块外巨噬细胞表面的CD44-HA的结合力的40倍。这表明动脉易损斑块内的巨噬细胞表面的CD44同样受到内环境中的诸如炎症因子等因素的影响而激活，与HA的亲和力显著增加。
CD44其他配体，与HA相似，易损斑块巨噬细胞表面的CD44与GAG的结合力积分是正常细胞的33倍，易损斑块巨噬细胞CD44与胶原的结合力积分是正常细胞的38倍，易损斑块巨噬细胞CD44与层黏连蛋白的结合力积分是正常细胞的37倍，易损斑块巨噬细胞CD44与纤黏连蛋白的结合力积分是正常细胞的35倍，易损斑块巨噬细胞CD44与选择蛋白的结合力积分是正常细胞的33倍，易损斑块巨噬细胞CD44与骨桥蛋白的结 合力积分是正常细胞的33倍。
CD44单克隆抗体也出现类似结果：易损斑块巨噬细胞表面的CD44与H144a的结合力积分是正常细胞的17倍，易损斑块巨噬细胞CD44与H1313的结合力积分是正常细胞的20倍，易损斑块巨噬细胞CD44与A3D8的结合力积分是正常细胞的16倍，易损斑块巨噬细胞CD44与H90的结合力积分是正常细胞的9倍，易损斑块巨噬细胞CD44与IM7的结合力积分是正常细胞的10倍。
综合上述实验的结果，可以得出如下结论：与正常细胞(诸如正常动脉血管壁内皮细胞、斑块外巨噬细胞)相比，易损斑块内的细胞(包括内皮细胞、巨噬细胞等，其对于动脉易损斑块的发展具有重要影响)表面上的CD44的密度显著提高，并且与配体的亲和力显著增强，从而导致动脉易损斑块内的CD44与配体的特异性亲和能力远远高于正常细胞，使其非常有利于作为本发明所述的靶向易损斑块的硅质体递送系统的优良靶点。
实施例6：本发明的硅质体递送系统对动脉易损斑块的影响的体内实验
本实施例的目的是验证本发明所述的负载治疗剂的硅质体递送系统对动脉易损斑块的体内治疗作用。
实验方法：
(1)配制游离瑞舒伐他汀的生理盐水溶液，并采用上述实施例2中所述的方法制备负载治疗剂的硅质体递送系统HA-CL1@R、负载治疗剂和小分子透明质酸的硅质体递送系统HACL1@R+LMHA、负载治疗剂和CD44活化剂的硅质体递送系统HA-CL1@R+S、负载治疗剂的脂质体递送系统HA-PL@R和透明质酸纳米胶束系统PDLLA/Chol-HA@R(作为对比例)。
透明质酸纳米胶束系统(PDLLA/Chol-HA@R)制备：将1g胆固醇溶解于30mL的丙酮中，加入1g丁二酸酐。反应溶液在70，℃下搅拌3h。减压蒸馏除去溶剂，粗产品溶解用水/无水乙醇(1∶10)的溶液重结晶得到胆固醇-丁二酸酯。称取500mg胆固醇-丁二酸酯溶于20mL无水氯仿中，滴加6mL含有1mL氯化亚砜的氯仿。滴加完成将体系升温至60℃，反应5h结束，减压蒸馏除去未反应完的二氯亚砜和三氯甲烷，得到淡绿色油状物。称取500mg透明质酸(10kD)溶解于60mL二甲基亚砜中加入1mL三乙胺，量取5mL胆固醇-丁二酸酯酰氯的二甲基亚砜溶液，在氮气保护下缓慢滴加胆固醇-丁二酸酯酰氯，将体系恒温80℃，反应7h，待反应结束以后，停止加热，反应产物在水中透析72h后冷冻干燥得到胆固醇改性透明质酸(Chol-HA)。
称取50mg Chol-HA和50mg聚乳酸(PDLLA)共同溶解于10mL DMF中，在磁力搅拌器上搅拌24h至充分混合，将所得高分子溶液装入截流相对分子质量为3000的透析袋中对500mL去离子水透析4h，然后将水相更换为质量分数为2％的瑞舒伐他汀溶液，继续透析48h，然后将透析袋立即放入到20mL瑞舒伐他汀溶液中(1mg mL-1)孵化24h，然后对1L去离子水透析4h，每小时换一次去离子水，以除去未包载的药物，将所得纳米囊泡溶液从透析袋中取出冻干，得到包载瑞舒伐他汀的纳米载体PDLLA/Chol-HA@R。
(2)ApoE-/-小鼠动脉易损斑块模型的建立：
取SPF级ApoE-/-小鼠(30只，5-6周龄，体重20±1g)作为实验动物。给予小鼠适应性高脂饮食(脂肪10％(w/w)，胆固醇2％(w/w)，胆酸钠0.5％(w/w)，其余部分为小鼠普通饲料)喂养4周后，用1％的戊巴比妥钠(配制方法为将1mg戊巴比妥钠加入至100ml的生理盐水中)以40mg/kg的剂量腹腔注射麻醉。然后，将小鼠以仰卧位固定于手术板上，用75％(v/v)酒精以颈部为中心进行消毒，纵向剪开颈部皮肤，钝性分离颈前腺体，在气管的左侧可见搏动的左颈总动脉。小心分离颈总动脉至分叉处，将长度为2.5mm、内径为0.3mm的硅胶管套置于左颈总动脉的外周，套管的近心段和远心段均以细丝线缩窄固定。局部紧缩造成近端血流湍流，剪切力增加，造成的血管内膜损伤。将颈动脉复位，间断缝合颈前皮肤。所有操作均在10倍体视显微镜下进行。术后待小鼠苏醒后将其放回笼中，维持环境温度在20～25℃，灯光保持开闭各12h。术后第4周开始腹腔注射脂多糖(LPS)(1mg/kg，在0.2ml磷酸盐缓冲盐水中，Sigma，USA)，每周2次，持续10周，诱导慢性炎症。术后8周将小鼠置入50ml注射器(预留充足通气孔)内造成限制性精神应激，6小时/天，每周5天，共持续6周。小鼠动脉粥样硬化易损斑块模型于术后14周造模完毕。
(3)实验动物分组及治疗：
将实验动物随机分为以下各组，每组6只：
易损斑块模型对照组：该组动物不进行任何治疗性处理；
瑞舒伐他汀灌胃组：以5mg瑞舒伐他汀/kg体重的剂量进行灌胃给药处理；
瑞舒伐他汀静脉注射组：以5mg瑞舒伐他汀/kg体重的剂量进行静脉注射给药处理；
HA-PL@R组：以5mg瑞舒伐他汀/kg体重的剂量进行静脉注射给药处理；
PDLLA/Chol-HA@R组：以5mg瑞舒伐他汀/kg体重的剂量进行静脉注射给药处理；
HA-CL1@R组：以5mg瑞舒伐他汀/kg体重的剂量进行静脉注射给药处理；
HA-CL1@R+LMHA组：以5mg瑞舒伐他汀和1.25mg分子量为3411Da的小分子透明质酸/kg体重的剂量进行静脉注射给药处理；
HA-CL1@R+S组：以5mg瑞舒伐他汀和1.75mgCD44抗体mAb/kg体重的剂量进行静脉注射给药处理。
除易损斑块模型对照组外，治疗组的治疗每隔一天进行1次，共治疗5次。对于各组动物，于治疗前后进行颈动脉MRI扫描以检测斑块和管腔面积，并计算斑块进展百分比。
斑块进展百分比＝(治疗后斑块面积-治疗前斑块面积)/管腔面积。
实验结果：
图14展示了本发明所述的负载治疗剂的硅质体递送系统对动脉易损斑块的体内治疗效果。如图所示，对于小鼠体内动脉易损斑块而言，无论是灌胃给药还是静脉注射给药，游离的瑞舒伐他汀都呈现出了一定的治疗效果，但是其无法阻止易损斑块的继续生长。与游离的瑞舒伐他汀相比，当将瑞舒伐他汀配制在脂质体递送系统或透明质酸纳米胶束 递送系统中给药时，其对于易损斑块的治疗效果有了一定程度的提高，但仍然无法阻止易损斑块的继续生长。然而，当将瑞舒伐他汀配制在本发明所述的硅质体递送系统中时，其对于易损斑块的治疗效果发生了显著的提升，并起到了逆转斑块生长(即，缩小斑块)的治疗效果。特别地，发明人意外地发现当将瑞舒伐他汀与小分子透明质酸或CD44活化剂--CD44抗体mAb一起配制在硅质体递送系统中联合给药时，对于小鼠体内动脉易损斑块产生了非常显著的治疗效果。综上所述，与游离药物和脂质体给系统相比，使用本发明所述的硅质体递送系统给药治疗剂可显著逆转动脉易损斑块的生长，具有更好的治疗作用。
另外，通过类似的研究发现本发明的硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R对动脉易损斑块的治疗效果相近，这进一步指示了本发明的三种硅质体递送系统HA-CL1@R、HA-CL2@R和HA-CL3@R具有类似的药物释放机理和特性。
实施例7：本发明的硅质体递送系统对动脉易损斑块的体内示踪实验
本实施例的目的是验证本发明所述的负载示踪剂的硅质体递送系统对动脉易损斑块的体内示踪效果。
实验方法：
(1)利用市售的纳米金溶液，采用上述实施例2中所述的方法制备负载CT示踪剂纳米金的硅质体递送系统HA-CL1@AuNPs、负载示踪剂和CD44活化剂的硅质体递送系统HACL1@AuNPs+S和非靶向的负载示踪剂的硅质体递送系统CL1@AuNPs(作为对比例)。
(2)按照上述实施例4中所述的方法构建小鼠动脉粥样硬化易损斑块模型。
(3)将模型小鼠用高脂饮食(同实施例4)喂养16周。取24只模型小鼠，随机分成游离纳米金颗粒组(6只，给予市售的纳米金溶液，纳米金的给药剂量为0.1mg/kg体重)，CL1@AuNPS组(6只，给予CL1@AuNPS，纳米金的给药剂量为0.1mg/kg体重)，HA-CL1@AuNPs组(6只，给予HA-CL1@AuNPs，纳米金的给药剂量为0.1mg/kg体重)和HA-CL1@AuNPs+S组(6只，给予HA-CL1@AuNPs+S，纳米金的给药剂量为0.1mg/kg体重，CD44活化剂--CD44抗体mAb的给药剂量为约0.07mg/kg体重)。各实验组分别经尾静脉注入相应的示踪剂，并于给药前以及给药后4h进行CT成像，观察各组动物的动脉粥样硬化易损斑块的识别情况。
实验结果：
图15展示了本发明所述的负载示踪剂的硅质体递送系统对动脉易损斑块的体内示踪效果。
如图所示，游离的纳米金颗粒对于小鼠体内动脉易损斑块而言呈现出了一定的示踪效果。与游离的纳米金颗粒相比，当将纳米金配制在非靶向的硅质体递送系统中时，其对于易损斑块的示踪效果有了一定程度的提高。当将纳米金配制在本发明所述的表面修饰有靶向配体透明质酸的硅质体递送系统中时，其对于易损斑块的示踪效果发生了显著的提升。特别地，发明人意外地发现当将纳米金与CD44活化剂--CD44抗体mAb一起配制 在硅质体递送系统中联合给药时，对于小鼠体内动脉易损斑块产生了非常显著的示踪效果。综上所述，与游离纳米金颗粒和非靶向的硅质体递送系统相比，使用本发明所述的表面修饰有靶向配体透明质酸的硅质体递送系统给药可显著提高纳米金对动脉易损斑块的识别作用，产生更好的示踪效果。
另外，通过类似的研究发现本发明的负载示踪剂的硅质体递送系统HACL1@AuNPs、HA-CL2@AuNPs和HA-CL3@AuNPs对动脉易损斑块的示踪效果相近，这进一步指示了本发明的三种硅质体递送系统HA-CL1@AuNPs、HA-CL2@AuNPs和HACL3@AuNPs具有类似的药物释放机理和特性。
实施例8：本发明的硅质体-HA-碘克沙醇递送系统对动脉易损斑块的体内示踪实验(CT示踪)
本实施例的目的是验证本发明所述的负载CT示踪剂的硅质体递送系统对动脉易损斑块的体内示踪效果。
实验方法：
(1)利用市售的碘克沙醇原料药，采用上述实施例1中所述的方法制备负载CT示踪剂的硅质体递送系统HA-CL1@I、负载示踪剂和CD44活化剂的硅质体递送系统HA-CL1@I+S和非靶向的负载示踪剂的硅质体递送系统CL1@I(作为对比例)。
(2)按照上述实施例6中所述的方法构建小鼠动脉粥样硬化易损斑块模型。
(3)将模型小鼠用高脂饮食(同实施例6)喂养16周。取30只模型小鼠，随机分成空白组(6只)，碘克沙醇组(6只，给予市售的碘克沙醇原料药，碘克沙醇的给药剂量为0.66mg/kg体重)，CL1@I组(6只，给予CL1@I，碘克沙醇的给药剂量为0.66mg/kg体重)，HA-CL1@I组(6只，给予HA-CL1@I，碘克沙醇的给药剂量为0.66mg/kg体重)和HA-CL1@I+S组(6只，给予HA-CL1@I+S，碘克沙醇的给药剂量为0.66mg/kg体重，CD44活化剂--LPS的给药剂量为约0.023mg/kg体重)。各实验组分别经尾静脉注入相应的示踪剂，并于给药前以及给药后72h进行CT成像，观察各组动物的动脉粥样硬化易损斑块的识别情况。
实验结果：
图16展示了本发明所述的负载示踪剂的硅质体递送系统对动脉易损斑块的体内示踪效果。如图所示，静脉注射碘克沙醇对于小鼠体内动脉易损斑块而言未出现任何的示踪效果。与静脉注射碘克沙醇相比，当将其负载在非靶向的硅质体递送系统中时，其对于易损斑块的示踪效果有了一定程度的提高。当将碘克沙醇配制在本发明所述的表面修饰有靶向配体透明质酸的硅质体递送系统中时，其对于易损斑块的示踪效果发生了显著的提升。特别地，发明人意外地发现当将碘克沙醇与CD44活化剂--LPS一起配制在硅质体递送系统中联合给药时，对于小鼠体内动脉易损斑块产生了非常显著的示踪效果。综上所述，与游离碘克沙醇和非靶向的硅质体递送系统相比，使用本发明所述的表面修饰有靶向配体透明质酸的硅质体递送系统给药可显著提高碘克沙醇对动脉易损斑块的识别作用，产生更好的示踪效果。
实施例9：本发明的硅质体-HA-钆特酸葡胺递送系统对动脉易损斑块的体内示踪实验(MRI示踪)
本实施例的目的是验证本发明所述的负载MRI示踪剂的硅质体递送系统对动脉易损斑块的体内示踪效果。
实验方法：
(1)利用市售的原料药钆特酸葡胺，采用上述实施例1中所述的方法制备负载MRI示踪剂的硅质体递送系统和空白硅质体递送系统(作为对比例)。
(2)按照上述实施例6中所述的方法构建小鼠动脉粥样硬化易损斑块模型。
(3)将模型小鼠用高脂饮食(同实施例6)喂养16周。取18只模型小鼠，随机分成空白硅质体组(6只)，低浓度钆特酸葡胺硅质体组(6只，给予市售的钆特酸葡胺原料药，钆特酸葡胺的给药浓度为0.5mg/ml，给药量为10ml/kg)，高浓度钆特酸葡胺硅质体组(6只，给予市售的钆特酸葡胺原料药，钆特酸葡胺的给药浓度为0.5mg/ml，给药量为10ml/kg)。各实验组分别经尾静脉注入相应的示踪剂，并于给药前以及给药后多时间点进行MRI成像，观察各组动物的动脉粥样硬化易损斑块的识别情况。
实验结果：
图17展示了本发明所述的负载示踪剂的硅质体递送系统对动脉易损斑块的体内示踪效果。如图所示，空白硅质体对于小鼠体内动脉易损斑块而言未出现任何的示踪效果。与空白硅质体组相比，当将钆特酸葡胺负载在靶向的硅质体递送系统中时，其对于易损斑块的示踪效果有了显著的提高，并呈剂量依赖性提高。综上所述，与空白硅质体递送系统相比，使用本发明所述的硅质体-HA-钆特酸葡胺递送系统给药可显示对动脉易损斑块的识别作用，产生较好的MRI示踪效果。
实施例10：本发明的CD44多种单克隆抗体-硅质体-钆特酸葡胺递送系统对动脉易损斑块的体内示踪实验(MRI示踪)
本实施例的目的是验证本发明所述的负载MRI示踪剂的硅质体表面分别装配多种不同CD44单克隆抗体构成的纳米递送系统对动脉易损斑块的体内示踪效果。
实验方法：
(1)利用市售的原料药钆特酸葡胺，采用上述实施例1中所述的方法制备不同CD44单克隆抗体作为靶向探针的，负载MRI示踪剂的硅质体纳米递送系统。
(2)按照上述实施例6中所述的方法构建小鼠动脉粥样硬化易损斑块模型。
(3)将模型小鼠用高脂饮食(同实施例5)喂养16周。取24只模型小鼠，随机分成空白硅质体组(6只)，钆特酸葡胺-硅质体-H144a组(6只，给予市售的钆特酸葡胺原料药，钆特酸葡胺的给药浓度为0.5mg/ml，给药量为10ml/kg)，钆特酸葡胺-硅质体-A3D8组(6只，给予市售的钆特酸葡胺原料药，钆特酸葡胺的给药浓度为0.5mg/ml，给药量为10ml/kg)，钆特酸葡胺-硅质体-H90组(6只，给予市售的钆特酸葡胺原料药，钆特酸葡胺的给药浓度为 0.5mg/ml，给药量为10ml/kg)。各实验组分别经尾静脉注入相应的示踪剂，并于给药前以及给药后多时间点进行MRI成像，观察各组动物的动脉粥样硬化易损斑块的识别情况。
实验结果：
图18展示了本发明所述的CD44多种单克隆抗体作为探针的纳米递送系统对动脉易损斑块的体内示踪效果。如图所示，与空白硅质体组相比，使用本发明所述的CD44多种单克隆抗体作为靶向探针的硅质体纳米系统(包括钆特酸葡胺-硅质体-H144a纳米系统，钆特酸葡胺-硅质体-A3D8纳米系统，钆特酸葡胺-硅质体-H90纳米系统)均可显示对动脉易损斑块的识别作用，产生较好的MRI示踪效果。
实施例11：本发明的CD44多种配体-硅质体-钆双胺递送系统对动脉易损斑块的体内示踪实验(MRI示踪)
本实施例的目的是验证本发明所述的负载MRI示踪剂的硅质体表面分别装配多种不同CD44配体构成的纳米递送系统对动脉易损斑块的体内示踪效果。
实验方法：
(1)利用市售的原料药钆双胺，采用上述实施例1中所述的方法制备不同CD44配体作为靶向探针的，负载MRI示踪剂的硅质体纳米递送系统。
(2)按照上述实施例6中所述的方法构建小鼠动脉粥样硬化易损斑块模型。
(3)将模型小鼠用高脂饮食(同实施例5)喂养16周。取42只模型小鼠，随机分成空白硅质体组(6只)，HA-硅质体组(6只)，胶原-钆双胺-硅质体组(6只，给予市售的钆双胺原料药，钆双胺的给药浓度为0.5mg/ml，给药量为10ml/kg)，层黏连蛋白-钆双胺-硅质体组(6只，给予市售的钆双胺原料药，钆双胺的给药浓度为0.5mg/ml，给药量为10ml/kg)，纤黏连蛋白-钆双胺-硅质体组(6只，给予市售的钆双胺原料药，钆双胺的给药浓度为0.5mg/ml，给药量为10ml/kg)，选择蛋白-钆双胺-硅质体组(6只，给予市售的钆双胺原料药，钆双胺的给药浓度为0.5mg/ml，给药量为10ml/kg)，骨桥蛋白-钆双胺-硅质体组(6只，给予市售的钆双胺原料药，钆双胺的给药浓度为0.5mg/ml，给药量为10ml/kg)。各实验组分别经尾静脉注入相应的示踪剂，并于给药前以及给药后多时间点进行MRI成像，观察各组动物的动脉粥样硬化易损斑块的识别情况。
实验结果：
图19展示了本发明所述的CD44多种配体作为探针的纳米递送系统对动脉易损斑块的体内示踪效果。如图所示，与空白硅质体组相比，使用本发明所述的CD44多种配体(包括HA、胶原、层黏连蛋白、纤黏连蛋白、选择蛋白、骨桥蛋白)作为靶向探针的硅质体纳米系统均可显示对动脉易损斑块的识别作用，产生较好的MRI示踪效果。
实施例12：本发明的HA-多种粒径硅质体-碘克沙醇递送系统的组织分布
本实施例的目的是验证本发明所述的负载CT示踪剂的不同粒径的硅质体纳米递送 系统的组织分布。
实验方法：
(1)利用市售的原料碘克沙醇，采用下列方法制备不同粒径的硅质体纳米递送系统(HA分子量10万Da)：
(2)按照上述实施例6中所述的方法构建小鼠动脉粥样硬化易损斑块模型。
(3)将模型小鼠用高脂饮食(同实施例5)喂养16周。取36只模型小鼠，随机分成粒径280nm硅质体组(12只)，粒径200nm硅质体组(12只)，粒径160nm硅质体组(12只)，载体中负载市售的碘克沙醇原料药，碘克沙醇的给药浓度为0.5mg/ml，给药量为10ml/kg，各实验组分别经尾静脉注入相应的示踪剂，并于给药前以及给药后4个时间点进行组织采集(每个时间点3只动物)，观察各组动物的碘克沙醇组织分布。
实验结果：
表3展示了本发明所述的负载CT示踪剂的不同粒径的硅质体纳米递送系统的组织分布。如表所示，其他粒径的硅质体组相比，使用本发明所述的200nm硅质体可在动脉易损斑块中更好的富集。
表3 单次静脉注射不同粒径硅质体后组织中的浓度
Figure PCTCN2018082850-appb-000010
实施例13：本发明的多种分子量HA-硅质体-碘克沙醇递送系统的组织分布
本实施例的目的是验证本发明所述的负载CT示踪剂的不同分子量HA作为识别配体的硅质体纳米递送系统的组织分布。
实验方法：
(1)利用市售的原料碘克沙醇，采用下列方法制备硅质体纳米递送系统(硅质体粒径200nm)：1g透明质酸钠HA1，HA2，HA3(分子量分别为：20万Da，10万Da，3万Da)充分溶解于超纯水中，加入0.1g 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC.HCl)和0.12g N-羟基硫代琥珀酰亚胺(sulfo-NHS)偶联剂活化羧基。在室温搅拌反应1小时后，加入丙酮沉淀活化的HA。将沉淀过滤、用乙醇洗涤并真空干燥，得到活化的HA。将其配置成为0.1mg mL-1的水溶液，吸取0.2mL溶液溶于上面的步骤中得到的硅质体囊泡悬 浮液中，使活化的HA中的活化的羧基和硅质体囊泡的脂质双分子层中掺杂的DSPE分子具有的氨基通过形成酰胺键实现偶联，从而得到三种负载治疗剂的硅质体递送系统HA1-CL1@R、HA2-CL1@R和HA3-CL1@R。
(2)按照上述实施例6中所述的方法构建小鼠动脉粥样硬化易损斑块模型。
(3)将模型小鼠用高脂饮食(同实施例5)喂养16周。取36只模型小鼠，随机分成HA1-CL1@R组(12只)，HA2-CL1@R组(12只)，HA3-CL1@R组(12只)，载体中负载市售的碘克沙醇原料药，碘克沙醇的给药浓度为0.5mg/ml，给药量为10ml/kg，各实验组分别经尾静脉注入相应的示踪剂，并于给药前以及给药后4个时间点进行组织采集(每个时间点3只动物)，观察各组动物的碘克沙醇组织分布。
实验结果：
表4展示了本发明所述的负载CT示踪剂的不同分子量HA-硅质体纳米递送系统的组织分布。如表所示，其他分子量HA相比，使用本发明所述的10万DaHA可在动脉易损斑块中更好的富集。
表4 单次静脉注射不同分子量HA的硅质体后组织中的浓度
Figure PCTCN2018082850-appb-000011
已经通过上述实施例对本发明的各个方面进行了例示。显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (47)

  1. 一种用于靶向活化的CD44分子的硅质体递送系统，其特征在于，所述硅质体的表面部分地被靶向配体修饰，所述靶向配体是能与活化的CD44分子特异性结合的配体。
  2. 一种用于靶向易损斑块的硅质体递送系统，其特征在于，所述硅质体的表面部分地被靶向配体修饰，所述靶向配体是能与易损斑块处的细胞表面上的CD44分子特异性结合的配体。
  3. 权利要求1-2中任一项的硅质体递送系统，其特征在于，所述硅质体递送系统包括硅质体囊泡，其中所述硅质体囊泡的表面部分地被靶向配体修饰。
  4. 权利要求3所述的硅质体递送系统，其特征在于，所述硅质体囊泡是由脂质双分子层形成的，内部具有亲水性空腔的闭合囊泡，其中该囊泡的表面具有无机聚硅氧烷网状结构和偶联的靶向配体。
  5. 权利要求4所述的硅质体递送系统，其特征在于，所述脂质双分子层是由包括硅质体单体分子、与靶向配体通过共价键偶联在一起的二硬脂酰磷脂酰乙醇胺(DSPE)分子和任选存在的其它脂质分子在内的组分形成。
  6. 权利要求5所述的硅质体递送系统，其特征在于，所述硅质体单体分子、与靶向配体通过共价键偶联在一起的二硬脂酰磷脂酰乙醇胺(DSPE)分子和任选存在的其它脂质分子之间的重量配比为1-10∶0.2-1∶1-9，或3-7∶0.5-1∶1.5-2.5，或4-6∶0.5∶2。
  7. 权利要求5-6中任一项所述的硅质体递送系统，其特征在于，所述硅质体单体分子是能够形成硅质体的无机-有机复合脂质分子，所述无机-有机复合脂质分子由具有硅氧烷结构的头部和疏水性的尾部构成，其中所述疏水性的尾部是有机疏水双分子链。
  8. 权利要求5-7中任一项所述的硅质体递送系统，其特征在于，所述硅质体单体分子为具有如下结构通式的单体分子：
    (R 1O) 3Si-L-C(O)-N(R 2)(R 3)
    其中：
    R 1代表C 1-6烷基；
    L是由4-12个碳原子(优选4-10个碳原子)和1-2个氮原子构成的连接基，其中所述连接基中有0-1个碳原子被氧代基取代，即形成羰基，条件是(1)如果所述连接基中存在羰基，则所述羰基与氮原子毗邻；(2)所述连接基中的1个氮原子可以被季铵化，并且该季铵化的氮原子与适当的抗衡离子形成盐；
    R 2和R 3彼此独立地代表C 10-24烷基或C 10-24烯基。
  9. 权利要求5-8中任一项所述的硅质体递送系统，其特征在于，所述硅质体单体分子选自以下化合物中的一种或多种：
    Figure PCTCN2018082850-appb-100001
    Figure PCTCN2018082850-appb-100002
  10. 权利要求5-9中任一项所述的硅质体递送系统，其特征在于，所述脂质双分子层的组分包括其它脂质分子。
  11. 权利要求10所述的硅质体递送系统，其特征在于，所述其它脂质分子选自中性磷脂、负电荷磷脂和正电荷脂质中的一种或多种。
  12. 权利要求10所述的硅质体递送系统，其特征在于，所述其它脂质分子为正电荷。
  13. 权利要求12所述的硅质体递送系统，其特征在于，所述正电荷脂质选自3β-[N-(N′，N′-二甲基胺乙基)胺基甲酰基]胆固醇(DC-chol)、N-[1-(2，3-二油酰基)丙基-]-N，N，N-三乙胺氯(DOTMA)、2，3-二油酰氧-N-[2(精氨酸基酰胺)乙基]-N，N-二甲基-1-丙基-三氧乙酸胺(DOSPA)和1，2-二油酰氧丙基-N，N，N-三甲基溴化铵(DOTAP)中的一种或多种，优选为1，2-二油酰氧丙基-N，N，N-三甲基溴化铵(DOTAP)。
  14. 权利要求10所述的硅质体递送系统，其特征在于，所述其他脂质选自磷脂胆碱、甘油磷脂、乙醇胺磷脂、丝氨酸磷脂、磷脂酸中的一种或多种。
  15. 权利要求10所述的硅质体递送系统，其特征在于，所述其他脂质为磷脂胆碱。
  16. 权利要求3-15中任一项所述的硅质体递送系统，其特征在于，所述硅质体囊泡的粒径范围为50nm～400nm，优选50nm～300nm，优选150nm～250nm，优选180nm～220nm。
  17. 权利要求1-16中任一项所述的硅质体递送系统，其特征在于，所述靶向配体选自GAG、胶原、层黏连蛋白、纤黏连蛋白、选择蛋白、骨桥蛋白(OPN)以及单克隆抗体HI44a，HI313，A3D8，H90，IM7，或透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物。
  18. 权利要求1-17中任一项所述的硅质体递送系统，其特征在于，其中所述靶向配体具有5万～40万Da的分子量，优选具有8万～15万Da的分子量，优选具有约10万Da的分子量。
  19. 权利要求1-18中任一项所述的硅质体递送系统，其特征在于，所述硅质体负载有用于诊断、预防和/或治疗与出现CD44分子活化状况相关的疾病的物质。
  20. 权利要求1-9中任一项所述的硅质体递送系统，其特征在于，所述硅质体负载有用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质。
  21. 权利要求20中所述的硅质体递送系统，其特征在于，所述物质是用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的药物、多肽、核酸和细胞因子中的一种 或多种。
  22. 权利要求20所述的硅质体递送系统，其特征在于，所述物质是用于诊断易损斑块或与易损斑块相关的疾病的物质。
  23. 权利要求22所述的硅质体递送系统，其特征在于，所述用于诊断易损斑块或与易损斑块相关的疾病的物质是示踪剂。
  24. 权利要求23所述的硅质体递送系统，其特征在于，所述示踪剂选自CT示踪剂和MRI示踪剂。
  25. 权利要求24所述的硅质体递送系统，其特征在于，所述CT示踪剂选自碘纳米造影剂、金纳米造影剂、氧化钽纳米造影剂、铋纳米造影剂、镧系纳米造影剂，或其他类似结构的示踪剂，优选地，所述CT示踪剂选自碘化造影剂或纳米金，或其他类似结构的示踪剂，优选地，所述CT示踪剂选自碘海醇、碘卡酸、碘佛醇、碘克沙醇、碘普罗胺、碘比醇、碘美普尔、碘帕醇、碘昔兰、醋碘苯酸、胆影酸、碘苯扎酸、碘甘卡酸、泛影酸、碘他拉酸钠、碘苯酯、碘番酸、碘阿芬酸、醋碘苯酸钠、碘多啥、丙碘酮、碘奥酮、碘曲仑、碘吡多、胆影酸葡甲胺、碘他拉酸、泛影葡胺、甲泛影酸、甲泛葡铵、碘化油或乙碘油，或其他类似结构的示踪剂。
  26. 权利要求24所述的硅质体递送系统，其特征在于，所述MRI示踪剂选自纵向弛豫造影剂和横向弛豫造影剂。
  27. 权利要求24所述的硅质体递送系统，其特征在于，所述MRI示踪剂选自顺磁性造影剂、铁磁性造影剂和超磁性造影剂。
  28. 权利要求24所述的硅质体递送系统，其特征在于，所述MRI示踪剂选自Gd-DTPA及其线型、环型多胺多羧类螯合物和锰的卟啉螯合物，大分子钆螯合物、生物大分子修饰的钆螯合物、叶酸修饰的钆螯合物、树状大分子显影剂、脂质体修饰的显影剂和含钆富勒烯，或其他类似结构的示踪剂，优选地，所述MRI示踪剂选自钆喷酸葡胺、钆特酸葡胺、钆贝葡胺、钆双胺、枸橼酸铁铵泡腾颗粒、顺磁性氧化铁，或其他类似结构的示踪剂。
  29. 权利要求19-22中任一项所述的硅质体递送系统，其特征在于，所述物质是CD44活化剂，优选地，所述CD44活化剂是CD44抗体mAb或IL5、IL12、IL18、TNF-α、LPS。
  30. 权利要求19-22中任一项所述的硅质体递送系统，其特征在于，所述物质是分子量范围为2000～5000Da的小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物，优选地，所述小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物的分子量范围为2500～4500Da，优选3000～4000Da，优选3411Da。
  31. 权利要求19-22中任一项所述的硅质体递送系统，其特征在于，所述硅质体同时负载有用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质和CD44活化剂。
  32. 权利要求19-22中任一项所述的硅质体递送系统，其特征在于，所述硅质体同时 负载有用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质和分子量范围为2000～5000Da的小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物。
  33. 权利要求19-22中任一项所述的硅质体递送系统，其特征在于，所述硅质体同时负载有用于诊断易损斑块或与易损斑块相关的疾病的物质、用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质、任选的CD44活化剂和任选的分子量范围为2000～5000Da的小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物。
  34. 权利要求19-22或31-33中任一项所述的硅质体递送系统，其特征在于，所述物质是用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质，优选地，所述用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质选自他汀类药物、贝特类药物、抗血小板药物、PCSK9抑制剂、抗凝药物、血管紧张素转换酶抑制剂(ACEI)、钙离子拮抗剂、MMPs抑制剂、β受体阻滞剂，以及它们的药学上可接受的盐中的一种或多种，包括这些种类药物的活性结构片段，优选地，所述用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质选自洛伐他汀、阿托伐他汀、瑞舒伐他汀、辛伐他汀、氟伐他汀、匹伐他汀、普伐他汀、苯扎贝特、环丙贝特、氯贝特、吉非贝齐、非诺贝特、普罗布考，抗PCSK9抗体如evolocumab、alirocumab、bococizumab、RG7652、LY3015014和LGT-209，反义RNAi寡核苷酸如ALN-PCSsc，核酸如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反义链以及它们的核酸类似物如锁核酸，或adnectin如BMS-962476，阿司匹林、阿西美辛、曲克芦丁、双嘧达莫、西洛他唑、盐酸噻氯匹定、奥扎格雷钠、氯吡格雷、普拉格雷、西洛他唑、替罗非班、贝列前素钠、替格瑞洛、坎格瑞洛、替罗非班、依替巴肽、阿昔单抗、IIb/IIIa受体拮抗剂、普通肝素、克赛、速碧林、黄达肝葵钠、华法林、达比加群、利伐沙班、阿哌沙班、依度沙班、比伐卢定、依诺肝素、替他肝素、阿地肝素、双香豆素、硝酸香豆素、枸杞酸钠、水蛭素、阿加曲班，贝那普利、卡托普利、依那普利、培多普利、福辛普利、赖诺普利、莫昔普利、西拉普利、培哚普利、喹那普利、雷米普利、群多普利、坎地沙坦，依普罗沙坦、厄贝沙坦、氯沙坦、替米沙坦、缬沙坦、奥美沙坦或他索沙坦、硝苯地平、尼卡地平、尼群地平、氨氯地平、尼莫地平、尼索地平、尼伐地平、伊拉地平、非洛地平、拉西地平、地尔硫卓、维拉帕米、氯己定、米诺环素、MMI-166、美托洛尔、阿替洛尔、比索洛尔、普萘洛尔、卡维地络、巴马司他、马立马司他、普啉司他、BMS-279251、BAY 12-9566、TAA211、AAJ996A、nacetrapib、evacetrapib、Torcetrapib和Dalcetrapib以及它们的药效片段或药学上可接受的盐中的一种或多种，以及它们的药学上可接受的盐中的一种或多种，包括这些种类药物的活性结构片段。
  35. 一种药物，其包含权利要求1-34中任一项所述的硅质体递送系统，以及药学上可接受的载体。
  36. 一种诊断制剂，其包含权利要求1-34中任一项所述的硅质体递送系统。
  37. 权利要求1-34中任一项所述的硅质体递送系统在制备用于预防和/或治疗与出现CD44分子活化状况相关的疾病的药物中的用途。
  38. 权利要求1-34中任一项所述的硅质体递送系统在制备用于预防和/或治疗与易损斑块或与易损斑块相关的疾病的药物中的用途。
  39. 权利要求1-34中任一项所述的硅质体递送系统在制备用于诊断易损斑块或与易损斑块相关的疾病的诊断制剂中的用途。
  40. 权利要求38或39所述的用途，其中所述易损斑块选自破裂斑块、侵蚀性斑块和部分钙化结节性病变中的一种或多种。
  41. 权利要求38或39所述的用途，其中所述与易损斑块相关的疾病选自动脉粥样硬化症、冠状动脉粥样硬化性心脏病(包括急性冠脉综合征、无症状心肌缺血-隐匿性冠心病、心绞痛、心肌梗死、缺血性心脏病、猝死、支架内再狭窄)、脑动脉粥样硬化症(包括脑卒中)、外周血管动脉粥样硬化症(包括颈动脉粥样硬化症、肾动脉粥样硬化症、下肢动脉粥样硬化症、上肢动脉粥样硬化症)、主动脉夹层、血管瘤、血栓栓塞、心力衰竭和心源性休克中的一种或多种。
  42. 一种用于预防和/或治疗与出现CD44分子活化状况相关的疾病的方法，其特征在于，所述方法给予权利要求1-34中任一项所述的硅质体递送系统。
  43. 一种用于预防和/或治疗易损斑块或与易损斑块相关的疾病的方法，其特征在于，所述方法给予权利要求1-34中任一项所述的硅质体递送系统。
  44. 一种用于诊断与出现CD44分子活化状况相关的疾病的方法，其特征在于，所述方法包括给予权利要求1-34中任一项所述的硅质体递送系统。
  45. 一种用于诊断易损斑块或与易损斑块相关的疾病的方法，其特征在于，所述方法包括给予权利要求1-34中任一项所述的硅质体递送系统。
  46. 权利要求43或45所述的方法，其中所述易损斑块选自破裂斑块、侵蚀性斑块和部分钙化结节性病变中的一种或多种。
  47. 权利要求43或45所述的方法，其中所述与易损斑块相关的疾病选自动脉粥样硬化症、冠状动脉粥样硬化性心脏病(包括急性冠脉综合征、无症状心肌缺血-隐匿性冠心病、心绞痛、心肌梗死、缺血性心脏病、猝死、支架内再狭窄)、脑动脉粥样硬化症(包括脑卒中)、外周血管动脉粥样硬化症(包括颈动脉粥样硬化症、肾动脉粥样硬化症、下肢动脉粥样硬化症、上肢动脉粥样硬化症)、主动脉夹层、血管瘤、血栓栓塞、心力衰竭和心源性休克中的一种或多种。
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