WO2022068927A1 - 具有肺靶向的肝素-多肽双重接枝环糊精骨架组合物及其制备方法与应用 - Google Patents

具有肺靶向的肝素-多肽双重接枝环糊精骨架组合物及其制备方法与应用 Download PDF

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WO2022068927A1
WO2022068927A1 PCT/CN2021/122163 CN2021122163W WO2022068927A1 WO 2022068927 A1 WO2022068927 A1 WO 2022068927A1 CN 2021122163 W CN2021122163 W CN 2021122163W WO 2022068927 A1 WO2022068927 A1 WO 2022068927A1
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cof
lmwh
polypeptide
composition
group
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French (fr)
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张继稳
何亚平
熊婷
伍丽
何思钰
周咏
周盼盼
陈加才
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中国科学院上海药物研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers

Definitions

  • the invention relates to the field of medical biomaterials, in particular to an anti-tumor heparin-polypeptide double-grafted cyclodextrin skeleton composition with lung-targeting effect and its preparation and application.
  • lung cancer is the tumor with the highest morbidity and mortality in the world, and the targeted therapy of lung cancer is a scientific problem that needs to be solved urgently. Due to its complex pathogenesis, difficult early diagnosis, easy metastasis, high degree of malignancy, and high mortality, chemotherapy is still a common method for tumor treatment.
  • chemotherapy drugs are mostly cytotoxic drugs. There are often strong toxic and side effects on normal tissues and organs. Therefore, there is an urgent need to develop targeted and intelligent drug delivery systems with low toxicity and low doses.
  • lung cancer nano-targeted nano-delivery systems liposomes, polymer nanoparticles, polymer micelles, solid lipid nanoparticles, magnetic nanoparticles and metal nanoparticles, etc.
  • EPR effect to achieve lung targeting, most of which target highly expressed cell surface receptors at tumor sites, do not have the specificity of lung tissue targeting, often the lung targeting effect is not ideal, and also has high toxicity and refractory degradation. Wait for the short board.
  • designing a safe and efficient targeted drug delivery system for lung cancer is a scientific problem that needs to be broken through in the treatment of lung cancer.
  • the purpose of the present invention is to provide an LMWH-polypeptide-COF composition and a drug-loaded LMWH-polypeptide-COF composition with excellent safety, high-efficiency targeting, and excellent anti-tumor efficacy.
  • a first aspect of the present invention provides a LMWH-polypeptide-COF composition, the composition comprising the following components:
  • Low molecular weight heparin LMWH the low molecular weight heparin LMWH is covalently linked to the cross-linked cyclodextrin organic framework COF.
  • the cross-linked cyclodextrin organic framework COF has lung targeting properties.
  • the cross-linked cyclodextrin organic framework COF has a cubic shape.
  • the size of the cross-linked cyclodextrin organic framework COF is 10-1000 nm, preferably 50-800 nm, more preferably 100-500 nm.
  • polypeptide is selected from the group consisting of integrin-binding peptide (RGD), CD13 metallopeptidase-binding peptide (NGR), or a combination thereof.
  • RGD integrin-binding peptide
  • NGR CD13 metallopeptidase-binding peptide
  • the RGD is selected from the group consisting of linear group A substances, annular c(RGDfK), or a combination thereof.
  • the group A substance is selected from the group consisting of RGD, DRGDS, GRGD, RGDS, GRGDS, GRGDSP, GRADSPK, GRGDSPK, or a combination thereof.
  • the NGR is selected from the group consisting of linear group B substances, cyclic group C substances, or a combination thereof.
  • the group B substance is selected from the group consisting of TNGRGP, NGRSRF, RSRNGR, NGRNTV, or a combination thereof.
  • the group C substance is selected from the group consisting of GGCNGRC, CNGRC, or a combination thereof.
  • the polypeptide is connected to the cross-linked cyclodextrin organic framework COF as follows: the carboxyl group or amino group at one end of the polypeptide is connected with the hydroxyl group on the surface of the cross-linked cyclodextrin organic framework COF. Covalently linked.
  • the low molecular weight heparin LMWH is selected from the group consisting of low molecular weight heparin, low molecular weight heparin sodium, low molecular weight heparin calcium, or a combination thereof.
  • the molecular weight of the low molecular weight heparin LMWH is 2000-8000 Da, preferably 3000-6000 Da, more preferably 4000-5000 Da.
  • the low molecular weight heparin LMWH and the cross-linked cyclodextrin organic framework COF are connected as follows:
  • a2) Covalently link the amino group at one end of the cystamine-modified low-molecular-weight heparin LMWH obtained in step a1) with the hydroxyl group on the surface of the cross-linked cyclodextrin organic framework COF.
  • the size of the composition is 10-1000 nm, preferably 50-800 nm, more preferably 100-500 nm.
  • the mass ratio of the cross-linked cyclodextrin organic framework COF to the polypeptide is 1:0.001-0.1 (preferably 1:0.01-0.1, more preferably 1:0.02-0.08); and / or
  • the mass ratio of the cross-linked cyclodextrin organic framework COF to the low molecular weight heparin LMWH is 1:0.001-0.05 (preferably 1:0.005-0.02, more preferably 1:0.008-0.01).
  • composition of the first aspect of the present invention comprising the steps of:
  • Activating the polypeptide-modified cross-linked cyclodextrin organic framework COF comprising the steps of: activating the polypeptide-modified cross-linked cyclodextrin organic framework COF at a first temperature in the presence of a third solvent, an activated linker, and an activated catalyst The first time, the activated polypeptide-modified cross-linked cyclodextrin organic framework COF is obtained;
  • composition of the first aspect of the present invention is prepared, comprising the steps of: in a fourth solvent, the cystamine-modified low-molecular-weight heparin LMWH obtained in step 2) and the activated polypeptide-modified cross-linked cyclodextrin obtained in step 3) are made
  • the organic framework COF is reacted at a second temperature for a second time to obtain the composition of the first aspect of the present invention.
  • the method has one or more features selected from the group consisting of:
  • the first solvent is selected from the group consisting of dimethylformamide, acetonitrile, acetone, or a combination thereof;
  • the activator A is selected from the following group: 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, triethylamine, N,N '-disuccinimidyl carbonate, N-hydroxysuccinimide, N,N'-carbonyldiimidazole, or a combination thereof;
  • the second solvent is selected from the group consisting of phosphate buffered saline, water, or a combination thereof;
  • the activator B is selected from the following group: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 1-hydroxybenzotrioxide azole, or a combination thereof;
  • the mass ratio of cystamine to low molecular weight heparin LMWH is 1:10-20 (preferably 1:12-18, more preferably 1:12-16) ;
  • the third solvent is selected from the group consisting of acetonitrile, formamide, dimethylformamide, acetone, methanol, or a combination thereof;
  • the activated linking arm is selected from the group consisting of N,N'-disuccinimidyl carbonate, N,N'-carbonyldiimidazole, succinyl chloride, isocyanate, or a combination thereof;
  • the activation catalyst is selected from the group consisting of triethylamine, pyridine, N-hydroxysuccinimide, or a combination thereof;
  • the molar ratio of the polypeptide-modified cross-linked cyclodextrin organic framework COF, the activation linker and the activation catalyst is 1:1-10:1-7 (preferably 1:1-8:1-5) );
  • the first temperature is 10-100°C (preferably 20-80°C);
  • the first time is 3-50h (preferably 5-25h);
  • the fourth solvent is selected from the group consisting of formamide, dimethylformamide, acetonitrile, acetone, methanol, or a combination thereof;
  • the second temperature is 10-100°C (preferably 20-80°C);
  • the second time is 8-60h (preferably 10-50h).
  • a pharmaceutical active ingredient is provided, and the pharmaceutical active ingredient comprises:
  • an anti-tumor drug or a drug targeting the lung is loaded in the drug carrier.
  • the anti-tumor drug includes an anti-tumor drug for treating lung cancer.
  • the pharmaceutical active ingredient has lung targeting properties.
  • the antitumor drug is selected from the group consisting of cytotoxic drugs, molecularly targeted drugs, adjuvant drugs, or a combination thereof.
  • the cytotoxic drug is selected from the group consisting of doxorubicin, doxorubicin hydrochloride, epirubicin, carboplatin, oxaliplatin, 5-fluoropyrimidine, capecitabine , gemcitabine, paclitaxel, docetaxel, topotecan, 10-hydroxycamptothecin, irinotecan, or a combination thereof.
  • the molecular targeted drug is selected from the group consisting of gefitinib, imatinib, erlotinib, or a combination thereof.
  • the auxiliary drug is selected from the group consisting of resveratrol, quercetin, baicalein, curcumin, or a combination thereof.
  • a pharmaceutical composition comprising:
  • the dosage form of the pharmaceutical composition includes: injection, freeze-dried preparation, oral preparation, liquid dosage form, solid dosage form, or a combination thereof.
  • step (i) after mixing the drug carrier and the antitumor drug, they are incubated at a suitable temperature (eg, 10-80° C., preferably 20-65° C.), so that the The tumor drug is loaded in the drug carrier.
  • a suitable temperature eg, 10-80° C., preferably 20-65° C.
  • the incubation time is 0.5-72 hours, preferably 1-36 hours, more preferably 2-24 hours.
  • a cross-linked cyclodextrin organic framework COF and/or the LMWH-polypeptide-COF composition of the first aspect of the present invention and/or the use of the pharmaceutical active ingredient described in the third aspect they are used in the preparation of drugs for preventing and/or treating tumors or for targeting the lungs.
  • the tumor includes lung cancer.
  • the pulmonary-targeting drug is used to treat a disease selected from the group consisting of pulmonary infection, pulmonary inflammation, pulmonary fibrosis, COPD, or a combination thereof.
  • the pulmonary infection includes bacterial infection, viral infection (eg, influenza virus infection, coronavirus infection, or infection caused by other pathogens).
  • viral infection eg, influenza virus infection, coronavirus infection, or infection caused by other pathogens.
  • FIG. 1 is a fluorescence image of tissue distribution of the cyclodextrin matrix composition COF in Example 1 with high lung targeting.
  • Figure 2 is a flow chart of the synthesis of low molecular weight heparin LMWH and GS5 peptide double modified cubic cyclodextrin backbone (LMWH-GS5-COF) in Example 2.
  • FIG. 3 is an electron microscope image of the double-modified cubic cyclodextrin framework (LMWH-GS5-COF) of low molecular weight heparin LMWH and GS5 peptide in Example 2.
  • LMWH-GS5-COF double-modified cubic cyclodextrin framework
  • FIG. 4 is the infrared spectrum of low molecular weight heparin LMWH and GS5 peptide double modified cubic cyclodextrin backbone (LMWH-GS5-COF) in Example 2.
  • FIG. 4 is the infrared spectrum of low molecular weight heparin LMWH and GS5 peptide double modified cubic cyclodextrin backbone (LMWH-GS5-COF) in Example 2.
  • FIG. 5 is the nuclear magnetic spectrum of low molecular weight heparin LMWH and GS5 peptide double modified cubic cyclodextrin framework (LMWH-GS5-COF) in Example 2.
  • FIG. 5 is the nuclear magnetic spectrum of low molecular weight heparin LMWH and GS5 peptide double modified cubic cyclodextrin framework (LMWH-GS5-COF) in Example 2.
  • Example 6 is the blood compatibility evaluation of the low molecular weight heparin LMWH and GS5 peptide double modified cubic cyclodextrin skeleton LMWH-GS5-COF and the low molecular weight heparin LMWH modified cubic cyclodextrin skeleton LMWH-COF in Example 2.
  • A Micrographs of erythrocytes after different sample treatments;
  • B Sample images after centrifugation;
  • Figure 7 shows the B16F10 cytotoxicity evaluation of the low molecular weight heparin LMWH and GS5 peptide double modified cubic cyclodextrin skeleton LMWH-GS5-COF and the low molecular weight heparin LMWH modified cubic cyclodextrin skeleton LMWH-COF in Example 2.
  • Figure 8 shows the A549 cytotoxicity evaluation of the double modified cubic cyclodextrin skeleton LMWH-GS5-COF with low molecular weight heparin LMWH and GS5 peptide and the low molecular weight heparin LMWH modified cubic cyclodextrin skeleton LMWH-COF in Example 2.
  • Figure 9 shows the redox-responsive in vitro release evaluation of the doxorubicin-loaded low-molecular-weight heparin LMWH and GS5 peptide double-modified cubic cyclodextrin backbone (RCLD) in Example 3,
  • FIG. 10 shows the cytotoxicity evaluation of the doxorubicin-loaded low-molecular-weight heparin LMWH-modified cubic cyclodextrin scaffold CLD and the adriamycin-loaded low-molecular-weight heparin LMWH and GS5 peptide double-modified cubic cyclodextrin scaffold RCLD in Example 3.
  • Figure 11 shows the effect of doxorubicin-loaded low molecular weight heparin LMWH modified cubic cyclodextrin skeleton CLD and doxorubicin-loaded low molecular weight heparin LMWH and GS5 peptide double modified cubic cyclodextrin skeleton RCLD in Example 3 on tumor cell migration and invasion inhibitory effect.
  • A and
  • Figure 12 shows the lung cancer targeting and RCLD of doxorubicin-loaded low-molecular-weight heparin LMWH modified cubic cyclodextrin skeleton CLD and doxorubicin-loaded low-molecular-weight heparin LMWH and GS5 peptide double modified cubic cyclodextrin skeleton RCLD in Example 3 biodistribution.
  • Figure 13 shows the comparison of the doxorubicin-loaded low-molecular-weight heparin LMWH modified cubic cyclodextrin skeleton CLD and the doxorubicin-loaded low-molecular-weight heparin LMWH and GS5 peptide double modified cubic cyclodextrin skeleton RCLD in the A549 lung cancer model in Example 3 Antitumor effect.
  • A pictures of lungs collected from A549 lung cancer mouse model;
  • B treatment regimen for RCLD;
  • D lung tissue H&E staining analysis.
  • *p ⁇ 0.05, ***p ⁇ 0.001, ns indicates no significant difference between the two groups.
  • FIG. 14 shows the body weight change of A549-bearing BALB/c mice in Example 3.
  • Fig. 15 shows the expression of the doxorubicin-loaded low-molecular-weight heparin LMWH modified cubic cyclodextrin skeleton CLD and the doxorubicin-loaded low-molecular-weight heparin LMWH and GS5 peptide double modified cubic cyclodextrin skeleton RCLD in B16F10 metastatic lung cancer model in Example 3 in the treatment effect.
  • A Lung pictures collected from a mouse model of B16F10 metastatic lung cancer;
  • B treatment regimen for RCLD;
  • Figure 16 shows the H&E staining analysis of major organs in the B16F10 metastatic lung cancer model mice in Example 3 after treatment. Black arrows point to atrophic cardiomyocytes.
  • Figure 18 shows the in vitro release evaluation of topotecan (TPT)-loaded low-molecular-weight heparin LMWH and GS5 peptide double-modified cubic cyclodextrin framework RCLT and topotecan-loaded cyclodextrin organic framework composition TPT@COF in Example 50 .
  • TPT topotecan
  • Figure 20 shows that the topotecan-loaded low-molecular-weight heparin LMWH and GS5 peptide double-modified cubic cyclodextrin framework RCLT and topotecan-loaded cyclodextrin framework composition TPT@COF in the B16F10 metastatic lung cancer model in Example 50 the therapeutic effect.
  • A Pictures of lungs collected in a mouse model of B16F10 metastatic lung cancer treated with RCLT;
  • B TPT@COF and RCLT treatment regimens;
  • C Area of B16F10 metastatic lung cancer treated with RCLT;
  • D Lung images collected in a mouse model of B16F10 metastatic lung cancer treated with TPT@COF;
  • Figure 21 shows the B16F10-loaded C57BL treated with topotecan-loaded low-molecular-weight heparin LMWH and GS5 peptide double-modified cubic cyclodextrin framework RCLT and topotecan-loaded cyclodextrin framework composition TPT@COF in Example 50 Body weight change in /6 mice.
  • A Body weight change of B16F10-bearing C57BL/6 mice treated with RCLT
  • B Body weight change of B16F10-bearing C57BL/6 mice treated with TPT@COF.
  • the inventors unexpectedly found that the nano-scale cyclodextrin skeleton composition COF has high lung targeting. And after further drug loading, a composition with excellent safety, high-efficiency targeting, and excellent curative effect (eg, anti-tumor curative effect) is obtained. On this basis, the inventors have completed the present invention.
  • RGD polypeptide refers to a polypeptide containing arginine-glycine-aspartic acid tripeptide sequence (Arg-Gly-Asp, RGD), which can specifically recognize integrin ⁇ v ⁇ 3 receptors. Integrin ⁇ v ⁇ 3 receptor protein plays an important role in the process of tumor formation and metastasis. It is highly expressed on the surface of various tumor cells and tumor neovascular endothelium, but is low or not expressed in normal cells. Therefore, RGD polypeptide-containing Vectors can target tumor sites through ligand-receptor-mediated active targeting.
  • RGD polypeptides containing the "RGD” sequence are called RGD polypeptides, and the terms “RGD”, “DRGDS”, “GRGD”, “RGDS”, “GRGDS” (also known as “GS5"), “GRGDSP”, “GRADSPK”, “GRGDSPK” all belong to the RGD polypeptide series of the present invention.
  • NGR polypeptides refer to polypeptides containing asparagine-glycine-arginine tripeptide sequence (Asn-Gly-Arg, NGR), which can bind to CD13 metallopeptidase in tumor cells and tumor neovascular endothelial cells in a heterotropic manner. To the tumor site, but also inhibit tumor angiogenesis.
  • Polypeptides comprising "NGR” sequences are referred to as NGR polypeptides, and the terms "TNGRGP”, "NGRSRF”, “RSRNGR”, “NGRNTV”, “GGCNGRC”, and “CNGRC” all belong to the NRG polypeptide series of the present invention.
  • LMWH Low molecular weight heparin
  • LMWH Low molecular weight heparin
  • LMWH can not only inhibit the interaction between tumor cells and platelets and hinder the epithelial-mesenchymal transition of tumor cells, but also affect the arrangement of the actin cytoskeleton of tumor cells.
  • the heparin-polypeptide double-grafted cyclodextrin scaffold composition is a covalent attachment of a polypeptide and a low molecular weight heparin LMWH to a cross-linked cyclodextrin organic scaffold COF.
  • GSH Reduced glutathione
  • Lung cancer is a tumor with the highest morbidity and mortality in the world, and lung metastasis is a major challenge in the clinical treatment of cancer.
  • Existing treatment methods are mostly chemotherapy, which often affects normal tissues and organs while obtaining therapeutic effects. There are strong toxic side effects.
  • Most lung cancer-targeted nano-delivery systems use the tumor microenvironment or the high-permeability and long-retention effect (EPR effect) to achieve lung targeting, and most of them only target highly expressed cell surface receptors at the tumor site, and do not have lung tissue targeting. specificity.
  • the invention carries out double grafting of polypeptide and low molecular weight heparin on its surface, endows the carrier with the effect of active targeting and synergistic therapy, and has high safety. , Good biocompatibility, high targeting, meet clinical needs.
  • the cyclodextrin-metal-organic framework (CD-MOF) formed with the pharmaceutical excipient cyclodextrin as the organic ligand and potassium ion as the center of the inorganic metal ion not only retains the biological safety of cyclodextrin, but also has a biological phase. It has the advantages of good compatibility, cubic shape, and surface modifiability, and can be used in the field of drug delivery.
  • the applicant of the present invention carried out cross-linking of nano-scale CD-MOF in the early stage to obtain a cross-linked cubic cyclodextrin organic framework (COF) stably existing in the water system, using the arginine-glycine-aspartic acid tripeptide sequence (Arg-Gly-Asp, RGD) modifies the surface of COF, and the synthesized RGD-COF material can be used as an artificial platelet carrier and has the effect of high hemostasis.
  • the present invention also finds and confirms that nano-scale COF has high lung targeting characteristics. After COF is injected intravenously, the lung targeting coefficients of COF in vivo are 31.7 (mice), 10.1 (rats), and 129.0 (home), respectively.
  • the lung targeting coefficient of other methods reported in the literature is generally lower than 10.
  • the uptake of disk-shaped polymer particles in the lung is 8.7 in the liver. times.
  • the present invention will have lung tissue targeting properties for COF, and construct a novel and efficient targeted drug delivery system on the basis of RGD-COF.
  • Polypeptide-mediated ligand-receptor active targeted therapy utilizes the high affinity between polypeptide ligands and overexpressed receptors in tumor cells to deliver drugs to specific tumor tissues and increase the accumulation of drugs at tumor sites. Reduce toxic side effects on normal tissues.
  • Integrin ⁇ v ⁇ 3 receptor protein plays an important role in the process of tumorigenesis and metastasis. It is highly expressed on the surface of various tumor cells and tumor neovascular endothelium, while low or no expression in normal cells.
  • RGD-containing vectors can be specific
  • the RGD polypeptide-containing carrier can mediate an active targeted drug delivery system by recognizing the integrin ⁇ v ⁇ 3 receptor. The growth and metastasis of tumor cells require new blood vessels to improve nutrients.
  • CD13 metallopeptidase is an important regulator of new blood vessels, promoting cell proliferation and migration and the formation of new blood vessels.
  • the NGR-containing carrier can specifically recognize the CD13 metallopeptidase, so the NGR carrier can increase the concentration of the drug in the tumor tissue through the targeting-mediated function.
  • Low-molecular-weight heparin is a heparin with lower molecular weight that is degraded from unfractionated heparin by physical, chemical, biological and other methods. characteristics of biocompatibility.
  • the LMWH skeleton contains a large number of active and modifiable free radicals, which can be chemically modified to endow it with new properties.
  • LMWH can inhibit the activity of tumor angiogenesis-related growth factors by binding to overexpressed neovascularization at the tumor site, thereby inhibiting tumor neovascularization growth.
  • the affinity of LMWH with heparinase and P-/L-selectin makes it inhibit key processes such as tumor cell escape and metastasis, and achieve the purpose of tumor treatment.
  • LMWH reduced glutathione
  • carboxyl, hydroxyl, amino and other groups on the LMWH molecule which can be grafted on the carrier through a simple chemical reaction to endow the carrier with the characteristics of biological modification. Since the concentration of reduced glutathione (GSH) in the tumor site is 100 times higher than that in normal tissues, under the stimulation of high concentration of GSH in tumor cells, it can specifically break the disulfide bond in cystamine, which is specific in tumor tissue. LMWH is released in a targeted manner to achieve targeted therapeutic efficacy.
  • GSH reduced glutathione
  • the present invention provides a LMWH-polypeptide-COF composition, such as LMWH-RGD-COF, whose interior is a COF material with a cubic structure, and the surface is biologically modified by LMWH and RGD.
  • COF as the basic drug carrier unit, RGD as the target can specifically bind to integrin ⁇ v ⁇ 3 receptors on the surface of various tumor cells and neovascular epidermis, LMWH as the coating layer, and LMWH with cystamine as the linking arm
  • the encapsulated COF can specifically shed the shell LMWH to expose the loaded drug.
  • Nano-scale intravenously injectable cubic LMWH-RGD-COF composition can escape the phagocytosis and clearance of macrophages, and reach the lung through autonomous lung targeting, thereby reducing the dosage of administration, reducing the toxic and side effects of chemotherapeutic drugs, and promoting the use of drugs in the lungs.
  • the enrichment of tumor sites can improve the anti-tumor efficacy.
  • the LMWH-RGD-COF composition of the present invention is different from the existing reports of functional nanoparticles.
  • LMWH and RGD are simultaneously modified on the surface of gold nanoparticles, which has a highly specific apoptotic activity on cancer cells;
  • PLGA nanoparticles modified with LMWH function it was found that the preparation could improve the accumulation of nanoparticles in tumors in vitro, but these two nanoparticles did not show lung targeting.
  • the LMWH-RGD-COF composition of the present invention has good biocompatibility, safety and lung targeting.
  • a lung-targeted anti-drug including anti-tumor drugs and/or lung-targeted drugs
  • drug delivery system based on cubic cyclodextrin skeleton materials.
  • the inventors will use reduction-sensitive chemical bonds to graft LMWH on RGD-COF as a carrier to achieve targeted drug delivery, and perform anti-lung cancer treatment with the help of the synergistic effect of the therapeutic drug LMWH and the drug.
  • heparin and RGD are simultaneously modified on the surface of gold nanoparticles, which has a highly specific apoptotic activity on cancer cells that selectively overexpress RGD receptors, but it does not have lung targeting.
  • the carrier designed by the present invention can utilize its own lung tissue targeting characteristics, and can significantly inhibit the growth of lung cancer when the dose of the drug is reduced (for example, the dose of DOX is reduced from 2.5 mg/kg to 1 mg/kg). and metastasis (A549 cells had almost no lung metastasis nodules, and the area of melanoma lung metastasis was reduced by 70%).
  • the invention provides a low molecular weight heparin LMWH with lung targeting, a polypeptide double grafted cubic cyclodextrin skeleton composition (LMWH-polypeptide-COF) and a preparation method and application thereof.
  • LMWH-polypeptide-COF polypeptide double grafted cubic cyclodextrin skeleton composition
  • the present invention combines polypeptides, low-molecular-weight heparin with anti-tumor activity and anti-tumor drugs in a cubic cyclodextrin skeleton COF to form a multi-component composition, aiming to construct a long-circulation, lung targeting and other functions in one. Smart drug delivery system.
  • the LMWH-polypeptide-COF nanoparticles of the present invention can escape the phagocytosis and clearance of macrophages, have good safety and high-efficiency targeting, enrich the drug at the tumor site, and release the drug through redox responsiveness at the tumor site. It greatly reduces the toxic and side effects on normal tissues and enhances the therapeutic effect of the drug.
  • an anti-tumor LMWH-polypeptide-COF composition provided by the present invention is characterized in that the hydroxyl group (-OH) in CD-MOF is covalently linked by a suitable cross-linking agent to form an anti-tumor LMWH-polypeptide-COF composition in water.
  • COF materials with good stability and then the carboxyl group (-COOH) or amino group (-NH 2 ) of the polypeptide is covalently linked to the hydroxyl group (-OH) on the outer surface of the COF by using a linker arm, and an activator is further used to modify the cystamine.
  • COF can be used as a high-efficiency drug carrier for anti-tumor drugs, and peptides are used as targets to specifically bind to receptors highly expressed by tumor cells and actively target tumor sites; Drug co-treatment.
  • the invention provides a preparation method for preparing LMWH-polypeptide-COF, the method comprises the steps:
  • Synthesis of LMWH-cystamine After grafting LMWH with cystamine, a terminal amino group is introduced, and the amino group forms an amide bond with the carbonyl group of the activated polypeptide-COF.
  • This method can improve the substitution degree of LMWH on the polypeptide-COF.
  • EDC 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • step (III) the polypeptide-COF is dispersed in an organic solvent, a certain amount of linker and catalyst are added, and the reaction is carried out at a certain temperature for a period of time to obtain an activated cubic polypeptide-COF composition reaction solution;
  • LMWH-polypeptide-COF the reaction solution in step (2) is slowly added dropwise to the organic solution of LMWH-cystamine, a certain amount of catalyst is added, and the reaction is carried out at a certain temperature for a period of time, and the reaction solution is dialyzed and freeze-dried.
  • the double-grafted cubic cyclodextrin framework composition of LMWH and polypeptide was obtained.
  • the preparation method of the pharmaceutical composition is summarized as follows: the antitumor drug solution and the LMWH-polypeptide-COF are stirred and incubated at a certain temperature for a certain period of time according to a certain molar ratio of feeding materials, centrifuged, washed, and dried to obtain the final product.
  • the invention provides a preparation method of a heparin/polypeptide double-grafted cyclodextrin skeleton composition with lung targeting, and a preparation method and application thereof.
  • the LMWH/RGD double-grafted cyclodextrin skeleton composition of the present invention has a lung-targeting function, and after being loaded with cytotoxic drugs (such as doxorubicin), it can significantly reduce the survival rate of B16F10 tumor cells in vitro, and can also Significantly inhibits the ability of tumor cells to migrate and invade.
  • cytotoxic drugs such as doxorubicin
  • the vector can reduce the dose of doxorubicin by 5 times under the same efficacy of doxorubicin, and reduce the cardiotoxicity and side effects of doxorubicin , is expected to achieve attenuation and synergistic effects of lung cancer treatment.
  • the nano-lung tissue delivery system has good biosafety, no acute toxicity or tissue necrosis occurs in major organs after intravenous injection, and hematological studies have shown that it does not affect the main blood cell levels in mice, which has good clinical translation value. .
  • the present invention has the following main advantages:
  • the preparation process of the nanomaterial prepared by the present invention is simple and controllable, does not require expensive equipment, can be produced on a large scale, and has high carrier safety, good biocompatibility, and no immunogenicity. Good lung targeting.
  • the nanomaterial prepared by the present invention has a regular cubic shape and a small particle size, which breaks through the limitation of the spherical shape of the previous carrier, effectively escapes the phagocytosis and clearance of macrophages, and prolongs the circulation time in the body.
  • the nanomaterial prepared by the present invention adopts polypeptide sequence modification in the construction process, endows the cubic cyclodextrin organic skeleton composition with COF active targeting characteristics, realizes the targeted therapy of tumor drugs, and reduces the toxic and side effects to normal organs and tissues.
  • the nanomaterial prepared by the present invention is coated with low molecular weight heparin LMWH in the construction process, and the nanoparticle is positioned to release the drug through a redox-sensitive release mechanism.
  • the nano-formulation prepared by the present invention simultaneously loads anti-tumor drugs and low molecular weight heparin, realizes synergistic administration, and greatly improves the therapeutic effect.
  • COF has high lung targeting in mice: Cy5-labeled COF nanoparticle suspension (40 mg/kg, 10 mL/kg) was injected into the tail vein of Kunming mice, 5 minutes before and 0 minutes after administration, respectively. , 5min, 10min, 15min, 1h, 2h, 4h, 8h and 24h, the mice were sacrificed, and the main organs of the heart, liver, spleen, lung, and kidney were taken out. Fluorescence intensity to study lung targeting of COF nanoparticles in mice.
  • COF has high lung targeting in rats: Cy5-labeled COF nanoparticle suspension (28 mg/kg, 10 mL/kg) was injected into the tail vein of SD rats, 5 min before administration and 0 min after administration, respectively. , 5min, 10min, 15min, 1h, 2h, 4h, 8h and 24h, the rats were sacrificed, and the main organs of the heart, liver, spleen, lung, and kidney were taken out. Fluorescence intensity to study the lung targeting of COF nanoparticles in rats.
  • COF has high lung targeting in rabbits: Cy5-labeled COF nanoparticle suspension was injected into the ear vein of rabbits, 5min before administration, 0min, 5min, 10min, 15min, 1h, After 2h, 4h, 8h and 24h, the rabbits were sacrificed, and the main organs of the heart, liver, spleen, lung and kidney were taken out, and the fluorescence intensity of each organ at different time points was measured by a small animal in vivo imager. Lung targeting in rabbits. After 15 minutes of injection of COF nanoparticles through the ear vein, the distribution of COF in the lung has reached the highest value. At this time, the fluorescence signal in the lung is 120 times that of the liver, which indicates that COF also has a significant effect in rabbits. Lung targeting function.
  • A is the tissue fluorescence intensity map of COF in mice, rats and rabbits
  • B is the in vivo tissue distribution map of COF in mice at different time points
  • C is COF in mice at different times. Fluorescence intensity graph.
  • COF can achieve high targeting to lung tissue in mice, rats and rabbits, and the fluorescence signal in the lung is 30 times, 10 times, and 120 times that of the liver.
  • GS5-COF Preparation of nano-scale GS5-COF: Weigh 230 mg of nano-scale COF prepared in Example 1 and 10 mg of GRGDS (also known as GS5) pentapeptide (the molar ratio of COF and GS5 is 1:1) into a round bottom flask, add 5 mL Dimethylformamide, stir well and then add 5mg 4-dimethylaminopyridine (DMAP) and 6mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), It was placed on a magnetic stirrer at 37°C and stirred at 600rpm for 24h to fully couple COF and GS5 polypeptide.
  • DMAP 4-dimethylaminopyridine
  • EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • GS5-COF is a regular cubic shape with a size of 100-500 nm, and the mass percentage of cyclodextrin skeleton material and GS5 polypeptide measured by high performance liquid chromatography (HPLC) is 1:0.05.
  • LMWH low molecular weight heparin
  • COF GS5 peptide double modified cubic cyclodextrin backbone
  • LMWH-CYS low molecular weight heparin-cystamine
  • the crude product was first dialyzed with 0.1M NaCl solution (5.85g of NaCl was dissolved in pure water and then adjusted to 1000mL) for 12h (the molecular weight cut-off of the dialysis bag was 3500Da), and then dialyzed with pure water for 48h.
  • the liquid obtained after dialysis was filtered through a 0.22 ⁇ m filter membrane and freeze-dried to obtain the intermediate product LMWH-CYS.
  • DSC N,N'-disuccinimidyl carbonate
  • ACN acetonitrile
  • TOA triethylamine
  • LMWH-GS5-COF Preparation of LMWH-GS5-COF: Weigh 300 mg of LMWH-CYS, add 18 mL of formamide, and dissolve under magnetic stirring to obtain LMWH-CYS solution. The activated GS5-COF solution was added to the LMWH-CYS solution, and the reaction was carried out at 25 °C for 48 h under magnetic stirring at 400 rpm to obtain a crude product of LMWH-GS5-COF.
  • the crude product was first dialyzed against acetonitrile/water mixed solvent (4:1, v:v) for three days, then continued to be dialyzed against pure water for two days, centrifuged, and frozen at -50°C Dry to obtain LMWH-GS5-COF.
  • Figure 3 is an electron microscope image of LMWH-GS5-COF in Example 2. It can be seen from Figure 3 that LMWH-GS5-COF still maintains a regular cubic shape with a particle size of 100-500 nm.
  • Nano-scale cross-linked cyclodextrin framework (COF) was prepared in the same way as in Example 1, and low molecular weight heparin-cystamine ((LMWH-CYS) was prepared in the same way as in Example 2.
  • LMWH-CYS low molecular weight heparin-cystamine
  • LMWH-COF heparin-cross-linked cyclodextrin framework composition
  • the crude product was first dialyzed against acetonitrile/water mixed solvent (4:1, v:v) for three days, then continued to be dialyzed against pure water for two days, centrifuged, and frozen at -50°C Dry to obtain LMWH-COF.
  • Example 4 is the infrared spectrum of LMWH-GS5-COF in Example 2.
  • Characteristic peaks of double-modified carriers were analyzed by 1H NMR spectroscopy: The samples were analyzed by 1H NMR spectroscopy using a Bruker AVANCE NEO 500 type 1H NMR spectrometer. GS5 polypeptide and LMWH were dissolved in 600 ⁇ L of D 2 O, LMWH-GS5-COF was dispersed in 600 ⁇ L of D 2 O, and 10 ⁇ L of NaOD was added to degrade it. Each sample solution was then placed in a capped NMR tube, and spectra were collected under a magnetic field of 500 MHz.
  • Example 5 is the nuclear magnetic spectrum of LMWH-GS5-COF in Example 2.
  • Hemolytic toxicity of the carrier take 2 mL of fresh C57BL/6 fresh blood in a test tube infiltrated with 3.2% sodium citrate, centrifuge at 2500 rpm for 10 min to precipitate red blood cells, discard the upper plasma and wash the red blood cells three times with normal saline.
  • the erythrocytes were diluted with physiological saline into a 2% erythrocyte suspension.
  • the LMWH-GS5-COF and LMWH-COF prepared in the above Example 2 were prepared into 5, 10, 25, 100, 200, 400 ⁇ g/mL suspensions with physiological saline, and an equal volume of 2% red blood cell suspension was added to shake. Homogenize and incubate in a 37°C water bath for 1 h.
  • FIG. 6 is the blood compatibility evaluation of LMWH-GS5-COF and LMWH-COF in Example 2.
  • Figures 6A-C showed that when the concentrations of LMWH-GS5-COF and LMWH-COF were increased to 600 ⁇ g/mL, the hemolysis rate was still less than 1%. A hemolysis rate of less than 5% is generally considered safe, indicating that LMWH-MOF and LMWH-GS5-COF nanoparticles have good blood compatibility.
  • B16F10 cells were cultured in DMEM medium (containing 10% fetal bovine serum) at 37° C., 5% CO2 in a constant temperature and humidity incubator. The cells in the logarithmic growth phase were selected for the experiment, and the cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 5 ⁇ 10 3 cells/well, and cultured for 12 h. After removing the supernatant medium, 200 ⁇ L of LMWH, LMWH-COF, and LMWH-GS5-COF samples with different concentrations were added respectively, and a blank group (containing only medium) and a control group (containing cells and medium) were set at the same time.
  • DMEM medium containing 10% fetal bovine serum
  • a Sample is the absorbance value of the sample
  • a control is the absorbance value of the control group
  • a blank is the absorbance value of the blank group.
  • Figure 7 is the B16F10 cytotoxicity evaluation of LMWH-GS5-COF and LMWH-COF in Example 2, wherein A is the B16F10 cytotoxicity of LMWH, and B is the B16F10 cytotoxicity of LMWH-COF and LMWH-GS5-COF nanoparticles.
  • A549 cytotoxicity of the vector was cultured in RPMI 1640 medium (containing 10% fetal bovine serum) at 37°C, 5% CO 2 in a constant temperature and humidity incubator. The cells in the logarithmic growth phase were selected for the experiment, and the cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 2 ⁇ 10 4 cells/well, and cultured for 24 hours. After removing the supernatant culture medium, 200 ⁇ L of LMWH-COF and LMWH-GS5-COF samples with different concentrations were added respectively, and a blank group (containing only culture medium) and a control group (containing cells and culture medium) were set at the same time.
  • RPMI 1640 medium containing 10% fetal bovine serum
  • Figure 8 shows the A549 cytotoxicity evaluation of LMWH-GS5-COF and LMWH-COF in Example 2.
  • the results show that the cell activity of LMWH-COF and LMWH-GS5-COF nanocarriers is basically in the range of 0.0067-13.3 ⁇ g ⁇ mL -1 were close to 100%, and there was no obvious cytotoxicity.
  • doxorubicin hydrochloride-loaded double-modified carrier composition Preparation of doxorubicin hydrochloride-loaded double-modified carrier composition: Weigh 12 mg of doxorubicin hydrochloride (DOX) and dissolve it in 2 mL of pure water, ultrasonicate for 10 min to dissolve, and then add the LMWH-GS5 prepared in Example 2 -COF nanoparticles 30 mg, the molar ratio of drug and LMWH-GS5-COF nanoparticles is 1:1, and the drug is incubated at 25°C under 300 rpm stirring for 24 h in the dark. After the drug loading is completed, centrifuge at 4000 rpm for 5 min, wash with pure water to remove free DOX, and obtain the lower layer of doxorubicin hydrochloride-loaded double modified carrier composition (RCLD).
  • DOX doxorubicin hydrochloride
  • doxorubicin hydrochloride-loaded carrier composition without GS5 grafted with low molecular weight heparin Weigh 12 mg of DOX and dissolve it in 2 mL of pure water, ultrasonicate for 10 min to dissolve, and then add the prepared in Example 2 respectively.
  • LMWH-COF nanoparticles were 30 mg, the molar ratio of drug to LMWH-COF was 1:1, and the drug was incubated at 25°C for 24 h at 300 rpm in the dark.
  • RCLD nanoparticles under different concentrations of reduced glutathione was investigated by dynamic membrane dialysis bag method.
  • Figure 9 shows the in vitro release evaluation of RCLD in Example 3.
  • the results show that compared with RCLD in PBS release medium without GSH release 50% in 72h, RCLD nanoparticles can rapidly DOX in 10mM GSH release medium, and 50% in the first 12h. % drug release, 70% drug release within 72h, which is attributed to the high concentration of GSH cutting off the connection between heparin and COF, accelerating the release of DOX.
  • the release behaviors of RCLD nanoparticles in the release medium containing 1 mM GSH and without GSH were similar, indicating that RCLD nanoparticles have good stability in the circulation in vivo, which can avoid the drug being released in advance before reaching the tumor site.
  • B16F10 cells were cultured in DMEM medium (containing 10% fetal bovine serum) at 37°C, 5% CO 2 in a constant temperature and humidity incubator. The cells in the logarithmic growth phase were selected for the experiment, and the cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 5 ⁇ 10 3 cells/well, with a final volume of 200 ⁇ L per well, and cultured for 12 h.
  • DMEM medium containing 10% fetal bovine serum
  • FIG. 10 is the cytotoxicity evaluation of CLD and RCLD in Example 3.
  • FIG. The results in Figure 10 show that: RCLD has strong cytotoxicity and can significantly inhibit the growth of tumor cells in vitro. Compared with free DOX, the cytotoxicity of RCLD is weaker.
  • B16F10 cells were seeded into 6-well plates, and when the cells had grown to 90% confluence of the well bottom area, scratch with a 200 ⁇ L sterile pipette tip A wound was made and rinsed once with PBS to create a cell-free scratch. Subsequently, cells were incubated with PBS, free DOX, free LMWH, COF prepared in Example 1, RCLD or CLD prepared in Example 3 for 24 h at 37°C. Using an inverted optical microscope to observe and photograph at 0h and 24h, and calculate the wound healing rate.
  • FIG. 11A and Figure 11C show that the B16F10 cells in the PBS control group covered almost the entire scratch after 24 hours of incubation, and the wound healing rate was 82.3%.
  • the wound healing rate of the LMWH group was significantly lower than that of the PBS group, indicating that LMWH could reduce the migration ability of B16F10 cells.
  • the wound healing rate also decreased in the DOX group due to the cytocidal effect of DOX.
  • the combined action of LMWH and DOX, RCLD or CLD nanoparticles showed the strongest inhibitory effect on tumor cell migration.
  • Transwell cell invasion assay of two doxorubicin-loaded carrier compositions B16F10 cells in exponential growth phase were digested, the cell suspension was centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the lower cells were dispersed in 0.5% FBS. in the culture medium.
  • the upper layer of the transwell chamber was pre-coated with Matrigel, then 1 ⁇ 10 5 B16F10 cells were added, and 100 ⁇ L of samples were added, which were DOX, LMWH, LMWH-COF and LMWH-GS5-COF prepared in Example 2, respectively,
  • Example 3 Prepared RCLD or CLD (to make the final concentration of DOX 0.5 ⁇ g/mL).
  • Figure 12 is the lung cancer targeting and biodistribution of CLD and RCLD in Example 3, wherein Figure 12A is the in vivo distribution of RCLD and CLD in B16F10 lung cancer metastasis, Figure 12B is the fluorescence intensity analysis of lung tissue, and C is the mouse In vitro imaging of organs, Figure 12D shows the quantitative fluorescence intensity analysis of RCLD and CLD in mouse heart, liver, spleen, lung, and kidney.
  • Figures 12A-D show that CLD achieves high targeting to lung cancer without GS5 surface functionalization, because the COF carrier itself has a high lung-targeting function.
  • the fluorescence intensity in the lungs of the RCLD group was 1.9 times higher than that of the CLD group, showing a more efficient lung cancer targeting ability due to the dual targeting effect of COF carrier and GS5.
  • RCLD nanoparticles were mainly distributed in the lung, and the fluorescence intensity in lung tissue was 5.8 times higher than that in liver.
  • A549 cells were dispersed in 200 ⁇ L of PBS and injected into BALB/c mice via tail vein to construct a human lung cancer metastasis model.
  • the mice were randomly divided into 6 groups and were given PBS, free DOX2.5 (dose of 2.5 mg/kg), free LMWH, CLD (dose of DOX of 1 mg/kg), RCLD (DOX doses were 1 mg/kg and 0.5 mg/kg, respectively), administered once every 3 days for 5 consecutive doses.
  • the weight changes of the mice were monitored daily throughout the experiment.
  • the mice were sacrificed, the lung tissue was dissected out, the number of lung tumor metastatic nodules was counted, and photographs were recorded. Lung tissue and other major organs were also analyzed by H&E staining.
  • FIG. 13 shows the anti-tumor effects of CLD and RCLD in the A549 lung cancer model in Example 3.
  • (C) quantitative analysis of the number of A549 metastatic nodules in the lungs (n 5);
  • Figures 13A-D showed that the DOX-loaded CLD and RCLD groups exhibited strong anti-tumor effects, not only because LMWH could inhibit the migration and invasion of cancer cells, but also DOX could directly kill tumor cells.
  • the RCLD group (the dose of DOX is 1 mg/kg) has the best therapeutic effect on lung cancer, and the metastatic nodules in the lungs are hardly seen in the RCLD group (the dose of DOX is 1 mg/kg).
  • Benefiting from RGD target modification can increase the targeted distribution and retention time of RCLD at the tumor site.
  • FIG. 14 shows the body weight change of A549-bearing BALB/c mice in Example 3.
  • Two carrier compositions loaded with doxorubicin hydrochloride in the treatment of lung metastatic lung cancer of B16F10 melanoma B16F10 cells in exponential growth phase were digested, the cell suspension was centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the cells in the lower layer were Sterile PBS was added to bring the cell concentration to 2 x 105 cells/mL. 100 ⁇ L of the cell suspension was drawn with an insulin syringe and injected into the C57BL/6 mice via the tail vein to construct a mouse melanoma lung metastasis model.
  • mice On the second day after tumor cell inoculation, the mice were randomly divided into 6 groups: PBS group, LMWH group, 2.5 mg/kg DOX group, CLD group (DOX dose of 1 mg/kg), RCLD group (DOX dose of 1 mg/kg, respectively). 1 mg/kg and 0.5 mg/kg), 200 ⁇ L of the drug was administered by tail vein injection, once every three days. After administration, the mice were raised normally, and the body weight of the mice was weighed every day. After administration of five times, the animals were sacrificed, and the lung tissue was dissected out, photographed, and the area of lung metastasis was calculated by Image-Pro-Plus software.
  • FIG. 15 shows the therapeutic effects of RCLD and CLD in the B16F10 metastatic lung cancer model in Example 3.
  • FIG. (A) Lung pictures collected from a mouse model of B16F10 metastatic lung cancer;
  • (C) area of B16F10 metastatic lung cancer (n 5). **p ⁇ 0.01, ***p ⁇ 0.001, ns means no significant difference between the two groups.
  • Figure 16 shows the H&E staining analysis of major organs in the B16F10 metastatic lung cancer model mice in Example 3 after treatment. Black arrows point to atrophic cardiomyocytes.
  • LMWH-GS5-COF contains different antitumor drugs: Weigh an appropriate amount of antitumor drugs such as paclitaxel, capecitabine, gemcitabine, carboplatin, oxaliplatin, gefitinib, topotecan, etc., add an appropriate amount of solvent for ultrasound10 Min to dissolve it, then add the LMWH-GS5-COF prepared in Example 2 respectively, the molar ratio of drug to LMWH-GS5-COF is 2:1, stir at 400 rpm at 37°C for 24 h, and incubate drug loading. After the drug loading was completed, centrifuge at 4000 rpm for 5 min, and use the incubation solvent to wash off the free drug on the surface. After drying, the lower drug-loaded LMWH-GS5-COF nanoparticles were obtained.
  • antitumor drugs such as paclitaxel, capecitabine, gemcitabine, carboplatin, oxaliplatin, gefitinib, topotecan,
  • the anti-tumor drug-loaded carrier composition in the above embodiment has a high drug-loading amount, strong cytotoxicity in vitro, and good anti-lung cancer efficacy in vivo.
  • Preparation of tumor-targeting polypeptide grafted COF carrier Weigh COF and functional polypeptide (RGD peptide, NGR peptide) in a 1:1 molar ratio in the same molar ratio of Example 1, put them in a round-bottomed flask, and add a certain volume of the first A certain amount of activator A (DMAP, EDC, TEA) was added to a solvent (DMF, ACN) after stirring evenly, and it was placed on a magnetic stirrer at 37 °C and stirred at 400 rpm for 24 h to fully couple COF and functional peptides.
  • DMAP activator A
  • the functional polypeptide composition is in cubic form, and the particle size is about 100-500 nm.
  • the mass percentage of the cross-linked cyclodextrin organic framework COF and the functional polypeptide measured by HPLC is 1:0.001-0.1.
  • Activation of GS5-COF Weigh a certain amount of activated linker (N,N'-disuccinimidyl carbonate (DSC), N,N'-carbonyldiimidazole (CDI), succinyl chloride), dissolve by ultrasonic In the third solvent (acetonitrile (ACN), formamide, dimethylformamide (DMF)), and add GS5-COF powder and activation catalyst (triethylamine (TEA), pyridine, N- Hydroxysuccinimide (NHS)), reacted at the first temperature (25°C, 40°C, 60°C) for the first time (6h, 12h, 48h).
  • DSC activated linker
  • CDI N,N'-carbonyldiimidazole
  • succinyl chloride dissolve by ultrasonic In the third solvent (acetonitrile (ACN), formamide, dimethylformamide (DMF)
  • GS5-COF powder and activation catalyst triethyl
  • LMWH-COF Preparation of LMWH-COF: Weigh 300 mg of LMWH-CYS, add an appropriate amount of the fourth solvent (acetonitrile (ACN), dimethylformamide (DMF), formamide) to dissolve, and dissolve under magnetic stirring to obtain LMWH-CYS solution. Add the activated GS5-COF or COF solution to the LMWH-CYS solution, and react at the second temperature (25°C, 40°C, 60°C) for a second time (12h, 24h, 48h) to obtain crude LMWH-COF. product.
  • ACN acetonitrile
  • DMF dimethylformamide
  • formamide formamide
  • the crude product was first dialyzed against acetonitrile/water mixed solvent (4:1, v:v) for three days, then continued to be dialyzed against pure water for two days, centrifuged, and frozen at -50°C Dry to obtain LMWH-COF.
  • the final product LMWH-COF the percentage content of low molecular weight heparin LMWH measured by the toluidine blue method is 0.1-1%, and the LMWH-COF composition is in cubic shape with a particle size of about 100-500nm.
  • cyclodextrin organic framework composition loaded with topotecan hydrochloride Weigh 100 mg of topotecan hydrochloride (TPT) and dissolve it in 2.5 mL of pure water, ultrasonicate for 10 min to dissolve, and then add the prepared in Example 1.
  • the cyclodextrin organic framework COF nanoparticles were 34 mg, and the molar ratio of the drug to the LMWH-GS5-COF nanoparticles was 2:1.
  • TPT@COF Drug loading was determined by UV spectrophotometry. An appropriate amount of TPT@COF was weighed and dissolved in a 0.1M sodium hydroxide solution. The liquid was filtered through a 0.22 ⁇ m filter membrane and the absorbance was measured at 422 nm. The drug loading of TPT was determined by formula (2) in Example 3. The results showed that the drug loading of TPT@COF was 12%.
  • Drug loading was determined by UV spectrophotometry. An appropriate amount of RCLT was weighed and dissolved in 0.1M sodium hydroxide solution, and the liquid was filtered through a 0.22 ⁇ m filter membrane to measure the absorbance at 422 nm, and the drug loading of TPT was determined by formula (2) in Example 3. The results showed that the drug loading of RCLT was 18%.
  • GS5-free low-molecular-weight heparin-grafted carrier composition loaded with topotecan hydrochloride Weigh 20 mg of TPT and dissolve it in 2 mL of pure water, ultrasonicate for 10 min to dissolve, and then add the prepared in Example 2 respectively.
  • LMWH-COF nanoparticles were 34 mg, the molar ratio of drug to LMWH-COF was 1:2, and the mixture was stirred at 300 rpm in the dark at 25°C for 14 h, and incubated with drug loading.
  • Drug loading was determined by UV spectrophotometry. An appropriate amount of CLT was weighed and dissolved in 0.1M sodium hydroxide solution, and the liquid was filtered through a 0.22 ⁇ m filter membrane to measure the absorbance at 422 nm, and the drug loading of TPT was determined by formula (2) in Example 3. The results showed that the drug loading of CLT was 17%.
  • Figure 18 shows the in vitro release evaluation of TPT@COF and RCLT in Example 50.
  • the results show that TPT@COF releases faster in the early stage, and about 50% of the drug is released within 12 hours, which may be caused by the sudden release of TPT adsorbed on the outer surface of COF; The better sustained release effect is conducive to maintaining the drug concentration within the therapeutic window.
  • RCLT Compared with RCLT, which released 48% in 48h in PBS release medium without GSH, RCLT nanoparticles were faster in 1mM GSH release medium, with 50% drug release in 12h and 62% drug release in 48h; Rapid release of TPT, 60% drug release in the first 1h, and 80% drug release in 12h, which is attributed to the high concentration of GSH severing the connection between heparin and COF, accelerating the release of TPT, indicating that RCLT has a significant redox-responsive release characteristic .
  • B16F10 cells were cultured in DMEM medium (containing 10% fetal bovine serum) at 37°C, 5% CO 2 in a constant temperature and humidity incubator. The cells in the logarithmic growth phase were selected for the experiment, and the cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 2 ⁇ 10 4 cells/well, with a final volume of 200 ⁇ L per well, and cultured for 24 h.
  • DMEM medium containing 10% fetal bovine serum
  • Figure 19 shows the B16F10 cytotoxicity evaluation of the carrier composition loaded with topotecan in Example 50. The results show that: TPT@COF, RCLT and CLT have strong cytotoxicity and can significantly inhibit the growth of tumor cells in vitro.
  • Tumor inhibition evaluation of cyclodextrin organic framework composition loaded with topotecan hydrochloride B16F10 cells in exponential growth phase were digested, the cell suspension was centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and sterile PBS was added to the lower cells , the cell concentration was 2 ⁇ 10 5 cells/mL. 100 ⁇ L of the cell suspension was drawn with an insulin syringe and injected into the C57BL/6 mice via the tail vein to construct a mouse melanoma lung metastasis model.
  • mice On the third day after tumor cell inoculation, mice were randomly divided into 6 groups: normal saline group, 5mg/kg TPT group, and TPT@COF group (TPT doses were 2.5mg/kg and 1mg/kg, respectively). , 200 ⁇ L of the drug was administered by tail vein injection, once every three days, for a total of 5 times. After administration, the mice were raised normally, and their body weights were weighed every day. The animals were sacrificed 20 days after the tumor was implanted, and the lung tissue was dissected out, photographed and the lung metastasis area was calculated by Image-Pro-Plus software.
  • Tumor inhibition evaluation of the double-modified carrier composition loaded with topotecan hydrochloride B16F10 cells in exponential growth phase were digested, the cell suspension was centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and sterile PBS was added to the lower cells to make the cell suspension. The cell concentration was 2 ⁇ 10 5 cells/mL. 100 ⁇ L of the cell suspension was drawn with an insulin syringe and injected into the C57BL/6 mice via the tail vein to construct a mouse melanoma lung metastasis model.
  • mice On the third day after tumor cell inoculation, mice were randomly divided into 6 groups: normal saline group, LMWH group, 5 mg/kg TPT group, and RLCT group (TPT doses were 2.5 mg/kg and 1 mg/kg, respectively). kg), 200 ⁇ L of the drug was administered by tail vein injection, once every three days, for a total of 5 doses. After administration, the mice were raised normally, and their body weights were weighed every day. The animals were sacrificed 20 days after the tumor was implanted, and the lung tissue was dissected out, photographed and the lung metastasis area was calculated by Image-Pro-Plus software.
  • Figure 20 shows the therapeutic effects of TPT@COF and RCLT in the B16F10 metastatic lung cancer model in Example 50.
  • A Pictures of lungs collected in a mouse model of B16F10 metastatic lung cancer treated with RCLT;
  • B TPT@COF and RCLT treatment regimens;
  • C Area of B16F10 metastatic lung cancer treated with RCLT;
  • D Lung images collected in a mouse model of B16F10 metastatic lung cancer treated with TPT@COF;
  • RCLT 2.5 group (TPT dose of 2.5 mg/kg) can significantly inhibit the lung metastasis of melanoma, almost no lung metastasis nodules can be seen in the administration group, and the lung metastasis area of the control group is reduced by 67% %, derived from the synergistic antitumor effect of LMWH and TPT.
  • the therapeutic effect of RCLT 1 group (TPT dose of 1 mg/kg) was comparable to that of the free drug TPT group.
  • Figure 21 shows body weight changes in B16F10-bearing C57BL/6 mice treated with TPT@COF or RCLT.
  • A Body weight change of B16F10-bearing C57BL/6 mice treated with RCLT;
  • B Body weight change of B16F10-bearing C57BL/6 mice treated with TPT@COF.
  • Figure 21 shows that during TPT@COF or RCLT treatment, the body weight of mice did not change significantly, indicating that the treatment regimen is safe.

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Abstract

一种具有肺靶向的低分子肝素-多肽-交联环糊精有机骨架（LMWH-多肽-COF）组合物及其制备方法。一种载药的LMWH-多肽-COF组合物，包含LMWH-多肽-COF组合物，以及抗肿瘤药物或靶向肺部的药物。一种药物组合物，包含载药的LMWH-多肽-COF组合物，以及药学上可接受的载体。所述组合物可以靶向***，尤其是肺癌。

Description

具有肺靶向的肝素-多肽双重接枝环糊精骨架组合物及其制备方法与应用 技术领域
本发明涉及医学生物材料领域，具体地涉及具有肺靶向功效的抗肿瘤肝素-多肽双重接枝环糊精骨架组合物及其制备与应用。
背景技术
当前，肺癌是全球发病率和死亡率最高的肿瘤，肺癌的靶向治疗是亟待突破的科学难题。由于其具有发病机制复杂、早期诊断难、易转移、恶性程度高、死亡率高等特点，化学疗法依然是肿瘤治疗的常见手段，然而化疗药物多以细胞毒性药物为主，在获得治疗效果的同时往往对正常组织器官也存在着较强的毒副作用。因此亟需开发低毒低剂量的靶向、智能药物传递***。
大部分肺癌纳米靶向纳米传递***(脂质体、聚合物纳米粒、聚合物胶束、固体脂质纳米粒、磁性纳米粒及金属纳米粒等)是利用肿瘤微环境或高渗透长滞留效应(EPR效应)而实现肺靶向，大多是靶向肿瘤部位高表达的细胞表面受体，不具有肺组织靶向的特异性，往往肺部靶向效果不理想，且还具有毒性大和难降解等短板。
因此，为了解决上述难题，设计安全高效的肺癌靶向给药***是肺癌治疗亟待突破的科学难题。
发明内容
本发明的目的在于提供一种具备优异安全性、高效靶向性、优异抗肿瘤疗效的LMWH-多肽-COF组合物及载药的LMWH-多肽-COF组合物。
本发明的第一方面，提供了一种LMWH-多肽-COF组合物，所述组合物包含如下组分：
1)交联环糊精有机骨架COF；
2)多肽，所述多肽共价连接于所述交联环糊精有机骨架COF；和
3)低分子肝素LMWH，所述低分子肝素LMWH共价连接于所述交联环糊精有机骨架COF。
在另一优选例中，所述交联环糊精有机骨架COF具有肺靶向性。
在另一优选例中，所述交联环糊精有机骨架COF具有立方体形态。
在另一优选例中，所述交联环糊精有机骨架COF的尺寸为10-1000nm，较佳地50-800nm，更佳地100-500nm。
在另一优选例中，所述多肽选自下组：整合素结合肽(RGD)、CD13金属肽酶结合肽(NGR)、或其组合。
在另一优选例中，所述RGD选自下组：线性的组A物质、环形的c(RGDfK)、或其组合。
在另一优选例中，所述组A物质选自下组：RGD、DRGDS、GRGD、RGDS、GRGDS、GRGDSP、GRADSPK、GRGDSPK、或其组合。
在另一优选例中，所述NGR选自下组：线性组B物质、环状组C物质、或其组合。
在另一优选例中，所述组B物质选自下组：TNGRGP、NGRSRF、RSRNGR、NGRNTV、或其组合。
在另一优选例中，所述组C物质选自下组：GGCNGRC、CNGRC、或其组合。
在另一优选例中，所述多肽与所述交联环糊精有机骨架COF是如下连接的：通过所述多肽一端的羧基或氨基与所述交联环糊精有机骨架COF表面的羟基进行共价连接。
在另一优选例中，所述低分子肝素LMWH选自下组：低分子肝素、低分子肝素钠、低分子肝素钙、或其组合。
在另一优选例中，所述低分子肝素LMWH的分子量为2000-8000Da，较佳地3000-6000Da，更佳地4000-5000Da。
在另一优选例中，所述低分子肝素LMWH与所述交联环糊精有机骨架COF是如下连接的：
a1)采用胱胺修饰所述低分子肝素LMWH，得到经胱胺修饰的低分子肝素LMWH；
a2)通过步骤a1)所得经胱胺修饰的低分子肝素LMWH一端的氨基与所述交联环糊精有机骨架COF表面的羟基进行共价连接。
在另一优选例中，a2)中，所述氨基经活化连接臂的羰基与所述羟基反应连接，得到-O-(C＝O)-NH-连接键，从而实现所述经胱胺修饰的低分子肝素LMWH与所述交联环糊精有机骨架COF的连接。
在另一优选例中，所述组合物的尺寸为10-1000nm，较佳地50-800nm，更佳地100-500nm。
在另一优选例中，所述交联环糊精有机骨架COF与所述多肽的质量比为1:0.001-0.1(较佳地1:0.01-0.1，更佳地1:0.02-0.08)；和/或
所述交联环糊精有机骨架COF与所述低分子肝素LMWH的质量比为1:0.001-0.05(较佳地1:0.005-0.02，更佳地1:0.008-0.01)。
在本发明的第二方面，提供了一种本发明第一方面的组合物的制备方法，包括如下步骤：
1)制备多肽修饰的交联环糊精有机骨架COF，包括步骤：在第一溶剂、活化剂A存在下，使多肽与交联环糊精有机骨架COF充分反应，得到多肽修饰的交联环糊精有 机骨架COF；
2)制备胱胺修饰的低分子肝素LMWH，包括步骤：在第二溶剂、活化剂B存在下，使低分子肝素LMWH和胱胺充分反应，得到胱胺修饰的低分子肝素LMWH；
3)活化多肽修饰的交联环糊精有机骨架COF，包括步骤：在第三溶剂、活化连接臂、活化催化剂存在下，在第一温度下活化处理多肽修饰的交联环糊精有机骨架COF第一时间，得到经活化的多肽修饰的交联环糊精有机骨架COF；
4)制备本发明第一方面的组合物，包括步骤：在第四溶剂中，使步骤2)所得胱胺修饰的低分子肝素LMWH和步骤3)所得经活化的多肽修饰的交联环糊精有机骨架COF在第二温度下反应第二时间，得到本发明第一方面的组合物。
在另一优选例中，所述方法具有选自下组的一个或多个特征：
1)所述第一溶剂选自下组：二甲基甲酰胺、乙腈、丙酮、或其组合；
2)所述活化剂A选自下组：4-二甲氨基吡啶、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、三乙胺、N,N'-二琥珀酰亚胺基碳酸酯、N-羟基琥珀酰亚胺、N,N'-羰基二咪唑、或其组合；
3)所述第二溶剂选自下组：磷酸盐缓冲液、水、或其组合；
4)所述活化剂B选自下组：1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺、1-羟基苯并***、或其组合；
5)所述胱胺修饰的低分子肝素LMWH中，胱胺与低分子肝素LMWH的投料质量比为1:10-20(较佳地1:12-18，更佳地1:12-16)；
6)所述第三溶剂选自下组：乙腈、甲酰胺、二甲基甲酰胺、丙酮、甲醇、或其组合；
7)所述活化连接臂选自下组：N,N'-二琥珀酰亚胺基碳酸酯、N,N'-羰基二咪唑、丁二酰氯、异氰酸酯、或其组合；
8)所述活化催化剂选自下组：三乙胺、吡啶、N-羟基丁二酰亚胺、或其组合；
9)多肽修饰的交联环糊精有机骨架COF、所述活化连接臂和所述活化催化剂的摩尔比例为1:1-10:1-7(较佳地1:1-8:1-5)；
10)所述第一温度为10-100℃(较佳地20-80℃)；
11)所述第一时间为3-50h(较佳地5-25h)；
12)所述第四溶剂选自下组：甲酰胺、二甲基甲酰胺、乙腈、丙酮、甲醇、或其组合；
13)所述第二温度为10-100℃(较佳地20-80℃)；
14)所述第二时间为8-60h(较佳地10-50h)。
在本发明第三方面，提供了一种药物活性成分，所述的药物活性成分包含：
(1)作为药物载体的本发明第一方面的LMWH-多肽-COF组合物；和
(2)抗肿瘤药物或靶向肺部的药物，所述抗肿瘤药物或靶向肺部的药物负载于所述药物载体中。
在另一优选例中，所述的抗肿瘤药物包括用于治疗肺癌的抗肿瘤药物。
在另一优选例中，所述的药物活性成分具有肺靶向性。
在另一优选例中，所述抗肿瘤药物选自下组：细胞毒药物、分子靶向药物、辅助药物、或其组合。
在另一优选例中，所述细胞毒药物选自下组：阿霉素、阿霉素盐酸盐、表阿霉素、卡铂、奥沙利铂、5-氟嘧啶、卡培他滨、吉西他滨、紫杉醇、多西紫杉醇、拓扑替康、10-羟基喜树碱、伊立替康、或其组合。
在另一优选例中，所述分子靶向药物选自下组：吉非替尼、伊马替尼、埃罗替尼、或其组合。
在另一优选例中，所述辅助药物选自下组：白藜芦醇、槲皮素、黄芩素、姜黄素、或其组合。
在本发明的第四方面，提供了一种药物组合物，包含：
(a)本发明第三方面的药物活性成分；和
(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物的剂型包括：注射剂、冻干制剂、口服制剂、液体剂型、固体剂型、或其组合。
在本发明第五方面，提供了一种本发明第三方面所述的药物活性成分的制备方法，包括步骤：
(i)混合作为药物载体的本发明第一方面的LMWH-多肽-COF组合物和抗肿瘤药物，使所述肿瘤药物负载于所述的药物载体，从而得到第三方面所述的药物活性成分。
在另一优选例中，在步骤(i)中，将药物载体和抗肿瘤药物混合后，在合适的温度(如10-80℃，较佳地20-65℃)下孵育，从而使所述肿瘤药物负载于所述的药物载体。
在另一优选例中，在步骤(i)中，所述的孵育时间为0.5-72小时，较佳地1-36小时，更佳地2-24小时。
在本发明的第六方面，提供了一种交联环糊精有机骨架COF和/或本发明第一方面的LMWH-多肽-COF组合物和/或第三方面所述的药物活性成分的用途，它们被用于制备预防和/或***的药物或靶向肺部的药物。
在另一优选例中，所述的肿瘤包括肺癌。
在另一优选例中，所述的靶向肺部的药物用于治疗选自下组的疾病：肺部感染、肺部炎症、肺部纤维化、COPD、或其组合。
在另一优选例中，所述的肺部感染包括：细菌感染、病毒感染(如流感病毒感染、 冠状病毒感染、或其他病原体导致的感染)。
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中环糊精骨架组合物COF具有高度肺靶向性的组织分布荧光图。
图2为实施例2中低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架(LMWH-GS5-COF)合成流程图。
图3为实施例2中低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架(LMWH-GS5-COF)的电镜图。
图4为实施例2中低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架(LMWH-GS5-COF)的红外图谱。
图5为实施例2中低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架(LMWH-GS5-COF)核磁图谱。
图6为实施例2中低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架LMWH-GS5-COF与低分子肝素LMWH修饰立方体环糊精骨架LMWH-COF的血液相容性评价。(A)不同样品处理后红细胞的显微镜图；(B)离心后的样品图；(C)溶血比例(n＝3)。
图7为实施例2中低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架LMWH-GS5-COF与低分子肝素LMWH修饰立方体环糊精骨架LMWH-COF的B16F10细胞毒性评价。
图8为实施例2中低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架LMWH-GS5-COF与低分子肝素LMWH修饰立方体环糊精骨架LMWH-COF的A549细胞毒性评价。
图9为实施例3中负载阿霉素的低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架(RCLD)的氧化还原响应型体外释放评价，
图10为实施例3中负载阿霉素的低分子肝素LMWH修饰立方体环糊精骨架CLD与负载阿霉素的低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架RCLD的细胞毒性评价。图11为实施例3中负载阿霉素的低分子肝素LMWH修饰立方体环糊精骨架CLD与负载阿霉素的低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架RCLD对肿瘤细胞迁移和侵袭的抑制作用。(A)和(C)伤口愈合实验的代表性图片和伤口愈合率分析(n＝3)。(B)和(D)细胞侵袭实验的代表性图片和定量相对侵袭率(n＝3)。***p<0.001。
图12为实施例3中负载阿霉素的低分子肝素LMWH修饰立方体环糊精骨架CLD与负载阿霉素的低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架RCLD的肺癌靶向性和生物分布。
图13为实施例3中负载阿霉素的低分子肝素LMWH修饰立方体环糊精骨架CLD与负载阿霉素的低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架RCLD在A549肺癌模型中的抗肿瘤效果。(A)从A549肺癌小鼠模型中收集的肺部图片；(B)RCLD的治疗方案；(C)肺部A549转移性结节数目的定量分析(n＝5)；(D)肺组织的H&E染色分析。*p<0.05,***p<0.001,ns表示两组之间没有显著性差异。
图14为实施例3中荷A549的BALB/c小鼠的体重变化。
图15为实施例3中负载阿霉素的低分子肝素LMWH修饰立方体环糊精骨架CLD与负载阿霉素的低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架RCLD在B16F10转移性肺癌模型中的治疗效果。(A)从B16F10转移性肺癌小鼠模型中收集的肺部图片；(B)RCLD的治疗方案；(C)B16F10转移性肺癌的面积(n＝5)。**p<0.01,***p<0.001,ns代表两组之间没有显著性差异。
图16为实施例3中B16F10转移性肺癌模型小鼠治疗结束后，主要器官的H&E染色分析。黑色箭头指向萎缩的心肌细胞。
图17为实施例3中静脉注射负载阿霉素的低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架RCLD五次后，小鼠的血细胞分析和血清生化分析(n＝4)。*p<0.05,**p<0.01,***p<0.001。
图18为实施例50中负载拓扑替康(TPT)的低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架RCLT与负载拓扑替康的环糊精有机骨架组合物TPT@COF的体外释放评价。
图19为实施例50中负载拓扑替康的低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架RCLT与负载拓扑替康的环糊精有机骨架组合物TPT@COF的B16F10细胞毒性评价。
图20为实施例50中负载拓扑替康的低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架RCLT与负载拓扑替康的环糊精有骨架组合物TPT@COF在B16F10转移性肺癌模型中的治疗效果。(A)经RCLT治疗的B16F10转移性肺癌小鼠模型中收集的肺部图片；(B)TPT@COF与RCLT的治疗方案；(C)经RCLT治疗的B16F10转移性肺癌的面积；(D)经TPT@COF治疗的B16F10转移性肺癌小鼠模型中收集的肺部图片；(E)经TPT@COF治疗的B16F10转移性肺癌的面积(n＝5)。**p<0.01,***p<0.001。
图21为实施例50中经负载拓扑替康的低分子肝素LMWH和GS5肽双重修饰立方体环糊精骨架RCLT与负载拓扑替康的环糊精有骨架组合物TPT@COF治疗的荷 B16F10的C57BL/6小鼠的体重变化。(A)经RCLT治疗的荷B16F10的C57BL/6小鼠的体重变化；(B)经TPT@COF治疗的荷B16F10的C57BL/6小鼠的体重变化。
具体实施方式
本发明人经过长期而深入的研究，意外地发现纳米级环糊精骨架组合物COF具有高度的肺靶向性，基于此，发明人同时采用功能性多肽和低分子肝素对COF进行双重修饰，并经进一步载药后获得了一种具备优异安全性、高效靶向性、优异疗效(如抗肿瘤疗效)的组合物。在此基础上，发明人完成了本发明。
多肽
RGD多肽是指含有精氨酸-甘氨酸-天冬氨酸三肽序列(Arg-Gly-Asp，RGD)多肽，可特异性识别整合素α vβ 3受体。整合素α vβ 3受体蛋白在肿瘤生成、转移过程中发挥重要作用，其在多种肿瘤细胞表面和肿瘤新生血管内皮高度表达，而在正常细胞低表达或不表达，故含有RGD多肽的载体可通过配体-受体介导的主动靶向作用靶向肿瘤部位。包含“RGD”序列的多肽均称为RGD多肽，术语“RGD”、“DRGDS”、“GRGD”、“RGDS”、“GRGDS”(又称“GS5”)、“GRGDSP”、“GRADSPK”、“GRGDSPK”均属于本发明的RGD多肽系列。
NGR多肽指含天冬酰胺-甘氨酸-精氨酸三肽序列(Asn-Gly-Arg，NGR)多肽，可与肿瘤细胞和肿瘤新生血管内皮细胞中CD13金属肽酶持异性结合，不仅可主动靶向肿瘤部位，又能抑制肿瘤新生血管的生成。包含“NGR”序列的多肽均称为NGR多肽，术语“TNGRGP”、“NGRSRF”、“RSRNGR”、“NGRNTV”、“GGCNGRC”、“CNGRC”均属于本发明的NRG多肽系列。
肝素
低分子肝素(Low molecular weight heparin,LMWH)是普通肝素通过物理、化学、生物等方式降解而成的分子量较低的肝素。LMWH不仅能抑制肿瘤细胞和血小板之间的相互作用，阻碍肿瘤细胞的上皮间质转化，而且也会影响肿瘤细胞肌动蛋白细胞骨架的排列。
肝素-多肽双重接枝环糊精骨架组合物
如本文所用，肝素-多肽双重接枝环糊精骨架组合物是将多肽与低分子肝素LMWH共价连接于交联环糊精有机骨架COF。
还原型谷胱甘肽
还原型谷胱甘肽(Glutathione,GSH)是由谷氨酸、半胱氨酸和甘氨酸组成的含 活性巯基的三肽，GSH在肿瘤部位的浓度高于正常组织(甚至可高100倍)，且可特异性断裂二硫键。
肺癌是全球发病率和死亡率最高的肿瘤，且肺部转移是癌症临床治疗中的一大挑战，现有治疗手段多以化学疗法为主，在获得治疗效果的同时往往也对正常组织器官也存在着较强的毒副作用。大部分肺癌靶向纳米传递***是利用肿瘤微环境或高渗透长滞留效应(EPR效应)而实现肺靶向，大多只是靶向肿瘤部位高表达的细胞表面受体，不具有肺组织靶向的特异性。本发明在纳米级环糊精骨架材料具有高度肺组织靶向性的基础上，对其表面进行多肽与低分子肝素的双重接枝，赋予载体主动靶向与协同治疗的功效，其安全性高、生物相容性好、靶向性高，满足临床需求。
以药用辅料环糊精为有机配体、钾离子为无机金属离子中心形成的环糊精-金属有机骨架(CD-MOF)，不仅保留了环糊精的生物安全性，同时又具有生物相容性良好、立方体形态、表面可修饰等优势，可应用于药物递送领域。
LMWH-多肽-COF组合物及其制法和应用
本发明申请人前期对纳米级CD-MOF进行交联，得到在水体系中稳定存在的交联立方体环糊精有机骨架(COF)，采用含精氨酸-甘氨酸-天冬氨酸三肽序列(Arg-Gly-Asp，RGD)对COF进行表面修饰，合成得到的RGD-COF材料，可作为人工血小板载体，具有高效止血的功效。本发明还发现并确证了纳米级COF具有高度的肺靶向性特征，COF经静脉注射后，COF的体内的肺靶向系数分别为31.7(小鼠)、10.1(大鼠)、129.0(家兔)，而文献报道的其他方式给药的肺靶向系数一般低于10，如文献(Biomaterials,2019,218:119331)中报道的盘状聚合物微粒在肺部的摄取量是肝脏的8.7倍。本发明将针对COF具有肺组织靶向特性，在RGD-COF的基础上构建新型高效的靶向给药***。
多肽介导的配体-受体主动靶向治疗是利用多肽配体与肿瘤细胞中过度表达受体间的高度亲和力，将药物定向输送到特定的肿瘤组织内，增加药物在肿瘤部位的蓄积，减轻对正常组织的毒副作用。整合素α vβ 3受体蛋白在肿瘤生成、转移过程中发挥重要作用，其在多种肿瘤细胞表面和肿瘤新生血管内皮高度表达，而在正常细胞低表达或不表达，含RGD载体可特异性识别整合素α vβ 3受体，由此含RGD多肽的载体可介导主动靶向给药***。肿瘤细胞的生长与转移需要新生血管提高养分，CD13金属肽酶是新生血管生成的重要调节因子，促进细胞的增殖与迁移以及新生血管的形成。含NGR载体可特异性识别CD13金属肽酶，故NGR载体可通过靶向介导功能提高药物在肿瘤组织内浓度。
低分子肝素(LMWH)是普通肝素通过物理、化学、生物等方式降解而成的分子量较低的肝素，在具有传统的抗凝血功能同时，还具有抑制肿瘤转移、抑制肿瘤细 胞增殖、较好的生物相容性等特点。LMWH骨架中含有大量活泼的可修饰的游离基团，可通过化学修饰赋予其新的性能。LMWH可以通过与肿瘤部位过度表达的新血管生成有关的生长因子结合而抑制其活性，从而抑制肿瘤新生血管生长。LMWH与肝素酶、P-/L-选择蛋白的亲和力，使之抑制肿瘤细胞逃逸转移等关键过程，达到肿瘤治疗的目的。加之LMWH分子上有重复的羧基、羟基、氨基等基团，可以通过简单的化学反应对其接枝在载体上，赋予载体生物修饰的特性。由于还原型谷胱甘肽(GSH)在肿瘤部位的浓度高于正常组织的100倍，在肿瘤细胞高浓度GSH的刺激下，可特异性断裂胱胺中的二硫键，在肿瘤组织内特异性地释放LMWH，以此达到定向治疗功效。
本发明提供了一种LMWH-多肽-COF组合物，如LMWH-RGD-COF，其内部为具有立方结构的COF材料，表面采用LMWH与RGD进行生物学修饰。COF作为基本药物载体单元，RGD作为靶头，可以特异性结合多种肿瘤细胞表面和新生血管表皮上的整合素α vβ 3受体，LMWH作为包衣层，以胱胺为连接臂的LMWH包裹的COF在肿瘤细胞内高浓度GSH的刺激下，可特异性将外壳LMWH脱落，暴露出负载的药物。纳米级可静脉注射立方体LMWH-RGD-COF组合物能够逃避巨噬细胞的吞噬和清除，通过自主的肺靶向性到达肺，进而可以降低给药剂量，降低化疗药物的毒副作用，促进药物在肿瘤部位的富集，提高抗肿瘤疗效。
本发明所述的LMWH-RGD-COF组合物不同于已有的功能性纳米粒报道，现有在金纳米粒表面同时修饰LMWH与RGD，其对癌细胞具有高度特异性的凋亡活性；现有用LMWH功能修饰的PLGA纳米颗粒，发现该制剂在体外可改善纳米粒在肿瘤积累，但这两纳米粒并不体现肺靶向性。本发明所述的LMWH-RGD-COF组合物具有良好的生物相容性和安全性及肺靶向性。
目前尚未见报道以立方体的环糊精骨架材料为基础，构建肺靶向抗药物(包括抗肿瘤药物和/或靶向肺部的药物)递药***。在前期基础上，本发明人将采用还原敏感型的化学键将LMWH接枝在RGD-COF上，作为载体实现靶向给药，且借助治疗药物LMWH和药物的协同作用，进行抗肺癌治疗。现有在金纳米粒表面同时修饰肝素与RGD，对选择性过量表达RGD受体的癌细胞具有高度特异性的凋亡活性，但其不具有肺靶向性。现有采用负载阿霉素(DOX)的低分子肝素的纳米粒(LH-DOX)进行体内抗黑色素肺转移，结果表明静脉注射与游离DOX相同剂量治疗(2.5mg/kg)，疗效大于游离药物DOX和LMWH组，与对照组相比，黑色素瘤肺转移面积减少为70.92％。现有用低分子量肝素包裹的阿霉素脂质体(LMWH-DOX-Lip)，小鼠肺黑色素瘤转移模型结果表明，相比于DOX与LMWH组，同等DOX(5mg/kg)剂量下，LMWH-DOX-Lip能有效抑制转移灶。相比于上述纳米制剂，本发明设计的载体可以利用自身的肺组织靶向特性，在降低药物剂量(如DOX剂量从2.5mg/kg降低到1mg/kg)情况下，可显著抑制肺癌的生长和转移(A549细胞肺转移结节几乎没 有，黑色素瘤肺转移面积减少70％)。
本发明提供了一种具有肺靶向的低分子肝素LMWH、多肽双重接枝立方体环糊精骨架组合物(LMWH-多肽-COF)及其制备方法与应用。具体地，本发明将与多肽、具有抗肿瘤活性的低分子肝素和抗肿瘤药物组装在立方体环糊精骨架COF中形成多元组合物，旨在构建具长循环、肺靶向等功能于一体的智能药物递送***。本发明的LMWH-多肽-COF纳米粒子能够逃避巨噬细胞的吞噬和清除，具有良好的安全性和高效的靶向性，将药物富集在肿瘤部位，并通过肿瘤部位氧化还原响应性释放，大大降低对正常组织的毒副作用，增强药物的治疗效果。
以抗肿瘤药物为例，本发明提供的一种抗肿瘤LMWH-多肽-COF组合物，其结构特征在于的CD-MOF内羟基(-OH)被合适的交联剂共价连接，形成在水中稳定性良好的COF材料，然后采用连接臂将多肽的羧基(-COOH)或氨基(-NH 2)与COF外表面的羟基(-OH)共价连接，进一步采用活化剂将胱胺修饰的低分子肝素LMWH的氨基(-NH 2)与COF外表面活化的羟基(-OH)共价连接，实现对COF的表面的双重生物学修饰。COF可作为高效负载抗肿瘤药物药用载体，多肽作为靶头，与肿瘤细胞高度表达的受体特异性结合，主动靶向肿瘤部位；含二硫键敏感释放的LMWH作为包衣层，与化疗药物协同治疗。
本发明提供了一种制备LMWH-多肽-COF的制备方法，所述方法包括步骤：
(I)提供纳米级CD-MOF材料；
(II)通过交联剂将所述的纳米级CD-MOF的羟基(-OH)化学交联，得到在水中稳定良好的COF；
(III)在所述COF的表面共价连接功能性多肽，得到所述的多肽-COF组合物；
(Ⅳ)在所述多肽-COF组合物表面包裹胱胺修饰的低分子肝素LMWH，从而得到所述的LMWH-多肽-COF。
LMWH-胱胺的合成：将LMWH接枝胱胺后，引入末端氨基，该氨基与活化后多肽-COF的羰基形成酰胺健，此种方法可以提高LMWH在多肽-COF上的取代度。具体方法为：将LMWH溶于磷酸盐缓冲液中(pH＝7.4)，向其中依次加入一定比例的1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)与N-羟基琥珀酰亚胺(NHS)，室温活化20-30min后，加入胱胺溶解，经透析、冻干得LMWH-胱胺。
多肽-COF的活化：将步骤(III)中多肽-COF分散于有机溶剂中，加入一定量的连接臂与催化剂，一定温度下反应一段时间，得活化立方体多肽-COF组合物反应液；
LMWH-多肽-COF的制备：将步骤(2)中反应液缓慢滴加至LMWH-胱胺的有机溶液中，加入一定量催化剂，一定温度下反应一段时间，反应液经透析、冷冻干燥后即得LMWH与多肽双重接枝立方体环糊精骨架组合物。
所述的药物组合物的制备方法概括为：将抗肿瘤药物溶液与LMWH-多肽-COF按 一定投料摩尔比在一定温度下搅拌孵育一定时间，离心，洗涤，干燥，即得。
本发明提供了一种具有肺靶向的肝素/多肽双重接枝环糊精骨架组合物制备方法及其制备方法与应用。具体地，本发明的LMWH/RGD双重接枝环糊精骨架组合物具有肺靶向功能，负载细胞毒性药物(如阿霉素)后，在体外能够显著降低B16F10肿瘤细胞的存活率，还可显著抑制肿瘤细胞迁移和侵袭的能力。对于小鼠A549人肺腺癌转移模型和B16F10黑色素瘤转移性肺癌模型，该载体在阿霉素同等疗效下，可以将阿霉素的剂量降低5倍，减少了阿霉素的心脏毒性和副作用，有望实现肺癌治疗的减毒和增效作用。该纳米肺组织递送***具有良好的生物安全性，静脉注射后主要器官没有发生急性毒性或组织坏死，且血液学研究表明其不会影响小鼠体内主要的血细胞水平，具有较好的临床转化价值。
与现有技术相比，本发明具有以下主要优点：
(1)本发明制备的纳米材料(即本发明组合物)制备工艺简单可控、无需昂贵的设备、可大规模生产，且载体安全性高、生物相容性好、无免疫原性，具有良好的肺靶向性。
(2)本发明制备的纳米材料具有规则的立方体形态，粒径较小，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，延长体内循环时间。
(3)本发明制备的纳米材料在构建过程中采用多肽序列修饰，赋予立方体环糊精有机骨架组合物COF主动靶向特性，实现肿瘤药物靶向治疗，减少对正常器官与组织的毒副作用。
(4)本发明制备的纳米材料在构建过程中采用低分子肝素LMWH包衣，通过氧化还原敏感释放机制将纳米粒定位释放药物。
(5)本发明制备的纳米制剂同时负载抗肿瘤药物和低分子肝素，实现协同给药，大大提高治疗效果。
下面结合具体实施例，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。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1
纳米级CD-MOF的制备：使用溶剂热的方式，将γ-CD、KOH水溶液与甲醇溶剂 混合体系进行加热。按摩尔比为1:8称取163.0mgγ-CD和56.0mg KOH溶解于5mL水中，经0.8μm滤膜过滤，获得γ-CD-KOH母液。在母液中加入3mL甲醇，60℃水浴加热20min后，加入等体积的甲醇，再加入64mg PEG 20000，继续加热20min。将反应体系静置2h后，4000rpm离心5min，分别用乙醇(10mL×2次)、甲醇(10mL×2次)洗涤，将所得产物80℃真空干燥2h，即得纳米级CD-MOF晶体，扫描电镜和动态光散射结果显示得到的CD-MOF为规则的立方体形态，尺寸为100-500nm。
纳米级COF的制备：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，400rpm搅拌条件下，加入771mg交联剂碳酸二苯酯(CD-MOF与碳酸二苯酯的摩尔比为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次)、纯水(10mL×2次)和丙酮(10mL×2次)各洗涤2次，将所得晶体40℃真空干燥4h即得水中稳定的COF，产率约为85％，扫描电镜和动态光散射结果结果显示得到的COF为规则的立方体形态，尺寸为100-500nm。
COF在小鼠中具有高度的肺靶向性：给昆明小鼠尾静脉注射Cy5标记的COF纳米粒混悬液(40mg/kg,10mL/kg)，分别于给药前5min，给药后0min、5min、10min、15min、1h、2h、4h、8h和24h后，将小鼠处死，取出心、肝、脾、肺、肾主要器官，采用小动物活体成像仪测定不同时间点各个脏器的荧光强度，以研究COF纳米粒在小鼠体内的肺靶向性。小鼠经尾静脉注射荧光染料Cy5及荧光染料Cy5标记的COF纳米粒1h后，荧光染料Cy5主要分布在肾脏和肝脏，而COF纳米粒主要分布在肺部；给药后15min，COF开始在肺部聚集；给药后2h，COF纳米粒在肺部的分布达到最高值，此时小鼠肺部的荧光信号是肝脏部位的30倍，由此说明COF对小鼠具有高度的肺靶向性。
COF在大鼠中具有高度的肺靶向性：给SD大鼠尾静脉注射Cy5标记的COF纳米粒混悬液(28mg/kg,10mL/kg),分别于给药前5min，给药后0min、5min、10min、15min、1h、2h、4h、8h和24h后，将大鼠处死，取出心、肝、脾、肺、肾主要器官，采用小动物活体成像仪测定不同时间点各个脏器的荧光强度，研究COF纳米粒在大鼠体内的肺靶向性。大鼠经尾静脉注射荧光染料Cy5、Cy5标记的γ-CD和COF纳米粒1h后，γ-CD和Cy5荧光染料主要分布在肾脏和肝脏；COF纳米粒主要分布在肺部；给药后15min，COF开始在肺部聚集；给药后2h，COF纳米粒在肺部的分布达到最高值，此时大鼠肺部的荧光信号是肝脏部位的10倍，说明COF对大鼠具有高度的肺靶向性。
COF在家兔中具有高度的肺靶向性：给家兔耳缘静脉注射Cy5标记的COF纳米粒混悬液，分别于给药前5min，给药后0min、5min、10min、15min、1h、 2h、4h、8h和24h后，将家兔处死，取出心、肝、脾、肺、肾主要器官，采用小动物活体成像仪测定不同时间点各个脏器的荧光强度，研究COF纳米粒在家兔体内的肺靶向性。家兔经耳缘静脉注射COF纳米粒15min后，COF在肺部的分布已经达到最高值，此时肺部的荧光信号是肝脏部位的120倍，由此说明COF在家兔体内也具有显著的肺靶向功能。
具体结果见图1，其中A为COF在小鼠、大鼠和家兔中组织荧光强度图，B为COF在小鼠体内不同时间点的体内组织分布图，C为COF在小鼠体内不同时间的荧光强度图。
从图1A可知：COF在小鼠、大鼠和家兔体内均能实现对肺组织的高度靶向，肺部的荧光信号是肝脏部位的30倍、10倍、120倍。
从图1B可知：小鼠尾静脉注射COF后，主要分布在肺部。
从图1C可知：小鼠尾静脉注射COF 2h后，COF在肺部的分布达到最高值，但随着时间延长，COF在肺部的富集渐渐减弱。
实施例2
(1)双重修饰的组合物载体的制备
纳米级GS5-COF的制备：称取230mg实施例1制备的纳米级COF和10mg GRGDS(又称GS5)五肽(COF和GS5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(DMAP)和6mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)，置于磁力搅拌器上37℃，600rpm搅拌24h，使COF与GS5多肽充分偶联。反应完成后，4000rpm离心5min，分别用10mL二甲基甲酰胺和10mL纯水各洗涤2次，-50℃冷冻干燥24h即得GS5修饰的COF(产物简写为GS5-COF)，扫描电镜和动态光散射结果显示得到的
GS5-COF为规则的立方体形态，尺寸为100-500nm，高效液相色谱法(HPLC)测得环糊精骨架材料与GS5多肽的质量百分比为1:0.05。
低分子肝素(LMWH)和GS5肽双重修饰立方体环糊精骨架(COF)，即LMWH-GS5-COF的制备：
低分子肝素-胱胺(LMWH-CYS)的合成：称取400mg的低分子肝素LMWH(0.71mmol)超声溶解于100mL磷酸盐缓冲液(PBS，pH＝7.4)，然后加入相当于LMWH中羧基5倍摩尔当量的3.56mmol的1-乙基-(1-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)681.60mg和N-羟基琥珀酰亚胺(NHS)409.20mg，在25℃磁力搅拌15min，以充分活化LMWH上的羧基。接着加入5.6g的胱胺CYS(24.8mmol)，充分溶解后，在25℃下继续搅拌48h后得到低分子肝素-胱胺(LMWH-CYS)粗产物。将粗产物先经0.1M NaCl溶液(5.85g的NaCl用纯水溶解后定容至1000mL)透析12h(透析袋截留分子量为3500Da)，再用纯水透析48h。将透析后得到的液体经 0.22μm滤膜过滤并冷冻干燥，即可得到中间产物LMWH-CYS。
GS5-COF的活化：称取N,N'-二琥珀酰亚胺基碳酸酯(DSC)34.13mg(0.133mol)超声溶解于3mL乙腈(ACN)中，并加入28μL三乙胺(TEA)，向上述溶液中加入GS5-COF粉末96.33mg(0.067mol)，摩尔比为COF:DSC:TEA＝1:2:3，25℃下400rpm搅拌活化12h。
LMWH-GS5-COF的制备：称取300mg LMWH-CYS，加入18mL甲酰胺，磁力搅拌下溶解，得到LMWH-CYS溶液。将活化后的GS5-COF溶液加入至LMWH-CYS溶液中，400rpm磁力搅拌下25℃反应48h，得到LMWH-GS5-COF粗产物。为了除去未反应的小分子及其副产物，该粗产物先对乙腈/水混合溶剂(4:1,v:v)透析三天，再继续对纯水透析两天，离心，-50℃冷冻干燥即得LMWH-GS5-COF。
具体LMWH-GS5-COF制备过程见图2。
图3为实施例2中LMWH-GS5-COF的电镜图,从图3可知：LMWH-GS5-COF仍然保持规则的立方体形态，粒径100-500nm。
(2)不含RGD的低分子肝素接枝的载体制备：
同实施例1制备纳米级交联环糊精骨架(COF)及同实施例2制备低分子肝素-胱胺((LMWH-CYS)。
COF的活化：称取DSC 34.13mg(0.133mol)超声溶解于3mL ACN中，并加入TEA 28μL，向上述溶液中加入COF粉末96.33mg(0.067mol)，摩尔比为COF:DSC:TEA＝1:2:3，25℃下400rpm搅拌活化12h。
肝素-交联环糊精骨架组合物(LMWH-COF)的制备：称取300mg LMWH-CYS，加入18mL甲酰胺，磁力搅拌下溶解，得到LMWH-CYS溶液。将活化后的COF溶液加入至LMWH-CYS溶液中，400rpm磁力搅拌下25℃反应48h，得到肝素-交联环糊精骨架组合物(LMWH-COF)粗产物。为了除去未反应的小分子及其副产物，该粗产物先对乙腈/水混合溶剂(4:1,v:v)透析三天，再继续对纯水透析两天，离心，-50℃冷冻干燥即得LMWH-COF。
(3)载体的表征
傅里叶变换红外光谱分析双重修饰载体组合物的官能团：使用Thermo Nicolet IS 5红外光谱仪测样，测样时将样品与溴化钾按照质量比1:10的比例进行混合，研磨，压片，测定样品在波数4000-400cm -1范围内的红外吸收光谱。
图4为实施例2中LMWH-GS5-COF的红外图谱。
图4红外结果表明，产物LMWH-GS5-COF含有COF在1751cm -1处的特征峰，也保留了GS5在1540cm -1和1203cm -1处的特征峰，说明成功将GS5修饰到COF表面。同时，合成的LMWH-GS5-COF也表现出了LMWH在1622cm -1处的红外特征吸收峰，证明GS5-COF已经成功修饰了LMWH。
核磁共振氢谱分析双重修饰载体的特征峰：采用Bruker AVANCE NEO 500型核 磁共振氢谱仪对样品进行核磁共振氢谱分析。将GS5多肽与LMWH溶解在600μL的D 2O中，LMWH-GS5-COF分散在600μL的D 2O中，再加入10μL的NaOD使其降解。然后各样品溶液装在有盖核磁管中，在500MHz的磁场下收集图谱。
图5为实施例2中LMWH-GS5-COF核磁图谱。
从图5可知产物LMWH-GS5-COF的 1H-NMR中既含有LMWH的特征峰(δ＝2.1ppm；δ＝5.1～5.8ppm)和GS5的特征峰(δ＝3.2ppm)，也保留了COF中环糊精质子在δ＝5.0ppm特征峰，说明成功合成以上两种载体。
(4)载体的生物安全性评价
载体的溶血毒性考察：取新鲜C57BL/6新鲜血液2mL于3.2％柠檬酸钠浸润的试管中，2500rpm离心10min沉淀红细胞，弃去上层血浆后用生理盐水洗涤红细胞3次。将红细胞用生理盐水稀释成2％的红细胞混悬液。将以上实施例2制备的LMWH-GS5-COF与LMWH-COF用生理盐水配制成5、10、25、100、200、400μg/mL的的混悬液，加入等体积2％红细胞混悬液摇匀，于37℃水浴中孵育1h。另取同等体积的纯水与生理盐水分别作为阳性对照和阴性对照。孵育后经1500rpm离心10min取上清液，采用紫外分光光度法测定540nm处的吸光度值，平行操作3次。
图6为实施例2中LMWH-GS5-COF与LMWH-COF的血液相容性评价。(A)不同样品处理后红细胞的显微镜图；(B)离心后的样品图；(C)溶血比例(n＝3)。
图6A-C结果表明：当LMWH-GS5-COF与LMWH-COF的浓度增加至600μg/mL时，溶血率仍然小于1％。溶血率小于5％通常认为是安全的，表明LMWH-MOF和LMWH-GS5-COF纳米粒具有良好的血液相容性。
载体的B16F10细胞毒性评价：B16F10细胞培养在DMEM培养基(含10％胎牛血清)中，在37℃，5％的CO2恒温恒湿孵育器中培养。选择对数生长期的细胞进行实验，将处于对数生长期的细胞以5×10 3个/孔的密度接种于96孔板，培养12h。去掉上层培养液后，分别加入200μL不同浓度的LMWH、LMWH-COF、LMWH-GS5-COF样品，同时设置空白组(仅含培养液)和对照组(含有细胞和培养液)。实验设置8个不同浓度，分别为0.0067、0.033、0.067、0.33、0.67、1.33、13.3和133μg·mL -1，在培养箱中继续培养24h后，每孔加入15μL的CCK-8溶液，37℃继续孵育2h。终止培养，采用酶标仪检测450nm处的吸光度值，按公式(1)计算细胞存活率。
Figure PCTCN2021122163-appb-000001
式中，A Sample为样品的吸光度值；A control为对照组的吸光度值；和A blank为空白组的吸光度值。
图7为实施例2中LMWH-GS5-COF与LMWH-COF的B16F10细胞毒性评价，其中 A为LMWH的B16F10细胞毒性，B为LMWH-COF与LMWH-GS5-COF纳米粒的B16F10细胞毒性。
图7A结果表明当LMWH浓度增加至133μg/mL时，细胞存活率仍然高达100％，说明LMWH本身不影响细胞活性。图7B结果表明LMWH-COF和LMWH-GS5-COF纳米载体的细胞存活率约100％，无明显的细胞毒性。
载体的A549细胞毒性评价：A549细胞培养在RPMI 1640培养基(含10％胎牛血清)中，在37℃，5％的CO 2恒温恒湿孵育器中培养。选择对数生长期的细胞进行实验，将处于对数生长期的细胞以2×10 4个/孔的密度接种于96孔板，培养24h。去掉上层培养液后，分别加入200μL不同浓度的LMWH-COF、LMWH-GS5-COF样品，同时设置空白组(仅含培养液)和对照组(含有细胞和培养液)。实验设置8个不同浓度，分别为0.0067、0.033、0.067、0.33、0.67、1.33、13.3和133μg·mL -1，在培养箱中继续培养24h后，每孔加入15μL的CCK-8溶液，37℃继续孵育1.5h。终止培养，采用酶标仪检测450nm处的吸光度值，按公式(1)计算细胞存活率。
图8为实施例2中LMWH-GS5-COF与LMWH-COF的A549细胞毒性评价，结果表明LMWH-COF和LMWH-GS5-COF纳米载体浓度在0.0067-13.3μg·mL -1范围内细胞活性基本上均接近100％，无明显的细胞毒性。
实施例3
(1)负载盐酸阿霉素
载盐酸阿霉素的双重修饰载体组合物的制备：称取12mg的盐酸阿霉素(DOX)溶解于2mL的纯水中，超声10min使其溶解，然后加入实施例2中制备的LMWH-GS5-COF纳米粒30mg，药物与LMWH-GS5-COF纳米粒的摩尔比为1:1，25℃下避光300rpm搅拌24h，孵育载药。载药完成后，4000rpm离心5min，用纯水洗涤以除去游离的DOX后，得到下层载盐酸阿霉素的双重修饰载体组合物(RCLD)。
药物的载药量通过荧光分光光度法测定，称取适量RCLD分散于纯水中，加入1M氢氧化钠溶液溶解后，再加入同体积1M盐酸中和，溶液在Ex＝470nm和Em＝598nm处测定DOX的荧光强度，按照公式(2)测定其载药量。结果表明RCLD载药量为15％。
Figure PCTCN2021122163-appb-000002
载盐酸阿霉素的不含GS5的低分子肝素接枝的载体组合物的制备：称取12mg的DOX溶解于2mL的纯水中，超声10min使其溶解，然后分别加入实施例2中制备的LMWH-COF纳米粒30mg，药物与LMWH-COF的摩尔比为1:1，25℃下避光300rpm搅拌24h，孵育载药。载药完成后，4000rpm离心5min，用纯水洗涤 以除去游离的DOX后，得到下层载盐酸阿霉素的不含RGD的低分子肝素接枝的载体组合物(CLD)。
CLD的载药量通过荧光分光光度法测定，称取适量CLD分散于纯水中，加入1M氢氧化钠溶液溶解后，再加入同体积1M盐酸中和，溶液在Ex＝470nm和Em＝598nm处测定DOX的荧光强度，按照公式(2)测定其载药量。结果表明CLD载药量为15％。
(2)负载阿霉素的载体的体外释放考察
采用动态膜透析袋法考察RCLD纳米粒在不同浓度还原型谷胱甘肽(GSH)条件下的释药行为。将5mg RCLD(DOX剂量为750μg)的5mL分散液装入透析袋(截止分子量为14kDa)中。将每个透析袋浸入具有不同GSH浓度(0、1、10mM)的PBS(pH＝7.4，100mL)中，并在摇床中以75rpm和37℃孵育，在预定的时间点(0.25、0.5、1、2、4、6、8、12、24、36、48、72h)收集5mL的释放介质，并补充同等体积的介质。样品使用荧光分光光度法在Ex＝470nm和Em＝598nm处确定释放的DOX量。
图9为实施例3中RCLD的体外释放评价，结果表明相比于RCLD在不含GSH的PBS释放介质中72h释放50％，RCLD纳米粒在10mM GSH释放介质中可以快速DOX，前12h内50％药物释放，72h内释放70％药物，这归功于高浓度GSH切断了肝素与COF间的连接，加快DOX释放。除此之外，在含1mM GSH与不含GSH的释放介质中，其释放行为相似，说明RCLD纳米粒在体内循环具有良好的稳定性，可以避免药物在到达肿瘤部位前被提前释放。
(3)两种负载阿霉素的载体组合物的细胞毒性评价
采用CCK-8法考察了以上两种负载阿霉素的载体组合物纳米粒(RCLD和CLD)对B16F10肿瘤细胞的细胞毒性。B16F10细胞培养在DMEM培养基(含10％胎牛血清)中，在37℃，5％的CO 2恒温恒湿孵育器中培养。选择对数生长期的细胞进行实验，将处于对数生长期的细胞以5×10 3个/孔的密度接种于96孔板，每孔终体积200μL，培养12h。去掉培养液，分别加入DOX、以上两种负载阿霉素的载体组合物RCLD或CLD，共设置8个不同的浓度，按照DOX的载药量换算使DOX的终浓度设置为0.1、0.2、0.5、1、2、5、10和20μg·mL -1。同时设置空白组(仅含培养液)和对照组(细胞和培养液)。在培养箱中继续培养24h后，每孔加入CCK-8溶液15μL，37℃继续孵育2h。终止培养，采用酶标仪检测450nm处的吸光度(A)值，按照实施例2的公式计算细胞存活率(n＝6)。
图10为实施例3中CLD与RCLD的细胞毒性评价。图10结果表明：RCLD具有较强的细胞毒性，在体外能够显著抑制肿瘤细胞的生长，且与游离DOX相比，RCLD的细胞毒性较弱。
(4)两种负载阿霉素的载体组合物的抑制肿瘤细胞的迁移和侵袭评价
两种负载盐酸阿霉素的载体组合物的伤口愈合实验：将B16F10细胞接种到6孔板中，当细胞生长至铺满90％的孔底面积时，用200μL无菌移液器吸头划出一道伤口，并用PBS冲洗一次，形成一道不含细胞的划痕。随后，将细胞与PBS、游离DOX、游离LMWH、实例1制备的COF、实例3制备的RCLD或CLD在37℃孵育24h。使用倒置光学显微镜分别在0h和24h观察拍照，并计算伤口愈合率。
图11A和图11C为实施例3中CLD与RCLD对伤口愈合实验的代表性图片和伤口愈合率分析(n＝3)。***p<0.001。
图11A和图11C结果表明，PBS对照组的B16F10细胞孵育24h后几乎铺满了整个划痕，伤口愈合率为82.3％。LMWH组的伤口愈合率较PBS组明显降低，表明LMWH可以降低B16F10细胞的迁移能力。由于DOX的杀细胞效应，DOX组的伤口愈合速度也下降了。此外LMWH和DOX的共同作用，RCLD或CLD纳米粒对肿瘤细胞的迁移表现出最强的抑制作用。
两种负载阿霉素的载体组合物的Transwell细胞侵袭实验：将处于指数生长期的B16F10细胞消化，将细胞悬液1000rpm离心3min，弃去上清液，将下层细胞分散在含0.5％FBS的培养基中。在transwell小室的上层预先包被上基质胶，然后加入1×10 5个B16F10细胞，再加入100μL样品，分别为DOX、LMWH、实施例2制备的LMWH-COF与LMWH-GS5-COF,实施例3制备的RCLD或CLD(使DOX的终浓度为0.5μg/mL)。在小室的下层加入600μL含20％FBS的培养基作为趋化因子。37℃孵育48h后，吸取上室内的培养液，用棉签擦去上室内的基质胶和细胞，再用PBS洗涤2遍。将小室的膜用4％多聚甲醛室温下固定10min，PBS洗涤2遍。将膜风干后，用0.1％结晶紫室温下染色30min，侵袭到下层的细胞会被结晶紫染色。将小室的膜风干后，在倒置显微镜下拍照，放大倍数为5倍，定性观察侵袭到膜下层的细胞。然后用33％的乙酸将下层膜上的结晶紫溶解下来，在570nm处测定OD值，定量研究侵袭到膜下层的细胞，评价两种负载阿霉素的载体组合物对细胞侵袭能力的影响。
图11B和图11D为CLD与RCLD细胞侵袭实验的代表性图片和定量相对侵袭率(n＝3)。***p<0.001。
图11B和图11D结果表明RCLD或CLD能显著抑制B16F10肿瘤细胞的侵袭，比同等剂量的DOX的抑制效果好。
(5)两种负载盐酸阿霉素的载体组合物的药效学研究
两种负载盐酸阿霉素的载体组合物的肺癌靶向性和生物分布研究：使用Cy5探针对CLD和RCLD纳米粒进行荧光标记。然后构建转移性黑色素肺癌模型研究纳米粒以上两种负载阿霉素的载体组合物在体内的肺癌靶向性和生物分布。具体地，将2×10 5个B16F10细胞分散到100μL的PBS中，尾静脉注射到C57BL/6小鼠体内，以构建黑色素瘤肺癌转移模型。在注射B16F10细胞后的第15天，分别向 小鼠尾静脉注射PBS、Cy5标记的RCLD或CLD纳米粒(n＝3)。注射RCLD或CLD纳米粒2h后，采用异氟烷将小鼠麻醉，采用IVIS活体观察RCLD对肺部肿瘤的靶向性。然后将小鼠处死，解剖取出心、肝、脾、肺和肾器官，并进行离体的荧光成像以研究纳米粒以上两种负载阿霉素的载体组合物的体内生物分布。
图12为实施例3中CLD与RCLD的肺癌靶向性和生物分布，其中图12A为RCLD和CLD在B16F10肺癌转移中的体内分布图，图12B为肺组织的荧光强度分析，C为小鼠器官的离体成像，图12D为RCLD与CLD在小鼠心、肝、脾、肺、肾的定量荧光强度分析。
图12A-D结果表明在未经GS5表面功能化的情况下，CLD实现了对肺癌的高度靶向，这是因为COF载体自身具有高度的肺靶向功能。经过GS5修饰之后，RCLD组在肺部的荧光强度是CLD组的1.9倍，由于具有COF载体和GS5的双重靶向作用而表现出更高效的肺癌靶向能力。此外，静脉注射之后，RCLD纳米粒主要分布在肺部，其在肺组织中的荧光强度是肝脏的5.8倍。
两种负载盐酸阿霉素的载体组合物的肺癌治疗效果评价：
两种负载盐酸阿霉素的载体组合物治疗A549转移性肺癌：将1×10 6个A549细胞分散到200μL的PBS中，尾静脉注射到BALB/c小鼠体内，构建人肺癌转移瘤模型。在注射A549细胞后第7天，将小鼠随机分为6组，分别给予PBS、游离DOX2.5(剂量为2.5mg/kg)、游离LMWH、CLD(DOX的剂量为1mg/kg)、RCLD(DOX剂量分别为1mg/kg和0.5mg/kg)，每3天给药一次，连续给药5次。整个实验过程每天监测小鼠的体重变化。在第32天，将小鼠处死，解剖取出肺组织，统计肺部肿瘤转移结节的数量，拍照记录。同时对肺组织和其他主要器官进行H&E染色分析。
图13为实施例3中CLD与RCLD在A549肺癌模型中的抗肿瘤效果。(A)从A549肺癌小鼠模型中收集的肺部图片；(B)RCLD的治疗方案；(C)肺部A549转移性结节数目的定量分析(n＝5)；(D)肺组织的H&E染色分析。*p<0.05,***p<0.001,ns表示两组之间没有显著性差异。
图13A-D结果表明，负载DOX的CLD和RCLD组表现出较强的抗肿瘤作用，这不仅是因为LMWH能够抑制癌细胞的迁移和侵袭，DOX也能直接杀伤肿瘤细胞。值得一提的是，RCLD组(DOX的剂量为1mg/kg)对肺癌的治疗效果最好，在RCLD组(DOX的剂量为1mg/kg)中几乎看不到肺部的转移结节，得益于RGD靶头修饰能够增加RCLD在肿瘤部位的靶向分布和滞留时间。即使DOX 2.5组(DOX剂量为2.5mg/kg)与RCLD 0.5组(DOX剂量为0.5mg/kg)的肺部转移结节的数量相近(图C)，但是H&E染色结果显示，RCLD 0.5组肺转移结节的尺寸明显小于DOX组(图D)。
图14为实施例3中荷A549的BALB/c小鼠的体重变化。其中，除了DOX 2.5剂量组之外的各组小鼠的体重无明显区别。DOX 2.5剂量组小鼠的体重有所下降， 可能是因为DOX的毒性所致。
两种负载盐酸阿霉素的载体组合物治疗B16F10黑色素瘤的肺部转移性肺癌：将处于指数生长期的B16F10细胞消化，将细胞悬液1000rpm离心3min，弃去上清液，向下层细胞内加入无菌PBS，使细胞浓度为2×10 5个/mL。用胰岛素注射器吸取100μL细胞悬液，尾静脉注射到C57BL/6小鼠体内，构建小鼠黑色素瘤肺转移模型。待肿瘤细胞接种的第2天，将小鼠随机分为6组：PBS组、LMWH组、2.5mg/kg DOX组、CLD组(DOX的剂量为1mg/kg)、RCLD组(DOX剂量分别为1mg/kg和0.5mg/kg)，尾静脉注射给药200μL药物，每三天给药一次。给药后小鼠正常饲养，每天称量小鼠体重，给药五次后处死动物，解剖取出肺组织，拍照并用Image-Pro-Plus软件计算肺转移面积。
图15为实施例3中RCLD与CLD在B16F10转移性肺癌模型中的治疗效果。(A)从B16F10转移性肺癌小鼠模型中收集的肺部图片；(B)RCLD的治疗方案；(C)B16F10转移性肺癌的面积(n＝5)。**p<0.01,***p<0.001,ns代表两组之间没有显著性差异。
图15结果表明：RCLD组(DOX剂量为1mg/kg)能显著抑制黑色素瘤的肺转移，给药组几乎看不到肺转移结节，将肺转移面积减少70％，源于LMWH和DOX的协同抗肿瘤作用。
图16为实施例3中B16F10转移性肺癌模型小鼠治疗结束后，主要器官的H&E染色分析。黑色箭头指向萎缩的心肌细胞。
图16结果表明：游离DOX显示出明显的心脏毒性，而RCLD组的主要器官并未发生组织结构上的变化，说明给予RCLD多次治疗不会造成器官的急性毒性或组织坏死，这是因为RCLD靶向递药平台降低了DOX的剂量和心脏毒性。
(6)两种负载阿霉素的载体组合物的体内安全性评价：分别向健康的雄性C57BL/6小鼠尾静脉注射PBS、游离DOX(剂量为2.5mg/kg)、游离LMWH、CLD(DOX的剂量为1mg/kg)、RCLD(DOX剂量分别为1mg/kg和0.5mg/kg)，每3天给药一次，连续给药5次。最后一次给药24h之后，以EDTAK 2为抗凝剂收集全血，进行血常规检查，并采集血清用于生化分析。
图17为实施例3中静脉注射RCLD和CLD五次后，小鼠的血细胞分析和血清生化分析(n＝4)。*p<0.05,**p<0.01,***p<0.001。
图17结果表明小鼠的白细胞，红细胞和血小板计数都没有明显的变化，表明以上两种负载阿霉素的载体组合物递送***具有良好的生物安全性。
实施例4
LMWH-GS5-COF载不同抗肿瘤药物：分别称取适量紫杉醇、卡培他滨、吉西他滨、卡铂、奥沙利铂、吉非替尼、拓扑替康等抗肿瘤药物，加入适量溶剂超声10 min使其溶解，然后分别加入实施例2中制备的LMWH-GS5-COF，药物与LMWH-GS5-COF的摩尔比为2:1，37℃下400rpm搅拌24h，孵育载药。载药完成后，4000rpm离心5min，用孵育溶剂洗去表面游离的药物，干燥后即得到下层载药LMWH-GS5-COF纳米粒，采用高效液相法测定载药量如表1。
表1 LMWH-GS5-COF负载抗肿瘤药物的载药量
Figure PCTCN2021122163-appb-000003
上述实施例中负载抗肿瘤药物的载体组合物具有较高的载药量，在体外具有较强的细胞毒性，体内具有良好的抗肺癌功效。
实施例5-18
肿瘤靶向多肽接枝COF载体的制备：按摩尔比为1:1称取实施例1中等摩尔比COF与功能性多肽(RGD肽、NGR肽)置于圆底烧瓶中，加入一定体积的第一溶剂(DMF、ACN)中，搅拌均匀后加入一定量的活化剂A(DMAP、EDC、TEA)，置于磁力搅拌器上37℃，400rpm搅拌24h，使COF与功能性多肽充分偶联。反应完成后，4000rpm离心5min，分别用同等体积的第一溶剂和纯水各洗涤2次，-50℃冷冻干燥24h即得功能性多肽修饰的COF，具体制备方案见表2。
表2 肿瘤靶向多肽接枝COF载体的制备
Figure PCTCN2021122163-appb-000004
Figure PCTCN2021122163-appb-000005
上述实施例中功能性多肽组合物为立方形态，粒径约100-500nm。通过HPLC法测得交联环糊精有机骨架COF与所述功能性多肽的质量百分比为1：0.001-0.1。
实施例19-49
按表3所示进行COF的活化和LMWH-COF的制备。
GS5-COF的活化：称取一定量的活化连接臂(N,N'-二琥珀酰亚胺碳酸酯(DSC)、N,N'-羰基二咪唑(CDI)、丁二酰氯)，超声溶解于第三溶剂(乙腈(ACN)、甲酰胺、二甲基甲酰胺(DMF))中，并加入按一定摩尔比加入GS5-COF粉末与活化催化剂(三乙胺(TEA)、吡啶、N-羟基琥珀酰亚胺(NHS))，在第一温度(25℃、40℃、60℃)下反应第一时间(6h、12h、48h)。
LMWH-COF的制备：称取300mg LMWH-CYS，加入适量第四溶剂(乙腈(ACN)、二甲基甲酰胺(DMF)、甲酰胺)溶解，磁力搅拌下溶解，得到LMWH-CYS溶液。将活化后的GS5-COF或COF溶液加入至LMWH-CYS溶液中，在第二温度(25℃、40℃、60℃)下反应第二时间(12h、24h、48h)，得到LMWH-COF粗产物。为了除去未反应的小分子及其副产物，该粗产物先对乙腈/水混合溶剂(4:1,v:v)透析三天，再继续对纯水透析两天，离心，-50℃冷冻干燥即得LMWH-COF。
表3 COF的活化和LMWH-COF的制备
Figure PCTCN2021122163-appb-000006
Figure PCTCN2021122163-appb-000007
上述实施例中终产物LMWH-COF，通过甲苯胺蓝法测得低分子肝素LMWH的百分含量为0.1-1％，且LMWH-COF组合物为立方形态，粒径约100-500nm。
实施例50
(1)负载盐酸拓扑替康
载盐酸拓扑替康的环糊精有机骨架组合物的制备：称取100mg的盐酸拓扑替康(TPT)溶解于2.5mL的纯水中，超声10min使其溶解，然后加入实施例1中制备的环糊精有机骨架COF纳米粒34mg，药物与LMWH-GS5-COF纳米粒的摩尔比为2:1，37℃下避光300rpm搅拌12h，孵育载药。载药完成后，4000rpm离心5min，用纯水洗涤以除去游离的TPT后，得到下层载盐酸拓扑替康的双重修饰载体组合物(TPT@COF)。
药物的载药量通过紫外分光光度法测定。称取适量TPT@COF溶解于0.1M氢氧化钠溶液，液体经0.22μm滤膜过滤后于422nm处测定吸光度，通过实施例3公式(2)测定TPT载药量。结果表明TPT@COF载药量为12％。
载盐酸拓扑替康的双重修饰载体组合物的制备：称取20mg的TPT溶解于2mL的纯水中，超声10min使其溶解，然后加入实施例2中制备的LMWH-GS5-COF纳米粒34mg，药物与LMWH-GS5-COF纳米粒的摩尔比为2:1，25℃下避光300rpm搅拌12h，孵育载药。载药完成后，4000rpm离心5min，用纯水洗涤以除去游离的TPT后，得到下层载盐酸拓扑替康的双重修饰载体组合物(RCLT)。
药物的载药量通过紫外分光光度法测定。称取适量RCLT溶解于0.1M氢氧化钠溶液，液体经0.22μm滤膜过滤后于422nm处测定吸光度，通过实施例3公式(2)测定TPT载药量。结果表明RCLT载药量为18％。
载盐酸拓扑替康的不含GS5的低分子肝素接枝的载体组合物的制备：称取20mg的TPT溶解于2mL的纯水中，超声10min使其溶解，然后分别加入实施例2中制备的LMWH-COF纳米粒34mg，药物与LMWH-COF的摩尔比为1:2，25℃下避 光300rpm搅拌14h，孵育载药。载药完成后，4000rpm离心5min，用纯水洗涤以除去游离的TPT后，得到下层载盐酸拓扑替康的不含RGD的低分子肝素接枝的载体组合物(CLT)。
药物的载药量通过紫外分光光度法测定。称取适量CLT溶解于0.1M氢氧化钠溶液，液体经0.22μm滤膜过滤后于422nm处测定吸光度，通过实施例3公式(2)测定TPT载药量。结果表明CLT载药量为17％。
(2)负载拓扑替康的载体的体外释放考察
载盐酸拓扑替康的环糊精有机骨架组合物体外释放：将一定量的TPT@COF纳米粒分散在40mL磷酸盐缓冲液(PBS，pH＝7.4)中，并在黑暗环境下中以50rpm和37℃孵育不同时间，在预定的时间点(0.25、0.5、1、2、4、6、8、12、24、36、48h)收集1mL的上清液，并补充同等体积的释放介质。纳米粒释放的药物含量通过使用高效液相方法经行评估(n＝3)。
载盐酸拓扑替康的双重修饰载体组合物在不同浓度还原型谷胱甘肽(GSH)条件下的释药行为：将一定量的RCLT纳米粒分散在40mL不同GSH浓度(0、1、10mM)的PBS(pH＝7.4)中，并在黑暗环境下中以50rpm和37℃孵育不同时间，在预定的时间点(0.25、0.5、1、2、4、6、8、12、24、36、48h)收集1mL的上清液，并补充同等体积的释放介质。纳米粒释放的药物含量通过使用高效液相方法经行评估(n＝3)。
图18为实施例50中TPT@COF与RCLT的体外释放评价，结果表明TPT@COF前期释放较快，12h内释放约50％药物，这可能COF外表面吸附的TPT突释所致；后期具有较好的缓释效果，有利于药物浓度维持在治疗窗内。相比于RCLT在不含GSH的PBS释放介质中48h释放48％，RCLT纳米粒在1mM GSH释放介质较快，12h内有50％药物释放，48h释放62％药物；在10mM GSH释放介质中可以快速释放TPT，前1h内60％药物释放，在12h内有80％药物释放，这归功于高浓度GSH切断了肝素与COF间的连接，加快TPT释放，表明RCLT具有显著氧化还原响应性释放特征。
(3)负载拓扑替康的载体组合物的细胞毒性评价
采用CCK-8法考察了以上负载拓扑替康的载体组合物纳米粒(TPT@COF、RCLT和CLT)对B16F10肿瘤细胞的细胞毒性。B16F10细胞培养在DMEM培养基(含10％胎牛血清)中，在37℃，5％的CO 2恒温恒湿孵育器中培养。选择对数生长期的细胞进行实验，将处于对数生长期的细胞以2×10 4个/孔的密度接种于96孔板，每孔终体积200μL，培养24h。去掉培养液，分别加入TPT、TPT@COF、RCLT或CLT，共设置8个不同的浓度，按照TPT的载药量换算使TPT的终浓度设置为0.01、0.1、1、2、5、10、20和50μg·mL -1。同时设置空白组(仅含培养液)和对照组(细胞和培养液)。在培养箱中继续培养24h后，每孔加入CCK-8溶液15μL，37℃ 继续孵育1.5h。终止培养，采用酶标仪检测450nm处的吸光度(A)值，按照实施例2的公式(1)计算细胞存活率(n＝6)。
图19为实施例50中负载拓扑替康的载体组合物的B16F10细胞毒性评价，结果表明：TPT@COF、RCLT与CLT具有较强的细胞毒性，在体外能够显著抑制肿瘤细胞的生长。
(4)负载盐酸拓扑替康的载体组合物治疗B16F10黑色素瘤的肺部转移性肺癌
载盐酸拓扑替康的环糊精有机骨架组合物的抑瘤评价：将处于指数生长期的B16F10细胞消化，将细胞悬液1000rpm离心3min，弃去上清液，向下层细胞内加入无菌PBS，使细胞浓度为2×10 5个/mL。用胰岛素注射器吸取100μL细胞悬液，尾静脉注射到C57BL/6小鼠体内，构建小鼠黑色素瘤肺转移模型。待肿瘤细胞接种的第3天，将小鼠随机分为6组：生理盐水组(Normal saline)、5mg/kg TPT组、TPT@COF组(TPT剂量分别为2.5mg/kg和1mg/kg)，尾静脉注射给药200μL药物，每三天给药一次，共给药5次。给药后小鼠正常饲养，每天称量小鼠体重，种瘤第20后处死动物，解剖取出肺组织，拍照并用Image-Pro-Plus软件计算肺转移面积。
载盐酸拓扑替康的双重修饰载体组合物的抑瘤评价：将处于指数生长期的B16F10细胞消化，将细胞悬液1000rpm离心3min，弃去上清液，向下层细胞内加入无菌PBS，使细胞浓度为2×10 5个/mL。用胰岛素注射器吸取100μL细胞悬液，尾静脉注射到C57BL/6小鼠体内，构建小鼠黑色素瘤肺转移模型。待肿瘤细胞接种的第3天，将小鼠随机分为6组：生理盐水组(Normal sal ine)、LMWH组、5mg/kg TPT组、RLCT组(TPT剂量分别为2.5mg/kg和1mg/kg)，尾静脉注射给药200μL药物，每三天给药一次，共给药5次。给药后小鼠正常饲养，每天称量小鼠体重，种瘤第20后处死动物，解剖取出肺组织，拍照并用Image-Pro-Plus软件计算肺转移面积。
图20为实施例50中TPT@COF与RCLT在B16F10转移性肺癌模型中的治疗效果。(A)经RCLT治疗的B16F10转移性肺癌小鼠模型中收集的肺部图片；(B)TPT@COF与RCLT的治疗方案；(C)经RCLT治疗的B16F10转移性肺癌的面积；(D)经TPT@COF治疗的B16F10转移性肺癌小鼠模型中收集的肺部图片；(E)经TPT@COF治疗的B16F10转移性肺癌的面积(n＝5)。**p<0.01,***p<0.001,ns代表两组之间没有显著性差异。
图20A与图20C结果表明：RCLT 2.5组(TPT剂量为2.5mg/kg)能显著抑制黑色素瘤的肺转移，给药组几乎看不到肺转移结节，将对照组的肺转移面积减少67％，源于LMWH和TPT的协同抗肿瘤作用。RCLT 1组(TPT剂量为1mg/kg)治疗效果与游离药TPT组疗效相当。
图20D与图20E结果表明：TPT@COF 1组(TPT剂量为1mg/kg)治疗效果与游 离药TPT 5mg/kg组疗效相当。而TPT@COF 2.5组(TPT剂量为2.5mg/kg)能显著抑制黑色素瘤的肺转移，将对照组的肺转移面积减少52％，主要归功于COF的肺靶向。
图21显示了经TPT@COF或RCLT治疗的荷B16F10的C57BL/6小鼠的体重变化。(A)经RCLT治疗的荷B16F10的C57BL/6小鼠的体重变化；(B)经TPT@COF治疗的荷B16F10的C57BL/6小鼠的体重变化。
图21显示，在TPT@COF或RCLT治疗期间，小鼠体重变化不明显，表明该治疗方案安全性较好。
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 (10)

  1. 一种LMWH-多肽-COF组合物，其特征在于，所述组合物包含如下组分：
    1)交联环糊精有机骨架COF；
    2)多肽，所述多肽共价连接于所述交联环糊精有机骨架COF；和
    3)低分子肝素LMWH，所述低分子肝素LMWH共价连接于所述交联环糊精有机骨架COF。
  2. 如权利要求1所述的组合物，其特征在于，所述多肽选自下组：整合素结合肽(RGD)、CD13金属肽酶结合肽(NGR)、或其组合。
  3. 如权利要求1所述的组合物，其特征在于，所述低分子肝素LMWH选自下组：低分子肝素、低分子肝素钠、低分子肝素钙、或其组合。
  4. 如权利要求1所述的组合物，其特征在于，所述交联环糊精有机骨架COF与所述多肽的质量比为1:0.001-0.1(较佳地1:0.01-0.1，更佳地1:0.02-0.08)；和/或
    所述交联环糊精有机骨架COF与所述低分子肝素LMWH的质量比为1:0.001-0.05(较佳地1:0.005-0.02，更佳地1:0.008-0.01)。
  5. 一种权利要求1所述的组合物的制备方法，其特征在于，包括如下步骤：
    1)制备多肽修饰的交联环糊精有机骨架COF，包括步骤：在第一溶剂、活化剂A存在下，使多肽与交联环糊精有机骨架COF充分反应，得到多肽修饰的交联环糊精有机骨架COF；
    2)制备胱胺修饰的低分子肝素LMWH，包括步骤：在第二溶剂、活化剂B存在下，使低分子肝素LMWH和胱胺充分反应，得到胱胺修饰的低分子肝素LMWH；
    3)活化多肽修饰的交联环糊精有机骨架COF，包括步骤：在第三溶剂、活化连接臂、活化催化剂存在下，在第一温度下活化处理多肽修饰的交联环糊精有机骨架COF第一时间，得到经活化的多肽修饰的交联环糊精有机骨架COF；
    4)制备LMWH-多肽-COF组合物，包括步骤：在第四溶剂中，使步骤2)所得胱胺修饰的低分子肝素LMWH和步骤3)所得经活化的多肽修饰的交联环糊精有机骨架COF在第二温度下反应第二时间，得到权利要求1所述的组合物。
  6. 如权利要求5所述的方法，其特征在于，所述方法具有选自下组的一个或多个特征：
    1)所述第一溶剂选自下组：二甲基甲酰胺、乙腈、丙酮、或其组合；
    2)所述活化剂A选自下组：4-二甲氨基吡啶、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、三乙胺、N,N'-二琥珀酰亚胺基碳酸酯、N-羟基琥珀酰亚胺、N,N'-羰基二咪唑、或其组合；
    3)所述第二溶剂选自下组：磷酸盐缓冲液、水、或其组合；
    4)所述活化剂B选自下组：1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟 基琥珀酰亚胺、1-羟基苯并***、或其组合；
    5)所述胱胺修饰的低分子肝素LMWH中，胱胺与低分子肝素LMWH的投料质量比为1:10-20(较佳地1:12-18，更佳地1:12-16)；
    6)所述第三溶剂选自下组：乙腈、甲酰胺、二甲基甲酰胺、丙酮、甲醇、或其组合；
    7)所述活化连接臂选自下组：N,N'-二琥珀酰亚胺基碳酸酯、N,N'-羰基二咪唑、丁二酰氯、异氰酸酯、或其组合；
    8)所述活化催化剂选自下组：三乙胺、吡啶、N-羟基丁二酰亚胺、或其组合；
    9)多肽修饰的交联环糊精有机骨架COF、所述活化连接臂和所述活化催化剂的摩尔比例为1:1-10:1-7(较佳地1:1-8:1-5)；
    10)所述第一温度为10-100℃(较佳地20-80℃)；
    11)所述第一时间为3-50h(较佳地5-25h)；
    12)所述第四溶剂选自下组：甲酰胺、二甲基甲酰胺、乙腈、丙酮、甲醇、或其组合；
    13)所述第二温度为10-100℃(较佳地20-80℃)；
    14)所述第二时间为8-60h(较佳地10-50h)。
  7. 一种药物活性成分，其特征在于，所述的药物活性成分包含：
    (1)作为药物载体的权利要求1所述的LMWH-多肽-COF组合物；和
    (2)抗肿瘤药物或靶向肺部的药物，所述抗肿瘤药物或靶向肺部的药物负载于所述药物载体中。
  8. 一种药物组合物，其特征在于，包含：
    (a)权利要求7所述的药物活性成分；和
    (b)药学上可接受的载体。
  9. 一种权利要求7所述的药物活性成分的制备方法，其特征在于，包括步骤：(i)混合作为药物载体的权利要求1所述的LMWH-多肽-COF组合物和抗肿瘤药物，使所述肿瘤药物负载于所述的药物载体，从而得到权利要求7所述的药物活性成分。
  10. 一种交联环糊精有机骨架COF和/或权利要求1所述的LMWH-多肽-COF组合物和/或权利要求7所述的药物活性成分的用途，其特征在于，用于制备预防和/或***的药物或靶向肺部的药物。
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