WO2020147754A1 - 立方形环糊精骨架-rgd组合物及其制备方法 - Google Patents

立方形环糊精骨架-rgd组合物及其制备方法 Download PDF

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WO2020147754A1
WO2020147754A1 PCT/CN2020/072261 CN2020072261W WO2020147754A1 WO 2020147754 A1 WO2020147754 A1 WO 2020147754A1 CN 2020072261 W CN2020072261 W CN 2020072261W WO 2020147754 A1 WO2020147754 A1 WO 2020147754A1
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rgd
cyclodextrin
cof
composition
cubic
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PCT/CN2020/072261
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English (en)
French (fr)
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张继稳
何亚平
苏勇
徐建
伍丽
孙娴
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中国科学院上海药物研究所
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Priority to JP2021541608A priority Critical patent/JP2022523006A/ja
Priority to US17/423,505 priority patent/US20220073653A1/en
Priority to EP20740833.7A priority patent/EP3913003A4/en
Publication of WO2020147754A1 publication Critical patent/WO2020147754A1/zh

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Definitions

  • the present invention relates to the field of biological materials, and more specifically to a cubic cyclodextrin skeleton-RGD composition and a preparation method thereof.
  • Non-targeted agents such as urokinase and other thrombolytic drugs have problems such as bleeding side effects and excessive exposure of the system.
  • urokinase and other thrombolytic drugs Due to the complex hemodynamics of the blood circulatory system and the rapid clearance of general carriers, targeted delivery of therapeutic drugs or imaging agents to the site of vascular injury still faces great challenges.
  • the physical and chemical properties of the carrier such as the shape will affect the fate and biological function of the nanocarrier in vivo.
  • Recent studies have shown that non-spherical drug carriers can evade the body's clearance mechanism, prolong circulation time, and increase adhesion to the surface of blood vessels.
  • Research on morphologically controllable, targeted and non-spherical morphological carriers has brought new hopes for targeted diagnosis and efficient treatment of vascular-related diseases.
  • the mortality rate caused by uncontrolled blood loss is high in hospitals and on the battlefield. In the case of severe injuries, the normal physiological process of hemostasis is far from enough, such as a car accident or war trauma. Effective and rapid hemostasis and reduction of bleeding time have become important measures to reduce patient mortality.
  • the current clinically commonly used hemostatic materials such as hemostatic gauze, hemostatic fibers, and hemostatic bandages all have limitations. These hemostatic materials can only be used for external bleeding, and have a long time to stop bleeding. They are easy to adhere to the wound and are not easy to change the dressing. Suppuration is powerless. Most of the internal bleeding is caused by organ rupture, and because external hemostatic drugs cannot be used, it is almost impossible to stop bleeding in the first time after injury.
  • Recombinant human coagulation factor VII (rFVII) is a representative of systemic hemostatic drugs, but rFVII has the disadvantages of expensive, easy to inactivate, and difficult to store, which greatly limits its clinical application.
  • the immunogenicity, difficulty in storage, and inactivation of platelet products also limit its application in emergency treatment. Therefore, there is a huge clinical demand for intravenously injectable hemostatic materials for the treatment of internal bleeding.
  • the specific binding of the RGD sequence and the GPIIb/IIIa receptor on the surface of activated platelets is the final pathway to form a hemostatic clot.
  • the RGD sequence can only bind to activated platelets at the bleeding site, while resting platelets in normal blood circulation do not have GPIIb/IIIa on the surface. Receptors, so there is no effect on circulating platelets.
  • researchers using polymer materials to load RGD peptides to construct artificial platelets have become a new research direction for hemostatic materials.
  • Lavik's team used PLGA-PLL as a polymer carrier, connected GRGDS pentapeptide to PLGA-PLL-PEG, and constructed a synthetic platelet.
  • the artificial platelets can be targeted to activate platelets through intravenous administration, promote platelet aggregation and further trigger the coagulation mechanism, and can reduce bleeding time by 45% in a rat femoral artery injury model.
  • this artificial platelet has disadvantages such as a complicated synthesis method.
  • the Anirban Sen Gupta team used circular RGD to modify nanoliposomes, and the artificial blood plate constructed by the tail vein injection reduced the bleeding time by about 50% in the mouse tail dock injury model.
  • the Mitragotri team used GRGDS to modify PAH-BSA nanoparticles to simulate the morphology and rheology of platelets. Synthetic artificial platelets can reduce the bleeding time of tail docking injuries in mice by 45%.
  • Zhang Jianxiang's team injected the positively charged nanoparticles synthesized from cholic acid and polyethyleneimine into the tail vein, which can reduce the bleeding time by about 40% in the rat femoral artery injury model.
  • Tan Yingxia and others invented the artificial platelet PLGA-PEG-RGD with RGD loaded on PLGA-PEG nanoparticles.
  • PLGA-PEG-RGD has a regular spherical shape, but the particle size is not uniform, although it can be used as a nano-hemostatic material for veins.
  • the hemostatic effect in the rat liver injury model is limited, and the hemostatic time can only be reduced by about 30%.
  • the hemostatic efficiency of artificial platelets largely depends on the surface biological functions and physical and mechanical properties of the nanoparticles, such as size and shape.
  • the morphology of nanocarriers not only affects their dynamic migration to the blood vessel wall, but also affects their adhesion and aggregation interaction with activated platelets.
  • carriers with anisotropic shapes such as ellipsoids and rods
  • the geometry of the carrier will also affect its circulation and clearance processes in the body. Therefore, regulating the shape of the carrier brings new hope for overcoming the physiological barrier and improving the efficiency of hemostasis.
  • the purpose of the present invention is to provide a cyclodextrin skeleton-RGD composition (RGD-COF) and its preparation method and application.
  • a cyclodextrin skeleton-RGD composition is provided.
  • the mass ratio of the cyclodextrin skeleton to RGD is 1:0.001 to 1:1, preferably 1. :0.005-1:0.5;
  • the particle size of the cyclodextrin skeleton-RGD composition is 10nm-50 ⁇ m, preferably 50nm-50 ⁇ m, more preferably 100-500nm or 1-5 ⁇ m.
  • the composition is a cubic cyclodextrin skeleton-RGD composition.
  • the mass ratio of the cyclodextrin skeleton to RGD is 1:0.001-1:1, preferably 1:0.005-1:0.5 , More preferably 1:0.04-1:0.5.
  • the mass ratio of the cyclodextrin skeleton to RGD is 1:0.005-1:0.1, preferably 1:0.05.
  • the mass ratio of the cyclodextrin skeleton to RGD in the cubic cyclodextrin skeleton-RGD composition is 1:0.05.
  • the mass ratio of the cyclodextrin skeleton to RGD is 1:0.049, 1:0.08, 1:0.005, 1:0.015, 1:0.05 , 1:0.016, 1:0.065, or 1:0.046.
  • the particle size of the cubic cyclodextrin skeleton-RGD composition is 50nm-50 ⁇ m.
  • the particle size of the cubic cyclodextrin skeleton-RGD composition is 100-500 nm.
  • the particle size of the cubic cyclodextrin skeleton-RGD composition is 100-300 nm, preferably 150-200 nm.
  • the particle size of the cubic cyclodextrin skeleton-RGD composition is 1-50 ⁇ m, preferably 30-50 ⁇ m, more preferably 10-30 ⁇ m.
  • the particle size of the cubic cyclodextrin skeleton-RGD composition is 1-5 ⁇ m.
  • the cubic cyclodextrin skeleton-RGD composition has a particle size of 200-500nm, 100-300nm, 200-400nm, 200-500nm, 1-10 ⁇ m, 1-5 ⁇ m, 30-50 ⁇ m, Or 10-30 ⁇ m.
  • the RGD includes a linear RGD and a circular RGD.
  • the RGD is a linear RGD.
  • the RGD is selected from the group consisting of RGD, GRGD, RGDS, RGDV, RGDF, GRGDV, GRGDF, GRGDS, RGDDSP, RGDDAP, other polypeptides containing RGD sequences, or a combination thereof.
  • the linear RGD is selected from the following group: linear RGD, linear GRGD, linear RGDS, linear GRGDS, or a combination thereof.
  • the ring-shaped RGD is selected from the following group: ring-shaped RGD, ring-shaped GRGD, ring-shaped RGDS, ring-shaped RGDV, ring-shaped RGDF, ring-shaped GRGDV, ring-shaped GRGDF, ring-shaped GRGDS, ring-shaped RGDDSP, circular RGDDAP, other polypeptides containing circular RGD sequence, or a combination thereof.
  • the cyclodextrin is selected from the following group: ⁇ -cyclodextrin (alpha cyclodextrin), ⁇ -cyclodextrin (beta cyclodextrin), ⁇ -cyclodextrin (gamma Cyclodextrin), hydroxypropyl- ⁇ -cyclodextrin, sulfobutyl- ⁇ -cyclodextrin, methyl- ⁇ -cyclodextrin, carboxymethyl- ⁇ -cyclodextrin, or a combination thereof.
  • the cyclodextrin is selected from the following group: ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin, and more preferably ⁇ -cyclodextrin.
  • the cyclodextrin is ⁇ -cyclodextrin.
  • the cyclodextrin skeleton-RGD composition can reduce the clot formation time by 30%, 40%, 50%, 60%, 70%, 80% or 90% or more.
  • the cyclodextrin skeleton-RGD composition can shorten the bleeding time by more than 50%, 60%, 70%, 80%, 85%, 90% or 95%.
  • the cyclodextrin skeleton-RGD composition can reduce blood loss by more than 50%, 60%, 70%, 80%, 85%, 90% or 95%.
  • CD-MOF cubic cyclodextrin-metal organic framework
  • RGD modification step modifying RGD on the cyclodextrin skeleton described in (2) to obtain a cubic cyclodextrin skeleton-RGD composition (RGD-COF).
  • the crosslinking is to crosslink the cuboidal cyclodextrin-hydroxyl (-OH) on the metal organic framework through a crosslinking agent.
  • the cyclodextrin skeleton is a cyclodextrin skeleton that is stable in an aqueous system.
  • the cyclodextrin skeleton described in (2) is surface-modified with RGD to obtain a cubic cyclodextrin skeleton-RGD composition.
  • step (2) further includes:
  • the cross-linking step includes the following sub-steps:
  • the dispersion 2d is centrifuged to obtain crystals 2e;
  • the dispersion 2a is a suspension, emulsion, suspension, or colloid.
  • the dispersion 2b is a suspension, emulsion, suspension, or colloid.
  • the dispersion 2d is a suspension, emulsion, suspension, or colloid.
  • the crosslinking reaction temperature T is 30-110°C, preferably 40-100°C, more preferably 60-90°C, most preferably 70-80°C.
  • the crosslinking reaction temperature T is 80°C, 40°C, 100°C, 50°C, 70°C, or 60°C.
  • the molar ratio of the cyclodextrin-metal organic framework in step (2a) to the crosslinking agent in step (2b) is 1:1-1:20, preferably 1:2-1:10 , More preferably 1:4-1:8.
  • the molar ratio of the cyclodextrin-metal organic framework in step (2a) to the cross-linking agent in step (2b) is 1:6, 1:2, 1:5, 1:10, 1 : 20, 1:8, 1:15, or 1:4.
  • the crosslinking agent and the catalyst are added under stirring conditions, and the stirring speed is 200-1000 rpm, preferably 300-800 rpm, more preferably 400-600 rpm.
  • the crosslinking agent and the catalyst are added under stirring conditions, and the stirring speed is 600rpm, 200rpm, 1000rpm, 900rpm, 700rpm, 400rpm, 500rpm, or 700rpm.
  • the hydroxyl groups in the cyclodextrin-metal organic framework are cross-linked by covalent bonds.
  • the reaction time t1 is 4-48h, preferably 8-24h, more preferably 12-16h.
  • the reaction time t1 is 24h, 48h, 4h, 12h, 8h, or 16h.
  • cooling is cooling to room temperature.
  • the reaction terminator is ethanol.
  • the reaction terminator is 95-100% ethanol or 70-90% ethanol.
  • the centrifugation is at 3000-4500 rpm for 3-15 min, preferably at 4000 rpm for 5 min.
  • the washing is washing with ethanol, pure water and/or acetone.
  • the COF has a cubic shape.
  • the COF has a particle size of 50nm-50 ⁇ m, preferably 50-500nm or 1-50 ⁇ m.
  • the particle size of the COF is 100-500 nm.
  • the particle size of the COF is 100-300 nm, preferably 150-200 nm.
  • the particle size of the COF is 1-50 ⁇ m, preferably 30-50 ⁇ m, more preferably 10-30 ⁇ m.
  • the COF has a particle size of 1-5 ⁇ m.
  • the particle size of the COF is 200-500nm, 100-300nm, 200-400nm, 200-500nm, 1-10 ⁇ m, 1-5 ⁇ m, 30-50 ⁇ m, or 10-30 ⁇ m.
  • the RGD modification step includes the following sub-steps:
  • the hydroxyl group on the surface of the cyclodextrin skeleton that is coupled into a cubic shape is coupled with the carboxyl group of RGD.
  • the dispersion 3a is a homogeneously mixed dispersion.
  • the dispersion 3a is a suspension, emulsion, suspension, or colloid
  • the coupling step is carried out under heating and stirring conditions.
  • the rotation speed of the magnetic stirrer is 200-1000 rpm during the stirring process.
  • the magnetic stirrer rotates at 200 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 900 rpm, or 1000 rpm.
  • the heating temperature is 20-40°C, preferably 37-38°C.
  • the heating temperature is 20°C, 25°C, 30°C, 35°C, 37°C, 40°C.
  • reaction time t2 is 4-48h, preferably 8-24h, more preferably 12-20h.
  • reaction time t2 is 4h, 6h, 8h, 12h, 18h, 24h, 48h.
  • step (3a) the molar ratio of COF to RGD is 1:0.1-1:10, preferably 1:0.2-1:5, preferably 1:1.
  • step (3a) the molar ratio of COF to RGD is 1:1, 1:2, 5:1, 4:1, 2:1, 1:3, or 1:5.
  • the cubic cyclodextrin skeleton-RGD composition (RGD-COF) has a particle size of 200-500nm, 100-300nm, 200-400nm, 200-500nm, 1 -10 ⁇ m, 1-5 ⁇ m, 30-50 ⁇ m, or 10-30 ⁇ m.
  • the crosslinking agent is selected from the following group: peroxides, polyisocyanates, glycidyl ethers, dibasic or polybasic acids, dibasic or polybasic aldehydes, carbonyl-containing compounds, epoxy Compounds, acrylates, acid chlorides, or combinations thereof.
  • the peroxide is selected from the group consisting of benzoyl peroxide, dicumyl peroxide, tert-butyl peroxide, or a combination thereof.
  • the polyisocyanate is selected from the group consisting of isocyanate, toluene diisocyanate, diphenylmethane diisocyanate, dicyclohexylmethane diisocyanate, hexamethylene diisocyanate, lysine diisocyanate, or Its combination.
  • the glycidyl ether is selected from the group consisting of ethylene glycol diglycidyl ether, polypropylene glycol glycidyl ether, trimethylolpropane triglycidyl ether, n-butyl glycidyl ether, or combination.
  • the dibasic or polybasic acid is selected from the group consisting of citric acid, malonic acid, succinic acid, phthalic acid, isophthalic acid, or a combination thereof.
  • the binary or polyhydric aldehydes are selected from the following group: glyoxal, glutaraldehyde, succinaldehyde, or a combination thereof.
  • the carbonyl-containing compound is selected from the group consisting of diphenyl carbonate, N,N'-carbonyldiimidazole, N,N'-dimethylimidazoline, and dicyclohexylcarbodiimide , Or a combination thereof.
  • the epoxide is selected from the group consisting of epichlorohydrin, propylene oxide, 1,4-dioxane, or a combination thereof.
  • the acrylic esters are selected from the group consisting of ethylene glycol dimethacrylate, hydroxyethyl acrylate, hydroxypropyl acrylate, methacrylic acid, hydroxyethyl methacrylate, and methacrylic acid. Hydroxypropyl ester, or a combination thereof.
  • the acid chlorides are selected from the group consisting of succinyl chloride, tetraisocyanate, or a combination thereof.
  • the crosslinking agent is diphenyl carbonate.
  • the crosslinking agent is epichlorohydrin.
  • the catalyst A is selected from the following group: 4-dimethylaminopyridine, triethylamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or Salt, N,N'-succinimidyl carbonate, N-hydroxysuccinimide, pyridine, or a combination thereof; preferably triethylamine;
  • the organic solvent A is selected from the following group: dimethylformamide, tetrahydrofuran, methanol, acetonitrile, acetone, isopropanol, ethyl acetate, chloroform, n-hexane, ethanol, dichloromethane.
  • the organic solvent A is selected from the following group: dimethylformamide, tetrahydrofuran, methanol, acetonitrile, acetone, isopropanol, ethyl acetate, chloroform, n-hexane, ethanol, dichloromethane , Or a combination thereof.
  • the organic solvent A is dimethylformamide.
  • the catalyst A is selected from the following group: 4-dimethylaminopyridine, triethylamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or Salt, N,N'-succinimidyl carbonate, N-hydroxysuccinimide, pyridine, or a combination thereof.
  • the catalyst A is triethylamine.
  • the organic solvent B is selected from the following group: dimethylformamide, tetrahydrofuran, methanol, acetonitrile, acetone, isopropanol, ethyl acetate, chloroform, n-hexane, ethanol, dichloromethane , Or a combination thereof.
  • the organic solvent B is dimethylformamide.
  • the catalyst B is selected from the following group: 4-dimethylaminopyridine, triethylamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or Salt, N-hydroxysuccinimide, N,N'-succinimidyl carbonate, pyridine, or a combination thereof.
  • the catalyst B is 4-dimethylaminopyridine.
  • the catalyst A is selected from the following group: 4-dimethylaminopyridine, triethylamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or Salt, N,N'-succinimidyl carbonate, N-hydroxysuccinimide, pyridine, or a combination thereof;
  • the catalyst B is selected from the following group: 4-dimethylaminopyridine, triethylamine , 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide or its salt, N-hydroxysuccinimide, N,N'-succinimidyl carbonate, pyridine, or Its combination.
  • the organic solvent A is selected from the following group: dimethylformamide, tetrahydrofuran, methanol, acetonitrile, acetone, isopropanol, ethyl acetate, chloroform, n-hexane, or a combination thereof;
  • the organic solvent B is selected from the following group: dimethylformamide, tetrahydrofuran, methanol, acetonitrile, acetone, isopropanol, ethyl acetate, chloroform, n-hexane, ethanol, methylene chloride, or a combination thereof.
  • the catalyst B is selected from the following group: 4-dimethylaminopyridine, triethylamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or Salt, N-hydroxysuccinimide, N,N'-succinimidyl carbonate, pyridine, or a combination thereof;
  • the organic solvent B is selected from the following group: dimethylformamide, tetrahydrofuran, methanol, acetonitrile, acetone, isopropanol, ethyl acetate, chloroform, n-hexane, ethanol, methylene chloride, or a combination thereof.
  • the organic solvent B is dimethylformamide.
  • the catalyst B is 4-dimethylaminopyridine.
  • the RGD sequence includes linear RGD, GRGD, RGDS, GRGDS, circular RGD, GRGD, RGDS, GRGDS, and preferably linear RGD sequence includes GRGD, RGDS, GRGDS.
  • a drug-loaded cuboidal cyclodextrin matrix-RGD composition is provided, and the cuboidal cyclodextrin matrix-RGD composition is the composition described in the first aspect of the present invention , Or prepared by the method described in the second aspect of the present invention, the drug is selected from the group consisting of antibacterial drugs, hemostatic drugs, antithrombotic drugs, anti-infective drugs, or combinations thereof.
  • the antibacterial drug is selected from the group consisting of nano silver, penicillin, cephalosporin, minocycline, doxycycline, tetracycline, chloramphenicol, lincomycin, vancomycin, Or a combination.
  • the hemostatic drug is selected from the group consisting of tranexamic acid, aminocaproic acid, vitamin K1, or a combination thereof.
  • the antithrombotic drug is selected from the group consisting of aspirin, clopidogrel, ticlopidine, cilostazol, tirofiban, ozagrel, rivaroxaban, or a combination thereof .
  • the anti-infective drug is selected from the group consisting of sulfadiazine, ceftriaxone, amoxicillin, levofloxacin, or a combination thereof.
  • the drug-loaded cuboidal cyclodextrin matrix-RGD composition can be used for intravenous injection (nano-level) and topical medication (micron-level).
  • composition further has one or more of the following characteristics:
  • the particle size of the cyclodextrin skeleton-RGD composition is 50nm-50 ⁇ m, preferably 100-500nm or 1-5 ⁇ m;
  • the drug loading of the composition is 1%-20%, preferably 5%-10%.
  • the particle size of the cyclodextrin skeleton-RGD composition is 100-500 nm; or the particle size of the cyclodextrin skeleton-RGD composition is 1-5 ⁇ m.
  • the drug loading of the composition is 2.1%-13.5%.
  • the drug loading of the composition is further preferably 2.7%-8.6%.
  • the cyclodextrin skeleton-RGD composition is in powder form.
  • the cyclodextrin skeleton-RGD composition is in the form of particles.
  • the drug-loaded cubic cyclodextrin matrix-RGD composition can reduce the clot formation time by 30%, 40%, 50%, 60%, 70%, 80% or 90% the above.
  • the drug-loaded cubic cyclodextrin matrix-RGD composition can shorten the bleeding time by 50%, 60%, 70%, 80%, 85%, 90% or 95% or more.
  • the drug-loaded cubic cyclodextrin matrix-RGD composition can reduce blood loss by 50%, 60%, 70%, 80%, 85%, 90% or 95% or more.
  • an active ingredient selected from the following group:
  • the active ingredients are used for:
  • the therapeutic and/or diagnostic reagent or kit is used for disease treatment and/or diagnosis.
  • the disease is selected from the group consisting of thrombus, atherosclerosis, stroke, tumor, hemorrhage, inflammation, and infection.
  • the diagnostic reagent or kit is used for medical CT imaging.
  • the therapeutic and/or diagnostic reagent or kit is used for anti-tumor, hemostasis, anti-inflammatory, and anti-infection.
  • the hemostatic drug and/or material can reduce the clot formation time by more than 30%, 40%, 50%, 60%, 70%, 80% or 90%.
  • the hemostatic drug and/or material can shorten the bleeding time by more than 50%, 60%, 70%, 80%, 85%, 90% or 95%.
  • the hemostatic drugs and/or materials can reduce blood loss by more than 50%, 60%, 70%, 80%, 85%, 90% or 95%.
  • a pharmaceutical composition comprising:
  • Active ingredient which is the cyclodextrin skeleton-RGD composition as described in the first aspect of the present invention or the drug-loaded cuboidal cyclodextrin skeleton as described in the third aspect of the present invention- RGD composition;
  • the pharmaceutical composition is a capsule, tablet, or granule.
  • the carrier is selected from the group consisting of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants Agent, or a combination thereof.
  • the pharmaceutical composition is formulated as a solid dosage form or a liquid dosage form, which is preferably suitable for oral administration, and more preferably for injection administration.
  • the solid dosage forms include capsules, tablets, pills, powders and granules.
  • the liquid dosage form includes a pharmaceutically acceptable emulsion, solution, suspension, syrup or tincture.
  • the pharmaceutical composition is a capsule, tablet, granule, or injection.
  • the pharmaceutical composition further comprises a surfactant, selected from the group consisting of polysorbate-80, polysorbate-60, polyethylene glycol glycerol fatty acid ester, and sorbitan fatty acid Esters and mixtures of two or more.
  • a surfactant selected from the group consisting of polysorbate-80, polysorbate-60, polyethylene glycol glycerol fatty acid ester, and sorbitan fatty acid Esters and mixtures of two or more.
  • the pharmaceutical composition can reduce the clot formation time by more than 30%, 40%, 50%, 60%, 70%, 80% or 90%.
  • the pharmaceutical composition can shorten the bleeding time by more than 50%, 60%, 70%, 80%, 85%, 90% or 95%.
  • the pharmaceutical composition can reduce blood loss by more than 50%, 60%, 70%, 80%, 85%, 90% or 95%.
  • Figure 1 is a scanning electron micrograph of the nano-CD-MOF in Example 1.
  • Figure 2 is a scanning electron micrograph of the nano COF in Example 1.
  • Figure 3 is a scanning electron micrograph of the nano-RGD-COF in Example 1.
  • FIG. 4 is a diagram showing the particle size distribution of nano RGD-COF dynamic light scattering in Example 1.
  • FIG. 4 is a diagram showing the particle size distribution of nano RGD-COF dynamic light scattering in Example 1.
  • Figure 5 shows the physical stability of nano-RGD-COF in Example 1 ( ⁇ : pure water; ⁇ : physiological saline; ⁇ : PBS pH 7.4; ⁇ : rat plasma).
  • Figure 6 shows the cytotoxicity results of nano-RGD-COF in Example 1.
  • Figure 7 is a scanning electron micrograph of the micron CD-MOF in Example 6.
  • Fig. 8 is a scanning electron microscope image of the micron COF in Example 6.
  • Fig. 9 is a scanning electron microscope image of micron RGD-COF in Example 6.
  • FIG. 10 is a scanning electron micrograph of the spherical RGD-NS in Example 9.
  • FIG. 11 shows the results of in vitro clot formation time of RGD-COF in Example 10.
  • Figure 12 shows that RGD-COF in Example 11 can significantly reduce the bleeding time in the mouse tail dock model.
  • Figure 13 shows that the RGD-COF in Example 11 can significantly reduce the blood loss in the mouse tail dock model.
  • Fig. 14 shows the time-antibacterial curve of silver-loaded RGD-COF etc. in Example 12.
  • Figure 15 shows the healing effect of silver-loaded RGD-COF in Example 12 on wounds in rats.
  • Figure 16 shows that RGD-COF in Example 16 can target mesenteric thrombus in vivo, and has a high degree of co-localization with activated platelets at the thrombus site.
  • the cubic cyclodextrin skeleton-RGD composition of the present invention contains a cyclodextrin skeleton with a cubic structure and RGD, which can avoid the phagocytosis and clearance of macrophages, enhance the migration and adhesion to damaged blood vessels, and is highly efficient Targeted and aggregated activated platelets at the site of vascular injury to achieve targeted therapy of vascular-related diseases (such as bleeding and thrombosis).
  • the nano-level cubic cyclodextrin skeleton-RGD composition of the present invention can reduce bleeding time and blood loss by 90% in a mouse tail dock injury model by intravenous injection; micron-level cyclodextrin skeleton -The RGD composition can be used locally and externally to reduce the bleeding time by 60% in the rat femoral artery injury model and greatly improve the hemostatic efficiency. On this basis, the present invention has been completed.
  • Metal organic framework is an inorganic-organic framework formed by self-assembly of metals (metal ions, metal ion clusters or metal chains) and organic bridging ligands under relatively mild conditions through coordination bonds.
  • Organic hybrid materials Due to the ultra-high porosity and huge specific surface area of MOF, and the combination of inorganic and organic components, its structure and composition are diverse, providing MOF applications in gas storage, adsorption and separation, catalysis, drug delivery and other fields A new research direction.
  • Cyclodextrin (CD) is the general term for a series of cyclic oligosaccharides produced by glucosyltransferase from amylose, usually containing 6-12 D-glucopyranose units. Among them, the molecules that have been studied more and have important practical significance are the molecules containing 6, 7, and 8 glucose units, called ⁇ , ⁇ - and ⁇ -cyclodextrins, respectively. Cyclodextrin is an ideal host molecule similar to an enzyme found so far, and it has the characteristics of an enzyme model.
  • cyclodextrin-metal organic framework and “CD-MOF” can be used interchangeably. They are a new type of cyclodextrin as the organic ligand and metal ions as the inorganic metal center.
  • the medicinal cube-shaped cyclodextrin-metal organic framework namely CD-MOF.
  • the cyclodextrin-metal organic framework is a framework material formed by cyclodextrin and alkali metal salts; alkali metals include but are not limited to Li + , K + , Rb + , Cs + , Na + , Mg 2+ , Cd 2+ , Sn 2+ , Ag + , Yb + , Ba 2+ , Sr 2+ , Ca 2+ , Pb 2+ , La 3+ , preferably K + .
  • the average particle size of the cyclodextrin-metal organic framework material is 50 nanometers to 50 microns, preferably 100 to 500 nanometers (nano-scale) or 1-5 microns (micron-scale).
  • a cyclodextrin-metal organic framework (refer to Patent 201610125456.X): the preparation method includes mixing a metal salt solution with a cyclodextrin aqueous solution, and pre-adding a part of an organic solvent at a certain temperature , Through the solvent vapor diffusion method, react for a certain period of time, and then add the size regulator to obtain the cyclodextrin-based metal organic framework material; or mix the metal salt solution with the cyclodextrin aqueous solution, pre-add a part of the organic solvent, use
  • the solvothermal/microwave/ultrasonic vibration reaction medium makes the reactants react quickly, and the size regulator is added after the reaction for a certain period of time, thereby obtaining the cyclodextrin-based metal organic framework material.
  • the concentration of the metal salt in the metal salt solution is 0.05-0.4M, preferably 0.2M.
  • the concentration of cyclodextrin in the cyclodextrin aqueous solution is 0.013-0.05M, preferably 0.025M.
  • the cyclodextrin is selected from the following group: ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin, hydroxypropyl- ⁇ -cyclodextrin, sulfobutyl- ⁇ -cyclodextrin Methine, methyl- ⁇ -cyclodextrin, carboxymethyl- ⁇ -cyclodextrin, or a combination thereof.
  • the cyclodextrin is ⁇ -cyclodextrin.
  • the cyclodextrin-metal organic framework is cubic.
  • the preparation of the cyclodextrin-metal organic framework includes the steps:
  • the volume ratio of the first organic solvent to the first mixed solution is (0.01-0.5):1, preferably (0.03-0.3):1, most preferably (0.05-0.2):1 ;
  • (3b1) A combination of solvothermal volatilization treatment and any treatment method selected from group A, where group A includes solvothermal treatment, microwave treatment, ultrasonic treatment, or a combination thereof;
  • step (4a) The cyclodextrin-metal organic framework material precipitated in step (4a) is optionally separated and/or dried.
  • step (3a) the solvent thermal volatilization treatment includes the steps:
  • step (iii) the entire closed system is heated to heat the organic solvent in the open container II
  • the heating treatment includes water bath heating and oil bath heating.
  • the temperature of the heating treatment is 25-100°C, preferably 30-80°C, more preferably 40-60°C.
  • the heating treatment time is 4-48h, preferably 6-24h.
  • cyclodextrin framework material cyclodextrin framework
  • cyclodextrin framework cubic cyclodextrin framework
  • cubic cyclodextrin framework cubic cyclodextrin framework
  • CD-MOF contains K + , cannot be injected directly intravenously, and will quickly disintegrate in an aqueous environment, and cannot ensure the stability of the porous crystal structure before reaching the target site.
  • Furukawa et al. used ethylene glycol diglycidyl ether to crosslink ⁇ -CD-MOF to produce ⁇ -CD-MOF hydrogel.
  • this cross-linking reaction is extremely time-consuming, requiring more than three days at 65°C and many steps to remove unreacted impurities.
  • C60 fullerene
  • the supramolecular assembly system can only maintain the structural integrity for a short period of time and degrade after 24 hours in water.
  • the occupation of ⁇ -CD cavity by C60 may also reduce the ability of ⁇ -CD-MOF to load drugs.
  • the synthesis of stable porous CD-MOF materials is still a huge challenge.
  • the present invention uses diphenyl carbonate, epichlorohydrin, etc. as cross-linking agents to cross-link the ordered CD in the CD-MOF, and successfully prepares a cyclodextrin skeleton (COF) that is stable in water.
  • COF cyclodextrin skeleton
  • the cube shape of CD-MOF is retained, and the coordinated K + can be removed.
  • COF based on nano-scale CD-MOF crosslinking can be used as a new type of nanocarrier for intravenous injection.
  • the inventors used diphenyl carbonate, epichlorohydrin, etc. as the crosslinking agent to successfully crosslink the CD in the CD-MOF.
  • the preparation method of the cyclodextrin framework is as follows: Weigh an appropriate amount of prepared CD-MOF powder into a round bottom flask, fix it on a magnetic stirrer, and add a certain volume of Organic solvent, under heating and stirring conditions, add a certain amount of crosslinking agent and catalyst, and react for a period of time to make the hydroxyl (-OH) in the CD-MOF crosslink by covalent bonds, and then cool to room temperature, using 95% After the reaction was terminated by ethanol, the precipitate was obtained by centrifugation, washed twice with 50% ethanol, pure water and acetone each, and dried in vacuum to obtain a stable cyclodextrin skeleton (COF) in water.
  • the RGD sequence is the arginine-glycine-aspartic acid (Arg-Gly-Asp) sequence.
  • the endogenous RGD sequence exists in a variety of adhesion proteins (such as fibrinogen, vitronectin, fibronectin, von Willebrand factor), and plays a variety of biological functions.
  • adhesion proteins such as fibrinogen, vitronectin, fibronectin, von Willebrand factor
  • the specific binding between the RGD sequence on the A ⁇ chain of fibrinogen and the membrane glycoprotein GPIIb/IIIa receptor on the surface of activated platelets is a common pathway for platelet aggregation and thrombus formation.
  • the RGD sequence is the active site where fibrinogen binds to the activated GPIIb/IIIa receptor.
  • the RGD sequence of the present invention includes linear RGD, linear GRGD, linear RGDS, linear RGDV, linear RGDF, linear GRGDV, linear GRGDF, linear GRGDS, linear RGDDSP, linear RGDDAP; circular RGD , Ring GRGD, ring RGDS, ring RGDV, ring RGDF, ring GRGDV, ring GRGDF, ring GRGDS, ring RGDDSP, ring RGDDAP.
  • the RGD sequence of the present invention is preferably a linear RGD sequence, including linear GRGD, linear RGDS, linear RGDF, and linear GRGDS.
  • the terms "cubic cyclodextrin backbone-RGD composition”, “RGD modified COF”, “RGD-COF”, “cubic cyclodextrin backbone-RGD polypeptide composition” are used interchangeably, and are Refers to the cubic cyclodextrin skeleton-RGD composition described in the first aspect of the present invention.
  • a cubic cyclodextrin framework-RGD composition is characterized in that the hydroxy (-OH) in the cyclodextrin-metal organic framework material with a cubic crystal structure is used by a suitable crosslinking agent Covalently linked together to form a stable cubic structure cyclodextrin skeleton in water.
  • An external activator is used to covalently link the hydroxyl (-OH) on the surface with the carboxyl group (-COOH) of RGD to carry out the biology of the cyclodextrin skeleton. Retouch.
  • the cuboidal cyclodextrin skeleton is used as an artificial platelet carrier, and RGD is used as the target head, which specifically binds to the GPIIb/IIIa receptor on the activated platelet surface to target the activated platelet at the vascular injury site.
  • RGD modified cyclodextrin skeleton material is used as a new and efficient artificial platelet. This feature can improve the migration ability of artificial platelets to damaged blood vessels, improve the targeting of bleeding sites, and promote the combination with activated platelets.
  • the present invention provides a cubic cyclodextrin skeleton-RGD composition composed of COF and RGD, the inside of which is a cyclodextrin skeleton with a cubic crystal structure, and the surface is biologically modified with RGD.
  • the cuboidal cyclodextrin skeleton is used as the carrier, and RGD is used as the target head to target the GPIIb/IIIa receptors on the platelet surface activated at the vascular injury site.
  • RGD-modified COF can improve the ability of migration to damaged blood vessels, improve the targeting of damaged parts, and promote the binding and aggregation of activated platelets.
  • the cubic cyclodextrin skeleton-RGD composition of the present invention is different from the existing artificial platelet reports.
  • the Anirban Sen Gupta team used RGD and collagen binding peptides to modify spherical liposomes to construct artificial platelets through the tail vein After injection, it has a certain hemostatic effect in the mouse tail docking model, but the targeting and hemostatic effect still need to be improved.
  • Tan Yingxia and others invented the artificial platelet PLGA-PEG-RGD with RGD loaded on PLGA-PEG nanoparticles.
  • PLGA-PEG-RGD is a regular spherical shape, but the particle size is not uniform. Although it can be used as a systemic nano-hemostatic material for intravenous use, it has limited hemostatic effect in the rat liver injury model and can only reduce the bleeding time by 30% about.
  • the nano-level cuboidal cyclodextrin skeleton-RGD composition of the present invention can reduce the bleeding time and blood loss by 90% in a mouse tail dock injury model through intravenous injection, and the micron-level cyclodextrin skeleton-RGD composition Through local external application, the bleeding time can be reduced by 60% in the rat femoral artery injury model, and the hemostatic efficiency is greatly improved.
  • the preparation method of the cubic cyclodextrin skeleton-RGD composition is as follows: Weigh an appropriate amount of COF and RGD into a round bottom flask, and add a certain volume of organic solvent B. After mixing uniformly, add appropriate amount of catalyst B and activator, and place it on a magnetic stirrer to heat and stir for an appropriate time to fully couple the hydroxyl group (-OH) on the COF surface with the carboxyl group (-COOH) of RGD.
  • the cubic cyclodextrin skeleton-RGD composition (RGD-COF) provided by the present invention has high-efficiency targeting performance. It can be injected intravenously for systemic blood loss and thrombosis, and can also be used for local medication to treat traumatic injuries. The treatment of trauma, surgical bleeding, stroke and tumor-related diseases provides more options and broad application prospects.
  • the invention provides a method for preparing a cubic cyclodextrin skeleton-RGD composition.
  • the pharmaceutically acceptable cyclodextrin is used as the organic linker, and K + is used as the inorganic metal center to prepare the CD-MOF with a cubic shape and uniform particle size.
  • the CD of the MOF is cross-linked to prepare a stable COF in the water phase.
  • the carboxyl group (-COOH) of the RGD sequence is bonded to the COF surface through an ester bond to form a cubic cyclodextrin skeleton-RGD composition (RGD-COF ).
  • the CD-MOF involved in the present invention is based on a method (201610125456.X) for preparing a cyclodextrin-metal organic framework material, which is fast, simple, adjustable in size and high in yield.
  • the preparation method of the cubic cyclodextrin skeleton-RGD composition includes the steps:
  • CD-MOF cubic cyclodextrin-metal organic framework
  • the specific cross-linking reaction process is: dispersing the cubic cyclodextrin-metal organic framework in the organic solvent A, adding the cross-linking agent and the catalyst A under stirring at a certain temperature to react, so that the cyclodextrin- The hydroxyl groups in the metal-organic framework are cross-linked by covalent bonds. After cooling, ethanol termination of the reaction, centrifugation, washing and drying, a cubic cyclodextrin framework (COF) is obtained;
  • COF cubic cyclodextrin framework
  • RGD-COF cubic cyclodextrin skeleton-RGD composition
  • the specific RGD modification process is: Cyclodextrin skeleton and RGD are added to organic solvent B according to a certain proportion. After mixing evenly, add catalyst B to heat and stir to fully couple the hydroxyl groups on the surface of the cuboidal cyclodextrin skeleton with the carboxyl groups of RGD. After the reaction is completed, it is washed and dried to cube Cyclodextrin-shaped skeleton-RGD composition (RGD-COF).
  • “Pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility” here means that the components of the composition can be blended with the compound of the present invention and between them without significantly reducing the efficacy of the compound.
  • pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and solid lubricants (such as stearic acid).
  • Magnesium stearate calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • vegetable oils such as soybean oil, sesame oil, peanut oil, olive oil, etc.
  • polyols such as propylene glycol, glycerin, mannitol, sorbitol, etc.
  • emulsifiers such as emulsifiers
  • wetting agents such as sodium lauryl sulfate
  • the invention provides a cubic cyclodextrin skeleton-RGD composition (RGD-COF) that can be used for intravenous injection (nano-level) or topical medication (micron-level) and a preparation method thereof, which has high safety and biocompatibility Good sex. It can be used for targeted therapy of vascular-related diseases, including traumatic blood loss, deep hemorrhage, atherosclerosis, stroke, thrombosis and tumors. It has the following technical advantages:
  • the RGD-COF of the present invention has a regular cubic shape, breaks through the limitation of the previous carrier spherical shape, effectively avoids the phagocytosis and clearance of macrophages, enhances the migration and adhesion to damaged blood vessels, and improves the targeting and Treatment efficiency.
  • the cubic RGD-COF can efficiently target and aggregate the activated platelets at the vascular injury site, realize the targeted therapy of vascular-related diseases, and reduce the impact on the normal blood circulation system. side effect.
  • the present invention takes full advantage of the controllable size of CD-MOF, uses nano- and micro-scale CD-MOF as the basic material, after cross-linking step and RGD modification step, to obtain intravenous injection and/or external use New and efficient carrier material.
  • Intravenous administration is suitable for trauma treatment under complicated conditions, and is suitable for the control of systemic blood loss and thrombotic diseases; micron RGD-COF can be used for hemostasis, anti-inflammatory and anti-infection treatment of external trauma such as car accidents and operations.
  • the RGD-COF in the present invention has good stability and can be used as a freeze-dried powder, which is conducive to storage and suitable for use in field conditions, such as equipping troops to improve the ability to treat complex war wounds.
  • the preparation method of the present invention is simple and controllable, does not require expensive equipment, can be mass-produced, is not immunogenic, and does not spread infectious diseases.
  • the preparation method of the present invention is simple, has high safety and good biocompatibility, and can be used for targeted diagnosis and treatment of uncontrolled bleeding and thrombotic diseases such as traumatic blood loss, internal bleeding, deep bleeding and the like.
  • Preparation of nano-scale CD-MOF using solvothermal method to directly heat the mixed system of ⁇ -CD and KOH aqueous solution and a part of organic solvent. Weigh 163.0mg of ⁇ -CD and 56.0mg KOH mixture (the molar ratio of ⁇ -CD to KOH is 1:8) and dissolve it in 5mL of water, pre-add 3mL of methanol to the mixed solution, heat it in a water bath at 50°C for 20 minutes, remove the solution, add, etc. Then add 64mg PEG 20000 to the volume of methanol.
  • CD-MOF-Nano nanometer Grade CD-MOF crystal
  • the obtained CD-MOF has a regular cubic shape with a particle size of 200-500nm ( Figure 1).
  • nano-level COF weigh 778.3 mg of nano-level CD-MOF powder in a round bottom flask, fix it on a magnetic stirrer, add 10 mL of dimethylformamide, heat at 80°C and stir at 600 rpm, add 771 mg of cross-linking agent Diphenyl carbonate (the molar ratio of CD-MOF to crosslinking agent is 1:6) and 450 ⁇ L of catalyst triethylamine. After reacting for 24h, cool to room temperature, stop the reaction with 20mL 95% ethanol, and centrifuge at 4000rpm for 5min.
  • COF has a regular cubic shape with a particle size of 200-500nm ( Figure 2).
  • PBS phosphate buffered solution
  • the MTT results showed that RGD-COF is non-cytotoxic, has good biocompatibility and safety (Figure 6).
  • nano-level COF weigh 778.3 mg of nano-level CD-MOF powder in a round bottom flask, fix it on a magnetic stirrer, add 10 mL of acetonitrile, heat at 40°C and stir at 200 rpm, add 194.6 mg of cross-linking agent N, N '-Carbonyl diimidazole (the molar ratio of CD-MOF to crosslinking agent is 1:2) and 450 ⁇ L of catalyst pyridine, react for 48h, and the rest is the same as in Example 1.
  • the COF obtained is a regular cubic shape with a particle size of 100-300nm .
  • micron COF Weigh 778.3mg of micron CD-MOF powder in a round bottom flask, fix it on a magnetic stirrer, add 10mL ethyl acetate, heat at 70°C, and stir at 700rpm, add 0.9mL crosslinker ring Oxychloropropane (the molar ratio of CD-MOF to the crosslinking agent is 1:20) and 5mg of the catalyst 4-dimethylaminopyridine. After the reaction for 16 hours, the rest is the same as in Example 1.
  • the obtained COF has a regular cubic shape with a particle size It is 1-10 ⁇ m.
  • RGD-COF Prepare micron RGD-COF: Weigh 230mg micron COF and 4mg RGDS (the molar ratio of COF to RGDS is 2:1), put them in a round bottom flask, add 6mL n-hexane, stir well and then add 5mg 4-dimethyl Aminopyridine and 0.2mL triethylamine were placed on a magnetic stirrer at 25°C and 700rpm for 24h, and the rest were the same as in Example 1.
  • the micron cubic cyclodextrin skeleton-RGD composition RGD-COF, SEM and DLS results were obtained It is shown that the obtained RGD-COF has a regular cubic shape with a size of 1-10 ⁇ m.
  • the mass ratio of the cyclodextrin framework material to RGD measured by the HPLC method is 1:0.05.
  • a micron-sized CD-MOF was prepared with a particle size of 1-5 ⁇ m ( Figure 7).
  • micron COF Prepare micron COF: Weigh 778.3mg of micron CD-MOF powder in a round bottom flask, fix it on a magnetic stirrer, add 10 mL of acetone, heat at 50°C and stir at 400 rpm, add 0.7 mL of crosslinking agent toluene diisocyanate (The molar ratio of CD-MOF to crosslinking agent is 1:8) and 450 ⁇ L of catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. After 8 hours of reaction, the rest is the same as in Example 1. The obtained COF has a regular cubic shape with a particle size of 1-5 ⁇ m ( Figure 8).
  • Example 5 As in Example 5, a micron-sized CD-MOF was prepared.
  • micron COF Prepare micron COF: Weigh 778.3mg of micron CD-MOF powder in a round bottom flask, fix it on a magnetic stirrer, add 10mL isopropanol, heat at 60°C and stir at 500rpm, add 1729mg crosslinker citric acid (The molar ratio of CD-MOF to crosslinking agent is 1:15) and 450 ⁇ L of catalyst pyridine. After reacting for 16 hours, the rest is the same as in Example 1. The obtained COF has a regular cubic shape with a particle size of 30-50 ⁇ m.
  • RGD-COF Preparing micron RGD-COF: Weigh 230mg micron COF and 30mg GRGDS pentapeptide (the molar ratio of COF to ring GRGDS is 1:3), put them in a round bottom flask, add 6mL n-hexane, stir well and then add 5mg 4- Dimethylaminopyridine and 6mg N,N'-succinimidyl carbonate were placed on a magnetic stirrer at 30°C and 500rpm for 24h. The rest were the same as in Example 1. The obtained RGD-COF was in a regular cubic shape. The diameter is 30-50 ⁇ m, and the mass ratio of cyclodextrin framework material to RGD measured by HPLC method is 1:0.065.
  • Example 5 As in Example 5, a micron-sized CD-MOF was prepared.
  • micron-level COF weigh 778.3 mg of micron-level CD-MOF powder in a round bottom flask, fix it on a magnetic stirrer, add 10 mL of chloroform, heat at 70°C, and stir at 700 rpm, add 403 mg of crosslinking agent succinyl chloride ( The molar ratio of CD-MOF to crosslinking agent is 1:4) and 450 ⁇ L of catalyst pyridine. After reacting for 16 hours, the rest is the same as in Example 1.
  • the obtained COF has a regular cubic shape with a particle size of 10-30 ⁇ m.
  • CD-NS spherical cyclodextrin nanosponges
  • GRGDS modified cyclodextrin nanosponge Weigh 230 mg spherical CD-NS and 10 mg GRGDS pentapeptide (the molar ratio of CD-NS to GRGDS is 1:1), place them in a round bottom flask, and add 5 mL of dimethyl Formamide, stir well, then add 5mg 4-dimethylaminopyridine and 6mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, place it on a magnetic stirrer at 37°C, Stir at 600rpm for 24h to fully couple CD-NS and GRGDS polypeptide.
  • the in vitro coagulation performance of the hemostatic material can be evaluated. Take an appropriate amount of fresh whole blood anticoagulated by healthy rats with 3.2% sodium citrate into a clean test tube, add 80 ⁇ L CaCl 2 (0.1M) to make the final concentration of Ca 2+ 10mM, and then add sample solution or physiological saline to make the reaction The final volume of the system is 800 ⁇ L and vortexed at 500 rpm/min for 10 seconds. Set up saline control group (blank control), RGD-COF group (20, 50, 100 ⁇ g/mL), COF group (100 ⁇ g/mL) and RGD-NS group (100 ⁇ g/mL).
  • the RGD-COF low, medium and high dose groups prepared in Example 2 reduced the clot formation time by 32%, 49% and 61%, respectively.
  • the low, medium, and high-dose RGD-COF groups prepared in Example 3 reduced the clot formation time by 36%, 55%, and 69%, respectively.
  • the RGD-COF low, medium and high dose groups prepared in Example 6 reduced the clot formation time by 37%, 48% and 65%, respectively.
  • Nano-level RGD-COF hemostatic drug for intravenous injection can be injected intravenously after being dispersed with saline.
  • the preparation method of nano-level RGD-COF hemostatic drug for intravenous injection includes the following steps :
  • Nanoparticle dispersion Suspend nanoparticles (1-10 mg/mL, preferably 2 mg/mL) in physiological saline to obtain a nanoparticle dispersion.
  • a mouse tail docking model was used to evaluate the hemostatic ability of RGD-COF nanoparticles in vivo.
  • Fifty healthy Kunming mice were randomly divided into 5 groups with 10 mice in each group. Set up a blank control group (only tail dock injury treatment, corresponding to the "damage treatment" in Figure 12 and Figure 13), physiological saline group, COF group, RGD-NS group, RGD-COF low-dose group (20mg/kg, That is, 20 mg/kg in Figure 12 and Figure 13) and the RGD-COF high-dose group (40 mg/kg, that is, 40 mg/kg in Figure 12 and Figure 13).
  • the administration method is tail vein injection, a single administration, the administration dose is 20-40 mg/kg body weight, and the administration volume: 10 mL/kg. 5min after administration, use sharp scissors to cut quickly 0.5cm away from the tail tip of the mouse.
  • the blood flows out by itself count the time immediately. Every 20s, touch the broken part of the tail with absorbent paper until there is no more blood in the tail. Outflow (no blood streaks appear), this period is recorded as bleeding time.
  • the Image Pro Plus software was used to calculate the area of the blood drop on the absorbent paper, and then to estimate the blood loss of the mouse.
  • the results of in vivo hemostasis experiments show that RGD-COF can significantly shorten the bleeding time of mice, and can greatly reduce the amount of bleeding in mice, and has a good hemostatic effect in vivo.
  • the RGD-COF low-dose group (20mg/kg) prepared in Example 1 can shorten the bleeding time from 400s to 150s, and shorten the bleeding time
  • the blood loss was reduced by 62.5%; the blood loss was reduced from 2mL/kg to 0.6mL/kg, and the blood loss was reduced by 70%.
  • the high-dose RGD-COF group (40 mg/kg) prepared in Example 1 can shorten the bleeding time from 400 s to 40 s, which is a reduction of 90%; and reduce blood loss to 0.2 mL/kg, which is a reduction of 90%.
  • the RGD-COF low-dose group prepared in Example 2 can shorten the bleeding time from 400s to 155s, and the bleeding time is reduced by 61%; the blood loss is reduced from 2mL/kg to 0.8mL/kg, and the blood loss is reduced by 60%.
  • the high-dose RGD-COF group prepared in Example 2 can shorten the bleeding time from 400s to 50s, which is a reduction of 87.5%; and reduce the blood loss to 0.12mL/kg, which is a reduction of 94%.
  • the spherical RGD-NS prepared in Example 9 only shortened the bleeding time by 23%, and could not reduce blood loss. Therefore, cubic RGD-COF has a more effective hemostatic effect in vivo than spherical RGD-NS.
  • the RGD-COF high-dose group prepared in Example 3 can shorten the bleeding time from 400s to 52s, which is a reduction of 87%; and reduce the blood loss to 0.15mL/kg, which is a reduction of 92.5%.
  • the RGD-COF high-dose group prepared in Example 4 can shorten the bleeding time from 400s to 47s, which is a reduction of 88.2%; and reduce the blood loss to 0.17 mL/kg, which is a reduction of 91.5%.
  • Example 6 As in Example 6, a micron-sized CD-MOF was prepared.
  • CD-MOF loaded silver Weigh 169 mg of silver nitrate, dilute with acetonitrile in a 100 mL volumetric flask, and prepare a 10 mmol/L silver nitrate solution. Weigh 600 mg of micron-grade CD-MOF into an EP tube, add 1.5 mL of acetonitrile and mix, ultrasonic for 10 minutes, then add 5 mL of 10 mmol/L silver nitrate solution, place in the dark for 72 hours, wash with acetonitrile (10 mL) 3 times, centrifuge at 4000 rpm 5min, put it in a vacuum drying oven at 40°C and dry for 12h.
  • Micron-level silver-loaded COF Weigh 1g of micron-level silver-loaded CD-MOF powder dried at 80°C in a round bottom flask, fix it on a magnetic stirrer, and add 12.85mL N,N-dimethylformamide, 80 Heat at °C and stir at 500rpm.
  • Silver-loaded RGD-COF Weigh 1g of micron-sized silver-loaded COF and 21.7mg 4-dimethylaminopyridine into a round bottom flask, add 21.7mL N,N-dimethylformamide, stir well and then add 21.7mg Linear GRGDS, 13.36mg carbodiimide and 86.95uL triethylamine, placed on a magnetic stirrer at 37°C, 500rpm for 24h in the dark, centrifuged at 4000rpm for 5min, and N,N-dimethylformamide (10mL) , Ethanol (10mL), pure water (10mL), acetone (10mL) each wash once, the obtained crystals are dried at 60°C under vacuum for 6h.
  • the obtained silver-loaded RGD-COF has a regular cubic shape with a particle size of 1-5 ⁇ m, and the mass ratio of the cyclodextrin framework material to RGD measured by the HPLC method is 1:0.015.
  • the MIC value of silver-loaded CD-MOF, silver-loaded COF, and silver-loaded RGD-COF on E. coli CMCC(B)44102 is 16 ⁇ g/mL, which is comparable to commercially available preparations In comparison, the antibacterial effect is better than that of commercially available preparations (128 ⁇ g/mL).
  • the time-kill curve ( Figure 14) shows that when the Ag concentration is between 4 ⁇ g/mL ⁇ 16 ⁇ g/mL, the bactericidal effect of nanosilver on E.
  • coli CMCC(B)44102 is weak, when the Ag concentration is greater than 16 ⁇ g/mL and gradually increases The bactericidal effect is obviously enhanced at high altitude, and 1.0MBC (32 ⁇ g/mL) nano-silver can well inhibit the growth of bacteria within 6-8h.
  • the MIC values of silver-loaded CD-MOF, silver-loaded COF, and silver-loaded RGD-COF against Staphylococcus aureus CMCC(B)26112 are all 128 ⁇ g/mL, and the antibacterial effect is similar to that of commercially available preparations (128 ⁇ g/mL).
  • RGD-COF antithrombotic drugs Weigh appropriate amounts of aspirin, ticlopidine hydrochloride, cilostazol, clopidogrel bisulfate type II, rivaroxaban, ozagrel hydrochloride, tirofiban hydrochloride, etc. 7 An antithrombotic drug was added to 30mL of absolute ethanol for 10 minutes to dissolve it, and then 500mg of RGD-COF prepared in Example 1 was added respectively. The molar ratio of the drug to RGD-COF was 2:1. Stir at 300rpm for 24h at room temperature and incubate Drug-loaded. After the drug loading is completed, centrifuge at 4000 rpm for 5 minutes to obtain the lower drug-loaded RGD-COF. The drug loading is determined by HPLC as shown in Table 1.
  • RGD-COF containing hemostatic drugs Weigh appropriate amounts of three hemostatic drugs, including tranexamic acid, aminocaproic acid, and vitamin K1, and add 30 mL of absolute ethanol for 10 minutes to dissolve them, and then add 500 mg of the RGD prepared in Example 1 respectively.
  • -COF the molar ratio of drug to RGD-COF is 2:1, stirring at 400 rpm for 12 hours at 37°C, and incubating for drug loading. After the drug loading was completed, centrifuged at 4000 rpm for 5 minutes to obtain the lower drug-loaded RGD-COF.
  • the drug loading was determined by HPLC to be 13.5%, 4.3% and 2.1%, respectively.
  • RGD-COF anti-infective drugs Weigh appropriate amounts of sulfadiazine, ceftriaxone, amoxicillin, and levofloxacin, add 30 mL of absolute ethanol and sonicate for 10 minutes to dissolve them, and then add 500 mg of each to prepare in Example 1.
  • RGD-COF the molar ratio of drug to RGD-COF is 1:1, stirred at 200 rpm at room temperature for 48 hours, and incubated for drug loading. After the drug loading was completed, centrifuged at 4000 rpm for 5 minutes to obtain the lower drug-loaded RGD-COF.
  • the drug loading was determined by HPLC to be 3.8%, 3.3%, 4.6%, and 6.7%, respectively.
  • RGD-COF targets thrombus in vivo: FeCl 3 is used to induce mesenteric thrombosis in mice, and pre-injected rhodamine B is used to label activated platelets at the thrombus site. After thrombosis, the tail vein is injected with red Cy5 fluorescently labeled as prepared in Example 3 RGD-COF (40mg/kg), observed under a fluorescence microscope, red Cy5 fluorescently labeled RGD-COF can target and enrich mesenteric thrombus, the co-localization coefficient of RGD-COF nanoparticles and activated platelets at the thrombus is as high as 0.65, far It is much higher than the unmodified COF and spherical RGD-NS group ( Figure 16), indicating that the cubic RGD-COF can highly target thrombus in vivo.
  • RGD-COF targets bleeding sites in vivo A mouse tail docking model is used to evaluate the targeting of RGD-COF prepared in Example 2 to bleeding sites in vivo. Kunming mice were injected with Cy5 fluorescent-labeled COF, RGD-NS and RGD-COF nanoparticles (40mg/kg) through the tail vein for 5 minutes, and then quickly cut them at 0.5 cm from the tail tip of the mouse with sharp scissors to establish a mouse segment. Tail model.
  • the fluorescence signal at the tail dock of the RGD-COF group is 4 times that of the COF group and 3 times that of the RGD-NS group, indicating that the cubic RGD-COF can be highly targeted And gather to the bleeding site of tail docking, which has better targeting in vivo than spherical RGD-NS.

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Abstract

一种立方形环糊精骨架-RGD组合物(RGD-COF)及其制备方法。具体地，环糊精骨架-RGD组合物含有具有立方形结构的环糊精骨架(COF)和RGD，能够逃避巨噬细胞的吞噬和清除，增强对受损血管的迁移性和粘附性，高效地靶向和聚集于受损血管部位的活化血小板，对失控性出血、动脉粥样硬化和脑卒中等血管相关疾病的靶向诊疗具有巨大的应用前景。利用环糊精-金属有机骨架(CD-MOF)尺寸可控的优势提供了一种纳米级可静脉注射用或微米级可局部外用的立方形环糊精骨架-RGD组合物。

Description

立方形环糊精骨架-RGD组合物及其制备方法 技术领域
本发明涉及生物材料领域，更具体地涉及一种立方形环糊精骨架-RGD组合物及其制备方法。
背景技术
血管相关疾病如心肌梗塞和脑卒中的发病率和死亡率在世界范围内均很高。非靶向制剂如尿激酶等溶栓药均存在出血副作用、系统暴露面过宽等问题。由于血液循环系统复杂的血流动力学和一般载体的快速清除，靶向递送治疗药物或显影剂至血管损伤部位仍面临巨大挑战。载体的理化性质如形状会影响纳米载体的体内命运和生物功能。近年来研究表明，非球形的药物载体可以逃避机体的清除机制，延长循环时间，并增加对血管表面的粘附性。研究形态可控、具有靶向性和非球形形态载体，为血管相关疾病的靶向诊断和高效治疗带来了新的希望。
失控性失血造成的死亡率在医院和战场都很高。在严重受伤的情况下，正常的生理止血过程是远远不够的，比如车祸意外或战争创伤，有效快速止血并降低出血时间成为降低患者死亡率的重要措施。目前临床常用的止血材料如止血纱布、止血纤维、止血绷带都存在局限性，这些止血材料只能用于外出血，且止血时间较长，易与伤口粘连而不易换药，对伤口的感染和化脓无能为力。内出血大部分是因脏器破裂造成，因无法使用外用止血药，使伤后“第一时间”止血几乎不可能。重组人凝血因子VII(rFVII)是全身用止血药的代表，但rFVII具有价格昂贵、易失活、不易保存等缺点，大大限制了其临床应用。血小板制品的免疫原性、不易储存、易失活等缺陷，也限制了其在紧急救治中的应用。因此，用于治疗内出血的可静脉注射的止血材料在临床上具有巨大的需求。综上所述，亟需开发出止血速度更快、效果更佳、且可用于内出血的止血材料。
RGD序列和活化血小板表面GPIIb/IIIa受体的特异性结合是形成止血块的最终通路，同时RGD序列只能与出血部位活化的血小板结合，而正常血液循环中的静息血小板表面没有GPIIb/IIIa受体，因此对循环血小板没有影响。近年来，研究者采用高分子材料负载RGD多肽构建人工血小板成为了止血材料新的研究方向。Lavik小组使用PLGA-PLL作为高分子载体，将GRGDS五肽接到PLGA-PLL-PEG上，构建了一种合成血小板。该人工血小板通过静脉给药，能够靶向激活血小板，促进血小板聚集并进一步触发凝血机制，在大鼠的股动脉损伤模型中能将出血时间降低45％。但是，该人工血小板存在合成方法复杂等缺点。Anirban Sen Gupta团队采用环形RGD修饰纳米脂质体，构建出的人工血 小板经尾静脉注射后在小鼠断尾损伤模型中将出血时间降低约50％。Mitragotri团队采用GRGDS修饰PAH-BSA纳米粒模拟血小板的形态和流变性，合成的人工血小板能将小鼠断尾损伤的出血时间降低45％。张建祥团队采用胆酸和聚乙烯亚胺合成的正电荷纳米粒尾静脉注射后，在大鼠的股动脉损伤模型中能将出血时间降低40％左右。檀英霞等人发明了PLGA-PEG纳米颗粒负载RGD的人工血小板PLGA-PEG-RGD。PLGA-PEG-RGD为规则的球形，但粒径大小不均一，虽然可以用作静脉用纳米止血材料。但在大鼠肝损伤模型中的止血效果有限，只能将止血时间降低30％左右。
人工血小板的止血效率，很大程度上取决于纳米粒的表面生物学功能和物理力学性质，如尺寸和形状。纳米载体的形态不仅影响其向血管壁的动态迁移过程，而且还影响它们与活化血小板的粘附和聚集相互作用。与球形颗粒相比，具有各向异性形状的载体(如椭圆体和棒状)具有较高的向血管壁迁移的能力，在生理相关的流动环境中具有较强的粘附性。此外，载体的几何形状也会影响其在体内的循环和清除等过程。因此，调控载体的形状为克服生理屏障，提高止血效率带来了新的希望。
然而，以往大多数研究主要集中在球形载体上，目前还无人尝试将立方形态的载体用于出血的靶向治疗。
发明内容
本发明的目的在于提供一种环糊精骨架-RGD组合物(RGD-COF)及其制备方法和用途。
在本发明的第一方面，提供了一种环糊精骨架-RGD组合物，所述组合物中，环糊精骨架与RGD的质量比为1:0.001-1:1，较佳地为1:0.005-1:0.5；
所述的环糊精骨架-RGD组合物粒径为10nm-50μm，优选为50nm-50μm，更优选为100-500nm或1-5μm。
在另一优选例中，所述组合物为立方形环糊精骨架-RGD组合物。
在另一优选例中，所述立方形环糊精骨架-RGD组合物中，环糊精骨架与RGD的质量比为1:0.001-1:1，较佳地为1:0.005-1:0.5，更佳地为1:0.04-1:0.5。
在另一优选例中，所述立方形环糊精骨架-RGD组合物中，环糊精骨架与RGD的质量比为1:0.005-1:0.1，较佳地为1:0.05。
在另一优选例中，所述立方形环糊精骨架-RGD组合物中环糊精骨架与RGD的质量比为1:0.05。
在另一优选例中，所述立方形环糊精骨架-RGD组合物中，环糊精骨架与 RGD的质量比为1:0.049、1:0.08、1:0.005、1:0.015、1:0.05、1:0.016、1:0.065、或1:0.046。
在另一优选例中，所述立方形环糊精骨架-RGD组合物的粒径为50nm-50μm。
在另一优选例中，所述立方形环糊精骨架-RGD组合物的粒径为100-500nm。
在另一优选例中，所述立方形环糊精骨架-RGD组合物的粒径为100-300nm，较佳地为150-200nm。
在另一优选例中，所述立方形环糊精骨架-RGD组合物的粒径为1-50μm、优选30-50μm，更优选为10-30μm。
在另一优选例中，所述立方形环糊精骨架-RGD组合物的粒径为1-5μm。
在另一优选例中，所述立方形环糊精骨架-RGD组合物粒径为200-500nm、100-300nm、200-400nm、200-500nm、1-10μm、1-5μm、30-50μm、或10-30μm。
在另一优选例中，所述RGD包括线性的RGD和环形的RGD。
在另一优选例中，所述RGD为线性的RGD。
在另一优选例中，所述RGD选自下组：RGD、GRGD、RGDS、RGDV、RGDF、GRGDV、GRGDF、GRGDS、RGDDSP、RGDDAP、其他含RGD序列的多肽、或其组合。
在另一优选例中，所述线性的RGD选自下组：线性的RGD、线性的GRGD、线性的RGDS、线性的GRGDS、或其组合。
在另一优选例中，所述环形的RGD选自下组：环形的RGD、环形的GRGD、环形的RGDS、环形的RGDV、环形的RGDF、环形的GRGDV、环形的GRGDF、环形的GRGDS、环形的RGDDSP、环形的RGDDAP、其他含环形RGD序列的多肽、或其组合。
在另一优选例中，所述的环糊精选自下组：α-环糊精(阿尔法环糊精)、β-环糊精(贝塔环糊精)、γ-环糊精(伽马环糊精)、羟丙基-β-环糊精、磺丁基-β-环糊精、甲基-β-环糊精、羧甲基-β-环糊精、或其组合。
在另一优选例中，环糊精选自下组：α-环糊精、β-环糊精、γ-环糊精，进一步优选为γ-环糊精。
在另一优选例中，所述的环糊精为γ-环糊精。
在另一优选例中，所述的环糊精骨架-RGD组合物能将凝血块形成时间降低30％、40％、50％、60％、70％、80％或90％以上。
在另一优选例中，所述的环糊精骨架-RGD组合物能将出血时间缩短50％、60％、70％、80％、85％、90％或95％以上。
在另一优选例中，所述的环糊精骨架-RGD组合物能将失血量降低50％、60％、70％、80％、85％、90％或95％以上。
在本发明的第二方面，提供了一种立方形环糊精骨架-RGD组合物的制备方法，包括步骤：
(1)提供一立方形环糊精-金属有机骨架(CD-MOF)；
(2)交联步骤，通过交联剂对(1)中的立方形环糊精-金属有机骨架进行交联，得到环糊精骨架(COF)；
(3)RGD修饰步骤，在(2)中所述的环糊精骨架上修饰RGD，得到立方形环糊精骨架-RGD组合物(RGD-COF)。
在另一优选例中，所述步骤(2)中，所述交联为通过交联剂将所述的立方形环糊精-金属有机骨架上的羟基(-OH)交联。
在另一优选例中，所述步骤(2)中，所述环糊精骨架为在水相体系中稳定的环糊精骨架。
在另一优选例中，所述步骤(3)中，在(2)中所述的环糊精骨架表面修饰RGD，得到立方形环糊精骨架-RGD组合物。
在另一优选例中，所述步骤(2)中，还包括：
(2a)任选地，保留COF立方形态的步骤；
(2b)任选地，去除金属离子的步骤。
在另一优选例中，所述交联步骤包括下列子步骤：
(2a)分散步骤，将立方形的环糊精-金属有机骨架分散于有机溶剂A中，得到分散液2a；
(2b)交联剂和催化剂加入步骤，在交联反应温度T条件下向分散液2a中加入交联剂和催化剂A，反应时间t1之后，得到分散液2b；
(2c)任选地，冷却步骤，对分散液2b进行冷却，得到经冷却的分散液2b；
(2d)任选地，终止反应步骤，向经冷却的分散液2b中，加入反应终止剂，得到分散液2d；
(2e)任选地，离心步骤，对分散液2d进行离心，得到晶体2e；
(2f)任选地，洗涤步骤，洗涤晶体2e，得到经洗涤的晶体2f；
(2g)任选地，干燥步骤，对经洗涤的晶体2f进行干燥处理；
(2h)获得立方形的环糊精骨架(COF)。
在另一优选例中，所述分散液2a为混悬液、乳浊液、悬浮液、或胶体。
在另一优选例中，所述分散液2b为混悬液、乳浊液、悬浮液、或胶体。
在另一优选例中，所述分散液2d为混悬液、乳浊液、悬浮液、或胶体。
在另一优选例中，所述交联反应温度T为30-110℃，优选40-100℃，更优选 60-90℃，最优选70-80℃。
在另一优选例中，所述交联反应温度T为80℃、40℃、100℃、50℃、70℃、或60℃。
在另一优选例中，所述步骤(2a)中环糊精-金属有机骨架与步骤(2b)中交联剂的摩尔比为1:1-1:20，优选为1:2-1:10，更优选为1:4-1:8。
在另一优选例中，所述步骤(2a)中环糊精-金属有机骨架与步骤(2b)中交联剂的摩尔比为1：6，1：2，1：5，1：10，1：20，1：8，1：15，或1：4。
在另一优选例中，所述步骤(2b)中，交联剂和催化剂在搅拌条件下加入，搅拌速度为200-1000rpm、优选300-800rpm，更优选400-600rpm。
在另一优选例中，所述步骤(2b)中，交联剂和催化剂在搅拌条件下加入，搅拌速度为600rpm、200rpm、1000rpm、900rpm、700rpm、400rpm、500rpm、或700rpm。
在另一优选例中，所述步骤(2b)中，环糊精-金属有机骨架内的羟基通过共价键交联起来。
在另一优选例中，所述步骤(2b)中，反应时间t1为4-48h，优选8-24h，更优选12-16h。
在另一优选例中，所述步骤(2b)中，反应时间t1为24h、48h、4h、12h、8h、或16h。
在另一优选例中，所述步骤(2c)中，冷却为冷却至室温。
在另一优选例中，所述步骤(2d)中，反应终止剂为乙醇。
在另一优选例中，所述步骤(2d)中，反应终止剂为95-100％乙醇或70-90％乙醇。
在另一优选例中，所述步骤(2e)中，离心为在3000-4500rpm下离心3-15min，优选为在4000rpm离心5min。
在另一优选例中，所述步骤(2f)中，所述洗涤为使用乙醇、纯水和/或丙酮洗涤。
在另一优选例中，所述COF为立方体形态。
在另一优选例中，所述COF的粒径为50nm-50μm，优选50-500nm或1-50μm。
在另一优选例中，所述COF的粒径为100-500nm。
在另一优选例中，所述COF的粒径为100-300nm，较佳地为150-200nm。
在另一优选例中，所述COF的粒径为1-50μm、优选30-50μm，更优选为10-30μm。
在另一优选例中，所述COF的粒径为1-5μm。
在另一优选例中，所述COF的粒径为200-500nm、100-300nm、200-400nm、 200-500nm、1-10μm、1-5μm、30-50μm、或10-30μm。
在另一优选例中，所述RGD修饰步骤包括下列子步骤：
(3a)分散步骤，将立方形的环糊精骨架(COF)和RGD分散于有机溶剂B中，得到分散液3a；
(3b)偶联步骤，向分散液3a中加入催化剂B，使立方形的环糊精骨架与RGD偶联，反应时间为t2；
(3c)任选地，离心步骤；
(3d)任选地，洗涤步骤；
(3f)任选地，干燥步骤；
(3g)获得立方形环糊精骨架-RGD组合物(RGD-COF)。
在另一优选例中，所述步骤(3b)中，偶联为立方形的环糊精骨架表面的羟基与RGD的羧基偶联。
在另一优选例中，所述分散液3a为混合均匀的分散液。
在另一优选例中，所述分散液3a为混悬液、乳浊液、悬浮液、或胶体
在另一优选例中，所述偶联步骤在加热搅拌条件下进行。
在另一优选例中，所述搅拌过程中，磁力搅拌器转速为200-1000rpm。
在另一优选例中，所述搅拌过程中，磁力搅拌器转速为200rpm、400rpm、500rpm、600rpm、700rpm、900rpm、1000rpm。
在另一优选例中，所述加热温度为20-40℃，优选37-38℃。
在另一优选例中，所述加热温度为20℃、25℃、30℃、35℃、37℃、40℃。
在另一优选例中，所述反应时间t2为4-48h，较佳地为8-24h，更佳地为12-20h。
在另一优选例中，所述反应时间t2为4h、6h、8h、12h、18h、24h、48h。
在另一优选例中，步骤(3a)中，其中COF和RGD的摩尔比为1:0.1-1:10，优选1:0.2-1:5，优选1:1。
在另一优选例中，步骤(3a)中，COF和RGD的摩尔比为1:1、1:2、5:1、4:1、2:1、1:3或1:5。
在另一优选例中，步骤(3g)中，所述立方形环糊精骨架-RGD组合物(RGD-COF)粒径为200-500nm、100-300nm、200-400nm、200-500nm、1-10μm、1-5μm、30-50μm、或10-30μm。
在另一优选例中，所述的交联剂选自下组：过氧化物、多异氰酸酯、缩水甘油醚、二元或多元酸类、二元或多元醛类、含羰基的化合物、环氧化物类、丙烯酸酯类、酰氯类、或其组合。
在另一优选例中，所述过氧化物选自下组：过氧化苯甲酰、过氧化二异丙苯、叔丁基过氧化物、或其组合。
在另一优选例中，所述多异氰酸酯选自下组：异氰酸酯、甲苯二异氰酸酯、二苯基甲烷二异氰酸酯、二环己基甲烷二异氰酸酯、六亚甲基二异氰酸酯、赖氨酸二异氰酸酯、或其组合。
在另一优选例中，所述缩水甘油醚选自下组：乙二醇二缩水甘油醚、聚丙二醇缩水甘油醚、三羟甲基丙烷三缩水甘油醚、正丁基缩水甘油醚、或其组合。
在另一优选例中，所述二元或多元酸类选自下组：柠檬酸、丙二酸、丁二酸、邻苯二甲酸、间苯二甲酸、或其组合。
在另一优选例中，所述二元或多元醛类选自下组：乙二醛、戊二醛、丁二醛、或其组合。
在另一优选例中，所述含羰基的化合物选自下组：碳酸二苯酯、N,N’-羰基二咪唑、N,N’-二甲基咪唑啉、二环己基碳二亚胺、或其组合。
在另一优选例中，所述环氧化物选自下组：环氧氯丙烷、环氧丙烷、1，4-二氧六环、或其组合。
在另一优选例中，所述丙烯酸酯类选自下组：二甲基丙烯酸乙二醇酯、丙烯酸羟乙酯、丙烯酸羟丙酯、甲基丙烯酸、甲基丙烯酸羟乙酯、甲基丙烯酸羟丙酯、或其组合。
在另一优选例中，所述酰氯类选自下组：丁二酰氯、四异氰酸酯、或其组合。
在另一优选例中，所述的交联剂为碳酸二苯酯。
在另一优选例中，所述的交联剂为环氧氯丙烷。
在另一优选例中，所述的催化剂A选自下组：4-二甲氨基吡啶、三乙胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺或其盐、N,N'-琥珀酰亚胺基碳酸酯、N-羟基丁二酰亚胺、吡啶、或其组合；较佳地为三乙胺；
所述的有机溶剂A选自下组：二甲基甲酰胺、四氢呋喃、甲醇、乙腈、丙酮、异丙醇、乙酸乙酯、氯仿、正己烷、乙醇、二氯甲烷。
在另一优选例中，所述的有机溶剂A选自下组：二甲基甲酰胺、四氢呋喃、甲醇、乙腈、丙酮、异丙醇、乙酸乙酯、氯仿、正己烷、乙醇、二氯甲烷、或其组合。
在另一优选例中，所述的有机溶剂A为二甲基甲酰胺。
在另一优选例中，所述的催化剂A选自下组：4-二甲氨基吡啶、三乙胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺或其盐、N,N'-琥珀酰亚胺基碳酸酯、N-羟基丁二酰亚胺、吡啶、或其组合。
在另一优选例中，所述的催化剂A为三乙胺。
在另一优选例中，所述的有机溶剂B选自下组：二甲基甲酰胺、四氢呋喃、甲醇、乙腈、丙酮、异丙醇、乙酸乙酯、氯仿、正己烷、乙醇、二氯甲烷、或 其组合。
在另一优选例中，所述的有机溶剂B为二甲基甲酰胺。
在另一优选例中，所述的催化剂B选自下组：4-二甲氨基吡啶、三乙胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺或其盐、N-羟基丁二酰亚胺、N,N'-琥珀酰亚胺基碳酸酯、吡啶、或其组合。
在另一优选例中，所述的催化剂B为4-二甲氨基吡啶。
在另一优选例中，所述的催化剂A选自下组：4-二甲氨基吡啶、三乙胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺或其盐、N,N'-琥珀酰亚胺基碳酸酯、N-羟基丁二酰亚胺、吡啶、或其组合；所述的催化剂B选自下组：4-二甲氨基吡啶、三乙胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺或其盐、N-羟基丁二酰亚胺、N,N'-琥珀酰亚胺基碳酸酯、吡啶、或其组合。
在另一优选例中，所述的有机溶剂A选自下组：二甲基甲酰胺、四氢呋喃、甲醇、乙腈、丙酮、异丙醇、乙酸乙酯、氯仿、正己烷、或其组合；
所述的有机溶剂B选自下组：二甲基甲酰胺、四氢呋喃、甲醇、乙腈、丙酮、异丙醇、乙酸乙酯、氯仿、正己烷、乙醇、二氯甲烷、或其组合。
在另一优选例中，所述的催化剂B选自下组：4-二甲氨基吡啶、三乙胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺或其盐、N-羟基丁二酰亚胺、N,N'-琥珀酰亚胺基碳酸酯、吡啶、或其组合；
所述的有机溶剂B选自下组：二甲基甲酰胺、四氢呋喃、甲醇、乙腈、丙酮、异丙醇、乙酸乙酯、氯仿、正己烷、乙醇、二氯甲烷、或其组合。
在另一优选例中，所述的有机溶剂B为二甲基甲酰胺。
在另一优选例中，所述的催化剂B为4-二甲氨基吡啶。
在另一优选例中，所述RGD序列包括线性的RGD，GRGD、RGDS、GRGDS、环形的RGD，GRGD、RGDS、GRGDS，优选线性的RGD序列，包括GRGD、RGDS、GRGDS。
在本发明的第三方面，提供了一种负载药物的立方形环糊精骨架-RGD组合物，所述立方形环糊精骨架-RGD组合物为本发明的第一方面所述的组合物，或通过本发明的第二方面所述的方法制备，所述的药物选自下组：抗菌药物、止血药物、抗血栓药物、抗感染药物、或其组合。
在另一优选例中，所述抗菌药物选自下组：纳米银、青霉素、头孢霉素、米诺环素、多西环素、四环素、氯霉素、林可霉素、万古霉素、或其组合。
在另一优选例中，所述止血药物选自下组：氨甲环酸、氨基己酸、维生素K1、或其组合。
在另一优选例中，所述抗血栓药物选自下组：阿司匹林、氯吡格雷、噻氯 匹定、西洛他唑、替罗非班、奥扎格雷、利伐沙班、或其组合。
在另一优选例中，所述抗感染药物选自下组：磺胺嘧啶、头孢曲松、阿莫西林、左氧氟沙星、或其组合。
在另一优选例中，所述的负载药物的立方形环糊精骨架-RGD组合物可用于静脉注射(纳米级)和局部用药(微米级)。
在另一优选例中，所述组合物还具有以下一个或多个特征：
(1)所述的环糊精骨架-RGD组合物粒径为50nm-50μm，优选为100-500nm或1-5μm；
(2)所述组合物的载药量为1％-20％，优选为5％-10％。
在另一优选例中，所述的环糊精骨架-RGD组合物粒径为100-500nm；或者所述的环糊精骨架-RGD组合物粒径为1-5μm。
在另一优选例中，所述组合物的载药量为2.1％-13.5％。
在另一优选例中，所述组合物的载药量进一步优选为2.7％-8.6％。
在另一优选例中，所述的环糊精骨架-RGD组合物为粉体形式。
在另一优选例中，所述的环糊精骨架-RGD组合物为颗粒形式。
在另一优选例中，所述的负载药物的立方形环糊精骨架-RGD组合物能将凝血块形成时间降低30％、40％、50％、60％、70％、80％或90％以上。
在另一优选例中，所述的负载药物的立方形环糊精骨架-RGD组合物能将出血时间缩短50％、60％、70％、80％、85％、90％或95％以上。
在另一优选例中，所述的负载药物的立方形环糊精骨架-RGD组合物能将失血量降低50％、60％、70％、80％、85％、90％或95％以上。
在本发明的第四方面，提供了一种活性成分的用途，所述活性成分选自下组：
(i)本发明的第一方面所述环糊精骨架-RGD组合物；
(ii)本发明的第三方面所述的负载药物的立方形环糊精骨架-RGD组合物；
(iii)环糊精骨架(COF)；
(iv)上述(i)、(ii)或(iii)的组合；
所述活性成分被用于：
(a)制备载药材料；
(b)制备治疗和/或诊断试剂或试剂盒；
(c)制备止血药物和/或材料；
(d)制备抗感染药物和/或材料；
(e)制备抗菌药物和/或材料；
(f)制备促进伤口愈合药物和/或材料；
(g)制备预防和/或治疗血栓的药物和/或材料。
在另一优选例中，所述治疗和/或诊断试剂或试剂盒用于疾病治疗和/或诊断。
在另一优选例中，所述疾病选自下组：血栓、动脉粥样硬化、脑卒中、肿瘤、出血、炎症、感染。
在另一优选例中，所述诊断试剂或试剂盒用于医学CT显影。
在另一优选例中，所述治疗和/或诊断试剂或试剂盒用于抗肿瘤、止血、抗炎、抗感染。
在另一优选例中，所述的止血药物和/或材料能将凝血块形成时间降低30％、40％、50％、60％、70％、80％或90％以上。
在另一优选例中，所述的止血药物和/或材料能将出血时间缩短50％、60％、70％、80％、85％、90％或95％以上。
在另一优选例中，所述的止血药物和/或材料能将失血量降低50％、60％、70％、80％、85％、90％或95％以上。
在本发明的第五方面，提供了一种药物组合物，所述药物组合物包含：
(1)活性成分，所述活性成分为如本发明的第一方面所述的环糊精骨架-RGD组合物或如本发明的第三方面所述的负载药物的立方形环糊精骨架-RGD组合物；和
(2)药学上可接受的载体。
在另一优选例中，所述药物组合物为胶囊剂、片剂、颗粒剂。
在另一优选例中，所述的载体选自下组：稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂、或其组合。
在另一优选例中，所述的药物组合物配制为固体剂型或液体剂型，较佳地适用于口服给药，更佳地适用于注射给药。
在另一优选例中，固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。
在另一优选例中，液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。
在另一优选例中，所述药物组合物为胶囊剂、片剂、颗粒剂、注射剂。
在另一优选例中，所述药物组合物还包含表面活性剂，选自下组：聚山梨坦-80、聚山梨坦-60、聚乙二醇甘油脂肪酸酯、脱水山梨糖醇脂肪酸酯及两种以上的混合物。
在另一优选例中，所述的药物组合物能将凝血块形成时间降低30％、40％、 50％、60％、70％、80％或90％以上。
在另一优选例中，所述的药物组合物能将出血时间缩短50％、60％、70％、80％、85％、90％或95％以上。
在另一优选例中，所述的药物组合物能将失血量降低50％、60％、70％、80％、85％、90％或95％以上。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为实施例1中纳米CD-MOF的扫描电镜图。
图2为实施例1中纳米COF的扫描电镜图。
图3为实施例1中纳米RGD-COF的扫描电镜图。
图4为实施例1中纳米RGD-COF动态光散射粒径分布图。
图5为实施例1中纳米RGD-COF的物理稳定性(■：纯水；●：生理盐水；▲：PBS pH 7.4；◆：大鼠血浆)。
图6为实施例1中纳米RGD-COF的细胞毒性结果。
图7为实施例6中微米CD-MOF的扫描电镜图。
图8为实施例6中微米COF的扫描电镜图。
图9为实施例6中微米RGD-COF的扫描电镜图。
图10为实施例9中球形RGD-NS的扫描电镜图。
图11为实施例10中的RGD-COF的体外凝血块形成时间结果。
图12显示实施例11中RGD-COF可以显著降低小鼠断尾模型的体内出血时间。
图13显示实施例11中RGD-COF可以显著降低小鼠断尾模型的体内失血量。
图14显示实施例12中载银RGD-COF等的时间-抗菌曲线。
图15显示实施例12中载银RGD-COF等对大鼠创口的愈合效果。
图16显示实施例16中RGD-COF可以靶向体内肠系膜血栓，与血栓部位的活化血小板有高度共定位。
具体实施方式
本发明人经过广泛而深入地研究，首次开发了一种立方形环糊精骨架-RGD组合物(RGD-COF)及其制备方法。本发明的立方形环糊精骨架-RGD组 合物含有具有立方形结构的环糊精骨架和RGD，能够逃避巨噬细胞的吞噬和清除，增强对受损血管的迁移性和粘附性，高效地靶向和聚集于血管损伤部位的活化血小板，实现血管相关疾病(例如出血、血栓)的靶向治疗。利用CD-MOF尺寸可控的优势，以纳米级和微米级CD-MOF为基础材料，经交联步骤和RGD修饰步骤，获得了静脉注射用(纳米级)或外用(微米级)的新型高效的载体材料。具体而言，本发明所述的纳米级立方形环糊精骨架-RGD组合物通过静脉注射可以在小鼠断尾损伤模型中将出血时间和失血量均降低90％；微米级环糊精骨架-RGD组合物通过局部外用，可以在大鼠股动脉损伤模型中将出血时间降低60％，止血效率大大提高。在此基础上，完成本发明。
术语
金属有机骨架材料
金属有机骨架(metal organic frameworks,MOF)是一种由金属(金属离子、金属离子簇或金属链)和有机桥连配体在较温和的条件下以配位键方式通过自组装形成的无机-有机杂化材料。由于MOF超高的孔隙率和巨大的比表面积，且无机和有机等多种不同成分的组合使得其结构及组成多样，为MOF在气体储存、吸附和分离、催化、药物输送等领域的应用提供了新的研究方向。
环糊精
环糊精(CD)是由直链淀粉经葡萄糖基转移酶作用下生成的一系列环状低聚糖的总称，通常含有6～12个D-吡喃葡萄糖单元。其中研究得较多并且具有重要实际意义的是含有6、7、8个葡萄糖单元的分子，分别称为α、β-和γ-环糊精。环糊精是迄今所发现的类似于酶的理想宿主分子，并且其本身就有酶模型的特性。
环糊精-金属有机骨架
如本文所用，术语“环糊精-金属有机骨架”、“CD-MOF”可互换使用，是将环糊精作为有机配体，金属离子作为无机金属中心形成的新型、安全性较高、可药用的立方形环糊精-金属有机骨架，即CD-MOF。
典型地，所述环糊精-金属有机骨架为环糊精与碱金属盐形成的骨架材料；碱金属包括但不限于Li +、K +、Rb +、Cs +、Na +、Mg 2+、Cd 2+、Sn 2+、Ag +、Yb +、Ba 2+、Sr 2+、Ca 2+、Pb 2+、La 3+，优选K +。
典型地，所述环糊精-金属有机骨架材料的平均粒径为50纳米-50微米，较佳地为100-500纳米(纳米级)或1-5微米(微米级)。
典型地，制备环糊精-金属有机骨架(CD-MOF)(参考专利 201610125456.X)：所述制备方法包括将金属盐溶液与环糊精水溶液混合后，预加一部分有机溶剂，一定温度下，通过溶剂蒸汽扩散方法，反应一定时间，再加入尺寸调节剂，从而得到所述基于环糊精的金属有机骨架材料；或将金属盐溶液与环糊精水溶液混合，预加一部分有机溶剂，用溶剂热/微波/超声波振动反应介质，使得反应物快速反应，反应一定时间后加入尺寸调节剂，从而得到所述基于环糊精的金属有机骨架材料。
典型地，所述金属盐溶液中金属盐的浓度为0.05-0.4M，优选0.2M。
典型地，所述环糊精水溶液中环糊精的浓度为0.013-0.05M，优选0.025M。
典型地，所述的环糊精选自下组：α-环糊精、β-环糊精、γ-环糊精、羟丙基-β-环糊精、磺丁基-β-环糊精、甲基-β-环糊精、羧甲基-β-环糊精、或其组合。
在本发明的一个优选的实施方式中，所述的环糊精为γ-环糊精。
在本发明的一个优选的实施方式中，所述环糊精-金属有机骨架为立方形。
在本发明的一个优选的实施方式中，所述的环糊精-金属有机骨架的制备包括步骤：
(1a)提供第一混合溶液，所述第一混合溶液为含有金属离子和环糊精的溶液；
(2a)向所述的第一混合溶液中加入第一有机溶剂，获得第二混合溶液，
其中，所述第一有机溶剂与所述第一混合溶液的体积比为(0.01-0.5):1，较佳地为(0.03-0.3):1，最佳地为(0.05-0.2):1；
(3a)对所述第二混合溶液进行预处理，获得经预处理的第一混合物，其中所述的预处理选自下组：
(3a1)溶剂热挥发处理；
(3b1)溶剂热挥发处理与选自A组的任一处理方式的组合，其中A组包括溶剂热处理、微波处理、超声波处理、或其组合；
(4a)当第一混合物中含有析出的环糊精-金属有机骨架材料时，从所述第一混合物中分离获得析出的环糊精-金属有机骨架材料；
或者从所述第一混合物中分离出部分或全部的溶液，作为第三混合溶液；并向所述第三混合溶液中加入第二有机溶剂和/或尺寸调节剂，从而析出环糊精-金属有机骨架材料；和
(5a)任选地对步骤(4a)中析出的环糊精-金属有机骨架材料进行分离和/或干燥。
在另一优选例中，在步骤(3a)中，所述的溶剂热挥发处理包括步骤：
(i)将混合溶液置于一开口容器I中；
(ii)提供一装有有机溶剂的开口容器II，将所述开口容器I和开口容器II共同置于一封闭体系内；和
(iii)对所述开口容器II中的有机溶剂进行加热/保温处理，使得所述有机溶剂蒸发扩散至混合溶液中。
在另一优选例中，在步骤(iii)中，对所述封闭体系进行整体加热处理，从而加热所述开口容器II中的有机溶剂
在另一优选例中，在步骤(iii)中，所述加热处理包括水浴加热、和油浴加热。
在另一优选例中，在步骤(iii)中，所述加热处理的温度为25-100℃，较佳地为30-80℃，更佳地为40-60℃。
在另一优选例中，在步骤(iii)中，所述加热处理的时间为4-48h，较佳地为6-24h。
环糊精骨架材料
术语“环糊精骨架材料”、“环糊精骨架”、“立方形的环糊精骨架”、“立方形环糊精骨架”、“COF”可互换使用。
CD-MOF中含有K +，不能直接静脉注射，且在水性环境中会迅速崩解，在到达靶部位之前无法确保多孔晶体结构的稳定。为了增加CD-MOF在水中的稳定性，迄今为止只报道了三种策略。Furukawa等人使用乙二醇二缩水甘油醚交联γ-CD-MOF以产生γ-CD-MOF水凝胶。然而，此种交联反应极其耗时，在65℃下需要三天以上时间，且需要诸多步骤来除去未反应的杂质。Li等人为了提高γ-CD-MOF的水稳定性，将富勒烯(C60)掺入到γ-CD的疏水空腔中。然而，由于配体-环糊精较弱的相互作用，超分子组装体系只能在较短时间内保持结构的完整性，在水中24h后便降解。此外，C60对γ-CD空腔的占用也可能降低γ-CD-MOF负载药物的能力。综上所述，合成稳定的多孔性CD-MOF材料仍然是一个巨大的挑战。
为此，本发明采用碳酸二苯酯、环氧氯丙烷等作为交联剂将CD-MOF中有序组织的CD交联起来，成功制备了在水中稳定的环糊精骨架(COF)，仍然保留了CD-MOF的立方体形态，并且能将配位的K +除去，基于纳米级CD-MOF交联得到的COF可作为静脉注射用的新型纳米载体。
在本发明的一个优选的实施方式中，发明人采用碳酸二苯酯、环氧氯丙烷等作为交联剂成功地交联CD-MOF中的CD。使用三乙胺、吡啶等作为催化剂发明了一种简便的合成途径，能够在4h内合成水中稳定性好的COF。
在本发明的一个优选的实施方式中，所述环糊精骨架(COF)制备方法如下：称取适量制备的CD-MOF粉末于圆底烧瓶中，固定于磁力搅拌器上，加入一定体积的有机溶剂，加热搅拌条件下，再加入一定量的交联剂和催化剂，反应一段时间，使CD-MOF内的羟基(-OH)通过共价键交联起来，然后冷却至 室温，采用95％乙醇终止反应后，离心得沉淀，用50％乙醇、纯水和丙酮各洗涤两次后，真空干燥后即得水中稳定的环糊精骨架(COF)。
RGD
RGD序列即精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp)序列。内源性RGD序列存在于多种粘附蛋白中(如纤维蛋白原、玻连蛋白、纤维粘连蛋白、血管性血友病因子)，发挥着多种生物学功能。纤维蛋白原中Aα链上的RGD序列和活化血小板表面的膜糖蛋白GPIIb/IIIa受体的特异性结合，是血小板聚集和血栓块形成的共同通路。RGD序列作为纤维蛋白原中的特定序列，是纤维蛋白原和活化的GPIIb/IIIa受体结合的活性位点。
本发明所述RGD序列包括线性的RGD、线性的GRGD、线性的RGDS、线性的RGDV、线性的RGDF、线性的GRGDV、线性的GRGDF、线性的GRGDS、线性的RGDDSP、线性的RGDDAP；环形的RGD，环形的GRGD、环形的RGDS、环形的RGDV、环形的RGDF、环形的GRGDV、环形的GRGDF、环形的GRGDS，环形的RGDDSP、环形的RGDDAP。
典型地，本发明所述RGD序列优选线性的RGD序列，包括线性的GRGD、线性的RGDS、线性的RGDF、线性的GRGDS。
立方形环糊精骨架-RGD组合物
如本文所用，术语“立方形环糊精骨架-RGD组合物”、“RGD修饰的COF”、“RGD-COF”、“立方形环糊精骨架-RGD多肽组合物”可互换使用，是指本发明第一方面所述的立方形环糊精骨架-RGD组合物。
典型地，本发明提供的一种立方形环糊精骨架-RGD组合物，其结构特征在于具有立方晶体结构的环糊精-金属有机骨架材料内的羟基(-OH)被合适的交联剂共价连接起来，形成在水中稳定的立方结构的环糊精骨架，外部采用活化剂将表面的羟基(-OH)与RGD的羧基(-COOH)共价连接，对环糊精骨架进行生物学修饰。立方形环糊精骨架作为人工血小板载体，RGD作为靶头，和活化血小板表面GPIIb/IIIa受体的特异性结合，靶向血管损伤部位活化的血小板。RGD修饰后的环糊精骨架材料作为新型高效的人工血小板，该特征能提高人工血小板向受损血管的迁移能力，提高对出血部位的靶向性，并促进与活化血小板的结合。
具体地，本发明提供了一种由COF和RGD组成的立方形环糊精骨架-RGD组合物，其内部为具有立方晶体结构的环糊精骨架，表面采用RGD进行生物学修饰。立方形环糊精骨架作为载体，RGD作为靶头，靶向血管损伤部位活化的血小板表面的GPIIb/IIIa受体。RGD修饰的COF作为新型高效的功能材料， 该特征能提高向受损血管的迁移能力，提高对受损部位的靶向性，并促进与活化血小板的结合和聚集。
本发明所述的立方形环糊精骨架-RGD组合物不同于已有的人工血小板报道，Anirban Sen Gupta团队采用RGD和胶原蛋白结合肽修饰球形的脂质体，构建出的人工血小板经尾静脉注射后在小鼠断尾模型中具有一定的止血效果，但靶向性和止血效果仍待提高。檀英霞等人发明了PLGA-PEG纳米颗粒负载RGD的人工血小板PLGA-PEG-RGD。PLGA-PEG-RGD为规则的球形，但粒径大小不均一，虽然可以用作静脉用全身性纳米止血材料，但在大鼠肝损伤模型中的止血效果有限，只能将出血时间降低30％左右。
本发明所述的纳米级立方形环糊精骨架-RGD组合物通过静脉注射可以在小鼠断尾损伤模型中将出血时间和失血量均降低90％，微米级环糊精骨架-RGD组合物通过局部外用，可以在大鼠股动脉损伤模型中将出血时间降低60％，止血效率大大提高。
在本发明的一个优选的实施方式中，所述立方形环糊精骨架-RGD组合物(RGD-COF)制备方法如下：称取适量COF和RGD于圆底烧瓶中，加入一定体积的有机溶剂B，混合均匀后再加入适量的催化剂B和活化剂，置于磁力搅拌器上加热搅拌适当时间，使COF表面的羟基(-OH)与RGD的羧基(-COOH)充分偶联。反应完成后，分别使用等体积的无水DMF和纯水各洗涤2遍，冷冻干燥过夜即得立方形环糊精骨架-RGD组合物(产物简写为RGD-COF)，置于-20℃冷冻保存备用。
本发明提供的立方形环糊精骨架-RGD组合物(RGD-COF)具有高效的靶向性能，可静脉注射用于全身性失血和血栓，也可局部用药治疗外伤，为复杂战争创伤、意外创伤、外科手术出血、脑卒中和肿瘤相关疾病的救治提供了更多选择，应用前景广阔。
立方形环糊精骨架-RGD组合物的制备方法
本发明提供了立方形环糊精骨架-RGD组合物的制备方法。
在本发明的一个优选的实施方式中，采用可药用的环糊精作为有机连接体，K +作为无机金属中心，制备立方体形态、粒径均一的CD-MOF，采用交联剂将CD-MOF的CD通过交联，制备水相中稳定的COF，将RGD序列的羧基(-COOH)通过酯键的方式键合在COF表面，形成立方形环糊精骨架-RGD组合物(RGD-COF)。本发明所涉及的CD-MOF，是基于一种制备环糊精-金属有机骨架材料的方法(201610125456.X)，该方法快速、简便，尺寸可调节、产率高。
在本发明的又一个优选的实施方式中，所述立方形环糊精骨架-RGD组合 物(RGD-COF)的制备方法包括步骤：
(1)制备立方形环糊精-金属有机骨架(CD-MOF)；
(2)通过交联剂对所述的立方形环糊精-金属有机骨架(CD-MOF)进行交联，去除金属离子，保留立方形态，得到环糊精骨架(COF)；
较佳地，具体交联反应过程为：将立方形的环糊精-金属有机骨架分散于有机溶剂A中，一定温度下搅拌条件下加入交联剂和催化剂A进行反应，使环糊精-金属有机骨架内的羟基通过共价键交联起来，经冷却、乙醇终止反应、离心、洗涤和干燥，得到立方形的环糊精骨架(COF)；
(3)在所述的立方形环糊精骨架表面修饰RGD，得到所述的立方形环糊精骨架-RGD组合物(RGD-COF)；较佳地，具体的RGD修饰过程为：将立方形环糊精骨架和RGD按照一定比例加入有机溶剂B中，混合均匀后加入催化剂B加热搅拌，使立方形环糊精骨架表面的羟基与RGD的羧基充分偶联，反应完成后洗涤干燥得立方形环糊精骨架-RGD组合物(RGD-COF)。
药学上可接受的载体
“药学上可接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如
Figure PCTCN2020072261-appb-000001
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明的主要优点在于：
本发明提供了一种可静脉注射用(纳米级)或局部用药(微米级)的立方形环糊精骨架-RGD组合物(RGD-COF)及其制备方法，其安全性高、生物相容性好。可用于血管相关疾病的靶向治疗，包括创伤失血、深部出血、动脉粥样硬化、脑卒中、血栓和肿瘤等疾病，具有以下技术优点：
(1)本发明RGD-COF具有规则的立方体形态，突破以往载体球形形态的限制，有效逃避巨噬细胞的吞噬和清除，增强对受损血管的迁移性和粘附性，提高靶向性和治疗效率。
(2)本发明采用RGD序列修饰后，立方形的RGD-COF可以高效地靶向和聚集于血管损伤部位的活化血小板，实现血管相关疾病的靶向治疗，减少对正 常血液循环系统的影响和副作用。
(3)本发明充分发挥CD-MOF尺寸可控的优势，以纳米级和微米级CD-MOF为基础材料，经交联步骤和RGD修饰步骤，获得了可静脉注射用和/或可外用的新型高效的载体材料。静脉注射给药适于复杂条件下的创伤救治，适用于全身性失血的控制和血栓类疾病；微米级RGD-COF可用于车祸、手术等外部创伤的止血、抗炎和抗感染治疗。
(4)本发明中的RGD-COF具有良好的稳定性，可制成冻干粉使用，利于保存，适合在野外条件下使用，比如装备部队，提高复杂战创伤救治能力。
(5)本发明制备方法简单可控、无需昂贵的设备、可大规模生产、不具有免疫原性，不会传播传染性疾病。
(6)本发明其制备方法简单，安全性高、生物相容性好，可用于创伤失血、脏器内出血、深部出血等失控性出血和血栓类疾病的靶向诊断和治疗。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1
制备纳米级CD-MOF：使用溶剂热的方式，直接对γ-CD与KOH水溶液与一部分有机溶剂混合体系进行加热。称取163.0mgγ-CD和56.0mg KOH混合物(γ-CD和KOH摩尔比为1:8)溶解于5mL水中，预加3mL甲醇至混合溶液内，50℃水浴加热20min后，取出溶液，加入等体积的甲醇，再加入64mg PEG 20000，静置1h后，4000rpm离心5min，分别用乙醇(10mL×2)、二氯甲烷(10mL×2)洗涤，将所得晶体50℃真空干燥12h，即得纳米级CD-MOF晶体(CD-MOF-Nano)，得到的CD-MOF为规则的立方体形态，粒径为200-500nm(图1)。
制备纳米级COF：称取778.3mg纳米级CD-MOF粉末于圆底烧瓶中，固定于磁力搅拌器上，加入10mL二甲基甲酰胺，80℃加热，600rpm搅拌条件下，加入771mg交联剂碳酸二苯酯(CD-MOF与交联剂的摩尔比为1:6)和450μL催化剂三乙胺，反应24h后，冷却至室温，采用20mL 95％乙醇终止反应后，4000rpm离心5min，分别用50％乙醇(10mL×2)、纯水(10mL×2)和丙酮(10 mL×2)洗涤，将所得晶体50℃真空干燥12h即得水中稳定的纳米COF，产率为61％，得到的COF为规则的立方体形态，粒径为200-500nm(图2)。
制备纳米级环糊精骨架-RGD组合物(RGD-COF)：称取230mg纳米级COF和10mg线性GRGDS五肽(COF和GRGDS的摩尔比为1:1)置于圆底烧瓶中，加入5mL二甲基甲酰胺，搅拌均匀后再加入5mg 4-二甲氨基吡啶和6mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，置于磁力搅拌器上37℃，600rpm搅拌12h，使COF与GRGDS多肽充分偶联。反应完成后，4000rpm离心5min，分别用二甲基甲酰胺(10mL×2)和纯水(10mL×2)洗涤，-50℃冷冻干燥12h即得GRGDS修饰的COF，SEM(图3)和DLS(图4)结果显示得到的RGD-COF为规则的立方体形态，粒径为200-500nm，高效液相色谱法(HPLC)测得环糊精骨架材料与RGD的质量比为1:0.049。
制备的RGD-COF在纯水、磷酸盐缓冲溶液(PBS，pH＝7.4)、生理盐水和大鼠血浆中均具有良好的物理稳定性(图5)。MTT结果显示RGD-COF无细胞毒性，具有良好的生物相容性和安全性(图6)。
实施例2
同实施例1制备纳米级CD-MOF。
制备纳米级COF：称取778.3mg纳米级CD-MOF粉末于圆底烧瓶中，固定于磁力搅拌器上，加入10mL乙腈，40℃加热，200rpm搅拌条件下，加入194.6mg交联剂N,N'-羰基二咪唑(CD-MOF与交联剂的摩尔比为1:2)和450μL催化剂吡啶，反应48h，其余同实施例1，得到的COF为规则的立方体形态，粒径为100-300nm。
制备纳米级RGD-COF：称取230mg纳米级COF和14mg线性RGD(COF和RGD的摩尔比为1:2)置于圆底烧瓶中，加入5mL乙腈，搅拌均匀后再加入5mg N,N'-琥珀酰亚胺基碳酸酯和6mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，置于磁力搅拌器上20℃，200rpm搅拌48h，其余同实施例1，得到的RGD-COF为规则的立方体形态，粒径为100-300nm，HPLC法测得环糊精骨架与RGD的质量为1:0.08。
实施例3
同实施例1制备纳米级CD-MOF。
制备纳米级COF：称取778.3mg纳米级CD-MOF粉末于圆底烧瓶中，固定于磁力搅拌器上，加入10mL四氢呋喃，100℃加热，1000rpm搅拌条件下，加入0.5mL交联剂乙二醇二缩水甘油醚(CD-MOF与交联剂的摩尔比为1:5)和5mg催化剂N-羟基丁二酰亚胺，反应4h后，其余同实施例1,SEM和DLS结果显示得到的COF为规则的立方体形态，粒径为200-400nm。
制备纳米级RGD-COF：称取230mg纳米级COF和3mg环形GRGD多肽 (COF和环形GRGD的摩尔比为5:1)置于圆底烧瓶中，加入5mL甲醇，搅拌均匀后再加入5mg N-羟基丁二酰亚胺和6mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，置于磁力搅拌器上40℃，1000rpm搅拌4h，其余同实施例1，SEM和DLS结果显示得到的RGD-COF为规则的立方体形态，粒径为200-400nm，HPLC法测得环糊精骨架材料与RGD的质量比为1:0.005。
实施例4
同实施例1制备纳米级CD-MOF。
制备纳米级COF：称取778.3mg纳米级CD-MOF粉末于圆底烧瓶中，固定于磁力搅拌器上，加入10mL丙酮，50℃加热，900rpm搅拌条件下，加入0.6mL交联剂戊二醛(CD-MOF与交联剂的摩尔比为1:10)和5mg催化剂吡啶，反应12h后，其余同实施例1，SEM和DLS结果显示得到的COF为规则的立方体形态，粒径为200-500nm。
制备纳米级RGD-COF：称取200mg纳米级COF和3mg环形RGDS(COF和环形RGDS的摩尔比为4:1)置于圆底烧瓶中，加入5mL丙酮，搅拌均匀后再加入5mg N-羟基丁二酰亚胺和6mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，置于磁力搅拌器上40℃，900rpm搅拌6h，其余同实施例1，得到的RGD-COF为规则的立方体形态，尺寸为200-500nm，HPLC法测得环糊精骨架材料与RGD的质量比为1:0.015。
实施例5
制备微米级CD-MOF：使用溶剂热的方式，直接对γ-CD与KOH水溶液与一部分有机溶剂混合体系进行加热。称取163.0mgγ-CD和56.0mg KOH混合物(γ-CD和KOH摩尔比为1:8)溶解于5mL水中，预加3mL甲醇至混合溶液内，50℃水浴加热20min后，取出溶液，再加入64mg PEG 2000，静置半小时后，4000rpm离心5min，分别用乙醇(10mL×2)、二氯甲烷(10mL×2)洗涤，将所得晶体50℃真空干燥12h，即得微米级CD-MOF晶体，SEM和DLS结果显示得到的CD-MOF为规则的立方体形态，尺寸为1-10μm。
制备微米级COF：称取778.3mg微米级CD-MOF粉末于圆底烧瓶中，固定于磁力搅拌器上，加入10mL乙酸乙酯，70℃加热，700rpm搅拌条件下，加入0.9mL交联剂环氧氯丙烷(CD-MOF与交联剂的摩尔比为1:20)和5mg催化剂4-二甲氨基吡啶，反应16h后，其余同实施例1，得到的COF为规则的立方体形态，粒径为1-10μm。
制备微米级RGD-COF：称取230mg微米级COF和4mg RGDS(COF和RGDS的摩尔比为2:1)置于圆底烧瓶中，加入6mL正己烷，搅拌均匀后再加入5mg 4-二甲氨基吡啶和0.2mL三乙胺，置于磁力搅拌器上25℃，700rpm搅拌24h，其余同实施例1，得到的微米级立方形环糊精骨架-RGD组合物 RGD-COF，SEM和DLS结果显示得到的RGD-COF为规则的立方体形态，尺寸为1-10μm，HPLC法测得环糊精骨架材料与RGD的质量比为1:0.05。
实施例6
同实施例5制备微米级CD-MOF，粒径为1-5μm(图7)。
制备微米级COF：称取778.3mg微米级CD-MOF粉末于圆底烧瓶中，固定于磁力搅拌器上，加入10mL丙酮，50℃加热，400rpm搅拌条件下，加入0.7mL交联剂甲苯二异氰酸酯(CD-MOF与交联剂的摩尔比为1:8)和450μL催化剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺，反应8h后，其余同实施例1,得到的COF为规则的立方体形态，粒径为1-5μm(图8)。
制备微米级RGD-COF：称取230mg微米级COF和18.5mg环形RGD多肽(COF和环形RGDS的摩尔比为1:2)置于圆底烧瓶中，加入6mL异丙醇，搅拌均匀后再加入5mg 4-二甲氨基吡啶和6mg N-羟基丁二酰亚胺，置于磁力搅拌器上35℃，400rpm搅拌8h，其余同实施例1，得到的RGD-COF为规则的立方体形态，尺寸为1-5μm(图9)，HPLC法测得环糊精骨架材料与RGD的质量比为1:0.016。
实施例7
同实施例5制备微米级CD-MOF。
制备微米级COF：称取778.3mg微米级CD-MOF粉末于圆底烧瓶中，固定于磁力搅拌器上，加入10mL异丙醇，60℃加热，500rpm搅拌条件下，加入1729mg交联剂柠檬酸(CD-MOF与交联剂的摩尔比为1:15)和450μL催化剂吡啶，反应16h后，其余同实施例1，得到的COF为规则的立方体形态，粒径为30-50μm。
制备微米级RGD-COF：称取230mg微米级COF和30mg GRGDS五肽(COF和环形GRGDS摩尔比为1:3)置于圆底烧瓶中，加入6mL正己烷，搅拌均匀后再加入5mg 4-二甲氨基吡啶和6mg N,N'-琥珀酰亚胺基碳酸酯，置于磁力搅拌器上30℃，500rpm搅拌24h，其余同实施例1，得到的RGD-COF为规则的立方体形态，粒径为30-50μm，HPLC法测得环糊精骨架材料与RGD的质量比为1:0.065。
实施例8
同实施例5制备微米级CD-MOF。
制备微米级COF：称取778.3mg微米级CD-MOF粉末于圆底烧瓶中，固定于磁力搅拌器上，加入10mL氯仿，70℃加热，700rpm搅拌条件下，加入403mg交联剂丁二酰氯(CD-MOF与交联剂的摩尔比为1:4)和450μL催化剂吡啶，反应16h后，其余同实施例1，得到的COF为规则的立方体形态，粒径为10-30μm。
制备微米级RGD-COF：称取230mg微米级COF和25mg环形GRGD(COF和环形GRGD摩尔比为1:5)置于圆底烧瓶中，加入6mL氯仿，搅拌均匀后再加入5mg 4-二甲氨基吡啶和6mg N-羟基丁二酰亚胺，置于磁力搅拌器上37℃，700rpm搅拌18h，其余同实施例1，SEM和DLS结果显示得到的RGD-COF为规则的立方体形态，粒径为10-30μm，HPLC法测得环糊精骨架材料与RGD的质量比为1:0.46。
实施例9
GRGDS修饰的球形的环糊精纳米海绵(RGD-NS)的制备(对比实施例)
(1)制备球形的环糊精纳米海绵：称取3.891gγ-CD(3.000mmol)超声溶于20mL 0.1M氢氧化钾水溶液，0.8μm滤膜过滤，得到水相备用。称取1.297g(8.00mmol)N,N'-羰基二咪唑溶于20mL二氯甲烷中，得到有机相。在连续磁力搅拌(600rpm)下，将γ-CD氢氧化钾水溶液滴加到有机相中。反应30min后，分别用去离子水和无水乙醇洗涤沉淀物各两次，每次4000rpm离心5min，收集沉淀。冷冻干燥，即得球形的环糊精纳米海绵(CD-NS)，得到的CD-NS为球形，粒径为200-500nm。
(2)GRGDS修饰环糊精纳米海绵：称取230mg球形的CD-NS和10mg GRGDS五肽(CD-NS和GRGDS的摩尔比为1:1)置于圆底烧瓶中，加入5mL二甲基甲酰胺，搅拌均匀后再加入5mg 4-二甲氨基吡啶和6mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，置于磁力搅拌器上37℃，600rpm搅拌24h，使CD-NS与GRGDS多肽充分偶联。反应完成后，4000rpm离心5min，分别用二甲基甲酰胺(10mL×2)和纯水(10mL×2)洗涤，-50℃冷冻干燥12h即得GRGDS修饰的CD-NS(产物简写为RGD-NS)，SEM和DLS结果显示得到的GS5-NS为球形，粒径为200-500nm(图10)，HPLC法测得环糊精骨架材料与RGD的质量比为1:0.052。
实施例10
RGD-COF的体外凝血性能检测
通过测定体外凝血块形成时间，可以评价止血材料的体外凝血性能。取健康大鼠3.2％柠檬酸钠抗凝的新鲜全血适量于干净的试管中，加入80μL CaCl 2(0.1M)使Ca 2+的终浓度为10mM，随后加入样品溶液或生理盐水，使反应体系终体积为800μL，500rpm/min涡旋10s。分别设生理盐水对照组(是空白对照)、RGD-COF组(20、50、100μg/mL)、COF组(100μg/mL)和RGD-NS组(100μg/mL)。立即移取60μL上述处理过的全血于96孔板中，每个样品种12个孔。每隔30s，采用生理盐水洗涤一个样品孔，除去可溶性的血液成分，阻止该孔的凝血反应，直到洗涤液为无色，说明已经彻底清除可溶性的血液成分。当血凝块覆盖整个孔的底部，且在后续时间点冲洗时凝血块的大小保持不 变时，认为已经形成稳定的凝血块，该时间记为体外凝血块形成时间。
体外凝血块形成时间结果如图11所示，RGD-COF能显著缩短大鼠全血在体外的凝血时间，并且具有剂量依赖性。健康大鼠全血在体外的凝血块形成时间平均为5.5min，实施例1中制备的COF空白载体组的平均凝血块形成时间为5.4min，说明COF载体自身对凝血过程无影响。实施例1中制备的立方形RGD-COF低(20μg/mL)、中(50μg/mL)、高剂量组(100μg/mL)分别将凝血块形成时间降低了39％、52％和68％，而实施例9中制备的球形RGD-NS仅将凝血块形成时间降低了29％，体外止血作用远不如立方形的RGD-COF。
实施例2中制备的RGD-COF低、中、高剂量组分别将凝血块形成时间降低了32％、49％和61％。
实施例3中制备的RGD-COF低、中、高剂量组分别将凝血块形成时间降低了36％、55％和69％。
实施例6中制备的RGD-COF低、中、高剂量组分别将凝血块形成时间降低了37％、48％和65％。
实施例11
RGD-COF的体内止血效果评价：纳米级静脉注射用RGD-COF止血药，需用生理盐水分散后才可以进行静脉注射，供静脉注射用纳米级RGD-COF止血药的制备方法，包括以下步骤：
(1)按实施例1、实施例2、实施例3和实施例4的方法制备立方形RGD-COF纳米颗粒；按实施例9球形的RGD-NS纳米颗粒。
(2)纳米颗粒的分散：用生理盐水悬浮纳米颗粒(1-10mg/mL，优选为2mg/mL)，得到纳米颗粒分散液。
采用小鼠断尾模型评价RGD-COF纳米颗粒的体内止血能力。取健康昆明小鼠50只，随机分为5组，每组10只。分别设空白对照组(只断尾损伤处理，对应于图12和图13中的“损伤处理”)、生理盐水组、COF组、RGD-NS组、RGD-COF低剂量组(20mg/kg，即图12和图13中的20mg/kg)和RGD-COF高剂量组(40mg/kg，即图12和图13中40mg/kg)。给药方式为尾静脉注射，单次给药，给药剂量为20-40mg/kg体重,给药体积：10mL/kg。给药后5min，用锋利的剪刀在距小鼠尾尖0.5cm处快速剪断，当血液自行流出后立即计时，每隔20s，用吸水纸轻触尾部断处，直到断尾处不再有血液流出(无血丝出现)，这段时间记为出血时间。采用Image Pro Plus软件计算吸水纸上的血滴面积，进而估算小鼠的失血量。
体内止血实验结果表明，RGD-COF能显著缩短小鼠的出血时间，并能大大降低小鼠的出血量，具有良好的体内止血效果。如图12和图13所示，与生理盐水组或COF载体组相比，实施例1中制备的RGD-COF低剂量组(20mg/kg) 能将出血时间从400s缩短到150s，出血时间缩短了62.5％；将失血量从2mL/kg降低到0.6mL/kg，失血量减少了70％。实施例1中制备的RGD-COF高剂量组(40mg/kg)能将出血时间从400s缩短到40s，缩短了90％；将失血量降低到0.2mL/kg，降低了90％。
实施例2中制备的RGD-COF低剂量组能将出血时间从400s缩短到155s，出血时间缩短了61％；将失血量从2mL/kg降低到0.8mL/kg，失血量减少了60％。实施例2中制备的RGD-COF高剂量组能将出血时间从400s缩短到50s，缩短了87.5％；将失血量降低到0.12mL/kg，降低了94％。然而，实施例9中制备的球形RGD-NS仅仅将出血时间缩短了23％，且不能降低失血量。因此，立方形的RGD-COF比球形的RGD-NS具有更高效的体内止血效果。
实施例3中制备的RGD-COF高剂量组能将出血时间从400s缩短到52s，缩短了87％；将失血量降低到0.15mL/kg，降低了92.5％。
实施例4中制备的RGD-COF高剂量组能将出血时间从400s缩短到47s，缩短了88.2％；将失血量降低到0.17mL/kg，降低了91.5％。
实施例12
同实施例6制备微米级CD-MOF。
CD-MOF载银：称取169mg硝酸银，用乙腈定容在100mL容量瓶内，配制成10mmol/L的硝酸银溶液。称取微米级CD-MOF 600mg于EP管内，加入1.5mL乙腈混合后超声10min，然后加入5mL 10mmol/L的硝酸银溶液，放置于暗处72h后，用乙腈(10mL)洗涤3次，4000rpm离心5min，放入真空干燥箱40℃干燥12h。
微米级载银COF：称取80℃真空干燥过的微米级载银CD-MOF粉末1g于圆底烧瓶中，固定于磁力搅拌器上，加入12.85mL N,N-二甲基甲酰胺，80℃加热，500rpm搅拌，当反应液温度达到60℃时，加入0.99g交联剂碳酸二苯酯(CD-MOF与交联剂的摩尔比为1:6)和0.5mL催化剂三乙胺，反应24h后，4000rpm离心5min，分别用乙醇(10mL)、纯水(10mL)、丙酮(10mL)洗涤2次，真空箱60℃干燥6h。得到的微米级载银COF为规则的立方体形态，粒径为1-5μm。
载银RGD-COF：称取1g微米级载银COF和21.7mg 4-二甲氨基吡啶置于圆底烧瓶中，加入21.7mL N,N-二甲基甲酰胺，搅拌均匀后再加入21.7mg线形GRGDS，13.36mg碳酰二亚胺和86.95uL三乙胺，置于磁力搅拌器上37℃，500rpm避光搅拌24h，4000rpm离心5min，分别用N,N-二甲基甲酰胺(10mL)、乙醇(10mL)、纯水(10mL)、丙酮(10mL)各洗涤1遍，将所得晶体60℃真空干燥6h即得。得到的载银RGD-COF为规则的立方体形态，粒径为1-5μm，HPLC法测得环糊精骨架材料与RGD的质量比为1:0.015。
抗菌效果：载银CD-MOF、载银COF、载银RGD-COF对大肠杆菌CMCC(B)44102的MIC值均为16μg/mL，与市售制剂
Figure PCTCN2020072261-appb-000002
相比，抗菌效果均优于市售制剂(128μg/mL)。时间-杀菌曲线上(图14)可见，当Ag的浓度在4μg/mL～16μg/mL时，纳米银对大肠杆菌CMCC(B)44102的杀菌作用微弱，当Ag浓度大于16μg/mL并逐渐升高时杀菌作用明显增强，1.0MBC(32μg/mL)的纳米银在6-8h内能很好地抑制菌的生长。载银CD-MOF、载银COF、载银RGD-COF对金黄色葡萄球菌CMCC(B)26112的MIC值均为128μg/mL，抗菌效果类似于市售制剂(128μg/mL)。
创口愈合效果(图15)：与市售纳米银试剂(对照组)相比，载银RGD-COF显著加速大鼠表皮创口愈合的速度；同时相比于载银CD-MOF与载银COF组相比，载银RGD-COF对大鼠表皮创口愈合效果更佳。
实施例13
RGD-COF载抗血栓药：分别称取适量阿司匹林、盐酸噻氯匹定、西洛他唑、硫酸氢氯吡格雷II型、利伐沙班、盐酸奥扎格雷、盐酸替罗非班等7种抗血栓药物，加入30mL无水乙醇超声10min使其溶解，然后分别加入500mg实施例1中制备的RGD-COF，药物与RGD-COF的摩尔比为2:1，室温下300rpm搅拌24h，孵育载药。载药完成后，4000rpm离心5min，得到下层载药RGD-COF，采用HPLC法测定载药量如表1。
表1纳米级RGD-COF负载抗血栓药物的载药量
Figure PCTCN2020072261-appb-000003
实施例14
RGD-COF载止血药：分别称取适量氨甲环酸、氨基己酸、维生素K1等3种止血药物，加入30mL无水乙醇超声10min使其溶解，然后分别加入500mg实施例1中制备的RGD-COF，药物与RGD-COF的摩尔比为2:1，37℃温度下400rpm搅拌12h，孵育载药。载药完成后，4000rpm离心5min，得到下层载药RGD-COF，采用HPLC法测定载药量分别为13.5％、4.3％和2.1％。
实施例15
RGD-COF载抗感染药：分别称取适量磺胺嘧啶、头孢曲松、阿莫西林、左氧氟沙星4种抗感染药物，加入30mL无水乙醇超声10min使其溶解，然后分别加入500mg实施例1中制备的RGD-COF，药物与RGD-COF的摩尔比为1:1，室温下200rpm搅拌48h，孵育载药。载药完成后，4000rpm离心5min，得到下层载药RGD-COF，采用HPLC法测定载药量分别为3.8％、3.3％、4.6％和6.7％。
实施例16
RGD-COF靶向体内血栓：采用FeCl 3诱导形成小鼠肠系膜血栓，并采用预先注射的罗丹明B标记血栓部位的活化血小板，血栓形成后，尾静脉注射红色Cy5荧光标记的实施例3中制备的RGD-COF(40mg/kg)，荧光显微镜下观察到，红色Cy5荧光标记的RGD-COF可以靶向富集肠系膜血栓，RGD-COF纳米粒与血栓处活化血小板的共定位系数高达0.65，远远高于未修饰的COF和球形的RGD-NS组(图16)，说明立方形的RGD-COF可以高度靶向体内血栓。
实施例17
RGD-COF靶向体内出血部位：采用小鼠断尾模型评价实施例2中制备的RGD-COF对体内出血部位的靶向性。昆明小鼠经尾静脉注射Cy5荧光标记的COF、RGD-NS和RGD-COF纳米粒(40mg/kg)5min后，用锋利的剪刀在距小鼠尾尖0.5cm处快速剪断，建立小鼠断尾模型。断尾损伤10min后，当断尾处的出血已停止，在距第一次切口1cm处剪断小鼠尾巴，得到1cm长的小鼠尾巴样品，采用小动物活体成像仪测定断尾处聚集的RGD-COF的荧光信号。因为RGD-COF可以靶向出血部位聚集的活化血小板，RGD-COF组断尾处的荧光信号是COF组的4倍，是RGD-NS组的3倍，说明立方形RGD-COF可以高度靶向并聚集到断尾出血部位，比球形RGD-NS具有更好的体内靶向性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一种环糊精骨架-RGD组合物，其特征在于，所述组合物中，环糊精骨架与RGD的质量比为1:0.001-1:1，较佳地为1:0.005-1:0.5；
    所述的环糊精骨架-RGD组合物粒径为10nm-50μm，优选为50nm-50μm，更优选为100-500nm或1-5μm。
  2. 如权利要求1所述的组合物，其特征在于，所述组合物为立方形环糊精骨架-RGD组合物。
  3. 一种立方形环糊精骨架-RGD组合物的制备方法，其特征在于，包括步骤：
    (1)提供一立方形环糊精-金属有机骨架(CD-MOF)；
    (2)交联步骤，通过交联剂对(1)中的立方形环糊精-金属有机骨架进行交联，得到环糊精骨架(COF)；
    (3)RGD修饰步骤，在(2)中所述的环糊精骨架上修饰RGD，得到立方形环糊精骨架-RGD组合物(RGD-COF)。
  4. 如权利要求3所述的制备方法，其特征在于，所述交联步骤包括下列子步骤：
    (2a)分散步骤，将立方形的环糊精-金属有机骨架分散于有机溶剂A中，得到分散液2a；
    (2b)交联剂和催化剂加入步骤，在交联反应温度T条件下向分散液2a中加入交联剂和催化剂A，反应时间t1之后，得到分散液2b；
    (2c)任选地，冷却步骤，对分散液2b进行冷却，得到经冷却的分散液2b；
    (2d)任选地，终止反应步骤，向经冷却的分散液2b中，加入反应终止剂，得到分散液2d；
    (2e)任选地，离心步骤，对分散液2d进行离心，得到晶体2e；
    (2f)任选地，洗涤步骤，洗涤晶体2e，得到经洗涤的晶体2f；
    (2g)任选地，干燥步骤，对经洗涤的晶体2f进行干燥处理；
    (2h)获得立方形的环糊精骨架(COF)。
  5. 如权利要求3所述的制备方法，其特征在于，所述RGD修饰步骤包括下列子步骤：
    (3a)分散步骤，将立方形的环糊精骨架(COF)和RGD分散于有机溶剂B中，得到分散液3a；
    (3b)偶联步骤，向分散液3a中加入催化剂B，使立方形的环糊精骨架与RGD偶联，反应时间为t2；
    (3c)任选地，离心步骤；
    (3d)任选地，洗涤步骤；
    (3f)任选地，干燥步骤；
    (3g)获得立方形环糊精骨架-RGD组合物(RGD-COF)。
  6. 如权利要求3所述的方法，其特征在于，所述的交联剂选自下组：过氧化物、多异氰酸酯、缩水甘油醚、二元或多元酸类、二元或多元醛类、含羰基的化合物、环氧化物类、丙烯酸酯类、酰氯类、或其组合。
  7. 如权利要求3所述的方法，其特征在于，
    所述的催化剂A选自下组：4-二甲氨基吡啶、三乙胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺或其盐、N,N'-琥珀酰亚胺基碳酸酯、N-羟基丁二酰亚胺、吡啶、或其组合；较佳地为三乙胺；
    所述的有机溶剂A选自下组：二甲基甲酰胺、四氢呋喃、甲醇、乙腈、丙酮、异丙醇、乙酸乙酯、氯仿、正己烷、乙醇、二氯甲烷。
  8. 如权利要求3所述的方法，其特征在于，
    所述的催化剂B选自下组：4-二甲氨基吡啶、三乙胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺或其盐、N-羟基丁二酰亚胺、N,N'-琥珀酰亚胺基碳酸酯、吡啶、或其组合；
    所述的有机溶剂B选自下组：二甲基甲酰胺、四氢呋喃、甲醇、乙腈、丙酮、异丙醇、乙酸乙酯、氯仿、正己烷、乙醇、二氯甲烷、或其组合。
  9. 一种负载药物的立方形环糊精骨架-RGD组合物，其特征在于，所述立方形环糊精骨架-RGD组合物为权利要求1或2所述的组合物，或通过权利要求3所述的方法制备，所述的药物选自下组：抗菌药物、止血药物、抗血栓药物、抗感染药物、或其组合。
  10. 一种活性成分的用途，其特征在于，所述活性成分选自下组：
    (i)权利要求1或2所述环糊精骨架-RGD组合物；
    (ii)权利要求9所述的负载药物的立方形环糊精骨架-RGD组合物；
    (iii)环糊精骨架(COF)；
    (iv)上述(i)、(ii)或(iii)的组合；
    所述活性成分被用于：
    (a)制备载药材料；
    (b)制备治疗和/或诊断试剂或试剂盒；
    (c)制备止血药物和/或材料；
    (d)制备抗感染药物和/或材料；
    (e)制备抗菌药物和/或材料；
    (f)制备促进伤口愈合药物和/或材料；
    (g)制备预防和/或治疗血栓的药物和/或材料。
  11. 一种药物组合物，其特征在于，所述药物组合物包含：
    (1)活性成分，所述活性成分为如权利要求1或2所述的环糊精骨架-RGD组合物或如权利要求9所述的负载药物的立方形环糊精骨架-RGD组合物；和
    (2)药学上可接受的载体。
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